U.S. patent application number 13/608337 was filed with the patent office on 2015-07-16 for psma antibodies and uses thereof.
This patent application is currently assigned to PSMA Development Company, LLC. The applicant listed for this patent is Gerald P. Donovan, Jason Gardner, Larry Green, Jaspal S. Kang, Dangshe Ma, Paul J. Maddon, William C. Olson, Norbert Schuelke. Invention is credited to Gerald P. Donovan, Jason Gardner, Larry Green, Jaspal S. Kang, Dangshe Ma, Paul J. Maddon, William C. Olson, Norbert Schuelke.
Application Number | 20150197577 13/608337 |
Document ID | / |
Family ID | 46303153 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150197577 |
Kind Code |
A9 |
Maddon; Paul J. ; et
al. |
July 16, 2015 |
PSMA ANTIBODIES AND USES THEREOF
Abstract
The invention includes stable multimeric, particularly dimeric,
forms of PSMA protein, compositions and kits containing dimeric
PSMA protein as well as methods of producing, purifying and using
these compositions. Such methods include methods for eliciting or
enhancing an immune response to cells expressing PSMA, including
methods of producing antibodies to dimeric PSMA, as well as methods
of treating cancer, such as prostate cancer.
Inventors: |
Maddon; Paul J.; (Scarsdale,
NY) ; Donovan; Gerald P.; (Kittery, ME) ;
Olson; William C.; (Yorktown Heights, NY) ; Schuelke;
Norbert; (East Walpole, MA) ; Gardner; Jason;
(Wayne, PA) ; Ma; Dangshe; (Millwood, NY) ;
Kang; Jaspal S.; (Surrey, CA) ; Green; Larry;
(San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Maddon; Paul J.
Donovan; Gerald P.
Olson; William C.
Schuelke; Norbert
Gardner; Jason
Ma; Dangshe
Kang; Jaspal S.
Green; Larry |
Scarsdale
Kittery
Yorktown Heights
East Walpole
Wayne
Millwood
Surrey
San Francisco |
NY
ME
NY
MA
PA
NY
CA |
US
US
US
US
US
US
CA
US |
|
|
Assignee: |
PSMA Development Company,
LLC
Tarrytown
NY
Amgen Fremont Inc.
Thousand Oaks
CA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20140286859 A1 |
September 25, 2014 |
|
|
Family ID: |
46303153 |
Appl. No.: |
13/608337 |
Filed: |
September 10, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12845686 |
Jul 28, 2010 |
8470330 |
|
|
13608337 |
|
|
|
|
10976352 |
Oct 27, 2004 |
|
|
|
12845686 |
|
|
|
|
10695667 |
Oct 27, 2003 |
|
|
|
10976352 |
|
|
|
|
10395894 |
Mar 21, 2003 |
7850971 |
|
|
10695667 |
|
|
|
|
PCT/US02/33944 |
Oct 23, 2002 |
|
|
|
10395894 |
|
|
|
|
60335215 |
Oct 23, 2001 |
|
|
|
60362747 |
Mar 7, 2002 |
|
|
|
60412618 |
Sep 20, 2002 |
|
|
|
Current U.S.
Class: |
424/1.49 ;
424/158.1; 424/178.1; 435/184; 435/320.1; 435/338; 435/375;
435/7.23; 530/389.7; 530/391.3; 530/391.7 |
Current CPC
Class: |
A61K 51/1072 20130101;
C07K 16/40 20130101; A61K 38/1709 20130101; C07K 2317/622 20130101;
G01N 33/57434 20130101; A61K 51/1093 20130101; A61K 51/1096
20130101; A61K 39/0011 20130101; C07K 2317/565 20130101; A61K
39/39558 20130101; A61K 2039/505 20130101; A61K 47/6825 20170801;
C07K 16/3069 20130101; A61K 39/001195 20180801; C07K 14/4748
20130101; A61P 35/00 20180101; C07K 2317/21 20130101; A61K 47/6869
20170801; A61P 37/04 20180101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; A61K 51/10 20060101 A61K051/10; G01N 33/574 20060101
G01N033/574 |
Claims
1. An isolated antibody or an antigen-binding fragment thereof
which specifically binds to an epitope on prostate specific
membrane antigen (PSMA), wherein the antibody or the
antigen-binding fragment thereof competitively inhibits the
specific binding of a second antibody to its target epitope on
PSMA, wherein the second antibody is selected from the group
consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4,
PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA
A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix
4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix
4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix
4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix
4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix
4.304.1, Abgenix 4.78.1, Abgenix 4.152.1, and antibodies
comprising: (a) a heavy chain encoded by a nucleic acid molecule
comprising the coding region or regions of a nucleotide sequence
selected from the group consisting of nucleotide sequences set
forth as SEQ ID NOs: 2-7, and (b) a light chain encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as SEQ ID NOs: 8-13.
2. The isolated antibody or antigen-binding fragment of claim 1,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 2, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
8.
3. The isolated antibody or antigen-binding fragment of claim 1,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 3, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
9.
4. The isolated antibody or antigen-binding fragment of claim 1,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 4, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
10.
5. The isolated antibody or antigen-binding fragment of claim 1,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 5, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
11.
6. The isolated antibody or antigen-binding fragment of claim 1,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 6, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
12.
7. The isolated antibody or antigen-binding fragment of claim 1,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 7, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
13.
8. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody or antigen-binding
fragment thereof is selected from the group consisting of PSMA 3.7,
PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA
10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2,
Abgenix 4.360.3, Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3,
Abgenix 4.16.1, Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2,
Abgenix 4.48.3, Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3,
Abgenix 4.288.1, Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1,
Abgenix 4.232.3, Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1,
Abgenix 4.152.1 and antigen-binding fragments thereof.
9. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody or antigen-binding
fragment thereof is selected from the group consisting of
antibodies comprising: (a) a heavy chain encoded by a nucleic acid
molecule comprising the heavy chain coding region or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as SEQ ID NOs: 2-7, and (b) a light
chain encoded by a nucleic acid molecule comprising the light chain
coding region or regions of a nucleotide sequence selected from the
group consisting of nucleotide sequences set forth as SEQ ID NOs:
8-13, and antigen-binding fragments thereof.
10. The isolated antibody or antigen-binding fragment of claim 9,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 2, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
8.
11. The isolated antibody or antigen-binding fragment of claim 9,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 3, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
9.
12. The isolated antibody or antigen-binding fragment of claim 9,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 4, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
10.
12. The isolated antibody or antigen-binding fragment of claim 9,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 5, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
11.
13. The isolated antibody or antigen-binding fragment of claim 9,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 6, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
12.
14. The isolated antibody or antigen-binding fragment of claim 9,
wherein the second antibody comprises: (a) a heavy chain encoded by
a nucleic acid molecule comprising the coding region or regions of
a nucleotide sequence set forth as SEQ ID NO: 7, and (b) a light
chain encoded by a nucleic acid molecule comprising the coding
region or regions of a nucleotide sequence set forth as SEQ ID NO:
13.
15. An isolated antibody which specifically binds to an
extracellular domain of prostate specific membrane antigen, wherein
the antibody is encoded by a nucleic acid molecule comprising a
nucleotide sequence that is at least about 90% identical to the
nucleotide sequence encoding the antibody of claim 9.
16. The isolated antibody of claim 15, wherein the antibody is
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 95% identical.
17. The isolated antibody of claim 15, wherein the antibody is
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 97% identical.
18. The isolated antibody of claim 15, wherein the antibody is
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 98% identical.
19. The isolated antibody of claim 15, wherein the antibody is
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 99% identical.
20. An antigen-binding fragment of the isolated antibody of claim
9, comprising: (a) a heavy chain variable region encoded by a
nucleic acid molecule comprising the coding regions or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as: SEQ ID NOs: 14, 18, 22, 26 and
30, and (b) a light chain variable region encoded by a nucleic acid
molecule comprising the coding region or region of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as: SEQ ID NOs: 16, 20, 24, 28 and 32.
21. An antigen-binding fragment of the isolated antibody of claim
20, comprising: (a) a heavy chain variable region encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence set forth as SEQ ID NO: 14, and (b) a light
chain variable region encoded by a nucleic acid molecule comprising
the coding region or regions of a nucleotide sequence set forth as
SEQ ID NO: 16.
22. An antigen-binding fragment of the isolated antibody of claim
20, comprising: (a) a heavy chain variable region encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence set forth as SEQ ID NO: 18, and (b) a light
chain variable region encoded by a nucleic acid molecule comprising
the coding region or regions of a nucleotide sequence set forth as
SEQ ID NO: 20.
23. An antigen-binding fragment of the isolated antibody of claim
20, comprising: (a) a heavy chain variable region encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence set forth as SEQ ID NO: 22, and (b) a light
chain variable region encoded by a nucleic acid molecule comprising
the coding region or regions of a nucleotide sequence set forth as
SEQ ID NO: 24.
24. An antigen-binding fragment of the isolated antibody of claim
20, comprising: (a) a heavy chain variable region encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence set forth as SEQ ID NO: 26, and (b) a light
chain variable region encoded by a nucleic acid molecule comprising
the coding region or regions of a nucleotide sequence set forth as
SEQ ID NO: 28.
25. An antigen-binding fragment of the isolated antibody of claim
20, comprising: (a) a heavy chain variable region encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence set forth as SEQ ID NO: 30, and (b) a light
chain variable region encoded by a nucleic acid molecule comprising
the coding region or regions of a nucleotide sequence set forth as
SEQ ID NO: 32.
26. The antigen-binding fragment of the isolated antibody of claim
9, comprising: (a) a heavy chain variable region comprising an
amino acid sequence selected from the group consisting of amino
acid sequences set forth as: SEQ ID NOs: 15, 19, 23, 27 and 31, and
(b) a light chain variable region comprising an amino acid sequence
selected from the group consisting of nucleotide sequences set
forth as: SEQ ID NOs: 17, 21, 25, 29 and 33.
27. An antigen-binding fragment of the isolated antibody of claim
26, comprising: (a) a heavy chain variable region comprising an
amino acid sequence set forth as SEQ ID NO: 15, and (b) a light
chain variable region comprising an amino acid set forth as SEQ ID
NO: 17.
28. An antigen-binding fragment of the isolated antibody of claim
26, comprising: (a) a heavy chain variable region comprising an
amino acid sequence set forth as SEQ ID NO: 19, and (b) a light
chain variable region comprising an amino acid set forth as SEQ ID
NO: 21.
29. An antigen-binding fragment of the isolated antibody of claim
26, comprising: (a) a heavy chain variable region comprising an
amino acid sequence set forth as SEQ ID NO: 23, and (b) a light
chain variable region comprising an amino acid set forth as SEQ ID
NO: 25.
30. An antigen-binding fragment of the isolated antibody of claim
26, comprising: (a) a heavy chain variable region comprising an
amino acid sequence set forth as SEQ ID NO: 27, and (b) a light
chain variable region comprising an amino acid set forth as SEQ ID
NO: 29.
31. An antigen-binding fragment of the isolated antibody of claim
26, comprising: (a) a heavy chain variable region comprising an
amino acid sequence set forth as SEQ ID NO: 31, and (b) a light
chain variable region comprising an amino acid set forth as SEQ ID
NO: 33.
32. An isolated antigen-binding fragment which comprises a CDR of
the antigen-binding fragment according to claim 20 or claim 26.
33. The isolated antigen-binding fragment of claim 32, wherein the
CDR is CDR3.
34. An expression vector comprising an isolated nucleic acid
molecule encoding the isolated antibody or antigen-binding fragment
of any one of claims 1-33.
35. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy chain of AB-PG1-XG1-006 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 2.
36. An expression vector comprising an isolated nucleic acid
molecule encoding the light chain of AB-PG1-XG1-006 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 8.
37. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy and light chains of AB-PG1-XG1-006
encoded by the nucleic acid molecules comprising the coding region
or regions of the nucleotide sequences set forth as SEQ ID NOs: 2
and 8.
38. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy chain of AB-PG1-XG1-026 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 3.
39. An expression vector comprising an isolated nucleic acid
molecule encoding the light chain of AB-PG1-XG1-026 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 9.
40. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy and light chains of AB-PG1-XG1-026
encoded by the nucleic acid molecules comprising the coding region
or regions of the nucleotide sequences set forth as SEQ ID NOs: 3
and 9.
41. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy chain of AB-PG1-XG1-051 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 4.
42. An expression vector comprising an isolated nucleic acid
molecule encoding the light chain of AB-PG1-XG1-051 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 10.
43. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy and light chains of AB-PG1-XG1-051
encoded by the nucleic acid molecules comprising the coding region
or regions of the nucleotide sequences set forth as SEQ ID NOs: 4
and 10.
44. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy chain of AB-PG1-XG1-069 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 5.
45. An expression vector comprising an isolated nucleic acid
molecule encoding the light chain of AB-PG1-XG1-069 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 11.
46. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy and light chains of AB-PG1-XG1-069
encoded by the nucleic acid molecules comprising the coding region
or regions of the nucleotide sequences set forth as SEQ ID NOs: 5
and 11.
47. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy chain of AB-PG1-XG1-077 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 6.
48. An expression vector comprising an isolated nucleic acid
molecule encoding the light chain of AB-PG1-XG1-077 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 12.
49. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy and light chains of AB-PG1-XG1-077
encoded by the nucleic acid molecules comprising the coding region
or regions of the nucleotide sequences set forth as SEQ ID NOs: 6
and 12.
50. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy chain of AB-PG1-XG1-006 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 7.
51. An expression vector comprising an isolated nucleic acid
molecule encoding the light chain of AB-PG1-XG1-006 encoded by a
nucleic acid molecules comprising the coding region or regions of
the nucleotide sequence set forth as SEQ ID NO: 13.
52. An expression vector comprising an isolated nucleic acid
molecule encoding the heavy and light chains of AB-PG1-XG1-006
encoded by the nucleic acid molecules comprising the coding region
or regions of the nucleotide sequences set forth as SEQ ID NOs: 7
and 13.
53. A host cell transformed or transfected by the expression vector
of claim 34.
54. A plasmid which produces the antibody or antigen binding
fragments of any one of claims 1-33
55. The plasmid of claim 54, wherein the plasmid is selected from
the group consisting of: AB-PG1-XG1-006 Heavy Chain (SEQ ID NO: 2),
AB-PG1-XG1-006 Light Chain (SEQ ID NO: 8), AB-PG1-XG1-026 Heavy
Chain (SEQ ID NO: 3), AB-PG1-XG1-026 Light Chain (SEQ ID NO: 9),
AB-PG1-XG1-051 Heavy Chain (SEQ ID NO: 4), AB-PG1-XG1-051 Light
Chain (SEQ ID NO: 10), AB-PG1-XG1-069 Heavy Chain (SEQ ID NO: 5),
AB-PG1-XG1-069 Light Chain (SEQ ID NO: 11), AB-PG1-XG1-077 Heavy
Chain (SEQ ID NO: 6), AB-PG1-XG1-077 Light Chain (SEQ ID NO: 12),
PSMA 10.3 Heavy Chain (SEQ ID NO: 7), and PSMA 10.3 Kappa (SEQ ID
NO: 13).
56. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody or antigen-binding
fragment thereof is selected for its ability to bind live
cells.
57. The isolated antibody or antigen-binding fragment thereof of
claim 56, wherein the cell is a tumor cell.
58. The isolated antibody or antigen-binding fragment thereof of
claim 57, wherein the tumor cell is a prostate tumor cell.
59. The isolated antibody or antigen-binding fragment thereof of
claim 58, wherein the tumor cell is a LNCaP cell.
60. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody or antigen-binding
fragment thereof mediates cytolysis of cells expressing PSMA.
61. The isolated antibody or antigen-binding fragment thereof of
claim 60 wherein cytolysis of cells expressing PSMA is mediated by
effector cells.
62. The isolated antibody or antigen-binding fragment thereof of
claim 60 wherein cytolysis of cells expressing PSMA is complement
mediated in the presence of effector cells.
63. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody or antigen-binding
fragment thereof inhibits the growth of cells expressing PSMA.
64. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody or antigen-binding
fragment thereof does not require cell lysis to bind to the epitope
on PSMA.
65. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody or antigen-binding
fragment thereof is selected from the group consisting of IgG1,
IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE or has
immunoglobulin constant and/or variable domain of IgG1, IgG2, IgG3,
IgG4, IgM, IgA1, IgA2, IgAsec, IgD or IgE.
66. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody is a recombinant
antibody.
67. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody is a polyclonal
antibody.
68. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody is a monoclonal
antibody.
69. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody is a humanized
antibody.
70. The isolated antibody or antigen-binding fragment thereof
according to claim 69, wherein said antibody is a monoclonal
antibody.
71. The isolated antibody or antigen-binding fragment thereof
according to claim 69, wherein said antibody is a polyclonal
antibody.
72. The isolated antibody or antigen-binding fragment thereof
according to claim 69, wherein said antibody is a mixture of
monoclonal and/or polyclonal antibodies.
73. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody is a chimeric
antibody.
74. The isolated antibody or antigen-binding fragment thereof
according to claim 73, wherein said antibody is a monoclonal
antibody.
75. The isolated antibody or antigen-binding fragment thereof
according to claim 73, wherein said antibody is a polyclonal
antibody.
76. The isolated antibody or antigen-binding fragment thereof
according to claim 73, wherein said antibody is a mixture of
monoclonal and/or polyclonal antibodies.
77. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody is a human
antibody.
78. The isolated antibody or antigen-binding fragment thereof
according to claim 77, wherein said antibody is a monoclonal
antibody.
79. The isolated antibody or antigen-binding fragment thereof
according to claim 77, wherein said antibody is a polyclonal
antibody.
80. The isolated antibody or antigen-binding fragment thereof
according to claim 77, wherein said antibody is a mixture of
monoclonal and/or polyclonal antibodies.
81. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein said antibody is a bispecific or
multispecific antibody.
82. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein the isolated antigen-binding fragment
is selected from the group consisting of a Fab fragment, a
F(ab').sub.2 fragment, and a Fv fragment CDR3.
83. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein the antibody is a monoclonal antibody
produced by a hybridoma cell line selected from the group
consisting of PSMA 3.7 (PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258),
PSMA 3.11 (PTA-3269), PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292),
PSMA 7.3 (PTA-3293), PSMA 10.3 (PTA-3247), PSMA 1.8.3 (PTA-3906),
PSMA A3.1.3 (PTA-3904), PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2
(PTA-4427), Abgenix 4.360.3 (PTA-4428), Abgenix 4.7.1 (PTA-4429),
Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3 (PTA-4557), Abgenix
4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358), Abgenix 4.28.3
(PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix 4.48.3 (PTA-4361),
Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3 (PTA-4365), Abgenix
4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367), Abgenix 4.333.1
(PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix 4.153.1 (PTA-4388),
Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3 (PTA-4390), Abgenix
4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652), and Abgenix 4.152.1
(PTA-4653).
84. The isolated antibody or antigen-binding fragment thereof
according to claim 1, wherein the antibody or antigen-binding
fragment thereof binds to a conformational epitope.
85. The isolated monoclonal antibody or antigen-binding fragment
thereof according to claim 1, wherein the antibody or
antigen-binding fragment thereof is internalized into a cell with
the prostate specific membrane antigen.
86. A hybridoma cell line that produces an antibody selected from
the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11,
PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3,
PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1,
Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3,
Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1,
Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1,
Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3,
Abgenix 4.304.1, Abgenix 4.78.1 and Abgenix 4.152.1.
87. The hybridoma cell line of claim 86, wherein the hybridoma cell
line is selected from the group consisting of PSMA 3.7 (PTA-3257),
PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269), PSMA 5.4
(PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA 10.3
(PTA-3247), PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904), PSMA
A3.3.1 (PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3
(PTA-4428), Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556),
Abgenix 4.177.3 (PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix
4.22.3 (PTA-4358), Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2
(PTA-4360), Abgenix 4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362),
Abgenix 4.209.3 (PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix
4.288.1 (PTA-4367), Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1
(PTA-4363), Abgenix 4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389),
Abgenix 4.292.3 (PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix
4.78.1 (PTA-4652), and Abgenix 4.152.1 (PTA-4653).
88. A composition comprising: an antibody or antigen-binding
fragment thereof according to any one of claims 1-33 and a
pharmaceutically acceptable carrier, excipient, or stabilizer.
89. The composition of claim 88, further comprising an antitumor
agent, an immunostimulatory agent, an immunomodulator, or a
combination thereof.
90. The composition of claim 89, wherein the antitumor agent is a
cytotoxic agent, an agent that acts on tumor neovasculature, or a
combination thereof.
91. The composition of claim 89, wherein the immunomodulator is
.alpha.-interferon, .gamma.-interferon, tumor necrosis
factor-.alpha. or a combination thereof.
92. The composition of claim 89, wherein the immunostimulatory
agent is interleukin-2, immunostimulatory oligonucleotides, or a
combination thereof.
93. A composition comprising: a combination of two or more
antibodies or antigen-binding fragments thereof according to any
one of claims 1-33 and a pharmaceutically acceptable carrier,
excipient, or stabilizer.
94. The composition of claim 93, further comprising an antitumor
agent, an immunostimulatory agent, an immunomodulator, or a
combination thereof.
95. The composition of claim 94, wherein the antitumor agent is a
cytotoxic agent, an agent that acts on tumor neovasculature, or a
combination thereof.
96. The composition of claim 94, wherein the immunomodulator is
.alpha.-interferon, .gamma.-interferon, tumor necrosis
factor-.alpha. or a combination thereof.
97. The composition of claim 94, wherein the immunostimulatory
agent is interleukin-2, immunostimulatory oligonucleotides, or a
combination thereof.
98. The isolated antibody or antigen-binding fragment thereof of
claim 1, bound to a label.
99. The isolated monoclonal antibody or antigen-binding fragment
thereof according to claim 98, wherein the label is selected from
the group consisting of a fluorescent label, an enzyme label, a
radioactive label, a nuclear magnetic resonance active label, a
luminescent label, and a chromophore label.
100. A composition comprising: an antibody or antigen-binding
fragment thereof according to claim 98 and a pharmaceutically
acceptable carrier, excipient, or stabilizer.
101. The isolated antibody or antigen-binding fragment thereof of
claim 1, wherein the antibody or antigen-binding fragment thereof
specifically binds cell-surface PSMA and/or rsPSMA with a binding
affinity of about 1.times.10.sup.-9M or less.
102. The isolated antibody or antigen-binding fragment thereof of
claim 101, wherein the binding affinity is about
1.times.10.sup.-10M or less.
103. The isolated antibody or antigen-binding fragment thereof of
claim 102, wherein the binding affinity is about
1.times.10.sup.-11M or less.
104. The isolated antibody or antigen-binding fragment thereof of
claim 101, wherein the binding affinity is less than about
5.times.10.sup.-10M.
105. The isolated antibody or antigen-binding fragment thereof of
claim 1, bound to at least one therapeutic moiety.
106. The isolated antibody or antigen-binding fragment thereof of
claim 105, wherein the antibody or antigen-binding fragment thereof
mediates specific cell killing of PSMA-expressing cells with an
IC.sub.50s of less than about 1.times.10.sup.-10M.
107. The isolated antibody or antigen-binding fragment thereof of
claim 106, wherein the IC.sub.50s is less than about
1.times.10.sup.-11M.
108. The isolated antibody or antigen-binding fragment thereof of
claim 107, wherein the IC.sub.50s is less than about
1.times.10.sup.-12M.
109. The isolated antibody or antigen-binding fragment thereof of
claim 106, wherein the IC.sub.50s is less than about
1.5.times.10.sup.-11M.
110. The isolated antibody or antigen-binding fragment thereof of
claim 105, wherein the therapeutic moiety is a drug.
111. The isolated antibody or antigen-binding fragment thereof of
claim 105, wherein the therapeutic moiety is a replication
selective virus.
112. The isolated antibody or antigen-binding fragment thereof of
claim 110, wherein the drug is a cytotoxic drug.
113. The isolated antibody or antigen-binding fragment thereof of
claim 112, wherein the cytotoxic drug is selected from the group
consisting of: calicheamicin, esperamicin, methotrexate,
doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin
C, cis-platinum, etoposide, bleomycin, 5-fluorouracil,
estramustine, vincristine, etoposide, doxorubicin, paclitaxel,
docetaxel, dolastatin 10, auristatin E and auristatin PHE.
114. The isolated antibody or antigen-binding fragment thereof of
claim 105, wherein the therapeutic moiety is a toxin or a fragment
thereof.
115. The isolated antibody or antigen-binding fragment thereof of
claim 105, wherein the therapeutic moiety is an enzyme or a
fragment thereof.
116. The isolated antibody or antigen-binding fragment thereof of
claim 105, wherein the therapeutic moiety is an immunostimulatory
or immunomodulating agent.
117. The isolated antibody or antigen-binding fragment thereof of
claim 116, wherein the immunostimulatory or immunomodulating agent
is selected from the group consisting of: a cytokine, chemokine and
adjuvant.
118. A composition comprising: the antibody or antigen-binding
fragment of claim 105 and a pharmaceutically acceptable carrier,
excipient, or stabilizer.
119. The isolated antibody or antigen-binding fragment thereof of
1, bound to a radioisotope.
120. The isolated antibody or antigen-binding fragment thereof
according to claim 119, wherein the radioisotope emits .alpha.
radiations.
121. The isolated antibody or antigen-binding fragment thereof
according to claim 119, wherein the radioisotope emits .beta.
radiations.
122. The isolated antibody or antigen-binding fragment thereof
according to claim 119, wherein the radioisotope emits .gamma.
radiations.
123. The isolated antibody or antigen-binding fragment thereof
according to claim 119, wherein the radioisotope is selected from
the group consisting of .sup.225Ac, .sup.211At, .sup.212Bi,
.sup.213Bi, .sup.186Rh, .sup.188Rh, .sup.177Lu, .sup.90Y, .sup.131,
.sup.67Cu, .sup.125I, .sup.123I, .sup.77Br, .sup.153Sm, .sup.166Ho,
.sup.64Cu, .sup.212Pb, .sup.224Ra and .sup.223Ra.
124. A composition comprising the isolated antibody or
antigen-binding fragment thereof of claim 119 and a
pharmaceutically acceptable carrier, excipient, or stabilizer.
125. A kit for detecting prostate cancer for diagnosis, prognosis
or monitoring comprising: the isolated labeled antibody or
antigen-binding fragment thereof of claim 98, and one or more
compounds for detecting the label.
126. A kit according to claim 125, wherein the label is selected
from the group consisting of a fluorescent label, an enzyme label,
a radioactive label, a nuclear magnetic resonance active label, a
luminescent label, and a chromophore label.
127. The isolated antibody or antigen-binding fragment thereof of
any of claims 1, 98, 105 or 119 packaged in lyophilized form.
128. The isolated antibody or antigen-binding fragment thereof of
any of claims 1, 98, 105 or 119 packaged in an aqueous medium.
129. An isolated antibody or an antigen-binding fragment thereof
which specifically binds to an epitope on prostate specific
membrane antigen (PSMA) defined by an antibody selected from the
group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA
5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA B3.1.3, PSMA
B3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix
4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix
4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix
4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix
4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix
4.304.1, Abgenix 4.78.1, Abgenix 4.152.1, and antibodies
comprising: (a) a heavy chain encoded by a nucleic acid molecule
comprising the coding region or regions of a nucleotide sequence
selected from the group consisting of nucleotide sequences set
forth as SEQ ID NOs: 2-7, and (b) a light chain encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as SEQ ID NOs: 8-13.
130. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody or antigen-binding
fragment thereof is selected from the group consisting of PSMA 3.7,
PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA
10.3, PSMA 1.8.3, PSMA B3.1.3, PSMA B3.3.1, Abgenix 4.248.2,
Abgenix 4.360.3, Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3,
Abgenix 4.16.1, Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2,
Abgenix 4.48.3, Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3,
Abgenix 4.288.1, Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1,
Abgenix 4.232.3, Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1,
Abgenix 4.152.1 and antigen-binding fragments thereof.
131. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody or antigen-binding
fragment thereof is selected from the group consisting of
antibodies comprising: (a) a heavy chain encoded by a nucleic acid
molecule comprising the heavy chain coding region or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as SEQ ID NOs: 2-7, and (b) a light
chain encoded by a nucleic acid molecule comprising the light chain
coding region or regions of a nucleotide sequence selected from the
group consisting of nucleotide sequences set forth as SEQ ID NOs:
8-13, and antigen-binding fragments thereof.
132. An isolated antibody which specifically binds to an epitope on
prostate specific membrane antigen, wherein the antibody is encoded
by a nucleic acid molecule comprising a nucleotide sequence that is
at least about 90% identical to the nucleotide sequence encoding
the antibody of claim 131.
133. The isolated antibody of claim 132, wherein the antibody is
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 95% identical.
134. The isolated antibody of claim 132, wherein the antibody is
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 97% identical.
135. The isolated antibody of claim 132, wherein the antibody is
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 98% identical.
136. The isolated antibody of claim 132, wherein the antibody is
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 99% identical.
137. An antigen-binding fragment of the isolated antibody of claim
131, comprising: (a) a heavy chain variable region encoded by a
nucleic acid molecule comprising the coding regions or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as: SEQ ID NOs: 14, 18, 22, 26 and
30, and (b) a light chain variable region encoded by a nucleic acid
molecule comprising the coding region or region of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as: SEQ ID NOs: 16, 20, 24, 28 and 32.
138. The antigen-binding fragment of the isolated antibody of claim
131, comprising: (a) a heavy chain variable region comprising an
amino acid sequence selected from the group consisting of amino
acid sequences set forth as: SEQ ID NOs: 15, 19, 23, 27 and 31, and
(b) a light chain variable region comprising an amino acid sequence
selected from the group consisting of nucleotide sequences set
forth as: SEQ ID NOs: 17, 21, 25, 29 and 33.
139. An isolated antigen-binding fragment which comprises a CDR of
the antigen-binding fragment according to claim 137 or claim
138.
140. The isolated antigen-binding fragment of claim 139, wherein
the CDR is CDR3.
141. An expression vector comprising an isolated nucleic acid
molecule encoding the isolated antibody or antigen-binding fragment
of any one of claims 129-137.
142. A host cell transformed or transfected by the expression
vector of claim 141.
143. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody or antigen-binding
fragment thereof is selected for its ability to bind live
cells.
144. The isolated antibody or antigen-binding fragment thereof of
claim 143, wherein the cell is a tumor cell.
145. The isolated antibody or antigen-binding fragment thereof of
claim 144, wherein the tumor cell is a prostate tumor cell.
146. The isolated antibody or antigen-binding fragment thereof of
claim 145, wherein the tumor cell is a LNCaP cell.
147. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody or antigen-binding
fragment thereof mediates cytolysis of cells expressing PSMA.
148. The isolated antibody or antigen-binding fragment thereof of
claim 147 wherein cytolysis of cells expressing PSMA is mediated by
effector cells.
149. The isolated antibody or antigen-binding fragment thereof of
claim 147 wherein cytolysis of cells expressing PSMA is complement
mediated in the presence of effector cells.
150. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody or antigen-binding
fragment thereof inhibits the growth of cells expressing PSMA.
151. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody or antigen-binding
fragment thereof does not require cell lysis to bind to the epitope
on PSMA.
152. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody or antigen-binding
fragment thereof is selected from the group consisting of IgG1,
IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE or has
immunoglobulin constant and/or variable domain of IgG1, IgG2, IgG3,
IgG4, IgM, IgA1, IgA2, IgAsec, IgA or IgE.
153. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody is a recombinant
antibody.
154. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody is a polyclonal
antibody.
155. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody is a monoclonal
antibody.
156. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody is a humanized
antibody.
157. The isolated antibody or antigen-binding fragment thereof
according to claim 156, wherein said antibody is a monoclonal
antibody.
158. The isolated antibody or antigen-binding fragment thereof
according to claim 156, wherein said antibody is a polyclonal
antibody.
159. The isolated antibody or antigen-binding fragment thereof
according to claim 156, wherein said antibody is a mixture of
monoclonal and/or polyclonal antibodies.
160. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody is a chimeric
antibody.
161. The isolated antibody or antigen-binding fragment thereof
according to claim 160, wherein said antibody is a monoclonal
antibody.
162. The isolated antibody or antigen-binding fragment thereof
according to claim 160, wherein said antibody is a polyclonal
antibody.
163. The isolated antibody or antigen-binding fragment thereof
according to claim 160, wherein said antibody is a mixture of
monoclonal and/or polyclonal antibodies.
164. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody is a human
antibody.
165. The isolated antibody or antigen-binding fragment thereof
according to claim 164, wherein said antibody is a monoclonal
antibody.
166. The isolated antibody or antigen-binding fragment thereof
according to claim 164, wherein said antibody is a polyclonal
antibody.
167. The isolated antibody or antigen-binding fragment thereof
according to claim 164, wherein said antibody is a mixture of
monoclonal and/or polyclonal antibodies.
168. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein said antibody is a bispecific or
multispecific antibody.
169. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein the isolated antigen-binding
fragment is selected from the group consisting of a Fab fragment, a
F(ab')2 fragment, and a Fv fragment CDR3.
170. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein the antibody is a monoclonal
antibody produced by a hybridoma cell line selected from the group
consisting of PSMA 3.7 (PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258),
PSMA 3.11 (PTA-3269), PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292),
PSMA 7.3 (PTA-3293), PSMA 10.3 (PTA-3247), PSMA 1.8.3 (PTA-3906),
PSMA B3.1.3 (PTA-3904), PSMA B3.3.1 (PTA-3905), Abgenix 4.248.2
(PTA-4427), Abgenix 4.360.3 (PTA-4428), Abgenix 4.7.1 (PTA-4429),
Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3 (PTA-4557), Abgenix
4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358), Abgenix 4.28.3
(PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix 4.48.3 (PTA-4361),
Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3 (PTA-4365), Abgenix
4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367), Abgenix 4.333.1
(PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix 4.153.1 (PTA-4388),
Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3 (PTA-4390), Abgenix
4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652), and Abgenix 4.152.1
(PTA-4653).
171. The isolated antibody or antigen-binding fragment thereof
according to claim 129, wherein the antibody or antigen-binding
fragment thereof binds to a conformational epitope.
172. The isolated monoclonal antibody or antigen-binding fragment
thereof according to claim 129, wherein the antibody or
antigen-binding fragment thereof is internalized into a cell with
the prostate specific membrane antigen.
173. A hybridoma cell line that produces an antibody selected from
the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11,
PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA B3.1.3,
PSMA B3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1,
Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3,
Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1,
Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1,
Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3,
Abgenix 4.304.1, Abgenix 4.78.1 and Abgenix 4.152.1.
174. The hybridoma cell line of claim 173, wherein the hybridoma
cell line is selected from the group consisting of PSMA 3.7
(PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269),
PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA
10.3 (PTA-3247), PSMA 1.8.3 (PTA-3906), PSMA B3.1.3 (PTA-3904),
PSMA B3.3.1 (PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3
(PTA-4428), Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556),
Abgenix 4.177.3 (PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix
4.22.3 (PTA-4358), Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2
(PTA-4360), Abgenix 4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362),
Abgenix 4.209.3 (PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix
4.288.1 (PTA-4367), Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1
(PTA-4363), Abgenix 4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389),
Abgenix 4.292.3 (PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix
4.78.1 (PTA-4652), and Abgenix 4.152.1 (PTA-4653).
175. A composition comprising: an antibody or antigen-binding
fragment thereof according to any one of claims 129-140 and a
pharmaceutically acceptable carrier, excipient, or stabilizer.
176. The composition of claim 175, further comprising an antitumor
agent, an immunostimulatory agent, an immunomodulator, or a
combination thereof.
177. The composition of claim 176, wherein the antitumor agent is a
cytotoxic agent, an agent that acts on tumor neovasculature, or a
combination thereof.
178. The composition of claim 176, wherein the immunomodulator is
.alpha.-interferon, .gamma.-interferon, tumor necrosis
factor-.alpha. or a combination thereof.
179. The composition of claim 176, wherein the immunostimulatory
agent is interleukin-2, immunostimulatory oligonucleotides, or a
combination thereof.
180. A composition comprising: a combination of two or more
antibodies or antigen-binding fragments thereof according to any
one of claims 129-140 and a pharmaceutically acceptable carrier,
excipient, or stabilizer.
181. The composition of claim 180, further comprising an antitumor
agent, an immunostimulatory agent, an immunomodulator, or a
combination thereof.
182. The composition of claim 181, wherein the antitumor agent is a
cytotoxic agent, an agent that acts on tumor neovasculature, or a
combination thereof.
183. The composition of claim 181, wherein the immunomodulator is
.alpha.-interferon, .gamma.-interferon, tumor necrosis
factor-.alpha. or a combination thereof.
184. The composition of claim 181, wherein the immunostimulatory
agent is interleukin-2, immunostimulatory oligonucleotides, or a
combination thereof.
185. The isolated antibody or antigen-binding fragment thereof of
claim 129, bound to a label.
186. The isolated monoclonal antibody or antigen-binding fragment
thereof according to claim 185, wherein the label is selected from
the group consisting of a fluorescent label, an enzyme label, a
radioactive label, a nuclear magnetic resonance active label, a
luminescent label, and a chromophore label.
187. A composition comprising: an antibody or antigen-binding
fragment thereof according to claim 185 and a pharmaceutically
acceptable carrier, excipient, or stabilizer.
188. The isolated antibody or antigen-binding fragment thereof of
claim 129, wherein the antibody or antigen-binding fragment thereof
specifically binds cell-surface PSMA and/or rsPSMA with a binding
affinity of about 1.times.10.sup.-9M or less.
189. The isolated antibody or antigen-binding fragment thereof of
claim 188, wherein the binding affinity is about
1.times.10.sup.-10M or less.
190. The isolated antibody or antigen-binding fragment thereof of
claim 189, wherein the binding affinity is about
1.times.10.sup.-11M or less.
191. The isolated antibody or antigen-binding fragment thereof of
claim 188, wherein the binding affinity is less than about
5.times.10.sup.-10M.
192. The isolated antibody or antigen-binding fragment thereof of
claim 129, bound to at least one therapeutic moiety.
193. The isolated antibody or antigen-binding fragment thereof of
claim 192, wherein the antibody or antigen-binding fragment thereof
mediates specific cell killing of PSMA-expressing cells with an
IC.sub.50s of less than about 1.times.10.sup.-10M.
194. The isolated antibody or antigen-binding fragment thereof of
claim 193, wherein the IC.sub.50s is less than about
1.times.10.sup.-11M.
195. The isolated antibody or antigen-binding fragment thereof of
claim 194, wherein the IC.sub.50s is less than about
1.times.10.sup.-12M.
196. The isolated antibody or antigen-binding fragment thereof of
claim 193, wherein the IC.sub.50s is less than about
1.5.times.10.sup.-11M.
197. The isolated antibody or antigen-binding fragment thereof of
claim 192, wherein the therapeutic moiety is a drug.
198. The isolated antibody or antigen-binding fragment thereof of
claim 192, wherein the therapeutic moiety is a
replication-selective virus.
199. The isolated antibody or antigen-binding fragment thereof of
claim 197, wherein the drug is a cytotoxic drug.
200. The isolated antibody or antigen-binding fragment thereof of
claim 199, wherein the cytotoxic drug is selected from the group
consisting of: calicheamicin, esperamicin, methotrexate,
doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin
C, cis-platinum, etoposide, bleomycin, 5-fluorouracil,
estramustine, vincristine, etoposide, doxorubicin, paclitaxel,
docetaxel, dolastatin 10, auristatin E and auristatin PHE.
201. The isolated antibody or antigen-binding fragment thereof of
claim 192, wherein the therapeutic moiety is a toxin or a fragment
thereof.
202. The isolated antibody or antigen-binding fragment thereof of
claim 192, wherein the therapeutic moiety is an enzyme or a
fragment thereof.
203. The isolated antibody or antigen-binding fragment thereof of
claim 192, wherein the therapeutic moiety is an immunostimulatory
or immunomodulating agent.
204. The isolated antibody or antigen-binding fragment thereof of
claim 203, wherein the immunostimulatory or immunomodulating agent
is selected from the group consisting of: a cytokine, chemokine and
adjuvant.
205. A composition comprising: the antibody or antigen-binding
fragment of claim 192 and a pharmaceutically acceptable carrier,
excipient, or stabilizer.
206. The isolated antibody or antigen-binding fragment thereof of
129, bound to a radioisotope.
207. The isolated antibody or antigen-binding fragment thereof
according to claim 206, wherein the radioisotope emits .alpha.
radiations.
208. The isolated antibody or antigen-binding fragment thereof
according to claim 206, wherein the radioisotope emits .beta.
radiations.
209. The isolated antibody or antigen-binding fragment thereof
according to claim 206, wherein the radioisotope emits .gamma.
radiations.
210. The isolated antibody or antigen-binding fragment thereof
according to claim 206, wherein the radioisotope is selected from
the group consisting of .sup.225Ac, .sup.211At, .sup.212Bi,
.sup.213Bi, .sup.186Rh, 188Rh, .sup.177Lu, .sup.90Y, .sup.131I,
.sup.67Cu, .sup.125I, .sup.123I, .sup.77Br, .sup.153Sm, .sup.166Ho,
.sup.64Cu, .sup.212Pb, .sup.224Ra and .sup.223Ra.
211. A composition comprising the isolated antibody or
antigen-binding fragment thereof of claim 206 and a
pharmaceutically acceptable carrier, excipient, or stabilizer.
212. A kit for detecting prostate cancer for diagnosis, prognosis
or monitoring comprising: the isolated labeled antibody or
antigen-binding fragment thereof of claim 185, and one or more
compounds for detecting the label.
213. A kit according to claim 212, wherein the label is selected
from the group consisting of a fluorescent label, an enzyme label,
a radioactive label, a nuclear magnetic resonance active label, a
luminescent label, and a chromophore label.
214. The isolated antibody or antigen-binding fragment thereof of
any of claims 129, 185, 192 or 206 packaged in lyophilized
form.
215. The isolated antibody or antigen-binding fragment thereof of
any of claims 129, 185, 192 or 206 packaged in an aqueous
medium.
216. A method for detecting the presence of PSMA, or a cell
expressing PSMA, in a sample comprising: contacting the sample with
an antibody or antigen-binding fragment thereof according to claim
1 or 129 for a time sufficient to allow the formation of a complex
between the antibody or antigen-binding fragment thereof and PSMA,
and detecting the PSMA-antibody complex or PSMA-antigen-binding
fragment complex, wherein the presence of a complex in the sample
is indicative of the presence in the sample of PSMA or a cell
expressing PSMA.
217. A method for diagnosing a PSMA-mediated disease in a subject
comprising: administering to a subject suspected of having or
previously diagnosed with PSMA-mediated disease an isolated amount
of an antibody or antigen-binding fragment thereof according to
claim 1 or 129, allowing the formation of a complex between the
antibody or antigen-binding fragment thereof and PSMA, detecting
the formation of the PSMA-antibody complex or PSMA-antigen-binding
fragment complex to the target epitope, wherein the presence of a
complex in the subject suspected of having or previously diagnosed
with PSMA-mediated disease is indicative of the presence of a
PSMA-mediated disease.
218. The method of claim 217 wherein the PSMA-mediated disease is
prostate cancer.
219. The method of claim 217 wherein the PSMA-mediated disease is a
non-prostate cancer.
220. The method of claim 219 wherein the non-prostate cancer is
selected from the group consisting of bladder cancer including
transitional cell carcinoma; pancreatic cancer including pancreatic
duct carcinoma; lung cancer including non-small cell lung
carcinoma; kidney cancer including conventional renal cell
carcinoma; sarcoma including soft tissue sarcoma; breast cancer
including breast carcinoma; brain cancer including glioblastoma
multiforme; neuroendocrine carcinoma; colon cancer including
colonic carcinoma; testicular cancer including testicular embryonal
carcinoma; and melanoma including malignant melanoma.
221. The method of claim 216 or claim 217 wherein the antibody or
antigen-binding fragment thereof is labeled.
222. The method of claim 216 or claim 217 wherein a second antibody
is administered to detect the first antibody or antigen-binding
fragment thereof.
223. A method for assessing the prognosis of a subject with a
PSMA-mediated disease: administering to a subject suspected of
having or previously diagnosed with PSMA-mediated disease an
effective amount of an antibody or antigen-binding fragment thereof
according to claim 1 or 129, allowing the formation of a complex
between the antibody or antigen-binding fragment thereof and PSMA,
detecting the formation of the complex to the target epitope,
wherein the amount of the complex in the subject suspected of
having or previously diagnosed with PSMA-mediated disease is
indicative of the prognosis.
224. A method for assessing the effectiveness of a treatment of a
subject with a PSMA-mediated disease: administering to a subject
suspected treated for a PSMA-mediated disease an effective amount
of an antibody or antigen-binding fragment thereof according to
claim 1 or 129, allowing the formation of a complex between the
antibody or antigen-binding fragment thereof and PSMA, detecting
the formation of the complex to the target epitope, wherein the
amount of the complex in the subject suspected of having or
previously diagnosed with PSMA-mediated disease is indicative of
the effectiveness of the treatment.
225. The method of claim 223 or 224 wherein the PSMA-mediated
disease is prostate cancer.
226. The method of claim 223 or 224 wherein the PSMA-mediated
disease is a non-prostate cancer.
227. The method of claim E4 wherein the non-prostate cancer is
selected from the group consisting of bladder cancer including
transitional cell carcinoma; pancreatic cancer including pancreatic
duct carcinoma; lung cancer including non-small cell lung
carcinoma; kidney cancer including conventional renal cell
carcinoma; sarcoma including soft tissue sarcoma; breast cancer
including breast carcinoma; brain cancer including glioblastoma
multiforme; neuroendocrine carcinoma; colon cancer including
colonic carcinoma; testicular cancer including testicular embryonal
carcinoma; and melanoma including malignant melanoma.
228. The method of claim 223 or claim 224 wherein the antibody or
antigen-binding fragment thereof is labeled.
229. The method of claim 223 or claim 224 wherein a second antibody
is administered to detect the first antibody or antigen-binding
fragment thereof.
230. A method for inhibiting the growth of a cell expressing PSMA
comprising: contacting a cell expressing PSMA with an amount of an
antibody or antigen-binding fragment thereof according to claim 1
or 129 which specifically binds to an extracellular domain of PSMA
effective to inhibit the growth of the cell expressing PSMA.
231. A method for inducing cytolysis of a cell expressing PSMA
comprising: contacting a cell expressing PSMA with an amount of an
antibody or antigen-binding fragment thereof according to claim 1
or 129 which specifically binds to an extracellular domain of PSMA
effective to induce cytolysis of the cell expressing PSMA.
232. The method of claim 231 wherein the cytolysis occurs in the
presence of an effector cell.
233. The method of claim 231 wherein the cytolysis is complement
mediated.
234. A method for treating or preventing a PSMA-mediated disease
comprising: administering to a subject having a PSMA-mediated
disease or at risk of having a PSMA-mediated disease an effective
amount of an antibody or antigen-binding fragment thereof according
to claim 1 or 129 to treat or prevent the PSMA-mediated
disease.
235. The method of claim 234 wherein the PSMA-mediated disease is a
cancer.
236. The method for claim 235 wherein the cancer is a prostate
cancer.
237. The method for claim 235 wherein the cancer is a non-prostate
cancer.
238. A method for treating or preventing a PSMA-mediated disease
comprising: administering to a subject having a PSMA-mediated
disease or at risk of having a PSMA-mediated disease an amount of
an antibody or antigen-binding fragment thereof according to claim
1 or 129 effective to treat or prevent the PSMA-mediated
disease.
239. The method of claim 238, wherein the PSMA-mediated disease is
a cancer.
240. The method of claim 239, wherein the cancer is prostate
cancer.
241. The method of claim 239 wherein the cancer is a non-prostate
cancer.
242. The method of claim 241, wherein the non-prostate cancer is
selected from the group consisting of: bladder cancer including
transitional cell carcinoma; pancreatic cancer including pancreatic
duct carcinoma; lung cancer including non-small cell lung
carcinoma; kidney cancer including conventional renal cell
carcinoma; sarcoma including soft tissue sarcoma; breast cancer
including breast carcinoma; brain cancer including glioblastoma
multiforme; neuroendocrine carcinoma; colon cancer including
colonic carcinoma; testicular cancer including testicular embryonal
carcinoma; and melanoma including malignant melanoma.
243. The method of claim 238, further comprising administering
another therapeutic agent to treat or prevent the PSMA-mediated
disease at any time before, during or after the administration of
the antibody or antigen-binding fragment thereof.
244. The method of claim 243, wherein the therapeutic agent is a
vaccine.
245. The method of claim 244, wherein the vaccine immunizes the
subject against PSMA.
246. The method of claim 243, wherein the antibody or
antigen-binding fragment thereof is bound to at least one
therapeutic moiety.
247. The method of claim 246, wherein the therapeutic moiety is a
cytotoxic drug, a drug which acts on the tumor neovasculature and
combinations thereof.
248. The method of claim 247, wherein the cytotoxic drug is
selected from the group consisting of: calicheamicin, esperamicin,
methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C,
vindesine, mitomycin C, cis-platinum, etoposide, bleomycin,
5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin,
paclitaxel, docetaxel, dolastatin 10, auristatin E and auristatin
PHE.
249. The method of claim 246, wherein the antibody or
antigen-binding fragment thereof is bound to a radioisotope,
wherein the radiations emitted by the radioisotope is selected from
the group consisting of .alpha., .beta. and .gamma. radiations.
250. The method of claim 249, wherein the radioisotope is selected
from the group consisting of .sup.225Ac, .sup.211At, .sup.212Bi,
.sup.213Bi, .sup.186Rh, .sup.188Rh, .sup.177Lu, .sup.90Y,
.sup.131I, .sup.67Cu, .sup.125I, .sup.123I, .sup.77Br, .sup.153Sm,
.sup.166Ho, .sup.64Cu, .sup.212Pb, .sup.224Ra and .sup.223Ra.
251. A method for inhibiting folate hydrolase activity comprising:
contacting a folate hydrolase polypeptide with an amount of
isolated antibody or antigen-binding fragment thereof according to
claim 1 or claim 129 under conditions wherein the isolated antibody
or antigen-binding fragment thereof inhibits the folate hydrolase
activity.
252. The method of claim 251 wherein the folate hydrolase
polypeptide is isolated.
253. The method of claim 251 wherein the folate hydrolase
polypeptide is contained in a sample selected from the group
consisting of a cell, a cell homogenate, a tissue, or a tissue
homogenate.
254. The method of claim 251 wherein the folate hydrolase
polypeptide is contained in an organism.
255. The method of claim 254 wherein the organism is an animal.
256. The method of claim 255 wherein the animal is a mammal.
257. A method for enhancing folate hydrolase activity comprising:
contacting a folate hydrolase polypeptide with an amount of
isolated antibody or antigen-binding fragment thereof according to
claim 1 or claim 129 under conditions wherein the isolated antibody
or antigen-binding fragment thereof enhances the folate hydrolase
activity.
258. The method of claim 257 wherein the folate hydrolase
polypeptide is isolated.
259. The method of claim 257 wherein the folate hydrolase
polypeptide is contained in a sample selected from the group
consisting of a cell, a cell homogenate, a tissue, or a tissue
homogenate.
260. The method of claim 257 wherein the folate hydrolase
polypeptide is contained in an organism.
261. The method of claim 260 wherein the organism is an animal.
262. The method of claim 261 wherein the animal is a mammal.
263. A method for inhibiting N-acetylated .alpha.-linked acidic
dipeptidase (NAALADase) activity comprising: contacting a NAALADase
polypeptide with an amount of an isolated antibody or
antigen-binding fragment thereof according to claim 1 or claim 129
under conditions wherein the isolated antibody or antigen-binding
fragment thereof inhibits NAALADase activity.
264. The method for claim 263 wherein the NAALADase polypeptide is
isolated.
265. The method for claim 263 wherein the NAALADase polypeptide is
contained in a sample selected from the group consisting of a cell,
a cell homogenate, a tissue, or a tissue homogenate.
266. The method for claim 263 wherein the NAALADase polypeptide is
contained in an organism.
267. The method for claim 266 wherein the organism is an
animal.
268. The method for claim 267 wherein the animal is a mammal.
269. A method for enhancing N-acetylated .alpha.-linked acidic
dipeptidase (NAALADase) activity comprising: contacting a NAALADase
polypeptide with an amount of an isolated antibody or
antigen-binding fragment thereof according to claim 1 or claim 129
under conditions wherein the isolated antibody or antigen-binding
fragment thereof enhances NAALADase activity.
270. The method for claim 269 wherein the NAALADase polypeptide is
isolated.
271. The method for claim 269 wherein the NAALADase polypeptide is
contained in a sample selected from the group consisting of a cell,
a cell homogenate, a tissue, or a tissue homogenate.
272. The method for claim 269 wherein the NAALADase polypeptide is
contained in an organism.
273. The method for claim 272 wherein the organism is an
animal.
274. The method for claim 273 wherein the animal is a mammal.
275. A method for inhibiting dipeptidyl dipeptidase IV activity
comprising: contacting a dipeptidyl dipeptidase IV polypeptide with
an amount of an isolated antibody or antigen-binding fragment
thereof according to claim 1 or claim 129 under conditions wherein
the isolated antibody or antigen-binding fragment thereof inhibits
dipeptidyl dipeptidase IV activity.
276. The method for claim 275 wherein the dipeptidyl dipeptidase IV
polypeptide is isolated.
277. The method for claim 275 wherein the dipeptidyl dipeptidase IV
polypeptide is contained in a sample selected from the group
consisting of a cell, a cell homogenate, a tissue, or a tissue
homogenate.
278. The method for claim 275 wherein the dipeptidyl dipeptidase IV
polypeptide is contained in an organism.
279. The method for claim 278 wherein the organism is an
animal.
280. The method for claim 279 wherein the animal is a mammal.
281. A method for inhibiting dipeptidyl dipeptidase IV activity
comprising: contacting a dipeptidyl dipeptidase IV polypeptide with
an amount of an isolated antibody or antigen-binding fragment
thereof according to claim 1 or claim 129 under conditions wherein
the isolated antibody or antigen-binding fragment thereof inhibits
dipeptidyl dipeptidase IV activity.
282. The method for claim 281 wherein the dipeptidyl dipeptidase IV
polypeptide is isolated.
283. The method for claim 281 wherein the dipeptidyl dipeptidase IV
polypeptide is contained in a sample selected from the group
consisting of a cell, a cell homogenate, a tissue, or a tissue
homogenate.
284. The method for claim 281 wherein the dipeptidyl dipeptidase IV
polypeptide is contained in an organism.
285. The method for claim 284 wherein the organism is an
animal.
286. The method for claim 285 wherein the animal is a mammal.
287. A method for inhibiting .gamma.-glutamyl hydrolase activity
comprising: contacting a .gamma.-glutamyl hydrolase polypeptide
with an amount of an isolated antibody or antigen-binding fragment
thereof according to claim 1 or claim 129 under conditions wherein
the isolated antibody or antigen-binding fragment thereof inhibits
.gamma.-glutamyl hydrolase activity.
288. The method for claim 287 wherein the .gamma.-glutamyl
hydrolase polypeptide is isolated.
289. The method for claim 287 wherein the .gamma.-glutamyl
hydrolase polypeptide is contained in a sample selected from the
group consisting of a cell, a cell homogenate, a tissue, or a
tissue homogenate.
290. The method for claim 287 wherein the .gamma.-glutamyl
hydrolase polypeptide is contained in an organism.
291. The method for claim 290 wherein the organism is an
animal.
292. The method for claim 291 wherein the animal is a mammal.
293. A method for inhibiting .gamma.-glutamyl hydrolase activity
comprising: contacting a .gamma.-glutamyl hydrolase polypeptide
with an amount of an isolated antibody or antigen-binding fragment
thereof according to claim 1 or claim 129 under conditions wherein
the isolated antibody or antigen-binding fragment thereof inhibits
.gamma.-glutamyl hydrolase activity.
294. The method for claim 293 wherein the .gamma.-glutamyl
hydrolase polypeptide is isolated.
295. The method for claim 293 wherein the .gamma.-glutamyl
hydrolase polypeptide is contained in a sample selected from the
group consisting of a cell, a cell homogenate, a tissue, or a
tissue homogenate.
296. The method for claim 293 wherein the .gamma.-glutamyl
hydrolase polypeptide is contained in an organism.
297. The method for claim 296 wherein the organism is an
animal.
298. The method for claim 297 wherein the animal is a mammal.
299. A method of specific delivery of at least one therapeutic
agent to PSMA-expressing cells, comprising: administering an
effective amount of an antibody or antigen-binding fragment thereof
according to claim 1 or 129 conjugated to the at least one
therapeutic agent.
300. The method of claim 299, wherein the therapeutic agent is a
nucleic acid molecule.
301. The method of claim 299, wherein the therapeutic agent is an
antitumor drug.
302. The method of claim 301, wherein the antitumor drug is
selected from the group consisting of: a cytotoxic drug, a drug
which acts on the tumor neovasculature and combinations
thereof.
303. The method of claim 302, wherein the cytotoxic drug is
selected from the group consisting of: calicheamicin, esperamicin,
methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C,
vindesine, mitomycin C, cis-platinum, etoposide, bleomycin,
5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin,
paclitaxel, docetaxel, dolastatin 10, auristatin E and auristatin
PHE.
304. The method of claim 299, wherein the therapeutic moiety is a
toxin or a fragment thereof.
305. The method of claim 299, wherein the therapeutic moiety is an
enzyme or a fragment thereof.
306. The method of claim 299, wherein the therapeutic moiety is a
replication-selective virus.
307. The method of claim 299, wherein the therapeutic moiety is an
immunostimulatory or immunomodulating agent.
308. The method of claim 307, wherein the immunostimulatory or
immunomodulating agent is selected from the group consisting of: a
cytokine, chemokine and adjuvant.
309. An isolated antibody or antigen-binding fragment thereof that
selectively binds a PSMA protein multimer.
310. The isolated antibody or antigen-binding fragment thereof of
claim 309, wherein the PSMA protein multimer is a dimer.
311. The isolated antibody or antigen-binding fragment thereof of
claim 310, wherein at least one of the PSMA proteins forming the
multimer is a recombinant, soluble PSMA (rsPSMA) polypeptide.
312. The isolated antibody or antigen-binding fragment thereof of
claim 311, wherein the rsPSMA polypeptide consists essentially of
amino acids 44-750 of SEQ ID NO: 1.
313. An isolated antibody or antigen-binding fragment thereof that
selectively binds a PSMA protein multimer, wherein the isolated
antibody inhibits at least one enzymatic activity of the PSMA
protein multimer.
314. The isolated antibody or antigen-binding fragment thereof of
claim 313, wherein the enzymatic activity is selected from the
group consisting of folate hydrolase activity, NAALADase activity,
dipeptidyl dipeptidase IV activity, .gamma.-glutamyl hydrolase
activity and combinations thereof.
315. The isolated antibody or antigen-binding fragment thereof of
claim 313, wherein the enzymatic activity is in the extracellular
domain of the PSMA molecule.
316. The isolated antibody or antigen-binding fragment thereof of
claim 313, wherein the antibody or antigen-binding fragment thereof
specifically binds to an extracellular domain of PSMA.
