U.S. patent application number 10/900399 was filed with the patent office on 2005-07-21 for method of using a cytokine that induces apoptosis.
This patent application is currently assigned to IMMUNEX CORPORATION. Invention is credited to Goodwin, Raymond G., Wiley, Steven R..
Application Number | 20050158823 10/900399 |
Document ID | / |
Family ID | 27535054 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158823 |
Kind Code |
A1 |
Wiley, Steven R. ; et
al. |
July 21, 2005 |
Method of using a cytokine that induces apoptosis
Abstract
A novel cytokine designated TRAIL induces apoptosis of certain
target cells, including cancer cells and virally infected cells.
Isolated DNA sequences encoding TRAIL are disclosed, along with
expression vectors and transformed host cells useful in producing
TRAIL polypeptides. Antibodies that specifically bind TRAIL are
provided as well.
Inventors: |
Wiley, Steven R.; (Seattle,
WA) ; Goodwin, Raymond G.; (Seattle, WA) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
IMMUNEX CORPORATION
|
Family ID: |
27535054 |
Appl. No.: |
10/900399 |
Filed: |
July 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10900399 |
Jul 28, 2004 |
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09796581 |
Feb 27, 2001 |
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09796581 |
Feb 27, 2001 |
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09320424 |
May 26, 1999 |
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6284236 |
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09320424 |
May 26, 1999 |
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09190046 |
Nov 10, 1998 |
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09190046 |
Nov 10, 1998 |
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09048641 |
Mar 26, 1998 |
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09048641 |
Mar 26, 1998 |
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08670354 |
Jun 25, 1996 |
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5763223 |
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08670354 |
Jun 25, 1996 |
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08548368 |
Nov 1, 1995 |
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08548368 |
Nov 1, 1995 |
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08496632 |
Jun 29, 1995 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 530/351; 536/23.5 |
Current CPC
Class: |
C07K 16/24 20130101;
C07K 2317/74 20130101; C07K 2317/34 20130101; C07K 16/2875
20130101; C07K 14/70575 20130101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/350; 530/351; 536/023.5 |
International
Class: |
A61K 038/19; C07K
014/715; C07K 014/53; C07H 021/04 |
Claims
1-28. (canceled)
29. A method of inducing death of cancer cells, comprising
contacting TRAIL-sensitive cancer cells with TRAIL polypeptide,
wherein said polypeptide comprises an amino acid sequence that is
at least 90% identical to an amino acid sequence selected from the
group consisting of amino acids 1 to 281 of SEQ ID NO:2 and amino
acids 1 to 291 of SEQ ID NO:6, wherein said TRAIL polypeptide is
capable of inducing apoptosis of Jurkat cells.
30. A method of inducing death of cancer cells, comprising
contracting TRAIL-sensitive cancer cells with an oligomer
comprising at least two soluble TRAIL polypeptides, wherein each of
said soluble TRAIL polypeptides comprises an amino acid sequence
that is at least 90% identical to a sequence selected from the
group consisting of: a) the extracellular domain of human TRAIL
(amino acids 39 to 281 of SEQ ID NO:2); and b) a fragment of said
extracellular domain; wherein said oligomer is capable of inducing
apoptosis of Jurkat cells.
31. A method of claim 29, wherein said TRAIL polypeptide comprises
an amino acid sequence selected from the group consisting of amino
acids 1 to 281 of SEQ ID NO:2 and amino acids 1 to 291 of SEQ ID
NO:6.
32. A method of inducing death of cancer cells, comprising
contacting TRAIL-sensitive cancer cells with a soluble polypeptide
comprising an amino acid sequence that is at least 90% identical to
a sequence selected from the group consisting of: a) the
extracellular domain of human TRAIL (amino acids 39 to 281 of SEQ
ID NO:2); and b) a fragment of said extracellular domain; wherein
said polypeptide is capable of inducing apoptosis of Jurkat
cells.
33. A method of claim 32, wherein said polypeptide comprises an
amino acid sequence selected from the group consisting of: a) the
extracellular domain of human TRAIL (amino acids 39 to 281 of SEQ
ID NO:2); and b) a fragment of said extracellular domain; wherein
said polypeptide is capable of inducing apoptosis of Jurkat
cells.
34. A method of claim 32, wherein said polypeptide comprises an
amino acid sequence that is at least 90% identical to the sequence
of amino acids 95 to 281 of SEQ ID NO:2, wherein said polypeptide
is capable of inducing apoptosis of Jurkat cells.
35. A method of claim 33, wherein said polypeptide comprises the
sequence of amino acids x to 281 of SEQ ID NO:2, wherein x
represents an integer from 39 to 95.
36. A method of claim 35, wherein said polypeptide comprises amino
acids 95 to 281 of SEQ ID NO:2.
37. A method of inducing death of cancer cells, comprising
contacting TRAIL-sensitive cancer cells with a polypeptide that
comprises a fragment of the human TRAIL protein of SEQ ID NO:2,
wherein said fragment kills TRAIL-sensitive cancer cells.
38. A method of claim 37, wherein said polypeptide consists of a
soluble fragment of the human TRAIL protein of SEQ ID NO:2.
39. A method of inducing death of cancer cells, comprising
contacting TRAIL-sensitive cancer cells with a polypeptide
comprising amino acids 124 to 276 of SEQ ID NO:2.
40. A method of claim 39, wherein said polypeptide is a fragment of
the TRAIL protein of SEQ ID NO:2, wherein the N-terminal amino acid
of said fragment is selected from residues 39 to 124 of SEQ ID
NO:2, and the C-terminal amino acid of said fragment is selected
from residues 276 to 281 of SEQ ID NO:2.
41. A method of claim 37, wherein said polypeptide comprises the
amino acid sequence presented in SEQ ID NO:2 or SEQ ID NO:6, with
the proviso that said polypeptide lacks a transmembrane region.
42. A method of claim 32, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
43. A method of claim 33, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
44. A method of claim 37, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
45. A method of claim 39, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
46. A method of claim 40, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
47. A method of claim 30, wherein each of said soluble TRAIL
polypeptides comprises an amino acid sequence selected from the
group consisting of: a) the extracellular domain of human TRAIL
(amino acids 39 to 281 of SEQ ID NO:2); and b) a fragment of said
extracellular domain; wherein said oligomer is capable of inducing
apoptosis of Jurkat cells.
48. A method of claim 30, wherein each of said soluble TRAIL
polypeptides comprises an amino acid sequence that is at least 90%
identical to the sequence of amino acids 95 to 281 of SEQ ID NO:2,
wherein said polypeptide induces apoptosis of Jurkat cells.
49. A method of claim 47, wherein each of said soluble TRAIL
polypeptides comprises the sequence of amino acids x to 281 of SEQ
ID NO:2, wherein x represents an integer from 39 to 95.
50. A method of claim 49, wherein each of said soluble TRAIL
polypeptides comprises amino acids 95 to 281 of SEQ ID NO:2.
51. A method of killing cancer cells, contacting TRAIL-sensitive
cancer cells with an oligomer comprising at least two TRAIL
polypeptides, wherein each of said TRAIL polypeptides is a soluble
fragment of the protein of SEQ ID NO:2, wherein said oligomer kills
TRAIL-sensitive cancer cells.
52. A method of inducing death of cancer cells, comprising
contacting TRAIL-sensitive cancer cells with an oligomer comprising
at least two TRAIL polypeptides, wherein each of said polypeptides
comprises amino acids 124 to 276 of SEQ ID NO:2.
53. A method of claim 52, wherein each of said polypeptides is a
fragment of the TRAIL protein of SEQ ID NO:2, wherein the
N-terminal amino acid of said fragment is selected from residues 39
to 124 of SEQ ID NO:2, and the C-terminal amino acid of said
fragment is selected from residues 276 to 281 of SEQ ID NO:2.
54. A method of claim 30, wherein each of said polypeptides
additionally comprises a leucine zipper peptide.
55. A method of claim 47, wherein each of said polypeptides
additionally comprises a leucine zipper peptide.
56. A method of claim 30, wherein said oligomer additionally
comprises an Fc polypeptide fused to each of two of said TRAIL
polypeptides.
57. A method of claim 47, wherein said oligomer additionally
comprises an Fc polypeptide fused to each of two of said TRAIL
polypeptides.
58. A method of claim 51, wherein said oligomer additionally
comprises an Fc polypeptide fused to each of two of said TRAIL
polypeptides.
59. A method of claim 30, wherein the oligomer comprises two or
three of said TRAIL polypeptides.
60. A method of claim 47, wherein the oligomer comprises two or
three of said TRAIL polypeptides.
61. A method of claim 59, wherein the oligomer comprises three of
said TRAIL polypeptides.
62. A method of claim 60, wherein the oligomer comprises three of
said TRAIL polypeptides.
63. A method of claim 51, wherein the oligomer comprises three of
said TRAIL polypeptides.
64. A method of claim 52, wherein the oligomer comprises three of
said TRAIL polypeptides.
65. A method of claim 53, wherein the oligomer comprises three of
said TRAIL polypeptides.
66. A method of claim 30, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
67. A method of claim 47, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
68. A method of claim 51, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
69. A method of claim 52, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
70. A method of claim 53, wherein said cancer cells are selected
from the group consisting of leukemia cells, melanoma cells, and
lymphoma cells.
71. A method of claim 30, wherein the method comprises
administering said oligomer to a human who has cancer.
72. A method of claim 32, wherein the method comprises
administering said polypeptide to a human who has cancer.
73. A method of claim 37, wherein the method comprises
administering said polypeptide to a human who has cancer.
74. A method of claim 39, wherein the method comprises
administering said polypeptide to a human who has cancer.
75. A method of claim 47, wherein the method comprises
administering said oligomer to a human who has cancer.
76. A method of claim 51, wherein the method comprises
administering said oligomer to a human who has cancer.
77. A method of claim 52, wherein the method comprises
administering said oligomer to a human who has cancer.
78. A method of claim 53, wherein the method comprises
administering said oligomer to a human who has cancer.
79. A method of treating a mammal having cancer, comprising
administering to the mammal a soluble, extracellular TRAIL
polypeptide in an amount effective to induce cell death in the
mammal's cancer cells, wherein the TRAIL polypeptide comprises a
soluble fragment of the TRAIL protein of SEQ ID NO:2, wherein the
N-terminal amino acid of said fragment is selected from residues 39
to 124 of SEQ ID NO:2, and the C-terminal amino acid of said
fragment is selected from residues 276 to 281 of SEQ ID NO:2.
80. A method of claim 79, wherein said mammal is a human, and said
TRAIL polypeptide consists of a soluble fragment of the TRAIL
protein of SEQ ID NO:2, wherein the N-terminal amino acid of said
fragment is selected from residues 39 to 124 of SEQ ID NO:2, and
the C-terminal amino acid of said fragment is selected from
residues 276 to 281 of SEQ ID NO:2.
81. A method of claim 79, wherein said method additionally
comprises administering another agent that exerts a cytotoxic
effect on the cancer cells.
82. A method of claim 80, wherein said method additionally
comprises administering another agent that exerts a cytotoxic
effect on the cancer cells.
83. A method of claim 79, wherein said polypeptide is in the form
of a composition comprising the polypeptide and a physiologically
acceptable carrier, diluent, or excipient.
84. A method of treating a mammal having cancer, comprising
administering to the mammal a soluble, extracellular TRAIL
polypeptide in an amount effective to induce cell death in the
mammal's cancer cells, wherein the TRAIL polypeptide comprises
amino acids 124 to 281 of SEQ ID NO:2.
85. A method of claim 84, wherein said mammal is a human.
86. A method comprising administering an oligomer to a human who
has cancer, wherein said oligomer comprises at least two TRAIL
polypeptides, wherein each of said polypeptides is a soluble
fragment of the protein of SEQ ID NO:2, wherein said oligomer kills
TRAIL-sensitive cancer cells.
87. A method of claim 86, wherein the N-terminal amino acid of said
fragment is selected from residues 39 to 124 of SEQ ID NO:2, and
the C-terminal amino acid of said fragment is selected from
residues 276 to 281 of SEQ ID NO:2.
88. A method of claim 86, wherein each of said polypeptides
comprises amino acids 124 to 276 of SEQ ID NO:2.
89. A method of claim 86, wherein the oligomer comprises two or
three of said TRAIL polypeptides.
90. A method of claim 87, wherein the oligomer comprises three of
said TRAIL polypeptides.
91. A method of killing target cells selected from the group
consisting of cancer cells and virally-infected cells, comprising
contacting the target cells with an oligomer comprising at least
two TRAIL polypeptides, wherein each of said TRAIL polypeptides is
a soluble fragment of the protein of SEQ ID NO:2, wherein said
oligomer kills TRAIL-sensitive target cells.
92. A method of claim 91, wherein the N-terminal amino acid of said
fragment is selected from residues 39 to 124 of SEQ ID NO:2, and
the C-terminal amino acid of said fragment is selected from
residues 276 to 281 of SEQ ID NO:2.
93. A method of claim 91, wherein each of said polypeptides
comprises amino acids 124 to 276 of SEQ ID NO:2.
94. A method of claim 92, wherein the oligomer comprises three of
said TRAIL polypeptides.
95. A method of claim 91, wherein said method additionally
comprises administering another agent that exerts a cytotoxic
effect on the target cells.
96. A method of claim 86, wherein said oligomer is in the form of a
composition comprising the oligomer and a physiologically
acceptable carrier, diluent, or excipient.
97. A method of claim 91, wherein said oligomer is in the form of a
composition comprising the oligomer and a physiologically
acceptable carrier, diluent, or excipient.
98. A method of inducing death of cancer cells in vitro, comprising
contacting TRAIL-sensitive cancer cells with a soluble polypeptide
comprising an amino acid sequence that is at least 90% identical to
a sequence selected from the group consisting of: a) the
extracellular domain of human TRAIL (amino acids 39 to 281 of SEQ
ID NO:2); and b) a fragment of said extracellular domain; wherein
said polypeptide is capable of inducing apoptosis of Jurkat
cells.
99. The method of claim 98, wherein said polypeptide comprises an
amino acid sequence selected from the group consisting of: a) the
extracellular domain of human TRAIL (amino acids 39 to 281 of SEQ
ID NO:2); and b) a fragment of said extracellular domain; wherein
said polypeptide is capable of inducing apoptosis of Jurkat
cells.
100. A method of killing target cells in vitro wherein the target
cells are cancer cells or virally-infected cells, comprising
contacting the target cells with an oligomer comprising at least
two TRAIL polypeptides, wherein each of said TRAIL polypeptides is
a soluble fragment of the protein of SEQ ID NO:2, wherein said
oligomer kills TRAIL-sensitive target cells.