317. An isolated antibody or antigen-binding fragment thereof that
selectively binds a PSMA protein multimer, wherein the isolated
antibody enhances at least one enzymatic activity of the PSMA
protein multimer.
318. The isolated antibody or antigen-binding fragment thereof of
claim 317, wherein the enzymatic activity is selected from the
group consisting of folate hydrolase activity, NAALADase activity,
dipeptidyl dipeptidase IV activity, .gamma.-glutamyl hydrolase
activity and combinations thereof.
319. The isolated antibody or antigen-binding fragment thereof of
claim 317, wherein the enzymatic activity is in the extracellular
domain of the PSMA molecule.
320. The isolated antibody or antigen-binding fragment thereof of
claim 317, wherein the antibody or antigen-binding fragment thereof
specifically binds to an extracellular domain of PSMA.
321. A composition comprising an isolated antibody or
antigen-binding fragment thereof as in any of claims 309-320, and
an immunostimulatory oligonucleotide.
322. A composition comprising an isolated antibody or
antigen-binding fragment thereof as in any of claims 309-320, and a
cytokine.
323. The composition of claim 322, wherein the cytokine is selected
from the group consisting of IL-2, IL-12, IL-18 and GM-CSF.
324. The composition of any of claims 321-323, further comprising a
pharmaceutically-acceptable carrier.
325. A method for inducing an immune response comprising
administering to a subject in need of such treatment an effective
amount of the isolated antibody or composition of any of claims
309-324.
326. An isolated antibody or antigen-binding fragment thereof that
selectively binds a PSMA protein multimer and inhibits at least one
enzymatic activity of PSMA.
327. The isolated antibody or antigen-binding fragment thereof of
claim 326, wherein the enzyme is selected from the group consisting
of hydrolases and peptidases.
328. The isolated antibody or antigen-binding fragment thereof of
claim 327, wherein the hydrolase is selected from the group
consisting of folate hydrolase and .gamma.-glutamyl hydrolase.
329. The isolated antibody or antigen-binding fragment thereof of
claim 328, wherein the hydrolase is folate hydrolase and the
antibody is mAb 5.4 or mAb 3.9.
330. The isolated antibody or antigen-binding fragment thereof of
claim 327, wherein the peptidase is selected from the group
consisting of NAALADase and dipeptidyl dipeptidase IV.
331. The isolated antibody or antigen-binding fragment thereof of
claim 326, wherein the enzyme is active in cancer cells and has
lesser activity in normal cells than in cancer cells or no activity
in normal cells.
332. The isolated antibody or antigen-binding fragment thereof of
claim 331, wherein the cancer cells are prostate cancer cells.
333. An isolated antibody or antigen-binding fragment thereof that
selectively binds a PSMA protein multimer and enhances at least one
enzymatic activity of PSMA.
334. The isolated antibody or antigen-binding fragment thereof of
claim 333, wherein the enzyme is selected from the group consisting
of hydrolases and peptidases.
335. The isolated antibody or antigen-binding fragment thereof of
claim 334, wherein the hydrolase is selected from the group
consisting of folate hydrolase and .gamma.-glutamyl hydrolase.
336. The isolated antibody or antigen-binding fragment thereof of
claim 334, wherein the peptidase is selected from the group
consisting of NAALADase and dipeptidyl dipeptidase IV.
337. The isolated antibody or antigen-binding fragment thereof of
claim 333, wherein the enzyme is active in normal cells and has
lesser activity in cancer cells than in normal cells or no activity
in cancer cells.
338. The isolated antibody or antigen-binding fragment thereof of
claim 337, wherein the cancer cells are prostate cancer cells.
339. An isolated antibody or antigen-binding fragment thereof that
selectively binds a PSMA protein multimer, wherein the isolated
antibody is raised by immunizing an animal with a preparation
comprising a PSMA protein multimer.
340. A composition comprising the isolated antibody or
antigen-binding fragment thereof of claims 326-339, and a
pharmaceutically acceptable carrier.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 12/845,686 filed Jul. 28, 2010, pending, which
is a continuation application of U.S. application Ser. No.
10/976,352 filed Oct. 27, 2004, abandoned, which is a
continuation-in-part of U.S. application Ser. No. 10/695,667 filed
Oct. 27, 2003, abandoned, which is a continuation-in-part of U.S.
application Ser. No. 10/395,894 filed Mar. 21, 2003, granted, which
is a continuation-in-part of International Application No.
PCT/US02/33944 designating the United States, filed Oct. 23, 2002,
which claims the benefit under 35 U.S.C. .sctn.119 of U.S.
Application No. 60/335,215 filed Oct. 23, 2001, expired, U.S.
Application No. 60/362,747 filed Mar. 7, 2002, expired, and U.S.
Application No. 60/412,618 filed Sep. 20, 2002, expired, the entire
contents of each of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to the field of cancer
associated polypeptides and formulations of and kits including
these polypeptides. In particular, the invention relates, in part,
to formulations of multimeric forms of PSMA proteins, particularly
dimeric PSMA, and methods of their processing, purification,
production and use.
BACKGROUND OF THE INVENTION
[0003] Prostate cancer is the most prevalent type of cancer and the
second leading cause of death from cancer in American men, with an
estimated 179,000 cases and 37,000 deaths in 1999, (Landis, S. H.
et al. CA Cancer J. Clin. 48:6-29 (1998)). The number of men
diagnosed with prostate cancer is steadily increasing as a result
of the increasing population of older men as well as a greater
awareness of the disease leading to its earlier diagnosis (Parker
et al., 1997, CA Cancer J. Clin. 47:5-280). The life time risk for
men developing prostate cancer is about 1 in 5 for Caucasians, 1 in
6 for African Americans. High risk groups are represented by those
with a positive family history of prostate cancer or African
Americans.
[0004] Over a lifetime, more than 2/3 of the men diagnosed with
prostate cancer die of the disease (Wingo et al., 1996, CA Cancer
J. Clin. 46:113-25). Moreover, many patients who do not succumb to
prostate cancer require continuous treatment to ameliorate symptoms
such as pain, bleeding and urinary obstruction. Thus, prostate
cancer also represents a major cause of suffering and increased
health care expenditures.
[0005] Where prostate cancer is localized and the patient's life
expectancy is 10 years or more, radical prostatectomy offers the
best chance for eradication of the disease. Historically, the
drawback of this procedure is that most cancers had spread beyond
the bounds of the operation by the time they were detected.
Patients with bulky, high-grade tumors are less likely to be
successfully treated by radical prostatectomy.
[0006] Radiation therapy has also been widely used as an
alternative to radical prostatectomy. Patients generally treated by
radiation therapy are those who are older and less healthy and
those with higher-grade, more clinically advanced tumors.
Particularly preferred procedures are external-beam therapy which
involves three dimensional, confocal radiation therapy where the
field of radiation is designed to conform to the volume of tissue
treated; interstitial-radiation therapy where seeds of radioactive
compounds are implanted using ultrasound guidance; and a
combination of external-beam therapy and interstitial-radiation
therapy.
[0007] For treatment of patients with locally advanced disease,
hormonal therapy before or following radical prostatectomy or
radiation therapy has been utilized. Hormonal therapy is the main
form of treating men with disseminated prostate cancer. Orchiectomy
reduces serum testosterone concentrations, while estrogen treatment
is similarly beneficial. Diethylstilbestrol from estrogen is
another useful hormonal therapy which has a disadvantage of causing
cardiovascular toxicity. When gonadotropin-releasing hormone
agonists are administered testosterone concentrations are
ultimately reduced. Flutamide and other nonsteroidal, anti-androgen
agents block binding of testosterone to its intracellular
receptors. As a result, it blocks the effect of testosterone,
increasing serum testosterone concentrations and allows patients to
remain potent--a significant problem after radical prostatectomy
and radiation treatments.
[0008] Cytotoxic chemotherapy is largely ineffective in treating
prostate cancer. Its toxicity makes such therapy unsuitable for
elderly patients. In addition, prostate cancer is relatively
resistant to cytotoxic agents.
[0009] Relapsed or more advanced disease is also treated with
anti-androgen therapy. Unfortunately, almost all tumors become
hormone-resistant and progress rapidly in the absence of any
effective therapy.
[0010] Accordingly, there is a need for effective therapeutics for
prostate cancer which are not overwhelmingly toxic to normal
tissues of a patient, and which are effective in selectively
eliminating prostate cancer cells.
SUMMARY OF THE INVENTION
[0011] The present invention relates, in part, to multimeric,
particularly dimeric, forms of PSMA protein, compositions and kits
containing dimeric PSMA protein as well as methods of producing,
purifying, processing and using these compositions.
[0012] In one aspect compositions comprising multimeric forms of
PSMA protein are provided. In some embodiments, these compositions
contain isolated PSMA protein, at least 5% of which is in the form
of PSMA protein multimer. In other embodiments at least 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or more of the isolated PSMA protein is in the form
of a PSMA protein multimer. In other embodiments the PSMA protein
multimer is a PSMA protein dimer, wherein the PSMA protein dimer is
formed by the covalent or non-covalent association of two PSMA
proteins. In some embodiments the PSMA protein dimer is engineered
to form a stable PSMA dimer through covalent bonds. In some
embodiments the covalent bonds are disulfide bonds. Preferably, the
PSMA protein dimer is associated in the same way as that of native
PSMA dimer or is associated in such a way as to form at least one
antigenic epitope that can be used to generate antibodies that
recognize the native PSMA dimer. These antibodies, preferably,
recognize the native PSMA dimer and not PSMA monomer or recognize
the native PSMA dimer with greater specificity. In some embodiments
of the invention the percent dimer can be calculated in terms of
the number of PSMA protein molecules in the dimeric form versus the
total number of PSMA protein (monomer, dimer or other multimer). In
other embodiments the percent dimer can be calculated in terms of
the number of PSMA dimers relative to the number of PSMA monomers,
PSMA dimers and PSMA multimers.
[0013] In some embodiments the PSMA protein multimers comprise the
full-length PSMA protein (SEQ ID NO: 1) or a fragment thereof. In
other embodiments the PSMA protein multimer comprises the
extracellular portion of PSMA (amino acids 44-750 of SEQ ID NO: 1)
or a fragment thereof. In still other embodiments the PSMA protein
multimer comprises the amino acids 58-750 of SEQ ID NO: 1 or a
fragment thereof. In yet other embodiments the PSMA protein
multimer comprises the amino acids 610-750 of SEQ ID NO: 1 or a
fragment thereof. The fragments are capable of forming a PSMA
multimer that can be used to generate antibodies that recognize
PSMA, preferably native PSMA dimer. Typically, the PSMA multimers
are homomultimers, meaning that the two or more PSMA molecules are
the same. In other embodiments, the PSMA multimers are
heteromultimers, whereby at least two of the PSMA proteins are not
the same. In still other embodiments the PSMA proteins can be
functionally equivalent proteins, whereby the PSMA protein is
conservatively substituted.
[0014] In another aspect of the invention compositions comprising
isolated multimeric PSMA protein, wherein the composition comprises
less than 35% of a monomeric PSMA protein are provided. In still
other embodiments the composition comprises less than 20% of the
monomeric PSMA protein. In yet other embodiments the composition
comprises less than 15% of the monomeric PSMA protein. In still
other embodiments the composition comprises less than 5% of the
monomeric PSMA protein. In some preferred embodiments the isolated
multimeric PSMA protein is an isolated dimeric PSMA protein.
[0015] In some aspects of the invention, agents and compositions
thereof that preserve or promote multimeric association of PSMA,
particularly dimeric association, are provided. In some embodiments
these agents include metal ions, salts, or pH adjusting agents.
These agents that preserve or promote multimeric PSMA associations
can do so individually or do so in combination. Therefore, in
another aspect of the invention, a composition comprising PSMA
protein multimers in conjunction with metal ion are provided. In
some embodiments these compositions comprise at least 0.25 molar
equivalents of metal ion to PSMA protein (total PSMA protein
regardless of its form). In other embodiments at least 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.3, 1.5, 1.7, 2, 3, 4, 5, or more
molar equivalents of metal ion to PSMA protein are present in the
composition. In other embodiments the metal ion is in molar excess
to PSMA protein. In some preferred embodiments the compositions
provided are free of chelating agents.
[0016] In yet another aspect of the invention compositions
comprising PSMA protein in a solution that promotes or preserves
multimeric association of PSMA protein are provided. In some
embodiments the solution that promotes or preserves multimeric
association of PSMA protein is a solution that promotes or
preserves dimeric association of PSMA protein. In other embodiments
the solution that promotes or preserves dimeric association of PSMA
protein has a pH that ranges from 4 to 8. In still other
embodiments the solution that promotes or preserves dimeric
association of PSMA protein has a pH that ranges from 5 to 7. Other
embodiments include compositions wherein the solution that promotes
or preserves dimeric association of PSMA protein has a pH that
ranges from 5.5 to 7. In still other embodiments the solution that
promotes or preserves dimeric association of PSMA protein has a pH
of 6.
[0017] In still another aspect of the invention compositions
comprising PSMA protein in a solution that promotes or preserves
multimeric association of PSMA protein, wherein the solution
comprises a salt, are provided. In some embodiments, the cationic
component of the salt is sodium, potassium, ammonium, magnesium,
calcium, zinc or a combination thereof, and the anionic component
of the salt is chloride, sulfate, acetate or a combination thereof.
In preferred embodiments the salt is sodium chloride, sodium
sulfate, sodium acetate or ammonium sulfate. In some embodiments
the salt is present at a concentration in the range of 50 mM to 2M.
In other embodiments the salt is present at a concentration in the
range of 100 mM to 300 mM. In still other embodiments the salt is
present at a concentration of 150 mM.
[0018] In yet another aspect of the invention a composition
comprising PSMA protein in a solution that promotes or preserves
dimeric association of PSMA protein, wherein the solution comprises
metal ions, are provided. In some embodiments the metal ions are
zinc ions, calcium ions, magnesium ions, cobalt ions, manganese
ions or a combination thereof.
[0019] In still other embodiments the metal ions are zinc ions and
calcium ions. In yet other embodiments the zinc ions and calcium
ions are present at a concentration in the range of 0.1 mM to 5 mM.
In still other embodiments the zinc ions are present at a
concentration that is lower than the concentration of the calcium
ions. In some embodiments the zinc ions are present at a
concentration of 0.1 mM and the calcium ions are present at a
concentration of 1 mM. In other embodiments the metal ions are
magnesium ions. In some of these embodiments the magnesium ions are
present at a concentration in the range of 0.1 mM to 5 mM. In other
embodiments the magnesium ions are present at a concentration of
0.5 mM. In another embodiment the metal ions are magnesium and
calcium ions. In a preferred embodiment the compositions are free
of chelating agents.
[0020] In still a further aspect of the invention a composition
comprising isolated PSMA protein in a solution that promotes or
preserves dimeric association of PSMA protein wherein the solution
comprises (a) 5 to 20 mM of sodium phosphate, sodium acetate or a
combination thereof, (b) 100 to 300 mM sodium chloride or sodium
sulfate, and (c) 0.1 to 2 mM of at least one metal ion is provided.
In one embodiment the solution has a pH in the range of 4 to 8. In
another embodiment the solution has a pH in a range of 5 to 7. In
still another embodiment the solution has a pH in a range of 6 to
6.5. The metal ion in some embodiments is a zinc ion, calcium ion,
magnesium ion, cobalt ion, manganese ion or a combination
thereof.
[0021] In still another aspect of the invention a composition
comprising isolated PSMA protein in a solution that promotes or
preserves dimeric association of PSMA protein wherein the solution
comprises (a) 1.47 mM potassium phosphate, monobasic, (b) 8.1 mM
sodium phosphate, dibasic, (c) 2.68 mM potassium chloride, (d) 0.14
M sodium chloride, (e) 0.9 mM calcium chloride, and (f) 0.49 mM
magnesium chloride; and wherein the solution has a pH of 7.0 is
provided. In one embodiment the isolated PSMA protein is at a
concentration of between 0.2 mg/mL and 10 mg/mL. In still a further
embodiment the isolated PSMA protein is at a concentration of
between 2 mg/mL and 5 mg/mL. In other embodiments the isolated PSMA
protein is at a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.5, 3, 3.5, 4,
4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14,
15, 17, 20, 22, 25, 30, 35, 40, 45, 50 mg/mL or more. In another
embodiment the isolated PSMA protein is at a concentration of 0.2
mg/mL. In still a further embodiment the isolated PSMA protein is
at a concentration of 2 mg/mL. In some embodiments the compositions
provided further comprise an adjuvant. In one embodiment the
adjuvant is a saponin-based adjuvant. In another embodiment the
saponin-based adjuvant is QS-21. The adjuvant in some embodiments
is in an amount of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 175, 200,
225, 250, 300 .mu.g or more. In one embodiment the QS-21 is in an
amount of between 50 .mu.g and 150 .mu.g. In another embodiment the
QS-21 is in an amount of 50 .mu.g. In another embodiment the QS-21
is in an amount of 100 .mu.g. When such compositions are
administered to a subject, in some embodiments, the amount of the
adjuvant is the amount of the adjuvant per dose to the subject.
[0022] In another aspect of the invention a composition comprising
PSMA protein which also comprises an agent that promotes or
preserves multimeric association, particularly dimeric association
of PSMA protein, is provided, wherein the composition is stable
when stored at -80.degree. C. In other aspects of the invention the
composition is stable when stored at -20.degree. C. In still other
aspects the composition is stable when stored at 4.degree. C. In
yet another aspect of the invention the composition is stable when
stored at room temperature.
[0023] Another aspect of the invention provides a method of
promoting or preserving dimeric association of PSMA protein in a
solution by obtaining a solution of PSMA protein, and adjusting the
pH to be in the range of 4 to 8. In some embodiments the pH is
adjusted to be in the range of 5 to 7. In other embodiments the pH
is adjusted to be in the range of 5.5 to 7. In yet other
embodiments the pH is adjusted to be 6.
[0024] In another aspect of the invention a method of processing a
PSMA protein by contacting the PSMA protein in a solution with a
first agent that promotes or preserves dimeric association of PSMA
protein in an amount effective to promote or preserve PSMA protein
dimer formation is provided. In some embodiments the amount
effective to promote or preserve PSMA protein dimer formation is
enough to promote or maintain at least 5% of the PSMA protein in
the solution in dimer form. In other embodiments at least 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or more of the PSMA protein in the solution is in
dimer form. The percentage of the dimer form of PSMA is calculated
in terms of the total amount of the various forms of PSMA protein.
In other words the percentage is calculated according to the number
of PSMA dimers relative to the number of PSMA monomers, dimers and
other multimers. In some embodiments the first agent that promotes
or preserves dimeric association of PSMA protein is a salt, metal
ion or a pH adjusting agent. The cationic components of the salt
can include sodium, potassium, ammonium, magnesium, calcium, zinc
or a combination thereof, while the anionic component of the salt
can include chloride, sulfate, acetate or a combination thereof. In
some embodiments the salt is sodium chloride, sodium sulfate,
sodium acetate or ammonium sulfate. In other embodiments the salt
is present at a concentration in the range of 50 mM to 2M. In still
other embodiments the salt is present at a concentration in the
range of 100 mM to 300 mM. In yet other embodiments the salt is
present at a concentration of 150 mM. In some embodiments of the
invention the method further includes combining the PSMA protein
solution with an adjuvant or diluent. The adjuvant or diluent can
be combined with the PSMA protein in an amount to dilute the salt
concentration to 100 mM to 300 mM. In some embodiment the salt
concentration is diluted to 150 mM. In certain embodiments this is
done prior to administering the solution to a subject. In other
embodiments the first agent is a metal ion and the metal ion is a
zinc ion, calcium ion, magnesium ion, cobalt ion, manganese ion or
a combination thereof. In some embodiments the metal ion is a
combination of zinc ion and calcium ion. In still other embodiments
the zinc ion and calcium ion are present at a concentration in the
range of 0.1 mM to 5 mM. In yet other embodiments the zinc ion is
present at a concentration that is lower than the concentration of
the calcium ion. In still further embodiments the zinc ion is
present at a concentration of 0.1 mM, and the calcium ion is
present at a concentration of 1 mM. In other embodiments the metal
ion is a magnesium ion. In some of these embodiments the magnesium
ion is present at a concentration in the range of 0.1 mM to 5 mM.
In still other of these embodiments the magnesium ion is present at
a concentration of 0.5 mM. In the embodiments where the first agent
is a solution of a certain pH, the pH of the solution can be
adjusted to be in the range of 4 to 8. In some embodiments the pH
of the solution is adjusted to be in the range of 5 to 7. In still
other embodiments the pH of the solution is adjusted to be in the
range of 5.5 to 7. In yet other embodiments the pH of the solution
is adjusted to be 6.
[0025] In some embodiments the method further comprises contacting
the PSMA protein with a second agent that promotes or preserves
dimeric association of PSMA protein, wherein the second agent is
different than the first agent. A second agent that is different
than the first agent includes agents that are of a different type
or different class. The second agent, therefore, can be a metal
ion, salt or pH adjusting agent. In some embodiments where the
first agent is a metal ion the second agent can be a salt, pH
adjusting agent or a solution with a certain pH. In other
embodiments the first agent is a salt, and the second agent is a
metal ion, pH adjusting agent or a solution with a certain pH. In
still another embodiment the first agent is a pH adjusting agent or
a solution with a certain pH and the second agent is a metal ion or
a salt. In yet other embodiments the first agent can be a salt,
metal ion, pH adjusting agent or a solution with a certain pH and
the second agent can be of the same class but a different type
within the same class of agents. For instance if the first agent is
a salt such as sodium chloride, the second agent can also be a salt
but a different type, e.g., ammonium sulfate.
[0026] In another aspect of the invention a method of purifying a
sample containing PSMA protein by subjecting the sample containing
PSMA to chromatography in the presence of an agent that preserves
or promotes the dimeric association of PSMA is provided. In some
embodiments the agent that promotes or preserves the dimeric
association of PSMA is a metal ion, a salt or a solution with a pH
in the range of 4 to 8 or a combination thereof. In a preferred
embodiment the metal ion is a combination of calcium ion and
magnesium ion. In one such embodiment the calcium ion and magnesium
ion are each present at a concentration in the range of 0.1 mM to 5
mM. In a further embodiment the calcium ion and magnesium ion are
present at a concentration of 1 mM and 0.5 mM, respectively. In
other embodiments wherein the agent that promotes or preserves the
dimeric association of PSMA is a salt, the salt is present at a
concentration in the range of 50 mM to 2M. In some of these
embodiments the salt is present at a concentration of 2M. In still
other embodiments where the agent that promotes or preserves the
dimeric association of PSMA is a solution with a pH in the range of
4 to 8, the pH of the solution is in the range of 5 to 7. In still
other embodiments the pH of the solution is in the range of 6 to
7.5.
[0027] In other aspects of the invention a method of purifying a
sample containing PSMA protein by applying the sample to a first
column, washing the first column with a first wash solution
containing salt and metal ions, and collecting the PSMA protein
that elutes from the first column is provided. In some embodiments
the salt is ammonium sulfate at a saturation of no more than 35% in
the wash solution.
[0028] In embodiments of the invention the method further comprises
dialyzing or diafiltering the eluted PSMA protein with a first salt
solution at a pH in the range of 6 to 7.5 to yield a dialyzed or
diafiltrated solution containing PSMA protein. In some of these
embodiments the first salt solution has a salt concentration of at
least 5 mM. In still other of these embodiments the first salt
solution is a 10 mM sodium phosphate solution with a pH of 7.
[0029] In still other embodiments of the invention the method
further comprises loading the eluted PSMA protein, dialyzed or
diafiltrated solution containing PSMA protein onto a second column,
washing the second column with a second salt solution, and
collecting the PSMA eluted by the second salt solution. In some
embodiments the second salt solution has a salt concentration of
100 mM to 2M. In certain of these embodiments the second salt
solution is 2M sodium chloride in 10 mM sodium phosphate. In still
other embodiments the second salt solution has a pH in the range of
6 to 7.5.
[0030] In yet another embodiment of the invention the method
further comprises dialyzing or diafiltrating the PSMA eluted by the
second salt solution with a metal ion solution, applying the
dialyzed or diafiltrated PSMA eluted by the second salt solution
onto a third column, washing the third column with a second wash
solution containing salt and metal ions and collecting the PSMA
eluted. In some of these embodiments the pH is maintained in the
range of 6 to 7.5 through all of the purification steps.
[0031] In other embodiments the method further comprises separating
the different forms of PSMA protein, wherein the different forms of
PSMA protein are monomeric, dimeric or other multimeric forms of
PSMA. In some of these embodiments the different forms of PSMA
protein are separated by size exclusion chromatography.
[0032] In yet another aspect of the invention a method of
identifying an agent which promotes or preserves dimeric
association of PSMA protein by determining the amount of a form of
PSMA protein in a sample prior to exposure to a candidate agent,
exposing the sample to the candidate agent, determining the amount
of the form of PSMA protein in the sample after the exposure, and
comparing the amount of the form of PSMA protein in the sample
prior to and after the exposure is provided. In some embodiments
the form of PSMA protein is monomer or dimer. In other embodiments
the form of PSMA can be another multimer form with three or more
associated PSMA proteins.
[0033] In another aspect of the invention a method of treating a
subject to elicit or enhance an immune response to cells in the
subject expressing PSMA, comprising administering to the subject an
effective amount of any of the compositions given herein is
provided. In some embodiments the expressed PSMA is expressed on
the cell surface. In other embodiments the method further comprises
administering one or more booster doses of a composition comprising
PSMA protein. In some of these embodiments the composition
comprising PSMA protein is a composition of PSMA protein dimer. In
still other embodiments the booster dose composition further
comprises an adjuvant. In yet other embodiments the booster dose
composition can be any of the compositions provided herein. In
still other embodiments the composition is administered by
intravenous, intramuscular, subcutaneous, parenteral, spinal,
intradermal or epidermal administration. In this aspect of the
invention the subject has cancer or is at risk of having cancer. In
some embodiments the subject has also been treated for cancer. In
some embodiments the cancer is a primary tumor or is metastatic
cancer. In a preferred embodiment the subject has prostate cancer.
In some embodiments the subject is a non-castrate patient who,
preferably, has received primary therapy, such as prostatectomy or
radiation therapy. In one embodiment the non-castrate patient has a
serum testosterone level that is greater than or equal to 180
ng/mL. In other embodiments the subject is a castrate patient who,
preferably, has completed a course of hormonal therapy. In one
embodiment the castrate patient has a serum testosterone level of
less than 50 ng/mL. In still other embodiments the subject is a
patient who has received a conventional cancer therapy for prostate
cancer.
[0034] In another aspect of the invention methods of treating a
subject with cancer, such as prostate cancer, are provided. Such
methods comprise administering to a subject at least one of the
compositions provided herein. In one embodiment the method
comprises administering to the subject a therapeutically effective
amount of a composition comprising isolated PSMA protein in a
solution that promotes or preserves dimeric association of the PSMA
protein, wherein the composition is effective in treating prostate
cancer. In another embodiment the method further includes the
administration of an adjuvant, which preferably, is contained in
the composition comprising the isolated PSMA protein. In some
embodiments the adjuvant is a saponin-based adjuvant. In other
embodiments the methods further include administering to the
subject a conventional cancer therapy. Conventional cancer therapy
includes, but is not limited to, surgery, radiation, cryosurgery,
thermotherapy, hormone therapy or chemotherapy.
[0035] In still another aspect of the invention a method of
inhibiting metastasis in a subject with cancer is also provided.
One example of such a method includes administering to the subject
a therapeutically effective amount of a composition comprising
isolated PSMA protein in a solution that promotes or preserves
dimeric association of the PSMA protein, wherein the composition is
effective in inhibiting metastasis. In one embodiment the method
also includes the administration of an adjuvant, which preferably,
is a component of the composition of the isolated PSMA protein. In
another embodiment the method further comprises administering to
the subject a conventional prostate cancer therapy.
[0036] In another aspect of the invention a method of eliciting an
immune response by administering to a subject an effective amount
of any of the compositions provided is given. In some embodiments
the method further comprises administering one or more booster
doses of a composition comprising PSMA protein. In certain of these
embodiments the composition comprising PSMA protein is a
composition comprising PSMA protein dimer. In still other
embodiments the booster dose composition is any of the compositions
given herein. In yet another embodiment the booster dose
compositions can also include an adjuvant.
[0037] In other aspects of the invention kits which contain any of
the compositions provided and instructions for use are provided. In
some aspects the kit contains a multimeric composition provided
herein, an adjuvant and instructions for mixing. In other aspects
the kit includes one of the compositions provided herein, a diluent
and instructions for mixing. In some embodiments the composition is
provided in a vial or ampoule with a septum or a syringe. In other
embodiments the composition is in lyophilized form.
[0038] The compositions provided herein can further comprise a
therapeutic agent (e.g., a cytokine, an anti-cancer agent, an
adjuvant, etc.). In some embodiments the adjuvant is alum;
monophosphoryl lipid A; a saponin; a saponin fraction; a
saponin-based adjuvant, such as SAPONIMMUNE; a chemically modified
saponin; QS-7; QS-17; QS-18; QS-21; a polysaccharide-based
adjuvant, such as POLYSACClMMUNE; a synthetic adjuvant, such as
SYNTHIMMUNE; an immunostimulatory oligonucleotide; incomplete
Freund's adjuvant; complete Freund's adjuvant; vitamin E; a
water-in-oil emulsion prepared from a biodegradable oil; MONTANIDE,
such as MONTANIDE ISA51 and MONTANIDE ISA720; Quil A; micellular
mixtures of Quil A and cholesterol known as immunostimulating
complexes (ISCOMS); a MPL and mycobacterial cell wall skeleton
combination; ENHANZYN; RC-529; RC-552; CRL-1005; L-121;
alpha-galactosylceramide; a composition of biodegradable particles
composed of poly-lactide-co-glycolide (PLG) or other similar
polymers; a composition of aluminum or iron oxide beads or a
combination thereof. Other specific examples of adjuvants include
QS-21 fractions, such as crude QA-21, a QA-21H form, QA-21-V1;
QA-21-V2; a combination of QA-21-V1 and QA-21-V2, and chemically
modified forms or combinations thereof. In some embodiments the
preferred adjuvant is QS-21.
[0039] As used herein a "saponin-based adjuvant" is any adjuvant
that is based on or includes a saponin or portion thereof.
Therefore, saponin-based adjuvants include saponins, saponin
fractions and modified saponins (e.g., chemically modified
saponins).
[0040] In another embodiment the compositions provided herein can
also include a cancer therapeutic agent. Cancer therapeutic agents
include any agent used to treat cancer in a subject. In some
embodiments the cancer therapeutic agent is a chemotherapeutic
agent, such as, for example, docetaxel. Cancer therapeutic agents
also include anti-inflammatory agents and immunomodulatory agents.
The anti-inflammatory agent in one embodiment is prednisone. In
another embodiment the immunomodulatory agent is a cytokine.
[0041] In other embodiments the compositions provided can also
include at least one buffer. Buffers include PBS (phosphate
buffered saline), citric acid, sodium citrate, sodium acetate,
acetic acid, sodium phosphate, phosphoric acid, sodium ascorbate,
tartartic acid, maleic acid, glycine, sodium lactate, lactic acid,
ascorbic acid, imidazole, sodium bicarbonate, carbonic acid, sodium
succinate, succinic acid, histidine, sodium benzoate, benzoic acid
and combinations thereof.
[0042] In some embodiments the compositions provided further
include a free amino acid. These free amino acids can be naturally
or non-naturally occurring. In some embodiments the free amino
acids are non-acidic free amino acids. Examples of non-acidic free
amino acids include glycine, proline, isoleucine, leucine, alanine,
arginine and combinations thereof.
[0043] Compositions of PSMA protein multimers including a
surfactant are also provided. Such surfactants include TWEEN20,
TWEEN80, Triton X-100, dodecylmaltoside, cholic acid, CHAPS and
combinations thereof.
[0044] Also provided are compositions of PSMA protein multimers
that comprise a cryoprotectant, an antioxidant, a preservative or a
combination thereof. Examples of cryoprotectants include a sugar, a
polyol, an amino acid, a polymer, an inorganic salt, an organic
salt, trimethylamine N-oxide, sarcosine, betaine,
gamma-aminobutyric acid, octapine, alanopine, strombine,
dimethylsulfoxide and ethanol. When the cryoprotectant is a sugar
the sugar can be sucrose, lactose, glucose, trehalose or maltose.
In other embodiments when the cryoprotectant is a polyol the polyol
can be inositol, ethylene glycol, glycerol, sorbitol, xylitol,
mannitol or 2-methyl-2,4-pentane-diol. When the cryoprotectant is
an amino acid the amino acid can be Na glutamate, proline,
alpha-alanine, beta-alanine, glycine, lysine-HCl or
4-hydroxyproline. When the cryoprotectant is a polymer the polymer
can be polyethylene glycol, dextran or polyvinylpyrrolidone. When
the cryoprotectant is an inorganic salt the cryoprotectant can be
sodium sulfate, ammonium sulfate, potassium phosphate, magnesium
sulfate or sodium fluoride. Finally, when the cryoprotectant is an
organic salt the organic salt can be sodium acetate, sodium
polyethylene, sodium caprylate, proprionate, lactate or succinate.
Examples of antioxidants that are part of these composition in some
embodiments include ascorbic acid, an ascorbic acid derivative,
butylated hydroxy anisole, butylated hydroxy toluene, alkylgallate,
dithiothreitol (DTT), sodium meta-bisulfite, sodium bisulfite,
sodium dithionite, sodium thioglycollic acid, sodium formaldehyde
sulfoxylate, tocopherol, a tocopherol derivative, monothioglycerol
and sodium sulfite. Ascorbic acid derivatives, in some embodiments,
include ascorbylpalmitate, ascorbylstearate, sodium ascorbate and
calcium ascorbate, while tocopherol derivatives include d-alpha
tocopherol, d-alpha tocopherol acetate, dl-alpha tocopherol
acetate, d-alpha tocopherol succinate, beta tocopherol, delta
tocopherol, gamma tocopherol and d-alpha tocopherol polyoxyethylene
glycol 1000 succinate. Examples of preservatives present in the
compositions in some embodiments include benzalkonium chloride,
chlorobutanol, parabens, thimerosal, benzyl alcohol and phenol.
[0045] The composition in some embodiments are physiologically
acceptable compositions.
[0046] The compositions provided are, in some embodiments, in
liquid or lyophilized form.
[0047] In some other embodiments the compositions provided are
sterile.
[0048] In other aspects of the invention pharmaceutical
compositions are provided which contain any of the compositions
provided herein and a pharmaceutically acceptable carrier.
[0049] The present invention also relates, in part, to antibodies
or antigen-binding fragments thereof which specifically bind the
extracellular domain of prostate specific membrane antigen (PSMA),
compositions containing one or a combination of such antibodies or
antigen-binding fragments thereof, hybridoma cell lines that
produce the antibodies, and methods of using the antibodies or
antigen-binding fragments thereof for cancer diagnosis and
treatment.
[0050] According to one aspect of the invention, isolated
antibodies or an antigen-binding fragments thereof are provided.
The antibodies or fragments thereof specifically bind to an
extracellular domain of prostate specific membrane antigen (PSMA),
and competitively inhibit the specific binding of a second antibody
to its target epitope on PSMA. In a second aspect of the invention,
isolated antibodies or antigen-binding fragments thereof are
provided which specifically bind to an epitope on prostate specific
membrane antigen (PSMA) defined by a second antibody. In each of
the forgoing aspects of the invention, the second antibody is
selected from the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9,
PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3,
PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix
4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix
4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix
4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix
4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix
4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, Abgenix 4.152.1, and
antibodies comprising (a) a heavy chain encoded by a nucleic acid
molecule comprising the coding region or regions of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as SEQ ID NOs: 2-7, and (b) a light chain encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as SEQ ID NOs: 8-13.
[0051] In certain embodiments, the antibody or antigen-binding
fragment thereof is selected from the group consisting of PSMA 3.7,
PSMA 3.8, PSMA 3.9, PSMA 3.11 PSMA 5.4, PSMA 7.3, PSMA 10.3, PSMA
1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3,
Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1,
Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3,
Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1,
Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3,
Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, and Abgenix
4.152.1. In other embodiments, the antibody or antigen-binding
fragment thereof is selected from the group consisting of
antibodies comprising (a) a heavy chain encoded by a nucleic acid
molecule comprising the coding region or regions of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as SEQ ID NOs: 2-7, and (b) a light chain encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as SEQ ID NOs: 8-13, and
antigen-binding fragments thereof.
[0052] In further embodiments, the antibody or antigen-binding
fragments thereof is encoded by a nucleic acid molecule comprising
a nucleotide sequence that is at least about 90% identical to the
nucleotide sequence encoding the foregoing antibodies, preferably
at least about 95% identical, more preferably at least about 97%
identical, still more preferably at least about 98% identical, and
most preferably is at least about 99% identical.
[0053] In some embodiments of the foregoing aspects,
antigen-binding fragments of the isolated antibodies are provided.
The antigen-binding fragments include (a) a heavy chain variable
region encoded by a nucleic acid molecule comprising the coding
regions or regions of a nucleotide sequence selected from the group
consisting of nucleotide sequences set forth as: SEQ ID NOs: 14,
18, 22, 26 and 30, and (b) a light chain variable region encoded by
a nucleic acid molecule comprising the coding region or region of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as: SEQ ID NOs: 16, 20, 24, 28 and
32. In other embodiments, the antigen-binding fragment includes (a)
a heavy chain variable region comprising an amino acid sequence
selected from the group consisting of amino acid sequences set
forth as: SEQ ID NOs: 15, 19, 23, 27 and 31, and (b) a light chain
variable region comprising an amino acid sequence selected from the
group consisting of nucleotide sequences set forth as: SEQ ID NOs:
17, 21, 25, 29 and 33. In a further embodiments of the invention,
isolated antigen-binding fragments of antibodies, which include a
CDR of the foregoing antigen-binding fragments are provided.
Preferably the CDR is CDR3.
[0054] According to another aspect of the invention, expression
vectors including an isolated nucleic acid molecule encoding the
foregoing isolated antibodies or antigen-binding fragments are
provided. Host cells transformed or transfected by these expression
vectors also are provided.
[0055] In certain embodiments, the antibody or antigen-binding
fragment thereof is selected for its ability to bind live cells,
such as a tumor cell or a prostate cell, preferably LNCaP cells. In
other embodiments, the antibody or antigen-binding fragment thereof
mediates cytolysis of cells expressing PSMA. Preferably cytolysis
of cells expressing PSMA is mediated by effector cells or is
complement mediated in the presence of effector cells.
[0056] In other embodiments, the antibody or antigen-binding
fragment thereof inhibits the growth of cells expressing PSMA.
Preferably the antibody or antigen-binding fragment thereof does
not require cell lysis to bind to the extracellular domain of
PSMA.
[0057] In further embodiments, the antibody or antigen-binding
fragment thereof is selected from the group consisting of IgG1,
IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE or has
immunoglobulin constant and/or variable domain of IgG1, IgG2, IgG3,
IgG4, IgM, IgA 1, IgA2, IgAsec, IgD or IgE. In other embodiments,
the antibody is a bispecific or multispecific antibody.
[0058] In still other embodiments, the antibody is a recombinant
antibody, a polyclonal antibody, a monoclonal antibody, a humanized
antibody or a chimeric antibody, or a mixture of these. In
particularly preferred embodiments, the antibody is a human
antibody, e.g., a monoclonal antibody, polyclonal antibody or a
mixture of monoclonal and polyclonal antibodies. In still other
embodiments, the antibody is a bispecific or multispecific
antibody.
[0059] Preferred antigen-binding fragments include a Fab fragment,
a F(ab').sub.2 fragment, and a Fv fragment CDR3.
[0060] In further embodiments, the isolated antibody or
antigen-binding fragment is a monoclonal antibody produced by a
hybridoma cell line selected from the group consisting of PSMA 3.7
(PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269),
PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA
10.3 (PTA-3247), PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904),
PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3
(PTA-4428), Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556),
Abgenix 4.177.3 (PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix
4.22.3 (PTA-4358), Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2
(PTA-4360), Abgenix 4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362),
Abgenix 4.209.3 (PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix
4.288.1 (PTA-4367), Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1
(PTA-4363), Abgenix 4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389),
Abgenix 4.292.3 (PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix
4.78.1 (PTA-4652), and Abgenix 4.152.1 (PTA-4653).
[0061] In certain other embodiments, the antibody or
antigen-binding fragment thereof binds to a conformational epitope
and/or is internalized into a cell along with the prostate specific
membrane antigen. In other embodiments, the isolated antibody or
antigen-binding fragment thereof is bound to a label, preferably
one selected from the group consisting of a fluorescent label, an
enzyme label, a radioactive label, a nuclear magnetic resonance
active label, a luminescent label, and a chromophore label.
[0062] In still other embodiments, the isolated antibody or
antigen-binding fragment thereof is bound to at least one
therapeutic moiety, such as a drug, preferably a cytotoxic drug, a
replication-selective virus, a toxin or a fragment thereof, or an
enzyme or a fragment thereof. Preferred cytotoxic drug include:
calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan,
chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum,
etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine,
etoposide, doxorubicin, paclitaxel, docetaxel, dolastatin 10,
auristatin E and auristatin PHE. In other embodiments, the
therapeutic moiety is an immunostimulatory or immunomodulating
agent, preferably one selected from the group consisting of: a
cytokine, chemokine and adjuvant.
[0063] In some embodiments, the antibodies or antigen-binding
fragments of the invention specifically bind cell-surface PSMA
and/or rsPSMA with a binding affinity of about 1.times.10.sup.-9M
or less. Preferably, the binding affinity is about
1.times.10.sup.-10M or less, more preferably the binding affinity
is about 1.times.10.sup.-11M or less. In other embodiments the
binding affinity is less than about 5.times.10.sup.-10M.
[0064] In additional embodiments, the antibodies or antigen-binding
fragments of the invention mediate specific cell killing of
PSMA-expressing cells with an IC.sub.50 of less than about
1.times.10.sup.-10M. Preferably the IC.sub.50 is less than about
1.times.10.sup.-11M. More preferably the IC.sub.50 is less than
about 1.times.10.sup.-12M. In other embodiments the IC.sub.50 is
less than about 1.5.times.10.sup.-11M.
[0065] In yet other embodiments, the isolated antibody or
antigen-binding fragment thereof is bound to a radioisotope. The
radioisotope can emit .alpha. radiations, .beta. radiations, or
.gamma. radiations. Preferably the radioisotope is selected from
the group consisting of .sup.225Ac, .sup.211At, .sup.212Bi,
.sup.213Bi, .sup.186Rh, .sup.188Rh, .sup.177Lu, .sup.90Y,
.sup.131I, .sup.67Cu, .sup.125I, .sup.123I, .sup.123I, .sup.77Br,
.sup.153Sm, .sup.166Ho, .sup.64Cu, .sup.212Pb, .sup.224Ra and
.sup.223Ra.
[0066] According to another aspect of the invention, hybridoma cell
lines are provided that produce an antibody selected from the group
consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4,
PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA
A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix
4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix
4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix
4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix
4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix
4.304.1, Abgenix 4.78.1 and Abgenix 4.152.1. In some embodiments,
the hybridoma cell line is selected from the group consisting of
PSMA 3.7 (PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11
(PTA-3269), PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3
(PTA-3293), PSMA 10.3 (PTA-3247), PSMA 1.8.3 (PTA-3906), PSMA
A3.1.3 (PTA-3904), PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2
(PTA-4427), Abgenix 4.360.3 (PTA-4428), Abgenix 4.7.1 (PTA-4429),
Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3 (PTA-4557), Abgenix
4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358), Abgenix 4.28.3
(PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix 4.48.3 (PTA-4361),
Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3 (PTA-4365), Abgenix
4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367), Abgenix 4.333.1
(PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix 4.153.1 (PTA-4388),
Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3 (PTA-4390), Abgenix
4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652), and Abgenix 4.152.1
(PTA-4653).
[0067] According to a further aspect of the invention, compositions
are provided that include the foregoing antibodies or
antigen-binding fragments thereof and a pharmaceutically acceptable
carrier, excipient, or stabilizer. Other compositions include a
combination of two or more of the foregoing antibodies or
antigen-binding fragments thereof and a pharmaceutically acceptable
carrier, excipient, or stabilizer. In some embodiments, the
compositions also include an antitumor agent, an immunostimulatory
agent, an immunomodulator, or a combination thereof. Preferred
antitumor agents include a cytotoxic agent, an agent that acts on
tumor neovasculature, or a combination thereof. Preferred
immunomodulators include .alpha.-interferon, .gamma.-interferon,
tumor necrosis factor-.alpha. or a combination thereof. Preferred
immunostimulatory agents include interleukin-2, immunostimulatory
oligonucleotides, or a combination thereof.
[0068] According to another aspect of the invention antibodies or
antigen-binding fragments thereof that mediate antibody-dependent
cellular cytotoxicity (ADCC) are provided. In some embodiments
these antibodies or antigen-binding fragments thereof mediate ADCC
of human prostate cancer cells. In other embodiments the antibodies
are human antibodies. In still other embodiments the antibodies are
capable of causing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
50%, 75% or more cell lysis in vitro with an effector to target
ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1 or more. In
other embodiments these antibodies mediate more ADCC than control
antibodies.
[0069] According to another aspect of the invention, kits for
detecting prostate cancer for diagnosis, prognosis or monitoring
are provided. The kits include the foregoing isolated labeled
antibody or antigen-binding fragment thereof, and one or more
compounds for detecting the label. Preferably the label is selected
from the group consisting of a fluorescent label, an enzyme label,
a radioactive label, a nuclear magnetic resonance active label, a
luminescent label, and a chromophore label.
[0070] The invention in another aspect provides one or more of the
foregoing isolated antibodies or antigen-binding fragments thereof
packaged in lyophilized form, or packaged in an aqueous medium.
[0071] In another aspect of the invention, methods for detecting
the presence of PSMA, or a cell expressing PSMA, in a sample are
provided. The methods include contacting the sample with any of the
foregoing antibodies or antigen-binding fragments thereof which
specifically bind to an extracellular domain of PSMA, for a time
sufficient to allow the formation of a complex between the antibody
or antigen-binding fragment thereof and PSMA, and detecting the
PSMA-antibody complex or PSMA-antigen-binding fragment complex. The
presence of a complex in the sample is indicative of the presence
in the sample of PSMA or a cell expressing PSMA.
[0072] In another aspect, the invention provides other methods for
diagnosing a PSMA-mediated disease in a subject. The methods
include administering to a subject suspected of having or
previously diagnosed with PSMA-mediated disease an amount of any of
the foregoing antibodies or antigen-binding fragments thereof which
specifically bind to an extracellular domain of prostate specific
membrane antigen. The method also includes allowing the formation
of a complex between the antibody or antigen-binding fragment
thereof and PSMA, and detecting the formation of the PSMA-antibody
complex or PSMA-antigen-binding fragment antibody complex to the
target epitope. The presence of a complex in the subject suspected
of having or previously diagnosed with prostate cancer is
indicative of the presence of a PSMA-mediated disease.
[0073] In certain embodiments of the methods, the PSMA-mediated
disease is prostate cancer. In other embodiments, the PSMA-mediated
disease is a non-prostate cancer, such as those selected from the
group consisting of bladder cancer including transitional cell
carcinoma; pancreatic cancer including pancreatic duct carcinoma;
lung cancer including non-small cell lung carcinoma; kidney cancer
including conventional renal cell carcinoma; sarcoma including soft
tissue sarcoma; breast cancer including breast carcinoma; brain
cancer including glioblastoma multiforme; neuroendocrine carcinoma;
colon cancer including colonic carcinoma; testicular cancer
including testicular embryonal carcinoma; and melanoma including
malignant melanoma.
[0074] In preferred embodiments of the foregoing methods, the
antibody or antigen-binding fragment thereof is labeled. In other
embodiments of the foregoing methods, a second antibody is
administered to detect the first antibody or antigen-binding
fragment thereof.
[0075] In a further aspect of the invention, methods for assessing
the prognosis of a subject with a PSMA-mediated disease are
provided. The methods include administering to a subject suspected
of having or previously diagnosed with PSMA-mediated disease an
effective amount of an antibody or antigen-binding fragment
thereof, allowing the formation of a complex between the antibody
or antigen-binding fragment thereof and PSMA, and detecting the
formation of the complex to the target epitope. The amount of the
complex in the subject suspected of having or previously diagnosed
with PSMA-mediated disease is indicative of the prognosis.
[0076] In another aspect of the invention, methods for assessing
the effectiveness of a treatment of a subject with a PSMA-mediated
disease are provided. The methods include administering to a
subject suspected treated for a PSMA-mediated disease an effective
amount of the foregoing antibodies or antigen-binding fragments
thereof, allowing the formation of a complex between the antibody
or antigen-binding fragment thereof and PSMA, and detecting the
formation of the complex to the target epitope. The amount of the
complex in the subject suspected of having or previously diagnosed
with PSMA-mediated disease is indicative of the effectiveness of
the treatment.
[0077] In certain embodiments of these two aspects of the
invention, the PSMA-mediated disease is prostate cancer. In other
embodiments, the PSMA-mediated disease is a non-prostate cancer. In
those embodiments, the non-prostate cancer preferably is selected
from the group consisting of bladder cancer including transitional
cell carcinoma; pancreatic cancer including pancreatic duct
carcinoma; lung cancer including non-small cell lung carcinoma;
kidney cancer including conventional renal cell carcinoma; sarcoma
including soft tissue sarcoma; breast cancer including breast
carcinoma; brain cancer including glioblastoma multiforme;
neuroendocrine carcinoma; colon cancer including colonic carcinoma;
testicular cancer including testicular embryonal carcinoma; and
melanoma including malignant melanoma. In still other embodiments,
the antibody or antigen-binding fragment thereof is labeled. In
further embodiments, a second antibody is administered to detect
the first antibody or antigen-binding fragment thereof.
[0078] According to yet another aspect of the invention, methods
for inhibiting the growth of a cell expressing PSMA are provided.
The methods include contacting a cell expressing PSMA with an
amount of at least one of the foregoing antibodies or
antigen-binding fragments thereof which specifically binds to an
extracellular domain of PSMA effective to inhibit the growth of the
cell expressing PSMA.
[0079] According to another aspect of the invention, methods for
inducing cytolysis of a cell expressing PSMA are provided. The
methods include contacting a cell expressing PSMA with an amount of
at least one of the foregoing antibodies or antigen-binding
fragments thereof which specifically binds to an extracellular
domain of PSMA effective to induce cytolysis of the cell expressing
PSMA. In certain embodiments, the cytolysis occurs in the presence
of an effector cell. In other embodiments, the cytolysis is
complement mediated.
[0080] According to still another aspect of the invention, methods
for treating or preventing a PSMA-mediated disease are provided.
The methods include administering to a subject having a
PSMA-mediated disease an effective amount of at least one of the
forgoing antibodies or antigen-binding fragments thereof to treat
or prevent the PSMA-mediated disease. In some embodiments, the
PSMA-mediated disease is a cancer, such as prostate cancer or a
non-prostate cancer (including the nonprostate cancers described
elsewhere herein).
[0081] In yet a further aspect of the invention, methods for
treating or preventing a PSMA-mediated disease are provided. The
methods include administering to a subject having a PSMA-mediated
disease or at risk of having a PSMA-mediated disease an amount of
at least one of the foregoing antibodies or antigen-binding
fragments thereof effective to treat or prevent the PSMA-mediated
disease.
[0082] In some embodiments, the PSMA-mediated disease is a cancer,
such as prostate cancer or a non-prostate cancer (including the
nonprostate cancers described elsewhere herein).
[0083] In other embodiments, the method also includes administering
another therapeutic agent to treat or prevent the PSMA-mediated
disease at any time before, during or after the administration of
the antibody or antigen-binding fragment thereof. In some of these
embodiments, the therapeutic agent is a vaccine, and preferably the
vaccine immunizes the subject against PSMA.
[0084] In still other embodiments, the antibody or antigen-binding
fragment thereof is bound to at least one therapeutic moiety,
preferably a cytotoxic drug, a drug which acts on the tumor
neovasculature and combinations thereof. Preferred cytotoxic drugs
are selected from the group consisting of: calicheamicin,
esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil,
ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin,
5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin,
paclitaxel, docetaxel, dolastatin 10, auristatin E and auristatin
PHE.
[0085] In other embodiments, the antibody or antigen-binding
fragment thereof is bound to a radioisotope and the radiations
emitted by the radioisotope is selected from the group consisting
of .alpha., .beta. and .gamma. radiations. Preferably, the
radioisotope is selected from the group consisting of .sup.225Ac,
.sup.211At, .sup.212Bi, .sup.213Bi, .sup.186Rh, .sup.188Rh, 177Lu,
.sup.90Y, .sup.131I, .sup.67Cu, 125I, .sup.123I, .sup.77Br,
.sup.153Sm, .sup.166Ho, .sup.64Cu, .sup.212Pb, .sup.224Ra and
.sup.223Ra.
[0086] The present invention provides methods for modulating at
least one enzymatic activity of PSMA. As used in preferred
embodiments of the methods, "modulating" an enzymatic activity of
PSMA means enhancing or inhibiting the enzymatic activity. Thus in
certain aspects of the invention, methods for inhibiting an
enzymatic activity of PSMA are provided, and in other aspects of
the invention, methods for enhancing an enzymatic activity of PSMA
are provided. The terms "enhancing` and "inhibiting" in this
context indicate that the enzymatic activity of PSMA is enhanced or
inhibited in the presence of an antibody that specifically binds
PSMA, or antigen-binding fragment thereof, relative to the level of
activity in the absence of such an antibody or antigen-binding
fragment thereof. Enzymatic activities of PSMA include folate
hydrolase activity, N-acetylated .alpha.-linked acidic dipeptidase
(NAALADase) activity, dipeptidyl dipeptidase IV activity and
.gamma.-glutamyl hydrolase activity.
[0087] Thus the invention in another aspect provides methods for
modulating folate hydrolase activity. In certain embodiments of
these methods, the activity is inhibited and in other embodiments,
the activity is enhanced. The methods include contacting a folate
hydrolase polypeptide with an amount of the foregoing isolated
antibody or antigen-binding fragment thereof, under conditions
wherein the isolated antibody or antigen-binding fragment thereof
modulates the folate hydrolase activity. The folate hydrolase
polypeptide can be isolated, contained in a sample such as a cell,
a cell homogenate, a tissue, or a tissue to homogenate, or
contained in an organism. The organism preferably is an animal,
particularly preferably a mammal.
[0088] In another aspect of the invention, methods for modulating
N-acetylated .alpha.-linked acidic dipeptidase (NAALADase) activity
are provided. In certain embodiments of these methods, the activity
is inhibited and in other embodiments, the activity is enhanced.
The methods include contacting a NAALADase polypeptide with an
amount of the foregoing isolated antibody or antigen-binding
fragment thereof under conditions wherein the isolated antibody or
antigen-binding fragment thereof modulates NAALADase activity. The
NAALADase polypeptide can be isolated, contained in a sample such
as a cell, a cell homogenate, a tissue, or a tissue homogenate, or
contained in an organism. The organism preferably is an animal,
particularly preferably a mammal.
[0089] In yet another aspect of the invention, methods for
modulating dipeptidyl dipeptidase IV activity are provided. In
certain embodiments of these methods, the activity is inhibited and
in other embodiments, the activity is enhanced. The methods include
contacting a dipeptidyl dipeptidase IV polypeptide with an amount
of the foregoing isolated antibody or antigen-binding fragment
thereof under conditions wherein the isolated antibody or
antigen-binding fragment thereof modulates dipeptidyl dipeptidase
IV activity. The dipeptidyl dipeptidase IV polypeptide can be
isolated, contained in a sample such as a cell, a cell homogenate,
a tissue, or a tissue homogenate, or contained in an organism. The
organism preferably is an animal, particularly preferably a
mammal.
[0090] In yet another aspect of the invention, methods for
modulating .gamma.-glutamyl hydrolase activity are provided. In
certain embodiments of these methods, the activity is inhibited and
in other embodiments, the activity is enhanced. The methods include
contacting a .gamma.-glutamyl hydrolase polypeptide with an amount
of the foregoing isolated antibody or antigen-binding fragment
thereof under conditions wherein the isolated antibody or
antigen-binding fragment thereof modulates .gamma.-glutamyl
hydrolase activity. The .gamma.-glutamyl hydrolase polypeptide can
be isolated, contained in a sample such as a cell, a cell
homogenate, a tissue, or a tissue homogenate, or contained in an
organism. The organism preferably is an animal, particularly
preferably a mammal.
[0091] Methods of specific delivery of at least one therapeutic
agent to PSMA-expressing cells are provided according to another
aspect of the invention. The methods include administering an
effective amount of at least one of the foregoing antibodies or
antigen-binding fragments thereof conjugated to the at least one
therapeutic agent. In some embodiments, the therapeutic agent is a
nucleic acid molecule, an antitumor drug, a toxin or a fragment
thereof, an enzyme or a fragment thereof, a replication-selective
virus, or an immunostimulatory or immunomodulating agent. Preferred
antitumor drugs include cytotoxic drugs, drugs which act on the
tumor neovasculature and combinations thereof. Preferred cytotoxic
drugs include calicheamicin, esperamicin, methotrexate,
doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin
C, cis-platinum, etoposide, bleomycin, 5-fluorouracil,
estramustine, vincristine, etoposide, doxorubicin, paclitaxel,
docetaxel, dolastatin 10, auristatin E and auristatin PHE.
Preferred immunostimulatory or immunomodulating agent included
cytokines, chemokines and adjuvants.
[0092] In still another aspect of the invention, isolated
antibodies that selectively bind a PSMA protein multimer are
provided. In preferred embodiments, the PSMA protein multimer is a
dimer, and preferably at least one of the PSMA proteins forming the
multimer is a recombinant, soluble PSMA (rsPSMA) polypeptide.
Preferably the rsPSMA polypeptide consists essentially of amino
acids 44-750 of SEQ ID NO: 1.
[0093] In a further aspect of the invention, isolated antibodies
are provided that selectively bind a PSMA protein multimer and
modulate one or more enzymatic activities of the PSMA protein
multimer. As used in preferred embodiments of this aspect of the
invention, "modulating" an enzymatic activity of a PSMA multimer
means enhancing or inhibiting the enzymatic activity. Thus in
certain aspects of the invention, antibodies that inhibit an
enzymatic activity of PSMA multimers are provided, and in other
aspects of the invention, antibodies that inhibit an enzymatic
activity of PSMA multimers are provided. The terms "enhancing` and
"inhibiting" in this context indicate that the enzymatic activity
of a PSMA multimer is enhanced or inhibited in the presence of an
antibody that specifically binds the PSMA multimers, or
antigen-binding fragment thereof, relative to the level of activity
in the absence of such an antibody or antigen-binding fragment
thereof. In some embodiments, the enzymatic activity is selected
from the group consisting of folate hydrolase activity, NAALADase
activity, dipeptidyl dipeptidase IV activity and .gamma.-glutamyl
hydrolase activity. In other embodiments, the enzymatic activity is
in the extracellular domain of the PSMA molecule. In still other
embodiments, the antibody or antigen-binding fragment thereof
specifically binds to an extracellular domain of PSMA.
[0094] In a further aspect, an isolated antibody or antigen-binding
fragment thereof is provided that selectively binds a PSMA protein
multimer. In this aspect, the isolated antibody is raised by
immunizing an animal with a preparation comprising a PSMA protein
multimer. Preferred preparations used in raising the antibody
include those having at least about 10%, 20%, 30%, 40%, 50%, 75%,
90%, or 95% PSMA protein multimer. Preferably the PSMA protein
multimer is a dimer.
[0095] In yet another aspect of the invention, compositions are
provided that include one or more of the foregoing isolated
antibodies, and an immunostimulatory molecule, such as an adjuvant
and/or and a cytokine. Preferably the immunostimulatory molecule is
IL-2 or an immunostimulatory oligonucleotide. In certain
embodiments, the foregoing compositions also include a
pharmaceutically-acceptable carrier.
[0096] The invention also includes methods for inducing an immune
response, including administering to a subject in need of such
treatment an effective amount of the foregoing isolated antibodies
or compositions.
[0097] The invention provides, in another aspect, isolated
antibodies or antigen-binding fragments thereof that selectively
bind a PSMA protein multimer and modulate at least one enzymatic
activity of PSMA. As used in preferred embodiments of this aspect
of the invention, "modulating" an enzymatic activity of a PSMA
means enhancing or inhibiting the enzymatic activity. Thus in
certain aspects of the invention, antibodies that inhibit an
enzymatic activity of PSMA are provided, and in other aspects of
the invention, antibodies that inhibit an enzymatic activity of
PSMA are provided. The terms "enhancing` and "inhibiting" in this
context indicate that the enzymatic activity of PSMA is enhanced or
inhibited in the presence of an antibody that specifically binds
PSMA, or antigen-binding fragment thereof, relative to the level of
activity in the absence of such an antibody or antigen-binding
fragment thereof. The enzyme, in certain embodiments, is selected
from the group consisting of hydrolases and peptidases. Preferred
hydrolases include folate hydrolase and .gamma.-glutamyl hydrolase.
In a particularly preferred embodiment of PSMA inhibition, the
hydrolase is folate hydrolase and the antibody is mAb 5.4 or mAb
3.9.
[0098] Preferred peptidases include NAALADase and dipeptidyl
dipeptidase IV. In some embodiments, the enzyme is active in cancer
cells and has lesser activity in normal cells than in cancer cells
or, preferably, no activity in normal cells. In preferred
embodiments, the cancer cells in which the enzyme is active are
prostate cancer cells. Compositions including the foregoing
isolated antibodies or antigen-binding fragments thereof, and a
pharmaceutically acceptable carrier, also are provided by the
invention.
[0099] In another aspect of the invention, compositions are
provided that include an isolated PSMA protein multimer. Preferably
the PSMA protein multimer is a dimer. In certain embodiments, the
compositions include at least about 10%, 20%, 30%, 40%, 50%, 75%,
90%, or 95% PSMA protein multimer. In other embodiments, the PSMA
protein multimer comprises noncovalently associated PSMA proteins.
The PSMA proteins preferably are noncovalently associated under
nondenaturing conditions.
[0100] In certain embodiments of the foregoing compositions, at
least one of the PSMA proteins forming the multimer is a
recombinant, soluble PSMA (rsPSMA) polypeptide. In other
embodiments, the PSMA protein multimer is reactive with a
conformation-specific antibody that specifically recognizes PSMA.
Preferably, the PSMA protein multimer comprises PSMA proteins in a
native conformation and/or the PSMA multimer is enzymatically
active. In preferred embodiments, the enzymatic activity is folate
hydrolase activity, NAALADase activity, dipeptidyl dipeptidase IV
activity and/or .gamma.-glutamyl hydrolase activity.
[0101] In still other embodiments, the foregoing compositions also
include an adjuvant and/or a cytokine or other immunostimulatory
molecule. Preferred cytokines include IL-2, IL-12, IL-18 and
GM-CSF. In further embodiments, the foregoing compositions also
include a pharmaceutically acceptable carrier.
[0102] According to yet another aspect of the invention, methods
for inducing an immune response are provided. The methods include
administering to a subject in need of such treatment an effective
amount of one or more of the foregoing compositions.
[0103] In a further aspect, the invention includes isolated
recombinant soluble PSMA (rsPSMA) protein multimers and isolated
rsPSMA protein dimers. In some embodiments, the dimer includes
noncovalently associated rsPSMA proteins, and preferably the rsPSMA
proteins are noncovalently associated under nondenaturing
conditions. In other embodiments, the isolated rsPSMA dimer is
reactive with a conformation-specific antibody that specifically
recognizes PSMA.
[0104] In a certain preferred embodiment, the isolated rsPSMA dimer
is enzymatically active, with the enzymatic activity selected from
the group consisting of folate hydrolase activity, NAALADase
activity, dipeptidyl dipeptidase IV activity and .gamma.-glutamyl
hydrolase activity.
[0105] In still another aspect of the invention, methods of
screening for a candidate agent that modulates at least one
enzymatic activity of a PSMA enzyme are provided. As used in
preferred embodiments of the methods, "modulating" an enzymatic
activity of PSMA means enhancing or inhibiting the enzymatic
activity. Thus in certain aspects of the invention, methods for
screening for a candidate agent that inhibits an enzymatic activity
of PSMA are provided, and in other aspects of the invention,
methods for screening for a candidate agent that enhances an
enzymatic activity of PSMA are provided. The terms "enhancing" and
"inhibiting" in this context indicate that the enzymatic activity
of PSMA is enhanced or inhibited in the presence of a candidate
agent relative to the level of activity in the absence of such an
agent. The methods include mixing the candidate agent with an
isolated PSMA protein multimer to form a reaction mixture, followed
by adding a substrate for the PSMA enzyme to the reaction mixture,
and determining the amount of a product formed from the substrate
by the PSMA enzyme. A change in the amount of product formed in
comparison to a control is indicative of an agent capable of
modulating at least one enzymatic activity of the PSMA enzyme. A
decrease in the amount of product formed in comparison to a control
is indicative of an agent capable of inhibiting at least one
enzymatic activity of the PSMA enzyme. An increase in the amount of
product formed in comparison to a control is indicative of an agent
capable of enhancing at least one enzymatic activity of the PSMA
enzyme. In some embodiments the PSMA enzyme is selected from the
group consisting of NAALADase, folate hydrolase, dipeptidyl
dipeptidase IV and .gamma.-glutamyl hydrolase. In other embodiments
the PSMA multimer comprises recombinant, soluble PSMA. In yet other
embodiments the candidate agent is selected from the group
consisting of an antibody, a small organic compound, or a
peptide.
[0106] In another aspect of the invention, candidate agents that
modulate at least one enzymatic activity of PSMA are provided. The
candidate agents are identified according to the foregoing methods.
Thus in certain aspects of the invention, candidate agents that
inhibit an enzymatic activity of PSMA are provided, and in other
aspects of the invention, candidate agents that enhance an
enzymatic activity of PSMA are provided. In certain embodiments,
the agent is selected from a combinatorial antibody library, a
combinatorial protein library, or a small organic molecule
library.
[0107] The invention also provides methods for identifying
compounds that promote dissociation of PSMA dimers. The methods
include contacting a PSMA dimer with a compound under conditions
that do not promote dissociation of the PSMA dimer in the absence
of the compound, measuring the amount of PSMA monomer and/or dimer;
and comparing the amount of PSMA monomer and/or dimer measured in
the presence of the compound with that observed in the absence of
the compound. An increase in the amount of PSMA monomer measured in
the presence of the compound indicates that the compound is capable
of promoting dissociation of the PSMA dimer. A decrease in the
amount of PSMA dimer measured in the presence of the compound
indicates that the compound is capable of promoting dissociation of
the PSMA dimer. When the amounts of PSMA monomer and PSMA dimer are
measured, the methods can include calculating a ratio of PSMA
monomer to PSMA dimer and comparing the ratio obtained in the
presence of the compound with that obtained in the absence of the
compound. In such methods, an increase in the ratio measured in the
presence of the compound indicates that the compound is capable of
promoting dissociation of the PSMA dimer.
[0108] The use of the foregoing compositions, molecules and agents
in the preparation of medicaments also is provided. In preferred
embodiments, the medicaments are useful in the treatment of
conditions related to hyperproliferative diseases including cancer,
and diseases of inappropriate NAALADase activity, folate hydrolase
activity, dipeptidyl dipeptidase IV activity and/or
.gamma.-glutamyl hydrolase activity.
[0109] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention.
[0110] These and other aspects of the invention will be described
in further detail in connection with the detailed description of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0111] FIG. 1 depicts PSMA reactivity of mAbs as determined by flow
cytometry. Anti-PSMA mAbs (3.7, 3.9, 3.11, 3.12, 5.4, and 10.3)
incubated with either parental 3T3 cells (denoted by black lines)
or 3T3 cells engineered to express cell-surface PSMA (3T3-PSMA;
gray lines).
[0112] FIG. 2 shows a digitized image of immunoprecipitation of
PSMA by mAbs. Lysates from 3T3-PSMA cells or parental 3T3 cells
were incubated with each mAb and then precipitated using Protein
A/G agarose beads. After washing, proteins were resolved on a
polyacrylamide gel, blotted onto nitrocellulose membranes and
visualized using the MAB544 anti-PSMA mAb.
[0113] FIG. 3 shows the recognition of non-denatured PSMA by
several PSMA antibodies that recognize PSMA conformation.
[0114] FIG. 4 is a digitized image of a Western blot that shows the
recognition of denatured PSMA by two PSMA antibodies and shows that
antibodies that recognize PSMA conformation do not recognize
denatured PSMA.
[0115] FIG. 5 is a digitized image of a polyacrylamide gel that
shows an analysis of purified recombinant, soluble PSMA (rsPSMA)
and of full-length PSMA from 3T3 cells (3T3 PSMA) or LNCaP cells
(LNCaP PSMA) by reduced and non-reduced SDS-PAGE.
[0116] FIGS. 6A and 6B provide the results for the determination of
the dimeric structure of PSMA. FIG. 6A is a digitized image of a
polyacrylamide gel that depicts a Blue Native PAGE analysis of
purified recombinant, soluble PSMA (Purified rsPSMA) and of
full-length PSMA extracted from 3T3 cells (3T3 PSMA) or LNCaP cells
(LNCaP PSMA). FIG. 6B shows the results of the analytical size
exclusion chromatography (SEC) of purified rsPSMA in neutral PBS
buffer. The arrows indicate the retention times of protein
standards. The retention time of 260 kDa for rsPSMA is consistent
with that of a homodimer.
[0117] FIGS. 7A and 7B illustrate that the dimeric but not
monomeric rsPSMA (also referred to as PSMA.sub.ECTO) is
enzymatically active. Dimeric and monomeric PSMA were tested for
folate hydrolase activity (FIG. 7A) and NAALADase activity (FIG.
7B). The background activity observed for PSMA monomer is
consistent with residual amount (approximately 4%) of dimer present
in the preparation.
[0118] FIG. 8 shows the effect of four antibodies (mAb 3.9, mAb
5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate
hydrolase through measuring the rate of cleavage of glutamate from
methotrexate di-gamma glutamate by folate hydrolase present in
0.0002 .mu.g rsPSMA #7.
[0119] FIG. 9 shows the effect of four antibodies (mAb 3.9, mAb
5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate
hydrolase through measuring the rate of cleavage of glutamate from
methotrexate di-gamma glutamate by folate hydrolase present in
0.0002 .mu.g rsPSMA #8.
[0120] FIG. 10 shows the effect of four antibodies (mAb 3.9, mAb
5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate
hydrolase through measuring the rate of cleavage of glutamate from
methotrexate di-gamma glutamate by folate hydrolase present in
lysates of C4-2 cells.
[0121] FIGS. 11A-11D show the impact of four antibodies (human mAbs
006 (FIG. 11A), 026 (FIG. 11B) and 4.40.2 (FIG. 11D) as well as
murine mAb 5.4 (FIG. 11C) on PSMA folate hydrolase activity.
[0122] FIGS. 12A and 12B illustrate the rapid and efficient
internalization of .sup.111In labeled mAb 026 incubated with C4-2
cells (FIG. 12A) as well as the total binding to the cells (FIG.
12B).
[0123] FIG. 13 depicts the cloning protocol for IgG1 antibody
cloning into pcDNA. The first four primers shown are set forth as
SEQ ID NOs: 34-37, respectively, and the first two amino acid
sequences shown are set forth as SEQ ID NOs: 38 and 39,
respectively.
[0124] FIG. 14 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PG1-XG1-006.
[0125] FIG. 15 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PG1-XG1-026.
[0126] FIG. 16 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PG1-XG1-051.
[0127] FIG. 17 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PG1-XG1-069.
[0128] FIG. 18 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PG1-XG1-077.
[0129] FIG. 19 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody PSMA 10.3.
[0130] FIG. 20 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-006.
[0131] FIG. 21 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-026.
[0132] FIG. 22 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-051.
[0133] FIG. 23 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-069.
[0134] FIG. 24 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-077.
[0135] FIG. 25 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody PSMA 10.3.
[0136] FIG. 26 depicts the cytotoxicity of .sup.2 Ac-3.9 on LNCaP
target cells.
[0137] FIGS. 27A-27F illustrate the reactivity of anti-PSMA
monoclonal antibodies XG-006, XG-051, 4.40.1, 4.49.1, 4.292.1 and
4.304.1 incubated with either parent 3T3 cells (black histogram) or
3T3 cells engineered to express cell-surface human PSMA (red
histogram) and analyzed by flow cytometry. FIG. 27A, FIG. 27B, FIG.
27C, FIG. 27D, FIG. 27E and FIG. 27F provide results for each of
the aforementioned antibodies.
[0138] FIGS. 28A-28C illustrate the binding of the anti-PSMA Abs.
FIG. 28A shows that anti-PSMA mAbs bind to 3T3-PSMA cells and not
3T3 cells. One representative experiment from at least ten
determinations is shown. FIG. 28B illustrates that binding to
cell-surface PSMA using serial dilutions of anti-PSMA
mAb-containing culture supernatants occurred. One representative
experiment from five is shown. FIG. 28C shows binding to
cell-surface PSMA using serial dilutions of purified anti-PSMA
mAbs, XG-006 and 10.3. One representative experiment is shown.
[0139] FIGS. 29A-29C illustrate the immunotoxin cytotoxicity of
murine anti-PSMA antibodies on C4-2 prostate cancer cells. SJ25C-1
as a control antibody is a murine anti-CD19 IgG. The LD 50s (M) for
5.4 (FIG. 29A), 3.9 (FIG. 29B), and mJ591 (FIG. 29C) antibodies
were 2.27.times.10.sup.-11, 2.29.times.10.sup.-11 and
8.82.times.10.sup.-11, respectively.
[0140] FIGS. 30A-30C illustrate the immunotoxin cytotoxicity of
murine anti-PSMA antibodies on PSMA-3T3 cells. SJ25C-1 as a control
antibody is a murine anti-CD 9 IgG. The LD 50s (M) for 5.4 (FIG.
30A), 3.9 (FIG. 30B), and mJ591 (FIG. 30C) antibodies were
1.64.times.10.sup.-11, 1.96.times.10.sup.-11 and
8.90.times.10.sup.-11, respectively.
[0141] FIG. 31 provides the cytotoxicity of direct conjugated human
4.304 anti-PSMA antibodies with saporin on PSMA-3T3. The LD50 was
1.48.times.10.sup.-11 M for direct conjugated 4.304 anti-PSMA
antibodies with saporin.
[0142] FIG. 32 illustrates the results of the competition assay of
unmodified 4.304, 4.40, mJ591 anti-PSMA antibodies used to compete
with In-111 radiolabeled 4.40 and 4.304 anti-PSMA antibodies.
[0143] FIG. 33 illustrates the results of the competition assay of
unmodified 4.304, mJ591 anti-PSMA antibodies used to compete with
In-111 radiolabeled mJ591 anti-PSMA antibodies.
[0144] FIG. 34 shows an analysis of antibody PRGX1-XG-006 in
association phase and dissociation phase at different
concentrations of rsPSMA from 100 nM to 6.25 nM.
[0145] FIG. 35 shows the results of the comparison of the fully
human anti-PSMA antibodies 4.40.1, 4.49.1, 051 and 006 and the
murine anti-PSMA antibody 3.9 performed using BIACORE analysis.
[0146] FIG. 36 provides results from the Scatchard analysis using
In-111 labeled anti-PSMA antibody 3.9 of the PSMA-3T3, LNCaP and
C4-2 cell lines.
[0147] FIG. 37 shows in vitro cytotoxicity of Ac-225 labeled human
anti-PSMA antibody 4.40 on prostate cancer cells.
[0148] FIG. 38 shows the specific killing of PSMA expressing cells
(C4-2) vs. PSMA non-expressing cells (PC-3) treated with .sup.225Ac
labeled mAb 026.
[0149] FIGS. 39A and 39B show the in vitro cytotoxicity of
.sup.225Ac labeled mAb 026 on human prostate cancer cell lines
(C4-2 (FIG. 39A) and LNCaP (FIG. 39B)).
[0150] FIG. 40 shows the in vitro cytotoxicity of .sup.225Ac
labeled mAb 026 on human prostate cancer cell line, C4-2, evaluated
by .sup.3H thymidine incorporation.
[0151] FIG. 41 shows the results of in vivo radioimmunotherapy with
Lu-177 labeled human anti-PSMA antibodies.
[0152] FIGS. 42A and 42B provide the radio-HPLC profile (FIG. 42A)
and cell-based immunoreactivity (FIG. 42B) of .sup.117Lu labeled
antibodies (006, 026, mJ591 and HuIgG (control)).
[0153] FIGS. 43A and 43B show the specific binding of .sup.177Lu
labeled antibodies (006, 026, mJ591 and IgG (control)) to PSMA
positive tumors in vivo. FIG. 43A shows results by % ID/g, and FIG.
43B shows results by PSMA+/PSMA- ratios.
[0154] FIGS. 44A and 44B show the preferential retention of
radiolabeled antibodies (006, 026, mJ591 and HuIgG) in PSMA+ (FIG.
44A) tumors vs. PSMA- (FIG. 44B) tumors as assessed by the percent
activity in the tumors.
[0155] FIGS. 45A-45H provide data for normal organ (blood (FIG.
45A), liver (FIG. 45B), kidneys (FIG. 45C), spleen (FIG. 45D),
lungs (FIG. 45E), bone (FIG. 45F), heart (FIG. 45G) and muscle
(FIG. 45H)) uptake (injected dose per gram of tissue, % ID/g) for
the antibodies (006, 026, mJ591 and HuIgG).
[0156] FIGS. 46A and 46B illustrate the therapeutic efficacy of
.sup.177Lu labeled mAb 026 in PSMA-3T3 and 3T3 tumor-bearing mice.
FIG. 46A provides results for tumor size. FIG. 46B provides results
for percent survival.
[0157] FIG. 47 shows the preferential binding of mAb 006 to rsPSMA
dimer.
[0158] FIG. 48 shows the preferential binding of mAb 026 to rsPSMA
dimer.
[0159] FIG. 49 shows the binding of mAb 4.40 to rsPSMA dimer and
monomer.
[0160] FIG. 50 shows the binding of mAb mJ591 to rsPSMA dimer and
monomer.
[0161] FIG. 51 is a series of graphs that show flow cytometry data
for the binding of anti-PSMA antisera to PSMA-3T3 cells. Antisera
from mice immunized with a rsPSMA dimer preparation (ABIM151,
ABIM152, ABIM153, ABIM154 and ABIM155) exhibited strong binding to
PSMA-expressing cells. Antisera from mice immunized with a rsPSMA
monomer preparation (ABIM156, ABIM157, ABIM158, ABIM159 and
ABIM160) exhibited little or no binding to PSMA-expressing
cells.
[0162] FIG. 52 provides the results showing antibody dependent
cell-mediated cytotoxicity (ADCC) of human prostate cancer cells
mediated by mAbs 006 and 026.
[0163] FIGS. 53A and 53B show the results of PSMA monomer-dimer
equilibrium analysis. Purified dimeric (FIG. 53A) and monomeric
(FIG. 53B) rsPSMA were subjected to various buffer conditions and
analyzed for size by analytical size exclusion chromatography
(SEC). The percentages of monomer (M) and dimer (D) are indicated.
The monomer and dimer were initially contained in PBS+ buffer at a
concentration of 0.2 mg/ml. The buffer conditions were adjusted as
indicated, and the proteins were incubated at ambient temperature
for the indicated time periods before SEC analysis.
DETAILED DESCRIPTION OF THE INVENTION
[0164] The present invention provides, in part, multimeric,
particularly dimeric, forms of PSMA protein, compositions and kits
containing dimeric PSMA protein as well as methods of producing,
purifying, processing and using these compositions. Such methods
include methods for eliciting or enhancing an immune response to
PSMA and/or cells expressing PSMA. Such methods include methods of
producing antibodies to dimeric PSMA as well as methods of treating
cancer, such as prostate cancer.
[0165] Prostate specific membrane antigen (PSMA) is a 100 kD Type
II membrane glycoprotein expressed in prostate tissues and was
originally identified by reactivity with a monoclonal antibody
designated 7E11-C5 (Horoszewicz et al., 1987, Anticancer Res.
7:927-935; U.S. Pat. No. 5,162,504). PSMA was obtained in purified
form (Wright et al., 1990, Antibody Immunoconjugates and Radio
Pharmaceuticals 3:Abstract 193) and characterized as a type II
transmembrane protein having sequence identity with the transferrin
receptor (Israeli et al., 1994, Cancer Res. 54:1807-1811) and with
NAALADase activity (Carter et al., 1996, Proc. Natl. Acad. Sci.
U.S.A. 93:749-753). More importantly, PSMA is expressed in
increased amounts in prostate cancer, and elevated levels of PSMA
are also detectable in the sera of these patients (Horoszewicz et
al., 1987; Rochon et al., 1994, Prostate 25:219-223; Murphy et al.,
1995, Prostate 26:164-168; and Murphy et al., 1995, Anticancer Res.
15:1473-1479). PSMA expression increases with disease progression,
becoming highest in metastatic, hormone-refractory disease for
which there is no present therapy. Provocative recent data
indicates that PSMA is also abundantly expressed on the
neovasculature of a variety of other important tumors, including
bladder, pancreas, sarcoma, melanoma, lung, and kidney tumor cells,
but not on normal vasculature.
[0166] It has been discovered that PSMA in its native form is a
homodimer. When ordinary isolation techniques are followed,
however, the native form of PSMA is not typically maintained.
Compositions of isolated PSMA protein that include isolated
multimeric PSMA, particularly dimeric PSMA, therefore, are
provided. These compositions include isolated PSMA protein, wherein
at least about 5% of the isolated PSMA protein is in multimeric
form. Other compositions are provided where at least about 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95% or more of the isolated PSMA protein is in
multimeric form. In a preferred embodiment, the PSMA protein
multimer composition contains substantially pure PSMA protein
multimer, with substantially no PSMA protein monomer. It is
understood that the list of specific percentages includes by
inference all of the unnamed percentages between the recited
percentages. It has further been discovered that certain agents
preserve or promote the multimeric, particularly the dimeric,
association of isolated PSMA. Compositions of isolated PSMA protein
that include these agents as well as methods of purifying and
processing isolated PSMA protein compositions are, therefore, also
provided.
[0167] As used herein "PSMA protein" includes the full-length PSMA
protein (provided as SEQ ID NO: 1) or a portion thereof. These
proteins are capable of forming multimers or aggregates of PSMA
protein. As used herein, a "multimer or aggregate of PSMA protein"
refers to the association of two or more PSMA proteins. Preferably,
the PSMA proteins described herein are those that are capable of
forming a dimer like that of native PSMA by non-covalent
interactions or engineered to form a stable native-like dimer
through covalent bonds, such as disulfide bonds. "A dimer like that
of native PSMA" includes two PSMA proteins that are associated in
the same way as the protein as found in nature or in such a way as
to allow for the generation of antibodies that recognize at least
one antigenic epitope of the native dimer (i.e., associate in a way
such as to form an antigenic region as found in the native PSMA
dimer or one capable of generating cross-reacting antibodies). The
antibodies generated to the dimers provided herein are, therefore,
capable of recognizing the native dimer. Preferably, the antibodies
generated recognize native PSMA dimer but not PSMA monomer or have
greater specificity for the native PSMA dimer than the monomer. In
some embodiments, the PSMA proteins provided herein are larger
aggregates of PSMA (i.e., three or more PSMA protein that are
associated). These aggregates are likewise capable of generating
antibodies that recognize PSMA. In some embodiments, these
antibodies do not recognize PSMA monomer but do recognize native
PSMA dimer. In other embodiments, these antibodies have greater
specificity for the native PSMA dimer rather than PSMA monomer.
[0168] PSMA multimers are typically homomultimers (i.e., the
associated PSMA proteins are the same). However, in some
embodiments the PSMA multimers can be heteromultimers, particularly
heterodimers. As used herein a "PSMA heteromultimer" is a multimer
of PSMA proteins that is composed of at least two different PSMA
proteins. Examples include two PSMA fragments, where one is
slightly longer than the other or when one has a conservative amino
acid substitution and the other does not. The heteromultimers
provided herein, like homomultimers, are capable of generating
antibodies that recognize native PSMA dimer. In preferred
embodiments the antibodies raised against the PSMA heteromultimers
recognize native PSMA dimer but not PSMA monomer. In still other
preferred embodiments these antibodies have greater specificity for
native PSMA dimer rather than PSMA monomer.
[0169] PSMA protein capable of forming multimers, particularly
dimers, include the full-length protein (SEQ ID NO: 1). In some
embodiments the PSMA protein capable of forming a multimer is the
extracellular portion of PSMA (amino acids 44-750 of SEQ ID NO: 1).
In other embodiments the PSMA protein capable of forming a multimer
is PSM' (amino acids 58-750 of SEQ ID NO: 1), an alternatively
spliced form of PSMA. In yet other embodiments fragments of the
full-length protein, the extracellular portion or PSM' are capable
of forming multimers. For example, these fragments include
truncated PSMA proteins that begin at amino acid 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, etc. of SEQ ID NO: 1 and end at amino acid 750 of SEQ ID NO: 1.
Other such truncated proteins begin at amino acid 44 of SEQ ID NO:
1 and end at amino acid 749, 748, 747, 746, 745, 744, 743, 742,
741, 740, etc. of SEQ ID NO: 1. Still other truncated proteins
include those that begin at amino acid 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, etc. of
SEQ ID NO: 1 and end at amino acid 749, 748, 747, 746, 745, 744,
743, 742, 741, 740, etc. of SEQ ID NO: 1. In some embodiments the
truncated PSMA protein includes the amino acids 601-750 of SEQ ID
NO: 1 or a functional portion thereof capable of forming dimers. As
provided herein, the PSMA proteins are intended to encompass any
fragment of the PSMA protein that is capable of forming a multimer
as provided herein. Therefore, any portion of SEQ ID NO: 1 is
included in this definition as well as its functional variant.
Functional variants are described further herein below.
[0170] In some embodiments the isolated PSMA protein is not
full-length PSM' (amino acids 58-750 of SEQ ID NO: 1) or the
full-length extracellular portion of PSMA (amino acids 44-750 of
SEQ ID NO: 1). In other instances, the isolated PSMA protein is not
full-length PSMA (SEQ ID NO: 1). The fragment can have a size of at
least about 25, 50, 100, 125, 150, 175, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, or 749 amino acids and every integer
length therebetween. In some embodiments, these fragments include
amino acids 63-68, 132-137 or 482-487 of SEQ ID NO:1. In some other
preferred instances, the PSMA protein is not membrane-bound.
[0171] Compositions of PSMA protein with agents and/or solutions
that preserve or promote the multimeric association, particularly
the dimeric association, of PSMA also are provided. In some
instances the agents are in a solution along with the PSMA protein
but are not necessarily so. An agent or solution that "preserves or
promotes the dimeric association of PSMA" is one that either
maintains the dimeric association (dimerization) of PSMA over time
or facilitates the dimeric association of monomeric forms of the
PSMA protein. For example, any solution that increases the amount
of PSMA dimers, maintains the amount of PSMA dimers or retards the
disassociation of PSMA dimers is encompassed by the above
definition. Although the dimeric state is specifically recited,
these terms are also intended to encompass other multimeric states
of PSMA, and therefore, compositions, kits and methods of
production and use of other multimers of PSMA.
[0172] Preferably, the "preservation or promotion of dimeric PSMA"
refers to the maintenance of the dimeric state of PSMA protein for
at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%
or more of the PSMA dimers initially present in a composition.
Preferred compositions comprising the dimeric form of PSMA have
less than about 35% of the monomeric form of PSMA, preferably less
than about 20%, more preferably less than about 15% of the
monomeric form. In one embodiment the composition has less than
about 5% of the monomeric PSMA protein. The preservation or
promotion of dimeric PSMA also refers to the conversion of about
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the
initially monomeric PSMA to dimeric form.
[0173] The promotion of dimerization or maintenance of the dimeric
state can occur at any of a number of experimental or storage
temperatures. In some instances the promotion or preservation of
dimeric PSMA occurs at a temperature of about 45.degree. C. or
lower. In other instances the promotion or preservation of dimeric
PSMA is at a temperature of about 37.degree. C. The promotion or
preservation can also be at a temperature range of about 20.degree.
C. to about 30.degree. C. or about or below room temperature. In
other instances the promotion or preservation is at a range of
about 4.degree. C. to about 20.degree. C. In still other instances
the promotion or preservation is at about -20.degree. C. to about
4.degree. C. or about -80.degree. C. to about -20.degree. C. The
promotion or preservation of the dimeric state of PSMA can also
occur in a composition of PSMA protein that is in solution or in a
freeze-dried form, e.g., lyophilized form. The dimeric state can
also be promoted or preserved over any period of time. In some
instances the period of time is at least about 1, 2, 3, 4, 5, 6,
10, 15, 20, 24, 48, 72 or more hours. In other instances the period
of time is at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 or more
days. In still other instances the period of time is at least about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30 or more weeks. In
yet other instances, the period of time is at least about 1, 2, 3,
6, 9, 12 or more months or as long as 2 years or more. The
formulations provided herein are stable during long-term storage,
i.e., the formulations preserve or promote the dimeric PSMA
state.
[0174] It was surprisingly discovered that pH alone can influence
the dimeric state of PSMA. As described below in the Examples
section, the pH at which a PSMA solution is incubated can influence
the multimeric form of PSMA as well as its recovery. Incubation at
various pHs for 4 days at a temperature of about 45.degree. C.
influenced the dimerization or aggregation of PSMA protein as well
as the recovery of PSMA protein by analytical TSK gel filtration
chromatography. The benefits of pH on the preservation of dimeric
rsPSMA (2 mg/ml in PBS+) are retained when the protein solution is
diluted 10-fold in a variety of buffer solutions, each containing 2
mM glycine, 2 mM citric acid, 2 mM Hepes, 2 mM MES and 2 mM Tris
Base.
[0175] The dimeric structure of PSMA according to the invention is
preserved at a pH in the range of about 4 to about 8. Therefore, a
solution that preserves or promotes the dimerization of PSMA is one
with a pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8. Recovery
of dimeric PSMA from a column was better at a pH in the range of
about 5 to about 7, and these pH values are preferred. In some
instances, a pH of about 6 is preferred. Thus, the invention
provides formulations of PSMA in solution, wherein the pH is in the
range from about 4 to about 8, preferably in the range from about 5
to about 7, more preferably in the range from about 5.5 to about 7,
and most preferably in the range from about 6 to about 7.
[0176] An "agent that preserves or promotes the dimeric association
of PSMA" is meant to encompass an agent that promotes or maintains
the dimerization of PSMA. Such agents have been found to include pH
adjusting agents (as discussed above), metal ions and salts. It has
been discovered that these agents, individually or in combination,
are able to preserve or promote dimeric association of PSMA. In
some embodiments it is the combination of the metal ion, salt or pH
adjusting agent that can promote or preserve dimeric association of
PSMA, while the individual metal ion, salt or pH adjusting agent
cannot. As provided in the Examples, the use of chelating agents,
such as EDTA, converted dimeric PSMA into the monomer. This result
indicated that the presence of metal ions can positively affect the
stability of the dimer. Additionally, PSMA shares modest sequence
and structural homology with human transferrin receptor (TfR),
which contains additional metal-binding sites within its helical
domains (Lawrence, C. M., et al. (1999) Science 286, 779-782).
Therefore, metal ions are considered to be agents which promote or
preserve the dimeric state of PSMA protein. Such metals ions
include, but are not limited to, zinc ions (e.g., Zn.sup.2+),
calcium ions (e.g., Ca.sup.2+), magnesium ions (e.g., Mg.sup.2+),
cobalt ions (e.g., Co.sup.2+), manganese ions (e.g., Mn.sup.2+) or
combinations thereof.
[0177] In some instances these metal ions can be added to a
composition of PSMA protein in the form of a salt. Such salts
include zinc chloride, calcium chloride, magnesium chloride, cobalt
chloride or manganese chloride. It has been further determined that
compositions of PSMA protein, wherein the dimeric state is promoted
or preserved, include at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1, 2, 3, 4, 5 or more molar equivalents of metal ion
to PSMA protein (total PSMA protein, i.e., total amount of PSMA
protein molecules). In some instances, the molar equivalent of
metal ions should be in molar excess to PSMA protein. In some
specific solutions of PSMA protein (2 mg/ml in PBS+; diluted
10-fold), as provided in the Examples, it has further been found,
that the metal ions are preferably present at a concentration in
the range of about 0.1 mM to about 5 mM. The metal ions in some
instances are present at a concentration in the range of about 0.1
mM to about 1 mM. In other embodiments the metal ions are present
at a concentration in the range of about 0.1 mM to about 0.5 mM. In
solutions where there is a combination of one or more types of
metal ions, the one or more metal ions can be at the same
concentration or at a different concentration. For example, one
such solution can contain a concentration of calcium ions of about
0.5 mM and a concentration of zinc ions of a concentration that is
greater than 0.1 mM but less than 0.5 mM. Because of the importance
of metal ions in the dimerization of PSMA in some compositions, in
some instances, it is preferred that the compositions do not
contain a chelating agent.
[0178] It has also been found that salts preserve or promote PSMA
dimerization. As shown below in the Examples, a dimer preparation
that contained approximately 5% monomer initially was converted to
100% dimer upon incubation for 72 hours at ambient temperature in
PBS+ (phosphate-buffered saline containing 1 mM Ca .sup.2 and 0.5
mM Mg.sup.2+, pH 7.2) supplemented with 2M sodium chloride. For a
preparation that initially comprised >95% monomer, high salt
similarly drove the equilibrium to mostly (81%) dimer within 72
hours.
[0179] Salts that preserve or promote PSMA dimerization can include
those with a cationic component selected from the group consisting
of sodium, potassium, ammonium, magnesium, calcium, zinc and
combinations thereof, and those with an anionic component selected
from the group consisting of chloride, sulfate, acetate and
combinations thereof. In preferred embodiments the salt is sodium
chloride, sodium sulfate, sodium acetate or ammonium sulfate. The
salt can be present in a PSMA-containing composition at any
concentration that preserves or promotes the dimerization of PSMA.
In some instances the salt is present at a concentration in the
range of about 50 mM to about 2M. The salt preferably is present at
a concentration of about 100 mM to 300 mM. The salt more preferably
is present at a concentration of about 150 mM.
[0180] In some cases where a high salt concentration is used to
promote or preserve PSMA dimerization, the salt concentration can
be diluted to within a physiologically acceptable range suitable
for parenteral use prior to administration. As an example, the salt
concentration can be diluted with an adjuvant or a diluent.
Diluents and adjuvants are both well known in the art. An adjuvant
is a substance which potentiates the immune response. Specific
examples of adjuvants include monophosphoryl lipid A (MPL,
SmithKline Beecham); saponins, including QS-7, QS-17, QS-18, QS-21
(Antigenics, New York, N.Y.; U.S. Pat. Nos. 6,524,584 and
6,645,495); saponin-based adjuvants, such as SAPONIMMUNE (GPI-0100)
Series (Galenica Pharmaceuticals, Birmingham, Ala.; U.S. Pat. Nos.
5,977,081 and 6,080,725) and chemically modified saponins (Galenica
Pharmaceuticals, U.S. Pat. No. 6,262,029); polysaccharide-based
adjuvants, such as POLYSACClMMUNE (GPI-0200) Series (Galenica
Pharmaceuticals); synthetic adjuvants, such as SYNTHIMMUNE
(GPI-0300) Series (Galenica Pharmaceuticals); biodegradable
particles composed of poly-lactide-co-glycolide (PLG) or other
similar polymers; immunostimulatory oligonucleotides (e.g., CpG
oligonucleotides described by Kreig et al., Nature 374:546-9,
1995); incomplete Freund's adjuvant; complete Freund's adjuvant;
vitamin E and various water-in-oil emulsions prepared from
biodegradable oils such as squalene and/or tocopherol; MONTANIDE,
such as MONTANIDE ISA51 and MONTANIDE ISA720, which are
water-in-oil emulsions provided by Seppic (Paris, France); Quil A;
micellular mixtures of Quil A and cholesterol known as
immunostimulating complexes (ISCOMS); MPL and cell wall skeleton
from mycobacterium combinations such as ENHANZYN (Corixa, Seattle,
Wash.); RC-529 (Corixa); RC-552 (Corixa); CRL-1005; L-121;
alpha-galactosylceramide (Fujii et al., J. Exp. Med., 2003, Jul.
21; 198(2): 267-79); aluminum or iron oxide beads and combinations
thereof. Other specific examples of adjuvants include QS-21
fractions, such as crude QA-21; a QA-21H form; QA-21-V1; QA-21-V2;
a combination of QA-21-V1 and QA-21-V2; and chemically modified
forms or combinations thereof. Preferred adjuvants include alum and
QS-21. Other diluents include water suitable for injection, saline,
PBS, solubilizing agents and emulsifiers such as ethyl alcohol,
isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,
benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl
formamide, oils (in particular, cottonseed, groundnut, corn, germ,
olive, castor and sesame oils), glycerol, tetrahydrofurfuryl
alcohol, polyethylene glycols and fatty acid esters of sorbitan and
mixtures thereof.
[0181] Therefore, in some aspects of the invention a preferred
composition comprising isolated PSMA protein is a solution that
promotes or preserves multimeric, particularly dimeric, association
of PSMA protein comprising 5 to 20 mM sodium phosphate, sodium
acetate or a combination thereof; 100 to 300 mM sodium chloride or
sodium sulfate; and 0.1 to 2 mM of at least one metal ion. The
metal ions can be chosen from zinc ions, calcium ions, magnesium
ions, cobalt ions, manganese ions or a combination thereof. The pH
of such a solution can also be adjusted to be in a range of about 4
to 8, preferable 5 to 7 and most preferable 6 to 6.5. Such a
solution can also, optionally, include an adjuvant such as alum or
a saponin-based adjuvant, such as QS-21.
[0182] Agents that preserve or promote PSMA dimerization can be
used in compositions of PSMA protein or methods of processing such
compositions. Furthermore, a method for identifying such agents is
provided herein. Such a method includes the following steps:
determining the amount of a form of PSMA in a sample prior to
exposure to a candidate agent; exposing the sample to the candidate
agent; determining the amount of the form of PSMA in the sample
after the exposure; and comparing the amount of the form of PSMA in
the sample prior to and after the exposure to the candidate agent.
The form of PSMA can be a monomer or multimer, preferably the
dimer. An agent which preserves and/or promotes dimer formation of
PSMA protein is suitable for use in the compositions comprising
PSMA protein dimers.
[0183] As described below the effect of buffering agent on the
ability of PSMA to dimerize or maintain its dimerization was also
tested. It was found that many can be used in a solution of PSMA
without negatively impacting the dimeric state of PSMA. The sole
exception for solutions of PSMA protein with 150 mM NaCl at a pH of
6 was citrate buffer. Interestingly, citrate buffer is known to
function as a chelating agent. Therefore the formulations of PSMA
described herein can include any buffer so long as the buffer is
not one with a chelating effect that outweighs the preservation or
promoting effect of the other properties of the formulation.
Preferably optimal buffers include those with buffering capacity at
a pH in the range of about 4 to about 8. More preferably, buffers
are those with buffering capacity at a pH in the range of about 5
to about 7. Most preferably the buffers are those that have
buffering capacity at a pH in the range of about 5.5 to about 7.
Buffers in general are well known to those of ordinary skill in the
art. Buffer systems include citrate buffers, acetate buffers,
borate buffers, and phosphate buffers. Specific examples of buffers
include citric acid, sodium citrate, sodium acetate, acetic acid,
sodium phosphate and phosphoric acid, sodium ascorbate, tartartic
acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic
acid, imidazole, sodium bicarbonate and carbonic acid, sodium
succinate and succinic acid, histidine, and sodium benzoate and
benzoic acid. Buffers also include PBS and Hepes.
[0184] The effect of free amino acids on the dimeric state of
rsPSMA (2 mg/ml in PBS+) dialyzed into 20 mM sodium acetate and 150
mM NaCl at a pH of about 6 was also tested. In general it was found
that free amino acids did not have a strong negative effect on
dimer association of PSMA and/or column recovery, with the
exception of histidine, glutamic acid and aspartic acid used
individually at the specific experimental conditions. Therefore,
the formulations provided herein can also include a free amino acid
or combination of free amino acids, provided that the free amino
acid does not have a negative effect that outweighs the dimeric
association promoting or preserving nature of the specific
formulation. Such free amino acids can be naturally occurring,
modified or non-naturally occurring free amino acids (i.e.,
compounds that do not occur in nature but that can be incorporated
into a polypeptide chain; for example, non-natural amino acids that
have been successfully incorporated into functional ion channels).
Modified or non-naturally occurring free amino acids also include
but are not limited to 2-aminoadipic acid; 3-aminoadipic acid;
beta-alanine, beta-aminopropionic acid; 2-aminobutyric acid;
4-aminobutyric acid, piperidinic acid; 6-aminocaproic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine;
2,2'-diaminopimelic acid; 2,3-diaminopropionic acid;
N-ethylglycine; N-ethylasparagine; hydroxylysine;
allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline;
isodesmosine; allo-isoleucine; N-methylglycine, sarcosine;
N-methylisoleucine; 6-N-methyllysine; N-methylvaline; norvaline;
norleucine and ornithine. In particular, free amino acids that do
not have a negative effect on dimeric association of PSMA and/or
column recovery include those that are non-acidic. Examples of
these non-acidic free amino acids include glycine, proline,
isoleucine, leucine, alanine and arginine.
[0185] In addition to free amino acids, surfactants and other
excipients were also found not to have a negative impact on the
dimeric state of PSMA. Therefore, surfactants as well as other
excipients can be included in the compositions provided herein.
Examples of surfactants include those known in the art and
described herein. For example, surfactants include Triton X-100,
dodecylmaltoside, cholic acid and CHAPS.
[0186] Examples of excipients include binders, coatings,
compression/encapsulation aids, disintegrants, creams and lotions,
lubricants, materials for chewable tablets, parenterals,
plasticizers, powder lubricants, soft gelatin capsules, spheres for
coating, spheronization agents, suspending/gelling agents,
sweeteners and wet granulation agents. Specific examples of such
excipients include acetyltriethyl citrate (ATEC); acetyltri-n-butyl
citrate (ATBC); aspartame; aspartame and lactose; alginates;
calcium carbonate; carbopol; carrageenan; cellulose acetate
phthalate-based coatings; cellulose-based coatings; cellulose and
lactose combinations; colorants for film coating systems;
croscarmellose sodium; crospovidone; dextrose; dibutyl sebacate;
ethylcellulose-based coatings; fructose; gellan gum; glyceryl
behenate; honey; lactose; anhydrous; lactose; monohydrate; lactose
and aspartame; lactose and cellulose; lactose and microcrystalline
cellulose; L-HPC (Low-substituted HydroxyPropyl Cellulose);
magnesium stearate; maltodextrin; maltose DC; mannitol DC;
methylcellulose-based coatings; microcrystalline cellulose;
methacrylate-based coatings; microcrystalline cellulose and
carrageenan; microcrystalline cellulose and guar gum;
microcrystalline cellulose and lactose; microcrystalline cellulose
and sodium carboxymethylcellulose; molasses DC; polyvinyl acetate
phathalate (PVAP); povidone; shellac; sodium starch glycolate;
sorbitol, crystalline; sorbitol, special solution; starch DC;
sucrose DC; sugar spheres; triacetin; triethylcitrate and xanthan
gum. Other excipients include antioxidants and cryoprotectants.
[0187] Antioxidants are substances capable of inhibiting oxidation
by removing free radicals from solution. Antioxidants are well
known to those of ordinary skill in the art and include materials
such as ascorbic acid, ascorbic acid derivatives (e.g.,
ascorbylpalmitate, ascorbylstearate, sodium ascorbate, calcium
ascorbate, etc.), butylated hydroxy anisole, butylated hydroxy
toluene, alkylgallate, dithiothreitol (DTT), sodium meta-bisulfite,
sodium bisulfite, sodium dithionite, sodium thioglycollic acid,
sodium formaldehyde sulfoxylate, tocopherol and derivatives thereof
(e.g., d-alpha tocopherol, d-alpha tocopherol acetate, dl-alpha
tocopherol acetate, d-alpha tocopherol succinate, beta tocopherol,
delta tocopherol, gamma tocopherol, and d-alpha tocopherol
polyoxyethylene glycol 1000 succinate) monothioglycerol, and sodium
sulfite. Such materials are typically added in ranges from about
0.01 to about 2%.
[0188] For a lyophilized product or a product stored in the cold,
one or more cryoprotectants can be added. Typical cryoprotectants
for proteins include but are not limited to: sugars such as
sucrose, lactose, glucose, trehalose, maltose, and the like;
polyols such as inositol, ethylene glycol, glycerol, sorbitol,
xylitol, mannitol, 2-methyl-2,4-pentane-diol and the like; amino
acids such as Na glutamate, proline, alpha-alanine, beta-alanine,
glycine, lysine-HCl, 4-hydroxyproline; polymers such as
polyethylene glycol, dextran, polyvinylpyrrolidone and the like;
inorganics salts such as sodium sulfate, ammonium sulfate,
potassium phosphate, magnesium sulfate, and sodium fluoride and the
like; organics salts such as sodium acetate, sodium polyethylene,
sodium caprylate, proprionate, lactate, succinate and the like; as
well as agents such as trimethylamine N-oxide, sarcosine, betaine,
gamma-aminobutyric acid, octapine, alanopine, strombine,
dimethylsulfoxide, and ethanol.
[0189] The invention also involves methods for preparing or
processing compositions of PSMA protein. Aqueous solutions of PSMA
protein are included in these methods. Some of these methods
include the step of adjusting the pH so that it is in the range of
about 4 to about 8. In some methods the pH is adjusted to be in the
range of about 5 to 7, and more preferably the pH is adjusted to be
in the range of about 5.5 to 7. Most preferably the pH is adjusted
to be about 6. The compositions can also contain any one or
combination of an isotonicity agent, a buffering agent, a
surfactant, an antioxidant, a cryoprotectant or other excipients.
Preferably the compositions do not include a chelating agent.
[0190] According to another aspect of the invention, a composition
of PSMA protein is processed by contacting the composition of PSMA
protein with an agent that promotes or preserves the dimeric
association of PSMA such as pH adjusting agents, metal ions and/or
salts as provided above. Compositions that include these agents can
also include agents selected from an isotonicity agent, a buffering
agent, a surfactant, an antioxidant, a cryoprotectant and other
excipients, but preferably, not a chelating agent. Such methods can
also include further steps of contacting the composition of PSMA
protein with other dimer promoting or preserving agents and/or pH
adjusting steps when the PSMA protein is in a solution.
[0191] Additionally, in another aspect of the invention, a method
of purifying PSMA protein is also provided. The methods of
purifying PSMA include the use of any of the agents and/or
solutions described herein that preserve or promote the multimeric,
particularly dimeric, association of PSMA in conjunction with any
of the separation techniques that are known to those in the art.
Such separation techniques include chromatography (e.g., TSK gel
filtration chromatography) and are described in more detail in the
Examples below. For instance, a separation technique encompassed
within this aspect of the invention can include the steps of
loading a sample onto a column, eluting or washing the sample from
the column and collecting the eluted fractions. Such steps can be
repeated any of a number of times to produce the desired PSMA
protein composition. These steps can, optionally, also include
steps whereby the sample containing PSMA protein is dialyzed.
Preferably, the sample containing PSMA protein is dialyzed into a
solution that preserves or promotes the multimeric association of
PSMA. In one embodiment, the solutions used in these methods
contain a metal ion or a salt. In other to embodiments, the
solution is at a pH that preserves or promotes PSMA
multimerization. The metal ion and salts, including concentration
ranges, as well as pH ranges that can be used in these purification
methods have been provided above. In some preferred embodiments,
the pH of the solution can be at about 7 or 7.5. In other preferred
embodiments, the metals are calcium ions, magnesium ions or
combinations thereof. The calcium and magnesium ions are present,
for instance, at a concentration of about 1 mM and of about 0.5 mM,
respectively. In other preferred embodiments the salt is present at
a concentration of about 2M.
[0192] The amount of dimeric PSMA in the compositions provided
herein is effective to elicit or enhance an immune response to
cells expressing PSMA. The compositions can, therefore, be used to
immunize an animal for the purpose of raising antibodies to dimeric
PSMA. The compositions provided herein can also be used to treat a
subject suffering from a cancer, wherein the cancer cells or
proximate neovasculature express PSMA. Such cancers can include
prostate, bladder, pancreas, lung, colon, kidney, melanomas and
sarcomas. In a preferred embodiment the cancer cell is a prostate
cancer cell. The cancer cells can be cells of a primary tumor or
can be those of a metastatic tumor.
[0193] The subject can be a non-castrate patient who has, in some
embodiments, received primary therapy, such as prostatectomy and/or
radiation therapy. As used herein, "non-castrate patient" refers to
a patient in some embodiments with a serum testosterone level that
is greater than or equal to about 180 ng/mL. The subject can also
be a castrate patient who has, in some embodiments, completed a
course of hormonal therapy. As used herein, the term "castrate
patient" refers to a patient in some embodiments with a serum
testosterone level of less than about 50 ng/mL. The compositions
provided herein can also be administered to a patient who has
received conventional cancer therapy.
[0194] Another aspect of the invention provides an isolated
antibody or an antigen-binding fragment thereof which specifically
binds to an extracellular domain of PSMA wherein the antibody or
the antigen-binding fragment thereof competitively inhibits the
specific binding of a second antibody to its target epitope on
PSMA, and wherein the second antibody is selected from the group
consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4,
PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA
A3.3.1, 4.248.2, 4.360.3, 4.7.1, 4.4.1, 4.177.3, 4.16.1, 4.22.3,
4.28.3, 4.40.2, 4.48.3, 4.49.1, 4.209.3, 4.219.3, 4.288.1, 4.333.1,
4.54.1, 4.153.1, 4.232.3, 4.292.3, 4.304.1, 4.78.1, and
4.152.1.
[0195] Another aspect of the invention provides an isolated
antibody or an antigen-binding fragment thereof that specifically
binds to an epitope on PSMA defined by an antibody selected from
the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11,
PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3,
PSMA A3.3.1, 4.248.2, 4.360.3, 4.7.1, 4.4.1, 4.177.3, 4.16.1,
4.22.3, 4.28.3, 4.40.2, 4.48.3, 4.49.1, 4.209.3, 4.219.3, 4.288.1,
4.333.1, 4.54.1, 4.153.1, 4.232.3, 4.292.3, 4.304.1, 4.78.1, and
4.152.1.
[0196] In particular embodiments, these antibodies are produced by
hybridomas referred to herein as PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA
3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA
A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix
4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix
4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix
4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix
4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix
4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, and Abgenix 4.152.1,
respectively. These hybridomas were deposited with ATCC as an
International Depository Authority and given the following Patent
Deposit Designations (Table 1):
TABLE-US-00001 TABLE 1 Patent Deposit Antibody Hybridoma/Plasmid
Designation Date of Deposit PSMA 3.7 PSMA 3.7 PTA-3257 Apr. 5, 2001
PSMA 3.9 PSMA 3.9 PTA-3258 Apr. 5, 2001 PSMA 3.11 PSMA 3.11
PTA-3269 Apr. 10, 2001 PSMA 5.4 PSMA 5.4 PTA-3268 Apr. 10, 2001
PSMA 7.1 PSMA 7.1 PTA-3292 Apr. 18, 2001 PSMA 7.3 PSMA 7.3 PTA-3293
Apr. 18, 2001 PSMA 10.3 PSMA 10.3 PTA-3347 May 1, 2001 PSMA 10.3 HC
in PTA-4413 May 29, 2002 pcDNA (SEQ ID NO: 7) PSMA 10.3 Kappa in
PTA-4414 May 29, 2002 pcDNA (SEQ ID NO: 13) PSMA 1.8.3 PSMA 1.8.3
PTA-3906 Dec. 5, 2001 PSMA A3.1.3 PSMA A3.1.3 PTA-3904 Dec. 5, 2001
PSMA A3.3.1 PSMA A3.3.1 PTA-3905 Dec. 5, 2001 Abgenix 4.248.2
Abgenix 4.248.2 PTA-4427 Jun. 4, 2002 Abgenix 4.360.3 Abgenix
4.360.3 PTA-4428 Jun. 4, 2002 Abgenix 4.7.1 Abgenix 4.7.1 PTA-4429
Jun. 4, 2002 Abgenix 4.4.1 Abgenix 4.4.1 PTA-4556 Jul. 18, 2002
Abgenix 4.177.3 Abgenix 4.177.3 PTA-4557 Jul. 18, 2002 Abgenix
4.16.1 Abgenix 4.16.1 PTA-4357 May 16, 2002 Abgenix 4.22.3 Abgenix
4.22.3 PTA-4358 May 16, 2002 Abgenix 4.28.3 Abgenix 4.28.3 PTA-4359
May 16, 2002 Abgenix 4.40.2 Abgenix 4.40.2 PTA-4360 May 16, 2002
Abgenix 4.48.3 Abgenix 4.48.3 PTA-4361 May 16, 2002 Abgenix 4.49.1
Abgenix 4.49.1 PTA-4362 May 16, 2002 Abgenix 4.209.3 Abgenix
4.209.3 PTA-4365 May 16, 2002 Abgenix 4.219.3 Abgenix 4.219.3
PTA-4366 May 16, 2002 Abgenix 4.288.1 Abgenix 4.288.1 PTA-4367 May
16, 2002 Abgenix 4.333.1 Abgenix 4.333.1 PTA-4368 May 16, 2002
Abgenix 4.54.1 Abgenix 4.54.1 PTA-4363 May 16, 2002 Abgenix 4.153.1
Abgenix 4.153.1 PTA-4388 May 23, 2002 Abgenix 4.232.3 Abgenix
4.232.3 PTA-4389 May 23, 2002 Abgenix 4.292.3 Abgenix 4.292.3
PTA-4390 May 23, 2002 Abgenix 4.304.1 Abgenix 4.304.1 PTA-4391 May
23, 2002 AB-PG1-XG1-006 AB-PG1-XG1-006 Heavy PTA-4403 May 29, 2002
Chain (SEQ ID NO: 2) AB-PG1-XG1-006 Light PTA-4404 Chain (SEQ ID
NO: 8) AB-PG1-XG1-026 AB-PG1-XG1-026 Heavy PTA-4405 May 29, 2002
Chain (SEQ ID NO: 3) AB-PG1-XG1-026 Light PTA-4406 Chain (SEQ ID
NO: 9) AB-PG1-XG1-051 AB-PG1-XG1-051 Heavy PTA-4407 May 29, 2002
Chain (SEQ ID NO: 4) AB-PG1-XG1-051 Light PTA-4408 Chain (SEQ ID
NO: 10) AB-PG1-XG1-069 AB-PG1-XG1-069 Heavy PTA-4409 May 29, 2002
Chain (SEQ ID NO: 5) AB-PG1-XG1-069 Light PTA-4410 Chain (SEQ ID
NO: 11) AB-PG1-XG1-077 AB-PG1-XG1-077 Heavy PTA-4411 May 29, 2002
Chain (SEQ ID NO: 6) AB-PG1-XG1-077 Light PTA-4412 Chain (SEQ ID
NO: 12)
[0197] In another aspect of the invention, antibodies having
particular sequences are provided. Specifically, the antibodies are
selected from the group consisting of antibodies comprising: a
heavy chain encoded by a nucleic acid molecule comprising the heavy
chain coding region or regions of a nucleotide sequence selected
from the group consisting of nucleotide sequences set forth as SEQ
ID NOs: 2-7, and a light chain encoded by a nucleic acid molecule
comprising the light chain coding region or regions of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as SEQ ID NOs: 8-13. Also provided are antigen-binding
fragments of the foregoing antibodies.
[0198] The plasmids encoding the heavy and light chains of
antibodies PSMA 10.3, AB-PG1-XG1-006, AB-PG1-XG1-026,
AB-PG1-XG1-051, AB-PG1-XG1-069, AB-PG1-XG1-077 were also deposited
with ATCC and are shown in Table 1 above. As used herein, the names
of the deposited hybridomas or plasmids may be used interchangeably
with the names of the antibodies. It would be clear to one of skill
in the art when the name is intended to refer to the antibody or
when it refers to the plasmids or hybridomas that encode or produce
the antibodies, respectively. Additionally, the antibody names may
be an abbreviated form of the name shown in Table 1. For instance
antibody AB-PG1-XG1-006 may be referred to as AB-PG1-XG1-006,
PG1-XG1-006, XG1-006, 006, etc. In another example, the antibody
name PSMA 4.232.3 may be referred to as PSMA 4.232.1, 4.232.3,
4.232.1, 4.232, etc. It is intended that all of the variations in
the name of the antibody refer to the same antibody and not a
different one.
[0199] Antibodies are also provided that are encoded by particular
sets of heavy and light chain sequences. In one embodiment an
antibody (AB-PG1-XG1-006) encoded by a nucleic acid molecule which
comprises the coding region or regions of the nucleic acid
sequences set forth as: SEQ ID NOs: 2 and 8 is provided. In another
embodiment the antibody (AB-PG1-XG1-026) is encoded by the nucleic
acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 3 and 9. In still
another embodiment the antibody (AB-PG1-XG1-051) is encoded by the
nucleic acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 4 and 10. In yet
another embodiment the antibody (AB-PG1-XG1-069) is encoded by the
nucleic acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 5 and 11. In another
embodiment the antibody (AB-PG1-XG1-077) is encoded by the nucleic
acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 6 and 12. In yet
another embodiment the antibody (PSMA 10.3) is encoded by the
nucleic acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 7 and 13.
[0200] In particularly preferred embodiments, the antibodies
include a heavy chain variable region encoded by a nucleic acid
molecule comprising the coding regions or regions of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as: SEQ ID NOs: 14, 18, 22, 26 and 30, and a light chain
variable region encoded by a nucleic acid molecule comprising the
coding region or region of a nucleotide sequence selected from the
group consisting of nucleotide sequences set forth as: SEQ ID NOs:
16, 20, 24, 28 and 32. As used herein, a "coding region" refers to
a region of a nucleotide sequence that encodes a polypeptide
sequence; the coding region can include a region coding for a
portion of a protein that is later cleaved off, such as a signal
peptide.
[0201] Those of skill in the art will appreciate that the invention
includes nucleic acids and polypeptides that include nucleotide and
amino acid sequences presented herein. In some instances, the
nucleotide and amino acid sequences may include sequences that
encode or that are signal peptides. The invention embraces each of
these sequences with, or without, the portion of the sequence that
encodes or is a signal peptide.