101. The method of claim 100, wherein the N-terminal amino acid of
said fragment is selected from residues 39 to 124 of SEQ ID NO:2,
and the C-terminal amino acid of said fragment is selected from
residues 276 to 281 of SEQ ID NO:2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 09/190,046, filed Nov. 10, 1998, currently pending, which
is a continuation-in-part of application Ser. No. 09/048,641, filed
Mar. 26, 1998, now abandoned, which is a continuation-in-part of
application Ser. No. 08/670,354, filed Jun. 25, 1996, now U.S. Pat.
No. 5,763,223, which is a continuation-in-part of application Ser.
No. 08/548,368, filed Nov. 1, 1995, now abandoned, which is a
continuation-in-part of application Ser. No. 08/496,632, filed Jun.
29, 1995, now abandoned.
BACKGROUND OF THE INVENTION
[0002] The programmed cell death known as apoptosis is distinct
from cell death due to necrosis. Apoptosis occurs in embryogenesis,
metamorphosis, endocrine-dependent tissue atrophy, normal tissue
turnover, and death of immune thymocytes (induced through their
antigen-receptor complex or by glucocorticoids) (Itoh et al., Cell
66: 233, 1991). During maturation of T-cells in the thymus, T-cells
that recognize self-antigens are destroyed through the apoptotic
process, whereas others are positively selected. The possibility
that some T-cells recognizing certain self epitopes (e.g.,
inefficiently processed and presented antigenic determinants of a
given self protein) escape this elimination process and
subsequently play a role in autoimmune diseases has been suggested
(Gammon et al., Immunology Today 12: 193, 1991).
[0003] A cell surface antigen known as Fas has been reported to
mediate apoptosis and is believed to play a role in clonal deletion
of self-reactive T-cells (Itoh et al., Cell 66: 233, 1991;
Watanabe-Fukunage et al., Nature 356: 314, 1992). Cross-linking a
specific monoclonal antibody to Fas has been reported to induce
various cell lines to undergo apoptosis (Yonehara et al., J. Exp.
Med., 169: 1747, 1989; Trauth et al., Science, 245: 301, 1989).
However, under certain conditions, binding of a specific monoclonal
antibody to Fas can have a costimulatory effect on freshly isolated
T cells (Alderson et al., J. Exp. Med. 178: 2231, 1993).
[0004] DNAs encoding a rat Fas ligand (Suda et al., Cell, 75: 1169,
1993) and a human Fas ligand (Takahashi et al., International
Immunology 6: 1567, 1994) have been isolated. Binding of the Fas
ligand to cells expressing Fas antigen has been demonstrated to
induce apoptosis (Suda et al., supra, and Takahashi et al.,
supra).
[0005] Investigation into the existence and identity of other
molecule(s) that play a role in apoptosis is desirable. Identifying
such molecules would provide an additional means of regulating
apoptosis, as well as providing further insight into the
development of self-tolerance by the immune system and the etiology
of autoimmune diseases.
SUMMARY OF THE INVENTION
[0006] The present invention provides a novel cytokine protein, as
well as isolated DNA encoding the cytokine and expression vectors
comprising the isolated DNA. Properties of the novel cytokine,
which is a member of the tumor necrosis factor (TNF) family of
ligands, include the ability to induce apoptosis of certain types
of target cells. This protein thus is designated TNF Related
Apoptosis Inducing Ligand (TRAIL). Among the types of cells that
are killed by contact with TRAIL are cancer cells such as leukemia,
lymphoma, and melanoma cells, and cells infected with a virus.
[0007] A method for producing TRAIL polypeptides involves culturing
host cells transformed with a recombinant expression vector that
contains TRAIL-encoding DNA under conditions appropriate for
expression of TRAIL, then recovering the expressed TRAIL
polypeptide from the culture. Antibodies directed against TRAIL
polypeptides are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 presents the results of an assay described in example
8. The assay demonstrated that a soluble human TRAIL polypeptide
induced death of Jurkat cells, which are a leukemia cell line.
[0009] FIG. 2 presents the results of an assay described in example
11. Contact with a soluble human TRAIL polypeptide induced death of
cytomegalovirus-infected human fibroblasts, whereas non-virally
infected fibroblasts were not killed.
[0010] FIG. 3 depicts a particular fusion protein encoded by an
expression vector of the present invention. The fusion protein
comprises (from N- to C-terminus) a growth hormone-derived leader
sequence (SEQ ID NO:19), followed by a tripeptide encoded by an
oligonucleotide employed in vector construction, a leucine zipper
peptide (SEQ ID NO:15), a tripeptide encoded by an oligonucleotide
employed in vector construction, and a soluble human TRAIL
polypeptide (amino acids 95 to 281 of SEQ ID NO:2). A DNA sequence
encoding the fusion protein, and the amino acid sequence of the
fusion protein, are presented in SEQ ID NO:10 and 11,
respectively.
[0011] FIG. 4 depicts a fusion protein encoded by another
expression vector of the present invention, comprising (from N- to
C-terminus) a cytomegalovirus-derived leader sequence (amino acids
1 to 29 of SEQ ID NO:9), followed by a tripeptide encoded by an
oligonucleotide employed in vector construction (amino acids 30 to
32 of SEQ ID NO:9), a leucine zipper peptide (SEQ ID NO:15), a
tripeptide encoded by an oligonucleotide employed in vector
construction, and a soluble human TRAIL polypeptide (amino acids 95
to 281 of SEQ ID NO:2). A DNA sequence encoding the fusion protein,
and the amino acid sequence of the fusion protein, are presented in
SEQ ID NO:12 and 13, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0012] A novel protein designated TRAIL is provided herein, along
with DNA encoding TRAIL and recombinant expression vectors
comprising TRAIL DNA. A method for producing recombinant TRAIL
polypeptides involves cultivating host cells transformed with the
recombinant expression vectors under conditions appropriate for
expression of TRAIL, and recovering the expressed TRAIL.
[0013] The present invention also provides antibodies that
specifically bind TRAIL proteins. In one embodiment, the antibodies
are monoclonal antibodies.
[0014] The TRAIL protein induces apoptosis of certain types of
target cells, such as transformed cells that include but are not
limited to cancer cells and virally-infected cells. As demonstrated
in examples 5, 8, 9, and 10 below, TRAIL induced apoptosis of human
leukemia, lymphoma, and melanoma cell lines. Among the uses of
TRAIL is use in killing cancer cells. TRAIL finds further use in
treatment of viral infections. Infection with cytomegalovirus (CMV)
rendered human fibroblasts susceptible to apoptosis when contacted
with TRAIL, whereas uninfected fibroblasts were not killed through
contact with TRAIL (see example 11).
[0015] Isolation of a DNA encoding human TRAIL is described in
example 1 below. The nucleotide sequence of the human TRAIL DNA
isolated in example 1 is presented in SEQ ID NO:1, and the amino
acid sequence encoded thereby is presented in SEQ ID NO:2. This
human TRAIL protein comprises an N-terminal cytoplasmic domain
(amino acids 1-18), a transmembrane region (amino acids 19-38), and
an extracellular domain (amino acids 39-281). The extracellular
domain contains a receptor-binding region.
[0016] E. coli strain DH10B cells transformed with a recombinant
vector containing this human TRAIL DNA were deposited with the
American Type Culture Collection on Jun. 14, 1995, and assigned
accession no. 69849. The deposit was made under the terms of the
Budapest Treaty. The recombinant vector in the deposited strain is
the expression vector pDC409 (described in example 5). The vector
was digested with SalI and NotI, and human TRAIL DNA that includes
the entire coding region shown in SEQ ID NO:1 was ligated into the
vector.
[0017] DNA encoding a second human TRAIL protein was isolated as
described in example 2. The nucleotide sequence of this DNA is
presented in SEQ ID NO:3, and the amino acid sequence encoded
thereby is presented in SEQ ID NO:4. The encoded protein comprises
an N-terminal cytoplasmic domain (amino acids 1-18), a
transmembrane region (amino acids 19-38), and an extracellular
domain (amino acids 39-101).
[0018] The DNA of SEQ ID NO:3 lacks a portion of the DNA of SEQ ID
NO:1, and is thus designated the human TRAIL deletion variant
(huTRAILdv) clone. Nucleotides 18 through 358 of SEQ ID NO:1 are
identical to nucleotides 8 through 348 of the huTRAILdv DNA of SEQ
ID NO:3. Nucleotides 359 through 506 of SEQ ID NO:1 are missing
from the cloned DNA of SEQ ID NO:3. The deletion causes a shift in
the reading frame, which results in an in-frame stop codon after
amino acid 101 of SEQ ID NO:4. The DNA of SEQ ID NO:3 thus encodes
a truncated protein. Amino acids 1 through 90 of SEQ ID NO:2 are
identical to amino acids 1 through 90 of SEQ ID NO:4. However, due
to the deletion, the C-terminal portion of the huTRAILdv protein
(amino acids 91 through 101 of SEQ ID NO:4) differs from the
residues in the corresponding positions in SEQ ID NO:2. In contrast
to the full length huTRAIL protein, the truncated huTRAILdv protein
does not exhibit the ability to induce apoptosis of the T cell
leukemia cells of the Jurkat cell line.
[0019] DNA encoding a mouse TRAIL protein has also been isolated,
as described in example 3. The nucleotide sequence of this DNA is
presented in SEQ ID NO:5 and the amino acid sequence encoded
thereby is presented in SEQ ID NO:6. The encoded protein comprises
an N-terminal cytoplasmic domain (amino acids 1-17), a
transmembrane region (amino acids 18-38), and an extracellular
domain (amino acids 39-291). This mouse TRAIL is 64% identical to
the human TRAIL of SEQ ID NO:2 at the amino acid level. The coding
region of the mouse TRAIL nucleotide sequence is 75% identical to
the coding region of the human nucleotide sequence of SEQ ID
NO:1.
[0020] One embodiment of the present invention is directed to human
TRAIL protein characterized by the N-terminal amino acid sequence
MetAlaMetMetGluValGlnGly GlyProSerLeuGlyGlnThr (amino acids 1-15 of
SEQ ID NOS:2 and 4). Mouse TRAIL proteins characterized by the
N-terminal amino acid sequence MetProSerSerGlyAla
LeuLysAspLeuSerPheSerGlnHis (amino acids 1-15 of SEQ ID NO:6) are
also provided herein.
[0021] The TRAIL of the present invention is distinct from the
protein known as Fas ligand (Suda et al., Cell, 75: 1169, 1993;
Takahashi et al., International Immunology 6: 1567, 1994). Fas
ligand induces apoptosis of certain cell types, via the receptor
known as Fas. As demonstrated in example 5, TRAIL-induced apoptosis
of target cells is not mediated through Fas. The human TRAIL amino
acid sequence of SEQ ID NO:2 is about 20% identical to the human
Fas ligand amino acid sequence that is presented in Takahashi et
al., supra. The extracellular domain of human TRAIL is about 28.4%
identical to the extracellular domain of human Fas ligand.
[0022] The amino acid sequences disclosed herein reveal that TRAIL
is a member of the TNF family of ligands (Smith et al. Cell, 73:
1349, 1993; Suda et al., Cell, 75: 1169, 1993; Smith et al., Cell,
76: 959, 1994). The percent identities between the human TRAIL
extracellular domain amino acid sequence and the amino acid
sequence of the extracellular domain of other proteins of this
family are as follows: 28.4% with Fas ligand, 22.4% with
lymphotoxin-.beta., 22.9% with TNF-.alpha., 23.1% with TNF-.beta.,
22.1% with CD30 ligand, and 23.4% with CD40 ligand.
[0023] TRAIL was tested for ability to bind receptors of the TNF-R
family of receptors. The binding analysis was conducted using the
slide autoradiography procedure of Gearing et al. (EMBO J. 8: 3667,
1989). The analysis revealed no detectable binding of human TRAIL
to human CD30, CD40, 4-1BB, OX40, TNF-R (p80 form), CD27, or
LT.beta.R (also known as TNFR-RP). The results in example 5
indicate that human TRAIL does not bind human Fas.
[0024] The TRAIL polypeptides of the present invention include
polypeptides having amino acid sequences that differ from, but are
highly homologous to, those presented in SEQ ID NOS:2 and 6.
Examples include, but are not limited to, homologs derived from
other mammalian species, variants (both naturally occurring
variants and those generated by recombinant DNA technology), and
TRAIL fragments that retain a desired biological activity. Such
polypeptides exhibit a biological activity of the TRAIL proteins of
SEQ ID NOS:2 and 6, and preferably comprise an amino acid sequence
that is at least 80% identical (most preferably at least 90%
identical) to the amino acid sequence presented in SEQ ID NO:2 or
SEQ ID NO:6. These embodiments of the present invention are
described in more detail below.
[0025] Conserved sequences located in the C-terminal portion of
proteins in the TNF family are identified in Smith et al. (Cell,
73: 1349, 1993, see page 1353 and FIG. 6); Suda et al. (Cell, 75:
1169, 1993, see FIG. 7); Smith et al. (Cell, 76: 959, 1994, see
FIG. 3); and Goodwin et al. (Eur. J. Immunol., 23: 2631, 1993, see
FIG. 7 and pages 2638-39), hereby incorporated by reference. Among
the amino acids in the human TRAIL protein that are conserved (in
at least a majority of TNF family members) are those in positions
124-125 (AH), 136 (L), 154 (W), 169 (L), 174 (L), 180 (G), 182 (Y),
187 (O), 190 (F), 193 (O), and 275-276 (FG) of SEQ ID NO:2. Another
structural feature of TRAIL is a spacer region between the
C-terminus of the trans-membrane region and the portion of the
extracellular domain that is believed to be most important for
biological activity. This spacer region, located at the N-terminus
of the extracellular domain, consists of amino acids 39 through 94
of SEQ ID NO:2. Analogous spacers are found in other family
members, e.g., CD40 ligand. Amino acids 138 through 153 correspond
to a loop between the .beta. sheets of the folded (three
dimensional) human TRAIL protein.
[0026] Provided herein are membrane-bound TRAIL proteins
(comprising a cytoplasmic domain, a transmembrane region, and an
extracellular domain) as well as TRAIL fragments that retain a
desired biological property of the full length TRAIL protein. In
one embodiment, TRAIL fragments are soluble TRAIL polypeptides
comprising all or part of the extracellular domain, but lacking the
transmembrane region that would cause retention of the polypeptide
on a cell membrane. Soluble TRAIL proteins are capable of being
secreted from the cells in which they are expressed.
Advantageously, a heterologous signal peptide is fused to the
N-terminus such that the soluble TRAIL is secreted upon
expression.
[0027] Soluble TRAIL may be identified (and distinguished from its
non-soluble membrane-bound counterparts) by separating intact cells
which express the desired protein from the culture medium, e.g., by
centrifugation, and assaying the medium (supernatant) for the
presence of the desired protein. The presence of TRAIL in the
medium indicates that the protein was secreted from the cells and
thus is a soluble form of the TRAIL protein. Naturally-occurring
soluble forms of TRAIL are encompassed by the present
invention.
[0028] The use of soluble forms of TRAIL is advantageous for
certain applications. Purification of the proteins from recombinant
host cells is facilitated, since the soluble proteins are secreted
from the cells. Further, soluble proteins are generally more
suitable for intravenous administration.
[0029] Examples of soluble TRAIL polypeptides are those containing
the entire extracellular domain (e.g., amino acids 39 to 281 of SEQ
ID NO:2 or amino acids 39 to 291 of SEQ ID NO:6). Fragments of the
extracellular domain that retain a desired biological activity are
also provided. Such fragments advantageously include regions of
TRAIL that are conserved in proteins of the TNF family of ligands,
as described above.
[0030] Additional examples of soluble TRAIL polypeptides are those
lacking not only the cytoplasmic domain and transmembrane region,
but also all or part of the above-described spacer region. Soluble
human TRAIL polypeptides thus include, but are not limited to,
polypeptides comprising amino acids x to 281, wherein x represents
any of the amino acids in positions 39 through 95 of SEQ ID NO:2.
In the embodiment in which residue 95 is the N-terminal amino acid,
the entire spacer region has been deleted.
[0031] TRAIL fragments, including soluble polypeptides, may be
prepared by any of a number of conventional techniques. A DNA
sequence encoding a desired TRAIL fragment may be subcloned into an
expression vector for production of the TRAIL fragment. The
TRAIL-encoding DNA sequence advantageously is fused to a sequence
encoding a suitable leader or signal peptide. The desired
TRAIL-encoding DNA fragment may be chemically synthesized using
known techniques. DNA fragments also may be produced by restriction
endonuclease digestion of a full length cloned DNA sequence, and
isolated by electrophoresis on agarose gels. If necessary,
oligonucleotides that reconstruct the 5' or 3' terminus to a
desired point may be ligated to a DNA fragment generated by
restriction enzyme digestion. Such oligonucleotides may
additionally contain a restriction endonuclease cleavage site
upstream of the desired coding sequence, and position an initiation
codon (ATG) at the N-terminus of the coding sequence.
[0032] The well known polymerase chain reaction (PCR) procedure
also may be employed to isolate and amplify a DNA sequence encoding
a desired protein fragment. Oligonucleotides that define the
desired termini of the DNA fragment are employed as 5' and 3'
primers. The oligonucleotides may additionally contain recognition
sites for restriction endonucleases, to faciliate insertion of the
amplified DNA fragment into an expression vector. PCR techniques
are described in Saiki et al., Science 239: 487 (1988); Recombinant
DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego
(1989), pp. 189-196; and PCR Protocols: A Guide to Methods and
Applications, Innis et al., eds., Academic Press, Inc. (1990).
[0033] As will be understood by the skilled artisan, the
transmembrane region of each TRAIL protein discussed above is
identified in accordance with conventional criteria for identifying
that type of hydrophobic domain. The exact boundaries of a
transmembrane region may vary slightly (most likely by no more than
five amino acids on either end) from those presented above.
Computer programs useful for identifying such hydrophobic regions
in proteins are available.
[0034] The TRAIL DNA of the present invention includes cDNA,
chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and
combinations thereof. Genomic TRAIL DNA may be isolated by
hybridization to the TRAIL cDNA disclosed herein using standard
techniques. RNA transcribed from the TRAIL DNA is also encompassed
by the present invention.
[0035] A search of the NCBI databank identified five expressed
sequence tags (ESTs) having regions of identity with TRAIL DNA.
These ESTs (NCBI accession numbers T90422, T82085, T10524, R31020,
and Z36726) are all human cDNA fragments. The NCBI records do not
disclose any polypeptide encoded by the ESTs, and do not indicate
what the reading frame, if any, might be. However, even if the
knowledge of the reading frame revealed herein by disclosure of
complete TRAIL coding regions is used to express the ESTs, none of
the encoded polypeptides would have the apoptosis-inducing property
of the presently-claimed TRAIL polypeptides. In other words, if
each of the five ESTs were inserted into expression vectors
downstream from an initiator methionine codon, in the reading frame
elucidated herein, none of the resulting expressed polypeptides
would contain a sufficient portion of the extracellular domain of
TRAIL to induce apoptosis of Jurkat cells.
[0036] Certain embodiments of the present invention provide
isolated DNA comprising a nucleotide sequence selected from the
group consisting of nucleotides 88 to 933 of SEQ ID NO:1 (human
TRAIL coding region); nucleotides 202 to 933 of SEQ ID NO:1
(encoding the human TRAIL extracellular domain); nucleotides 47 to
922 of SEQ ID NO:5 (mouse TRAIL coding region); and nucleotides 261
to 922 of SEQ ID NO:5 (encoding the mouse TRAIL extracellular
domain). DNAs encoding biologically active fragments of the
proteins of SEQ ID NOS:2 and 6 are also provided. Further
embodiments include sequences comprising nucleotides 370 to 930 of
SEQ ID NO:1 and nucleotides 341 to 919 of SEQ ID NO:5, which encode
the particular human and murine soluble TRAIL polypeptides,
respectively, described in example 7.
[0037] Due to degeneracy of the genetic code, two DNA sequences may
differ, yet encode the same amino acid sequence. The present
invention thus provides isolated DNA sequences encoding
biologically active TRAIL, selected from DNA comprising the coding
region of a native human or murine TRAIL cDNA, or fragments
thereof, and DNA which is degenerate as a result of the genetic
code to the native TRAIL DNA sequence.
[0038] Also provided herein are purified TRAIL polypeptides, both
recombinant and non-recombinant. Variants and derivatives of native
TRAIL proteins that retain a desired biological activity are also
within the scope of the present invention. In one embodiment, the
biological activity of an TRAIL variant is essentially equivalent
to the biological activity of a native TRAIL protein. One desired
biological activity of TRAIL is the ability to induce death of
Jurkat cells. Assay procedures for detecting apoptosis of target
cells are well known. DNA laddering is among the characteristics of
cell death via apoptosis, and is recognized as one of the
observable phenomena that distinguish apoptotic cell death from
necrotic cell death. Examples of assay techniques suitable for
detecting death or apoptosis of target cells include those
described in examples 5 and 8 to 11. Another property of TRAIL is
the ability to bind to Jurkat cells.
[0039] TRAIL variants may be obtained by mutations of native TRAIL
nucleotide sequences, for example. A TRAIL variant, as referred to
herein, is a polypeptide substantially homologous to a native
TRAIL, but which has an amino acid sequence different from that of
native TRAIL because of one or a plurality of deletions, insertions
or substitutions. TRAIL-encoding DNA sequences of the present
invention encompass sequences that comprise one or more additions,
deletions, or substitutions of nucleotides when compared to a
native TRAIL DNA sequence, but that encode an TRAIL protein that is
essentially biologically equivalent to a native TRAIL protein.
[0040] The variant amino acid or DNA sequence preferably is at
least 80% identical to a native TRAIL sequence, most preferably at
least 90% identical. The degree of homology (percent identity)
between a native and a mutant sequence may be determined, for
example, by comparing the two sequences using computer programs
commonly employed for this purpose. One suitable program is the GAP
computer program, version 6.0, described by Devereux et al. (Nucl.
Acids Res. 12: 387, 1984) and available from the University of
Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes
the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:
443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:
482, 1981). Briefly, the GAP program defines identity as the number
of aligned symbols (i.e., nucleotides or amino acids) which are
identical, divided by the total number of symbols in the shorter of
the two sequences. The preferred default parameters for the GAP
program include: (1) a unary comparison matrix (containing a value
of 1 for identities and 0 for non-identities) for nucleotides, and
the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids
Res. 14: 6745, 1986, as described by Schwartz and Dayhoff, eds.,
Atlas of Protein Sequence and Structure, National Biomedical
Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps.
[0041] Alterations of the native amino acid sequence may be
accomplished by any of a number of known techniques. Mutations can
be introduced at particular loci by synthesizing oligonucleotides
containing a mutant sequence, flanked by restriction sites enabling
ligation to fragments of the native sequence. Following ligation,
the resulting reconstructed sequence encodes an analog having the
desired amino acid insertion, substitution, or deletion.
[0042] Alternatively, oligonucleotide-directed site-specific
mutagenesis procedures can be employed to provide an altered gene
having particular codons altered according to the substitution,
deletion, or insertion required. Techniques for making such
alterations include those disclosed by Walder et al. (Gene 42: 133,
1986); Bauer et al. (Gene 37: 73, 1985); Craik (BioTechniques,
January 1985, 12-19); Smith et al. (Genetic Engineering: Principles
and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and
4,737,462, which are incorporated by reference herein.
[0043] Variants may comprise conservatively substituted sequences,
meaning that one or more amino acid residues of a native TRAIL
polypeptide are replaced by different residues, but that the
conservatively substituted TRAIL polypeptide retains a desired
biological activity that is essentially equivalent to that of a
native TRAIL polypeptide. Examples of conservative substitutions
include substitution of amino acids that do not alter the secondary
and/or tertiary structure of TRAIL. Other examples involve
substitution of amino acids outside of the receptor-binding domain,
when the desired biological activity is the ability to bind to a
receptor on target cells and induce apoptosis of the target cells.
A given amino acid may be replaced by a residue having similar
physiochemical characteristics, e.g., substituting one aliphatic
residue for another (such as Ile, Val, Leu, or Ala for one
another), or substitution of one polar residue for another (such as
between Lys and Arg; Glu and Asp; or Gln and Asn). Other such
conservative substitutions, e.g., substitutions of entire regions
having similar hydrophobicity characteristics, are well known.
TRAIL polypeptides comprising conservative amino acid substitutions
may be tested in one of the assays described herein to confirm that
a desired biological activity of a native TRAIL is retained. DNA
sequences encoding TRAIL polypeptides that contain such
conservative amino acid substitutions are encompassed by the
present invention.
[0044] Conserved amino acids located in the C-terminal portion of
proteins in the TNF family, and believed to be important for
biological activity, have been identified. These conserved
sequences are discussed in Smith et al. (Cell, 73: 1349, 1993, see
page 1353 and FIG. 6); Suda et al. (Cell, 75: 1169, 1993, see FIG.
7); Smith et al. (Cell, 76: 959, 1994, see FIG. 3); and Goodwin et
al. (Eur. J. Immunol., 23: 2631, 1993, see FIG. 7 and pages
2638-39). Advantageously, the conserved amino acids are not altered
when generating conservatively substituted sequences. If altered,
amino acids found at equivalent positions in other members of the
TNF family are substituted.
[0045] TRAIL also may be modified to create TRAIL derivatives by
forming covalent or aggregative conjugates with other chemical
moieties, such as glycosyl groups, lipids, phosphate, acetyl groups
and the like. Covalent derivatives of TRAIL may be prepared by
linking the chemical moieties to functional groups on TRAIL amino
acid side chains or at the N-terminus or C-terminus of a TRAIL
polypeptide or the extracellular domain thereof. Other derivatives
of TRAIL within the scope of this invention include covalent or
aggregative conjugates of TRAIL or its fragments with other
proteins or polypeptides, such as by synthesis in recombinant
culture as N-terminal or C-terminal fusions. For example, the
conjugate may comprise a signal or leader polypeptide sequence
(e.g. the .alpha.-factor leader of Saccharomyces) at the N-terminus
of a TRAIL polypeptide. The signal or leader peptide
co-translationally or post-translationally directs transfer of the
conjugate from its site of synthesis to a site inside or outside of
the cell membrane or cell wall.
[0046] TRAIL polypeptide fusions can comprise peptides added to
facilitate purification and identification of TRAIL. Such peptides
include, for example, poly-His or the antigenic identification
peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al.,
Bio/Technology 6: 1204, 1988. One such peptide is the FLAG.RTM.
peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Ly- s (DYKDDDDK) (SEQ ID
NO:7), which is highly antigenic and provides an epitope reversibly
bound by a specific monoclonal antibody, thus enabling rapid assay
and facile purification of expressed recombinant protein. This
sequence is also specifically cleaved by bovine mucosal
enterokinase at the residue immediately following the Asp-Lys
pairing. Fusion proteins capped with this peptide may also be
resistant to intracellular degradation in E. coli.
[0047] A murine hybridoma designated 4E11 produces a monoclonal
antibody that binds the peptide DYKDDDDK (SEQ ID NO:7) in the
presence of certain divalent metal cations (as described in U.S.
Pat. No. 5,011,912), and has been deposited with the American Type
Culture Collection under accession no HB 9259. Expression systems
useful for producing recombinant proteins fused to the FLAG.RTM.
peptide, as well as monoclonal antibodies that bind the peptide and
are useful in purifying the recombinant proteins, are available
from Eastman Kodak Company, Scientific Imaging Systems, New Haven,
Conn.
[0048] The present invention further includes TRAIL polypeptides
with or without associated native-pattern glycosylation. TRAIL
expressed in yeast or mammalian expression systems may be similar
to or significantly different from a native TRAIL polypeptide in
molecular weight and glycosylation pattern, depending upon the
choice of expression system. Expression of TRAIL polypeptides in
bacterial expression systems, such as E. coli, provides
non-glycosylated molecules.
[0049] Glycosylation sites in the TRAIL extracellular domain can be
modified to preclude glycosylation while allowing expression of a
homogeneous, reduced carbohydrate analog using yeast or mammalian
expression systems. N-glycosylation sites in eukaryotic
polypeptides are characterized by an amino acid triplet Asn-X-Y,
wherein X is any amino acid except Pro and Y is Ser or Thr.
Appropriate modifications to the nucleotide sequence encoding this
triplet will result in substitutions, additions or deletions that
prevent attachment of carbohydrate residues at the Asn side chain.
Known procedures for inactivating N-glycosylation sites in proteins
include those described in U.S. Pat. No. 5,071,972 and EP 276,846.
A potential N-glycosylation site is found at positions 109-111 in
the human protein of SEQ ID NO:2 and at positions 52-54 in the
murine protein of SEQ ID NO:6.