[0202] Antibodies also are provided that include particular sets of
heavy and light chain variable sequences. In one embodiment an
antibody (AB-PG1-XG1-006) includes an immunoglobulin variable
sequence encoded by nucleic acid molecules which included the
coding region or regions of the nucleic acid sequences set forth
as: SEQ ID NOs: 14 and 16 is provided. Likewise the antibody may
include an immunoglobulin variable sequence which comprises the
amino acid sequences set forth as SEQ ID NOs: 15 and 17. In another
embodiment the antibody (AB-PG1-XG1-026) includes an immunoglobulin
variable sequence encoded by nucleic acid molecules comprising the
coding region or regions of nucleotide sequences set forth as: SEQ
ID NOs: 18 and 20 or includes an immunoglobulin variable sequence
which comprises the amino acid sequences set forth as SEQ ID NOs:
19 and 21. In still another embodiment the antibody
(AB-PG1-XG1-051) includes an immunoglobulin variable sequence
encoded by the nucleic acid molecules comprising the coding region
or regions of nucleotide sequences set forth as: SEQ ID NOs: 22 and
24 or includes an immunoglobulin variable sequence which comprises
the amino acid sequences set forth as SEQ ID NOs: 23 and 25. In yet
another embodiment the antibody (AB-PG1-XG1-069) includes an
immunoglobulin variable sequence encoded by the nucleic acid
molecules comprising the coding region or regions of nucleotide
sequences set forth as: SEQ ID NOs: 26 and 28 or includes an
immunoglobulin variable sequence which comprises the amino acid
sequences set forth as SEQ ID NOs: 27 and 29. In another embodiment
the antibody (AB-PG1-XG1-077) includes an immunoglobulin variable
sequence encoded by the nucleic acid molecules comprising the
coding region or regions of nucleotide sequences set forth as: SEQ
ID NOs: 30 and 32 or includes an immunoglobulin variable sequence
which comprises the amino acid sequences set forth as SEQ ID NOs:
31 and 33.
[0203] In certain embodiments, the antibody is encoded by a nucleic
acid molecule that is highly homologous to the foregoing nucleic
acid molecules. Preferably the homologous nucleic acid molecule
comprises a nucleotide sequence that is at least about 90%
identical to the nucleotide sequence provided herein. More
preferably, the nucleotide sequence is at least about 95%
identical, at least about 97% identical, at least about 98%
identical, or at least about 99% identical to the nucleotide
sequence provided herein. The homology can be calculated using
various, publicly available software tools well known to one of
ordinary skill in the art. Exemplary tools include the BLAST system
available from the website of the National Center for Biotechnology
Information (NCBI) at the National Institutes of Health.
[0204] One method of identifying highly homologous nucleotide
sequences is via nucleic acid hybridization. Thus the invention
also includes antibodies having the PSMA-binding properties and
other functional properties described herein, which are encoded by
nucleic acid molecules that hybridize under high stringency
conditions to the foregoing nucleic acid molecules. Identification
of related sequences can also be achieved using polymerase chain
reaction (PCR) and other amplification techniques suitable for
cloning related nucleic acid sequences. Preferably, PCR primers are
selected to amplify portions of a nucleic acid sequence of
interest, such as a CDR.
[0205] The term "high stringency conditions" as used herein refers
to parameters with which the art is familiar. Nucleic acid
hybridization parameters may be found in references that compile
such methods, e.g. Molecular Cloning: A Laboratory Manual, J.
Sambrook, et al., eds., Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current
Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York. One example of high-stringency
conditions is hybridization at 65.degree. C. in hybridization
buffer (3.5.times.SSC, 0.02% FICOLL, 0.02% polyvinyl pyrrolidone,
0.02% Bovine Serum Albumin, 2.5 mM NaH.sub.2PO.sub.4(pH7), 0.5%
SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium
citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is
ethylenediaminetetracetic acid. After hybridization, a membrane
upon which the nucleic acid is transferred is washed, for example,
in 2.times.SSC at room temperature and then at
0.1-0.5.times.SSC/0.1.times.SDS at temperatures up to 68.degree.
C.
[0206] In other preferred embodiments, the antibodies include a
heavy chain variable region comprising an amino acid sequence
selected from the group consisting of amino acid sequences set
forth as: SEQ ID NOs: 15, 19, 23, 27 and 31, and a light chain
variable region comprising an amino acid sequence selected from the
group consisting of nucleotide sequences set forth as: SEQ ID NOs:
17, 21, 25, 29 and 33. Antigen-binding fragments of the foregoing
also are provided, as described elsewhere herein.
[0207] As used herein, the term "antibody" refers to a glycoprotein
comprising at least two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds. Each heavy chain is comprised
of a heavy chain variable region (abbreviated herein as HCVR or
V.sub.H) and a heavy chain constant region. The heavy chain
constant region is comprised of three domains, C.sub.H1, C.sub.H2
and C.sub.H3. Each light chain is comprised of a light chain
variable region (abbreviated herein as LCVR or V.sub.L) and a light
chain constant region. The light chain constant region is comprised
of one domain, CL. The V.sub.H and V.sub.L regions can be further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each V.sub.H and V.sub.L
is composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy
and light chains contain a binding domain that interacts with an
antigen. The constant regions of the antibodies may mediate the
binding of the immunoglobulin to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (C1q) of the classical complement system.
[0208] The term "antigen-binding fragment" of an antibody as used
herein, refers to one or more portions of an antibody that retain
the ability to specifically bind to an antigen (e.g., PSMA). It has
been shown that the antigen-binding function of an antibody can be
performed by fragments of a full-length antibody. Examples of
binding fragments encompassed within the term "antigen-binding
fragment" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the V.sub.H and CH1
domains; (iv) a Fv fragment consisting of the V.sub.L and V.sub.H
domains of a single arm of an antibody, (v) a dAb fragment (Ward et
al., (1989) Nature 341:544-546) which consists of a V.sub.H domain;
and (vi) an isolated complementarity determining region (CDR).
Furthermore, although the two domains of the Fv fragment, V and
V.sub.H, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the V.sub.L and V.sub.H
regions pair to form monovalent molecules (known as single chain Fv
(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and
Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such
single chain antibodies are also intended to be encompassed within
the term "antigen-binding portion" of an antibody. These antibody
fragments are obtained using conventional procedures, such as
proteolytic fragmentation procedures, as described in J. Goding,
Monoclonal Antibodies: Principles and Practice, pp 98-118 (N.Y.
Academic Press 1983), which is hereby incorporated by reference as
well as by other techniques known to those with skill in the art.
The fragments are screened for utility in the same manner as are
intact antibodies.
[0209] An "isolated antibody", as used herein, is intended to refer
to an antibody which is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds to PSMA is substantially free of
antibodies that specifically bind antigens other than PSMA). An
isolated antibody that specifically binds to an epitope, isoform or
variant of PSMA may, however, have cross-reactivity to other
related antigens, e.g., from other species (e.g., PSMA species
homologs). Moreover, an isolated antibody may be substantially free
of other cellular material and/or chemicals. As used herein,
"specific binding" refers to antibody binding to a predetermined
antigen. Typically, the antibody binds with an affinity that is at
least two-fold greater than its affinity for binding to a
non-specific antigen (e.g., BSA, casein) other than the
predetermined antigen or a closely-related antigen.
[0210] The isolated antibodies of the invention encompass various
antibody isotypes, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2,
IgAsec, IgD, IgE. As used herein, "isotype" refers to the antibody
class (e.g. IgM or IgG1) that is encoded by heavy chain constant
region genes. The antibodies can be full length or can include only
an antigen-binding fragment such as the antibody constant and/or
variable domain of IgG1, IgG2, IgG3, IgG4, IgM, IgA 1, IgA2,
IgAsec, IgD or IgE or could consist of a Fab fragment, a
F(ab').sub.2 fragment, and a Fv fragment.
[0211] The antibodies of the present invention can be polyclonal,
monoclonal, or a mixture of polyclonal and monoclonal antibodies.
The antibodies can be produced by a variety of techniques well
known in the art. Procedures for raising polyclonal antibodies are
well known. For example anti-PSMA polyclonal antibodies are raised
by administering PSMA protein subcutaneously to New Zealand white
rabbits which have first been bled to obtain pre-immune serum. The
PSMA can be injected at a total volume of 100 .mu.l per site at six
different sites, typically with one or more adjustments. The
rabbits are then bled two weeks after the first injection and
periodically boosted with the same antigen three times every six
weeks. A sample of serum is collected 10 days after each boost.
Polyclonal antibodies are recovered from the serum, preferably by
affinity chromatography using PSMA to capture the antibody. This
and other procedures for raising polyclonal antibodies are
disclosed in E. Harlow, et. al., editors, Antibodies: A Laboratory
Manual (1988), which is hereby incorporated by reference.
[0212] Monoclonal antibody production may be effected by techniques
which are also well known in the art. The term "monoclonal
antibody," as used herein, refers to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
displays a single binding specificity and affinity for a particular
epitope. The process of monoclonal antibody production involves
obtaining immune somatic cells with the potential for producing
antibody, in particular B lymphocytes, which have been previously
immunized with the antigen of interest either in vivo or in vitro
and that are suitable for fusion with a B-cell myeloma line.
[0213] Mammalian lymphocytes typically are immunized by in vivo
immunization of the animal (e.g., a mouse) with the desired protein
or polypeptide, e.g., with PSMA in the present invention. Such
immunizations are repeated as necessary at intervals of up to
several weeks to obtain a sufficient titer of antibodies. Once
immunized, animals can be used as a source of antibody-producing
lymphocytes. Following the last antigen boost, the animals are
sacrificed and spleen cells removed. Mouse lymphocytes give a
higher percentage of stable fusions with the mouse myeloma lines
described herein. Of these, the BALB/c mouse is preferred. However,
other mouse strains, rabbit, hamster, sheep and frog may also be
used as hosts for preparing antibody-producing cells. See; Goding
(in Monoclonal Antibodies: Principles and Practice, 2d ed., pp.
60-61, Orlando, Fla., Academic Press, 1986). In particular, mouse
strains that have human immunoglobulin genes inserted in the genome
(and which cannot produce mouse immunoglobulins) are preferred.
Examples include the HUMAB-MOUSE strains produced by
Medarex/GenPharm International, and the XENOMOUSE strains produced
by Abgenix. Such mice produce fully human immunoglobulin molecules
in response to immunization.
[0214] Those antibody-producing cells that are in the dividing
plasmablast stage fuse preferentially. Somatic cells may be
obtained from the lymph nodes, spleens and peripheral blood of
antigen-primed animals, and the lymphatic cells of choice depend to
a large extent on their empirical usefulness in the particular
fusion system. The antibody-secreting lymphocytes are then fused
with (mouse) B cell myeloma cells or transformed cells, which are
capable of replicating indefinitely in cell culture, thereby
producing an immortal, immunoglobulin-secreting cell line. The
resulting fused cells, or hybridomas, are cultured, and the
resulting colonies screened for the production of the desired
monoclonal antibodies. Colonies producing such antibodies are
cloned, and grown either in vivo or in vitro to produce large
quantities of antibody. A description of the theoretical basis and
practical methodology of fusing such cells is set forth in Kohler
and Milstein, Nature 256:495 (1975), which is hereby incorporated
by reference.
[0215] Alternatively, human somatic cells capable of producing
antibody, specifically B lymphocytes, are suitable for fusion with
myeloma cell lines. While B lymphocytes from biopsied spleens,
tonsils or lymph nodes of an individual may be used, the more
easily accessible peripheral blood B lymphocytes are preferred. The
lymphocytes may be derived from patients with diagnosed prostate
carcinomas or another PSMA-expressing cancer. In addition, human B
cells may be directly immortalized by the Epstein-Barr virus (Cole
et al., 1995, Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96). Although somatic cell hybridization
procedures are preferred, in principle, other techniques for
producing monoclonal antibodies can be employed such as viral or
oncogenic transformation of B lymphocytes.
[0216] Myeloma cell lines suited for use in hybridoma-producing
fusion procedures preferably are non-antibody-producing, have high
fusion efficiency, and enzyme deficiencies that render them
incapable of growing in certain selective media which support the
growth of the desired hybridomas. Examples of such myeloma cell
lines that may be used for the production of fused cell lines
include P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4.1, Sp2/0-Ag14, FO,
NSO/U, MPC-11, MPC11-X45-GTG 1.7, S194/5XX0 Bul, all derived from
mice; R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210 derived from rats
and U-266, GM1500-GRG2, LICR-LON-HMy2, UC729-6, all derived from
humans (Goding, in Monoclonal Antibodies: Principles and Practice,
2d ed., pp. 65-66, Orlando, Fla., Academic Press, 1986; Campbell,
in Monoclonal Antibody Technology, Laboratory Techniques in
Biochemistry and Molecular Biology Vol. 13, Burden and Von
Knippenberg, eds. pp. 75-83, Amsterdam, Elseview, 1984).
[0217] Fusion with mammalian myeloma cells or other fusion partners
capable of replicating indefinitely in cell culture is effected by
standard and well-known techniques, for example, by using
polyethylene glycol ("PEG") or other fusing agents (See Milstein
and Kohler, Eur. J. Immunol. 6:511 (1976), which is hereby
incorporated by reference).
[0218] In other embodiments, the antibodies can be recombinant
antibodies. The term "recombinant antibody", as used herein, is
intended to include antibodies that are prepared, expressed,
created or isolated by recombinant means, such as antibodies
isolated from an animal (e.g., a mouse) that is transgenic for
another species' immunoglobulin genes, antibodies expressed using a
recombinant expression vector transfected into a host cell,
antibodies isolated from a recombinant, combinatorial antibody
library, or antibodies prepared, expressed, created or isolated by
any other means that involves splicing of immunoglobulin gene
sequences to other DNA sequences.
[0219] In yet other embodiments, the antibodies can be chimeric or
humanized antibodies. As used herein, the term "chimeric antibody"
refers to an antibody, that combines the murine variable or
hypervariable regions with the human constant region or constant
and variable framework regions. As used herein, the term "humanized
antibody" refers to an antibody that retains only the
antigen-binding CDRs from the parent antibody in association with
human framework regions (see, Waldmann, 1991, Science 252:1657).
Such chimeric or humanized antibodies retaining binding specificity
of the murine antibody are expected to have reduced immunogenicity
when administered in vivo for diagnostic, prophylactic or
therapeutic applications according to the invention.
[0220] According to an alternative embodiment, the monoclonal
antibodies of the present invention can be modified to be in the
form of a bispecific antibody, or a multispecific antibody. The
term "bispecific antibody" is intended to include any agent, e.g.,
a protein, peptide, or protein or peptide complex, which has two
different binding specificities which bind to, or interact with (a)
a cell surface antigen and (b) an Fc receptor on the surface of an
effector cell. The term "multispecific antibody" is intended to
include any agent, e.g., a protein, peptide, or protein or peptide
complex, which has more than two different binding specificities
which bind to, or interact with (a) a cell surface antigen, (b) an
Fc receptor on the surface of an effector cell, and (c) at least
one other component. Accordingly, the invention includes, but is
not limited to, bispecific, trispecific, tetraspecific, and other
multispecific antibodies which are directed to cell surface
antigens, such as PSMA, and to Fc receptors on effector cells. The
term "bispecific antibodies" further includes diabodies. Diabodies
are bivalent, bispecific antibodies in which the V.sub.H and
V.sub.L domains are expressed on a single polypeptide chain, but
using a linker that is too short to allow for pairing between the
two domains on the same chain, thereby forcing the domains to pair
with complementary domains of another chain and creating two
antigen-binding sites (see e.g., Holliger, P., et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448; Poijak, R. J., et al. (1994)
Structure 2:1121-1123).
[0221] A bispecific antibody can be formed of an antigen-binding
region specific for the extracellular domain of PSMA and an
antigen-binding region specific for an effector cell which has
tumoricidal or tumor inhibitory activity. The two antigen-binding
regions of the bispecific antibody are either chemically linked or
can be expressed by a cell genetically engineered to produce the
bispecific antibody. (See generally, Fanger et al., 1995 Drug News
& Perspec. 8(3):133-137). Suitable effector cells having
tumoricidal activity include but are not limited to cytotoxic
T-cells (primarily CD8.sup.+ cells), natural killer cells, etc. An
effective amount of a bispecific antibody according to the
invention is administered to a prostrate cancer patient and the
bispecific antibody kills and/or inhibits proliferation of the
malignant cells after localization at sites of primary or
metastatic tumors bearing PSMA.
[0222] In certain embodiments, the antibodies are human antibodies.
The term "human antibody", as used herein, is intended to include
antibodies having variable and constant regions derived from human
germline immunoglobulin sequences. The human antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). However, the term "human antibody", as used herein, is
not intended to include antibodies in which CDR sequences derived
from the germline of another mammalian species, such as a mouse
have been grafted onto human framework sequences (referred to
herein as "humanized antibodies"). Human antibodies directed
against PSMA are generated using transgenic mice carrying parts of
the human immune system rather than the mouse system.
[0223] Fully human monoclonal antibodies also can be prepared by
immunizing mice transgenic for large portions of human
immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat.
Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and
references cited therein, the contents of which are incorporated
herein by reference. These animals have been genetically modified
such that there is a functional deletion in the production of
endogenous (e.g., murine) antibodies. The animals are further
modified to contain all or a portion of the human germ-line
immunoglobulin gene locus such that immunization of these animals
results in the production of fully human antibodies to the antigen
of interest. Following immunization of these mice (e.g., XENOMOUSE
(Abgenix), HUMAB-MOUSE mice (Medarex/GenPharm)), monoclonal
antibodies are prepared according to standard hybridoma technology.
These monoclonal antibodies have human immunoglobulin amino acid
sequences and therefore will not provoke human anti-mouse antibody
(HAMA) responses when administered to humans.
[0224] Preferably, the mice are 6-16 weeks of age upon the first
immunization. For example, a purified or enriched preparation of
PSMA antigen (e.g., recombinant PSMA, PSMA-expressing cells,
dimeric PSMA) is used to immunize the mice intraperitoneally (IP),
although other routes of immunization known to one of ordinary
skill in the art are also possible. PSMA antigen is injected in
combination with an adjuvant, such as complete Freund's adjuvant,
and preferably the initial injection is followed by booster
immunizations with antigen in an adjuvant, such as incomplete
Freund's adjuvant. The immune response is monitored over the course
of the immunization protocol with plasma samples obtained by, for
example, retroorbital bleeds. The plasma is screened by ELISA (as
described below), and mice with sufficient titers of anti-PSMA
human immunoglobulin are used for fusions. Mice are boosted
intravenously with antigen 3 days before sacrifice and removal of
the spleen.
[0225] In particular embodiments, the antibodies are produced by
hybridomas referred to herein as PSMA 3.7 (PTA-3257), PSMA 3.8,
PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269), PSMA 5.4 (PTA-3268),
PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA 10.3 (PTA-3247),
PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904), PSMA A3.3.1
(PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3 (PTA-4428),
Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3
(PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358),
Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix
4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3
(PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367),
Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix
4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3
(PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652),
and Abgenix 4.152.1 (PTA-4653). These hybridomas were deposited
pursuant to, and in satisfaction of, the requirements of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure with the
American Type Culture Collection ("ATCC") as an International
Depository Authority and given the Patent Deposit Designations
shown above and in Table 1.
[0226] The present invention further provides nucleic acid
molecules encoding anti-PSMA antibodies and vectors comprising the
nucleic acid molecules as described herein. The vectors provided
can be used to transform or transfect host cells for producing
anti-PSMA antibodies with the specificity of antibodies described
herein. In a preferred embodiment the antibodies produced will have
the specificity of the antibodies AB-PG1-XG1-006, AB-PG1-XG1-026,
AB-PG1-XG1-051, AB-PG1, XG1-069, AB-PG1-XG1-077 and PSMA 10.3. In
one embodiment the vectors can comprise an isolated nucleic acid
molecule encoding the heavy chain of the antibodies listed above
encoded by a nucleic acid molecules comprising the coding region or
regions of the nucleic acid sequences set forth as SEQ ID NO: 2-7.
In another embodiment, the vectors can comprise the nucleic acid
sequences encoding the light chain of the antibodies set forth as
SEQ ID NOs: 8-13. In a further embodiment the vectors of the
invention may comprise a heavy chain and a light chain sequence. In
a further embodiment, plasmids are given which produce the
antibodies or antigen binding fragments described herein. Plasmids
of the invention include plasmids selected from the group
consisting of: AB-PG1-XG1-006 Heavy Chain (SEQ ID NO: 2),
AB-PG1-XG1-006 Light Chain (SEQ ID NO: 8), AB-PG1-XG1-026 Heavy
Chain (SEQ ID NO: 3), AB-PG1-XG1-026 Light Chain (SEQ ID NO: 9),
AB-PG1-XG1-051 Heavy Chain (SEQ ID NO: 4), AB-PG1-XG1-051 Light
Chain (SEQ ID NO: 10), AB-PG1-XG1-069 Heavy Chain (SEQ ID NO: 5),
AB-PG1-XG1-069 Light Chain (SEQ ID NO: 11), AB-PG1-XG1-077 Heavy
Chain (SEQ ID NO: 6), AB-PG1-XG1-077 Light Chain (SEQ ID NO: 12),
PSMA 10.3 Heavy Chain (SEQ ID NO: 7), and PSMA 10.3 Kappa (SEQ ID
NO: 13).
[0227] The isolated antibody or antigen-binding fragment thereof
preferably is selected for its ability to bind live cells
expressing PSMA. In order to demonstrate binding of monoclonal
antibodies to live cells expressing the PSMA, flow cytometry can be
used. For example, cell lines expressing PSMA (grown under standard
growth conditions) or prostate cancer cells that express PSMA are
mixed with various concentrations of monoclonal antibodies in PBS
containing 0.1% TWEEN 80 and 20% mouse serum, and incubated at
37.degree. C. for 1 hour. After washing, the cells are reacted with
fluorescein-labeled anti-human IgG secondary antibody (if human
anti-PSMA antibodies were used) under the same conditions as the
primary antibody staining. The samples can be analyzed by a
fluorescence activated cell sorter (FACS) instrument using light
and side scatter properties to gate on single cells. An alternative
assay using fluorescence microscopy may be used (in addition to or
instead of) the flow cytometry assay. Cells can be stained exactly
as described above and examined by fluorescence microscopy. This
method allows visualization of individual cells, but may have
diminished sensitivity depending on the density of the antigen.
[0228] Binding of the antibody or antigen-binding fragment thereof
to live cells expressing PSMA can inhibit the growth of the cells
or mediate cytolysis of the cells. Cytolysis can be complement
mediated or can be mediated by effector cells. In a preferred
embodiment, the cytolysis is carried out in a living organism,
preferably a mammal, and the live cell is a tumor cell. Examples of
tumors which can be targeted by the antibodies of the invention
include, any tumor that expresses PSMA, such as, prostate, bladder,
pancreas, lung, colon, kidney, melanomas and sarcomas. In a
preferred embodiment the tumor cell is a prostate cancer cell.
[0229] The testing of antibody cytolytic activity in vitro by
chromium release assay can provide an initial screening prior to
testing in vivo models. This testing can be carried out using
standard chromium release assays. Briefly, polymorphonuclear cells
(PMN), or other effector cells, from healthy donors can be purified
by FICOLL Hypaque density centrifugation, followed by lysis of
contaminating erythrocytes. Washed PMNs can be suspended in RPMI
supplemented with 10% heat-inactivated fetal calf serum and mixed
with .sup.51Cr labeled cells expressing PSMA, at various ratios of
effector cells to tumor cells (effector cells:tumor cells).
Purified anti-PSMA IgGs can then be added at various
concentrations. Irrelevant IgG can be used as negative control.
Assays can be carried out for 0-120 minutes at 37.degree. C.
Samples can be assayed for cytolysis by measuring .sup.51Cr release
into the culture supernatant. Anti-PSMA monoclonal antibodies can
also be tested in combinations with each other to determine whether
cytolysis is enhanced with multiple monoclonal antibodies.
[0230] Antibodies which bind to PSMA also can be tested in an in
vivo model (e.g., in mice) to determine their efficacy in mediating
cytolysis and killing of cells expressing PSMA, e.g., tumor cells.
These antibodies can be selected, for example, based on the
following criteria, which are not intended to be exclusive: [0231]
1) binding to live cells expressing PSMA; [0232] 2) high affinity
of binding to PSMA; [0233] 3) binding to a unique epitope on PSMA
(to eliminate the possibility that antibodies with complimentary
activities when used in combination would compete for binding to
the same epitope); [0234] 4) opsonization of cells expressing PSMA;
[0235] 5) mediation of growth inhibition, phagocytosis and/or
killing of cells expressing PSMA in the presence of effector cells;
[0236] 6) modulation (inhibition or enhancement) of NAALADase,
folate hydrolase, dipeptidyl peptidase IV and/or .gamma.-glutamyl
hydrolase activities; [0237] 7) growth inhibition, cell cycle
arrest and/or cytotoxicity in the absence of effector cells; [0238]
8) internalization of PSMA; [0239] 9) binding to a conformational
epitope on PSMA; [0240] 10) minimal cross-reactivity with cells or
tissues that do not express PSMA; and [0241] 11) preferential
binding to dimeric forms of PSMA rather than monomeric forms of
PSMA.
[0242] Preferred antibodies of the invention meet one or more, and
preferably all, of these criteria. In a particular embodiment, the
antibodies are used in combination, e.g., as a pharmaceutical
composition comprising two or more different anti-PSMA antibodies
or binding fragments thereof. For example, anti-PSMA antibodies
having different, but complementary activities can be combined in a
single therapy to achieve a desired therapeutic or diagnostic
effect. An illustration of this would be a composition containing
an anti-PSMA antibody that mediates highly effective killing of
target cells in the presence of effector cells, combined with
another anti-PSMA antibody that inhibits the growth of cells
expressing PSMA.
[0243] In a preferred aspect of the invention, the antibody or
antigen-binding fragment thereof binds to a conformational epitope
within the extracellular domain of the PSMA molecule. To determine
if the selected human anti-PSMA antibodies bind to conformational
epitopes, each antibody can be tested in assays using native
protein (e.g., non-denaturing immunoprecipitation, flow cytometric
analysis of cell surface binding) and denatured protein (e.g.,
Western blot, immunoprecipitation of denatured proteins). A
comparison of the results will indicate whether the antibodies bind
conformational epitopes. Antibodies that bind to native protein but
not denatured protein are those antibodies that bind conformational
epitopes, and are preferred antibodies.
[0244] In another preferred aspect of the invention, the antibody
or antigen-binding fragment thereof binds to a dimer-specific
epitope on PSMA. Generally, antibodies or antigen-binding fragments
thereof which bind to a dimer-specific epitope preferentially bind
the PSMA dimer rather than the PSMA monomer. To determine if the
selected human anti-PSMA antibodies bind preferentially (i.e.,
selectively and/or specifically) to a PSMA dimer, each antibody can
be tested in assays (e.g., immunoprecipitation followed by Western
blotting) using native dimeric PSMA protein and dissociated
monomeric PSMA protein. A comparison of the results will indicate
whether the antibodies bind preferentially to the dimer or to the
monomer. Antibodies that bind to the PSMA dimer but not to the
monomeric PSMA protein are preferred antibodies.
[0245] Preferred antibodies include antibodies that competitively
inhibit the specific binding of a second antibody to its target
epitope on PSMA. To determine competitive inhibition, a variety of
assays known to one of ordinary skill in the art can be employed.
For example, the cross-competition assays set forth in Examples 4
and 21 can be used to determine if an antibody competitively
inhibits binding to PSMA by another antibody. These examples
provide cell-based methods employing flow cytometry or solid phase
binding analysis. Other assays that evaluate the ability of
antibodies to cross-compete for PSMA molecules that are not
expressed on the surface of cells, in solid phase or in solution
phase, also can be used. These assays preferably use the PSMA
multimers described herein.
[0246] Certain preferred antibodies competitively inhibit the
specific binding of a second antibody to its target epitope on PSMA
by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
or 99%. Inhibition can be assessed at various molar ratios or mass
ratios; for example competitive binding experiments can be
conducted with a 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold or
more molar excess of the first antibody over the second
antibody.
[0247] Other preferred antibodies include antibodies that
specifically (i.e., selectively) bind to an epitope on PSMA defined
by a second antibody. To determine the epitope, one can use
standard epitope mapping methods known in the art. For example,
fragments (peptides) of PSMA antigen (preferably synthetic
peptides) that bind the second antibody can be used to determine
whether a candidate antibody binds the same epitope. For linear
epitopes, overlapping peptides of a defined length (e.g., 8 or more
amino acids) are synthesized. The peptides preferably are offset by
1 amino acid, such that a series of peptides covering every 8 amino
acid fragment of the PSMA protein sequence are prepared. Fewer
peptides can be prepared by using larger offsets, e.g., 2 or 3
amino acids. In addition, longer peptides (e.g., 9-, 10- or
11-mers) can be synthesized. Binding of peptides to antibodies can
be determined using standard methodologies including surface
plasmon resonance (BIACORE; see Example 22) and ELISA assays. For
examination of conformational epitopes, larger PSMA fragments can
be used. Other methods that use mass spectrometry to define
conformational epitopes have been described and can be used (see,
e.g., Baerga-Ortiz et al., Protein Science 11:1300-1308, 2002 and
references cited therein). Still other methods for epitope
determination are provided in standard laboratory reference works,
such as Unit 6.8 ("Phage Display Selection and Analysis of B-cell
Epitopes") and Unit 9.8 ("Identification of Antigenic Determinants
Using Synthetic Peptide Combinatorial Libraries") of Current
Protocols in Immunology, Coligan et al., eds., John Wiley &
Sons. Epitopes can be confirmed by introducing point mutations or
deletions into a known epitope, and then testing binding with one
or more antibodies to determine which mutations reduce binding of
the antibodies.
[0248] In one embodiment of the invention the antibody or
antigen-binding fragment thereof binds to and is internalized with
PSMA expressed on cells. The mechanism by which the antibody or
antigen-binding fragment thereof is internalized with the prostate
specific membrane antigen is not critical to the practice of the
present invention. For example, the antibody or antigen-binding
fragment thereof can induce internalization of PSMA. Alternatively,
internalization of the antibody or antigen-binding fragment thereof
can be the result of routine internalization of PSMA. The antibody
or antigen-binding fragment thereof can be used in an unmodified
form, alone or in combination with other compositions.
Alternatively, the antibody or antigen-binding fragment thereof can
be bound to a substance effective to kill the cells upon binding of
the antibody or antigen-binding fragment thereof to prostate
specific membrane antigen and upon internalization of the
biological agent with the prostate specific membrane antigen.
[0249] The human PSMA antibodies of the present invention
specifically bind cell-surface PSMA and/or rsPSMA with
sub-nanomolar affinity. The human PSMA antibodies of the present
invention have binding affinities of about 1.times.10.sup.-9M or
less, preferably about 1.times.10.sup.-10M or less, more preferably
1.times.10.sup.-11M or less. In a particular embodiment the binding
affinity is less than about 5.times.10.sup.-10M.
[0250] An antibody can be linked to a detectable marker, an
antitumor agent or an immunomodulator. Antitumor agents can include
cytotoxic agents and agents that act on tumor neovasculature.
Detectable markers include, for example, radioactive or fluorescent
markers. Cytotoxic agents include cytotoxic radionuclides, chemical
toxins and protein toxins.
[0251] The cytotoxic radionuclide or radiotherapeutic isotope
preferably is an alpha-emitting isotope such as .sup.225Ac,
.sup.211At, .sup.212Bi, .sup.213Bi, .sup.212Pb, .sup.224Ra or
.sup.223Ra. Alternatively, the cytotoxic radionuclide may a
beta-emitting isotope such as .sup.186Rh, .sup.188Rh, .sup.177Lu,
.sup.90Y, .sup.131I, .sup.67Cu, .sup.64Cu, .sup.153Sm or
.sup.166Ho. Further, the cytotoxic radionuclide may emit Auger and
low energy electrons and include the isotopes .sup.125I, .sup.123I
or .sup.77Br.
[0252] Suitable chemical toxins or chemotherapeutic agents include
members of the enediyne family of molecules, such as calicheamicin
and esperamicin. Chemical toxins can also be taken from the group
consisting of methotrexate, doxorubicin, melphalan, chlorambucil,
ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin
and 5-fluorouracil. Other antineoplastic agents that may be
conjugated to the anti-PSMA antibodies of the present invention
include dolastatins (U.S. Pat. Nos. 6,034,065 and 6,239,104) and
derivatives thereof. Of particular interest is dolastatin 10
(dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine) and the
derivatives auristatin PHE
(dolavaline-valine-dolaisoleuine-dolaproine-phenylalanine-methyl
ester) (Pettit, G. R. et al., Anticancer Drug Des. 13(4):243-277,
1998; Woyke, T. et al., Antimicrob. Agents Chemother.
45(12):3580-3584, 2001), and aurastatin E and the like. Toxins that
are less preferred in the compositions and methods of the invention
include poisonous lectins, plant toxins such as ricin, abrin,
modeccin, botulina and diphtheria toxins. Of course, combinations
of the various toxins could also be coupled to one antibody
molecule thereby accommodating variable cytotoxicity. Other
chemotherapeutic agents are known to those skilled in the art.
[0253] Toxin-conjugated forms of the PSMA antibodies of the present
invention mediate specific cell killing of PSMA-expressing cells at
picomolar concentrations. The toxin-conjugated PSMA antibodies of
the present invention exhibit IC.sub.50s at concentrations of less
than about 1.times.10.sup.-10M, preferably less than about
1.times.10.sup.-10M, more preferably less than about
1.times.10.sup.-12M. In a particular embodiment an IC.sub.50 is
achieved at a concentration of less than about
1.5.times.10.sup.-11M.
[0254] Agents that act on the tumor vasculature can include
tubulin-binding agents such as combrestatin A4 (Griggs et al.,
Lancet Oncol. 2:82, 2001), angiostatin and endostatin (reviewed in
Rosen, Oncologist 5:20, 2000, incorporated by reference herein) and
interferon inducible protein 10 (U.S. Pat. No. 5,994,292). A number
of antiangiogenic agents currently in clinical trials are also
contemplated. Agents currently in clinical trials include: 2ME2,
Angiostatin, Angiozyme, Anti-VEGF RhuMAb, Apra (CT-2584), AVICINE,
Benefin, BMS275291, Carboxyamidotriazole, CC4047, CC5013, CC7085,
CDC801, CGP-41251 (PKC 412), CM101, Combretastatin A-4 Prodrug, EMD
121974, Endostatin, Flavopiridol, Genistein (GCP), Green Tea
Extract, IM-862, ImmTher, Interferon alpha, Interleukin-12, IRESSA
(ZD 1839), Marimastat, METASTAT (Col-3), NEOVASTAT, Octreotide,
Paclitaxel, Penicillamine, PHOTOFRIN, PHOTOPOINT, PI-88,
Prinomastat (AG-3340), PTK787 (ZK22584), R0317453, Solimastat,
Squalamine, SU 101, SU 5416, SU-6668, Suradista (FCE 26644),
Suramin (Metaret), Tetrathiomolybdate, Thalidomide, TNP-470 and
VITAXIN. Additional antiangiogenic agents are described by Kerbel,
J. Clin. Oncol. 19(18s):45s-51s, 2001, which is incorporated by
reference herein. Immunomodulators suitable for conjugation to
anti-PSMA antibodies include .alpha.-interferon,
.gamma.-interferon, and tumor necrosis factor alpha
(TNF.alpha.).
[0255] The coupling of one or more toxin molecules to the anti-PSMA
antibody is envisioned to include many chemical mechanisms, for
instance covalent binding, affinity binding, intercalation,
coordinate binding, and complexation. The toxic compounds used to
prepare the anti-PSMA immunotoxins are attached to the antibodies
or PSMA-binding fragments thereof by standard protocols known in
the art.
[0256] The covalent binding can be achieved either by direct
condensation of existing side chains or by the incorporation of
external bridging molecules. Many bivalent or polyvalent agents are
useful in coupling protein molecules to other proteins, peptides or
amine functions, etc. For example, the literature is replete with
coupling agents such as carbodiimides, diisocyanates,
glutaraldehyde, diazobenzenes, and hexamethylene diamines. This
list is not intended to be exhaustive of the various coupling
agents known in the art but, rather, is exemplary of the more
common coupling agents.
[0257] In preferred embodiments, it is contemplated that one may
wish to first derivatize the antibody, and then attach the toxin
component to the derivatized product. Suitable cross-linking agents
for use in this manner include, for example, SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate), and SMPT,
4-succinimidyl-oxycarbonyl-methyl-(2-pyridyldithio)toluene.
[0258] In addition, protein toxins can be fused to the anti-PSMA
antibody or PSMA binding fragment by genetic methods to form a
hybrid immunotoxin fusion protein. To make a fusion immunotoxin
protein in accordance with the invention, a nucleic acid molecule
is generated that encodes an anti-PSMA antibody, a fragment of an
anti-PSMA antibody, a single chain anti-PSMA antibody, or a subunit
of an anti-PSMA antibody linked to a protein toxin. Such fusion
proteins contain at least a targeting agent (e.g., anti-PSMA
antibody subunit) and a toxin of the invention, operatively
attached. The fusion proteins may also include additional peptide
sequences, such as peptide spacers which operatively attach the
targeting agent and toxin compound, as long as such additional
sequences do not appreciably affect the targeting or toxin
activities of the fusion protein. The two proteins can be attached
by a peptide linker or spacer, such as a glycine-serine spacer
peptide, or a peptide hinge, as is well known in the art. Thus, for
example, the C-terminus of an anti-PSMA antibody or fragment
thereof can be fused to the N-terminus of the protein toxin
molecule to form an immunotoxin that retains the binding properties
of the anti-PSMA antibody. Other fusion arrangements will be known
to one of ordinary skill in the art. To express the fusion
immunotoxin, the nucleic acid encoding the fusion protein is
inserted into an expression vector in accordance with standard
methods, for stable expression of the fusion protein, preferably in
mammalian cells, such as CHO cells. The fusion protein can be
isolated and purified from the cells or culture supernatant using
standard methodology, such as a PSMA affinity column.
[0259] Radionuclides typically are coupled to an antibody by
chelation. For example, in the case of metallic radionuclides, a
bifunctional chelator is commonly used to link the isotope to the
antibody or other protein of interest. Typically, the chelator is
first attached to the antibody, and the chelator-antibody conjugate
is contacted with the metallic radioisotope. A number of
bifunctional chelators have been developed for this purpose,
including the diethylenetriamine pentaacetic acid (DTPA) series of
amino acids described in U.S. Pat. Nos. 5,124,471, 5,286,850 and
5,434,287, which are incorporated herein by reference. As another
example, hydroxamic acid-based bifunctional chelating agents are
described in U.S. Pat. No. 5,756,825, the contents of which are
incorporated herein. Another example is the chelating agent termed
p-SCN-Bz-HEHA
(1,4,7,10,13,16-hexaazacyclo-octadecane-N,N',N'',N''',N'''',N'''''-hexaac-
etic acid) (Deal et al., J. Med. Chem. 42:2988, 1999), which is an
effective chelator of radiometals such as .sup.225Ac. Yet another
example is DOTA (1,4,7,10-tetraazacyclododecane
N,N',N'',N'''-tetraacetic acid), which is a bifunctional chelating
agent (see McDevitt et al., Science 294:1537-1540, 2001) that can
be used in a two-step method for labeling followed by
conjugation.
[0260] In another aspect, the invention provides compositions
comprising a multimeric (e.g., dimeric) PSMA protein, an isolated
antibody, an antibody derivatized or linked to other functional
moieties, or an antigen-binding fragment thereof or a combination
of one or more of the aforementioned multimeric PSMA proteins,
antibodies or antigen-binding fragments thereof. The compositions
include a physiologically or pharmaceutically acceptable carrier,
excipient, or stabilizer mixed with the isolated multimeric PSMA
protein, antibody or antigen-binding fragment thereof. In a
preferred embodiment, the compositions include a combination of
multiple (e.g., two or more) isolated multimeric PSMA proteins,
antibodies or antigen-binding portions thereof of the invention.
Preferably, each of the antibodies or antigen-binding portions
thereof of the composition binds to a distinct conformational
epitope of PSMA. In one embodiment, anti-PSMA antibodies having
complementary activities are used in combination, e.g., as a
pharmaceutical composition, comprising two or more anti-PSMA
antibodies. For example, an antibody that mediates highly effective
cytolysis of target cells in the presence of effector cells can be
combined with another antibody that inhibits the growth of cells
expressing PSMA. As used herein, "target cell" shall mean any
undesirable cell in a subject (e.g., a human or animal) that can be
targeted by a composition of the invention. In preferred
embodiments, the target cell is a cell expressing or overexpressing
PSMA. Cells expressing PSMA typically include tumor cells, such as
prostate, bladder, pancreas, lung, kidney, colon tumor cells,
melanomas, and sarcomas.
[0261] Pharmaceutical compositions of the invention also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include a
composition of the present invention with at least one anti-tumor
agent, immunomodulator, immunostimulatory agent, or other
conventional therapy. For instance, the agent may be bound or
conjugated to or formed as a recombinant fusion molecule with the
PSMA antibodies of the present invention for directed targeting of
the agent to PSMA-expressing cells.
[0262] In some embodiments the various agents can be administered
concomitantly. In other embodiments the agents are administered
separately (prior to or subsequent to each other). The compositions
provided herein can be given to any patient in need thereof. As one
example, the compositions provided herein can be given to a
conventional cancer treatment-experienced patient. For instance a
composition of dimeric PSMA can be administered to such a patient
at some time subsequent to a conventional cancer therapy.
Conventional cancer therapy, such as for prostate cancer, includes
one or more of the following: surgery, radiation, cryosurgery,
thermotherapy, hormone treatment, chemotherapy, etc. In one
embodiment the therapy received prior to administration of a
composition of dimeric PSMA is at least prostatectomy and/or
radiation. In another embodiment the therapy received prior to
administration of a composition of dimeric PSMA is at least
castration and hormonal therapy. In yet another embodiment the
therapy received prior to administration of a composition of
dimeric PSMA is at least chemotherapy. In one embodiment for
prostate cancer the chemotherapy is preferably the administration
of the chemotherapeutic agent, docetaxel, alone or in combination
with an anti-inflammatory compound. The anti-inflammatory compound
in one embodiment is prednisone. Therefore, in some embodiments
compositions and methods are provided for treating patients with a
composition containing dimeric PSMA that is administered
concomitantly with, subsequent to, or prior to conventional cancer
therapy. In one such embodiment the methods provided include the
administration of docetaxel (75 mg/m.sup.2 q3 weeks) plus the
anti-inflammatory agent, prednisone (5 mg po bid), concomitantly
with, subsequent to, or prior to the administration of dimeric PSMA
compositions as provided herein.
[0263] In one embodiment patients amenable to treatment using
dimeric PSMA include those who have not received conventional
cancer treatment. In another embodiment patients amenable to
treatment using dimeric PSMA include those who have evidence of
cancer despite having received one or more conventional cancer
therapies. Patients therefore can include patients with
biochemically progressive prostate cancer such as non-castrate
patients (serum testosterone greater than or equal to 180 ng/mL).
In some embodiments these patients have received definitive primary
therapy such as prostatectomy or radiation. Patients can also
include castrate patients (serum testosterone less than 50 ng/mL),
who in some embodiments have completed a course of hormonal
therapy. Patients can also include patients having radiographic
evidence of disease progression. In one embodiment such a treatment
regimen is indicated in hormone-refractory prostate cancer
patients.
[0264] The PSMA antibodies of the present invention may be used as
a targeting moiety for delivery of replication-selective virus to
PSMA-expressing cells for tumor therapy. Replication-competent
virus such as the p53 pathway targeting adenovirus mutant d11520,
ONYX-015, kill tumor cells selectively (Biederer, C. et al., J.
Mol. Med. 80(3):163-175, 2002).
[0265] The compositions of the present invention may include or be
diluted into a pharmaceutically-acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" or "physiologically
acceptable carrier" means one or more compatible solid or liquid
fillers, diluents or encapsulating substances which are suitable
for administration to a human or other mammal such as a primate,
dog, cat, horse, cow, sheep, or goat. Such carriers include any and
all salts, solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. The term "carrier"
denotes an organic or inorganic ingredient, natural or synthetic,
with which the active ingredient is combined to facilitate the
application. The carriers are capable of being commingled with the
preparations of the present invention, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy or stability.
Preferably, the carrier is suitable for oral, intranasal,
intravenous, intramuscular, subcutaneous, parenteral, spinal,
intradermal or epidermal administration (e.g., by injection or
infusion). Suitable carriers can be found in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
Depending on the route of administration, the active compound,
i.e., antibody or PSMA multimer may be coated in a material to
protect the compound from the action of acids and other natural
conditions that may inactivate the compound.
[0266] When administered, the pharmaceutical preparations of the
invention are applied in pharmaceutically-acceptable amounts and in
pharmaceutically-acceptable compositions. The term
"pharmaceutically acceptable" means a non-toxic material that does
not interfere with the effectiveness of the biological activity of
the active ingredients. The components of the pharmaceutical
compositions also are capable of being co-mingled with the
molecules of the present invention, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy. Such preparations may
routinely contain salts, buffering agents, preservatives,
compatible carriers, and optionally other therapeutic agents, such
as supplementary immune potentiating agents including adjuvants,
chemokines and cytokines. When used in medicine, the salts should
be pharmaceutically acceptable, but non-pharmaceutically acceptable
salts may conveniently be used to prepare
pharmaceutically-acceptable salts thereof and are not excluded from
the scope of the invention.
[0267] A salt retains the desired biological activity of the parent
compound and does not impart any undesired toxicological effects
(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19).
Examples of such salts include acid addition salts and base
addition salts. Acid addition salts include those derived from
nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric,
sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as
well as from nontoxic organic acids such as aliphatic mono- and
dicarboxylic acids, phenyl substituted alkanoic acids, hydroxy
alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic
acids and the like. Base addition salts include those derived from
alkaline earth metals, such as sodium, potassium, magnesium,
calcium and the like, as well as from nontoxic organic amines, such
as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0268] The pharmaceutical preparations of the invention also may
include isotonicity agents. This term is used in the art
interchangeably with iso-osmotic agent, and is known as a compound
which is added to the pharmaceutical preparation to increase the
osmotic pressure to that of 0.9% sodium chloride solution, which is
iso-osmotic with human extracellular fluids, such as plasma.
Preferred isotonicity agents are sodium chloride, mannitol,
sorbitol, lactose, dextrose and glycerol.
[0269] Optionally, the pharmaceutical preparations of the invention
may further comprise a preservative, such as benzalkonium chloride.
Suitable preservatives also include but are not limited to:
chlorobutanol (0.3-0.9% W/V), parabens (0.01-5.0%), thimerosal
(0.004-0.2%), benzyl alcohol (0.5-5%), phenol (0.1-1.0%), and the
like.
[0270] The formulations provided herein also include those that are
sterile. Sterilization processes or techniques as used herein
include aseptic techniques such as one or more filtration (0.45 or
0.22 micron filters) steps.
[0271] An anti-PSMA antibody composition may be combined, if
desired, with a pharmaceutically-acceptable carrier.
[0272] The pharmaceutical compositions may contain suitable
buffering agents, including: acetic acid in a salt; citric acid in
a salt; boric acid in a salt; and phosphoric acid in a salt.
[0273] The pharmaceutical compositions may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well-known in the art of pharmacy. All methods include the
step of bringing the active agent into association with a carrier
which constitutes one or more accessory ingredients. In general,
the compositions are prepared by uniformly and intimately bringing
the active compound into association with a liquid carrier, a
finely divided solid carrier, or both, and then, if necessary,
shaping the product.
[0274] Compositions suitable for parenteral administration
conveniently comprise a sterile aqueous or non-aqueous preparation
of PSMA multimers and/or anti-PSMA antibodies, which is preferably
isotonic with the blood of the recipient. This preparation may be
formulated according to known methods using suitable dispersing or
wetting agents and suspending agents. The sterile injectable
preparation also may be a sterile injectable solution or suspension
in a non-toxic parenterally-acceptable diluent or solvent, for
example, as a solution in 1,3-butane diol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium.
[0275] For this purpose any bland fixed oil may be employed
including synthetic mono- or di-glycerides. In addition, fatty
acids such as oleic acid may be used in the preparation of
injectables. Carrier formulations suitable for oral, subcutaneous,
intravenous, intramuscular, etc. administration can be found in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa.
[0276] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0277] The therapeutics of the invention can be administered by any
conventional route, including injection or by gradual infusion over
time. The administration may, for example, be oral, subcutaneous,
intravenous, intraperitoneal, intramuscular, intracavity,
intratumor, or transdermal. In some embodiments subcutaneous or
intramuscular administration is preferred. When antibodies are used
therapeutically, preferred routes of administration include
intravenous and by pulmonary aerosol. Techniques for preparing
aerosol delivery systems containing antibodies are well known to
those of skill in the art. Generally, such systems should utilize
components which will not significantly impair the biological
properties of the antibodies, such as the paratope binding capacity
(see, for example, Sciarra and Cutie, "Aerosols," in Remington's
Pharmaceutical Sciences, 18th edition, 1990, pp. 1694-1712;
incorporated by reference). Those of skill in the art can readily
determine the various parameters and conditions for producing
antibody aerosols without resorting to undue experimentation.
[0278] The pharmaceutical preparations of the invention, when used
in alone or in cocktails, are administered in therapeutically
effective amounts. Effective amounts are well known to those of
ordinary skill in the art and are described in the literature. A
therapeutically effective amount will be determined by the
parameters discussed below; but, in any event, is that amount which
establishes a level of the drug(s) effective for treating a
subject, such as a human subject, having one of the conditions
described herein. An effective amount means that amount alone or
with multiple doses, necessary to delay the onset of, inhibit
completely or lessen the progression of or halt altogether the
onset or progression of the condition being treated. When
administered to a subject, effective amounts will depend, of
course, on the particular condition being treated; the severity of
the condition; individual patient parameters including age,
physical condition, size and weight; concurrent treatment;
frequency of treatment; and the mode of administration. These to
factors are well known to those of ordinary skill in the art and
can be addressed with no more than routine experimentation. It is
preferred generally that a maximum dose be used, that is, the
highest safe dose according to sound medical judgment.
[0279] An "effective amount" is that amount of an anti-PSMA
antibody or PSMA multimer that alone, or together with further
doses, produces the desired response, e.g. treats a malignancy in a
subject. The term is also meant to encompass the amount of an
anti-PSMA antibody and/or PSMA multimer that in combination with a
chemotherapeutic agent produces the desired response. This may
involve only slowing the progression of the disease temporarily,
although more preferably, it involves halting the progression of
the disease permanently. This can be monitored by routine methods.
The desired response to treatment of the disease or condition also
can be delaying the onset or even preventing the onset of the
disease or condition.
[0280] Such amounts will depend, of course, on the particular
condition being treated, the severity of the condition, the
individual patient parameters including age, physical condition,
size and weight, the duration of the treatment, the nature of
concurrent therapy (if any), the specific route of administration
and like factors within the knowledge and expertise of the health
practitioner. These factors are well known to those of ordinary
skill in the art and can be addressed with no more than routine
experimentation. It is generally preferred that a maximum dose of
the individual components or combinations thereof be used, that is,
the highest safe dose according to sound medical judgment. It will
be understood by those of ordinary skill in the art, however, that
a patient may insist upon a lower dose or tolerable dose for
medical reasons, psychological reasons or for virtually any other
reasons.
[0281] The pharmaceutical compositions used in the foregoing
methods preferably are sterile and contain an effective amount of
anti-PSMA antibodies or PSMA multimers for producing the desired
response in a unit of weight or volume suitable for administration
to a patient. The response can, for example, be measured by
determining the physiological effects of the anti-PSMA antibody or
PSMA multimer, such as regression of a tumor or decrease of disease
symptoms. Other assays will be known to one of ordinary skill in
the art and can be employed for measuring the level of the
response.
[0282] The doses of anti-PSMA antibodies or PSMA multimers
administered to a subject can be chosen in accordance with
different parameters, in particular in accordance with the mode of
administration used and the state of the subject. Other factors
include the desired period of treatment. In the event that a
response in a subject is insufficient at the initial doses applied,
higher doses (or effectively higher doses by a different, more
localized delivery route) may be employed to the extent that
patient tolerance permits.
[0283] A variety of administration routes are available. The
particular mode selected will depend of course, upon the particular
drug selected, the severity of the disease state being treated and
the dosage required for therapeutic efficacy. The methods of this
invention, generally speaking, may be practiced using any mode of
administration that is medically acceptable, meaning any mode that
produces effective levels of the active compounds without causing
clinically unacceptable adverse effects. Such modes of
administration include oral, rectal, sublingual, topical, nasal,
transdermal or parenteral routes. The term "parenteral" includes
subcutaneous, intravenous, intramuscular, or infusion.
[0284] In general, doses can range from about 10 .mu.g/kg to about
100,000 .mu.g/kg. In one embodiment, the dose is about 50 mg. In
another embodiment, the dose is about 250 mg. In still another
embodiment, the dose is about 500 mg, 1000 mg or greater. Based
upon the composition, the dose can be delivered once, continuously,
such as by continuous pump, or at periodic intervals. The periodic
interval may be weekly, bi-weekly, or monthly. The dosing can occur
over the period of one month, two months, three months or more to
elicit an appropriate humoral and/or cellular immune response.
Desired time intervals of multiple doses of a particular
composition can be determined without undue experimentation by one
skilled in the art. Other protocols for the administration of
anti-PSMA antibody or PSMA multimers will be known to one of
ordinary skill in the art, in which the dose amount, schedule of
administration, sites of administration, mode of administration and
the like vary from the foregoing.
[0285] Dosage may be adjusted appropriately to achieve desired drug
levels, locally or systemically. Generally, daily oral doses of
active compounds will be from about 0.1 mg/kg per day to 30 mg/kg
per day. It is expected that IV doses in the range of 0.01-1.00
mg/kg will be effective. In the event that the response in a
subject is insufficient at such doses, even higher doses (or
effective higher doses by a different, more localized delivery
route) may be employed to the extent that patient tolerance
permits. Continuous IV dosing over, for example, 24 hours or
multiple doses per day also are contemplated to achieve appropriate
systemic levels of compounds.
[0286] In general, doses of radionuclide delivered by the anti-PSMA
antibodies of the invention can range from about 0.01 mCi/Kg to
about 10 mCi/kg. Preferably the dose of radionuclide ranges from
about 0.1 mCi/Kg to about 1.0 mCi/kg. The optimal dose of a given
isotope can be determined empirically by simple routine titration
experiments well known to one of ordinary skill in the art.
[0287] Administration of anti-PSMA antibody or PSMA multimer
compositions to mammals other than humans, e.g. for testing
purposes or veterinary therapeutic purposes, is carried out under
substantially the same conditions as described above.
[0288] The compositions (antibodies to PSMA and
derivatives/conjugates thereof and PSMA multimers) of the present
invention have in vitro and in vivo diagnostic and therapeutic
utilities. For example, these molecules can be administered to
cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g.,
in vivo, to treat, prevent or diagnose a variety of disorders. As
used herein, the term "subject" is intended to include humans and
non-human animals. Preferred subjects include a human patient
having a disorder characterized by expression, typically aberrant
expression (e.g., overexpression) of PSMA. Other preferred subjects
include subjects that are treatable with the compositions of the
invention. This includes those who have or are at risk of having a
cancer or who would otherwise would benefit from an enhanced or
elicited immune response to cells expressing PSMA. In preferred
embodiments these cells express PSMA on their surface.
[0289] One aspect of the present invention relates to a method of
detecting cancerous cells or portions thereof in a biological
sample (e.g., histological or cytological specimens, biopsies and
the like), and, in particular, to distinguish malignant tumors from
normal tissues and non-malignant tumors. This method involves
providing an antibody or an antigen-binding binding fragment
thereof, probe, or ligand, which binds to an extracellular domain
of PSMA of such cells, e.g., an anti-PSMA antibody. The anti-PSMA
antibody is bound to a label that permits the detection of the
cells or portions thereof (e.g., PSMA or fragments thereof
liberated from such cancerous cells) upon binding of the anti-PSMA
antibody to the cells or portions thereof. The biological sample is
contacted with the labeled anti-PSMA antibody under conditions
effective to permit binding of the anti-PSMA antibody to the
extracellular domain of PSMA of any of the cells or portions
thereof in the biological sample. The presence of any cells or
portions thereof in the biological sample is detected by detection
of the label. In one preferred form, the contact between the
anti-PSMA antibody and the biological sample is carried out in a
living mammal and involves administering the anti-PSMA antibody to
the mammal under conditions that permit binding of the anti-PSMA
antibody to PSMA of any of the cells or portions thereof in the
biological sample. Again, such administration can be carried out by
any suitable method known to one of ordinary skill in the art.
[0290] In addition, the anti-PSMA antibodies of the present
invention can be used in immunofluorescence techniques to examine
human tissue, cell and bodily fluid specimens. In a typical
protocol, slides containing cryostat sections of frozen, unfixed
tissue biopsy samples or cytological smears are air dried, formalin
or acetone fixed, and incubated with the monoclonal antibody
preparation in a humidified chamber at room temperature. The slides
are then washed and further incubated with a preparation of a
secondary antibody directed against the monoclonal antibody,
usually some type of anti-mouse immunoglobulin if the monoclonal
antibodies used are derived from the fusion of a mouse spleen
lymphocyte and a mouse myeloma cell line. This secondary antibody
is tagged with a compound, for instance rhodamine or fluorescein
isothiocyanate, that fluoresces at a particular wavelength. The
staining pattern and intensities within the sample are then
determined by fluorescent light microscopy and optionally
photographically recorded.
[0291] As yet another alternative, computer enhanced fluorescence
image analysis or flow cytometry can be used to examine tissue
specimens or exfoliated cells, i.e., single cell preparations from
aspiration biopsies of tumors using the anti-PSMA antibodies of
this invention. The anti-PSMA antibodies of the invention are
particularly useful in quantitation of live tumor cells, i.e.,
single cell preparations from aspiration biopsies of prostate
tumors by computer enhanced fluorescence image analyzer or with a
flow cytometer. The antibodies of the invention are particularly
useful in such assays to differentiate benign from malignant
prostate tumors since the PSMA protein to which the anti-PSMA
antibodies bind is expressed in increased amounts by malignant
tumors as compared to benign prostate tumors. The percent PSMA
positive cell population, alone or in conjunction with
determination of other attributes of the cells (e.g., DNA ploidy of
these cells), may, additionally, provide very useful prognostic
information by providing an early indicator of disease
progression.
[0292] In yet another alternative embodiment, the antibodies of the
present invention can be used in combination with other known
antibodies to provide additional information regarding the
malignant phenotype of a cancer.
[0293] The method of the present invention can be used to screen
patients for diseases associated with the presence of cancerous
cells or portions thereof. Alternatively, it can be used to
identify the recurrence of such diseases, particularly when the
disease is localized in a particular biological material of the
patient. For example, recurrence of prostatic disease in the
prostatic fossa may be encountered following radical prostatectomy.
Using the method of the present invention, this recurrence can be
detected by administering a short range radiolabeled antibody to
the mammal and then detecting the label rectally, such as with a
transrectal detector probe.