[0050] Alternatively, known procedures such as mutagenesis may be
employed to add glycosylation sites to TRAIL, thereby promoting an
increase in the carbohydrate moieties attached to TRAIL. Such an
approach may be taken when slowing the clearance of TRAIL from the
body following in vivo administration is desired, for example.
[0051] In another example, sequences encoding Cys residues that are
not essential for biological activity can be altered to cause the
Cys residues to be deleted or replaced with other amino acids,
preventing formation of incorrect intramolecular disulfide bridges
upon renaturation. Cysteine residues are found in the human TRAIL
protein of SEQ ID NO:2 at positions 16, 30, 56, 77, and 230; and in
the murine TRAIL protein of SEQ ID NO:6 at positions 22, 60, 81,
and 240.
[0052] Among the soluble human TRAIL polypeptides disclosed herein
are fragments of the extracellular domain that lack the spacer
region, as described above. Such spacer-deleted soluble TRAIL
polypeptides include only one cysteine, corresponding to the
residue at position 230 of SEQ ID NO:2. Thus, any disulfide bonds
forming from the Cys-230 residue would be intermolecular, joining
two such soluble TRAIL polypeptides. In the fusion protein of FIG.
3 (SEQ ID NO:11), the TRAIL polypeptide moiety comprises only one
cysteine, at position 202 (which corresponds to the cysteine
residue at position 230 in the full length human TRAIL sequence of
SEQ ID NO:2). In the fusion protein of FIG. 4 (SEQ ID NO:13), the
TRAIL polypeptide comprises only one cysteine, at position 205
(which corresponds to the Cys-230 residue in SEQ ID NO:2).
[0053] One embodiment of the invention is directed to a TRAIL
polypeptide (or fusion protein comprising a TRAIL polypeptide), in
which the cysteine residue corresponding to the cysteine at
position 230 in SEQ ID NO:2 is deleted or substituted, in order to
prevent formation of disulfide bonds that involve the Cys-230
residue. If substituted, cysteine may be replaced by any suitable
amino acid, whereby a desired biological activity of TRAIL is
maintained. Examples include, but are not limited to, serine,
alanine, glycine, or valine. Altering the number of cysteine
residues to manipulate oligomer formation is discussed further
below.
[0054] Other variants are prepared by modification of adjacent
dibasic amino acid residues to enhance expression in yeast systems
in which KEX2 protease activity is present. EP 212,914 discloses
the use of site-specific mutagenesis to inactivate KEX2 protease
processing sites in a protein. KEX2 protease processing sites are
inactivated by deleting, adding or substituting residues to alter
Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of
these adjacent basic residues. Lys-Lys pairings are considerably
less susceptible to KEX2 cleavage, and conversion of Arg-Lys or
Lys-Arg to Lys-Lys represents a conservative and preferred approach
to inactivating KEX2 sites. Potential KEX2 protease processing
sites are found at positions 89-90 and 149-150 in the protein of
SEQ ID NO:2, and at positions 85-86, 135-136, and 162-163 in the
protein of SEQ ID NO:6.
[0055] Naturally occurring TRAIL variants are also encompassed by
the present invention. Examples of such variants are proteins that
result from alternative mRNA splicing events (since TRAIL is
encoded by a multi-exon gene) or from proteolytic cleavage of the
TRAIL protein, wherein a desired biological activity is retained.
Alternative splicing of mRNA may yield a truncated but biologically
active TRAIL protein, such as a naturally occurring soluble form of
the protein, for example. Variations attributable to proteolysis
include, for example, differences in the N- or C-termini upon
expression in different types of host cells, due to proteolytic
removal of one or more terminal amino acids from the TRAIL protein.
In addition, proteolytic cleavage may release a soluble form of
TRAIL from a membrane-bound form of the protein. Allelic variants
are also encompassed by the present invention.
[0056] Also provided herein are conjugates or fusion proteins
comprising a TRAIL polypeptide and a tumor-targeting moiety. Such
embodiments may be employed in cancer treatment, for example. The
TRAIL component may be any of the various forms of TRAIL disclosed
herein, with one example being a soluble TRAIL polypeptide.
Oligomers comprising such fusion proteins also are contemplated.
The conjugates or fusion proteins may additionally comprise other
components of TRAIL-containing fusions, compositions, and the like
that are described herein. Examples of such other components
include, but are not limited to, leucine zipper peptides.
[0057] The tumor-targeting moiety may be any compound that enhances
delivery of TRAIL to a tumor. Such compounds include, but are not
limited to, compounds that selectively bind to cancer cells
compared with normal cells, specifically bind to a particular type
of cancer that is to be treated, or enhance penetration into solid
tumors. In one embodiment, the tumor-targeting moiety is a
peptide.
[0058] Examples of tumor-targeting peptides are described in Arap
et al. (Science 279: 377, Jan. 16, 1998), and Pasqualini et al.
(Nature Biotechnology 15: 542, June 1997), which are hereby
incorporated by reference in their entirety. Arap et al. and
Pasqualini et al. report studies of peptides that "home" to tumors,
selectively binding to tumor vessels and/or to tumor cells. The
tripeptides Arg-Gly-Asp, Asn-Gly-Arg and Gly-Ser-Leu, or peptides
comprising such tripeptide sequences, are contemplated herein for
use as tumor targeting moieties as components of TRAIL fusion
proteins.
[0059] Arap et al. and Pasqualini et al., supra, disclose that
peptides comprising the sequence Arg-Gly-Asp (RGD) bind to
integrins, including but not limited to .alpha..sub.v integrins.
Such integrins have been detected in tumor vasculature and on a
number of tumor cell types.
[0060] Arap et al. additionally disclose that peptides comprising
the tripeptide Asn-Gly-Arg or Gly-Ser-Leu selectively bind to
tumors. Among the peptides studied by Arap et al. are the
RGD-containing peptide CDCRGDCFC, and the NGR-containing peptides
CNGRCVSGCAGRC, NGRAHA, CVLNGRMEC, and CNGRC.
[0061] One type of fusion protein provided herein comprises a TRAIL
polypeptide and a peptide that binds an integrin associated with a
tumor. Such integrin may be expressed on tumor cells or tumor
vessels, for example. An example of an integrin is an .alpha..sub.v
integrin. Arap et al., supra, note that human .alpha..sub.v
integrins are selectively expressed in human tumor blood
vessels.
[0062] Oligomers
[0063] The present invention encompasses TRAIL polypeptides in the
form of oligomers, such as dimers, trimers, or higher oligomers.
Oligomers may be formed by disulfide bonds between cysteine
residues on different TRAIL polypeptides, or by non-covalent
interactions between TRAIL polypeptide chains, for example. In
other embodiments, oligomers comprise from two to four TRAIL
polypeptides joined via covalent or non-covalent interactions
between peptide moieties fused to the TRAIL polypeptides. Such
peptides may be peptide linkers (spacers), or peptides that have
the property of promoting oligomerization. Leucine zippers and
certain polypeptides derived from antibodies are among the peptides
that can promote oligomerization of TRAIL polypeptides attached
thereto, as described in more detail below. The TRAIL polypeptides
preferably are soluble.
[0064] Preparation of fusion proteins comprising heterologous
polypeptides fused to various portions of antibody-derived
polypeptides (including the Fc domain) has been described, e.g., by
Ashkenazi et al. (PNAS USA 88: 10535, 1991); Byrn et al. (Nature
344: 667, 1990); and Hollenbaugh and Aruffo ("Construction of
Immunoglobulin Fusion Proteins", in Current Protocols in
Immunology, Supplement 4, pages 10.19.1-10.19.11, 1992), hereby
incorporated by reference. In one embodiment of the invention, an
TRAIL dimer is created by fusing TRAIL to an Fc region polypeptide
derived from an antibody. The term "Fc polypeptide" includes native
and mutein forms, as well as truncated Fc polypeptides containing
the hinge region that promotes dimerization. The Fc polypeptide
preferably is fused to a soluble TRAIL (e.g., comprising only the
extracellular domain).
[0065] A gene fusion encoding the TRAIL/Fc fusion protein is
inserted into an appropriate expression vector. In one embodiment,
the Fc polypeptide is fused to the N-terminus of a soluble TRAIL
polypeptide. The TRAIL/Fc fusion proteins are allowed to assemble
much like antibody molecules, whereupon interchain disulfide bonds
form between the Fc polypeptides, yielding divalent TRAIL. In other
embodiments, TRAIL may be substituted for the variable portion of
an antibody heavy or light chain. If fusion proteins are made with
both heavy and light chains of an antibody, it is possible to form
an TRAIL oligomer with as many as four TRAIL extracellular
regions.
[0066] One suitable Fc polypeptide is the native Fc region
polypeptide derived from a human IgG1, which is described in PCT
application WO 93/10151, hereby incorporated by reference. Another
useful Fc polypeptide is the Fc mutein described in U.S. Pat. No.
5,457,035. The amino acid sequence of the mutein is identical to
that of the native Fc sequence presented in WO 93/10151, except
that amino acid 19 has been changed from Leu to Ala, amino acid 20
has been changed from Leu to Glu, and amino acid 22 has been
changed from Gly to Ala. This mutein Fc exhibits reduced affinity
for immunoglobulin receptors.
[0067] Alternatively, oligomeric TRAIL may comprise two or more
soluble TRAIL polypeptides joined through peptide linkers. Examples
include those peptide linkers described in U.S. Pat. No. 5,073,627
(hereby incorporated by reference). Fusion proteins comprising
multiple TRAIL polypeptides separated by peptide linkers may be
produced using conventional recombinant DNA technology.
[0068] Another method for preparing oligomeric TRAIL polypeptides
involves use of a leucine zipper. Leucine zipper domains are
peptides that promote oligomerization of the proteins in which they
are found. Leucine zippers were originally identified in several
DNA-binding proteins (Landschulz et al., Science 240: 1759, 1988),
and have since been found in a variety of different proteins. Among
the known leucine zippers are naturally occurring peptides and
derivatives thereof that dimerize or trimerize.
[0069] Leucine zippers were originally identified in several
DNA-binding proteins (Landschulz et al., Science 240: 1759, 1988).
Zipper domain is a term used to refer to a conserved peptide domain
present in these (and other) proteins, which is responsible for
oligomerization of the proteins. The zipper domain (also referred
to herein as an oligomerizing, or oligomer-forming, domain)
comprises a repetitive heptad repeat, often with four or five
leucine residues interspersed with other amino acids. Examples of
zipper domains are those found in the yeast transcription factor
GCN4 and a heat-stable DNA-binding protein found in rat liver
(C/EBP; Landschulz et al., Science 243: 1681, 1989). Two nuclear
transforming proteins, fos and jun, also exhibit zipper domains, as
does the gene product of the murine proto-oncogene, c-myc
(Landschulz et al., Science 240: 1759, 1988). The products of the
nuclear oncogenes fos and jun comprise zipper domains
preferentially form a heterodimer (O'Shea et al., Science 245: 646,
1989; Turner and Tjian, Science 243: 1689, 1989). The zipper domain
is necessary for biological activity (DNA binding) in these
proteins.
[0070] The fusogenic proteins of several different viruses,
including paramyxovirus, coronavirus, measles virus and many
retroviruses, also possess zipper domains (Buckland and Wild,
Nature 338: 547, 1989; Britton, Nature 353: 394, 1991; Delwart and
Mosialos, AIDS Research and Human Retroviruses 6: 703, 1990). The
zipper domains in these fusogenic viral proteins are near the
transmembrane region of the proteins; it has been suggested that
the zipper domains could contribute to the oligomeric structure of
the fusogenic proteins. Oligomerization of fusogenic viral proteins
is involved in fusion pore formation (Spruce et al, Proc. Natl.
Acad. Sci. U.S.A. 88: 3523, 1991). Zipper domains have also been
recently reported to play a role in oligomerization of heat-shock
transcription factors (Rabindran et al., Science 259: 230,
1993).
[0071] Zipper domains fold as short, parallel coiled coils. (O'Shea
et al., Science 254: 539; 1991) The general architecture of the
parallel coiled coil has been well characterized, with a
"knobs-into-holes" packing as proposed by Crick in 1953 (Acta
Crystallogr. 6: 689). The dimer formed by a zipper domain is
stabilized by the heptad repeat, designated (abcdefg).sub.n
according to the notation of McLachlan and Stewart (J. Mol. Biol.
98: 293; 1975), in which residues a and d are generally hydrophobic
residues, with d being a leucine, which line up on the same face of
a helix. Oppositely-charged residues commonly occur at positions g
and e. Thus, in a parallel coiled coil formed from two helical
zipper domains, the "knobs" formed by the hydrophobic side chains
of the first helix are packed into the "holes" formed between the
side chains of the second helix.
[0072] The residues at position d (often leucine) contribute large
hydrophobic stabilization energies, and are important for oligomer
formation (Krystek et al., Int. J. Peptide Res. 38: 229, 1991).
Lovejoy et al. recently reported the synthesis of a triple-stranded
.alpha.-helical bundle in which the helices run up-up-down (Science
259: 1288, 1993). Their studies confirmed that hydrophobic
stabilization energy provides the main driving force for the
formation of coiled coils from helical monomers. These studies also
indicate that electrostatic interactions contribute to the
stoichiometry and geometry of coiled coils. Further discussion of
the structure of leucine zippers is found in Harbury et al.
(Science 262: 1401, 26 Nov. 1993).
[0073] Several studies have indicated that conservative amino acids
may be substituted for individual leucine residues with minimal
decrease in the ability to dimerize; multiple changes, however,
usually result in loss of this ability (Landschulz et al., Science
243: 1681, 1989; Turner and Tjian, Science 243: 1689, 1989; Hu et
al., Science 250: 1400, 1990). van Heekeren et al. reported that a
number of different amino residues can be substituted for the
leucine residues in the zipper domain of GCN4, and further found
that some GCN4 proteins containing two leucine substitutions were
weakly active (Nucl. Acids Res. 20: 3721, 1992). Mutation of the
first and second heptadic leucines of the zipper domain of the
measles virus fusion protein (MVF) did not affect syncytium
formation (a measure of virally-induced cell fusion); however,
mutation of all four leucine residues prevented fusion completely
(Buckland et al., J. Gen. Virol. 73: 1703, 1992). None of the
mutations affected the ability of MVF to form a tetramer.
[0074] Examples of leucine zipper domains suitable for producing
soluble oligomeric TRAIL proteins include, but are not limited to,
those described in PCT application WO 94/10308 and in U.S. Pat. No.