[0294] Alternatively, the contacting step can be carried out in a
sample of serum or urine or other body fluids, including but not
limited to seminal fluid, prostatic fluid, ejaculate, and the like,
such as to detect the presence of PSMA in the body fluid. When the
contacting is carried out in a serum or urine sample, it is
preferred that the biological agent recognize substantially no
antigens circulating in the blood other than PSMA. Since intact
cells do not excrete or secrete PSMA into the extracellular
environment, detecting PSMA in serum, urine, or other body fluids
generally indicates that cells are being lysed or shed. Thus, the
biological agents and methods of the present invention can be used
to determine the effectiveness of a cancer treatment protocol by
monitoring the level of PSMA in serum, urine or other body
fluids.
[0295] In a particularly preferred embodiment of the method of
detecting cancerous cells in accordance with the present invention,
the anti-PSMA antibodies or an antigen-binding fragment thereof,
binds to and is internalized with the prostate specific membrane
antigen of such cells. Again, the biological agent is bound to a
label effective to permit detection of the cells or portions
thereof upon binding of the biological agent to and internalization
of the biological agent with the prostate specific membrane
antigen.
[0296] Biological agents suitable for detecting cancerous cells
include anti-PSMA antibodies, such as monoclonal or polyclonal
antibodies. In addition, antibody fragments, half-antibodies,
hybrid derivatives, probes, and other molecular constructs may be
utilized.
[0297] These biological agents, such as antibodies, antigen-binding
fragments thereof, probes, or ligands, bind to extracellular
domains of prostate specific membrane antigens or portions thereof
in cancerous cells. As a result, the biological agents bind not
only to cells which are fixed or cells whose intracellular
antigenic domains are otherwise exposed to the extracellular
environment. Consequently, binding of the biological agents is
concentrated in areas where there are prostate cells, irrespective
of whether these cells are fixed or unfixed, viable or necrotic.
Additionally or alternatively, these biological agents bind to and
are internalized with prostate specific membrane antigens or
portions thereof in normal, benign hyperplastic, and to a greater
degree in cancerous cells.
[0298] The PSMA multimers and antibodies or antigen-binding
fragments thereof can also be utilized in in vivo therapy of
cancer. The PSMA multimers and antibodies or antigen-binding
fragments thereof can be used with a compound which kills and/or
inhibits proliferation of malignant cells or tissues. For instance,
the antibodies can be covalently attached, either directly or via
linker, to such a compound following administration and
localization of the conjugates. When the antibody is used by
itself, it may mediate tumor destruction by complement fixation or
antibody-dependent cellular cytotoxicity. Alternatively, the PSMA
multimer or antibody may be administered in combination with a
chemotherapeutic drug to result in synergistic therapeutic effects
(Baslya and Mendelsohn, 1994 Breast Cancer Res. and Treatment
29:127-138). A variety of different types of substances can be
directly conjugated for therapeutic uses, including radioactive
metal and non-metal isotopes, chemotherapeutic drugs, toxins, etc.
as described above and known in the art (see, e.g., Vitetta and
Uhr, 1985, Annu. Rev. Immunol. 3:197).
[0299] The antibodies or antigen-binding fragments thereof of the
invention can also be administered together with complement.
Accordingly, within the scope of the invention are compositions
comprising antibodies or antigen-binding fragments thereof and
serum or complement. These compositions are advantageous in that
the complement is located in close proximity to the human
antibodies or antigen-binding fragments thereof. Alternatively, the
antibodies or antigen-binding fragments thereof of the invention
and the complement or serum can be administered separately.
[0300] The PSMA multimers or antibodies can be administered with
one or more immunostimulatory agents to induce or enhance an immune
response, such as IL-2 and immunostimulatory oligonucleotides
(e.g., those containing CpG motifs). Preferred immunostimulatory
agents stimulate specific arms of the immune system, such as
natural killer (NK) cells that mediate antibody-dependent cell
cytotoxicity (ADCC).
[0301] As provided elsewhere herein, the compositions provided can
be administered with one or more adjuvants to induce or enhance an
immune response. An adjuvant is a substance which potentiates the
immune response. Adjuvants of many kinds are well known in the art.
Specific examples of adjuvants include monophosphoryl lipid A (MPL,
SmithKline Beecham); saponins including QS-21 (Antigenics);
immunostimulatory oligonucleotides (e.g., CpG oligonucleotides
described by Kreig et al., Nature 374:546-9, 1995); incomplete
Freund's adjuvant; complete Freund's adjuvant; MONTANIDE; vitamin E
and various water-in-oil emulsions prepared from biodegradable oils
such as squalene and/or tocopherol, Quil A, Ribi Detox, CRL-1005,
L-121, and combinations thereof.
[0302] Other agents which stimulate the immune response of the
subject to PSMA multimer antigens can also be administered to the
subject. For example, cytokines are also useful in vaccination
protocols as a result of their lymphocyte regulatory properties.
Many cytokines useful for such purposes will be known to one of
ordinary skill in the art, including interleukin-2 (IL-2); IL-4;
IL-5; IL-12, which has been shown to enhance the protective effects
of vaccines (see, e.g., Science 268: 1432-1434, 1995); GM-CSF;
IL-15; IL-18; combinations thereof, and the like. Thus cytokines
can be administered in conjunction with antibodies, antigens,
chemokines and/or adjuvants to increase an immune response.
[0303] Chemokines useful in increasing immune responses include but
are not limited to SLC, ELC, MIP3.alpha., MIP3.beta., IP-10, MIG,
and combinations thereof.
[0304] The PSMA multimers or antibodies or antigen-binding
fragments thereof of the present invention can be used in
conjunction with other therapeutic treatment modalities. Current
standard or conventional treatments for cancer, such as prostate
cancer, include surgery, radiation, cryosurgery, thermotherapy,
hormone treatment and chemotherapy. Subjects receiving one or more
of the standard treatments may be referred to as
treatment-experienced subjects. Hormone therapy includes treatment
with one or more of the following modalities: a leutinizing
hormone-releasing hormone agonist such as leuprolide, goserelin or
buserelin; an antiandrogen, such as flutaminde or bicalutamide; a
drug that prevents adrenal glands from making androgens, such as
ketoconazole or aminoglutethimide; estrogens; and orchiectomy
(castration). Chemotherapy may use any
chemotherapeutic/antineoplastic agent known in the art. In some
preferred embodiments the chemotherapeutic agent is a taxane, such
as paclitaxel (TAXOL) or docetaxel (TAXOTERE). Chemotherapy may be
used in combination with an anti-inflammatory compound such as a
corticosteroid. Corticosteroids include cortisone, hydrocortisone,
prednisone, prednisolone, triamcinolone, methylprednisolone,
dexamethasone, betamethasone and the like. A preferred
anti-inflammatory compound is prednisone. Other therapeutic
modalities that may be used in combination with PSMA multimers
include the use of other vaccines and immunotherapies.
[0305] Also encompassed by the present invention is a method which
involves using the PSMA multimers or antibodies or antigen-binding
fragments thereof for prophylaxis. For example, these materials can
be used to prevent or delay development or progression of
cancer.
[0306] Use of the cancer therapy of the present invention has a
number of benefits. Since the anti-PSMA antibodies or
antigen-binding fragments thereof according to the present
invention preferentially target prostate cancer cells, other tissue
is spared. As a result, treatment with such biological agents is
safer, particularly for elderly patients. Treatment according to
the present invention is expected to be particularly effective,
because it directs high levels of anti-PSMA antibodies or
antigen-binding fragments thereof to the bone marrow and lymph
nodes where prostate cancer metastases predominate. Moreover, tumor
sites for prostate cancer tend to be small in size and, therefore,
easily destroyed by cytotoxic agents. Treatment in accordance with
the present invention can be effectively monitored with clinical
parameters such as serum prostate specific antigen and/or
pathological features of a patient's cancer, including stage,
Gleason score, extracapsular, seminal, vesicle or perineural
invasion, positive margins, involved lymph nodes, etc.
Alternatively, these parameters can be used to indicate when such
treatment should be employed.
[0307] Because the antibodies or antigen-binding fragments thereof
of the present invention bind to living cells, therapeutic methods
using these biological agents are much more effective than those
which target lysed cells. For the same reasons, diagnostic and
imaging methods which determine the location of living normal,
benign hyperplastic, or cancerous cells are much improved by
employing the antibodies or antigen-binding fragments thereof of
the present invention. In addition, the ability to differentiate
between living and dead cells can be advantageous, especially to
monitor the effectiveness of a particular treatment regimen.
[0308] Also within the scope of the invention are kits comprising
the compositions of the invention and instructions for use. The
kits can further contain at least one additional reagent, such as
complement, or one or more additional antibodies of the invention
(e.g., an antibody having a complementary activity which binds to
an epitope in PSMA antigen distinct from the first antibody). Other
kits can include the PSMA multimers described herein below.
[0309] The kits provided herein include any of the compositions
described and instructions for the use of these compositions. The
instructions can include instructions for mixing a particular
amount of an agent or solution that preserves or promotes the
multimerization of PSMA with a particular amount of a PSMA
composition. The instructions can also include instructions for
mixing a particular amount of a diluent with a particular amount of
a PSMA dimeric composition, whereby a final formulation for
injection or infusion is prepared. Therefore, kits are also
provided, which include the compositions of the invention and,
optionally, an adjuvant (e.g., alum) or diluent and instructions
for mixing. Kits are also provided wherein the compositions of the
inventions are provided in a vial or ampoule with a septum or a
syringe. Other kits where the composition is in lyophilized form
are also provided. The instructions, therefore, would take a
variety of forms depending on the presence or absence of diluent or
other agents (e.g., therapeutic agents). The instructions can
include instructions for treating a patient with an effective
amount of dimeric PSMA. It also will be understood that the
containers containing the pharmaceutical preparation, whether the
container is a bottle, a vial with a septum, an ampoule with a
septum, an infusion bag, and the like, can contain indicia such as
conventional markings which change color when the pharmaceutical
preparation has been autoclaved or otherwise sterilized.
[0310] Kits containing the antibodies or antigen-binding fragments
thereof of the invention can be prepared for in vitro diagnosis,
prognosis and/or monitoring cancer by the immunohistological,
immunocytological and immunoserological methods described above.
The components of the kits can be packaged either in aqueous medium
or in lyophilized form. When the antibodies or antigen-binding
fragments thereof are used in the kits in the form of conjugates in
which a label moiety is attached, such as an enzyme or a
radioactive metal ion, the components of such conjugates can be
supplied either in fully conjugated form, in the form of
intermediates or as separate moieties to be conjugated by the user
or the kit.
[0311] A kit may comprise a carrier being compartmentalized to
receive in close confinement therein one or more container means or
series of container means such as test tubes, vials, flasks,
bottles, syringes, or the like. A first of said container means or
series of container means may contain one or more anti-PSMA
antibodies or antigen-binding fragments thereof or PSMA. A second
container means or series of container means may contain a label or
linker-label intermediate capable of binding to the primary
anti-PSMA antibodies (or fragment thereof).
[0312] It should be understood that the pharmaceutical preparations
of the invention will typically be held in bottles, vials,
ampoules, infusion bags, and the like, any one of which may be
sparged to eliminate oxygen or purged with nitrogen. In some
embodiments, the bottles vials and ampoules are opaque, such as
when amber in color. Such sparging and purging protocols are well
known to those of ordinary skill in the art and should contribute
maintaining the stability of the pharmaceutical preparations. The
pharmaceutical preparations also, in certain embodiments, are
expected to be contained within syringes.
[0313] Kits for use in in vivo tumor localization and therapy
method containing the anti-PSMA antibodies or antigen-binding
fragments thereof conjugated to other compounds or substances can
be prepared. The components of the kits can be packaged either in
aqueous medium or in lyophilized form. When the antibodies or
antigen-binding fragments thereof are used in the kits in the form
of conjugates in which a label or a therapeutic moiety is attached,
such as a radioactive metal ion or a therapeutic drug moiety, the
components of such conjugates can be supplied either in fully
conjugated form, in the form of intermediates or as separate
moieties to be conjugated by the user of the kit.
[0314] In one aspect of the invention, a method for modulating at
least one enzymatic activity of PSMA, the activity selected from
the group consisting of N-acetylated .alpha.-linked acidic
dipeptidase (NAALADase), folate hydrolase, dipeptidyl dipeptidase
IV and .gamma.-glutamyl hydrolase activity or combination thereof
in vitro or in vivo. The modulation may be enhancement or
inhibition of at least one enzymatic activity of PSMA.
[0315] In a preferred embodiment, the invention provides methods
for inhibiting at least one enzymatic activity of PSMA, the
activity selected from the group consisting of N-acetylated
.alpha.-linked acidic dipeptidase (NAALADase), folate hydrolase,
dipeptidyl dipeptidase IV and .gamma.-glutamyl hydrolase activity
or combination thereof in vitro or in vivo. The method comprises
contacting a NAALADase, a folate hydrolase, a dipeptidyl
dipeptidase IV and/or a .gamma.-glutamyl hydrolase with an amount
of an isolated antibody or antigen-binding fragment thereof of the
invention under conditions wherein the isolated monoclonal antibody
or antigen-binding fragment thereof inhibits NAALADase, folate
hydrolase, dipeptidyl dipeptidase IV or .gamma.-glutamyl hydrolase
activity.
[0316] Tissue levels of NAALADase can be determined by detergent
solubilizing homogenizing tissues, pelleting the insoluble material
by centrifugation and measuring the NAALADase activity in the
remaining supernatant. Likewise, the NAALADase activity in bodily
fluids can also be measured by first pelleting the cellular
material by centrifugation and performing a typical enzyme assay
for NAALADase activity on the supernatant. NAALADase enzyme assays
have been described by Frieden, 1959, J. Biol, Chem., 234:2891. In
this assay, the reaction product of the NAALADase enzyme is
glutamic acid. This is derived from the enzyme catalyzed cleavage
of N-acetylaspartylglutamate to yield N-acetylaspartic acid and
glutamic acid. Glutamic acid, in a NAD(P).sup.+ requiring step,
yields 2-oxoglutarate plus NAD(P)H in a reaction catalyzed by
glutamate dehydrogenase. Progress of the reaction can easily and
conveniently be measured by the change in absorbance at 340 nm due
to the conversion of NAD(P).sup.+ to NAD(P)H.
[0317] Folate hydrolase activity of PSMA can be measured by
performing enzyme assays as described by Heston and others (e.g.,
Clin. Cancer Res. 2(9):1445-51, 1996; Urology 49(3A Suppl):104-12,
1997). Folate hydrolases such as PSMA remove the gamma-linked
glutamates from polyglutamated folates. Folate hydrolase activity
can be measured using substrates such as methotrexate tri-gamma
glutamate (MTXGlu3), methotrexate di-gamma glutamate (MTXGlu2) and
pteroylpentaglutamate (PteGlu5), for example using capillary
electrophoresis (see Clin. Cancer Res. 2(9):1445-51, 1996). Timed
incubations of PSMA with polyglutamated substrates is followed by
separation and detection of hydrolysis products.
[0318] The invention also includes isolated antibodies and binding
fragments thereof that selectively bind PSMA multimers. As used
herein, particularly with respect to the binding of PSMA multimers
by the anti-PSMA antibodies and binding fragments, "selectively
binds" means that an antibody preferentially binds to a PSMA
protein multimer (e.g., with greater avidity, greater binding
affinity) rather than to a PSMA protein monomer. In preferred
embodiments, the antibodies of the invention bind to a PSMA protein
multimer with an avidity and/or binding affinity that is 1.1-fold,
1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,
1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 7-fold,
10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 70-fold, 100-fold,
200-fold, 300-fold, 500-fold, 1000-fold or more than that exhibited
by the antibody for a PSMA protein monomer. Preferably, the
antibody selectively binds a PSMA protein multimer, and not a PSMA
protein monomer, i.e., substantially exclusively binds to a PSMA
protein multimer. Most preferably, the antibody selectively binds a
PSMA protein dimer.
[0319] The isolated antibody or binding fragment that selectively
binds a PSMA protein multimer can, in some embodiments, modulate
enzymatic activity of the PSMA protein multimer. In one such
embodiment, the antibody inhibits at least one enzymatic activity
such as NAALADase activity, folate hydrolase activity, dipeptidyl
dipeptidase IV activity, .gamma.-glutamyl hydrolase activity, or
combinations thereof. In another embodiment, the antibody enhances
at least one enzymatic activity such as NAALADase activity, folate
hydrolase activity, dipeptidyl dipeptidase IV activity,
.gamma.-glutamyl hydrolase activity, or combinations thereof.
[0320] As described elsewhere herein, a PSMA protein multime is a
protein complex of at least two PSMA proteins or fragments thereof.
The PSMA protein multimers can be composed of various combinations
of full-length PSMA proteins (e.g., SEQ ID NO: 1), recombinant
soluble PSMA (rsPSMA, e.g., amino acids 44-750 of SEQ ID NO:1) and
fragments of the foregoing that form multimers (i.e., that retain
the protein domain required for forming dimers and/or higher order
multimers of PSMA). In preferred embodiments, at least one of the
PSMA proteins forming the multimer is a recombinant, soluble PSMA
(rsPSMA) polypeptide. Preferred PSMA protein multimers are dimers,
particularly those formed from recombinant soluble PSMA protein. A
particularly preferred embodiment is a rsPSMA homodimer.
[0321] The PSMA protein multimers referred to herein are believed
to assume a native conformation and preferably have such a
conformation. The PSMA proteins in certain embodiments are
noncovalently bound together to form the PSMA protein multimer. For
example, it has been discovered that PSMA protein noncovalently
associates to form dimers under non-denaturing conditions, as
described in the Examples below.
[0322] The PSMA protein multimers can, and preferably do, retain
the activities of PSMA. The PSMA activity may be an enzymatic
activity, such as folate hydrolase activity, NAALADase activity,
dipeptidyl peptidase IV activity and .gamma.-glutamyl hydrolase
activity. Methods for testing the PSMA activity of multimers are
well known in the art (reviewed by O'Keefe et al. in: Prostate
Cancer: Biology. Genetics. and the New Therapeutics, L. W. K.
Chung, W. B. Isaacs and J. W. Simons (eds.) Humana Press, Totowa,
N.J., 2000, pp. 307-326), some of which are described in the
Examples herein below.
[0323] As used herein with respect to polypeptides, proteins or
fragments thereof, "isolated" means separated from its native
environment and present in sufficient quantity to permit its
identification or use. Isolated, when referring to a protein or
polypeptide, means, for example: (i) selectively produced by
expression cloning or (ii) purified as by chromatography or
electrophoresis. Isolated proteins or polypeptides may be, but need
not be, substantially pure. The term "substantially pure" means
that the proteins or polypeptides are essentially free of other
substances with which they may be found in nature or in vivo
systems to an extent practical and appropriate for their intended
use. Substantially pure polypeptides may be produced by techniques
well known in the art. Because an isolated protein may be admixed
with a pharmaceutically acceptable carrier in a pharmaceutical
preparation, the protein may comprise only a small percentage by
weight of the preparation. The protein is nonetheless isolated in
that it has been separated from the substances with which it may be
associated in living systems, i.e. isolated from other
proteins.
[0324] Fragments of a PSMA protein preferably are those fragments
which retain a distinct functional capability of the PSMA protein.
Functional capabilities which can be retained in a fragment include
binding of other PSMA molecules to form dimers and higher order
multimers, interaction with antibodies, interaction with other
polypeptides or fragments thereof, and enzymatic activity. Other
PSMA protein fragments, e.g., other recombinant soluble fragments
of SEQ ID NO:1, can be selected according to their functional
properties. For example, one of ordinary skill in the art can
prepare PSMA fragments recombinantly and test those fragments
according to the methods exemplified below.
[0325] Modifications to a PSMA polypeptide are typically made to
the nucleic acid which encodes the PSMA polypeptide, and can
include deletions, point mutations, truncations, amino acid
substitutions and additions of amino acids or non-amino acid
moieties. Alternatively, modifications can be made directly to the
polypeptide, such as by cleavage, addition of a linker molecule,
addition of a detectable moiety, such as biotin, addition of a
fatty acid, and the like. Modifications also embrace fusion
proteins comprising all or part of the PSMA amino acid
sequence.
[0326] In general, modified PSMA polypeptides include polypeptides
which are modified specifically to alter a feature of the
polypeptide unrelated to its physiological activity. For example,
cysteine residues can be added or substituted or deleted to promote
or prevent unwanted disulfide linkages, respectively. Similarly,
certain amino acids can be changed to enhance expression of a PSMA
polypeptide by eliminating proteolysis by proteases in an
expression system (e.g., dibasic amino acid residues in yeast
expression systems in which KEX2 protease activity is present).
[0327] Modifications conveniently are prepared by altering a
nucleic acid molecule that encodes the PSMA polypeptide. Mutations
of a nucleic acid which encode a PSMA polypeptide preferably
preserve the amino acid reading frame of the coding sequence, and
preferably do not create regions in the nucleic acid which are
likely to hybridize to form secondary structures, such a hairpins
or loops, which can be deleterious to expression of the modified
polypeptide.
[0328] Modifications can be made by selecting an amino acid
substitution, or by random mutagenesis of a selected site in a
nucleic acid which encodes the PSMA polypeptide. Modified PSMA
polypeptides then can be expressed and tested for one or more
activities (e.g., antibody binding, enzymatic activity, multimeric
stability) to determine which mutation provides a modified
polypeptide with the desired properties. Further mutations can be
made to modified PSMA polypeptides (or to non-modified PSMA
polypeptides) which are silent as to the amino acid sequence of the
polypeptide, but which provide preferred codons for translation in
a particular host. The preferred codons for translation of a
nucleic acid in, e.g., E. coli, are well known to those of ordinary
skill in the art. Still other mutations can be made to the
noncoding sequences of a PSMA coding sequence or cDNA clone to
enhance expression of the polypeptide. The activity of modified
PSMA polypeptides can be tested by cloning the gene encoding the
modified PSMA polypeptide into a bacterial or mammalian expression
vector, introducing the vector into an appropriate host cell,
expressing the modified PSMA polypeptide, and testing for a
functional capability of the PSMA polypeptides as disclosed herein.
The foregoing procedures are well known to one of ordinary skill in
the art.
[0329] The skilled artisan will also realize that conservative
amino acid substitutions may be made in PSMA polypeptides to
provide functionally equivalent PSMA polypeptides, i.e., modified
PSMA polypeptides that retain the functional capabilities of PSMA
polypeptides. These functionally equivalent PSMA polypeptides
include those PSMA polypeptides or proteins that are capable of
associating to form multimers, particularly dimers. As used herein,
a "conservative amino acid substitution" refers to an amino acid
substitution which does not alter the relative charge or size
characteristics of the protein in which the amino acid substitution
is made. Modified PSMA polypeptides can be prepared according to
methods for altering polypeptide sequence known to one of ordinary
skill in the art such as are found in references which compile such
methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,
et al., eds., Second Edition, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular
Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc.,
New York. Exemplary functionally equivalent PSMA polypeptides
include conservative amino acid substitutions of SEQ ID NO: 1, or
fragments thereof, such as the recombinant soluble PSMA polypeptide
(amino acids 44-750 of SEQ ID NO: 1). Conservative substitutions of
amino acids include substitutions made amongst amino acids within
the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d)
A, G; (e) S, T; (f) Q, N; and (g) E, D.
[0330] Conservative amino-acid substitutions in PSMA polypeptides
typically are made by alteration of a nucleic acid encoding a PSMA
polypeptide. Conservatively substituted PSMA polypeptides include
those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more
substitutions. Such substitutions can be made by a variety of
methods known to one of ordinary skill in the art. For example,
amino acid substitutions may be made by PCR-directed mutation,
site-directed mutagenesis, or by chemical synthesis of a gene
encoding a PSMA polypeptide. Where amino acid substitutions are
made to a small fragment of a PSMA polypeptide, the substitutions
can be made by directly synthesizing the peptide. The activity of
functionally equivalent fragments of PSMA polypeptides can be
tested by cloning the gene encoding the altered PSMA polypeptide
into a bacterial or mammalian expression vector, introducing the
vector into an appropriate host cell, expressing the altered PSMA
polypeptide, and testing for a functional capability of the PSMA
polypeptides as disclosed herein.
[0331] The PSMA protein multimers as described herein have a number
of uses, some of which are described elsewhere herein. The
multimers are useful for testing of compounds that modulate PSMA
enzymatic activity or PSMA multimerization. The multimers can be
used to isolate antibodies that selectively bind PSMA, including
those selective for conformational epitopes, those selective for
binding PSMA multimers and not PSMA monomers, and those that
selectively modulate an enzymatic activity of PSMA. The multimers,
particularly dimeric PSMA, also can be used to induce or increase
immune responses to PSMA, as vaccine compositions.
[0332] Agents that selectively modulate an enzymatic activity of
PSMA include agents that inhibit or enhance at least one enzymatic
activity of PSMA, such as NAALADase activity, folate hydrolase
activity, dipeptidyl dipeptidase IV activity, .gamma.-glutamyl
hydrolase activity, or combinations thereof.
[0333] Thus methods of screening for candidate agents that modulate
at least one enzymatic activity of a PSMA enzyme are provided in
accordance with the invention. The methods can include mixing the
candidate agent with an isolated PSMA protein multimer to form a
reaction mixture, thereby contacting the PSMA enzyme with the
candidate agent. The methods also include adding a substrate for
the PSMA enzyme to the reaction mixture, and determining the amount
of a product formed from the substrate by the PSMA enzyme. Such
methods are adaptable to automated, high-throughput screening of
compounds. A decrease in the amount of product formed in comparison
to a control is indicative of an agent capable of inhibiting at
least one enzymatic activity of the PSMA enzyme. An increase in the
amount of product formed in comparison to a control is indicative
of an agent capable of enhancing at least one enzymatic activity of
the PSMA enzyme. The PSMA enzyme can be NAALADase, folate
hydrolase, dipeptidyl dipeptidase IV and/or .gamma.-glutamyl
hydrolase. The PSMA enzyme preferably is a PSMA multimer that
includes recombinant soluble PSMA, most preferably a noncovalently
associated dimer of PSMA in a native conformation.
[0334] The reaction mixture comprises a candidate agent. The
candidate agent is preferably an antibody, a small organic
compound, or a peptide, and accordingly can be selected from
combinatorial antibody libraries, combinatorial protein libraries,
or small organic molecule libraries. Typically, a plurality of
reaction mixtures are run in parallel with different agent
concentrations to obtain a different response to the various
concentrations. Typically, one of these concentrations serves as a
negative control, i.e., at zero concentration of agent or at a
concentration of agent below the limits of assay detection.
[0335] Candidate agents encompass numerous chemical classes,
although typically they are organic compounds, proteins or
antibodies (and fragments thereof that bind antigen). In some
preferred embodiments, the candidate agents are small organic
compounds, i.e., those having a molecular weight of more than 50
yet less than about 2500, preferably less than about 1000 and, more
preferably, less than about 500. Candidate agents comprise
functional chemical groups necessary for structural interactions
with polypeptides and/or nucleic acids, and typically include at
least an amine, carbonyl, hydroxyl, or carboxyl group, preferably
at least two of the functional chemical groups and more preferably
at least three of the functional chemical groups. The candidate
agents can comprise cyclic carbon or heterocyclic structure and/or
aromatic or polyaromatic structures substituted with one or more of
the above-identified functional groups. Candidate agents also can
be biomolecules such as peptides, saccharides, fatty acids,
sterols, isoprenoids, purines, pyrimidines, derivatives or
structural analogs of the above, or combinations thereof and the
like.
[0336] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides, synthetic organic
combinatorial libraries, phage display libraries of random or
non-random peptides, combinatorial libraries of proteins or
antibodies, and the like. Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant, and animal
extracts are available or readily produced. Additionally, natural
and synthetically produced libraries and compounds can be readily
be modified through conventional chemical, physical, and
biochemical means. Further, known agents may be subjected to
directed or random chemical modifications such as acylation,
alkylation, esterification, amidification, etc. to produce
structural analogs of the agents.
[0337] A variety of other reagents also can be included in the
mixture. These include reagents such as salts, buffers, neutral
proteins (e.g., albumin), detergents, etc. which may be used to
facilitate optimal protein-protein and/or protein-agent binding.
Such a reagent may also reduce non-specific or background
interactions of the reaction components. Other reagents that
improve the efficiency of the assay such as protease inhibitors,
nuclease inhibitors, antimicrobial agents, and the like may also be
used.
[0338] The mixture of the foregoing reaction materials is incubated
under conditions whereby, the candidate agent interacts with the
PSMA enzyme. The order of addition of components, incubation
temperature, time of incubation, and other parameters of the assay
may be readily determined. Such experimentation merely involves
optimization of the assay parameters, not the fundamental
composition of the assay. Incubation temperatures typically are
between 4.degree. C. and 40.degree. C. Incubation times preferably
are minimized to facilitate rapid, high throughput screening, and
typically are between 0.1 and 10 hours.
[0339] After incubation, the presence or absence of PSMA enzyme
activity is detected by any convenient method available to the
user. For example, the reaction mixture can contain a substrate for
the PSMA enzyme. Preferably the substrate and/or the product formed
by the action of the PSMA enzyme are detectable. The substrate
usually comprises, or is coupled to, a detectable label. A wide
variety of labels can be used, such as those that provide direct
detection (e.g., radioactivity, luminescence, optical, or electron
density, etc) or indirect detection (e.g., epitope tag such as the
FLAG epitope, enzyme tag such as horseradish peroxidase, etc.). The
label may be bound to the substrate, or incorporated into the
structure of the substrate.
[0340] A variety of methods may be used to detect the label,
depending on the nature of the label and other assay components.
For example, the label may be detected while bound to the substrate
or subsequent to separation from the substrate. Labels may be
directly detected through optical or electron density, radioactive
emissions, nonradiative energy transfers, etc. or indirectly
detected with antibody conjugates, strepavidin-biotin conjugates,
etc. Methods for detecting a variety of labels are well known in
the art.
[0341] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Materials and Methods
DNA Constructs.
[0342] All secreted PSMA constructs were derived from the original
human PSMA clone p55A provided by Dr. W. D. W. Heston (Israeli et
al., Cancer Res. 53: 227-230, 1993). The constructs were subcloned
into expression vector PPI4 (Trkola et al., Nature 384: 184-187,
1996) for high-level expression and secretion in mammalian cells.
Recombinant soluble PSMA (rsPSMA) corresponds to the entire
extracellular domain of PSMA (amino acids 44-750 of SEQ ID NO:1
(GENBANK Protein Accession number AAA60209)).
pcDNA Plasmid Constructs:
[0343] Nucleic acid molecules encoding the anti-PSMA antibodies
10.3, 006, 026, 051, 069 and 077 were cloned into plasmid pcDNA.
The cloning protocol is given in FIG. 13. Primers (SEQ ID NOs:
33-36, sense and anti-sense) used for the variable region
amplifications are also shown. The plasmids constructed for
anti-PSMA antibodies 006, 026, 051, 069, 077 and 10.3 contain
nucleotide sequences encoding the heavy chain of the antibodies
(SEQ ID NOs: 2-7; PTA-4403, PTA-4405, PTA-4407, PTA-4409, PTA-4411,
PTA-4413, respectively) or contain nucleotide sequences encoding
light chain of the antibodies (SEQ ID NOs: 8-13; PTA-4404,
PTA-4406, PTA-4408, PTA-4410, PTA-4412 and PTA-4414, respectively).
Plasmid maps are given in FIGS. 14-25.
Western Blots.
[0344] Cells were lysed in PBS containing 1 mM EDTA, 1% NP-40, 1%
Triton X-100, and 5 mg/ml aprotinin and cell debris was removed by
centrifugation at 3000 g for 30 min at 4.degree. C. Lysates were
separated on a 5-20% gradient gel before transfer to nitrocellulose
membranes. The resulting blots were blocked in PBS containing 5%
milk, 0.02% SDS and 0.1% Triton X-100 before incubation with MAB544
primary antibody (Maine Biotechnologies) at a concentration of 2
mg/ml. After three washes, blots were incubated with a goat
anti-mouse HRP-conjugated secondary antibody at a concentration of
0.2 mg/ml. Blots are visualized using the Renaissance
chemiluminescence system (Perkin-Elmer Life Sciences, Boston,
Mass.).
ELISA.
[0345] Cells were lysed in PBS containing 1 mM EDTA, 1% NP-40, 1%
Triton X-100, and 5 mg/ml aprotinin. The resulting cell membranes
were plated onto 96-well plates and dried in a sterile hood
overnight. The plates were then blocked with PBS containing casein
and TWEEN-20 before addition of mouse sera or hybridoma
supernatants, using purified MAB544 (Maine Biotechnologies) or 7E11
(Cytogen) as a standard. After washing in PBS, an alkaline
phosphatase conjugated secondary antibody (subclass specific) was
incubated and subsequently washed in PBS. The pNPP substrate was
then added for colorimetric detection at a wavelength of 405
nm.
Flow Cytometry.
[0346] Wild-type 3T3 or PSMA-expressing 3T3 cells (10.sup.6 cells
per condition) were washed in PBS containing 0.1% NaN.sub.3.
Antibodies or sera were then added (1:100 dilution in PBS) and
incubated on ice for 30 minutes. After washing in PBS+0.1%
NaN.sub.3, the cells were incubated with anti-mouse IgG+IgM
(Calbiotech) for 30 minutes on ice. Cells were washed again in
PBS+0.1% NaN.sub.3 and analyzed by flow cytometry.
Example 1
Generation of a Panel of Monoclonal Antibodies (mAbs) to
Conformational Epitopes on PSMA
[0347] A panel of anti-PSMA mAbs that represent promising
candidates for therapy was created. Briefly, the mAbs were
generated as follows: BALB/c mice were immunized subcutaneously
with recombinant PSMA at approximately three-week intervals. After
a total of 4 injections, mice were sacrificed and their splenocytes
fused with a myeloma cell line using standard techniques in order
to create hybridomas. Individual hybridoma supernatants were
screened by ELISA for reactivity with PSMA derived from either
LNCaP human prostate tumor cells or from 3T3 cells engineered to
express full-length human PSMA (3T3-PSMA cells). Positive clones
were secondarily screened by flow cytometry for specific reactivity
with intact 3T3-PSMA and LNCaP cells so as to select antibodies
that recognize native, cell-surface PSMA and thus have the greatest
therapeutic potential.
[0348] Mice having the ability to produce human antibodies
(XENOMOUSE, Abgenix; Mendez et al., Nature Genetics 15:146, 1997)
were immunized subcutaneously once or twice weekly with
5.times.10.sup.6 LNCaP cells adjuvanted with alum or TITERMAX Gold
(Sigma Chemical Co., St. Louis, Mo.). Animals were boosted twice
with 10 .mu.g of recombinant PSMA protein immunoaffinity captured
onto protein G magnetic microbeads (Miltenyi Biotec, Auburn,
Calif.). PSMA mAb 3.11 was used for capture. Splenocytes were fused
with NSO myeloma cells and the hybridomas that resulted were
screened as above by flow cytometry to detect clones producing
antibodies reactive with the extracellular portion of PSMA. One
clone, 10.3 (PTA-3347), produced such antibodies.
[0349] These methods have yielded a high proportion of mAbs that
react exclusively with conformation-specific epitopes on
cell-surface PSMA. As shown in FIG. 1, several (mAbs 3.7, 3.9,
3.11, 5.4, and 10.3) but not all (mAb 3.12) mAbs specifically bind
viable PSMA-expressing cells. Using recombinant soluble PSMA
proteins expressed in Chinese hamster ovary (CHO) cell lines, it
further was demonstrated that the mAbs bind epitopes in the
extracellular region of PSMA. The mAbs were also tested for their
ability to immunoprecipitate native PSMA from 3T3-PSMA cell
lysates. The mAbs positive in flow cytometry (FIG. 1) were also
effective in immunoprecipitation (FIG. 2), whereas mAb 3.12 was
unreactive. FIG. 3 shows the recognition of non-denatured
full-length PSMA and recombinant soluble PSMA by several PSMA
antibodies that recognize PSMA conformation. This further confirms
that these methods yield a preponderance of mAbs that efficiently
recognize native PSMA.
[0350] The mAbs were tested for reactivity with denatured PSMA by
Western blot analysis (FIG. 4). Lysates from the indicated cells
and samples (controls: 3T3 cells, PSMA-negative human prostate cell
lines PC-3 and DU145, mock supernatant; PSMA-positive samples:
PSMA-expressing 3T3 cells, PSMA-positive human prostate cell line
LNCaP, rsPSMA-positive supernatant) were resolved by SDS-PAGE,
electroblotted, and probed with anti-PSMA mAbs 3.1 and 3.12 (ATCC
Patent Deposit Designations PTA-3639 and PTA-3640, respectively).
Four mAbs tested in parallel (3.7, 3.8, 3.9, 3.11) showed no
reactivity to either full-length or secreted rsPSMA proteins. 7E11
mAb immunoprecipitated full-length but not secreted rsPSMA.
[0351] The mAbs reactive in flow cytometry and immunoprecipitation
(mAbs 3.7, 3.9, 3.11, 5.4, and 10.3) were all unreactive in Western
blot analysis, indicating that the mAbs do not recognize linear
epitopes. Taken together, the data strongly suggest that these 5
mAbs recognize conformation-specific epitopes located in the
extracellular domain of PSMA. Since mAbs to conformational epitopes
typically possess the greatest affinity and specificity for
antigen, they represent preferred candidates for therapy.
[0352] The reactivities of certain anti-PSMA antibodies are
described in Table 2:
TABLE-US-00002 TABLE 2 Anti-PSMA Antibody Properties Reactivity
Flow West- mAb ELISA Cytometry IP ern Epitope 3.1 + + + + Linear,
Extracellular, exposed on native PSMA 3.7 + + + - Conformational,
extracellular 3.8 + + + - Conformational, extracellular 3.9 + + + -
Conformational, extracellular 3.11 + + + - Conformational,
extracellular 3.12 + - - + Linear, Extracellular, not exposed on
native PSMA 5.4 + + + - Conformational, extracellular 7.1 + - - +
Linear, Extracellular, not exposed on native PSMA 7.3 + + + -
Conformational, extracellular 10.3 + + + - Conformational,
extracellular 1.8.3 + + - Extracellular A3.1.3 + + - Extracellular
A3.3.1 + + - Extracellular
[0353] The mAbs were determined by ELISA to be primarily of the
mouse IgG2a, mouse IgG2b and human IgG1 isotypes, which mediate
potent effector functions. Although a number of anti-PSMA mAbs have
been described over the years and evaluated for therapeutic
potential (see, e.g., Liu, H. et al. Cancer Res. 57: 3629-3634,
1997; Chang, S. S. et al. Cancer Res. 59: 3192-3198, 1999; Murphy,
G. P. et al. J Urology 160: 2396-2401, 1998), none inhibit the
enzymatic activity of PSMA and few recognize conformational
determinants on PSMA.
Example 2
Production of Anti-PSMA mAbs
[0354] To accurately and quantitatively assess the therapeutic
potential of these mAbs, the mAbs are produced in a quantity and
quality suitable for extensive in vitro and in vivo
characterization. Briefly, the mAb-secreting hybridomas are
cultured in roller bottles in DMEM/F12 medium supplemented with 10%
FBS that has been depleted of bovine IgG (Life Technologies).
During the production phase of the culture, cells are maintained at
5.times.10.sup.6 cells/mL via twice-weekly exchanges of media.
Collected media are clarified by filtration through a 0.22 micron
filter and stored at -95.degree. C. prior to purification. Given an
average antibody expression levels of .about.25 mg/L, approximately
3 L of roller bottle supernatants are required for each antibody to
allow for losses in purification.
[0355] Culture supernatants from a given hybridoma are pooled and
loaded onto a Protein A SEPHAROSE affinity column. Mouse IgG2a,
mouse IgG2b and human IgG1 antibodies are loaded directly, but
supernatants containing mouse IgG1 antibodies are adjusted to pH
8.5 and 1M NaCl prior to loading in order to promote binding. After
washing the column, the mAb is eluted with low pH buffer into
fractions using 1M Tris, pH 8.0. Elution peak fractions are pooled,
dialyzed against PBS buffer, concentrated to 5 mg/mL and stored in
sterile aliquots at -95.degree. C. All purification procedures are
carried out using endotoxin-free buffers and sanitized
chromatography columns. Purified mAbs are tested for purity by
reducing and nonreducing SDS-PAGE, for PSMA binding affinity by
ELISA, and for endotoxin levels by the limulus amebocyte lysate
assay. These procedures routinely yield "animal-grade" antibody at
>95% purity and <0.5 endotoxin units per milligram of
protein.
Example 3
Evaluation of the Therapeutic Potential of the Unlabeled mAbs In
Vitro
[0356] Purified mAbs are tested in a battery of assays for
therapeutically relevant properties, including affinity,
specificity, enzyme inhibitory activity and effector functions. The
ideal product candidate binds and inhibits PSMA activity at
subnanomolar concentrations and mediates potent cell-killing
through Fc-related effector functions.
[0357] First, the mAbs' affinity for cell-surface and secreted
forms of PSMA is measured by flow cytometry and ELISA,
respectively. In the flow cytometry assay, varying amounts of mAbs
are incubated with 5.times.10.sup.5 3T3-PSMA cells in FACS buffer
(PBS containing 1% FBS and 0.1% NaN.sub.3) for 2 hr to allow for
saturation binding. Cells are washed and incubated with a
phycoerythrin-coupled goat antibody to mouse IgG (ICN/Cappel) for
detection of bound mAb by flow cytometry. Specific binding is
calculated by subtracting the fluorescence intensity observed with
parental 3T3 cells.
[0358] For ELISA, CHO cell-derived recombinant soluble PSMA protein
(rsPSMA, Progenics, Tarrytown, N.Y.) is diluted to 1 .mu.g/ml in 50
mM carbonate buffer, pH 9.4, and coated overnight at 4.degree. C.
onto 96-well IMMULON II microtiter plates at 100 .mu.l/well. The
plates are then blocked for 2 hr with PBS buffer containing 5% BSA.
mAbs are added in a range of concentrations in ELISA buffer (PBS
buffer containing 2% BSA, 1% FBS and 0.5% TWEEN 20) for 2 hours at
room temperature. The plates are washed, and horseradish peroxidase
conjugated goat antibody to mouse IgG is added for 1 hr at room
temperature. The plates are washed again and
3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) substrate
(Pierce, Rockford, Ill.) is added for colorimetric readout at 450
nm using an ELISA plate reader (Molecular Devices, Sunnyvale,
Calif.).
Example 4
mAb Cross-Competition Binding Assay
[0359] To identify whether a given group of mAbs recognize distinct
or overlapping epitopes on PSMA, cross-competition binding assays
are performed (Liu, H. et al. Cancer Res 57: 3629-3634, 1997). In
this flow cytometry assay, a biotinylated test mAb is incubated
with 3T3-PSMA cells in the presence or absence of varying
concentrations of unlabeled competitor mAbs as described above.
Following washing, phycoerythrin-conjugated streptavidin is added
to determine the amount of bound biotinylated mAb. The percent
inhibition is defined relative to that observed in the presence of
an isotype-matched mAb of irrelevant specificity (0% inhibition)
and to that observed using excess unlabeled test mAb (100%
inhibition).
Example 5
Effects of mAbs on PSMA Enzymatic Activity
[0360] PSMA has been shown to possess both folate hydrolase
(pteroyl-glutamyl carboxypeptidase) and N-acetylated .alpha.-linked
acidic dipeptidase (NAALADase) enzymatic activities, which may
influence the proliferation and malignancies of the tumor cell
(Heston, W. D. W. Prostate: Basic and Clinical Aspects (R. K. Naz,
ed.). CRC Press, New York: 219-243, 1997). A first set of mAbs
described above (mAb 3.9, mAb 5.4 and mAb 7.3) and mAb J591 (ATCC
#HB-12126) were tested for folate hydrolase modulating activity
using previously described assays for measuring PSMA enzymatic
activity (Pinto, J. T. et al. Clinical Cancer Res 2: 1445-1451,
1996).
[0361] Briefly, folate hydrolase activity was measured as follows.
Fifty .mu.M methotrexate di-gamma glutamate and 10 .mu.g/ml rsPSMA
(premixed with anti-PSMA or irrelevant mAb) was incubated in pH 4.5
acetate buffer in a volume of 100 .mu.l for 2 hr at 37.degree. C.
Reactions were terminated by boiling for 5 minutes prior to
separation of free, mono- and di-gamma glutamate forms of
methotrexate by capillary electrophoresis on a Spectra Phoresis
1000 (Thermo Separation, San Jose, Calif.). The various
methotrexate derivatives were quantified based on their retention
times and absorbance at 300 nm.
[0362] The data show that mAb 5.4 potently blocks the enzymatic
activity of purified rsPSMA protein and in lysates of C4-2 cells.
C4-2 is an androgen independent derivative of the LNaCP cell line
(human prostate cancer line) which expresses endogenous PSMA. More
details regarding the C4-2 cell line may be found in O'Keefe D. S.
et al. Prostate 45: 149-157, 2000). FIGS. 8 and 9 provide the
results for two production lots of rsPSMA (rsPSMA #7 and rsPSMA
#8). The results for the C4-2 cell lysates are shown in FIG. 10.
The figures illustrate the effect of four antibodies (mAb 3.9, mAb
5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate
hydrolase by way of the rate of cleavage of glutamate from
methotrexate di-gamma glutamate (MTXGlu2) by folate hydrolase
present in the two production lots of rsPSMA and in the C4-2 cell
lysates. In addition to the inhibitory effects of mAb 5.4, mAb 3.9
was also found to inhibit folate hydrolase activity.
[0363] Another set of mAbs (mAb 4.40.2, mAb 006, mAb 026 and mAb
5.4) was also tested for folate hydrolase modulating activity. The
data confirm that mAb 5.4 potently blocks folate hydrolase activity
of PSMA (FIG. 11). The concentration of mAb 5.4 which inhibited
PSMA enzymatic activity by 50% (IC50, also referred to as EC50 or
"effective concentration") was determined to be
4.902.times.10.sup.-4 mg/mL. The data further show that mAb 006 and
mAb 026 also block PSMA folate hydrolase activity, while mAb 4.40.2
did not (FIG. 11). The IC50 values for mAb 006 and mAb 026 were
9.338.times.10.sup.-3 mg/mL and 1.385.times.10.sup.-3 mg/mL,
respectively.
[0364] For NAALADase activity assays, rsPSMA protein is incubated
with varying amounts of anti-PSMA or control mAbs in 50 mM Tris pH
7.4, 1 mM CoCl.sub.2 for 10 minutes at 37.degree. C. before adding
50 .mu.l of 0.6 .mu.M N-acetylaspartyl-[.sup.3H]glutamate. After 15
minutes, the reaction is stopped by adding 1 ml of 100 mM
NaPO.sub.4. Cleaved glutamate is separated from the substrate by
ion exchange chromatography and detected by scintillation counting.
Each measurement is performed in triplicate.
Example 6
Reactivity with Normal and Malignant Human Tissues by
Immunohistochemistry
[0365] Anti-PSMA mAbs are tested by immunohistochemistry for
reactivity with both normal and malignant human tissues using an
avidin-biotin peroxidase method (Silver, D. A. et al. Clin Cancer
Res 3: 81-85, 1997). Frozen or paraffin-embedded tissues can be
used. Paraffin-embedded tissue sections are deparaffinized and
endogenous peroxidase activity is blocked by incubation with 1%
H.sub.2O.sub.2 for 15 minutes. Sections are blocked in a 1:10
dilution of horse serum in 2% PBS-BSA (Sigma Chemical, St Louis,
Mo.) for 30 minutes before overnight incubation with 2 .mu.g/ml
anti-PSMA mAb in 2% PBS-BSA. After washing, sections are incubated
with biotinylated secondary antibody, washed, and incubated with
avidin:biotin peroxidase complexes (Vector Laboratories,
Burlingame, Calif.) diluted 1:25 in PBS for 30 minutes. After
washing, sections are visualized by immersion in PBS containing
0.05% diaminobenzidine tetrachloride, 0.01% H.sub.2O.sub.2, and
0.5% Triton X-100.
[0366] Negative control sections are incubated with isotype-matched
mAbs of irrelevant specificity. As a positive control, 7E11
(Cytogen, Princeton, N.J.), a well-characterized anti-PSMA mAb, is
used.
Example 7
Antibody-Dependent Cellular Cytotoxicity (ADCC)
[0367] In the ADCC assay, mAbs are serially diluted and combined
with .sup.51Cr-labeled 3T3-PSMA cells or human prostate PC-3 cells
that have been engineered to express human PSMA (PC-3-PSMA cells).
NK effector cells are purified from lymph nodes or spleens using
anti-NK microbeads (Miltenyi Biotec). Sera, NK effector cells, and
.sup.51Cr-loaded target cells are co-incubated at effector:target
cell ratios of 10:1, 20:1, and 40:1, with each condition performed
in triplicate. Cells are incubated 4-5 hours at 37.degree. C.
before supernatants are collected for measurement of .sup.51Cr
release by gamma counting. The percent specific lysis is determined
relative to that observed in the presence of isotype-matched
non-specific mAb (0% lysis) to that obtained using 10% sodium
dodecyl sulfate (100% lysis).
Example 8
Complement-Mediated Lysis (CML)
[0368] For CML, .sup.51Cr-loaded 3T3-PSMA or PC-3-PSMA cells serve
as target cells. Serial dilutions of mAbs are co-incubated with
rabbit complement and target cells for 4-5 hours at 37.degree. C.,
with each condition being performed in triplicate. Supernatants are
then collected and counted with a gamma counter. Specific lysis is
computed as previously done with the ADCC assay data.
Example 9
Anti-Proliferative Effects
[0369] To test anti-proliferative effects of these antibodies,
anti-PSMA mAbs are serially diluted and incubated with LNCaP,
PC-3-PSMA and parental PC-3 cells in log-phase growth. At 4 hr, 24
hr, and 72 hr intervals, cells are removed and analyzed for density
and viability by trypan blue staining and WST-1 assay (Roche
Biochemicals).
Example 10
Optimization of Chelation and Radiolabeling Procedures
[0370] The most promising mAbs identified using the procedures
described in the foregoing examples will be optimized for
biochemical and biological stability and activity after labeling
prior to evaluation in animals. Success in in vitro experiments is
defined as identification of a radiolabeled mAb that specifically
kills PSMA-expressing tumor cells at >10-fold lower
concentrations than unlabeled or similarly labeled isotype control
mAb.
[0371] Because the preferred .alpha.- and .beta.-emitting isotopes
are all radiometals, each of the mAbs is first conjugated with an
appropriate metal chelating agent. Based on the favorable in vivo
stability data and its proven use in human clinical trials, the
bifunctional chelating agent C-functionalized trans
cyclohexyldiethylenetriaminepentaacetic acid (p-SCN-CHX-A''-DTPA)
is the preferred agent for attaching either .sup.90Y or .sup.213Bi
to the antibody (Brechbiel, M. W. et al. J. Chem. Soc. Chem.
Commun. 1169-1170, 1991). A form of this chelate has previously
been tested in more than 70 doses in humans in ongoing trials at
Memorial-Sloan Kettering Cancer Center (McDevitt, M. R. et al. J.
Nucl. Med. 40:1722-1727, 1999). For .sup.225Ac, our initial studies
will examine a novel bifunctional chelating agent termed
p-SCN-Bz-HEHA
(1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N'''',N'''''-hexaace-
tic acid) (Deal, K. A. et al. J. Med. Chem. 42:2988-2992, 1999).
The objective is to optimize the antibody conjugation and chelation
ratios to maximize labeling yield and activity while maintaining
suitable stability for in vivo utilization. Additional chelating
agents also are used as they become available from the N.I.H. and
other sources.
[0372] Initially, the antibody is rendered metal-free by incubation
with a large molar excess of EDTA at pH=5. The EDTA and any metals
scavenged from the antibody preparation are removed via continuous
buffer exchange/dialysis so as to replace the pH=5 buffer with the
conjugation buffer (Nikula, T. K. et al. Nucl. Med. Biol.
22:387-390, 1995). Conditions that yield optimal chelator to
antibody ratio but still remain immunoreactive are identified by
systematically varying the chelator:antibody ratio, reaction time,
temperature, and/or buffer systems about initial conditions that
employ a 40-fold molar excess of chelator to antibody in HEPES
buffer, pH 8.5. The number of chelates bound per antibody is
determined using an established spectrophotometric method (Pippin,
C. G. et al. Bioconjugate Chemistry 3: 342-345, 1992).
[0373] For .sup.90Y and .sup.225Ac constructs, labeling efficiency
is measured directly. For .sup.213Bi, initial antibody constructs
are tested for chelation efficiency using .sup.111In, which has
similar chelation chemistry as .sup.213Bi but possesses the
advantages of a longer half life (t.sub.1/2=3 days), ready
availability, and traceable .gamma.-emission. Once optimized using
.sup.111In, labeling efficiency is determined for .sup.213Bi.
[0374] Radiolabeled mAb is purified over a BioRad 10DG desalting
column using 1% HSA as the mobile phase and evaluated by instant
thin layer liquid chromatography (ITLC) and/or high performance
liquid chromatography (HPLC) to determine the percent incorporation
of radionuclide (Zamora, P. O. et al. Biotechniques 16: 306-311,
1994). ITLC and HPLC provide a means of establishing purity and
identifying the percent of low molecular weight radiochemical
impurities (i.e., metal chelates, colloids, and free metal).
Duplicate ITLC strips for each mobile phase are developed, dried,
and cut at the R.sub.f of 0.5 mark and counted in a gamma counter.
The HPLC system is equipped with both an online UV absorption
detector and radioactivity detector. The HPLC elution profile
directly correlates radioactivity with protein and low molecular
weight species as a function of the elution time. A TSK
SW3000.sub.XL column (TosoHaas, Montgomeryville, Pa.) is used and
calibrated using a range of protein molecular weight standards.
Example 11
Affinity and Immunoreactivity of Radiolabeled mAbs
[0375] Once radiolabeled constructs are obtained, purified, and
assessed for biochemical and radiochemical purity, biological
activity is determined. Binding activity of the radioconstruct is
performed by Scatchard analysis of binding data obtained using
whole LNCaP and 3T3-PSMA cells and/or membrane fractions as
previously described (Scheinberg, D. A. et al. Leukemia 3: 440-445
(1991).
[0376] The immunoreactivity of the synthetic constructs is
evaluated in order to correlate the chelate:antibody molar ratio
with the biological activity. Briefly, 2 ng of labeled mAb is
incubated with a .about.25-fold excess of PSMA as expressed on
3T3-PSMA cells. After a 30 min incubation at 0.degree. C., the
cells are collected by centrifugation and the supernatant
containing unbound mAb is added to fresh 3T3-PSMA cells for an
additional 30 min at 0.degree. C. Both sets of cells are
centrifuged and washed twice with cold PBS. The cell pellets,
supernatant and wash fractions are counted for radioactivity.
Immunoreactivity is defined as the amount of radioactivity in the
cell pellets divided by the total radioactivity in the cell
pellets, supernatant and wash fractions.
Example 12
mAb Internalization
[0377] The activity of radiolabeled mAbs can be significantly
modulated by their internalization rates. Based upon previous
results by other groups (Smith-Jones P. M. et al. Cancer Res 60:
5237-5243, 2000), significant internalization of PSMA after binding
with one or more of the mAb constructs was expected.
Internalization of the cell surface antibody-antigen complex was
measured using .sup.111In radiolabeled antibody (mAb 026)
constructs (Caron, P. C. et al. Cancer Res 52: 6761-6767, 1992).
Briefly, 5.times.10.sup.5 C4-2 cells were incubated at 37.degree.
C. in 5% CO.sub.2 with .sup.111In radiolabeled antibody. At
different times, cells were washed with PBS and cell-surface bound
radiolabeled constructs were stripped with 1 ml of 50 mM
glycine/150 mM NaCl, pH=2.8. Total cell-associated radioactivity
and acid-resistant (internalized) radioactivity were determined by
.gamma.-counting. Percent internalization and total binding were
calculated. .sup.111In labeled mAb 026 was found to be rapidly and
efficiently internalized. FIG. 12 shows the percent internalization
and total binding of .sup.111In labeled mAb 026 as a function of
incubation time. Cells (such as parental 3T3 cells) that do not
express PSMA can be used as a control to determine non-specific
binding.
Example 13
In Vitro Cytotoxicity Studies
[0378] Assessment of in vitro cytotoxicity of .alpha.-labeled mAbs
was undertaken once the immunoreactivity of the
radioimmunoconjugate was established. Approximately 50,000 target
cells (either LNCaP or 3T3-PSMA cells) were treated in 96 well
plates and analyzed 24-96 hours later. Quantification of cell death
due to .sup.225Ac-labeled constructs (or .sup.213Bi) was
accomplished by determining the uptake of .sup.3H-thymidine by
surviving cells (Nikula, T. K. et al. J. Nucl. Med. 40: 166-176,
1999). Specificity was determined by use of control cells
(PSMA-negative human prostate cell lines PC-3 and DU-145, as well
as control 3T3 cells), blocking with excess unlabeled antibody, and
control radioconjugates.
[0379] The cytotoxic effects of antibody conjugate concentration,
specific activity, and time of exposure were then assessed.
Cytotoxicity was expressed relative to that seen with 1M HCl (100%
cell death) and media (background cell death). LD.sub.50 values
were calculated by plotting cell viability as a function of the
number of mAc atoms bound on the cells (McDevitt, M. R. et al.
(1998) Eur. J. Nucl. Med. 25: 1341-1351 (1998).
[0380] Multicellular spheroids of LNCaP-FGC cells had been
established and were used to investigate the potential of
radioimmunotherapy (RIT) to eradicate minimal disease in vitro.
[0381] These three-dimensional spheroids mimic tissue structures
more accurately than monolayer cultures and thus provide a more
relevant model of solid tumors (O'Connor, K. C. Pharm. Res. 16:
486-493, 1999). LNCaP-FGC is a fast growing clone of the original
LNCaP cell line, and the cells were grown using a liquid overlay
technique to a size of 200-600 .mu.m (Ballangrud, A. M. et al.
Clin. Cancer Res. 5: 3171s-3176s, 1999). In larger spheroids, the
inner mass of cells becomes necrotic, while the outer rim consists
of proliferating tumor cells. Antibody penetration was measured by
confocal microscopy, and prior results suggested that an anti-PSMA
antibody should penetrate to a depth of 40-50 .mu.m (Ballangrud, A.
M. et al. 7th Conference on Radioimmunodetection and
Radioimmunotherapy of Cancer, Princeton N.J., 1998). The in vitro
cytotoxicity of .sup.225Ac-3.9 on LNCaP target cells is shown in
FIG. 26. The percentage of viable PSMA.sup.+ LNCaP cells was
plotted as a function of activity of the radioconjugate. Addition
of a 100-fold excess of unlabeled antibody was used as a control
for specificity.
Example 14
Evaluation of the In Vivo Efficacy of Unlabeled and Radiolabeled
mAbs in Mouse Xenograft Models of Human Prostate Cancer
[0382] Antibodies that are successful in the foregoing assays
demonstrate significant specificity and functional properties that
suggest they will be useful for therapeutic use. The most promising
of these radiolabeled and "naked" mAb constructs are evaluated in
the best available mouse models of prostate cancer. The studies
employ an established xenograft model in which the LNCaP human
prostate tumor cell line is injected into immunocompromised nude
mice and allowed to form solid tumors (Ellis, W. J. et al. Clin
Cancer Res 2: 1039-1048 (1996), which then are treated with both
radiolabeled and unlabeled anti-PSMA mAb constructs. Follow-on
studies also utilize a mouse xenograft model, CWR22, which
reproduces many of the key biological features of human prostate
cancer.
Lncap Tumor Cell Xenograft Model
[0383] A construct showing high affinity and high specificity is
taken into the LNCaP tumor cell xenograft in vivo model for
biodistribution and pharmacokinetic analysis. .sup.111In-labeled
anti-PSMA antibody is used for these studies due to its favorable
chelation chemistry, radioactive half-life and traceable gamma
emission. Timepoints are evaluated as appropriate for the
half-lives of .sup.213Bi, 225Ac, .sup.177Lu and .sup.90Y, which are
the nuclides of therapeutic interest. Labeled radioconstructs (1-5
.mu.g) are injected i.v. into nude mice (normal and tumor bearing)
and the mice are sacrificed at 5 min, 15 min, 30 min, 60 min, 2
hrs, 4 hrs, 18 hrs, and 24 hrs post-injection. Blood and major
organs are taken from animals, weighed, and the percent
radioactivity injected per gram of tissue is determined (Nikula, T.
K. et al. J. Nucl. Med. 40: 166-176, 1999). Specificity is
addressed by pre-injection with excess unlabeled construct.
Macroscopic tumor volume and animal survival rates is recorded
throughout the experiments.
[0384] A dose-ranging study is also conducted to determine the
toxicity of the constructs when administered via i.v. or i.p.
injection to normal and tumor-bearing mice. These animals are
routinely examined for toxic side effects during the course of the
studies by blood chemistry and physical examination. Animals are
sacrificed during and at the conclusion of the study in order to
collect blood and body tissues for further evaluation. Previous
data has demonstrated an approximate maximum tolerated dose of 250
.mu.Ci/mouse, so total doses are kept below that level.
[0385] Once i.v. biodistribution and toxicity is documented,
radiotherapy of tumors is assessed. Groups of five mice are
injected with <1 .mu.g radiolabeled anti-PSMA mAb construct both
pre- and post-tumor challenge to assess anti-tumor activity.
Antigen negative (RAJI or RAMOS) xenografted tumors are also used
as a control. Other controls include (1) treatment with unlabeled
anti-PSMA mAb only and (2) excess unlabeled anti-PSMA mAb
pretreatment before .sup.213Bi, .sup.225Ac, .sup.177Lu and/or
.sup.90Y-labeled anti-PSMA to block specific targeting.
[0386] Groups of tumor bearing mice are injected with unlabeled
anti-PSMA mAbs (at equimolar concentrations) and several dose
levels of radiolabeled anti-PSMA or a similarly labeled isotype
control antibody. The effect on tumor growth is assessed over time.
Statistical differences between therapy groups is determined using
an analysis of variance (ANOVA) method and animal survival is
illustrated using Kaplan-Meier plots. The efficacy of .sup.213Bi,
.sup.225Ac, .sup.177Lu and/or .sup.90Y-labeled anti-PSMA constructs
is correlated to the data obtained in vitro. Success in these
experiments is defined as the ability to significantly (p<0.05)
increase life-span and/or decrease tumor volume as compared to a
radiolabeled isotype control mAb.
[0387] Furthermore, the tumor models are used to test whether
predosing with unlabeled antibody prior to injection of
radiolabeled antibody improves delivery of the radiolabeled
antibody to the tumor. The tumor-bearing mice are injected with
<1 .mu.g radiolabeled anti-PSMA antibody with or without a prior
single injection of 5-100 g of unlabeled antibody. After several
days, animals are sacrificed for evaluation of the distribution of
radioactivity in the tumor, normal tissue, and blood. If predosing
with unlabeled antibody improves delivery and targeting of
radiolabeled antibody to the tumors, this approach is applied and
optimized in toxicity and therapeutic studies.
[0388] In addition to overall survival, the role of timing of the
injection after tumor transplantation (Day 1 vs 3 vs 7), the role
of dosage (dose-response curves using 3-4 dose levels), the role of
schedule (single vs multiple divided daily injections) and the
specificity of the treatment (pre-treatment with unlabeled
anti-PSMA to block targeting) is examined.
[0389] These in vivo studies are designed to address the maximum
tolerated dose of radiolabeled antibody, the activity of the
antibody, the optimal dosing schedule (single or multiple
injections), and the effect on tumor size. Successful completion of
this work enables determination of the feasibility of PSMA-targeted
alpha particle radioimmunotherapy (RIT) of prostate cancer and
identifies the optimal .sup.213Bi and/or .sup.225Ac-labeled
constructs to enter into clinical development.
CWR22Mouse Xenograft Model
[0390] The most promising anti-PSMA mAbs in unlabeled,
toxin-labeled and/or radiolabeled form are tested in the CWR22
human prostate cancer xenograft mouse model, (Wainstein, M. A. et
al. Cancer Res 54:6049-6052 (1994); Nagabhushan, M. et al. Cancer
Res 56:3042-3046 (1996); Pretlow, T. G. et al. J Natl Cancer Inst
85:394-398 (1993)). This model has many features of the human
condition including a dependence on androgens, a correlation
between measured levels of PSA in serum and tumor size, and
high-level expression of PSMA. Following androgen withdrawal, PSA
levels decrease to nearly undetectable levels and tumor volume
decreases. Later, the tumor regrows as an androgen-independent
neoplasm, manifest initially by a rise in PSA and later, measurable
tumor growth. After androgen withdrawal, tumors regrow at variable
time periods.
[0391] Four to six week old nude athymic BALB/c male mice are
obtained from the National Cancer Institute-Frederick Cancer Center
and maintained in pressurized ventilated caging. While
immunodeficient in many respects, these mice mediate wild-type
levels of ADCC and CML. The CWR22 tumor line is propagated in the
animals by the injection of minced tumor tissue from an established
tumor into the subcutaneous tissue of the flanks of athymic nude
mice together with reconstituted basement membrane (MATRIGEL,
Collaborative Research, Bedford, Mass.). To maintain serum androgen
levels, the mice are administered 12.5-mg sustained-release
testosterone pellets (Innovative Research of America, Sarasota,
Fla.) subcutaneously before receiving tumors. Three to four weeks
after inoculation, tumors of approximately 1.5.times.1.0.times.1.0
cm are measured. Androgens are withdrawn by surgical castration
under pentobarbital anesthesia and removal of the sustained-release
testosterone pellets. Tumor size is determined by caliper
measurements of height, width and depth. PSA values are performed
on the serum of the mice after tail bleeding using a Tandem-R PSA
immuno-radiometric assay (Hybritech, San Diego, Calif.).
[0392] Groups of five mice are injected with anti-PSMA mAb or a
similar isotype control mAb at dosages from 5-100 .mu.g to assess
anti-tumor activity. The effect of scheduling single doses vs.
multiple divided daily injections is also examined. Macroscopic
tumor volume and animal survival rates are recorded throughout the
experiments. Statistical differences between therapy groups are
determined using an analysis of variance (ANOVA) method and animal
survival are illustrated using Kaplan-Meier plots, with success
defined as a difference of p<0.05. Similarly, the efficacy of
"naked" mAbs is compared to that seen with .sup.90Y, .sup.177Lu,
.sup.213Bi and/or .sup.225Ac-labeled anti-PSMA constructs.
[0393] These in vivo studies are designed to address the maximum
tolerated dose of mAb, the activity of the antibody, the optimal
dosage and dosing schedule (single or multiple divided injections),
and the effect of treatment on tumor size. Successful completion of
this work will enable determination of the feasibility of
PSMA-targeted immunotherapy of prostate cancer and identification
of the optimal constructs to enter into clinical development.
Example 15
Investigation of Native PSMA Protein Conformation
[0394] Extraction of PSMA from the Cell Surface of LNCaP and 3T3
Cells
[0395] LNCaP or 3T3 cells were grown to confluency in a T150 cell
culture flask, detached using cell dissociation solution
(Mediatech, Herndon, Va.) and transferred to a 15 ml conical tube.
The cells were washed twice with PBS and resuspended with 2 ml of
M-PER.TM. Mammalian Protein Extraction Reagent (Pierce, Rockford,
Ill.). Following incubation for 10 min at 4.degree. C., cell debris
and insoluble aggregates were removed by centrifugation at 15,000
rpm for 30 min at 4.degree. C. The supernatant was transferred to a
cryogenic vial and stored at -80.degree. C. until further use.
Production of Recombinant, Soluble PSMA (rsPSMA)
[0396] The extracellular domain of PSMA (amino acids 44-750 of the
full-length protein, SEQ ID NO: 1) was obtained as a secreted
protein from a DXB11 Chinese hamster ovary (CHO) cell line, stably
transfected with a rsPSMA expression vector. The cells were grown
in a Celligen Plus 2.2 L Packed Bed Bioreactor (New Brunswick
Scientific, Edison, N.J.) in protein-free media. The Bioreactor was
operated in perfusion mode, and supernatant was collected
aseptically into collection bags maintained at 4.degree. C. The
protease inhibitor aprotinin was added to the harvest supernatant,
which was concentrated 25-fold prior to storage at -90.degree. C.
In some instances for purification, the concentrate was thawed and
purified using subsequent steps of Concanavalin A lectin affinity
chromatography and Butyl-SEPHAROSE hydrophobic interaction
chromatography or according to the steps shown below.
[0397] The purified rsPSMA protein is dimeric, and possesses folate
hydrolase enzymatic activity when tested according to published
procedures (Pinto et al., Clinical Cancer Research 2:1445, 1996)
and reacts with each of a panel of conformation-specific monoclonal
antibodies, indicating that rsPSMA adopts a native
conformation.
Purification of Recombinant, Soluble PSMA (rsPSMA)
[0398] Cell culture supernatants were concentrated 25-fold by
tangential flow ultrafiltration and adjusted to 35% saturation with
ammonium sulfate. Under these conditions, rsPSMA remains in the
supernatant. Precipitated proteins were removed by centrifugation
(20,000.times.g for 30 min, SS-34, Sorvall, Inc.) and the clarified
supernatant was applied to a Butyl-SEPHAROSE resin (BioRad,
Hercules, Calif.) followed by a wash with 35% ammonium sulfate in
neutral phosphate-buffered saline containing 1 mM Ca.sup.2+ and 0.5
mM Mg.sup.2+ (PBS+). rsPSMA eluted in the flow-through and wash
fractions of the column. The fractions containing the rsPSMA
protein were pooled, dialyzed into 10 mM sodium phosphate, pH 7.0,
and loaded onto a Ceramic Hydroxyapatite column (BioRad, Hercules,
Calif.). rsPSMA was eluted from the resin using 2M sodium chloride
in 10 mM sodium phosphate, pH 7.0. The fractions containing the
protein were pooled, dialyzed into 20 mM Tris, pH 7.5 containing 1
mM Ca.sup.2+ and 0.5 mM Mg.sup.2+, and applied to a Q650-SEPHAROSE
column (TosoHaas, Montgomeryville, Pa.). rsPSMA was eluted from the
resin with 150 mM NaCl in 20 mM Tris, pH 7.5 containing 1 mM
Ca.sup.2+ and 0.5 mM Mg.sup.2+. Monomeric and dimeric forms of
rsPSMA present after this step were separated using preparative
size exclusion chromatography on a SUPERDEX 200 resin (Amersham
Biosciences, Piscataway, N.J.) and PBS+ (containing 1 mM Ca.sup.2+
and 0.5 mM Mg.sup.2+) as the running buffer. Purified rsPSMA was
stored at -80.degree. C. in PBS+. Unless otherwise indicated, PSMA
monomers represent spontaneously dissociated protein recovered over
SEC rather than forcibly denatured material.
Polyacrylamide Gel Electrophoresis (PAGE) and Western Blotting of
the Different PSMA Proteins
[0399] For each individual PAGE analysis, 15 .mu.l of each cell
lysate and 5 .mu.l of the purified rsPSMA were used.
[0400] SDS-PAGE was performed using standard procedures. Samples
were prepared by boiling for 5 minutes in the presence of Laemmli
sample buffer (with or without the reducing agent dithiothreitol
[DTT]). Samples were then applied on a 4-15% Tris-Glycine gel
(BioRad, Hercules, Calif.). After electrophoresis for 1 h at 200V,
the proteins were transferred onto nitrocellulose (BioRad) and
analyzed by Western blotting.
[0401] The oligomeric nature of the different PSMA proteins was
analyzed using Blue Native PAGE (BN-PAGE). Each sample was diluted
with an equal volume of 2.times. BN-PAGE sample buffer (0.1M
MOPS/0.1M Tris/40% glycerol/0.1% Coomassie G-250) prior to loading
onto the gel. BN-PAGE was performed using 4-12% BisTris gels
(Invitrogen, Carlsbad, Calif.) and 50 mM MOPS/50 mM Tris, pH 7.7 as
running buffer. Coomassie Blue was omitted from the cathode buffer
to avoid interference with protein binding during the transfer of
the proteins onto nitrocellulose. Following electrophoresis for 2.5
hrs at 125V, the proteins were transferred onto a nitrocellulose
membrane (BioRad) and analyzed by Western blotting.
[0402] Western blotting was performed as follows: Subsequent to
transfer, the nitrocellulose membrane was blocked with 5% milk in
PBS/0.1% Triton X-100/0.02% SDS, which was also used for the
subsequent wash and antibody incubation steps. PSMA proteins were
detected using the anti-PSMA mAbs 3.1 or 3.9 (Progenics
Pharmaceuticals) as primary antibody and HRP-labeled anti-mouse IgG
as secondary antibody and 1 h incubation at room temperature. The
membranes were colorimetrically developed using chemiluminescence
(NEN Plus, Perkin Elmer Life Sciences, Boston, Mass.).
Analytical size exclusion chromatography (SEC) was performed using
a TSK G3000SW.sub.XL (TosoHaas, Montgomeryville, Pa.) column
equilibrated in PBS+. The column was calibrated using bovine serum
albumin (67 kDa), immunoglobulin G (150 kDa), ferritin (440 kDa)
and thyroglobulin (670 kDa) as standards.
Results
[0403] Both full-length PSMA and recombinant, soluble PSMA (rsPSMA)
migrated on reducing and non-reducing SDS-PAGE with a molecular
weight of .about.100 kDa (FIG. 5). Thus, like full-length PSMA,
rsPSMA is a monomer in the presence of denaturing agents, and no
disulfide or other covalent bonds are present to mediate
oligomerization. The result for full-length PSMA is in accordance
with prior observations (Israeli et al., U.S. Pat. No. 5,538,866;
Murphy et al., U.S. Pat. No. 6,158,508; Israeli, et al., Cancer
Research 54:1807, 1994; Troyer et al. Int. J. Cancer 62:552, 1995;
Troyer et al., The Prostate 30:233, 1997; Grauer et al., Cancer
Research 58:4787, 1998). In each of these reports, full-length PSMA
migrated as a major band of 100-120 kDa, with a minor (typically
<5% of the total PSMA protein) 180-200 kDa band observed in a
subset of reports (U.S. Pat. No. 6,158,508; Troyer et al., 1995;
Troyer et al., 1997). Troyer et al. (1995) describe the 180-200 kDa
species as being a noncovalently associated PSMA dimer that can be
disrupted with increasing concentrations of SDS detergent.
[0404] rsPSMA contains 94% (707 of 750) of the amino acids present
in full-length PSMA, and the two proteins were not clearly resolved
in this analysis, as expected.
[0405] SDS-PAGE allows the analysis of denatured proteins only. In
order to examine native proteins in their native state, other
techniques have to be employed, such as Blue Native PAGE (BN-PAGE).
BN-PAGE is used to determine the native molecular weight of
proteins and their noncovalent complexes (Schagger & v. Jagow,
Anal. Biochem. 199:223-231, 1991; Schagger et al., Anal. Biochem.
217:220-230, 1994). The dye Coomassie Blue G-250 binds to the
hydrophobic domains on the surface of most proteins, enhances
solubility, and introduces a charge shift on the native proteins
resulting in migration towards the anode at pH 7.5 irrespective of
the isoelectric point of the protein. Although the migration
velocity of proteins in BN-PAGE varies somewhat, the molecular mass
of proteins can be determined by their respective end points of
migration due to the decreasing pore size of the acrylamide
gradient present in the gels.
[0406] When analyzed by BN-PAGE, full-length PSMA (extracted from
LNCaP or 3T3 cells with nonionic detergents) as well as purified
rsPSMA migrate with a molecular weight of .about.190 kDa (FIG. 6A).
This surprising observation for full-length PSMA indicates that the
predominant form of cell-surface PSMA is a noncovalently associated
dimer. This unexpected result can be contrasted with that of
previous reports (U.S. Pat. No. 6,158,508; Troyer et al. 1995;
Troyer et al., 1997), where the PSMA dimer represents a minor
species in SDS-PAGE analyses. Presumably, the noncovalent PSMA
dimer is largely dissociated by boiling in the presence of the
denaturing detergent SDS.
[0407] Moreover, the result for the purified rsPSMA protein
indicates that the dimer is stabilized via interactions between
extracellular amino acids in addition to or exclusive of amino
acids in the transmembrane or intracellular segments, which are not
present in rsPSMA.
[0408] rsPSMA was subjected to analytical size exclusion
chromatography (SEC) as a second sizing method. When analyzed in
neutral PBS+ buffer, purified rsPSMA eluted as a single major peak
with an apparent molecular mass of 260 kDa (FIG. 6B), slightly
higher than expected. However, glycoproteins (such as rsPSMA) are
typically nonglobular in shape and run at higher apparent molecular
mass than standard SEC calibration proteins (Schulke, N., et al.
(2002) J. Virol. 76, 7760-7776). Therefore, an apparent molecular
mass of 260 kDa is consistent with the proposed homodimeric
structure of rsPSMA. In contrast, purified monomeric rsPSMA eluted
with an apparent molecular mass of 130 kDa. The studies demonstrate
that the extracellular domain of PSMA is sufficient for
dimerization, and the similarities between rsPSMA (amino acids
44-750) and PSM' (amino acids 58-750) suggest that the latter
protein is likely to dimerize as well.
Example 16
Homodimerization is Required for Enzymatic Activity
Enzyme Assays
[0409] Pteroyl .gamma.-glutamyl carboxypeptidase (folate hydrolase)
activity was determined by monitoring the cleavage of poly
.gamma.-glutamylated methotrexate as described (Pinto, J. T., et
al. (1996) Clin. Cancer Res. 2, 1445-1451) with the following
exceptions. Di-.gamma.-glutamylated methotrexate (MTXglu2) was used
as substrate and HPLC was used rather than capillary
electrophoresis. At the completion of the incubation (50 .mu.M
methotrexate di-gamma glutamate and 10 .mu.g/ml rsPSMA in pH 4.5
acetate buffer in a volume of 100 .mu.l for 2 hr at 37.degree. C.),
100 .mu.l of 0.5M Na.sub.2HPO.sub.4 was added to stop the reaction.
Samples were loaded at a flow rate of 1.25 ml/min through a
50.times.4.6 mm, 3 .mu.m PRISM reversed-phase column (Thermo
Hypersil-Keystone, Bellefonte, Pa.) with a PRISM 10.times.4-mm
guard column, eluted with 15% methanol in 85% 0.5M
K.sub.2HPO.sub.4, pH 7.0, and quantitated based on relative peak
area observed at a wavelength of 313 nm.
[0410] For NAALADase assays, rsPSMA was incubated with
N-acetyl-.alpha.-L-aspartyl-L-glutamate for 22 h at 37.degree. C.
in the presence of 20 mM sodium phosphate, 50 mM NaCl, 10 mM
ZnCl.sub.2, pH 7.1. Released L-glutamic acid was quantitated by
using a commercial kit (R-Biopharm, Marshall, Mich.);
2-(phosphonomethyl) pentanedioic acid and
Gly-Pro-7-amido-4-methylcoumarin were purchased from Sigma. Porcine
kidney dipeptidyl peptidase IV (DPP IV) was used according to the
manufacturer's instructions (Sigma).
Results
[0411] PSMA has been reported to possess folate hydrolase,
NAALADase, and DPP IV activities (Pinto, J. T., et al. (1996) Clin.
Cancer Res. 2, 1445-1451; Carter, R. E., et al. (1996) Proc. Natl.
Acad. Sci. USA 93, 749-753; Pangalos, M. N., et al. (1999) J. Biol.
Chem. 274, 8470-8483). The first two activities involve the
hydrolysis of a carboxyl-terminal peptide bond to liberate a
glutamic acid residue, whereas DPP IV cleaves downstream of an
amino-terminal Aaa-Pro dipeptide sequence. The folate hydrolase
activities of purified monomeric and dimeric forms of rsPSMA were
evaluated. Whereas the dimer demonstrated high-level folate
hydrolase activity, the monomer was essentially inactive (FIG. 7A).
In fact, the residual activity of the monomer could be attributed
to the residual amount (approximately 4%) of dimeric rsPSMA present
in the preparation. High-level folate hydrolase activity was also
observed for LNCaP cell lysates, consistent with prior observations
(Pinto, J. T., et al. (1996) Clin. Cancer Res. 2, 1445-1451).
Similarly, dimeric but not monomeric forms of rsPSMA possessed
high-level NAALADase activity (FIG. 7B), which was abrogated by
using 5 nM of the inhibitor 2-(phosphonomethyl)pentanedioic acid.
Neither monomer nor dimer demonstrated DPP IV activity under
conditions where porcine DPP IV efficiently hydrolyzed the
substrate Gly-Pro-7-amido-4-methylcoumarin. This is consistent with
the results reported by Barinka et al. (Barinka, C., et al. (2002)
J. Neurochem. 80, 477-487), who similarly failed to confirm the DPP
IV activity previously reported for PSMA (Pangalos, M. N., et al.
(1999) J. Biol. Chem. 274, 8470-8483).
Example 16
Dissociation of PSMA Multimers
[0412] PSMA is a putative zinc metalloprotease, and site-directed
mutagenesis of amino acids implicated in zinc binding results in a
profound loss of enzymatic activity (Speno et al., Molecular
Pharmacology, 55:179, 1999). These amino acids include His-377,
Asp-387, Glu-425, Asp-453 and His-553. Ethylenediaminetetraacetic
acid (EDTA) is a strong chelating agent for Zn.sup.2+ and other
divalent cations, and thus has the potential to remove Zn.sup.2+ or
other coordinate divalent cations from PSMA. We have determined
that EDTA treatment causes the PSMA homodimer to dissociate into
monomeric subunits. Similar results can be expected for other
agents that possess similar chelating properties, such as
ethyleneglycol-bis(beta-aminoethyl ether) (EGTA).
[0413] The purified rsPSMA protein was incubated with or without 10
mM EDTA for 16 hr at 4.degree. C. and then analyzed by BN-PAGE.
Under these conditions, the EDTA-treated protein was monomeric,
whereas rsPSMA remained dimeric in the absence of EDTA. Although
the dissociation of the PSMA dimer into monomer was essentially
complete, any residual dimeric protein can be removed if desired by
gel filtration, ultracentrifugation or other size-based separation
methods that are well-known to those skilled in the art.
Example 17
Methods for Identifying Promoters of PSMA Dissociation
[0414] Compounds are screened for the ability to promote
dissociation of PSMA dimers using a method that includes:
[0415] (a) contacting a PSMA dimer with a compound under conditions
that do not promote dissociation of the PSMA dimer in the absence
of the compound;
[0416] (b) measuring the amount of PSMA monomer; and
[0417] (c) comparing the amount of PSMA monomer measured in the
presence of the compound with that observed in the absence of the
compound.
[0418] An increase in the amount of PSMA monomer measured in the
presence of the compound indicates that the compound is capable of
promoting dissociation of the PSMA dimer.
[0419] In a further embodiment, compounds are screened for the
ability to promote dissociation of PSMA dimers using a method that
includes:
[0420] (a) contacting a PSMA dimer with a compound under conditions
that do not promote dissociation of the PSMA dimer in the absence
of the compound;
[0421] (b) measuring the amount of PSMA dimer, and
[0422] (c) comparing the amount of PSMA dimer measured in the
presence of the compound with that observed in the absence of the
compound.
[0423] A decrease in the amount of PSMA dimer measured in the
presence of the compound indicates that the compound is capable of
promoting dissociation of the PSMA dimer.
[0424] In a further embodiment, compounds are screened for the
ability to promote dissociation of PSMA dimers using a method that
includes:
[0425] (a) contacting a PSMA dimer with a compound under conditions
that do not promote dissociation of the PSMA dimer in the absence
of the compound;
[0426] (b) measuring the amounts of PSMA monomer and PSMA
dimer,
[0427] (c) calculating a ratio of PSMA monomer to PSMA dimer;
and
[0428] (d) comparing the ratio obtained in (c) with that obtained
in the absence of the compound.
[0429] An increase in the ratio measured in the presence of the
compound indicates that the compound is capable of promoting
dissociation of the PSMA dimer.
Example 18
Cell Surface PSMA Binding Studies
Flow Cytometry
[0430] Parent 3T3 cells or PSMA-expressing 3T3 cells
(2.times.10.sup.5 cells per condition) were washed in PBS and
incubated with PBS containing goat serum (10% v/v) for 20 minutes
on ice to block non-specific binding sites. Anti-PSMA monoclonal
antibodies (unpurified form in supernatants or purified mAbs) were
added in serial dilutions to cells in 100 .mu.l PBS and incubated
on ice for 30 minutes. Control anti-human IgG (Caltag, Burlingame,
Calif.) was used to establish background binding. After two washes
in PBS, the cells were incubated with anti-human IgG (BD
Pharmingen, San Diego, Calif.) for 30 minutes on ice. Cells were
washed twice in PBS, resuspended in 250 .mu.l PBS and analyzed by
flow cytometry using a FACScan machine (Becton Dickinson, Franklin
Lakes, N.J.) and CellQuest software. Viable cells were gated by
forward scatter and side scatter parameters, and binding was
quantified using histogram plots of mean fluorescence intensity
(MFI) levels.
[0431] Anti-PSMA mAbs XG-006 (PTA-4403 and PTA-4404, heavy and
light chain plasmids), XG-051 (PTA-4407 and PTA-4408), 4.4-0.1
(PTA-4360; 4.40, 4.40.1 and 4.40.2 are the same antibody that
represent different stages of subcloning the hybridoma), 4.49.1,
4.292.1 (PTA-4390) and 4.304.1 were found to avidly bind to cell
surface PSMA (FIG. 27).
Maximal Binding
[0432] Flow cytometry data (mean fluorescence intensity v. antibody
concentration) were transposed and plotted using EXCEL software
(Microsoft, Redmond, Wash.). Results from representative
experiments of at least three determinations are depicted in FIGS.
28A-28C. Binding was compared by calculation of 50% effective
concentration (EC50) using the Forecast function in EXCEL. The EC50
value represents the concentration of antibody required for
half-maximal binding.
[0433] Anti-PSMA mAbs 10.3 (PSMA 10.3) and XG-006 were found to
bind to 3T3-PSMA cells and not 3T3 cells (FIG. 28A). Antibody (26
nM) was added to cells, which were analyzed by flow cytometry.
Binding to cell-surface PSMA using serial dilutions of anti-PSMA
mAb-containing culture supernatants of XG-006, 4.304.1, XG-026
(PTA-4405 and PTA-4406) and 4.49.1 also was demonstrated (FIG.
28B). Binding to cell-surface PSMA using serial dilutions of
purified anti-PSMA mAbs XG-006 and 10.3 is represented by FIG.
28C.
Example 19
Cytotoxicity of Toxin-Labeled Antibody
[0434] PSMA-3T3, LNCaP, and/or C4-2 cells (and control cell lines
3T3 and PC3 that do not express PSMA) were plated at 2,500
cells/100 .mu.L/well in 96-well microplates (Falcon) and were
incubated overnight at 37.degree. C. in the presence of 5%
CO.sub.2. The media used for PSMA-3T3 (and 3T3) and LNCaP (and C4-2
and PC3) was DMEM or RMPI 1640, respectively, containing 2 mM
L-glutamine, 10% FBS, and 1% penicillin-streptomycin. 50 ng (in 50
.mu.L) of Mab-Zap or Hum-ZAP (Advanced Targeting Systems, San
Diego, Calif.) in medium was added in each well. Mab-Zap and
Hum-Zap are goat anti-mouse IgG antibody or goat anti-human IgG
antibody covalently linked to saporin, the most potent of the plant
ribosome-inactivating proteins (RIP) from the seeds of the plant
Saponaria officinalis. Saporin induces cell death by apoptosis
(Bergamaschi, G., Perfetti, V., Tonon, L., Novella, A., Lucotti,
C., Danova, M., Glennie, M J., Merlini, G., Cazzola, M. Saporin, a
ribosome-inactivating protein used to prepare immunotoxins, induces
cell death via apoptosis. Br J Haematol 93, 789-94. (1996)). The
Mab-Zap did not bind to or internalize in cells in the absence of
an appropriate primary antibody.
[0435] Murine 3.9, 5.4, mJ591 (ATCC# HB-12126) and human 006, 4.40,
4.304 anti-PSMA antibodies (and control IgG antibodies) were added
into plates at different concentrations to bring the total volume
to 200 .mu.L in triplicate. The plates were kept cold on ice for at
least 30 min to maximize Map-Zap or Hum-Zap binding to PSMA
antibodies before internalization. The plates were incubated for 2
days and then the medium was changed and incubated for another 2
days. After 4 days incubation, the medium was withdrawn and fresh
medium containing 10% Alamar Blue (20 .mu.L, Bioscience, Camarillo,
Calif.) was added into each well and incubated for 2 hrs. A
CYTOFLUOR plate reader was used to measure fluorescence in 96-well
plates at wavelengths of 530 nm excitation and 590 nm emission.
Internalization of toxin was mediated by anti-PSMA antibodies. The
cell kill is illustrated in FIG. 29 on C4-2 cells and in FIG. 30 on
PSMA-3T3 cells.
[0436] Human 4.304 anti-PSMA antibody was directly conjugated with
saporin (Wrenn et al., Brain Res. 740:175-184, 1996), and its
cytotoxicity was demonstrated using a similar protocol as described
above (see FIG. 31).
Example 20
Immunoreactivity
[0437] PSMA-3T3, LNCaP and C4-2 were used as PSMA expressing cell
lines and 3T3 was used as a control cell line not expressing PSMA.
The cells were blocked with 10% goat serum on ice to reduce
non-specific binding in this assay.
[0438] A small amount (1-5 ng) of labeled mAb was added into a cell
pellet of 10 million cells and incubated at 0.degree. C. (on ice)
with gentle mixing. After a 1 hour incubation, the cells were
collected by centrifugation and the supernatant containing unbound
mAb was transferred to a fresh cell pellet for an additional 1 hour
incubation at 0.degree. C. Both sets of cells were centrifuged and
washed twice with cold PBS. The cell pellets, supernatant and wash
fractions were counted for radioactivity. Immunoreactivity is
defined as the amount of radioactivity in the cell pellets divided
by the total radioactivity in the cell pellets, supernatant and
wash fractions. These data are shown below in Table 3.
TABLE-US-00003 TABLE 3 Immunoreactivity of .sup.111In Radiolabeled
Antibody on PSMA Expressing Cells Radiolabeled mAb Immunoreactivity
(%) Cell line .sup.111In 4.304 92.6 (1.4) PSMA-3T3 (3T3) 92.6
PSMA-3T3 91.4 (1.7) PSMA-3T3 (3T3) 89.1 LNCaP 92.4 C4-2 Average =
91.6 .+-. 1.5 .sup.111In 4.40 87.7 (0.5) PSMA-3T3 (3T3) 86.8
PSMA-3T3 89.4 (1.5) PSMA-3T3 (3T3) Average = 88.0 .+-. 1.3
.sup.111In mJ591 58.5 PSMA-3T3 54.9 (1.1) PSMA-3T3 (3T3) Average =
56.7 .+-. 2.5 .sup.111In 3.9 88 LNCaP 87 C4-2 89 (2) PSMA-3T3 (3T3)
95.3 (0.5) PSMA-3T3 (3T3) 88.6 PSMA-3T3 84.8 C4-2 89.3 PSMA-3T3
Average = 88.6 .+-. 3.2
[0439] Antibodies 4.40, 4.304 and mJ591 were conjugated to the
bifunctional chelate CHX-A''-DTPA and antibody 3.9 was conjugated
to C-DOTA.
[0440] Immunoreactivity of .sup.225Ac radiolabeled antibody (026
and 4.40) was also assessed with a methodology similar to that
described above for the .sup.111In labeled antibodies. .sup.225Ac
was chelated with the bifunctional DOTA at 50.degree. C. for 30
minutes. The chelated .sup.225Ac was then conjugated to antibodies
026 and 4.40 at 35.degree. C. for 30 minutes. Unconjugated
.sup.225Ac was removed by a PD10 column (Amersham Biosciences,
Picataway, N.J.). The immunoreactivity of the radiolabeled
antibodies was then determined. The data are presented below in
Table 4. In addition to the assessment of the immunoreactivity of
these antibodies, the yield of the labeling procedure was also
assessed, and these data are also provided below in Table 4.
TABLE-US-00004 TABLE 4 Yield and Immunoreactivity of .sup.225Ac
Radiolabeled Antibody Antibody Yield Immunoreactivity 026 9.3 +/-
0.8 (n = 2) 61.3 +/- 1.1 (n = 2) 4.40 14.3 +/- 0.6 (n = 2) 78.1 +/-
0.1 (n = 2)
Example 21
Competitive Binding Assay to Identify Binding Epitopes
[0441] To identify whether a given group of mAbs recognize distinct
or overlapping epitopes on PSMA, competition binding assays were
performed with .sup.111In radiolabeled antibodies. 2.times.10.sup.5
cells (100 L) of PSMA-3T3 were plated into 96-well microplates, and
antibodies 4.40, 4.304 and mJ591 (100 .mu.L) at different
concentrations (series dilution) were added. The cells were
incubated at 0.degree. C. for 30 min. 20 .mu.L of In-111
radiolabeled CHX-A''-DTPA antibody constructs were added into each
well. After a 2 hour incubation on ice for competition binding, the
cells were washed 5 times using cold PBS. The cells containing
bound .sup.111In antibodies were recovered from microplates into
test tubes and counted in a gamma counter.
[0442] Results detailed in FIGS. 32 show that mJ591 blocked
.sup.111In 4.40 binding to PSMA-3T3 cells and did not block
.sup.111In 4.304. In addition, 4.40 and 4.304 did not block each
other. Unmodified antibodies 4.304 and mJ591 were also used to
compete with .sup.111In radiolabeled mJ591. Human 4.304 did not
compete with .sup.111In mJ591 for binding to PSMA-3T3 (FIG.
33).
Example 22
Binding Affinity Using BIACORE 3000
[0443] To determine the kinetics and affinity of the antibodies,
the antibodies in crude supernatants, in purified form and in
bifunctional chelate modified forms were analyzed using a BIACORE
3000 instrument (Biacore Inc., Piscataway, N.J.). BIACORE 3000 is a
fully automated surface plasmon resonance (SPR)-based biosensor
system that is designed to provide real-time kinetic data from
assay formats that require no tags or labeling of compounds for
biomolecular interactions. It is ideal for screening crude
supernatants.
[0444] The streptavidin-coated sensor chips (SA chips, Biacore,
Inc.) were used to capture biotinylated anti-human IgG antibody
(Sigma, St. Louis, Mo.). The entire sensor chip surface was
conditioned with five injections of conditioning solution (1 M
NaCl, 50 mM NaOH) and equilibrated with PBS buffer containing
0.005% polysorbate 20. Two to three thousand resonance units (RU)
of biotinylated anti-human IgG antibody (Sigma) were immobilized
onto the SA chip followed by an injection of regeneration buffer
(glycine-HCl, pH 2.2). Antibodies in supernatants were diluted to 2
.mu.g/mL in PBS buffer and captured onto one anti-human IgG flow
cell, while isotype-matched control human antibody (Sigma) was
similarly captured on a second flow cell. rsPSMA at different
concentrations in PBS buffer was flowed over the cells at 30
.mu.L/min for 3 min in an "association phase" followed by a
"dissociation phase" for 10 min. SPR was monitored and displayed as
a function of time. For each antibody at one concentration, the
chip was regenerated and equilibrated. Examples of the analysis of
antibody PRGX1-XG-006 in association phase and dissociation phase
at different concentrations of rsPSMA from 100 nM to 6.25 nM are
shown in FIG. 34. Thermodynamic and kinetic rate constants of
binding were calculated using the BIACORE Evaluation software. For
example, the affinity of XG-006 antibodies in a supernatant to
rsPSMA was determined to be 4.92.times.10.sup.-10 M with a K. of
1.3.times.10.sup.5 M.sup.-1 s.sup.-1 and a K.sub.d of
6.4.times.10.sup.-5 s.sup.-1. Selective data for several human PSMA
antibodies in crude supernatant, purified form, and modified with
bifunctional chelate is listed in Table 5 for comparison.
[0445] Binding activity of .sup.111In radiolabeled antibodies was
determined by Scatchard analysis of binding data obtained using
PSMA-expressing cells (LNCaP, C4-2, PSMA-3T3 and parental 3T3 as a
control). The experimental procedures and methods of data analysis
have been described previously (Scheinberg, D. A. et al. Leukemia
3: 440-445 (1991).
TABLE-US-00005 TABLE 5 Kinetic Rate Constants of Antibodies in
Crude Supernatant, Purified, Bifunctional Chelate Modified Forms
along with KD Determined Using .sup.111In Radiolabeled Scatchard
Analysis Ka Antibodies (M.sup.-1, s.sup.-1) Kd (s.sup.-1) KD
(M.sup.-1) Avg KD 006 Supernatant 1.30E+05 6.40E-05 4.92E-10
4.92E-10 Purified 006-1 2.94E+05 1.37E-04 4.66E-10 Purified 006-2
2.26E+05 1.27E-04 5.62E-10 5.14E-10 4.40 Supernatant 2.10E+05
1.25E-04 5.95E-10 5.95E-10 Purified 4.40-1 2.54E+05 1.52E-04
5.98E-10 Purified 4.40-2 2.43E+05 2.37E-04 9.75E-10 7.87E-10
CHX-4.40-1 2.57E+05 1.60E-04 6.23E-10 CHX-4.40-2 2.47E+05 1.55E-04
6.28E-10 6.25E-10 IN-111CHX-4.40-1 4.44E-09 IN-111CHX-4.40-2
4.95E-09 4.70E-09 4.304 Supernatant 1.40E+05 1.25E-04 8.93E-10
8.93E-10 Purified 4.304-1 8.31E+04 1.20E-04 1.44E-09 Purified
4.304-2 1.06E+05 6.33E-05 5.97E-10 1.02E-09 CHX-4.304-1 6.19E+04
1.21E-04 1.95E-09 CHX-4.304-2 6.79E+04 1.49E-04 2.19E-09 2.07E-09
IN-111CHX-4.304-1 9.63E-09 IN-111CHX-4.304-2 5.97E-09 7.80E-09 10.3
Supernatant 1.90E+05 3.63E-04 1.91E-09 1.91E-09 Purified 10.3-1
3.28E+05 6.32E-05 1.93E-10 Purified 10.3-2 2.96E+05 6.43E-05
2.17E-10 2.05E-10
[0446] A comparison of the fully human antibodies 4.40.1, 4.49.1,
051 and 006 and the murine antibody 3.9 was performed by BIACORE.
For each antibody for comparison, response was normalized to 100
RU. The graph of time vs. response difference for these antibodies
is given in FIG. 35. The binding affinities for these antibodies
were determined to be 6.1, 6.7, 5.8, 4.8 and
13.7.times.10.sup.-10M, respectively.
Example 23
Characterization of Cell Lines for In Vitro And In Vivo Studies
[0447] Results from a Scatchard analysis using .sup.111In labeled
anti-PSMA antibody 3.9 are represented in FIG. 36. Transfected
murine 3T3 cells express >1 million copies of PSMA per cell,
LNCAP cells (androgen dependent human prostate cancer cell line)
express 0.64 million copies, while C4-2 cells (androgen
independent) express 0.25 million copies per cell. The affinity of
3.9 for cell surface PSMA is 6.4 nM for PSMA-3T3, 4.0 nM for LNCAP
and 3.3 nM for C4-2 (4.6 nM is the average of these data).
[0448] A summary of the analyses of crude supernatants for the
human anti-PSMA antibodies is given in Table 6 below.
TABLE-US-00006 TABLE 6 Characterization of Anti-PSMA Monoclonal
Antibodies Binding to 3T3- Ab Conc PSMA (FACS) Biacore studies
(.mu.g/mL) AVG Anti- KD, Ka, Kd, Lysate PGNX Max AVG C4.2 PSMA M-1
M-1s-1 s-1 Supernatant PGNX EIA FACS binding EC50 FACS Western
(.times.10.sup.-10) (.times.10.sup.5) (.times.10-5) PRGX1- 4.7
.sup. ND.sup.1 ND 148 2.4 ND .sup. Conf..sup.2 2.0 1.5 2.9 XG1-026
4.4.1 4.7 0.08 7 8 ND 5.2 Conf. 4.2 2.3 9.7 PRGX1- 1.8 0.39 114 183
3.4 9.5 Conf. 4.8 1.3 6.4 XG1-006 PRGX1- 3.5 0.48 83 202 2.0 9.9
Conf. 5.8 1.4 8.2 XG1-051 4.40.1 4.3 0.33 53 163 2.3 10.8 Conf. 6.1
2.1 12.5 4.49.1 2.6 0.36 362 162 0.9 16.2 Conf. 6.7 3.1 20.7
4.292.1 2.7 0.18 75 195 6.0 9.2 Conf. 6.8 1.2 8.5 4.304.1 4.1 0.39
92 184 9.1 8.4 Conf. 8.7 1.4 12.5 4.232.1 2.4 0.49 97 138 2.7 6.0
Linear.sup.3 9.4 1.5 13.8 4.153.1 5.9 0.29 279 182 5.3 14.8 Conf.
9.5 1.2 11.8 4.333.1 2.9 0.18 82 168 3.1 6.6 Conf. 11 0.7 8.5
PRGX1- 3.9 0.45 392 227 6.0 12.4 Conf. 16 0.6 10.4 XG1-077 10.3 8.5
1.06 ND ND ND ND ND 19 1.9 36.4 pure 10.3 0.44 130 181 7.5 ND Conf.
ND 4.7 4.22.1 2.8 0.08 7 ND ND 4.7 ND 20 1.7 33 4.248.1 3.5 0.37 7
ND ND 4.1 Conf. 27 1.0 28 4.54.1 10 0.14 267 162 3.9 13.6 ND 30 1.9
56 4.7.1 5 0.23 156 141 1.6 10.2 Conf. 32 1.7 56 4.78.1 5.3 0.00
205 118 1.0 7.9 Conf. 53 2.4 125 4.48.1 4.9 0.06 14 ND ND 7.7 ND 62
0.9 59 4.209.1 3.5 0.22 60 ND ND 6.7 ND 142 0.9 125 4.177.1 1.1
0.15 236 174 2.4 10.6 ND 155 0.6 93 4.152.1 3.4 0.38 81 85 4.0 7.5
ND 163 0.8 126 4.28.1 4.2 0.04 112 155 4.2 11.3 ND 167 1.2 192
4.16.1 5.3 0.00 8 ND ND 7.8 ND 177 1.8 313 4.360.1 1.5 0.02 112 130
2.2 7.9 ND 197 1.0 201 4.288.1 15.4 0.02 67 141 4.1 6.5 ND 198 1.3
257 4.219.2 0.5 0.34 69 ND ND 5.9 ND ND PRGX1- 6.5 ND ND 71 7.9 ND
ND No Binding XG1-069 Murine 3.9 13.7 0.7 9.7 Control 6.34 2.24
14.2 .sup.1ND = not determined .sup.2conf. = conformational epitope
.sup.3linear = linear epitope
Example 24
Cytotoxicity of Radiolabeled Antibody
[0449] The in vitro cytotoxicity of .sup.225Ac labeled anti-PSMA
antibody (4.40 and 026) was determined using methodology similar to
that used in Example 19. Prostate cancer cells (100 .mu.L of C4-2,
LNCaP, and PC3 cells at a concentration of 2.times.10.sup.4
cells/mL) were placed into separate wells of a 96 well microplate.
For tests with the 026 antibody, C4-2 and PC3 cells were placed
into separate wells of a 96 well microplate. After overnight
incubation, the cells were treated with .sup.225Ac labeled human
anti-PSMA antibody at different concentrations for over 4 days.
Cell cytotoxicity was quantified using Alamar Blue (Biosource
International, Camarillo, Calif.).
[0450] FIG. 37 shows a plot of cell survival vs. .sup.225Ac
activity concentration using .sup.225Ac labeled 4.40 antibody. The
EC50 for PSMA expressing cells (C4-2 and LNCaP) was <2 nCi/mL.
However, the EC50 was 420 nCi/mL for PC3 cells, which do not
express PSMA on the cell surface. Therefore, the .sup.225Ac labeled
human anti-PSMA 4.40 antibody shows >200-fold selectivity in
killing PSMA expressing prostate cancer cells (C4-2 and LNCaP) vs.
control cells (PC3).
[0451] FIG. 38 shows a plot of cell survival vs. .sup.225Ac
activity concentration using .sup.225Ac labeled 026 antibody. The
.sup.225Ac labeled human anti-PSMA 026 antibody shows >50-fold
selectivity in killing PSMA expressing prostate cancer cells (C4-2)
vs. control cells (PC3).
Example 25
Cytotoxicity of .sup.225Ac Labeled Antibody vs. Control
Antibody
[0452] The in vitro cytotoxicity of .sup.225mAc labeled anti-PSMA
antibody was determined using methodology similar to that used in
Example 19 and Example 24 above. Human prostate cancer cells (100
.mu.L of C4-2 and LNCaP cells at a concentration of
2.times.10.sup.4 cells/mL) were placed into separate wells of a 96
well microplate. After overnight incubation, the cells were treated
with .sup.225Ac labeled human anti-PSMA 026 antibody at different
concentrations for 4 days. Cell cytotoxicity was quantified using
Alamar Blue (Biosource International, Camarillo, Calif.). Human IgG
(HuIgG) was used as a control. The cytotoxicity of an anti-PSMA mAb
026 "2 hour wash" was also determined. A 2 hour wash means that the
cells were incubated with .sup.225Ac labeled antibody for 2 hours.
After 2 hours, the media was removed and fresh media was added for
the 4 day incubation.
[0453] FIG. 39 shows a plot of cell survival vs. the .sup.225Ac
activity concentration for both C4-2 and LNCaP cells using
radiolabeled mAb 026, mAb 026 2 hour wash and HuIgG. .sup.225Ac
labeled mAb 026 showed an IC50 of <1 nCi/mL. Therefore, the mAc
labeled human anti-PSMA 026 antibody showed >50-fold selectivity
in killing the prostate cancer cells vs. the control antibody.
Example 26
Cytotoxicity of .sup.225Ac Labeled Antibody vs. Control Antibody
Evaluated by .sup.3H Thymidine Incorporation
[0454] Human prostate cancer cells (C4-2) in a 96 microplate were
treated with .sup.225Ac labeled mAbs at different concentrations
for 4 days. Cell survival was assessed using .sup.3H thymidine
incorporation (Nikula, T. K, et al. J. Nucl. Med. 40: 166-176,
1999).
[0455] FIG. 40 shows a plot of cell survival vs. the .sup.225Ac
activity concentration for C4-2 cells using radiolabeled mAb 026
and control mAb (HuM195). The IC50 was 0.12 nCi/mL using .sup.225Ac
labeled 026 vs. 13 nCi/ml with the control mAb (HuM195). The
radiolabeled 026 antibody, therefore, showed >100-fold
selectivity in killing the PSMA expressing C4-2 cells vs. the
control antibody.
Example 27
In Vivo Radioimmunotherapy with .sup.177Lu Labeled Antibodies
[0456] Athymic nude mice from the National Cancer Institute were
implanted subcutaneously with 2.times.10.sup.6 PSMA-3T3 cells.
After measurable tumors appeared at day 7 post implantation, the
mice were treated by injection with either a single 250 .mu.Ci dose
human anti-PSMA antibody 4.40 or 4.304 labeled with .sup.177Lu
(University of Missouri Research Reactor), or were injected with
buffer only as control. The tumor size of individual animals was
measured using an electronic caliper. FIG. 41 shows a plot of the
median tumor size in each group over time. Tumor growths were
substantially reduced in .sup.177Lu antibody treated groups
compared to the control group.
Example 28
In Vivo Biodistribution Study with .sup.177Lu Labeled
Antibodies
[0457] Athymic nude mice from the National Cancer Institute (male,
approximately 6 weeks old) were injected subcutaneously with
4.times.10.sup.6 PSMA-3T3 cells and 2.8.times.10.sup.6 3T3 cells in
0.2 mL in the right and left flank of each animal, respectively.
Anti-PSMA antibodies 006, 026, mJ591 and HuIgG (control) modified
with CHX-A''-DTPA were labeled with .sup.177Lu. FIG. 42 shows the
radio-HPLC profile of the radiolabeled antibodies as well as the
cell-based immunoreactivity performed as quality control. On day 6
after tumor implantation, .sup.177Lu labeled antibodies (10 .mu.Ci
and 1 .mu.g in 0.15 mL) were injected retro-orbitally. The animals
were randomized before antibody injection. Mice (30 per antibody, 5
per time point) were sacrificed at different times (days 0.17, 1,
2, 4, 7 and 12). Tumors and individual organs (PSMA+ tumor,
PSMA-tumor, blood, liver, kidneys, spleen, lungs, heart, bone,
muscle, carcass) were taken and weighed. Activity in each organ
along with standards prepared from injection solutions were counted
using a multi-channel gamma counter.
[0458] Results of this study show that .sup.177Lu labeled
antibodies specifically bound to tumors expressing PSMA in vivo in
the animal model. The percent injected dose per gram of tissue (%
ID/g) was calculated and plotted over time for the different
antibodies in the PSMA+ and PSMA- tumors (FIG. 43A). PSMA specific
tumor targeting (ratio of PSMA+/PSMA- tumor uptake) is provided in
FIG. 43B.
[0459] FIG. 44 shows the percent activity in the tumors with the
various radiolabeled antibodies (006, 026, mJ591 and HuIgG) over
time (% tumor retention vs. total body retention). The data again
illustrate the specificity by which the radiolabeled antibodies
target the PSMA expressing tumors. FIG. 44A shows the activity over
time in the PSMA+ tumors while FIG. 44B shows the percent activity
over time in the PSMA-tumors for the different antibodies. FIG. 45
shows the data for normal organ (blood, liver, kidneys, spleen,
lungs, bone, heart and muscle) uptake (% ID/g) plotted over
time.
Example 29
In Vivo Therapeutic Efficacy of .sup.177Lu Radiolabeled
Antibodies
[0460] Athymic nude mice from the National Cancer Institute (male,
approximately 6 weeks old) were injected subcutaneously with
4.times.10.sup.6 PSMA-3T3 cells and 2.8.times.10.sup.6 3T3 cells in
0.2 mL in the right and left flank of each animal, respectively.
.sup.177Lu labeled mAb 026 (0 .mu.Ci, n=5; 300 .mu.Ci and 10 .mu.g,
n=9; and 400 .mu.Ci and 13.5 .mu.g, n=5) were injected into the
mice on day 6 after tumor implantation. Animals were weighed and
tumors were measured over time. Tumor size (mm.sup.3) was
calculated using the formula: length.times.(width).sup.2/2. Mice
were sacrificed if tumor size reached 1000 mm.sup.3. Animal
survival was also assessed, and the Kaplan-Meier plot was
created.
[0461] The results of the study show that treatment decreased tumor
size and increased survival in the mice. FIG. 46A shows the tumor
size in the mice treated with the radiolabeled antibodies
(.sup.177Lu labeled mAb 026) at all three dose levels. The mice
treated with 300 .mu.Ci and 400 .mu.Ci had consistently smaller
tumors than the mice in the control group (0 .mu.Ci). FIG. 46B
shows that the mice treated with 300 .mu.Ci and 400 .mu.Ci had to
increased survival relative to the control mice. Median survival
was increased by 2.4-fold in mice treated with 300 .mu.Ci and
3.5-fold in mice treated with 400 .mu.Ci using time after
treatment. Treatment with 400 .mu.Ci was found to be non-toxic.
Additionally, at the end of the experiment (48 days after tumor
implantation), one animal from each treated group remained PSMA-3T3
tumor free but had large 3T3 tumors.
Example 30
Binding of Antibodies to rsPSMA Dimer and Monomer
[0462] A Biacore 3000 instrument was used to monitor, in real time,
binding of rsPSMA dimer and monomer to anti-PSMA mAbs. Antibodies
were immobilized at approximately 10,000 resonance units to CM5
sensor chips according to the manufacturer's instructions for amine
coupling (Biacore, Inc., Piscataway, N.J.). A reference surface of
isotype-matched antibody of irrelevant specificity was used as a
background control. Binding experiments were performed at
25.degree. C. in PBS buffer with 0.005% [vol/vol] Surfactant P20.
Purified rsPSMA dimer (50 nM) or monomer (100 nM) was passed over
control and test flow cells at a flow rate of 5 .mu.L/min. The
sensor surface was regenerated with two pulses of 20 nM HCl.
[0463] FIGS. 47 and 48, respectively, show that anti-PSMA mAbs 006
and 026 bind preferentially to the rsPSMA dimer rather than the
rsPSMA monomer. Anti-PSMA antibodies 4.40 and mJ591, however, were
shown to bind both the rsPSMA dimer and monomer at significant
levels (FIGS. 49 and 50, respectively). This study illustrates that
anti-PSMA mAbs 006 and 026 are PSMA dimer-specific antibodies and
bind dimer-specific epitopes on PSMA. The results also indicate
that the native conformation of PSMA is a homodimer, and that the
monomer possesses a partially denatured conformation or exposes
epitopes located at the dimer surface and/or dimer interface that
are not accessible in the dimer.
Example 31
Immunization with rsPSMA Dimer Preparations
Immunization
[0464] BALB/c mice were immunized by subcutaneous injection at days
0, 7, 14, and 42 with either 5 .mu.g clinical rsPSMA lot #4019-C001
(75% dimer/25% monomer) or 5 .mu.g rsPSMA batch #TD045-003 run
1/peak 2 (100% monomer) on alum (250 .mu.g per dose, Sigma) or
adjuvanted with 50 .mu.alhydrogel per dose. Serum was drawn 10 days
after the fourth immunization and analyzed by enzyme-linked
immunoassay (EIA) and flow cytometry.
EIA
[0465] rsPSMA lot #4019-C001 or rsPSMA batch #TD045-003 run 1/peak
2 was passively adsorbed to 96-well microtiter plates. Remaining
binding sites on the plate were blocked with a PBS/Casein/Tween 20
buffer. Serially diluted mouse serum or controls were added and
bound antibody was detected using a goat anti-mouse IgG antibody
conjugated to alkaline phosphatase. The EIA was developed with the
substrate pNPP which produces a color change that is directly
proportional to the amount of anti-PSMA antibody bound. Absorbance
was read at 405 nm with a correction of 620 nm. Antibody titer was
defined as the highest dilution of mouse serum yielding a blank
corrected absorbance of 0.1. Immune mouse serum with a known
anti-PSMA titer or normal mouse serum with no anti-PSMA reactivity
was used as controls.
Flow Cytometry Analysis
[0466] PSMA-3T3 cells were incubated with 200 .mu.L of immune serum
at a dilution of 1/50 in PBS with 0.1% sodium azide on ice for 30
minutes. Immune mouse serum with known anti-PSMA titer or normal
mouse serum with no anti-PSMA reactivity was used as controls. The
cells were washed twice with PBS with 0.1% sodium azide and
incubated for 30 minutes on ice with FITC-conjugated goat
anti-mouse IgG. Cells were washed once, resuspended in PBS with
0.1% sodium azide and subjected to flow cytometric analysis on
FACScaliber (Becton Dickinson).
Results
[0467] 5/5 mice immunized with rsPSMA lot #4019-C001 showed an
anti-PSMA antibody response by EIA. Antibody titer was similar for
assay plates coated with rsPSMA lot #4019-C001 (75% dimer/25%
monomer) and assay plates coated with rsPSMA batch #TD045-003 run
1/peak 2 (100% monomer). Median response for the group was
1/6400.
[0468] 4/5 mice immunized with rsPSMA batch #TD045-003 run 1/peak 2
showed an anti-PSMA antibody response by EIA. One mouse was
negative. Antibody titer was similar for assay plates coated with
rsPSMA lot #4019-C001 (75% dimer/25% monomer) and assay plates
coated with rsPSMA batch #TD045-003 run 1/peak 2 (100% monomer).
Median response for the group was 1/6400.
[0469] The results of the EIA analysis are provided in Table 7.
[0470] The results of the flow cytometry analysis are provided in
FIG. 51.
TABLE-US-00007 TABLE 7 Specificity of the Anti-PSMA Antibody
Response in Mice Vaccinated 4 Times with rsPSMA 5 .mu.g/dose and 50
.mu.g/dose Alhydrogel EIA Titer Median EIA Titer vs. Batch RFI vs.
Mouse vs. Lot TD045-003 PSMA-3T3 ID # Immunogen 4019-C001 run1/peak
2 cells ABIM151 4019-C001 Dimer 1/3200 1/3200 84 ABIM152 4019-C001
Dimer 1/3200 1/3200 41 ABIM153 4019-C001 Dimer 1/25600 1/25600 76
ABIM154 4019-C001 Dimer 1/12800 1/12800 63 ABIM155 4019-C001 Dimer
1/6400 1/6400 74 ABIM156 Monomer 1/1600 1/1600 5 ABIM157 Monomer
1/6400 1/12800 8 ABIM158 Monomer 0 0 6 ABIM159 Monomer 1/6400
1/6400 6 ABIM160 Monomer 1/6400 1/6400 12
[0471] When tested by ELISA, sera from both monomer and dimer
immunized animals showed similar levels of anti-PSMA antibodies,
indicating that each protein was immunogenic when formulated on
alum. For dimer immunized animals, the median endpoint titers were
1/6,400 (range 1/3,200 to 1/12,800) regardless of whether rsPSMA
monomer or dimer was used as the coating antigen. Similarly,
monomer-immunized animals had median endpoint titers of 1/6,400 in
both assay formats, although the range varied depending on whether
the monomer (range < 1/400 to 1/12,800) or dimer (range <
1/400 to 1/6,400) was used for coating.
[0472] However, a difference between sera was observed with
cell-based flow cytometry (FIG. 51). Anti-PSMA antibody in the
serum of mice immunized with a dimer preparation of rsPSMA (lot
#4019-C001) showed strong binding to PSMA-3T3 cells. Anti-PSMA
antibody in the serum of mice immunized with a 100% monomer
preparation of rsPSMA (batch #TD045-003 run 1/peak 2) showed no
binding to PSMA-3T3 cells.
[0473] Each dimer immunized animal elicited high-titered antibodies
to PSMA-3T3 cells (median mean fluorescence intensity (MFI)=74,
range 41-84), but such antibodies were very weak to absent in
monomer-immunized animals (median MFI=6, range 5-12). The level of
binding observed for monomer immunized animals was comparable to
that for naive animals. Similar background levels of binding to
parental 3T3 cells were observed for all sera (median MFI=6 in all
cases).