5,716,805, hereby incorporated by reference. Recombinant fusion
proteins comprising a soluble TRAIL polypeptide, fused to a peptide
that dimerizes or trimerizes in solution, are expressed in suitable
host cells, and the resulting soluble oligomeric TRAIL is recovered
from the culture supernatant. DNA encoding such fusion proteins is
provided herein.
[0075] Certain members of the TNF family of proteins are believed
to exist in trimeric form (Beutler and Huffel, Science 264: 667,
1994; Banner et al., Cell 73: 431, 1993). Thus, trimeric TRAIL may
offer the advantage of enhanced biological activity. Preferred
leucine zipper moieties are those that preferentially form trimers.
One example is a leucine zipper derived from lung surfactant
protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:
191, 1994) and in U.S. Pat. No. 5,716,805, hereby incorporated by
reference in their entirety. This lung SPD-derived leucine zipper
peptide comprises the amino acid sequence Pro Asp Val Ala Ser Leu
Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val Gln His Leu Gln Ala Ala
Phe Ser Gln Tyr (SEQ ID NO:14).
[0076] Another example of a leucine zipper that promotes
trimerization is a peptide comprising the amino acid sequence Arg
Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His
Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Arg
(residues 2 to 34 of SEQ ID NO:15), as described in U.S. Pat. No.
5,716,805. In an alternative embodiment, the peptide lacks the
N-terminal Arg residue, thus comprising residues 3 to 34 of SEQ ID
NO:15. In another embodiment, an N-terminal Asp residue is added,
such that the peptide comprises the sequence Asp Arg Met Lys Gln
Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn
Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Arg (SEQ ID NO:15).
[0077] Yet another example of a suitable leucine zipper peptide
comprises the amino acid sequence Ser Leu Ala Ser Leu Arg Gln Gln
Leu Glu Ala Leu Gln Gly Gln Leu Gln His Leu Gln Ala Ala Leu Ser Gln
Leu Gly Glu (SEQ ID NO:16). In an alternative peptide, the leucine
residues in the foregoing sequence are replaced with isoleucine:
Ser Ile Ala Ser Ile Arg Gln Gln Ile Glu Ala Ile Gln Gly Gln Ile Gln
His Ile Gln Ala Ala Ile Ser Gln Ile Gly Glu (SEQ ID NO:17).
Fragments of the foregoing zipper peptides that retain the property
of promoting oligomerization may be employed as well. Examples of
such fragments include, but are not limited to, peptides lacking
one or two of the N-terminal or C-terminal residues presented in
the foregoing amino acid sequences.
[0078] Other peptides derived from naturally occurring trimeric
proteins may be employed in preparing trimeric TRAIL.
Alternatively, synthetic peptides that promote oligomerization may
be employed. In particular embodiments, leucine residues in a
leucine zipper moiety are replaced by isoleucine residues. Such
peptides comprising isoleucine may be referred to as isoleucine
zippers, but are encompassed by the term "leucine zippers" as
employed herein.
[0079] As described in example 7, a soluble Flag.RTM.-TRAIL
polypeptide expressed in CV-1/EBNA cells spontaneously formed
oligomers believed to be a mixture of dimers and trimers. The
cytotoxic effect of this soluble Flag.RTM.-TRAIL in the assay of
example 8 was enhanced by including an anti-Flag.RTM. antibody,
possibly because the antibody facilitated cross-linking of
TRAIL/receptor complexes. In one embodiment of the invention,
biological activity of TRAIL is enhanced by employing TRAIL in
conjunction with an antibody that is capable of cross-linking
TRAIL. Cells that are to be killed may be contacted with both a
soluble TRAIL polypeptide and such an antibody.
[0080] As one example, cancer or virally infected cells are
contacted with an anti-Flag.RTM. antibody and a soluble
Flag.RTM.-TRAIL polypeptide. Preferably, an antibody fragment
lacking the Fc region is employed. Bivalent forms of the antibody
may bind the Flag.RTM. (moieties of two soluble Flag.RTM.-TRAIL
polypeptides that are found in separate dimers or trimers. The
antibody may be mixed or incubated with a Flag.RTM.-TRAIL
polypeptide prior to administration in vivo. When an LZ-TRAIL
protein is employed, an antibody directed against the leucine
zipper peptide may be substituted for the anti-Flag.RTM. antibody,
in the foregoing procedures.
[0081] Oligomerization is attributable to factors and mechanisms
that include, but are not limited to, inter-chain disulfide bonds,
and non-covalent interactions such as hydrophobic interactions, as
discussed above. Such factors and mechanisms influence the type of
oligomers that are formed, which may include higher order
oligomers, and may result in protein preparations comprising
multiple species of oligomers (e.g., dimers, trimers, hexamers,
12-mers, and so on).
[0082] Provided herein are methods for manipulating oligomerization
of TRAIL and TRAIL-containing fusion proteins. The products of
these methods also are provided.
[0083] One approach involves altering the number of cysteine
residues in a TRAIL polypeptide or fusion protein. The number of
cysteines may be increased or decreased, depending upon whether a
corresponding increase or decrease in disulfide bonds is
desired.
[0084] One may choose to inhibit or promote disulfide bond
formation, depending on the form of TRAIL that is desired for a
particular purpose. One reason for manipulating disulfide bond
formation may be to obtain a more homogeneous protein preparation,
by controlling one mechanism of oligomerization. The proportion of
oligomers that are of a desired species may be increased through
such an approach. Another reason may be to enhance the proportion
of a particularly advantageous form of TRAIL in a protein
preparation, such as an oligomeric form exhibiting enhanced
biological activity.
[0085] The amino acid sequence of a TRAIL protein or fusion protein
may be altered to increase or decrease the number of cysteine
residues. Such sequence alteration may be accomplished by
conventional procedures, such as mutagenesis techniques, as
discussed above. One alternative for increasing the number of
cysteine residues involves adding cysteine-containing peptides,
preferably fused to the N-terminus of a TRAIL polypeptide (or
included in a fusion protein comprising TRAIL, such as an LZ-TRAIL
fusion).
[0086] The cysteine residue at position 230 of SEQ ID NO:2 is
located within the extracellular domain, which contains the
receptor-binding region. One embodiment of the invention is
directed to TRAIL polypeptides in which the Cys-230 residue is
deleted or substituted. Formation of disulfide bonds involving the
Cys-230 residue, including intramolecular disulfides which would
occur in the extracellular domain containing the receptor-binding
function of the protein, thus is avoided.
[0087] Oligomers may be treated with chemical cross-linking
reagents. Reagents that stabilize the oligomers, without destroying
a desired biological activity, are chosen for use.
[0088] Expression Systems
[0089] The present invention provides recombinant expression
vectors for expression of TRAIL, and host cells transformed with
the expression vectors. Any suitable expression system may be
employed. The vectors include a DNA encoding a TRAIL polypeptide,
operably linked to suitable transcriptional or translational
regulatory nucleotide sequences, such as those derived from a
mammalian, microbial, viral, or insect gene. Examples of regulatory
sequences include transcriptional promoters, operators, or
enhancers, an mRNA ribosomal binding site, and appropriate
sequences which control transcription and translation initiation
and termination. Nucleotide sequences are operably linked when the
regulatory sequence functionally relates to the TRAIL DNA sequence.
Thus, a promoter nucleotide sequence is operably linked to an TRAIL
DNA sequence if the promoter nucleotide sequence controls the
transcription of the TRAIL DNA sequence. An origin of replication
that confers the ability to replicate in the desired host cells,
and a selection gene by which transformants are identified, are
generally incorporated into the expression vector.
[0090] In addition, a sequence encoding an appropriate signal
peptide can be incorporated into expression vectors. A DNA sequence
for a signal peptide (secretory leader) may be fused in frame to
the TRAIL sequence so that the TRAIL is initially translated as a
fusion protein comprising the signal peptide. A signal peptide that
is functional in the intended host cells promotes extracellular
secretion of the TRAIL polypeptide. The signal peptide is cleaved
from the TRAIL polypeptide upon secretion of TRAIL from the
cell.
[0091] Suitable host cells for expression of TRAIL polypeptides
include prokaryotes, yeast or higher eukaryotic cells. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described, for example, in
Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier,
N.Y., (1985). Cell-free translation systems could also be employed
to produce TRAIL polypeptides using RNAs derived from DNA
constructs disclosed herein.
[0092] Prokaryotes include gram negative or gram positive
organisms, for example, E. coli or Bacilli. Suitable prokaryotic
host cells for transformation include, for example, E. coli,
Bacillus subtilis, Salmonella typhimurium, and various other
species within the genera Pseudomonas, Streptomyces, and
Staphylococcus. In a prokaryotic host cell, such as E. coli, a
TRAIL polypeptide may include an N-terminal methionine residue to
facilitate expression of the recombinant polypeptide in the
prokaryotic host cell. The N-terminal Met may be cleaved from the
expressed recombinant TRAIL polypeptide.
[0093] Expression vectors for use in prokaryotic host cells
generally comprise one or more phenotypic selectable marker genes.
A phenotypic selectable marker gene is, for example, a gene
encoding a protein that confers antibiotic resistance or that
supplies an autotrophic requirement. Examples of useful expression
vectors for prokaryotic host cells include those derived from
commercially available plasmids such as the cloning vector pBR322
(ATCC 37017). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides simple means for identifying
transformed cells. An appropriate promoter and a TRAIL DNA sequence
are inserted into the pBR322 vector. Other commercially available
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,
USA).
[0094] Promoter sequences commonly used for recombinant prokaryotic
host cell expression vectors include .beta.-lactamase
(penicillinase), lactose promoter system (Chang et al., Nature 275:
615, 1978; and Goeddel et al., Nature 281: 544, 1979), tryptophan
(trp) promoter system (Goeddel et al., Nucl. Acids Res. 8: 4057,
1980; and EP-A-36776) and tac promoter (Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.
412, 1982). A particularly useful prokaryotic host cell expression
system employs a phage .lambda. P.sub.L promoter and a cI857ts
thermolabile repressor sequence. Plasmid vectors available from the
American Type Culture Collection which incorporate derivatives of
the .lambda. P.sub.L promoter include plasmid pHUB2 (resident in E.
coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1,
ATCC 53082).
[0095] TRAIL alternatively may be expressed in yeast host cells,
preferably from the Saccharomyces genus (e.g., S. cerevisiae).
Other genera of yeast, such as Pichia or Kluyveromyces, may also be
employed. Yeast vectors will often contain an origin of replication
sequence from a 21 yeast plasmid, an autonomously replicating
sequence (ARS), a promoter region, sequences for polyadenylation,
sequences for transcription termination, and a selectable marker
gene. Suitable promoter sequences for yeast vectors include, among
others, promoters for metallothionein, 3-phosphoglycerate kinase
(Hitzeman et al., J. Biol. Chem. 255: 2073, 1980) or other
glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7: 149, 1968;
and Holland et al., Biochem. 17: 4900, 1978), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phospho-glucose isomerase, and glucokinase. Other
suitable vectors and promoters for use in yeast expression are
further described in Hitzeman, EPA-73,657. Another alternative is
the glucose-repressible ADH2 promoter described by Russell et al.
(J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724,
1982). Shuttle vectors replicable in both yeast and E. coli may be
constructed by inserting DNA sequences from pBR322 for selection
and replication in E. coli (Amp.sup.r gene and origin of
replication) into the above-described yeast vectors.
[0096] The yeast .alpha.-factor leader sequence may be employed to
direct secretion of the TRAIL polypeptide. The .alpha.-factor
leader sequence is often inserted between the promoter sequence and
the structural gene sequence. See, e.g., Kurjan et al., Cell 30:
933, 1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330,
1984. Other leader sequences suitable for facilitating secretion of
recombinant polypeptides from yeast hosts are known to those of
skill in the art. A leader sequence may be modified near its 3' end
to contain one or more restriction sites. This will facilitate
fusion of the leader sequence to the structural gene.
[0097] Yeast transformation protocols are known to those of skill
in the art. One such protocol is described by Hinnen et al., Proc.
Natl. Acad. Sci. USA 75: 1929, 1978. The Hinnen et al. protocol
selects for Trp.sup.+ transformants in a selective medium, wherein
the selective medium consists of 0.67% yeast nitrogen base, 0.5%
casamino acids, 2% glucose, 10 .mu.g/ml adenine and 20 .mu.g/ml
uracil.
[0098] Yeast host cells transformed by vectors containing an ADH2
promoter sequence may be grown for inducing expression in a "rich"
medium. An example of a rich medium is one consisting of 1% yeast
extract, 2% peptone, and 1% glucose supplemented with 80 .mu.g/ml
adenine and 80 .mu.g/ml uracil. Derepression of the ADH2 promoter
occurs when glucose is exhausted from the medium.
[0099] Mammalian or insect host cell culture systems could also be
employed to express recombinant TRAIL polypeptides. Bacculovirus
systems for production of heterologous proteins in insect cells are
reviewed by Luckow and Summers, Bio/Technology 6: 47 (1988).
Established cell lines of mammalian origin also may be employed.
Examples of suitable mammalian host cell lines include the COS-7
line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell
23: 175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163),
Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL
10) cell lines, and the CVI/EBNA cell line derived from the African
green monkey kidney cell line CVI (ATCC CCL 70) as described by
McMahan et al. (EMBO J. 10: 2821, 1991).
[0100] CHO cells are preferred for use as host cells. One example
of a suitable CHO cell line is the cell line designated DX-B11,
which is deficient in dihydrofolate reductase (DHFR), as described
in Urlaub and Chasin (Proc. Natl. Acad. Sci USA 77: 4216-4220,
1980), hereby incorporated by reference. DX-B11 cells may be
transformed with expression vectors that encode DHFR, which serves
as a selectable marker (Kauffman et al., Meth. in Enzymology, 185:
487-511, 1990). The use of DHFR as a selectable marker, when cells
are cultured in medium containing methotrexate, and for amplifying
a heterologous DNA inserted into the expression vector, are well
known.
[0101] In other embodiments, the host cells are CHO cells that can
be grown in suspension culture, and that are adapted to grow in
media that does not contain serum. The cells may be further adapted
to grow in media lacking insulin-like growth factor (IGF-1) and/or
transferrin. The host cells may be adapted to grow in media that
does not contain any exogenous growth factors that are animal
proteins.
[0102] Such CHO cell lines may be generated by any suitable
procedure. One such procedure is conducted generally as follows.
DX-B11 cells are adapted to growth in serum free medium by a
gradual reduction of serum supplementation in the media, and
replacement of serum with low levels of the growth factors
transferrin and insulin-like growth factor (IGF-1), in an enriched
cell growth media. Cells adapted to serum-free medium then are
weaned off transferrin and insulin-like growth factor-1. The
resulting CHO cells maintain vigorous growth and high viability, as
well as a DHFR-deficient phenotype, in serum-free, essentially
protein-free, media.
[0103] Transformed host cells provided herein include, but are not
limited to, host cells in which heterologous DNA, including a
TRAIL-encoding sequence, is inserted into the cell's genomic DNA.
Procedures that result in integration of expression vectors (or
portions thereof) into host cell DNA are well known. Conventional
procedures may be employed to amplify, or increase the copy number
of, heterologous DNA integrated into the genomic DNA of transformed
host cells.
[0104] Transcriptional and translational control sequences for
mammalian host cell expression vectors may be excised from viral
genomes. Commonly used promoter sequences and enhancer sequences
are derived from Polyoma virus, Adenovirus 2, Simian Virus 40
(SV40), and human cytomegalovirus. DNA sequences derived from the
SV40 viral genome, for example, SV40 origin, early and late
promoter, enhancer, splice, and polyadenylation sites may be used
to provide other genetic elements for expression of a structural
gene sequence in a mammalian host cell. Viral early and late
promoters are particularly useful because both are easily obtained
from a viral genome as a fragment which may also contain a viral
origin of replication (Fiers et al., Nature 273: 113, 1978).
Smaller or larger SV40 fragments may also be used, provided the
approximately 250 bp sequence extending from the Hind III site
toward the Bgl I site located in the SV40 viral origin of
replication site is included.
[0105] Expression vectors for use in mammalian host cells can be
constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:
280, 1983), for example. A useful system for stable high level
expression of mammalian cDNAs in C127 murine mammary epithelial
cells can be constructed substantially as described by Cosman et
al. (Mol. Immunol. 23: 935, 1986). A high expression vector, PMLSV
N1/N4, described by Cosman et al., Nature 312: 768, 1984 has been
deposited as ATCC 39890. Additional mammalian expression vectors
are described in EP-A-0367566, and in WO 91/18982. As one
alternative, the vector may be derived from a retrovirus.
Additional suitable expression systems are described in the
examples below.
[0106] One preferred expression system employs Chinese hamster
ovary (CHO) cells and an expression vector designated PG5.7. This
expression vector is described in U.S. patent application Ser. No.
08/586,509, filed Jan. 11, 1996, and in PCT application publication
no. WO 97/25420, which are hereby incorporated by reference. PG5.7
components include a fragment of CHO cell genomic DNA, followed by
a CMV-derived promoter, which is followed by a sequence encoding an
adenovirus tripartite leader, which in turn is followed by a
sequence encoding dihydrofolate reductase (DHFR). These components
were inserted into the plasmid vector pGEM1 (Promega, Madison,
Wis.). DNA encoding a TRAIL polypeptide (or fusion protein
containing TRAIL) may be inserted between the sequences encoding
the tripartite leader and DHFR. Methotrexate may be added to the
culture medium to increase expression levels, as is recognized in
the field.
[0107] The fragment of CHO cell genomic DNA in vector PG5.7
enhances expression of TRAIL. A phage lysate containing a fragment
of genomic DNA isolated from CHO cells was deposited with the
American Type Culture Collection on Jan. 4, 1996, and assigned
accession number ATCC 97411. Vector PG5.7 contains nucleotides 8671
through 14507 of the CHO genomic DNA insert in strain deposit ATCC
97411.
[0108] A further example of a suitable expression vector is similar
to PG5.7, but comprises a multiple cloning site and an internal
ribosome binding site (IRES), positioned between the adenovirus
tripartite leader and DHFR-encoding sequences. The multiple cloning
site comprises several restriction endonuclease recognition sites,
at which heterologous DNA (e.g., TRAIL DNA) may be inserted into
the vector. The IRES, a 575 bp non-coding region derived from the
encephalomyocarditis virus, allows cap-independent internal binding
of the ribosome and initiation of translation. For discussion of
the use of IRES sequences in expression vectors, including the role
such sequences play in allowing dicistronic mRNAs to be translated
efficiently, see Kaufman R., Nucleic Acids Research 19: 4485, 1991;
Oh and Sarnow, Current Opinion in Genetics and Development 3:
295-300, 1993; and Ramesh et al., Nucleic Acids Research, 24:
2697-2700, 1996. In addition, the vector may comprise a truncated
CHO genomic DNA fragment, shorter than the fragment incorporated
into PG5.7, yet still functional in enhancing expression of TRAIL
(see WO 97/25420).
[0109] For expression of TRAIL, a type II protein lacking a native
signal sequence, a heterologous signal sequence or leader
functional in mammalian host cells may be added. Examples include
the signal sequence for interleukin-7 (IL-7) described in U.S. Pat.
No. 4,965,195, the signal sequence for interleukin-2 receptor
described in Cosman et al., Nature 312: 768 (1984); the
interleukin-1 receptor signal peptide described in EP 367,566; the
type I interleukin-1 receptor signal peptide described in U.S. Pat.
No. 4,968,607; and the type II interleukin-1 receptor signal
peptide described in EP 460,846. Another option is a leader derived
from Ig-kappa (cite), such as a leader comprising the amino acid
sequence
Met-Gly-Thr-Asp-Thr-Leu-Leu-Leu-Trp-Val-Leu-Leu-Leu-Trp-Val-Pro-Gly-Ser-T-
hr-Gly. Further alternatives are cytomegalovirus-derived leaders
and signal peptides derived from a growth hormone, as described in
more detail below.
[0110] A preferred expression system employs a leader sequence
derived from cytomegalovirus (CMV). Example 7 illustrates the use
of one such leader. In example 7, mammalian host cells were
transformed with an expression vector encoding the peptide Met Ala
Arg Arg Leu Trp Ile Leu Ser Leu Leu Ala Val Thr Leu Thr Val Ala Leu
Ala Ala Pro Ser Gln Lys Ser Lys Arg Arg Thr Ser Ser (SEQ ID NO:9)
fused to the N-terminus of an octapeptide designated FLAG.RTM. (SEQ
ID NO:7, described above), which in turn is fused to the N-terminus
of a soluble TRAIL polypeptide. Residues 1 through 29 of SEQ ID
NO:9 constitute a CMV-derived leader sequence, whereas residues 30
through 32 are encoded by oligonucleotides employed in constructing
the expression vector described in example 7. In one embodiment,
DNA encoding a poly-His peptide (e.g., a peptide containing six
histidine residues) is positioned between the sequences encoding
the CMV leader and the FLAG.RTM. peptide.
[0111] In another embodiment of the invention, the FLAG.RTM.
peptide in the fusion protein described immediately above is
replaced with a leucine zipper peptide. Thus, one recombinant
expression vector provided herein comprises DNA encoding a fusion
protein comprising a CMV leader, a leucine zipper peptide, and a
soluble TRAIL polypeptide. One example of such a fusion protein is
depicted in FIG. 4 (SEQ ID NO:13). The protein of FIG. 4 comprises
(from N- to C-terminus) a CMV leader (residues 1 through 29 of SEQ
ID NO:9); an optional tripeptide Thr-Ser-Ser encoded by
oligonucleotides employed in vector construction (residues 30
through 32 of SEQ ID NO:9); a leucine zipper (SEQ ID NO:11); an
optional tripeptide Thr-Arg-Ser encoded by oligonucleotides
employed in vector construction; and amino acids 95 to 281 of the
human TRAIL protein of SEQ ID NO:2.
[0112] Expression systems that employ such CMV-derived leader
peptides are useful for expressing proteins other than TRAIL.
Expression vectors comprising a DNA sequence that encodes amino
acids 1 through 29 of SEQ ID NO:9 are provided herein. In another
embodiment, the vector comprises a sequence that encodes amino
acids 1 through 28 of SEQ ID NO:9. DNA encoding a desired
heterologous protein is positioned downstream of, and in the same
reading frame as, DNA encoding the leader. Additional residues
(e.g., those encoded by linkers or primers) may be encoded by DNA
positioned between the sequences encoding the leader and the
desired heterologous protein, as illustrated by the vector
described in example 7. As is understood in the pertinent field,
the expression vectors comprise promoters and any other desired
regulatory sequences, operably linked to the sequences encoding the
leader and heterologous protein.
[0113] The leader peptide presented in SEQ ID NO:9 may be cleaved
after the arginine residue at position 29 to yield the mature
secreted form of a protein fused thereto. Alternatively or
additionally, cleavage may occur between amino acids 20 and 21, or
between amino acids 28 and 29, of SEQ ID NO:9.
[0114] The skilled artisan will recognize that the position(s) at
which the signal peptide is cleaved may vary according to such
factors as the type of host cells employed, whether murine or human
TRAIL is expressed by the vector, and the like. Analysis by
computer program reveals that the primary cleavage site may be
between residues 20 and 21 of SEQ ID NO:9. Cleavage between
residues 22 and 23, and between residues 27 and 28, is predicted to
be possible, as well. To illustrate, expression and secretion of a
soluble murine TRAIL polypeptide resulted in cleavage of a
CMV-derived signal peptide at multiple positions. The three most
prominent species of secreted protein (in descending order)
resulted from cleavage between amino acids 20 and 21 of SEQ ID
NO:9, cleavage between amino acids 22 and 23, and cleavage between
amino acids 27 and 28.
[0115] In one particular expression system, in which the fusion
protein of FIG. 4 (SEQ ID NO:13) was expressed in CHO cells, the
CMV leader was cleaved at two positions. Two forms of mature
protein resulted, one comprising amino acids 21 to 256, and the
other comprising amino acids 29 to 256, of SEQ ID NO:13.
[0116] A signal peptide comprising amino acids 1 to 20 of the CMV
leader of SEQ ID NO:9 is also provided herein. Such a signal
peptide may yield a more homogeneous preparation of mature protein,
since certain of the above-mentioned alternative signal peptidase
cleavage sites are omitted from the leader.
[0117] A method for producing a heterologous recombinant protein
involves culturing mammalian host cells transformed with such an
expression vector under conditions that promote expression and
secretion of the heterologous protein, and recovering the protein
from the culture medium. Expression systems employing CMV leaders
may be used to produce any desired protein, examples of which
include, but are not limited to, colony stimulating factors,
interferons, interleukins, other cytokines, and cytokine
receptors.
[0118] A particularly preferred signal peptide for expression of
TRAIL polypeptides is a signal peptide derived from a growth
hormone gene. One such signal peptide or leader, which is derived
from human growth hormone, comprises the following amino acid
sequence: Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly
Leu Leu Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala (SEQ ID NO:19). An
example of a DNA sequence that encodes this growth hormone leader
is as follows:
1 (SEQ ID NO: 18) ATGGCTACAGGCTCCCGGACGTCCCTGTCCTGGCTTTTGGC-
CTGCTCTGC CTGCCCTGGCTTCAAGAGGGCAGTGCA.
[0119] One expression system employing such a signal peptide is
described below, in example 14. In alternative embodiments of the
invention, CHO cells (described above) are employed as host cells,
in place of the CV1-EBNA cells described in example 14. Preferred
embodiments of the present invention are directed to expression
vectors encoding a fusion protein comprising (from N- to
C-terminus) a growth hormone leader, the leucine zipper peptide of
SEQ ID NO:15, and a soluble TRAIL polypeptide. In one embodiment,
the TRAIL polypeptide is a soluble human TRAIL polypeptide
comprising amino acids 95 to 281 of SEQ ID NO:2. Optionally,
peptide linkers (which may be encoded by DNA segments resulting
from the vector construction technique, for example) are positioned
between the growth hormone leader and the leucine zipper, or
between the leucine zipper and TRAIL. The leucine zipper moiety
promotes oligomerization of the fusion proteins.
[0120] One example of such a fusion protein is depicted in FIG. 3.
The fusion protein comprises (from N- to C-terminus) a growth
hormone-derived leader sequence (SEQ ID NO:19), followed by a
tripeptide encoded by an oligonucleotide employed in vector
construction (Thr-Ser-Ser), a leucine zipper peptide (SEQ ID
NO:15), a tripeptide encoded by an oligonucleotide employed in
vector construction (Thr-Arg-Ser), and a soluble human TRAIL
polypeptide (amino acids 95 to 281 of SEQ ID NO:2). A DNA sequence
encoding the fusion protein, and the amino acid sequence of the
fusion protein, are presented in SEQ ID NO:10 and 11,
respectively.
[0121] Purified TRAIL Protein
[0122] The present invention provides purified TRAIL proteins,
which may be produced by recombinant expression systems as
described above or purified from naturally occurring cells. The
desired degree of purity may depend on the intended use of the
protein. A relatively high degree of purity is desired when the
protein is to be administered in vivo, for example. Advantageously,
TRAIL polypeptides are purified such that no protein bands
corresponding to other proteins are detectable by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be
recognized by one skilled in the pertinent field that multiple
bands corresponding to TRAIL protein may be detected by SDS-PAGE,
due to differential glycosylation, variations in post-translational
processing, and the like, as discussed above. A preparation of
TRAIL protein is considered to be purified as long as no bands
corresponding to different (non-TRAIL) proteins are visualized.
TRAIL most preferably is purified to substantial homogeneity, as
indicated by a single protein band upon analysis by SDS-PAGE. The
protein band may be visualized by silver staining, Coomassie blue
staining, or (if the protein is radiolabeled) by
autoradiography.
[0123] One process for producing the TRAIL protein comprises
culturing a host cell transformed with an expression vector
comprising a DNA sequence that encodes TRAIL under conditions such
that TRAIL is expressed. The TRAIL protein is then recovered from
the culture (from the culture medium or cell extracts). As the
skilled artisan will recognize, procedures for purifying the
recombinant TRAIL will vary according to such factors as the type
of host cells employed and whether or not the TRAIL is secreted
into the culture medium.
[0124] For example, when expression systems that secrete the
recombinant protein are employed, the culture medium first may be
concentrated using a commercially available protein concentration
filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. Following the concentration step, the
concentrate can be applied to a purification matrix such as a gel
filtration medium. Alternatively, an anion exchange resin can be
employed, for example, a matrix or substrate having pendant
diethylaminoethyl (DEAE) groups. The matrices can be acrylamide,
agarose, dextran, cellulose or other types commonly employed in
protein purification. Alternatively, a cation exchange step can be
employed. Suitable cation exchangers include various insoluble
matrices comprising sulfopropyl or carboxymethyl groups.
Sulfopropyl groups are preferred. Finally, one or more
reversed-phase high performance liquid chromatography (RP-HPLC)
steps employing hydrophobic RP-HPLC media, (e.g., silica gel having
pendant methyl or other aliphatic groups) can be employed to
further purify TRAIL. Some or all of the foregoing purification
steps, in various combinations, can be employed to provide a
purified TRAIL protein.