[0474] An identical pattern of reactivity was observed with human
prostate cancer cell lines. Consistent, high-level reactivity with
PSMA-expressing C4-2 cells was observed for sera from dimer
immunized animals (median MFI=28.0, range 23.1-28.8) but not
monomer immunized (median MFI=12.8, range 11.2-14.5) or control
animals (median MFI=12.3, range 8.6-16.0). Background levels of
binding to PSMA-negative PC-3 cells were observed for all sera
(median MFI=7 in all cases).
[0475] Thus, while it is possible to elicit the production of
antibodies that recognize native PSMA using monomeric forms of the
PSMA protein or fragments thereof, these results speak to the
relative efficiency of eliciting an immune response to native PSMA
using dimeric forms of PSMA protein. Additionally, flow cytometry
but not ELISA was able to reveal the differences in the humoral
immune responses elicited by monomeric and dimeric forms of PSMA.
The inability of the ELISA to uncover such differences suggests
that rsPSMA adopts a partially denatured conformation upon
adsorption to plastic.
Example 32
mAbs 006 and 026 Mediate Efficient ADCC of Human Prostate Cancer
Cells
[0476] .sup.51Cr labeled C4-2 cells (1.times.10.sup.4/well, target
cells) were incubated in triplicates with 10 .mu.g/mL mAb at
4.degree. C. for 1 hour. Fresh human PBMCs (effector cells) were
added to washed target cells at effector to target (E/T) ratios of
40:1, 20:1, and 10:1 and incubated at 37.degree. C. overnight.
.sup.51Cr in harvested supernatants was measured using a
.gamma.-scintillation counter and % cell lysis was calculated. mAbs
006 and 026 demonstrated statistically significant antibody
dependent cell-mediated cytotoxicity (ADCC) of C4-2 cells compared
to isotype matched human IgG1 mAb control (FIG. 52). No effect was
observed when PSMA-negative human prostate tumor cells (PC-3) were
used.
Example 33
Monomer-Dimer Equilibrium
[0477] Purified dimeric and monomeric forms of rsPSMA were resolved
by preparative size exclusion chromatography (SEC) in PBS+ buffer
and collected in separate fractions. To assess whether dimer and
monomer exist in a reversible equilibrium, the buffer conditions
were perturbed, and the monomer-dimer ratio was analyzed by SEC. As
indicated in FIG. 53A, a dimer preparation that contained
approximately 5% monomer initially was converted to 100% dimer upon
incubation for 72 h at ambient temperature in PBS+ supplemented
with 2M sodium chloride (FIG. 53A). Conversely, the addition of 2
mM of the metal-chelating agent EDTA converted the dimer into
monomer with a half-life of approximately 2 days (FIG. 53A),
indicating that dimer stability is dependent upon the presence of
metal ions, such as Zn.sup.2+ in the active site of PSMA.
[0478] For a preparation that initially comprised >95% monomer,
high salt similarly drove the equilibrium to mostly (81%) dimer
within 72 h (FIG. 53B). EDTA had little influence on the oligomeric
state of the monomer. Thus, regardless of the initial oligomeric
state of the protein, high salt concentrations promoted
dimerization, whereas metal-chelating agents dissociated dimers
into monomers.
[0479] PSMA shares modest sequence and structural homology with
human transferrin receptor (TfR), which contains a vestigial
catalytic domain but lacks enzymatic activity. TfR is expressed as
a type II membrane protein that forms a disulfide-linked homodimer,
but the intermolecular disulfides are not required for dimerization
(Alvarez, E., et al. (1989) EMBO J. 8, 2231-2240.0). The
high-resolution crystal structure of the TfR ectodomain reveals
that the protein is organized into three distinct domains known as
the protease-like, apical, and helical domains, with the last
domain being principally responsible for dimerization (Lawrence, C.
M., et al. (1999) Science 286, 779-782). PSMA and TfR share 30%,
30%, and 24% sequence identity within these domains, respectively.
The helical dimerization domain of PSMA are amino acids 601-750 of
SEQ ID NO: 1.
Example 34
rsPSMA Formulation Studies
[0480] pH Stability of rsPSMA
[0481] Dimeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into a
broad-range base buffer solution (2 mM glycine, 2 mM citric acid, 2
mM Hepes, 2 mM MES, 2 mM Tris Base) that was adjusted to cover pH 4
to pH 8.5 in steps of 0.5 pH units. Following incubation for 4 days
at 45.degree. C., the individual samples were subjected to
analytical TSK gel filtration chromatography (run at pH 7.5) and
analyzed for protein recovery and the preservation of the dimeric
structure of rsPSMA. The findings are summarized in Table 8.
TABLE-US-00008 TABLE 8 Recovery and Structure of rsPSMA at Various
pHs Dimer Monomer Aggregate Recovery from pH Content.sup.1
Content.sup.1 Content.sup.1 column.sup.2 4.0 +++++ - - + 4.5 - - -
- 5.0 ++ - +++ ++ 5.5 ++++ + - +++ 6.0 +++++ - - +++++ 6.5 ++++ + -
++++ 7.0 ++++ + - ++++ 7.5 ++++ + - + 8.0 +++ + + + 8.5 - - - -
.sup.1of recovered protein .sup.2of total protein at t = 0 - <5%
+ 5%-25% ++ 25-50% +++ 50-75% ++++ 75-95% +++++ >95%
Base Buffer Evaluation
[0482] Dimeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into
the following buffer solutions: [0483] PBS+ [0484] 20 mM Hepes, pH
7.0 [0485] 20 mM sodium phosphate +150 mM NaCl, pH 6.5 [0486] 20 mM
histidine +150 mM NaCl, pH 6.0 [0487] 20 mM sodium phosphate +150
mM NaCl, pH 6.0 [0488] 20 mM sodium acetate+150 mM NaCl, pH 6.0
[0489] 20 mM sodium citrate+150 mM NaCl, pH 6.0 Each sample was
incubated for 3 or 4 days at 45.degree. C. and subsequently
analyzed by analytical TSK gel filtration chromatography for
protein recovery and the preservation of the dimeric structure of
rsPSMA. The findings are summarized in Table 9.
TABLE-US-00009 [0489] TABLE 9 Recovery and Structure of rsPSMA with
Various Buffers Base Dimer Monomer Aggregate Recovery from Buffer
Content.sup.1 Content.sup.1 Content.sup.1 column.sup.2 PBS+ +++ - +
++ Phosphate +++++ - - +++++ Acetate +++++ - - +++++ Citrate N/A
N/A N/A - Histidine +++ + ++ + .sup.1of recovered protein .sup.2of
total protein at t = 0 - <5% + 5%-25% ++ 25-50% +++ 50-75% ++++
75-95% +++++ >95%
Excipients
[0490] Dimeric rsPSMA (2 mg/ml in PBS+) was dialyzed over night
into 20 mM sodium acetate, pH 6.0 and 150 mM NaCl. To evaluate the
effect of the individual amino acids, the protein was diluted
8-fold into 20 mM sodium acetate, pH 6.0 and 150 mM NaCl containing
50 mM of either glycine, histidine, proline, isoleucine, leucine,
alanine, lysine, arginine, threonine, glutamic acid, or aspartic
acid as excipients. Following incubation for 5 days at 45.degree.
C., each sample was analyzed by analytical TSK gel filtration
chromatography for protein recovery and the preservation of the
dimeric structure of the protein. The findings are summarized in
Table 10.
TABLE-US-00010 TABLE 10 Recovery and Structure of rsPSMA with
Various Amino Acids Dimer Monomer Aggregate Recovery from Amino
Acid Content.sup.1 Content.sup.1 Content.sup.1 column.sup.2 Glycine
++++ + - ++++ Histidine N/A N/A N/A + Proline ++++ + - ++++
Isoleucine ++++ + - ++++ Leucine ++++ + - ++++ Alanine ++++ + -
++++ Arginine ++++ + - ++++ Threonine ++ N/A +++ ++++ Glutamic Acid
- - +++++ ++++ Aspartic Acid - - +++++ +++ .sup.1of recovered
protein .sup.2of total protein at t = 0 - <5% + 5%-25% ++ 25-50%
+++ 50-75% ++++ 75-95% +++++ >95%
Surfactants
[0491] Dimeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into
PBS+ containing 0.5% (w/v) of either Triton X-100,
dodecylmaltoside, cholic acid, or CHAPS and incubated for 4 days at
4.degree. C. Each sample was subsequently analyzed by analytical
TSK gel filtration chromatography for protein recovery and the
preservation of the dimeric structure of the protein. The findings
are summarized in Table 11.
TABLE-US-00011 TABLE 11 Recovery and Structure of rsPSMA with
Various Surfactants Dimer Monomer Aggregate Recovery from
Surfactant Content.sup.1 Content.sup.1 Content.sup.1 column.sup.2
Triton X-100 ++++ + + ++++ Dodecylmaltoside ++++ - + +++++ Cholic
Acid ++++ - + +++++ CHAPS ++++ + - +++++ .sup.1of recovered protein
.sup.2of total protein at t = 0 - <5% + 5%-25% ++ 25-50% +++
50-75% ++++ 75-95% +++++ >95%
Other Excipients
[0492] Dimeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into
PBS+ containing either 1.4 M (35% saturation) ammonium sulfate, 5
mM EDTA, 1 mM DTT, or 10% glycerol and incubated for 4 days at
4.degree. C. Each sample was subsequently analyzed by analytical
TSK gel filtration chromatography for protein recovery and the
preservation of the dimeric structure of the protein. The findings
are summarized in Table 12.
TABLE-US-00012 TABLE 12 Recovery and Structure of rsPSMA with
Various Excipients Dimer Monomer Aggregate Recovery Excipient
Content.sup.1 Content.sup.1 Content.sup.1 from column.sup.2
Ammonium Sulfate ++++ - + +++++ EDTA ++ ++++ - +++++ DTT ++++ + -
+++++ Glycerol ++++ + - +++++ .sup.1of recovered protein .sup.2of
total protein at t = 0 - <5% + 5%-25% ++ 25-50% +++ 50-75% ++++
75-95% +++++ >95%
Conversion of Monomers into Dimers
[0493] To evaluate the potential of reversing monomeric rsPSMA into
dimers, monomeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into
PBS+ containing either 1.4 M (35% saturation) ammonium sulfate, 2 M
NaCl, 1 mM DTT, 5 mM EDTA, or 10% glycerol and incubated for up to
4 days at 4.degree. C. Each sample was subsequently analyzed by
analytical TSK gel filtration chromatography for protein recovery
and the formation of the dimeric structure of the protein. The
findings are summarized in Table 13.
TABLE-US-00013 TABLE 13 Conversion of rsPSMA Monomers Dimer Monomer
Aggregate Recovery from Excipient Content.sup.1 Content.sup.1
Content.sup.1 column.sup.2 Ammonium Sulfate ++ +++ + +++++ NaCl +++
++ + +++++ DTT + ++++ - +++++ EDTA - +++++ - +++++ Glycerol + ++++
- +++++ .sup.1of recovered protein .sup.2of total protein at t = 0
- <5% + 5%-25% ++ 25-50% +++ 50-75% ++++ 75-95% +++++
>95%
[0494] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose and variations can be made by those skilled
in the art without departing from the spirit and scope of the
invention which is defined by the following claims.
[0495] The contents of all references, patents and published patent
applications cited throughout this application are incorporated
herein by reference.
Sequence CWU 1
1
391750PRTHomo sapiens 1Met Trp Asn Leu Leu His Glu Thr Asp Ser Ala
Val Ala Thr Ala Arg 1 5 10 15 Arg Pro Arg Trp Leu Cys Ala Gly Ala
Leu Val Leu Ala Gly Gly Phe 20 25 30 Phe Leu Leu Gly Phe Leu Phe
Gly Trp Phe Ile Lys Ser Ser Asn Glu 35 40 45 Ala Thr Asn Ile Thr
Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu 50 55 60 Leu Lys Ala
Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln Ile 65 70 75 80 Pro
His Leu Ala Gly Thr Glu Gln Asn Phe Gln Leu Ala Lys Gln Ile 85 90
95 Gln Ser Gln Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala His
100 105 110 Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro Asn
Tyr Ile 115 120 125 Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe Asn
Thr Ser Leu Phe 130 135 140 Glu Pro Pro Pro Pro Gly Tyr Glu Asn Val
Ser Asp Ile Val Pro Pro 145 150 155 160 Phe Ser Ala Phe Ser Pro Gln
Gly Met Pro Glu Gly Asp Leu Val Tyr 165 170 175 Val Asn Tyr Ala Arg
Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met 180 185 190 Lys Ile Asn
Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys Val 195 200 205 Phe
Arg Gly Asn Lys Val Lys Asn Ala Gln Leu Ala Gly Ala Lys Gly 210 215
220 Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val Lys
225 230 235 240 Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly Val
Gln Arg Gly 245 250 255 Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro
Leu Thr Pro Gly Tyr 260 265 270 Pro Ala Asn Glu Tyr Ala Tyr Arg Arg
Gly Ile Ala Glu Ala Val Gly 275 280 285 Leu Pro Ser Ile Pro Val His
Pro Ile Gly Tyr Tyr Asp Ala Gln Lys 290 295 300 Leu Leu Glu Lys Met
Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp Arg 305 310 315 320 Gly Ser
Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly Asn 325 330 335
Phe Ser Thr Gln Lys Val Lys Met His Ile His Ser Thr Asn Glu Val 340
345 350 Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val Glu
Pro 355 360 365 Asp Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser Trp
Val Phe Gly 370 375 380 Gly Ile Asp Pro Gln Ser Gly Ala Ala Val Val
His Glu Ile Val Arg 385 390 395 400 Ser Phe Gly Thr Leu Lys Lys Glu
Gly Trp Arg Pro Arg Arg Thr Ile 405 410 415 Leu Phe Ala Ser Trp Asp
Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr 420 425 430 Glu Trp Ala Glu
Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val Ala 435 440 445 Tyr Ile
Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val 450 455 460
Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu 465
470 475 480 Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr
Glu Ser 485 490 495 Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly
Met Pro Arg Ile 500 505 510 Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu
Val Phe Phe Gln Arg Leu 515 520 525 Gly Ile Ala Ser Gly Arg Ala Arg
Tyr Thr Lys Asn Trp Glu Thr Asn 530 535 540 Lys Phe Ser Gly Tyr Pro
Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu 545 550 555 560 Leu Val Glu
Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val 565 570 575 Ala
Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val 580 585
590 Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala
595 600 605 Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu Met
Lys Thr 610 615 620 Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val
Lys Asn Phe Thr 625 630 635 640 Glu Ile Ala Ser Lys Phe Ser Glu Arg
Leu Gln Asp Phe Asp Lys Ser 645 650 655 Asn Pro Ile Val Leu Arg Met
Met Asn Asp Gln Leu Met Phe Leu Glu 660 665 670 Arg Ala Phe Ile Asp
Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg 675 680 685 His Val Ile
Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu Ser 690 695 700 Phe
Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp 705 710
715 720 Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val Ala
Ala 725 730 735 Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu Val
Ala 740 745 750 27570DNAArtificial SequenceSynthetic
Oligonucleotide 2gacggatcgg gagatctccc gatcccctat ggtcgactct
cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag
gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg
ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt
aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
900ggtaccaagc ttggatctca ccatggagtt gggactgcgc tggggcttcc
tcgttgctct 960tttaagaggt gtccagtgtc aggtgcaatt ggtggagtct
gggggaggcg tggtccagcc 1020tgggaggtcc ctgagactct cctgtgcagc
gtctggattc gccttcagta gatatggcat 1080gcactgggtc cgccaggctc
caggcaaggg gctggagtgg gtggcagtta tatggtatga 1140tggaagtaat
aaatactatg cagactccgt gaagggccga ttcaccatct ccagagacaa
1200ttccaagaac acgcagtatc tgcaaatgaa cagcctgaga gccgaggaca
cggctgtgta 1260ttactgtgcg agaggcggtg acttcctcta ctactactat
tacggtatgg acgtctgggg 1320ccaagggacc acggtcaccg tctcctcagc
ctccaccaag ggcccatcgg tcttccccct 1380ggcaccctct agcaagagca
cctctggggg cacagcggcc ctgggctgcc tggtcaagga 1440ctacttcccc
gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca
1500caccttcccg gctgtcctac agtcctcagg actctactcc ctcagcagcg
tggtgaccgt 1560gccctccagc agcttgggca cccagaccta catctgcaac
gtgaatcaca agcccagcaa 1620caccaaggtg gacaagagag ttggtgagag
gccagcacag ggagggaggg tgtctgctgg 1680aagccaggct cagcgctcct
gcctggacgc atcccggcta tgcagtccca gtccagggca 1740gcaaggcagg
ccccgtctgc ctcttcaccc ggaggcctct gcccgcccca ctcatgctca
1800gggagagggt cttctggctt tttccccagg ctctgggcag gcacaggcta
ggtgccccta 1860acccaggccc tgcacacaaa ggggcaggtg ctgggctcag
acctgccaag agccatatcc 1920gggaggaccc tgcccctgac ctaagcccac
cccaaaggcc aaactctcca ctccctcagc 1980tcggacacct tctctcctcc
cagattccag taactcccaa tcttctctct gcagagccca 2040aatcttgtga
caaaactcac acatgcccac cgtgcccagg taagccagcc caggcctcgc
2100cctccagctc aaggcgggac aggtgcccta gagtagcctg catccaggga
caggccccag 2160ccgggtgctg acacgtccac ctccatctct tcctcagcac
ctgaactcct ggggggaccg 2220tcagtcttcc tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag 2280gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 2340gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
2400acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag 2460tacaagtgca aggtctccaa caaagccctc ccagccccca
tcgagaaaac catctccaaa 2520gccaaaggtg ggacccgtgg ggtgcgaggg
ccacatggac agaggccggc tcggcccacc 2580ctctgccctg agagtgaccg
ctgtaccaac ctctgtccct acagggcagc cccgagaacc 2640acaggtgtac
accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac
2700ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga
gcaatgggca 2760gccggagaac aactacaaga ccacgcctcc cgtgctggac
tccgacggct ccttcttcct 2820ctatagcaag ctcaccgtgg acaagagcag
gtggcagcag gggaacgtct tctcatgctc 2880cgtgatgcat gaggctctgc
acaaccacta cacgcagaag agcctctccc tgtctccggg 2940taaatgagaa
ttcctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
3000gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
tgaccctgga 3060aggtgccact cccactgtcc tttcctaata aaatgaggaa
attgcatcgc attgtctgag 3120taggtgtcat tctattctgg ggggtggggt
ggggcaggac agcaaggggg aggattggga 3180agacaatagc aggcatgctg
gggatgcggt gggctctatg gcttctgagg cggaaagaac 3240cagctggggc
tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
3300tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
ccgctccttt 3360cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
ccccgtcaag ctctaaatcg 3420gggcatccct ttagggttcc gatttagtgc
tttacggcac ctcgacccca aaaaacttga 3480ttagggtgat ggttcacgta
gtgggccatc gccctgatag acggtttttc gccctttgac 3540gttggagtcc
acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
3600tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct
attggttaaa 3660aaatgagctg atttaacaaa aatttaacgc gaattaattc
tgtggaatgt gtgtcagtta 3720gggtgtggaa agtccccagg ctccccaggc
aggcagaagt atgcaaagca tgcatctcaa 3780ttagtcagca accaggtgtg
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 3840catgcatctc
aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
3900aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
ttatttatgc 3960agaggccgag gccgcctctg cctctgagct attccagaag
tagtgaggag gcttttttgg 4020aggcctaggc ttttgcaaaa agctcccggg
agcttgtata tccattttcg gatctgatca 4080gcacgtgatg aaaaagcctg
aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa 4140gttcgacagc
gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag
4200cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
atggtttcta 4260caaagatcgt tatgtttatc ggcactttgc atcggccgcg
ctcccgattc cggaagtgct 4320tgacattggg gaattcagcg agagcctgac
ctattgcatc tcccgccgtg cacagggtgt 4380cacgttgcaa gacctgcctg
aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc 4440catggatgcg
atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc
4500gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
ctgatcccca 4560tgtgtatcac tggcaaactg tgatggacga caccgtcagt
gcgtccgtcg cgcaggctct 4620cgatgagctg atgctttggg ccgaggactg
ccccgaagtc cggcacctcg tgcacgcgga 4680tttcggctcc aacaatgtcc
tgacggacaa tggccgcata acagcggtca ttgactggag 4740cgaggcgatg
ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg
4800gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
agcttgcagg 4860atcgccgcgg ctccgggcgt atatgctccg cattggtctt
gaccaactct atcagagctt 4920ggttgacggc aatttcgatg atgcagcttg
ggcgcagggt cgatgcgacg caatcgtccg 4980atccggagcc gggactgtcg
ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac 5040cgatggctgt
gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag
5100ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct
atgaaaggtt 5160gggcttcgga atcgttttcc gggacgccgg ctggatgatc
ctccagcgcg gggatctcat 5220gctggagttc ttcgcccacc ccaacttgtt
tattgcagct tataatggtt acaaataaag 5280caatagcatc acaaatttca
caaataaagc atttttttca ctgcattcta gttgtggttt 5340gtccaaactc
atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
5400ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
caattccaca 5460caacatacga gccggaagca taaagtgtaa agcctggggt
gcctaatgag tgagctaact 5520cacattaatt gcgttgcgct cactgcccgc
tttccagtcg ggaaacctgt cgtgccagct 5580gcattaatga atcggccaac
gcgcggggag aggcggtttg cgtattgggc gctcttccgc 5640ttcctcgctc
actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
5700ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
agaacatgtg 5760agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
gcgttgctgg cgtttttcca 5820taggctccgc ccccctgacg agcatcacaa
aaatcgacgc tcaagtcaga ggtggcgaaa 5880cccgacagga ctataaagat
accaggcgtt tccccctgga agctccctcg tgcgctctcc 5940tgttccgacc
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
6000gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc
gctccaagct 6060gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
gccttatccg gtaactatcg 6120tcttgagtcc aacccggtaa gacacgactt
atcgccactg gcagcagcca ctggtaacag 6180gattagcaga gcgaggtatg
taggcggtgc tacagagttc ttgaagtggt ggcctaacta 6240cggctacact
agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
6300aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
gtggtttttt 6360tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
caagaagatc ctttgatctt 6420ttctacgggg tctgacgctc agtggaacga
aaactcacgt taagggattt tggtcatgag 6480attatcaaaa aggatcttca
cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 6540ctaaagtata
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
6600tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
tcgtgtagat 6660aactacgata cgggagggct taccatctgg ccccagtgct
gcaatgatac cgcgagaccc 6720acgctcaccg gctccagatt tatcagcaat
aaaccagcca gccggaaggg ccgagcgcag 6780aagtggtcct gcaactttat
ccgcctccat ccagtctatt aattgttgcc gggaagctag 6840agtaagtagt
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
6900ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
gatcaaggcg 6960agttacatga tcccccatgt tgtgcaaaaa agcggttagc
tccttcggtc ctccgatcgt 7020tgtcagaagt aagttggccg cagtgttatc
actcatggtt atggcagcac tgcataattc 7080tcttactgtc atgccatccg
taagatgctt ttctgtgact ggtgagtact caaccaagtc 7140attctgagaa
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
7200taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
cttcggggcg 7260aaaactctca aggatcttac cgctgttgag atccagttcg
atgtaaccca ctcgtgcacc 7320caactgatct tcagcatctt ttactttcac
cagcgtttct gggtgagcaa aaacaggaag 7380gcaaaatgcc gcaaaaaagg
gaataagggc gacacggaaa tgttgaatac tcatactctt 7440cctttttcaa
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
7500tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
gaaaagtgcc 7560acctgacgtc 757037597DNAArtificial SequenceSynthetic
Oligonucleotide 3gacggatcgg gagatctccc gatcccctat ggtcgactct
cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag
gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg
ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt
aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
900ggtaccaagc ttggatctca ccatggggtc aaccgccatc ctcaccatgg
agttggggct 960gcgctgggtt ctcctcgttg ctcttttaag aggtgtccag
tgtcaggtgc agctggtgga 1020gtctggggga ggcgtggtcc agcctgggag
gtccctgaga ctctcctgtg cagcgtctgg 1080attcaccttc agtaactatg
tcatgcactg ggtccgccag gctccaggca aggggctgga 1140gtgggtggca
attatatggt atgatggaag taataaatac tatgcagact ccgtgaaggg
1200ccgattcacc atctccagag acaattccaa gaacacgctg tatctgcaaa
tgaacagcct 1260gagagccgag gacacggctg tgtattactg tgcgggtgga
tataactgga actacgagta 1320ccactactac ggtatggacg tctggggcca
agggaccacg gtcaccgtct cctcagcctc 1380caccaagggc ccatcggtct
tccccctggc accctctagc aagagcacct ctgggggcac 1440agcggccctg
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa
1500ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct gtcctacagt
cctcaggact 1560ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc
ttgggcaccc agacctacat 1620ctgcaacgtg aatcacaagc ccagcaacac
caaggtggac aagagagttg gtgagaggcc 1680agcacaggga gggagggtgt
ctgctggaag ccaggctcag cgctcctgcc tggacgcatc 1740ccggctatgc
agtcccagtc cagggcagca aggcaggccc cgtctgcctc ttcacccgga
1800ggcctctgcc cgccccactc atgctcaggg agagggtctt ctggcttttt
ccccaggctc 1860tgggcaggca caggctaggt gcccctaacc caggccctgc
acacaaaggg gcaggtgctg 1920ggctcagacc tgccaagagc catatccggg
aggaccctgc ccctgaccta agcccacccc 1980aaaggccaaa ctctccactc
cctcagctcg gacaccttct ctcctcccag attccagtaa 2040ctcccaatct
tctctctgca gagcccaaat cttgtgacaa aactcacaca tgcccaccgt
2100gcccaggtaa gccagcccag gcctcgccct ccagctcaag gcgggacagg
tgccctagag 2160tagcctgcat ccagggacag gccccagccg ggtgctgaca
cgtccacctc catctcttcc 2220tcagcacctg aactcctggg gggaccgtca
gtcttcctct tccccccaaa acccaaggac 2280accctcatga tctcccggac
ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 2340gaccctgagg
tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca
2400aagccgcggg
aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg
2460caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa
agccctccca 2520gcccccatcg agaaaaccat ctccaaagcc aaaggtggga
cccgtggggt gcgagggcca 2580catggacaga ggccggctcg gcccaccctc
tgccctgaga gtgaccgctg taccaacctc 2640tgtccctaca gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggagga 2700gatgaccaag
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
2760cgccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt 2820gctggactcc gacggctcct tcttcctcta tagcaagctc
accgtggaca agagcaggtg 2880gcagcagggg aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac 2940gcagaagagc ctctccctgt
ctccgggtaa atgagaattc ctcgagtcta gagggcccgt 3000ttaaacccgc
tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
3060ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
cctaataaaa 3120tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
attctggggg gtggggtggg 3180gcaggacagc aagggggagg attgggaaga
caatagcagg catgctgggg atgcggtggg 3240ctctatggct tctgaggcgg
aaagaaccag ctggggctct agggggtatc cccacgcgcc 3300ctgtagcggc
gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
3360tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
ccacgttcgc 3420cggctttccc cgtcaagctc taaatcgggg catcccttta
gggttccgat ttagtgcttt 3480acggcacctc gaccccaaaa aacttgatta
gggtgatggt tcacgtagtg ggccatcgcc 3540ctgatagacg gtttttcgcc
ctttgacgtt ggagtccacg ttctttaata gtggactctt 3600gttccaaact
ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
3660tttggggatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
ttaacgcgaa 3720ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
ccccaggctc cccaggcagg 3780cagaagtatg caaagcatgc atctcaatta
gtcagcaacc aggtgtggaa agtccccagg 3840ctccccagca ggcagaagta
tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 3900gcccctaact
ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
3960tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
ctgagctatt 4020ccagaagtag tgaggaggct tttttggagg cctaggcttt
tgcaaaaagc tcccgggagc 4080ttgtatatcc attttcggat ctgatcagca
cgtgatgaaa aagcctgaac tcaccgcgac 4140gtctgtcgag aagtttctga
tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 4200ggagggcgaa
gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
4260ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
actttgcatc 4320ggccgcgctc ccgattccgg aagtgcttga cattggggaa
ttcagcgaga gcctgaccta 4380ttgcatctcc cgccgtgcac agggtgtcac
gttgcaagac ctgcctgaaa ccgaactgcc 4440cgctgttctg cagccggtcg
cggaggccat ggatgcgatc gctgcggccg atcttagcca 4500gacgagcggg
ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
4560tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
tggacgacac 4620cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
ctttgggccg aggactgccc 4680cgaagtccgg cacctcgtgc acgcggattt
cggctccaac aatgtcctga cggacaatgg 4740ccgcataaca gcggtcattg
actggagcga ggcgatgttc ggggattccc aatacgaggt 4800cgccaacatc
ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
4860cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata
tgctccgcat 4920tggtcttgac caactctatc agagcttggt tgacggcaat
ttcgatgatg cagcttgggc 4980gcagggtcga tgcgacgcaa tcgtccgatc
cggagccggg actgtcgggc gtacacaaat 5040cgcccgcaga agcgcggccg
tctggaccga tggctgtgta gaagtactcg ccgatagtgg 5100aaaccgacgc
cccagcactc gtccgagggc aaaggaatag cacgtgctac gagatttcga
5160ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
acgccggctg 5220gatgatcctc cagcgcgggg atctcatgct ggagttcttc
gcccacccca acttgtttat 5280tgcagcttat aatggttaca aataaagcaa
tagcatcaca aatttcacaa ataaagcatt 5340tttttcactg cattctagtt
gtggtttgtc caaactcatc aatgtatctt atcatgtctg 5400tataccgtcg
acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
5460aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
agtgtaaagc 5520ctggggtgcc taatgagtga gctaactcac attaattgcg
ttgcgctcac tgcccgcttt 5580ccagtcggga aacctgtcgt gccagctgca
ttaatgaatc ggccaacgcg cggggagagg 5640cggtttgcgt attgggcgct
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 5700tcggctgcgg
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
5760aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
ggaaccgtaa 5820aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
cctgacgagc atcacaaaaa 5880tcgacgctca agtcagaggt ggcgaaaccc
gacaggacta taaagatacc aggcgtttcc 5940ccctggaagc tccctcgtgc
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 6000cgcctttctc
ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag
6060ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
ttcagcccga 6120ccgctgcgcc ttatccggta actatcgtct tgagtccaac
ccggtaagac acgacttatc 6180gccactggca gcagccactg gtaacaggat
tagcagagcg aggtatgtag gcggtgctac 6240agagttcttg aagtggtggc
ctaactacgg ctacactaga aggacagtat ttggtatctg 6300cgctctgctg
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
6360aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
gcagaaaaaa 6420aggatctcaa gaagatcctt tgatcttttc tacggggtct
gacgctcagt ggaacgaaaa 6480ctcacgttaa gggattttgg tcatgagatt
atcaaaaagg atcttcacct agatcctttt 6540aaattaaaaa tgaagtttta
aatcaatcta aagtatatat gagtaaactt ggtctgacag 6600ttaccaatgc
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
6660agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
catctggccc 6720cagtgctgca atgataccgc gagacccacg ctcaccggct
ccagatttat cagcaataaa 6780ccagccagcc ggaagggccg agcgcagaag
tggtcctgca actttatccg cctccatcca 6840gtctattaat tgttgccggg
aagctagagt aagtagttcg ccagttaata gtttgcgcaa 6900cgttgttgcc
attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
6960cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
gcaaaaaagc 7020ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
ttggccgcag tgttatcact 7080catggttatg gcagcactgc ataattctct
tactgtcatg ccatccgtaa gatgcttttc 7140tgtgactggt gagtactcaa
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 7200ctcttgcccg
gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
7260catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
tgttgagatc 7320cagttcgatg taacccactc gtgcacccaa ctgatcttca
gcatctttta ctttcaccag 7380cgtttctggg tgagcaaaaa caggaaggca
aaatgccgca aaaaagggaa taagggcgac 7440acggaaatgt tgaatactca
tactcttcct ttttcaatat tattgaagca tttatcaggg 7500ttattgtctc
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
7560tccgcgcaca tttccccgaa aagtgccacc tgacgtc 759747579DNAArtificial
SequenceSynthetic Oligonucleotide 4gacggatcgg gagatctccc gatcccctat
ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg
cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca
acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag
gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggac
tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt
acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
gctggctaga 900ggtaccaagc ttggatctca ccatggagtt gggacttagc
tgggttttcc tcgttgctct 960tttaagaggt gtccagtgtc aggtccagct
ggtggagtct gggggaggcg tggtccagcc 1020tgggaggtcc ctgagactct
cctgtgcagc gtctggattc accttcagta gctatggcat 1080gcactgggtc
cgccaggctc caggcaaggg gctggactgg gtggcaatta tttggcatga
1140tggaagtaat aaatactatg cagactccgt gaagggccga ttcaccatct
ccagagacaa 1200ttccaagaag acgctgtacc tgcaaatgaa cagtttgaga
gccgaggaca cggctgtgta 1260ttactgtgcg agagcttggg cctatgacta
cggtgactat gaatactact tcggtatgga 1320cgtctggggc caagggacca
cggtcaccgt ctcctcagcc tccaccaagg gcccatcggt 1380cttccccctg
gcaccctcta gcaagagcac ctctgggggc acagcggccc tgggctgcct
1440ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg
ccctgaccag 1500cggcgtgcac accttcccgg ctgtcctaca gtcctcagga
ctctactccc tcagcagcgt 1560ggtgaccgtg ccctccagca gcttgggcac
ccagacctac atctgcaacg tgaatcacaa 1620gcccagcaac accaaggtgg
acaagagagt tggtgagagg ccagcacagg gagggagggt 1680gtctgctgga
agccaggctc agcgctcctg cctggacgca tcccggctat gcagtcccag
1740tccagggcag caaggcaggc cccgtctgcc tcttcacccg gaggcctctg
cccgccccac 1800tcatgctcag ggagagggtc ttctggcttt ttccccaggc
tctgggcagg cacaggctag 1860gtgcccctaa cccaggccct gcacacaaag
gggcaggtgc tgggctcaga cctgccaaga 1920gccatatccg ggaggaccct
gcccctgacc taagcccacc ccaaaggcca aactctccac 1980tccctcagct
cggacacctt ctctcctccc agattccagt aactcccaat cttctctctg
2040cagagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccaggt
aagccagccc 2100aggcctcgcc ctccagctca aggcgggaca ggtgccctag
agtagcctgc atccagggac 2160aggccccagc cgggtgctga cacgtccacc
tccatctctt cctcagcacc tgaactcctg 2220gggggaccgt cagtcttcct
cttcccccca aaacccaagg acaccctcat gatctcccgg 2280acccctgagg
tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc
2340aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg
ggaggagcag 2400tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc
tgcaccagga ctggctgaat 2460ggcaaggagt acaagtgcaa ggtctccaac
aaagccctcc cagcccccat cgagaaaacc 2520atctccaaag ccaaaggtgg
gacccgtggg gtgcgagggc cacatggaca gaggccggct 2580cggcccaccc
tctgccctga gagtgaccgc tgtaccaacc tctgtcccta cagggcagcc
2640ccgagaacca caggtgtaca ccctgccccc atcccgggag gagatgacca
agaaccaggt 2700cagcctgacc tgcctggtca aaggcttcta tcccagcgac
atcgccgtgg agtgggagag 2760caatgggcag ccggagaaca actacaagac
cacgcctccc gtgctggact ccgacggctc 2820cttcttcctc tatagcaagc
tcaccgtgga caagagcagg tggcagcagg ggaacgtctt 2880ctcatgctcc
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct
2940gtctccgggt aaatgagaat tcctcgagtc tagagggccc gtttaaaccc
gctgatcagc 3000ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
ccctcccccg tgccttcctt 3060gaccctggaa ggtgccactc ccactgtcct
ttcctaataa aatgaggaaa ttgcatcgca 3120ttgtctgagt aggtgtcatt
ctattctggg gggtggggtg gggcaggaca gcaaggggga 3180ggattgggaa
gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
3240ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
gcgcattaag 3300cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
cttgccagcg ccctagcgcc 3360cgctcctttc gctttcttcc cttcctttct
cgccacgttc gccggctttc cccgtcaagc 3420tctaaatcgg ggcatccctt
tagggttccg atttagtgct ttacggcacc tcgaccccaa 3480aaaacttgat
tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
3540ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
ctggaacaac 3600actcaaccct atctcggtct attcttttga tttataaggg
attttgggga tttcggccta 3660ttggttaaaa aatgagctga tttaacaaaa
atttaacgcg aattaattct gtggaatgtg 3720tgtcagttag ggtgtggaaa
gtccccaggc tccccaggca ggcagaagta tgcaaagcat 3780gcatctcaat
tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
3840tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
ctccgcccat 3900cccgccccta actccgccca gttccgccca ttctccgccc
catggctgac taattttttt 3960tatttatgca gaggccgagg ccgcctctgc
ctctgagcta ttccagaagt agtgaggagg 4020cttttttgga ggcctaggct
tttgcaaaaa gctcccggga gcttgtatat ccattttcgg 4080atctgatcag
cacgtgatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct
4140gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg
aagaatctcg 4200tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg
cgggtaaata gctgcgccga 4260tggtttctac aaagatcgtt atgtttatcg
gcactttgca tcggccgcgc tcccgattcc 4320ggaagtgctt gacattgggg
aattcagcga gagcctgacc tattgcatct cccgccgtgc 4380acagggtgtc
acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
4440cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
ggttcggccc 4500attcggaccg caaggaatcg gtcaatacac tacatggcgt
gatttcatat gcgcgattgc 4560tgatccccat gtgtatcact ggcaaactgt
gatggacgac accgtcagtg cgtccgtcgc 4620gcaggctctc gatgagctga
tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt 4680gcacgcggat
ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
4740tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
tcttcttctg 4800gaggccgtgg ttggcttgta tggagcagca gacgcgctac
ttcgagcgga ggcatccgga 4860gcttgcagga tcgccgcggc tccgggcgta
tatgctccgc attggtcttg accaactcta 4920tcagagcttg gttgacggca
atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc 4980aatcgtccga
tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
5040cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
gccccagcac 5100tcgtccgagg gcaaaggaat agcacgtgct acgagatttc
gattccaccg ccgccttcta 5160tgaaaggttg ggcttcggaa tcgttttccg
ggacgccggc tggatgatcc tccagcgcgg 5220ggatctcatg ctggagttct
tcgcccaccc caacttgttt attgcagctt ataatggtta 5280caaataaagc
aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
5340ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
cgacctctag 5400ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
tgaaattgtt atccgctcac 5460aattccacac aacatacgag ccggaagcat
aaagtgtaaa gcctggggtg cctaatgagt 5520gagctaactc acattaattg
cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 5580gtgccagctg
cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
5640ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
ggcgagcggt 5700atcagctcac tcaaaggcgg taatacggtt atccacagaa
tcaggggata acgcaggaaa 5760gaacatgtga gcaaaaggcc agcaaaaggc
caggaaccgt aaaaaggccg cgttgctggc 5820gtttttccat aggctccgcc
cccctgacga gcatcacaaa aatcgacgct caagtcagag 5880gtggcgaaac
ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
5940gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
tcccttcggg 6000aagcgtggcg ctttctcaat gctcacgctg taggtatctc
agttcggtgt aggtcgttcg 6060ctccaagctg ggctgtgtgc acgaaccccc
cgttcagccc gaccgctgcg ccttatccgg 6120taactatcgt cttgagtcca
acccggtaag acacgactta tcgccactgg cagcagccac 6180tggtaacagg
attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
6240gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
tgaagccagt 6300taccttcgga aaaagagttg gtagctcttg atccggcaaa
caaaccaccg ctggtagcgg 6360tggttttttt gtttgcaagc agcagattac
gcgcagaaaa aaaggatctc aagaagatcc 6420tttgatcttt tctacggggt
ctgacgctca gtggaacgaa aactcacgtt aagggatttt 6480ggtcatgaga
ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
6540taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
gcttaatcag 6600tgaggcacct atctcagcga tctgtctatt tcgttcatcc
atagttgcct gactccccgt 6660cgtgtagata actacgatac gggagggctt
accatctggc cccagtgctg caatgatacc 6720gcgagaccca cgctcaccgg
ctccagattt atcagcaata aaccagccag ccggaagggc 6780cgagcgcaga
agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
6840ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
ccattgctac 6900aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
ttcagctccg gttcccaacg 6960atcaaggcga gttacatgat cccccatgtt
gtgcaaaaaa gcggttagct ccttcggtcc 7020tccgatcgtt gtcagaagta
agttggccgc agtgttatca ctcatggtta tggcagcact 7080gcataattct
cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
7140aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
cggcgtcaat 7200acgggataat accgcgccac atagcagaac tttaaaagtg
ctcatcattg gaaaacgttc 7260ttcggggcga aaactctcaa ggatcttacc
gctgttgaga tccagttcga tgtaacccac 7320tcgtgcaccc aactgatctt
cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 7380aacaggaagg
caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
7440catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
tcatgagcgg 7500atacatattt gaatgtattt agaaaaataa acaaataggg
gttccgcgca catttccccg 7560aaaagtgcca cctgacgtc
757957558DNAArtificial SequenceSynthetic Oligonucleotide
5gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct
ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga
ctcactatag ggagacccaa gctggctaga 900ggtaccaagc ttggatccca
ccatggggtc aaccgtcatc ctcgccctcc tcctggctgt 960tctccaagga
gtctgtgccg aggtgcagct ggtgcagtct ggagcagagg tgaaaaagcc
1020cggggagtct ctgaagatct cctgtaaggg ttctggatac agctttacca
gttactggat 1080cggctgggtg cgccagatgc ccgggaaagg cctggagtgg
atggggatca tctatcctgg 1140tgactctgat accagataca gcccgtcctt
ccaaggccag gtcaccatct cagccgacaa 1200gtccatcagc accgcctacc
tgcagtggag cagcctgaag gcctcggaca ccgccatgta 1260ttactgtgcg
agacggatgg cagcagctgg cccctttgac tactggggcc agggaaccct
1320ggtcaccgtc tcctcagcct ccaccaaggg cccatcggtc ttccccctgg
caccctctag 1380caagagcacc tctgggggca cagcggccct gggctgcctg
gtcaaggact acttccccga 1440accggtgacg gtgtcgtgga actcaggcgc
cctgaccagc ggcgtgcaca ccttcccggc 1500tgtcctacag tcctcaggac
tctactccct cagcagcgtg gtgaccgtgc cctccagcag 1560cttgggcacc
cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga
1620caagagagtt ggtgagaggc cagcacaggg agggagggtg tctgctggaa
gccaggctca 1680gcgctcctgc ctggacgcat cccggctatg cagtcccagt
ccagggcagc aaggcaggcc 1740ccgtctgcct cttcacccgg aggcctctgc
ccgccccact catgctcagg gagagggtct 1800tctggctttt tccccaggct
ctgggcaggc acaggctagg tgcccctaac ccaggccctg 1860cacacaaagg
ggcaggtgct gggctcagac ctgccaagag ccatatccgg gaggaccctg
1920cccctgacct aagcccaccc caaaggccaa actctccact ccctcagctc
ggacaccttc 1980tctcctccca gattccagta actcccaatc ttctctctgc
agagcccaaa tcttgtgaca 2040aaactcacac atgcccaccg tgcccaggta
agccagccca ggcctcgccc tccagctcaa 2100ggcgggacag
gtgccctaga gtagcctgca tccagggaca ggccccagcc gggtgctgac
2160acgtccacct ccatctcttc ctcagcacct gaactcctgg ggggaccgtc
agtcttcctc 2220ttccccccaa aacccaagga caccctcatg atctcccgga
cccctgaggt cacatgcgtg 2280gtggtggacg tgagccacga agaccctgag
gtcaagttca actggtacgt ggacggcgtg 2340gaggtgcata atgccaagac
aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 2400gtcagcgtcc
tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag
2460gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc
caaaggtggg 2520acccgtgggg tgcgagggcc acatggacag aggccggctc
ggcccaccct ctgccctgag 2580agtgaccgct gtaccaacct ctgtccctac
agggcagccc cgagaaccac aggtgtacac 2640cctgccccca tcccgggagg
agatgaccaa gaaccaggtc agcctgacct gcctggtcaa 2700aggcttctat
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa
2760ctacaagacc acgcctcccg tgctggactc cgacggctcc ttcttcctct
atagcaagct 2820caccgtggac aagagcaggt ggcagcaggg gaacgtcttc
tcatgctccg tgatgcatga 2880ggctctgcac aaccactaca cgcagaagag
cctctccctg tctccgggta aatgagaatt 2940cctcgagtct agagggcccg
tttaaacccg ctgatcagcc tcgactgtgc cttctagttg 3000ccagccatct
gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
3060cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
ggtgtcattc 3120tattctgggg ggtggggtgg ggcaggacag caagggggag
gattgggaag acaatagcag 3180gcatgctggg gatgcggtgg gctctatggc
ttctgaggcg gaaagaacca gctggggctc 3240tagggggtat ccccacgcgc
cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 3300gcgcagcgtg
accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
3360ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
gcatcccttt 3420agggttccga tttagtgctt tacggcacct cgaccccaaa
aaacttgatt agggtgatgg 3480ttcacgtagt gggccatcgc cctgatagac
ggtttttcgc cctttgacgt tggagtccac 3540gttctttaat agtggactct
tgttccaaac tggaacaaca ctcaacccta tctcggtcta 3600ttcttttgat
ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat
3660ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
gtgtggaaag 3720tccccaggct ccccaggcag gcagaagtat gcaaagcatg
catctcaatt agtcagcaac 3780caggtgtgga aagtccccag gctccccagc
aggcagaagt atgcaaagca tgcatctcaa 3840ttagtcagca accatagtcc
cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 3900ttccgcccat
tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
3960cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
gcctaggctt 4020ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
tctgatcagc acgtgatgaa 4080aaagcctgaa ctcaccgcga cgtctgtcga
gaagtttctg atcgaaaagt tcgacagcgt 4140ctccgacctg atgcagctct
cggagggcga agaatctcgt gctttcagct tcgatgtagg 4200agggcgtgga
tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
4260tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
acattgggga 4320attcagcgag agcctgacct attgcatctc ccgccgtgca
cagggtgtca cgttgcaaga 4380cctgcctgaa accgaactgc ccgctgttct
gcagccggtc gcggaggcca tggatgcgat 4440cgctgcggcc gatcttagcc
agacgagcgg gttcggccca ttcggaccgc aaggaatcgg 4500tcaatacact
acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
4560gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
atgagctgat 4620gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
cacgcggatt tcggctccaa 4680caatgtcctg acggacaatg gccgcataac
agcggtcatt gactggagcg aggcgatgtt 4740cggggattcc caatacgagg
tcgccaacat cttcttctgg aggccgtggt tggcttgtat 4800ggagcagcag
acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
4860ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
ttgacggcaa 4920tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
atcgtccgat ccggagccgg 4980gactgtcggg cgtacacaaa tcgcccgcag
aagcgcggcc gtctggaccg atggctgtgt 5040agaagtactc gccgatagtg
gaaaccgacg ccccagcact cgtccgaggg caaaggaata 5100gcacgtgcta
cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
5160cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
tggagttctt 5220cgcccacccc aacttgttta ttgcagctta taatggttac
aaataaagca atagcatcac 5280aaatttcaca aataaagcat ttttttcact
gcattctagt tgtggtttgt ccaaactcat 5340caatgtatct tatcatgtct
gtataccgtc gacctctagc tagagcttgg cgtaatcatg 5400gtcatagctg
tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
5460cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
cattaattgc 5520gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
tgccagctgc attaatgaat 5580cggccaacgc gcggggagag gcggtttgcg
tattgggcgc tcttccgctt cctcgctcac 5640tgactcgctg cgctcggtcg
ttcggctgcg gcgagcggta tcagctcact caaaggcggt 5700aatacggtta
tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
5760gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
ggctccgccc 5820ccctgacgag catcacaaaa atcgacgctc aagtcagagg
tggcgaaacc cgacaggact 5880ataaagatac caggcgtttc cccctggaag
ctccctcgtg cgctctcctg ttccgaccct 5940gccgcttacc ggatacctgt
ccgcctttct cccttcggga agcgtggcgc tttctcaatg 6000ctcacgctgt
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
6060cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
ttgagtccaa 6120cccggtaaga cacgacttat cgccactggc agcagccact
ggtaacagga ttagcagagc 6180gaggtatgta ggcggtgcta cagagttctt
gaagtggtgg cctaactacg gctacactag 6240aaggacagta tttggtatct
gcgctctgct gaagccagtt accttcggaa aaagagttgg 6300tagctcttga
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
6360gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
ctacggggtc 6420tgacgctcag tggaacgaaa actcacgtta agggattttg
gtcatgagat tatcaaaaag 6480gatcttcacc tagatccttt taaattaaaa
atgaagtttt aaatcaatct aaagtatata 6540tgagtaaact tggtctgaca
gttaccaatg cttaatcagt gaggcaccta tctcagcgat 6600ctgtctattt
cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
6660ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
gctcaccggc 6720tccagattta tcagcaataa accagccagc cggaagggcc
gagcgcagaa gtggtcctgc 6780aactttatcc gcctccatcc agtctattaa
ttgttgccgg gaagctagag taagtagttc 6840gccagttaat agtttgcgca
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 6900gtcgtttggt
atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
6960ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
tcagaagtaa 7020gttggccgca gtgttatcac tcatggttat ggcagcactg
cataattctc ttactgtcat 7080gccatccgta agatgctttt ctgtgactgg
tgagtactca accaagtcat tctgagaata 7140gtgtatgcgg cgaccgagtt
gctcttgccc ggcgtcaata cgggataata ccgcgccaca 7200tagcagaact
ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
7260gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
actgatcttc 7320agcatctttt actttcacca gcgtttctgg gtgagcaaaa
acaggaaggc aaaatgccgc 7380aaaaaaggga ataagggcga cacggaaatg
ttgaatactc atactcttcc tttttcaata 7440ttattgaagc atttatcagg
gttattgtct catgagcgga tacatatttg aatgtattta 7500gaaaaataaa
caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
755867576DNAArtificial SequenceSynthetic Oligonucleotide
6gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct
ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga
ctcactatag ggagacccaa gctggctaga 900ggtaccaagc ttggatctca
ccatggagtt tgggctgtgc tggattttcc tcgttgctct 960tttaagaggt
gtccagtgtc aggtgcagct ggtggagtct gggggaggcg tggtccagcc
1020tgggaggtcc ctgagactct cctgtgcagc ctctggattc accttcatta
gctatggcat 1080gcactgggtc cgccaggctc caggcaaggg gctggagtgg
gtggcagtta tatcatatga 1140tggaagtaat aaatactatg cagactccgt
gaagggccga ttcaccatct ccagagacaa 1200ttccaagaac acgctgtatc
tgcaaatgaa cagcctgaga gctgaggaca cggctgtgta 1260ttactgtgcg
agagtattag tgggagcttt atattattat aactactacg ggatggacgt
1320ctggggccaa gggaccacgg tcaccgtctc ctcagcctcc accaagggcc
catcggtctt 1380ccccctggca ccctctagca agagcacctc tgggggcaca
gcggccctgg gctgcctggt 1440caaggactac ttccccgaac cggtgacggt
gtcgtggaac tcaggcgccc tgaccagcgg 1500cgtgcacacc ttcccggctg
tcctacagtc ctcaggactc tactccctca gcagcgtggt 1560gaccgtgccc
tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc
1620cagcaacacc aaggtggaca agagagttgg tgagaggcca gcacagggag
ggagggtgtc 1680tgctggaagc caggctcagc gctcctgcct ggacgcatcc
cggctatgca gtcccagtcc 1740agggcagcaa ggcaggcccc gtctgcctct
tcacccggag gcctctgccc gccccactca 1800tgctcaggga gagggtcttc
tggctttttc cccaggctct gggcaggcac aggctaggtg 1860cccctaaccc
aggccctgca cacaaagggg caggtgctgg gctcagacct gccaagagcc
1920atatccggga ggaccctgcc cctgacctaa gcccacccca aaggccaaac
tctccactcc 1980ctcagctcgg acaccttctc tcctcccaga ttccagtaac
tcccaatctt ctctctgcag 2040agcccaaatc ttgtgacaaa actcacacat
gcccaccgtg cccaggtaag ccagcccagg 2100cctcgccctc cagctcaagg
cgggacaggt gccctagagt agcctgcatc cagggacagg 2160ccccagccgg
gtgctgacac gtccacctcc atctcttcct cagcacctga actcctgggg
2220ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc 2280cctgaggtca catgcgtggt ggtggacgtg agccacgaag
accctgaggt caagttcaac 2340tggtacgtgg acggcgtgga ggtgcataat
gccaagacaa agccgcggga ggagcagtac 2400aacagcacgt accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc 2460aaggagtaca
agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc
2520tccaaagcca aaggtgggac ccgtggggtg cgagggccac atggacagag
gccggctcgg 2580cccaccctct gccctgagag tgaccgctgt accaacctct
gtccctacag ggcagccccg 2640agaaccacag gtgtacaccc tgcccccatc
ccgggaggag atgaccaaga accaggtcag 2700cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc gccgtggagt gggagagcaa 2760tgggcagccg
gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt
2820cttcctctat agcaagctca ccgtggacaa gagcaggtgg cagcagggga
acgtcttctc 2880atgctccgtg atgcatgagg ctctgcacaa ccactacacg
cagaagagcc tctccctgtc 2940tccgggtaaa tgagaattcc tcgagtctag
agggcccgtt taaacccgct gatcagcctc 3000gactgtgcct tctagttgcc
agccatctgt tgtttgcccc tcccccgtgc cttccttgac 3060cctggaaggt
gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
3120tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
agggggagga 3180ttgggaagac aatagcaggc atgctgggga tgcggtgggc
tctatggctt ctgaggcgga 3240aagaaccagc tggggctcta gggggtatcc
ccacgcgccc tgtagcggcg cattaagcgc 3300ggcgggtgtg gtggttacgc
gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 3360tcctttcgct
ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
3420aaatcggggc atccctttag ggttccgatt tagtgcttta cggcacctcg
accccaaaaa 3480acttgattag ggtgatggtt cacgtagtgg gccatcgccc
tgatagacgg tttttcgccc 3540tttgacgttg gagtccacgt tctttaatag
tggactcttg ttccaaactg gaacaacact 3600caaccctatc tcggtctatt
cttttgattt ataagggatt ttggggattt cggcctattg 3660gttaaaaaat
gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
3720cagttagggt gtggaaagtc cccaggctcc ccaggcaggc agaagtatgc
aaagcatgca 3780tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
tccccagcag gcagaagtat 3840gcaaagcatg catctcaatt agtcagcaac
catagtcccg cccctaactc cgcccatccc 3900gcccctaact ccgcccagtt
ccgcccattc tccgccccat ggctgactaa ttttttttat 3960ttatgcagag
gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
4020ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
ttttcggatc 4080tgatcagcac gtgatgaaaa agcctgaact caccgcgacg
tctgtcgaga agtttctgat 4140cgaaaagttc gacagcgtct ccgacctgat
gcagctctcg gagggcgaag aatctcgtgc 4200tttcagcttc gatgtaggag
ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 4260tttctacaaa
gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
4320agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
gccgtgcaca 4380gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
gctgttctgc agccggtcgc 4440ggaggccatg gatgcgatcg ctgcggccga
tcttagccag acgagcgggt tcggcccatt 4500cggaccgcaa ggaatcggtc
aatacactac atggcgtgat ttcatatgcg cgattgctga 4560tccccatgtg
tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
4620ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
acctcgtgca 4680cgcggatttc ggctccaaca atgtcctgac ggacaatggc
cgcataacag cggtcattga 4740ctggagcgag gcgatgttcg gggattccca
atacgaggtc gccaacatct tcttctggag 4800gccgtggttg gcttgtatgg
agcagcagac gcgctacttc gagcggaggc atccggagct 4860tgcaggatcg
ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
4920gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
gcgacgcaat 4980cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
gcccgcagaa gcgcggccgt 5040ctggaccgat ggctgtgtag aagtactcgc
cgatagtgga aaccgacgcc ccagcactcg 5100tccgagggca aaggaatagc
acgtgctacg agatttcgat tccaccgccg ccttctatga 5160aaggttgggc
ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
5220tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
atggttacaa 5280ataaagcaat agcatcacaa atttcacaaa taaagcattt
ttttcactgc attctagttg 5340tggtttgtcc aaactcatca atgtatctta
tcatgtctgt ataccgtcga cctctagcta 5400gagcttggcg taatcatggt
catagctgtt tcctgtgtga aattgttatc cgctcacaat 5460tccacacaac
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
5520ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
acctgtcgtg 5580ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
ggtttgcgta ttgggcgctc 5640ttccgcttcc tcgctcactg actcgctgcg
ctcggtcgtt cggctgcggc gagcggtatc 5700agctcactca aaggcggtaa
tacggttatc cacagaatca ggggataacg caggaaagaa 5760catgtgagca
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
5820tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
gtcagaggtg 5880gcgaaacccg acaggactat aaagatacca ggcgtttccc
cctggaagct ccctcgtgcg 5940ctctcctgtt ccgaccctgc cgcttaccgg
atacctgtcc gcctttctcc cttcgggaag 6000cgtggcgctt tctcaatgct
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 6060caagctgggc
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
6120ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
cagccactgg 6180taacaggatt agcagagcga ggtatgtagg cggtgctaca
gagttcttga agtggtggcc 6240taactacggc tacactagaa ggacagtatt
tggtatctgc gctctgctga agccagttac 6300cttcggaaaa agagttggta
gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 6360tttttttgtt
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
6420gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
ggattttggt 6480catgagatta tcaaaaagga tcttcaccta gatcctttta
aattaaaaat gaagttttaa 6540atcaatctaa agtatatatg agtaaacttg
gtctgacagt taccaatgct taatcagtga 6600ggcacctatc tcagcgatct
gtctatttcg ttcatccata gttgcctgac tccccgtcgt 6660gtagataact
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
6720agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
gaagggccga 6780gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
tctattaatt gttgccggga 6840agctagagta agtagttcgc cagttaatag
tttgcgcaac gttgttgcca ttgctacagg 6900catcgtggtg tcacgctcgt
cgtttggtat ggcttcattc agctccggtt cccaacgatc 6960aaggcgagtt
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
7020gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
cagcactgca 7080taattctctt actgtcatgc catccgtaag atgcttttct
gtgactggtg agtactcaac 7140caagtcattc tgagaatagt gtatgcggcg
accgagttgc tcttgcccgg cgtcaatacg 7200ggataatacc gcgccacata
gcagaacttt aaaagtgctc atcattggaa aacgttcttc 7260ggggcgaaaa
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
7320tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
gagcaaaaac 7380aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
cggaaatgtt gaatactcat 7440actcttcctt tttcaatatt attgaagcat
ttatcagggt tattgtctca tgagcggata 7500catatttgaa tgtatttaga
aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 7560agtgccacct gacgtc
757677561DNAArtificial SequenceSynthetic Oligonucleotide
7gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct
ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga
ctcactatag ggagacccaa gctggctaga 900ggtaccggat ctcaccatgg
agttggggct gagctgggtt ttcctcgttg ctcttttaag 960aggtgtccag
tgtcaggagc agctggtgga gtctggggga ggcgtggtcc agcctgggag
1020gtccctgaga ctctcctgtg cagcgtctgg attcaccttc agtacctatg
gcatgcactg 1080ggtccgccag gctccaggca aggggctgga gtgggtggca
gttacatggc atgatggaag 1140taataaatac tatgcagact ccgtgaaggg
ccgattcacc atctccagag acaactccaa 1200gaacacgctg tatctgcaaa
tgaacagcct gagagccgag gacacggctg tgtattactg 1260tgcgagagga
ggagtgggag caacttacta ctactactac ggtatggacg tctggggcca
1320agggaccacg gtcaccgtct cctcagcctc caccaagggc ccatcggtct
tccccctggc 1380accctctagc aagagcacct ctgggggcac agcggccctg
ggctgcctgg tcaaggacta 1440cttccccgaa ccggtgacgg tgtcgtggaa
ctcaggcgcc ctgaccagcg gcgtgcacac 1500cttcccggct gtcctacagt
cctcaggact ctactccctc agcagcgtgg tgaccgtgcc 1560ctccagcagc
ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac
1620caaggtggac aagagagttg gtgagaggcc agcacaggga gggagggtgt
ctgctggaag 1680ccaggctcag cgctcctgcc tggacgcatc ccggctatgc
agtcccagtc cagggcagca 1740aggcaggccc cgtctgcctc ttcacccgga
ggcctctgcc cgccccactc atgctcaggg 1800agagggtctt ctggcttttt
ccccaggctc tgggcaggca caggctaggt gcccctaacc 1860caggccctgc
acacaaaggg gcaggtgctg ggctcagacc tgccaagagc catatccggg
1920aggaccctgc ccctgaccta agcccacccc aaaggccaaa ctctccactc
cctcagctcg 1980gacaccttct ctcctcccag attccagtaa ctcccaatct
tctctctgca gagcccaaat 2040cttgtgacaa aactcacaca tgcccaccgt
gcccaggtaa gccagcccag gcctcgccct 2100ccagctcaag gcgggacagg
tgccctagag tagcctgcat ccagggacag gccccagccg 2160ggtgctgaca
cgtccacctc catctcttcc tcagcacctg aactcctggg gggaccgtca
2220gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac
ccctgaggtc 2280acatgcgtgg tggtggacgt gagccacgaa gaccctgagg
tcaagttcaa ctggtacgtg 2340gacggcgtgg aggtgcataa tgccaagaca
aagccgcggg aggagcagta caacagcacg 2400taccgtgtgg tcagcgtcct