[0125] In one example of a procedure for producing and purifying
TRAIL, Chinese Hamster Ovary (CHO) cells are stably transformed
with a recombinant expression vector encoding soluble TRAIL. In one
embodiment, the vector encodes a fusion protein comprising a
CMV-derived leader, a leucine zipper, and a soluble TRAIL
polypeptide, as described in more detail elsewhere herein. The
transformed cells are cultured to allow expression and secretion of
the soluble LZ-TRAIL protein into the culture supernatant. The
culture supernatant then is diluted 5-fold with 20 mM Tris buffer,
pH 8.5, and applied to a Q-Sepharose anion exchange column
(Pharmacia LKB, Uppsala, Sweden) at a ratio of 1 ml supernatant per
0.3 ml bead volume. The flow-through then is passed over a
Fractogel.RTM. S-Sepharose cation exhange column (EM Separations,
Gibbstown, N.J.) at a ratio of 1/0.06 ml (v/v), washed with five
column volumes of buffer, and eluted with a salt gradient of 0 to
1.0M NaCl in 20 mM Tris buffer, pH 8.5. Fractions containing the
LZ-TRAIL protein are pooled and dialyzed against Tris Buffered
Saline (TBS).
[0126] Another example of a protein purification procedure,
slightly modified from the procedure described immediately above,
is as follows. This procedure may be employed when a leader derived
from growth hormone is substituted for the CMV leader, for example.
Chinese Hamster Ovary (CHO) cells are stably transformed with a
recombinant expression vector encoding GH leader-LZ-TRAIL, and
cultured to allow expression and secretion of the soluble LZ-TRAIL
protein into the culture supernatant. The culture supernatant then
is diluted 5-fold with 25 mM Tris buffer, pH 7.0, and applied to a
Q-Sepharose anion exchange column (Pharmacia LKB, Uppsala, Sweden)
at a ratio of 1 ml supernatant per 0.3 ml bead volume. The
flow-through was concentrated and buffer exchanged into 10 mM Tris,
pH 7.0, then passed over a Fractogel.RTM. S-Sepharose cation
exhange column (EM Separations, Gibbstown, N.J.) at a ratio of
1/0.06 ml (v/v); washed with five column volumes of buffer; and
eluted with a 0 to 0.5M NaCl gradient in 10 mM Tris buffer, pH 7.0.
Fractions containing the LZ/TRAIL protein were concentrated and
applied to an S200 sizing column (Pharmacia) in 10 mM Tris, pH 7.0,
100 mM NaCl, 10% glycerol.
[0127] When bacterial host cells are employed, the recombinant
protein produced in bacterial culture may be isolated by initial
disruption of the host cells, centrifugation, extraction from cell
pellets if an insoluble polypeptide, or from the supernatant fluid
if a soluble polypeptide, followed by one or more concentration,
salting-out, ion exchange, affinity purification or size exclusion
chromatography steps. Finally, RP-HPLC can be employed for final
purification steps. Microbial cells can be disrupted by any
convenient method, including freeze-thaw cycling, sonication,
mechanical disruption, or use of cell lysing agents.
[0128] When transformed yeast host cells are employed, TRAIL
preferably is expressed as a secreted polypeptide. This simplifies
purification. Secreted recombinant polypeptide from a yeast host
cell fermentation can be purified by methods analogous to those
disclosed by Urdal et al. (J. Chromatog. 296: 171, 1984). Urdal et
al. describe two sequential, reversed-phase HPLC steps for
purification of recombinant human IL-2 on a preparative HPLC
column.
[0129] Alternatively, TRAIL polypeptides can be purified by
immunoaffinity chromatography. An affinity column containing an
antibody that binds TRAIL may be prepared by conventional
procedures and employed in purifying TRAIL. Example 4 describes a
procedure for generating monoclonal antibodies directed against
TRAIL.
[0130] Expression of various forms of TRAIL described herein in
particular expression systems may yield protein preparations
comprising multiple species of oligomers (e.g., dimers, trimers,
hexamers, 12-mers, and so on). To illustrate, a mixture of
oligomers, including hexamers and trimers, may result from
expression of the fusion protein of SEQ ID NO:11 in COS cells. In
another illustrative scenario, involving expression of a fusion
protein comprising a growth hormone leader, an isoleucine zipper
and a spacer-deleted soluble TRAIL polypeptide, 12-mers may be
among the resulting oligomers. If a particular species of oligomer
is desired for a particular use, that species may be isolated using
conventional procedures. An example of a suitable procedure employs
size exclusion chromatography.
[0131] A TRAIL protein (e.g., a fusion protein or oligomer)
prepared using a particular expression system may comprise inter-
or intra-molecular disulfide bonds that are disadvantageous for a
particular use of the protein. In such a case, the TRAIL protein
may be treated with a reducing agent in accordance with
conventional techniques. An example of a suitable procedures
comprises treating the protein with 5-10 mM DTT (dithiothreitol)
for 10 minutes at 37.degree. C. Other suitable reducing agents,
such as B-mercaptoethanol (preferably at a concentration of at
least 100 mM in the reaction soluttion), may be substituted for DTT
and used in accordance with standard procedures.
[0132] To inhibit reoxidation and formation of new disulfide bonds,
the protein may be stored in the presence of a reducing agent. One
alternative involves further treatment of the protein, after the
reducing step, with a sulfhydryl-specific modifying agent. Examples
of such agents are iodoacetamide or iodoacetic acid.
[0133] Treatment with a reducing agent may be conducted when
refolding of a protein into a different conformation is desired.
Disulfide bonds, including intramolecular disulfide bonds, are
reduced, and the reducing agent then removed to allow refolding of
the protein. If promoting disulfide bond formation is desired,
oxygen can be bubbled through the reaction solution.
[0134] Properties and Uses of TRAIL
[0135] Programmed cell death (apoptosis) occurs during
embryogenesis, metamorphosis, endocrine-dependent tissue atrophy,
normal tissue turnover, and death of immune thymocytes. Regulation
of programmed cell death is vital for normal functioning of the
immune system. To illustrate, T cells that recognize self-antigens
are destroyed through the apoptotic process during maturation of
T-cells in the thymus, whereas other T cells are positively
selected. The possibility that some T-cells recognizing certain
self epitopes (e.g., inefficiently processed and presented
antigenic determinants of a given self protein) escape this
elimination process and subsequently play a role in autoimmune
diseases has been proposed (Gammon et al., Immunology Today 12:
193, 1991).
[0136] Insufficient apoptosis has been implicated in certain
conditions, while elevated levels of apoptotic cell death have been
associated with other diseases. The desirability of identifying and
using agents that regulate apoptosis in treating such disorders is
recognized (Kromer, Advances in Immunology, 58: 211, 1995; Groux et
al., J. Exp. Med. 175: 331, 1992; Sachs and Lotem, Blood 82: 15,
1993).
[0137] Abnormal resistance of T cells toward undergoing apoptosis
has been linked to lymphocytosis, lymphadenopathy, splenomegaly,
accumulation of self-reactive T cells, autoimmune disease,
development of leukemia, and development of lymphoma (Kromer,
supra; see especially pages 214-215). Conversely, excessive
apoptosis of T cells has been suggested to play a role in
lymphopenia, systemic immunodeficiency, and specific
immunodeficiency, with specific examples being virus-induced
immunodeficient states associated with infectious mononucleosis and
cytomegalovirus infection, and tumor-mediated immunosuppression
(Kromer, supra; see especially page 214). Depletion of CD4.sup.+ T
cells in HIV-infected individuals may be attributable to
inappropriate activation-induced cell death (AICD) by apoptosis
(Groux et al., J. Exp. Med. 175: 331, 1992).
[0138] As demonstrated in examples 5 and 8, TRAIL induces apoptosis
of the acute T cell leukemia cell line designated Jurkat clone
E6-1. TRAIL thus is a research reagent useful in studies of
apoptosis, including the regulation of programmed cell death. Since
Jurkat cells are a leukemia cell line arising from T cells, the
TRAIL of the present invention finds use in studies of the role
TRAIL may play in apoptosis of other transformed T cells, such as
other malignant cell types arising from T cells.
[0139] TRAIL binds Jurkat cells, as well as inducing apoptosis
thereof. TRAIL did not cause death of freshly isolated murine
thymocytes, or peripheral blood T cells (PBTs) freshly extracted
from a healthy human donor. A number of uses flow from these
properties of TRAIL.
[0140] TRAIL polypeptides may be used to purify leukemia cells, or
any other cell type to which TRAIL binds. Leukemia cells may be
isolated from a patient's blood, for example. In one embodiment,
the cells are purified by affinity chromatography, using a
chromatography matrix having TRAIL bound thereto. The TRAIL
attached to the chromatography matrix may be a full length protein,
an TRAIL fragment comprising the extracellular domain, an
TRAIL-containing fusion protein, or other suitable TRAIL
polypeptide described herein. In one embodiment, a soluble TRAIL/Fc
fusion protein is bound to a Protein A or Protein G column through
interaction of the Fc moiety with the Protein A or Protein G.
Alternatively, TRAIL may be used in isolating leukemia cells by
flow cytometry.
[0141] The thus-purified leukemia cells are expected to die
following binding of TRAIL, but the dead cells will still bear cell
surface antigens, and may be employed as immunogens in deriving
anti-leukemia antibodies. The leukemia cells, or a desired cell
surface antigen isolated therefrom, find further use in vaccine
development.
[0142] Since TRAIL binds and kills leukemia cells (the Jurkat cell
line), TRAIL also may be useful in treating leukemia. A therapeutic
method involves contacting leukemia cells with an effective amount
of TRAIL. In one embodiment, a leukemia patient's blood is
contacted ex vivo with an TRAIL polypeptide. The TRAIL may be
immobilized on a suitable matrix. TRAIL binds the leukemia cells,
thus removing them from the patient's blood before the blood is
returned into the patient.
[0143] Alternatively or additionally, bone marrow extracted from a
leukemia patient may be contacted with an amount of TRAIL effective
in inducing death of leukemia cells in the bone marrow. Bone marrow
may be aspirated from the sternum or iliac crests, for example, and
contacted with TRAIL to purge leukemia cells. The thus-treated
marrow is returned to the patient.
[0144] TRAIL also binds to, and induces apoptosis of, lymphoma and
melanoma cells (see examples 5, 9, and 10). Thus, uses of TRAIL
that are analogous to those described above for leukemia cells are
applicable to lymphoma and melanoma cells. TRAIL polypeptides may
be employed in treating cancer, including, but not limited to,
leukemia, lymphoma, and melanoma. In one embodiment, the lymphoma
is Burkitt's lymphoma. Table I in example 9 shows that TRAIL had a
cytotoxic effect on several Burkitt's lymphoma cell lines.
Epstein-Barr virus is an etiologic agent of Burkitt's lymphoma.
[0145] TRAIL polypeptides also find use in treating viral
infections. Contact with TRAIL caused death of cells infected with
cytomegalovirus, but not of the same cell type when uninfected, as
described in example 11. The ability of TRAIL to kill cells
infected with other viruses can be confirmed using the assay
described in example 11. Such viruses include, but are not limited
to, encephalomyocarditis virus, Newcastle disease virus, vesicular
stomatitis virus, herpes simplex virus, adenovirus-2, bovine viral
diarrhea virus, HIV, and Epstein-Barr virus.
[0146] An effective amount of TRAIL is administered to a mammal,
including a human, afflicted with a viral infection. In one
embodiment, TRAIL is employed in conjunction with interferon to
treat a viral infection. In the experiment described in example 11,
pretreatment of CMV-infected cells with .gamma.-interferon enhanced
the level of killing of the infected cells that was mediated by
TRAIL. TRAIL may be administered in conjunction with other agents
that exert a cytotoxic effect on cancer cells or virus-infected
cells.
[0147] A wide variety of drugs have been employed in cancer
treatment. Examples include, but are not limited to, cisplatin,
taxol, etoposide, Novantrone.RTM. (mitoxantrone), actinomycin D,
camptothecin (or water soluble derivatives thereof), methotrexate,
mitomycin (e.g., mitomycin C), dacarbazine (DTIC), and
anti-neoplastic antibiotics such as doxorubicin and daunomycin.
Drugs employed in cancer therapy may have a cytotoxic or cytostatic
effect on cancer cells, or may reduce proliferation of the
malignant cells. Cancer treatment may include radiation therapy. In
particular embodiments, TRAIL may be co-administered with other
proteins in cancer therapy; one such protein is
.gamma.-interferon.
[0148] Among the texts providing guidance for cancer therapy is
Cancer, Principles and Practice of Oncology, 4th Edition, DeVita et
al., Eds. J. B. Lippincott Co., Philadelphia, Pa. (1993). An
appropriate therapeutic approach is chosen according to the
particular type of cancer, and other factors such as the general
condition of the patient, as is recognized in the pertinent
field.
[0149] TRAIL may be added to a standard chemotherapy regimen, in
treating a cancer patient. For those combinations in which TRAIL
and a second anti-cancer agent exert a synergistic effect against
cancer cells, the dosage of the second agent may be reduced,
compared to the standard dosage of the second agent when
administered alone. A method for increasing the sensitivity of
cancer cells to TRAIL comprises co-administering TRAIL with an
amount of a chemotherapeutic anti-cancer drug that is effective in
enhancing sensitivity of cancer cells to TRAIL.
[0150] Particular embodiments of the invention are directed to
co-administration of TRAIL and methotrexate, etoposide, or
mitoxantrone to a cancer patient, including but not limited to
prostate cancer patients. One such therapeutic method comprises
administration of TRAIL and mitoxantrone (Novantrone.RTM.; Immunex
Corporation, Seattle, Wash.) to a prostate cancer patient. For
descriptions of mitoxantrone or the use thereof in treating
prostate cancer, see U.S. Pat. Nos. 4,197,249 and 4,278,689; and
Moore et al. (J. Clinical Oncology 12: 689-694, 1994), which are
hereby incorporated by reference. In an in vitro assay in which a
prostate tumor cell line was contacted with various concentrations
of LZ-TRAIL and Novantrone.RTM., a synergistic effect was seen, in
that the combination of LZ-TRAIL and Novantrone.RTM. resulting in
enhanced tumor cell death. A synergistic effect also was seen when
TRAIL and methotrexate were employed in the assay. LZ-TRAIL is a
fusion protein comprising a leucine zipper peptide and a soluble
TRAIL polypeptide, as described in more detail above and in example
14.