caccgtcctg caccaggact ggctgaatgg caaggagtac 2460aagtgcaagg
tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc
2520aaaggtggga cccgtggggt gcgagggcca catggacaga ggccggctcg
gcccaccctc 2580tgccctgaga gtgaccgctg taccaacctc tgtccctaca
gggcagcccc gagaaccaca 2640ggtgtacacc ctgcccccat cccgggagga
gatgaccaag aaccaggtca gcctgacctg 2700cctggtcaaa ggcttctatc
ccagcgacat cgccgtggag tgggagagca atgggcagcc 2760ggagaacaac
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta
2820tagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct
catgctccgt 2880gatgcatgag gctctgcaca accactacac gcagaagagc
ctctccctgt ctccgggtaa 2940atgactcgag tctagagggc ccgtttaaac
ccgctgatca gcctcgactg tgccttctag 3000ttgccagcca tctgttgttt
gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 3060tcccactgtc
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
3120ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
aagacaatag 3180caggcatgct ggggatgcgg tgggctctat ggcttctgag
gcggaaagaa ccagctgggg 3240ctctaggggg tatccccacg cgccctgtag
cggcgcatta agcgcggcgg gtgtggtggt 3300tacgcgcagc gtgaccgcta
cacttgccag cgccctagcg cccgctcctt tcgctttctt 3360cccttccttt
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc ggggcatccc
3420tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
attagggtga 3480tggttcacgt agtgggccat cgccctgata gacggttttt
cgccctttga cgttggagtc 3540cacgttcttt aatagtggac tcttgttcca
aactggaaca acactcaacc ctatctcggt 3600ctattctttt gatttataag
ggattttggg gatttcggcc tattggttaa aaaatgagct 3660gatttaacaa
aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
3720aagtccccag gctccccagg caggcagaag tatgcaaagc atgcatctca
attagtcagc 3780aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
agtatgcaaa gcatgcatct 3840caattagtca gcaaccatag tcccgcccct
aactccgccc atcccgcccc taactccgcc 3900cagttccgcc cattctccgc
cccatggctg actaattttt tttatttatg cagaggccga 3960ggccgcctct
gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
4020cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
agcacgtgat 4080gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt
ctgatcgaaa agttcgacag 4140cgtctccgac ctgatgcagc tctcggaggg
cgaagaatct cgtgctttca gcttcgatgt 4200aggagggcgt ggatatgtcc
tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 4260ttatgtttat
cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
4320ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
tcacgttgca 4380agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
gtcgcggagg ccatggatgc 4440gatcgctgcg gccgatctta gccagacgag
cgggttcggc ccattcggac cgcaaggaat 4500cggtcaatac actacatggc
gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 4560ctggcaaact
gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct
4620gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
atttcggctc 4680caacaatgtc ctgacggaca atggccgcat aacagcggtc
attgactgga gcgaggcgat 4740gttcggggat tcccaatacg aggtcgccaa
catcttcttc tggaggccgt ggttggcttg 4800tatggagcag cagacgcgct
acttcgagcg gaggcatccg gagcttgcag gatcgccgcg 4860gctccgggcg
tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg
4920caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
gatccggagc 4980cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
gccgtctgga ccgatggctg 5040tgtagaagta ctcgccgata gtggaaaccg
acgccccagc actcgtccga gggcaaagga 5100atagcacgtg ctacgagatt
tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 5160aatcgttttc
cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
5220cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
gcaatagcat 5280cacaaatttc acaaataaag catttttttc actgcattct
agttgtggtt tgtccaaact 5340catcaatgta tcttatcatg tctgtatacc
gtcgacctct agctagagct tggcgtaatc 5400atggtcatag ctgtttcctg
tgtgaaattg ttatccgctc acaattccac acaacatacg 5460agccggaagc
ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
5520tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
tgcattaatg 5580aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
cgctcttccg cttcctcgct 5640cactgactcg ctgcgctcgg tcgttcggct
gcggcgagcg gtatcagctc actcaaaggc 5700ggtaatacgg ttatccacag
aatcagggga taacgcagga aagaacatgt gagcaaaagg 5760ccagcaaaag
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
5820cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
acccgacagg 5880actataaaga taccaggcgt ttccccctgg aagctccctc
gtgcgctctc ctgttccgac 5940cctgccgctt accggatacc tgtccgcctt
tctcccttcg ggaagcgtgg cgctttctca 6000atgctcacgc tgtaggtatc
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6060gcacgaaccc
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
6120caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
ggattagcag 6180agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
tggcctaact acggctacac 6240tagaaggaca gtatttggta tctgcgctct
gctgaagcca gttaccttcg gaaaaagagt 6300tggtagctct tgatccggca
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6360gcagcagatt
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
6420gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
gattatcaaa 6480aaggatcttc acctagatcc ttttaaatta aaaatgaagt
tttaaatcaa tctaaagtat 6540atatgagtaa acttggtctg acagttacca
atgcttaatc agtgaggcac ctatctcagc 6600gatctgtcta tttcgttcat
ccatagttgc ctgactcccc gtcgtgtaga taactacgat 6660acgggagggc
ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
6720ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
gaagtggtcc 6780tgcaacttta tccgcctcca tccagtctat taattgttgc
cgggaagcta gagtaagtag 6840ttcgccagtt aatagtttgc gcaacgttgt
tgccattgct acaggcatcg tggtgtcacg 6900ctcgtcgttt ggtatggctt
cattcagctc cggttcccaa cgatcaaggc gagttacatg 6960atcccccatg
ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
7020taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
ctcttactgt 7080catgccatcc gtaagatgct tttctgtgac tggtgagtac
tcaaccaagt cattctgaga 7140atagtgtatg cggcgaccga gttgctcttg
cccggcgtca atacgggata ataccgcgcc 7200acatagcaga actttaaaag
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 7260aaggatctta
ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
7320ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
ggcaaaatgc 7380cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
ctcatactct tcctttttca 7440atattattga agcatttatc agggttattg
tctcatgagc ggatacatat ttgaatgtat 7500ttagaaaaat aaacaaatag
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 7560c
756186082DNAArtificial SequenceSynthetic Oligonucleotide
8gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct
ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga
ctcactatag ggagacccaa gctggctaga 900aagcttggat ctcaccatga
gggtccctgc tcagctcctg ggactcctgc tgctctggct 960cccagatacc
agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt
1020aggagacaga gtcaccatca cttgccgggc gagtcagggc attagcaatt
atttagcctg 1080gtatcagcag aaaacaggga aagttcctaa gttcctgatc
tatgaagcat ccactttgca 1140atcaggggtc ccatctcggt tcagtggcgg
tggatctggg acagatttca ctctcaccat 1200cagcagcctg cagcctgaag
atgttgcaac ttattactgt caaaattata acagtgcccc 1260attcactttc
ggccctggga ccaaagtgga tatcaaacga actgtggctg caccctctgt
1320cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgctagcg
ttgtgtgcct 1380gctgaataac ttctatccca gagaggccaa agtacagtgg
aaggtggata acgccctcca 1440atcgggtaac tcccaggaga gtgtcacaga
gcaggacagc aaggacagca cctacagcct 1500cagcagcacc ctgacgctga
gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga 1560agtcacccat
cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta
1620ggaattcgcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca
gcctcgactg 1680tgccttctag ttgccagcca tctgttgttt gcccctcccc
cgtgccttcc ttgaccctgg 1740aaggtgccac tcccactgtc ctttcctaat
aaaatgagga aattgcatcg cattgtctga 1800gtaggtgtca ttctattctg
gggggtgggg tggggcagga cagcaagggg gaggattggg 1860aagacaatag
caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
1920ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
agcgcggcgg 1980gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
cgccctagcg cccgctcctt 2040tcgctttctt cccttccttt ctcgccacgt
tcgccggctt tccccgtcaa gctctaaatc 2100ggggcatccc tttagggttc
cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2160attagggtga
tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
2220cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
acactcaacc 2280ctatctcggt ctattctttt gatttataag ggattttggg
gatttcggcc tattggttaa 2340aaaatgagct gatttaacaa aaatttaacg
cgaattaatt ctgtggaatg tgtgtcagtt 2400agggtgtgga aagtccccag
gctccccagg caggcagaag tatgcaaagc atgcatctca 2460attagtcagc
aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
2520gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
atcccgcccc 2580taactccgcc cagttccgcc cattctccgc cccatggctg
actaattttt tttatttatg 2640cagaggccga ggccgcctct gcctctgagc
tattccagaa gtagtgagga ggcttttttg 2700gaggcctagg cttttgcaaa
aagctcccgg gagcttgtat atccattttc ggatctgatc 2760aagagacagg
atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
2820cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
atcggctgct 2880ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
ggttcttttt gtcaagaccg 2940acctgtccgg tgccctgaat gaactgcagg
acgaggcagc gcggctatcg tggctggcca 3000cgacgggcgt tccttgcgca
gctgtgctcg acgttgtcac tgaagcggga agggactggc 3060tgctattggg
cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
3120aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
gctacctgcc 3180cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
tactcggatg gaagccggtc 3240ttgtcgatca ggatgatctg gacgaagagc
atcaggggct cgcgccagcc gaactgttcg 3300ccaggctcaa ggcgcgcatg
cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 3360gcttgccgaa
tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
3420tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
gctgaagagc 3480ttggcggcga atgggctgac cgcttcctcg tgctttacgg
tatcgccgct cccgattcgc 3540agcgcatcgc cttctatcgc cttcttgacg
agttcttctg agcgggactc tggggttcga 3600aatgaccgac caagcgacgc
ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 3660ctatgaaagg
ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
3720cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
cttataatgg 3780ttacaaataa agcaatagca tcacaaattt cacaaataaa
gcattttttt cactgcattc 3840tagttgtggt ttgtccaaac tcatcaatgt
atcttatcat gtctgtatac cgtcgacctc 3900tagctagagc ttggcgtaat
catggtcata gctgtttcct gtgtgaaatt gttatccgct 3960cacaattcca
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
4020agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
cgggaaacct 4080gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
agaggcggtt tgcgtattgg 4140gcgctcttcc gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc 4200ggtatcagct cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg 4260aaagaacatg
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
4320ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca 4380gaggtggcga aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct 4440cgtgcgctct cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc 4500gggaagcgtg gcgctttctc
aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4560tcgctccaag
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
4620cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc 4680cactggtaac aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg 4740gtggcctaac tacggctaca ctagaaggac
agtatttggt atctgcgctc tgctgaagcc 4800agttaccttc ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag 4860cggtggtttt
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
4920tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
gttaagggat 4980tttggtcatg agattatcaa aaaggatctt cacctagatc
cttttaaatt aaaaatgaag 5040ttttaaatca atctaaagta tatatgagta
aacttggtct gacagttacc aatgcttaat 5100cagtgaggca cctatctcag
cgatctgtct atttcgttca tccatagttg cctgactccc 5160cgtcgtgtag
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
5220accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
cagccggaag 5280ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
atccagtcta ttaattgttg 5340ccgggaagct agagtaagta gttcgccagt
taatagtttg cgcaacgttg ttgccattgc 5400tacaggcatc gtggtgtcac
gctcgtcgtt tggtatggct tcattcagct ccggttccca 5460acgatcaagg
cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
5520tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
ttatggcagc 5580actgcataat tctcttactg tcatgccatc cgtaagatgc
ttttctgtga ctggtgagta 5640ctcaaccaag tcattctgag aatagtgtat
gcggcgaccg agttgctctt gcccggcgtc 5700aatacgggat aataccgcgc
cacatagcag aactttaaaa gtgctcatca ttggaaaacg 5760ttcttcgggg
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
5820cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
ctgggtgagc 5880aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
gcgacacgga aatgttgaat 5940actcatactc ttcctttttc aatattattg
aagcatttat cagggttatt gtctcatgag 6000cggatacata tttgaatgta
tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6060ccgaaaagtg
ccacctgacg tc 608296082DNAArtificial SequenceSynthetic
Oligonucleotide 9gacggatcgg gagatctccc gatcccctat ggtcgactct
cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag
gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg
ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt
aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
900aagcttggat ctcaccatga gggtccccgc tcagctcctg gggctcctgc
tgctctgttt 960cccaggtgcc agatgtgaca tccagatgac ccagtctcca
tcctcactgt ctgcatctgt 1020aggagacaga gtcaccatca cttgtcgggc
gagtcagggc attaccaatt atttagcctg 1080gtttcagcag aaaccaggga
aagcccctaa gtcccttatc tatgctgcat ccagtttgca 1140aagtggggtc
ccatcaaagt tcagcggcag tggatctggg acagatttca gtctcaccat
1200cagcagcctg cagcctgaag attttgcaac ttattactgc caacagtata
atagttaccc 1260gatcaccttc ggccaaggga cacgactgga gattaaacga
actgtggctg caccatctgt 1320cttcatcttc ccgccatctg atgagcagtt
gaaatctgga actgctagcg ttgtgtgcct 1380gctgaataac ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca 1440atcgggtaac
tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct
1500cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga 1560agtcacccat cagggcctga gctcgcccgt cacaaagagc
ttcaacaggg gagagtgtta 1620ggaattcgcg gccgctcgag tctagagggc
ccgtttaaac ccgctgatca gcctcgactg 1680tgccttctag ttgccagcca
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1740aaggtgccac
tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
1800gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
gaggattggg 1860aagacaatag caggcatgct ggggatgcgg tgggctctat
ggcttctgag gcggaaagaa 1920ccagctgggg ctctaggggg tatccccacg
cgccctgtag cggcgcatta agcgcggcgg 1980gtgtggtggt tacgcgcagc
gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2040tcgctttctt
cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
2100ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc
aaaaaacttg 2160attagggtga tggttcacgt agtgggccat cgccctgata
gacggttttt cgccctttga 2220cgttggagtc cacgttcttt aatagtggac
tcttgttcca aactggaaca acactcaacc 2280ctatctcggt ctattctttt
gatttataag ggattttggg gatttcggcc tattggttaa 2340aaaatgagct
gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
2400agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc
atgcatctca 2460attagtcagc aaccaggtgt ggaaagtccc caggctcccc
agcaggcaga agtatgcaaa 2520gcatgcatct caattagtca gcaaccatag
tcccgcccct aactccgccc atcccgcccc 2580taactccgcc cagttccgcc
cattctccgc cccatggctg actaattttt tttatttatg 2640cagaggccga
ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
2700gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
ggatctgatc 2760aagagacagg atgaggatcg tttcgcatga ttgaacaaga
tggattgcac gcaggttctc 2820cggccgcttg ggtggagagg ctattcggct
atgactgggc acaacagaca atcggctgct 2880ctgatgccgc cgtgttccgg
ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 2940acctgtccgg
tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
3000cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
agggactggc 3060tgctattggg
cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
3120aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
gctacctgcc 3180cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
tactcggatg gaagccggtc 3240ttgtcgatca ggatgatctg gacgaagagc
atcaggggct cgcgccagcc gaactgttcg 3300ccaggctcaa ggcgcgcatg
cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 3360gcttgccgaa
tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
3420tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
gctgaagagc 3480ttggcggcga atgggctgac cgcttcctcg tgctttacgg
tatcgccgct cccgattcgc 3540agcgcatcgc cttctatcgc cttcttgacg
agttcttctg agcgggactc tggggttcga 3600aatgaccgac caagcgacgc
ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 3660ctatgaaagg
ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
3720cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
cttataatgg 3780ttacaaataa agcaatagca tcacaaattt cacaaataaa
gcattttttt cactgcattc 3840tagttgtggt ttgtccaaac tcatcaatgt
atcttatcat gtctgtatac cgtcgacctc 3900tagctagagc ttggcgtaat
catggtcata gctgtttcct gtgtgaaatt gttatccgct 3960cacaattcca
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
4020agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
cgggaaacct 4080gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
agaggcggtt tgcgtattgg 4140gcgctcttcc gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc 4200ggtatcagct cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg 4260aaagaacatg
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
4320ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca 4380gaggtggcga aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct 4440cgtgcgctct cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc 4500gggaagcgtg gcgctttctc
aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4560tcgctccaag
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
4620cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc 4680cactggtaac aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg 4740gtggcctaac tacggctaca ctagaaggac
agtatttggt atctgcgctc tgctgaagcc 4800agttaccttc ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag 4860cggtggtttt
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
4920tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
gttaagggat 4980tttggtcatg agattatcaa aaaggatctt cacctagatc
cttttaaatt aaaaatgaag 5040ttttaaatca atctaaagta tatatgagta
aacttggtct gacagttacc aatgcttaat 5100cagtgaggca cctatctcag
cgatctgtct atttcgttca tccatagttg cctgactccc 5160cgtcgtgtag
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
5220accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
cagccggaag 5280ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
atccagtcta ttaattgttg 5340ccgggaagct agagtaagta gttcgccagt
taatagtttg cgcaacgttg ttgccattgc 5400tacaggcatc gtggtgtcac
gctcgtcgtt tggtatggct tcattcagct ccggttccca 5460acgatcaagg
cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
5520tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
ttatggcagc 5580actgcataat tctcttactg tcatgccatc cgtaagatgc
ttttctgtga ctggtgagta 5640ctcaaccaag tcattctgag aatagtgtat
gcggcgaccg agttgctctt gcccggcgtc 5700aatacgggat aataccgcgc
cacatagcag aactttaaaa gtgctcatca ttggaaaacg 5760ttcttcgggg
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
5820cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
ctgggtgagc 5880aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
gcgacacgga aatgttgaat 5940actcatactc ttcctttttc aatattattg
aagcatttat cagggttatt gtctcatgag 6000cggatacata tttgaatgta
tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6060ccgaaaagtg
ccacctgacg tc 6082106082DNAArtificial SequenceSynthetic
Oligonucleotide 10gacggatcgg gagatctccc gatcccctat ggtcgactct
cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag
gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg
ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt
aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
900aagcttggat ctcaccatga gggtccctgc tcagctcctg gggctcctgc
tgctctgttt 960cccaggtgcc agatgtgaca tccagatgac ccagtctcca
tcctcactgt ctgcatctgt 1020aggagacaga gtcaccatca cttgtcgggc
gagtcagggc attagccatt atttagcctg 1080gtttcagcag aaaccaggga
aagcccctaa gtccctgatc tatgctgcat ccagtttgca 1140aagtggggtc
ccatcaaagt tcagcggcag tggatctggg acagatttca ctctcaccat
1200cagcagccta cagcctgaag attttgcaac ttattactgc caacagtata
atagtttccc 1260gctcactttc ggcggaggga ccaaggtgga gatcaaacga
actgtggctg caccatctgt 1320cttcatcttc ccgccatctg atgagcagtt
gaaatctgga actgctagcg ttgtgtgcct 1380gctgaataac ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca 1440atcgggtaac
tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct
1500cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga 1560agtcacccat cagggcctga gctcgcccgt cacaaagagc
ttcaacaggg gagagtgtta 1620ggaattcgcg gccgctcgag tctagagggc
ccgtttaaac ccgctgatca gcctcgactg 1680tgccttctag ttgccagcca
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1740aaggtgccac
tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
1800gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
gaggattggg 1860aagacaatag caggcatgct ggggatgcgg tgggctctat
ggcttctgag gcggaaagaa 1920ccagctgggg ctctaggggg tatccccacg
cgccctgtag cggcgcatta agcgcggcgg 1980gtgtggtggt tacgcgcagc
gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2040tcgctttctt
cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
2100ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc
aaaaaacttg 2160attagggtga tggttcacgt agtgggccat cgccctgata
gacggttttt cgccctttga 2220cgttggagtc cacgttcttt aatagtggac
tcttgttcca aactggaaca acactcaacc 2280ctatctcggt ctattctttt
gatttataag ggattttggg gatttcggcc tattggttaa 2340aaaatgagct
gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
2400agggtgtgga aagtccccag gctccccagg caggcagaag tatgcaaagc
atgcatctca 2460attagtcagc aaccaggtgt ggaaagtccc caggctcccc
agcaggcaga agtatgcaaa 2520gcatgcatct caattagtca gcaaccatag
tcccgcccct aactccgccc atcccgcccc 2580taactccgcc cagttccgcc
cattctccgc cccatggctg actaattttt tttatttatg 2640cagaggccga
ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
2700gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
ggatctgatc 2760aagagacagg atgaggatcg tttcgcatga ttgaacaaga
tggattgcac gcaggttctc 2820cggccgcttg ggtggagagg ctattcggct
atgactgggc acaacagaca atcggctgct 2880ctgatgccgc cgtgttccgg
ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 2940acctgtccgg
tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
3000cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
agggactggc 3060tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
tcaccttgct cctgccgaga 3120aagtatccat catggctgat gcaatgcggc
ggctgcatac gcttgatccg gctacctgcc 3180cattcgacca ccaagcgaaa
catcgcatcg agcgagcacg tactcggatg gaagccggtc 3240ttgtcgatca
ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
3300ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
ggcgatgcct 3360gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
attcatcgac tgtggccggc 3420tgggtgtggc ggaccgctat caggacatag
cgttggctac ccgtgatatt gctgaagagc 3480ttggcggcga atgggctgac
cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 3540agcgcatcgc
cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
3600aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
ccgccgcctt 3660ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
ggctggatga tcctccagcg 3720cggggatctc atgctggagt tcttcgccca
ccccaacttg tttattgcag cttataatgg 3780ttacaaataa agcaatagca
tcacaaattt cacaaataaa gcattttttt cactgcattc 3840tagttgtggt
ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
3900tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
gttatccgct 3960cacaattcca cacaacatac gagccggaag cataaagtgt
aaagcctggg gtgcctaatg 4020agtgagctaa ctcacattaa ttgcgttgcg
ctcactgccc gctttccagt cgggaaacct 4080gtcgtgccag ctgcattaat
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 4140gcgctcttcc
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
4200ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
ataacgcagg 4260aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
cgtaaaaagg ccgcgttgct 4320ggcgtttttc cataggctcc gcccccctga
cgagcatcac aaaaatcgac gctcaagtca 4380gaggtggcga aacccgacag
gactataaag ataccaggcg tttccccctg gaagctccct 4440cgtgcgctct
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
4500gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg
tgtaggtcgt 4560tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
cccgaccgct gcgccttatc 4620cggtaactat cgtcttgagt ccaacccggt
aagacacgac ttatcgccac tggcagcagc 4680cactggtaac aggattagca
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 4740gtggcctaac
tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
4800agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
ccgctggtag 4860cggtggtttt tttgtttgca agcagcagat tacgcgcaga
aaaaaaggat ctcaagaaga 4920tcctttgatc ttttctacgg ggtctgacgc
tcagtggaac gaaaactcac gttaagggat 4980tttggtcatg agattatcaa
aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5040ttttaaatca
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
5100cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
cctgactccc 5160cgtcgtgtag ataactacga tacgggaggg cttaccatct
ggccccagtg ctgcaatgat 5220accgcgagac ccacgctcac cggctccaga
tttatcagca ataaaccagc cagccggaag 5280ggccgagcgc agaagtggtc
ctgcaacttt atccgcctcc atccagtcta ttaattgttg 5340ccgggaagct
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
5400tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
ccggttccca 5460acgatcaagg cgagttacat gatcccccat gttgtgcaaa
aaagcggtta gctccttcgg 5520tcctccgatc gttgtcagaa gtaagttggc
cgcagtgtta tcactcatgg ttatggcagc 5580actgcataat tctcttactg
tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 5640ctcaaccaag
tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
5700aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
ttggaaaacg 5760ttcttcgggg cgaaaactct caaggatctt accgctgttg
agatccagtt cgatgtaacc 5820cactcgtgca cccaactgat cttcagcatc
ttttactttc accagcgttt ctgggtgagc 5880aaaaacagga aggcaaaatg
ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 5940actcatactc
ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
6000cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
gcacatttcc 6060ccgaaaagtg ccacctgacg tc 6082116085DNAArtificial
SequenceSynthetic Oligonucleotide 11gacggatcgg gagatctccc
gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat
ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat
ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc
attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa
atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata
atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca
atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca
gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc
agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt
ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag
ggagacccaa gctggctaga 900aagcttggat ctcaccatga gggtccccgc
tcagcttctc ttccttctgc tactctggct 960cccagatacc actggaggaa
tagtgatgac gcagtctcca gccaccctgt ctgtgtctcc 1020aggggaaaga
gccaccctct cctgcaggac cagtcagagt attggctgga acttagcctg
1080gtaccaacag aaacctggcc aggctcccag gctcctcatc tatggtgcat
cttccaggac 1140cactggtatc ccagccaggt tcagtggcag tgggtctggg
acagagttca ctctcaccat 1200cagcagcctg cagtctgaag attctgcagt
ttattactgt cagcattatg ataactggcc 1260catgtgcagt tttggccagg
ggaccgagct ggagatcaaa cgaactgtgg ctgcaccatc 1320tgtcttcatc
ttcccgccat ctgatgagca gttgaaatct ggaactgcta gcgttgtgtg
1380cctgctgaat aacttctatc ccagagaggc caaagtacag tggaaggtgg
ataacgccct 1440ccaatcgggt aactcccagg agagtgtcac agagcaggac
agcaaggaca gcacctacag 1500cctcagcagc accctgacgc tgagcaaagc
agactacgag aaacacaaag tctacgcctg 1560cgaagtcacc catcagggcc
tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg 1620ttaggaattc
gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga
1680ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
tccttgaccc 1740tggaaggtgc cactcccact gtcctttcct aataaaatga
ggaaattgca tcgcattgtc 1800tgagtaggtg tcattctatt ctggggggtg
gggtggggca ggacagcaag ggggaggatt 1860gggaagacaa tagcaggcat
gctggggatg cggtgggctc tatggcttct gaggcggaaa 1920gaaccagctg
gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
1980cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
gcgcccgctc 2040ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
ctttccccgt caagctctaa 2100atcggggcat ccctttaggg ttccgattta
gtgctttacg gcacctcgac cccaaaaaac 2160ttgattaggg tgatggttca
cgtagtgggc catcgccctg atagacggtt tttcgccctt 2220tgacgttgga
gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
2280accctatctc ggtctattct tttgatttat aagggatttt ggggatttcg
gcctattggt 2340taaaaaatga gctgatttaa caaaaattta acgcgaatta
attctgtgga atgtgtgtca 2400gttagggtgt ggaaagtccc caggctcccc
aggcaggcag aagtatgcaa agcatgcatc 2460tcaattagtc agcaaccagg
tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 2520aaagcatgca
tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
2580ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
ttttttattt 2640atgcagaggc cgaggccgcc tctgcctctg agctattcca
gaagtagtga ggaggctttt 2700ttggaggcct aggcttttgc aaaaagctcc
cgggagcttg tatatccatt ttcggatctg 2760atcaagagac aggatgagga
tcgtttcgca tgattgaaca agatggattg cacgcaggtt 2820ctccggccgc
ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
2880gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
tttgtcaaga 2940ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
agcgcggcta tcgtggctgg 3000ccacgacggg cgttccttgc gcagctgtgc
tcgacgttgt cactgaagcg ggaagggact 3060ggctgctatt gggcgaagtg
ccggggcagg atctcctgtc atctcacctt gctcctgccg 3120agaaagtatc
catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
3180gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
atggaagccg 3240gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
gctcgcgcca gccgaactgt 3300tcgccaggct caaggcgcgc atgcccgacg
gcgaggatct cgtcgtgacc catggcgatg 3360cctgcttgcc gaatatcatg
gtggaaaatg gccgcttttc tggattcatc gactgtggcc 3420ggctgggtgt
ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
3480agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
gctcccgatt 3540cgcagcgcat cgccttctat cgccttcttg acgagttctt
ctgagcggga ctctggggtt 3600cgaaatgacc gaccaagcga cgcccaacct
gccatcacga gatttcgatt ccaccgccgc 3660cttctatgaa aggttgggct
tcggaatcgt tttccgggac gccggctgga tgatcctcca 3720gcgcggggat
ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
3780tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
tttcactgca 3840ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
catgtctgta taccgtcgac 3900ctctagctag agcttggcgt aatcatggtc
atagctgttt cctgtgtgaa attgttatcc 3960gctcacaatt ccacacaaca
tacgagccgg aagcataaag tgtaaagcct ggggtgccta 4020atgagtgagc
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
4080cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
gtttgcgtat 4140tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
tcggtcgttc ggctgcggcg 4200agcggtatca gctcactcaa aggcggtaat
acggttatcc acagaatcag gggataacgc 4260aggaaagaac atgtgagcaa
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 4320gctggcgttt
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
4380tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
ctggaagctc 4440cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
tacctgtccg cctttctccc 4500ttcgggaagc gtggcgcttt ctcaatgctc
acgctgtagg tatctcagtt cggtgtaggt 4560cgttcgctcc aagctgggct
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 4620atccggtaac
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
4680agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
agttcttgaa 4740gtggtggcct aactacggct acactagaag gacagtattt
ggtatctgcg ctctgctgaa 4800gccagttacc ttcggaaaaa gagttggtag
ctcttgatcc ggcaaacaaa ccaccgctgg 4860tagcggtggt ttttttgttt
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 4920agatcctttg
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
4980gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
attaaaaatg 5040aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
tctgacagtt accaatgctt 5100aatcagtgag gcacctatct cagcgatctg
tctatttcgt tcatccatag ttgcctgact 5160ccccgtcgtg tagataacta
cgatacggga gggcttacca tctggcccca gtgctgcaat 5220gataccgcga
gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
5280aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
ctattaattg 5340ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
ttgcgcaacg ttgttgccat 5400tgctacaggc atcgtggtgt cacgctcgtc
gtttggtatg gcttcattca gctccggttc 5460ccaacgatca aggcgagtta
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 5520cggtcctccg
atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
5580agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
tgactggtga 5640gtactcaacc aagtcattct gagaatagtg tatgcggcga
ccgagttgct cttgcccggc 5700gtcaatacgg gataataccg cgccacatag
cagaacttta aaagtgctca tcattggaaa 5760acgttcttcg
gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
5820acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
tttctgggtg 5880agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
agggcgacac ggaaatgttg 5940aatactcata ctcttccttt ttcaatatta
ttgaagcatt tatcagggtt attgtctcat 6000gagcggatac atatttgaat
gtatttagaa aaataaacaa ataggggttc cgcgcacatt 6060tccccgaaaa
gtgccacctg acgtc 6085126097DNAArtificial SequenceSynthetic
Oligonucleotide 12gacggatcgg gagatctccc gatcccctat ggtcgactct
cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag
gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg
ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc 420attgacgtca atgggtggac tatttacggt
aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
900aagcttggat ctcaccatga gggtccctgc tcagctcctg gggctgctaa
tgctctggat 960acctggatcc agtgcagata ttgtgatgac ccagactcca
ctctctctgt ccgtcacccc 1020tggacagccg gcctccatct cctgcaagtc
tagtcagagc ctcctgcata gtgatggaaa 1080gacctttttg tattggtatc
tgcagaagcc aggccagcct ccacagctcc tgatctatga 1140ggtttccaac
cggttctctg gagtgccaga taggttcagt ggcagcgggt cagggacaga
1200tttcacactg aaaatcagcc gggtggaggc tgaggatgtt gggctttatt
actgcatgca 1260aagtatacag cttccgctca ctttcggcgg agggaccaag
gtggagatca aacgaactgt 1320ggctgcacca tctgtcttca tcttcccgcc
atctgatgag cagttgaaat ctggaactgc 1380tagcgttgtg tgcctgctga
ataacttcta tcccagagag gccaaagtac agtggaaggt 1440ggataacgcc
ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga
1500cagcacctac agcctcagca gcaccctgac gctgagcaaa gcagactacg
agaaacacaa 1560agtctacgcc tgcgaagtca cccatcaggg cctgagctcg
cccgtcacaa agagcttcaa 1620caggggagag tgttaggaat tcgcggccgc
tcgagtctag agggcccgtt taaacccgct 1680gatcagcctc gactgtgcct
tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 1740cttccttgac
cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
1800catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
caggacagca 1860agggggagga ttgggaagac aatagcaggc atgctgggga
tgcggtgggc tctatggctt 1920ctgaggcgga aagaaccagc tggggctcta
gggggtatcc ccacgcgccc tgtagcggcg 1980cattaagcgc ggcgggtgtg
gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2040tagcgcccgc
tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
2100gtcaagctct aaatcggggc atccctttag ggttccgatt tagtgcttta
cggcacctcg 2160accccaaaaa acttgattag ggtgatggtt cacgtagtgg
gccatcgccc tgatagacgg 2220tttttcgccc tttgacgttg gagtccacgt
tctttaatag tggactcttg ttccaaactg 2280gaacaacact caaccctatc
tcggtctatt cttttgattt ataagggatt ttggggattt 2340cggcctattg
gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
2400gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccaggcaggc
agaagtatgc 2460aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
gtccccaggc tccccagcag 2520gcagaagtat gcaaagcatg catctcaatt
agtcagcaac catagtcccg cccctaactc 2580cgcccatccc gcccctaact
ccgcccagtt ccgcccattc tccgccccat ggctgactaa 2640ttttttttat
ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
2700gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
tgtatatcca 2760ttttcggatc tgatcaagag acaggatgag gatcgtttcg
catgattgaa caagatggat 2820tgcacgcagg ttctccggcc gcttgggtgg
agaggctatt cggctatgac tgggcacaac 2880agacaatcgg ctgctctgat
gccgccgtgt tccggctgtc agcgcagggg cgcccggttc 2940tttttgtcaa
gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
3000tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
gtcactgaag 3060cgggaaggga ctggctgcta ttgggcgaag tgccggggca
ggatctcctg tcatctcacc 3120ttgctcctgc cgagaaagta tccatcatgg
ctgatgcaat gcggcggctg catacgcttg 3180atccggctac ctgcccattc
gaccaccaag cgaaacatcg catcgagcga gcacgtactc 3240ggatggaagc
cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
3300cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
ctcgtcgtga 3360cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
tggccgcttt tctggattca 3420tcgactgtgg ccggctgggt gtggcggacc
gctatcagga catagcgttg gctacccgtg 3480atattgctga agagcttggc
ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg 3540ccgctcccga
ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
3600gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
gagatttcga 3660ttccaccgcc gccttctatg aaaggttggg cttcggaatc
gttttccggg acgccggctg 3720gatgatcctc cagcgcgggg atctcatgct
ggagttcttc gcccacccca acttgtttat 3780tgcagcttat aatggttaca
aataaagcaa tagcatcaca aatttcacaa ataaagcatt 3840tttttcactg
cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
3900tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
ttcctgtgtg 3960aaattgttat ccgctcacaa ttccacacaa catacgagcc
ggaagcataa agtgtaaagc 4020ctggggtgcc taatgagtga gctaactcac
attaattgcg ttgcgctcac tgcccgcttt 4080ccagtcggga aacctgtcgt
gccagctgca ttaatgaatc ggccaacgcg cggggagagg 4140cggtttgcgt
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
4200tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
ccacagaatc 4260aggggataac gcaggaaaga acatgtgagc aaaaggccag
caaaaggcca ggaaccgtaa 4320aaaggccgcg ttgctggcgt ttttccatag
gctccgcccc cctgacgagc atcacaaaaa 4380tcgacgctca agtcagaggt
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 4440ccctggaagc
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
4500cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta
ggtatctcag 4560ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
gaaccccccg ttcagcccga 4620ccgctgcgcc ttatccggta actatcgtct
tgagtccaac ccggtaagac acgacttatc 4680gccactggca gcagccactg
gtaacaggat tagcagagcg aggtatgtag gcggtgctac 4740agagttcttg
aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
4800cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
ccggcaaaca 4860aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
cagattacgc gcagaaaaaa 4920aggatctcaa gaagatcctt tgatcttttc
tacggggtct gacgctcagt ggaacgaaaa 4980ctcacgttaa gggattttgg
tcatgagatt atcaaaaagg atcttcacct agatcctttt 5040aaattaaaaa
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
5100ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
gttcatccat 5160agttgcctga ctccccgtcg tgtagataac tacgatacgg
gagggcttac catctggccc 5220cagtgctgca atgataccgc gagacccacg
ctcaccggct ccagatttat cagcaataaa 5280ccagccagcc ggaagggccg
agcgcagaag tggtcctgca actttatccg cctccatcca 5340gtctattaat
tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
5400cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
tggcttcatt 5460cagctccggt tcccaacgat caaggcgagt tacatgatcc
cccatgttgt gcaaaaaagc 5520ggttagctcc ttcggtcctc cgatcgttgt
cagaagtaag ttggccgcag tgttatcact 5580catggttatg gcagcactgc
ataattctct tactgtcatg ccatccgtaa gatgcttttc 5640tgtgactggt
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
5700ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
taaaagtgct 5760catcattgga aaacgttctt cggggcgaaa actctcaagg
atcttaccgc tgttgagatc 5820cagttcgatg taacccactc gtgcacccaa
ctgatcttca gcatctttta ctttcaccag 5880cgtttctggg tgagcaaaaa
caggaaggca aaatgccgca aaaaagggaa taagggcgac 5940acggaaatgt
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
6000ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
aaataggggt 6060tccgcgcaca tttccccgaa aagtgccacc tgacgtc
6097136094DNAArtificial SequenceSynthetic Oligonucleotide
13gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct
ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga
ctcactatag ggagacccaa gctggctaga 900aagcttggat ctcaccatgg
tgttgcagac ccaggtcttc atttctctgt tactctggat 960ctctggtgcc
tacggggaca tcgtgatgac ccagtctcca gactccctgg ctgtgtctct
1020gggcgagagg gccaccatca actgcaagtc caaccagagt gtcttacaca
gctccaacaa 1080taagaactat ttagcttggt accagcagaa accaggacag
cctcctaaat tgctcattta 1140ttgggcattc ctccgggaat ccggggtccc
tgaccgcttc agtggcagcg ggtctgggac 1200agatttcact ctcaccatca
gcagcctgca ggctgaagat gtggcagttt attactgtca 1260ccaatattat
tctactttat atactttcgg cggagggacc aaggtagaga tcaaacgaac
1320ygtggctgca ccatctgtct tcatcttccc gccatctgat gagcagttga
aatctggaac 1380tgctagcgtt gtgtgcctgc tgaataactt ctatcccaga
gaggccaaag tacagtggaa 1440ggtggataac gccctccaat cgggtaactc
ccaggagagt gtcacagagc aggacagcaa 1500ggacagcacc tacagcctca
gcagcaccct gacgctgagc aaagcagact acgagaaaca 1560caaagtctac
gcctgcgaag tcacccatca gggcctgagc tcgcccgtca caaagagctt
1620caacagggga gagtgttagg cggccgctcg agtctagagg gcccgtttaa
acccgctgat 1680cagcctcgac tgtgccttct agttgccagc catctgttgt
ttgcccctcc cccgtgcctt 1740ccttgaccct ggaaggtgcc actcccactg
tcctttccta ataaaatgag gaaattgcat 1800cgcattgtct gagtaggtgt
cattctattc tggggggtgg ggtggggcag gacagcaagg 1860gggaggattg
ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
1920aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
agcggcgcat 1980taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
tacacttgcc agcgccctag 2040cgcccgctcc tttcgctttc ttcccttcct
ttctcgccac gttcgccggc tttccccgtc 2100aagctctaaa tcggggcatc
cctttagggt tccgatttag tgctttacgg cacctcgacc 2160ccaaaaaact
tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
2220ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
caaactggaa 2280caacactcaa ccctatctcg gtctattctt ttgatttata
agggattttg gggatttcgg 2340cctattggtt aaaaaatgag ctgatttaac
aaaaatttaa cgcgaattaa ttctgtggaa 2400tgtgtgtcag ttagggtgtg
gaaagtcccc aggctcccca ggcaggcaga agtatgcaaa 2460gcatgcatct
caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
2520gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
ctaactccgc 2580ccatcccgcc cctaactccg cccagttccg cccattctcc
gccccatggc tgactaattt 2640tttttattta tgcagaggcc gaggccgcct
ctgcctctga gctattccag aagtagtgag 2700gaggcttttt tggaggccta
ggcttttgca aaaagctccc gggagcttgt atatccattt 2760tcggatctga
tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
2820acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
gcacaacaga 2880caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
gcaggggcgc ccggttcttt 2940ttgtcaagac cgacctgtcc ggtgccctga
atgaactgca ggacgaggca gcgcggctat 3000cgtggctggc cacgacgggc
gttccttgcg cagctgtgct cgacgttgtc actgaagcgg 3060gaagggactg
gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
3120ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
acgcttgatc 3180cggctacctg cccattcgac caccaagcga aacatcgcat
cgagcgagca cgtactcgga 3240tggaagccgg tcttgtcgat caggatgatc
tggacgaaga gcatcagggg ctcgcgccag 3300ccgaactgtt cgccaggctc
aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc 3360atggcgatgc
ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
3420actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
acccgtgata 3480ttgctgaaga gcttggcggc gaatgggctg accgcttcct
cgtgctttac ggtatcgccg 3540ctcccgattc gcagcgcatc gccttctatc
gccttcttga cgagttcttc tgagcgggac 3600tctggggttc gaaatgaccg
accaagcgac gcccaacctg ccatcacgag atttcgattc 3660caccgccgcc
ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
3720gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
tgtttattgc 3780agcttataat ggttacaaat aaagcaatag catcacaaat
ttcacaaata aagcattttt 3840ttcactgcat tctagttgtg gtttgtccaa
actcatcaat gtatcttatc atgtctgtat 3900accgtcgacc tctagctaga
gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 3960ttgttatccg
ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
4020gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
ccgctttcca 4080gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
caacgcgcgg ggagaggcgg 4140tttgcgtatt gggcgctctt ccgcttcctc
gctcactgac tcgctgcgct cggtcgttcg 4200gctgcggcga gcggtatcag
ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4260ggataacgca
ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
4320ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
acaaaaatcg 4380acgctcaagt cagaggtggc gaaacccgac aggactataa
agataccagg cgtttccccc 4440tggaagctcc ctcgtgcgct ctcctgttcc
gaccctgccg cttaccggat acctgtccgc 4500ctttctccct tcgggaagcg
tggcgctttc tcaatgctca cgctgtaggt atctcagttc 4560ggtgtaggtc
gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
4620ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
acttatcgcc 4680actggcagca gccactggta acaggattag cagagcgagg
tatgtaggcg gtgctacaga 4740gttcttgaag tggtggccta actacggcta
cactagaagg acagtatttg gtatctgcgc 4800tctgctgaag ccagttacct
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4860caccgctggt
agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
4920atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
acgaaaactc 4980acgttaaggg attttggtca tgagattatc aaaaaggatc
ttcacctaga tccttttaaa 5040ttaaaaatga agttttaaat caatctaaag
tatatatgag taaacttggt ctgacagtta 5100ccaatgctta atcagtgagg
cacctatctc agcgatctgt ctatttcgtt catccatagt 5160tgcctgactc
cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
5220tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
caataaacca 5280gccagccgga agggccgagc gcagaagtgg tcctgcaact
ttatccgcct ccatccagtc 5340tattaattgt tgccgggaag ctagagtaag
tagttcgcca gttaatagtt tgcgcaacgt 5400tgttgccatt gctacaggca
tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5460ctccggttcc
caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
5520tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
tatcactcat 5580ggttatggca gcactgcata attctcttac tgtcatgcca
tccgtaagat gcttttctgt 5640gactggtgag tactcaacca agtcattctg
agaatagtgt atgcggcgac cgagttgctc 5700ttgcccggcg tcaatacggg
ataataccgc gccacatagc agaactttaa aagtgctcat 5760cattggaaaa
cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
5820ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
tcaccagcgt 5880ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
aagggaataa gggcgacacg 5940gaaatgttga atactcatac tcttcctttt
tcaatattat tgaagcattt atcagggtta 6000ttgtctcatg agcggataca
tatttgaatg tatttagaaa aataaacaaa taggggttcc 6060gcgcacattt
ccccgaaaag tgccacctga cgtc 609414481DNAArtificial SequenceSynthetic
Oligonucleotide 14ggatctcacc atggagttgg gactgcgctg gggcttcctc
gttgctcttt taagaggtgt 60ccagtgtcag gtgcaattgg tggagtctgg gggaggcgtg
gtccagcctg ggaggtccct 120gagactctcc tgtgcagcgt ctggattcgc
cttcagtaga tatggcatgc actgggtccg 180ccaggctcca ggcaaggggc
tggagtgggt ggcagttata tggtatgatg gaagtaataa 240atactatgca
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac
300gcagtatctg caaatgaaca gcctgagagc cgaggacacg gctgtgtatt
actgtgcgag 360aggcggtgac ttcctctact actactatta cggtatggac
gtctggggcc aagggaccac 420ggtcaccgtc tcctcagcct ccaccaaggg
cccatcggtc ttccccctgg caccctctag 480c 48115142PRTHomo sapiens 15Met
Glu Leu Gly Leu Arg Trp Gly Phe Leu Val Ala Leu Leu Arg Gly 1 5 10
15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ala Phe 35 40 45 Ser Arg Tyr Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly
Ser Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Gln Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala
Arg Gly Gly Asp Phe Leu Tyr Tyr Tyr Tyr Tyr Gly 115 120 125 Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140
16463DNAArtificial SequenceSynthetic Oligonucleotide 16ggatctcacc
atgagggtcc ctgctcagct cctgggactc ctgctgctct ggctcccaga 60taccagatgt
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
120cagagtcacc atcacttgcc gggcgagtca gggcattagc aattatttag
cctggtatca 180gcagaaaaca gggaaagttc ctaagttcct gatctatgaa
gcatccactt tgcaatcagg 240 ggtcccatct cggttcagtg gcggtggatc
tgggacagat ttcactctca ccatcagcag 300cctgcagcct gaagatgttg
caacttatta ctgtcaaaat tataacagtg ccccattcac 360tttcggccct
gggaccaaag tggatatcaa acgaactgtg gctgcaccct ctgtcttcat
420cttcccgcca tctgatgagc agttgaaatc tggaactgct agc 46317127PRTHomo
sapiens 17Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
Leu Pro 1 5 10 15 Asp Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala
Ser Gln Gly 35 40 45 Ile Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
Thr Gly Lys Val Pro 50 55 60 Lys Phe Leu Ile Tyr Glu Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Gly Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu
Asp Val Ala Thr Tyr Tyr Cys Gln Asn Tyr Asn 100 105 110 Ser Ala Pro
Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 115 120 125
18508DNAArtificial SequenceSynthetic Oligonucleotide 18ggatctcacc
atggggtcaa ccgccatcct caccatggag ttggggctgc gctgggttct 60cctcgttgct
cttttaagag gtgtccagtg tcaggtgcag ctggtggagt ctgggggagg
120cgtggtccag cctgggaggt ccctgagact ctcctgtgca gcgtctggat
tcaccttcag 180taactatgtc atgcactggg tccgccaggc tccaggcaag
gggctggagt gggtggcaat 240tatatggtat gatggaagta ataaatacta
tgcagactcc gtgaagggcc gattcaccat 300ctccagagac aattccaaga
acacgctgta tctgcaaatg aacagcctga gagccgagga 360cacggctgtg
tattactgtg cgggtggata taactggaac tacgagtacc actactacgg
420tatggacgtc tggggccaag ggaccacggt caccgtctcc tcagcctcca
ccaagggccc 480atcggtcttc cccctggcac cctctagc 50819143PRTHomo
sapiens 19Met Glu Leu Gly Leu Arg Trp Val Leu Leu Val Ala Leu Leu
Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asn Tyr Val Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Ile Ile
Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110
Tyr Tyr Cys Ala Gly Gly Tyr Asn Trp Asn Tyr Glu Tyr His Tyr Tyr 115
120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
130 135 140 20463DNAArtificial SequenceSynthetic Oligonucleotide
20ggatctcacc atgagggtcc ccgctcagct cctggggctc ctgctgctct gtttcccagg
60tgccagatgt gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga
120cagagtcacc atcacttgtc gggcgagtca gggcattacc aattatttag
cctggtttca 180gcagaaacca gggaaagccc ctaagtccct tatctatgct
gcatccagtt tgcaaagtgg 240ggtcccatca aagttcagcg gcagtggatc
tgggacagat ttcagtctca ccatcagcag 300cctgcagcct gaagattttg
caacttatta ctgccaacag tataatagtt acccgatcac 360cttcggccaa
gggacacgac tggagattaa acgaactgtg gctgcaccat ctgtcttcat
420cttcccgcca tctgatgagc agttgaaatc tggaactgct agc 46321127PRTHomo
sapiens 21Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys
Phe Pro 1 5 10 15 Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly 35 40 45 Ile Thr Asn Tyr Leu Ala Trp Phe
Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Ser Leu Ile Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser 65 70 75 80 Lys Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser 85 90 95 Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110
Ser Tyr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 115 120
125 22490DNAArtificial SequenceSynthetic Oligonucleotide
22ggatctcacc atggagttgg gacttagctg ggttttcctc gttgctcttt taagaggtgt
60ccagtgtcag gtccagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct
120gagactctcc tgtgcagcgt ctggattcac cttcagtagc tatggcatgc
actgggtccg 180ccaggctcca ggcaaggggc tggactgggt ggcaattatt
tggcatgatg gaagtaataa 240atactatgca gactccgtga agggccgatt
caccatctcc agagacaatt ccaagaagac 300gctgtacctg caaatgaaca
gtttgagagc cgaggacacg gctgtgtatt actgtgcgag 360agcttgggcc
tatgactacg gtgactatga atactacttc ggtatggacg tctggggcca
420agggaccacg gtcaccgtct cctcagcctc caccaagggc ccatcggtct
tccccctggc 480accctctagc 49023145PRTHomo sapiens 23Met Glu Leu Gly
Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln
Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30
Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35
40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 50 55 60 Asp Trp Val Ala Ile Ile Trp His Asp Gly Ser Asn Lys
Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Lys 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ala Trp
Ala Tyr Asp Tyr Gly Asp Tyr Glu Tyr 115 120 125 Tyr Phe Gly Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 130 135 140 Ser 145
24463DNAArtificial SequenceSynthetic Oligonucleotide 24ggatctcacc
atgagggtcc ctgctcagct cctggggctc ctgctgctct gtttcccagg 60tgccagatgt
gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga
120cagagtcacc atcacttgtc gggcgagtca gggcattagc cattatttag
cctggtttca 180gcagaaacca gggaaagccc ctaagtccct gatctatgct
gcatccagtt tgcaaagtgg 240ggtcccatca aagttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag 300cctacagcct gaagattttg
caacttatta ctgccaacag tataatagtt tcccgctcac 360tttcggcgga
gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat
420cttcccgcca tctgatgagc agttgaaatc tggaactgct agc 46325127PRTHomo
sapiens 25Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys
Phe Pro 1 5 10 15 Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly 35 40 45 Ile Ser His Tyr Leu Ala Trp Phe
Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Ser Leu Ile Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser 65 70 75 80 Lys Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110
Ser Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120
125 26469DNAArtificial SequenceSynthetic Oligonucleotide
26ggatcccacc atggggtcaa ccgtcatcct cgccctcctc ctggctgttc tccaaggagt
60ctgtgccgag gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct
120gaagatctcc tgtaagggtt ctggatacag ctttaccagt tactggatcg
gctgggtgcg 180ccagatgccc gggaaaggcc tggagtggat ggggatcatc
tatcctggtg actctgatac 240cagatacagc ccgtccttcc aaggccaggt
caccatctca gccgacaagt ccatcagcac 300cgcctacctg cagtggagca
gcctgaaggc ctcggacacc gccatgtatt actgtgcgag 360acggatggca
gcagctggcc cctttgacta ctggggccag ggaaccctgg tcaccgtctc
420ctcagcctcc accaagggcc catcggtctt ccccctggca ccctctagc
46927138PRTHomo sapiens 27Met Gly Ser Thr Val Ile Leu Ala Leu Leu
Leu Ala Val Leu Gln Gly 1 5 10 15 Val Cys Ala Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Glu Ser Leu Lys
Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe 35 40 45 Thr Ser Tyr Trp
Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu 50 55 60 Glu Trp
Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser 65 70 75 80
Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser 85
90 95 Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala
Met 100 105 110 Tyr Tyr Cys Ala Arg Arg Met Ala Ala Ala Gly Pro Phe
Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130
135 28466DNAArtificial SequenceSynthetic Oligonucleotide
28ggatctcacc atgagggtcc ccgctcagct tctcttcctt ctgctactct ggctcccaga
60taccactgga ggaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga
120aagagccacc ctctcctgca ggaccagtca gagtattggc tggaacttag
cctggtacca 180acagaaacct ggccaggctc ccaggctcct catctatggt
gcatcttcca ggaccactgg 240tatcccagcc aggttcagtg gcagtgggtc
tgggacagag ttcactctca ccatcagcag 300cctgcagtct gaagattctg
cagtttatta ctgtcagcat tatgataact ggcccatgtg 360cagttttggc
caggggaccg agctggagat caaacgaact gtggctgcac catctgtctt
420catcttcccg ccatctgatg agcagttgaa atctggaact gctagc
46629128PRTHomo sapiens 29Met Arg Val Pro Ala Gln Leu Leu Phe Leu
Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Gly Ile Val Met
Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Val Ser Pro Gly Glu Arg
Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser 35 40 45 Ile Gly Trp Asn
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Leu
Leu Ile Tyr Gly Ala Ser Ser Arg Thr Thr Gly Ile Pro Ala 65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser 85
90 95 Ser Leu Gln Ser Glu Asp Ser Ala Val Tyr Tyr Cys Gln His Tyr
Asp 100 105 110 Asn Trp Pro Met Cys Ser Phe Gly Gln Gly Thr Glu Leu
Glu Ile Lys 115 120 125 30487DNAArtificial SequenceSynthetic
Oligonucleotide 30ggatctcacc atggagtttg ggctgtgctg gattttcctc
gttgctcttt taagaggtgt 60ccagtgtcag gtgcagctgg tggagtctgg gggaggcgtg
gtccagcctg ggaggtccct 120gagactctcc tgtgcagcct ctggattcac
cttcattagc tatggcatgc actgggtccg 180ccaggctcca ggcaaggggc
tggagtgggt ggcagttata tcatatgatg gaagtaataa 240atactatgca
gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac
300gctgtatctg caaatgaaca gcctgagagc tgaggacacg gctgtgtatt
actgtgcgag 360agtattagtg ggagctttat attattataa ctactacggg
atggacgtct ggggccaagg 420gaccacggtc accgtctcct cagcctccac
caagggccca tcggtcttcc ccctggcacc 480ctctagc 48731144PRTHomo sapiens
31Met Glu Phe Gly Leu Cys Trp Ile Phe Leu Val Ala Leu Leu Arg Gly 1
5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe 35 40 45 Ile Ser Tyr Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Ser Tyr Asp
Gly Ser Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys
Ala Arg Val Leu Val Gly Ala Leu Tyr Tyr Tyr Asn Tyr 115 120 125 Tyr
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135
140 32478DNAArtificial SequenceSynthetic Oligonucleotide
32ggatctcacc atgagggtcc ctgctcagct cctggggctg ctaatgctct ggatacctgg
60atccagtgca gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca
120gccggcctcc atctcctgca agtctagtca gagcctcctg catagtgatg
gaaagacctt 180tttgtattgg tatctgcaga agccaggcca gcctccacag
ctcctgatct atgaggtttc 240caaccggttc tctggagtgc cagataggtt
cagtggcagc gggtcaggga cagatttcac 300actgaaaatc agccgggtgg
aggctgagga tgttgggctt tattactgca tgcaaagtat 360acagcttccg
ctcactttcg gcggagggac caaggtggag atcaaacgaa ctgtggctgc
420accatctgtc ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgctagc
47833132PRTHomo sapiens 33Met Arg Val Pro Ala Gln Leu Leu Gly Leu
Leu Met Leu Trp Ile Pro 1 5 10 15 Gly Ser Ser Ala Asp Ile Val Met
Thr Gln Thr Pro Leu Ser Leu Ser 20 25 30 Val Thr Pro Gly Gln Pro
Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser 35 40 45 Leu Leu His Ser
Asp Gly Lys Thr Phe Leu Tyr Trp Tyr Leu Gln Lys 50 55 60 Pro Gly
Gln Pro Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe 65 70 75 80
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85
90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Leu Tyr
Tyr 100 105 110 Cys Met Gln Ser Ile Gln Leu Pro Leu Thr Phe Gly Gly
Gly Thr Lys 115 120 125 Val Glu Ile Lys 1303415DNAArtificial
SequenceSynthetic Oligonucleotide 34gaagatctca ccatg
153539DNAArtificial SequenceSynthetic Oligonucleotide 35aactagctag
cagttccaga tttcaactgc tcatcagat 393615DNAArtificial
SequenceSynthetic Oligonucleotide 36gaagatctca ccatg
153730DNAArtificial SequenceSynthetic Oligonucleotide 37gctctagagg
gtgccagggg gaagaccgat 303811PRTArtificial SequenceSynthetic Peptide
38Ser Ala Thr Gly Ser Lys Leu Gln Glu Asp Ser 1 5 10
399PRTArtificial SequenceSynthetic Peptide 39Arg Ser Pro Ala Leu
Pro Phe Val Ser 1 5
* * * * *