[0151] Another embodiment of the invention is directed to
contacting colorectal cancer cells (e.g., colon carcinoma cells)
with TRAIL and camptothecene. Alternatives include contacting
colorectal cancer cells with TRAIL in conjunction with adriamycin
(doxorubicin) or mitomycin.
[0152] For in vivo use, derivatives of camptothecene that are more
water soluble would be advantageous. Examples of such water soluble
derivatives are the drugs
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-campt-
othecin (CPT-11; irinotecan) and
9-dimethyl-aminomethyl-10-hydroxycamptoth- ecin (topotecan).
Camptothecene and the two above-described derivatives are DNA
topoisomerase I inhibitors.
[0153] A method for treatment of colorectal cancer (e.g., colon
carcinoma) comprises administering TRAIL in conjunction with a
water soluble derivative of camptothecene, such as irinotecan or
topotecan. For further description of the chemical structure of
irinotecan and topotecan, or the use thereof in treating cancer,
including colorectal cancer, see Rougier et al. (J. Clinical
Oncology, 15: 251-260, January 1997), Pommier (Seminars in Oncology
Vol. 23, No. 1, Suppl. 3, pp 3-10, February 1996), Lavelle et al.
(Seminars in Oncology Vol. 23, No. 1, Suppl. 3, pp 11-20, February
1996), Pitot et al. (J. Clinical Oncology, 15: 2910-2919, August
1997), Kunimoto et al. (Cancer Research, 47: 5944-5947, Nov. 15,
1987), and Jansen et al. (Int. J. of Cancer, 70: 33540, Jan. 27,
1997), all hereby incorporated by reference.
[0154] Also provided herein are methods for treating melanoma by
administering TRAIL in conjunction with other therapeutic agent(s).
In an in vitro assay, actinomycin D and cycloheximide were found to
enhance the sensitivity of certain melanoma cell lines to TRAIL.
For particular melanoma cell lines that were resistant to
TRAIL-mediated cytotoxicity, addition of the protein synthesis
inhibitors actinomycin D or cycloheximide rendered the cells more
sensitive to TRAIL-induced death. Thus, one method of the present
invention comprises co-administering TRAIL, together with
actinomycin D or cycloheximide, to a melanoma patient.
[0155] Experimental evidence suggests a correlation between the
intracellular concentration of an apoptosis inhibitor designated
FLIP, and sensitivity of certain tumor cells to TRAIL-mediated cell
death. Certain TRAIL-resistant melanoma cell lines expressed
relatively high levels of FLIP, whereas lower levels (or
undetectable levels) of FLIP were expressed in TRAIL-sensitive
melanoma cell lines. Further, addition of actinomycin D to certain
TRAIL-resistant melanoma cell lines resulted in a decrease in the
intracellular concentration of FLIP. For further discussion of FLIP
proteins (FLICE inhibitory proteins) and their involvement in
caspase signaling cascades, see Thome et al., (Nature, 386: 517, 3
Apr. 1997) and Irmler et al. (Nature 388: 190, 1997), hereby
incorporated by reference.
[0156] One approach toward increasing sensitivity of cancer cells
(including but not limited to melanoma cells) to TRAIL is
inhibiting expression of FLIP in the target cancer cells. Antisense
molecules that are derived from a FLIP DNA sequence, and that will
inhibit FLIP expression in target cells, may be employed in such an
approach.
[0157] As used herein, "co-administration" is not limited to
simultaneous administration. TRAIL may be administered along with
other therapeutic agents, during the course of a treatment regimen.
In one embodiment, administration of TRAIL and other therapeutic
agents is sequential. An appropriate time course may be chosen by
the physician, according to such factors as the nature of a
patient's illness, and the patient's condition.
[0158] In preferred embodiments of the therapeutic methods
described herein, soluble human TRAIL polypeptides, or oligomeric
forms thereof, are administered. Oligomers comprising leucine
zipper-TRAIL fusion proteins are especially preferred.
[0159] In another embodiment, TRAIL is used to kill virally
infected cells in cell preparations, tissues, or organs that are to
be transplanted. To illustrate, bone marrow may be contacted with
TRAIL to kill virus infected cells that may be present therein,
before the bone marrow is transplanted into the recipient.
[0160] The TRAIL of the present invention may be used in developing
treatments for any disorder mediated (directly or indirectly) by
defective or insufficient amounts of TRAIL. A therapeutically
effective amount of purified TRAIL protein is administered to a
patient afflicted with such a disorder. Alternatively, TRAIL DNA
sequences may be employed in developing a gene therapy approach to
treating such disorders. Disclosure herein of native TRAIL
nucleotide sequences permits the detection of defective TRAIL
genes, and the replacement thereof with normal TRAIL-encoding
genes. Defective genes may be detected in vitro diagnostic assays,
and by comparision of the native TRAIL nucleotide sequence
disclosed herein with that of a TRAIL gene derived from a person
suspected of harboring a defect in this gene.
[0161] The present invention provides pharmaceutical compositions
comprising purified TRAIL and a physiologically acceptable carrier,
diluent, or excipient. Suitable carriers, diluents, and excipients
are nontoxic to recipients at the dosages and concentrations
employed. Such compositions may comprise buffers, antioxidants such
as ascorbic acid, low molecular weight (less than about 10
residues) polypeptides, proteins, amino acids, carbohydrates
including glucose, sucrose or dextrins, chelating agents such as
EDTA, glutathione and other stabilizers and excipients commonly
employed in pharmaceutical compositions. Neutral buffered saline or
saline mixed with conspecific serum albumin are among the
appropriate diluents. The composition may be formulated as a
lyophilizate using appropriate excipient solutions (e.g. sucrose)
as diluents.
[0162] For therapeutic use, purified proteins of the present
invention are administered to a patient, preferably a human, for
treatment in a manner appropriate to the indication. Thus, for
example, the pharmaceutical compositions can be administered
locally, by intravenous injection, continuous infusion, sustained
release from implants, or other suitable technique. Appropriate
dosages and the frequency of administration will depend, of course,
on such factors as the nature and severity of the indication being
treated, the desired response, the condition of the patient and so
forth.
[0163] The TRAIL protein employed in the pharmaceutical
compositions preferably is purified such that the TRAIL protein is
substantially free of other proteins of natural or endogenous
origin, desirably containing less than about 1% by mass of protein
contaminants residual of production processes. Such compositions,
however, can contain other proteins added as stabilizers, carriers,
excipients or co-therapeutics.
[0164] The TRAIL-encoding DNAs disclosed herein find use in the
production of TRAIL polypeptides, as discussed above. Fragments of
the TRAIL nucleotide sequences are also useful. In one embodiment,
such fragments comprise at least about 17 consecutive nucleotides,
more preferably at least 30 consecutive nucleotides, of the human
or murine TRAIL DNA disclosed herein. DNA and RNA complements of
said fragments are provided herein, along with both single-stranded
and double-stranded forms of the TRAIL DNA of SEQ ID NOS:1, 3 and
5.
[0165] Among the uses of such TRAIL nucleic acid fragments are use
as a probe or as primers in a polymerase chain reaction (PCR). As
one example, a probe corresponding to the extracellular domain of
TRAIL may be employed. The probes find use in detecting the
presence of TRAIL nucleic acids in in vitro assays and in such
procedures as Northern and Southern blots. Cell types expressing
TRAIL can be identified as well. Such procedures are well known,
and the skilled artisan can choose a probe of suitable length,
depending on the particular intended application. For PCR, 5' and
3' primers corresponding to the termini of a desired TRAIL DNA
sequence are employed to isolate and amplify that sequence, using
conventional techniques.
[0166] Other useful fragments of the TRAIL nucleic acids are
antisense or sense oligonucleotides comprising a single-stranded
nucleic acid sequence (either RNA or DNA) capable of binding to
target TRAIL mRNA (sense) or TRAIL DNA (antisense) sequences. Such
a fragment generally comprises at least about 14 nucleotides,
preferably from about 14 to about 30 nucleotides. The ability to
create an antisense or a sense oligonucleotide, based upon a cDNA
sequence for a given protein is described in, for example, Stein
and Cohen, Cancer Res. 48: 2659, 1988 and van der Krol et al.,
BioTechniques 6: 958, 1988.
[0167] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block translation (RNA) or transcription (DNA) by one of several
means, including enhanced degradation of the duplexes, premature
termination of transcription or translation, or by other means. The
antisense oligonucleotides thus may be used to block expression of
TRAIL proteins.
[0168] Antisense or sense oligonucleotides further comprise
oligonucleotides having modified sugar-phosphodiester backbones (or
other sugar linkages, such as those described in WO91/06629) and
wherein such sugar linkages are resistant to endogenous nucleases.
Such oligonucleotides with resistant sugar linkages are stable in
vivo (i.e., capable of resisting enzymatic degradation) but retain
sequence specificity to be able to bind to target nucleotide
sequences. Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10448, and other
moieties that increases affinity of the oligonucleotide for a
target nucleic acid sequence, such as poly-(L-lysine). Further
still, intercalating agents, such as ellipticine, and alkylating
agents or metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oliginucleotide for the target nucleotide sequence.
[0169] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or other gene transfer vectors such
as Epstein-Barr virus. Antisense or sense oligonucleotides are
preferably introduced into a cell containing the target nucleic
acid sequence by insertion of the antisense or sense
oligonucleotide into a suitable retroviral vector, then contacting
the cell with the retrovirus vector containing the inserted
sequence, either in vivo or ex vivo. Suitable retroviral vectors
include, but are not limited to, the murine retrovirus M-MuLV, N2
(a retrovirus derived from M-MuLV), or or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see PCT Application WO
90/13641). Alternatively, other promotor sequences may be used to
express the oligonucleotide.
[0170] Sense or antisense oligonucleotides may also be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
[0171] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0172] Antibodies Immunoreactive with TRAIL
[0173] The TRAIL proteins of the present invention, or immunogenic
fragments thereof, may be employed in generating antibodies. The
present invention thus provides antibodies that specifically bind
TRAIL, i.e., the antibodies bind to TRAIL via the antigen-binding
sites of the antibody (as opposed to non-specific binding).
[0174] Polyclonal and monoclonal antibodies may be prepared by
conventional techniques. See, for example, Monoclonal Antibodies,
Hybridomas: A New Dimension in Biological Analyses, Kennet et al.
(eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory
Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., (1988). Production of monoclonal
antibodies that are immunoreactive with TRAIL is further
illustrated in example 4 below.
[0175] Antigen-binding fragments of such antibodies, which may be
produced by conventional techniques, are also encompassed by the
present invention. Examples of such fragments include, but are not
limited to, Fab, F(ab'), and F(ab').sub.2 fragments. Antibody
fragments and derivatives produced by genetic engineering
techniques are also provided.
[0176] The monoclonal antibodies of the present invention include
chimeric antibodies, e.g., humanized versions of murine monoclonal
antibodies. Such humanized antibodies may be prepared by known
techniques, and offer the advantage of reduced immunogenicity when
the antibodies are administered to humans. In one embodiment, a
humanized monoclonal antibody comprises the variable region of a
murine antibody (or just the antigen binding site thereof) and a
constant region derived from a human antibody. Alternatively, a
humanized antibody fragment may comprise the antigen binding site
of a murine monoclonal antibody and a variable region fragment
(lacking the antigen-binding site) derived from a human antibody.
Procedures for the production of chimeric and further engineered
monoclonal antibodies include those described in Riechmann et al.
(Nature 332: 323, 1988), Liu et al. (PNAS 84: 3439, 1987), Larrick
et al. (Bio/Technology 7: 934, 1989), and Winter and Harris (TIPS
14: 139, May, 1993).
[0177] Among the uses of the antibodies is use in assays to detect
the presence of TRAIL polypeptides, either in vitro or in vivo. The
antibodies find further use in purifying TRAIL by affinity
chromatography.
[0178] Those antibodies that additionally can block binding of
TRAIL to target cells may be used to inhibit a biological activity
of TRAIL. A therapeutic method involves in vivo administration of
such an antibody in an amount effective in inhibiting a
TRAIL-mediated biological activity. Disorders mediated or
exacerbated by TRAIL, directly or indirectly, are thus treated.
Monoclonal antibodies are generally preferred for use in such
therapeutic methods.
[0179] Antibodies directed against TRAIL may be useful for treating
thrombotic microangiopathies. One such disorder is thrombotic
thrombocytopenic purpura (TTP) (Kwaan, H. C., Semin. Hematol., 24:
71, 1987; Thompson et al., Blood, 80: 1890, 1992). Increasing
TTP-associated mortality rates have been reported by the U.S.
Centers for Disease Control (Torok et al., Am. J. Hematol. 50: 84,
1995).
[0180] Plasma from patients afflicted with TTP (including HIV.sup.+
and HIV.sup.- patients) induces apoptosis of human endothelial
cells of dermal microvascular origin, but not large vessel origin
(Laurence et al., Blood, 87: 3245, Apr. 15, 1996). Plasma of TTP
patients thus is thought to contain one or more factors that
directly or indirectly induce apoptosis. In the assay described in
example 13 below, polyclonal antibodies raised against TRAIL
inhibited TTP plasma-induced apoptosis of dermal microvascular
endothelial cells. The data presented in example 13 suggest that
TRAIL is present in the serum of TTP patients, and may play a role
in inducing apoptosis of microvascular endothelial cells.
[0181] Another thrombotic microangiopathy is hemolytic-uremic
syndrome (HUS) (Moake, J. L., Lancet, 343: 393, 1994; Melnyk et
al., (Arch. Intern. Med., 155: 2077, 1995; Thompson et al., supra).
One embodiment of the invention is directed to use of an anti-TRAIL
antibody to treat the condition that is often referred to as "adult
HUS" (even though it can strike children as well). A disorder known
as childhood/diarrhea-associat- ed HUS differs in etiology from
adult HUS.
[0182] Other conditions characterized by clotting of small blood
vessels may be treated using anti-TRAIL antibodies. Such conditions
include but are not limited to the following. Cardiac problems seen
in about 5-10% of pediatric AIDS patients are believed to involve
clotting of small blood vessels. Breakdown of the microvasculature
in the heart has been reported in multiple sclerosis patients. As a
further example, treatment of systemic lupus erythematosus (SLE) is
contemplated.
[0183] In one embodiment, a patient's blood or plasma is contacted
with an anti-TRAIL antibody ex vivo. The antibody (preferably a
monoclonal antibody) may be bound to a suitable chromatography
matrix by conventional procedures. The patient's blood or plasma
flows through a chromatography column containing the antibody bound
to the matrix, before being returned to the patient. The
immobilized antibody binds TRAIL, thus removing TRAIL protein from
the patient's blood.
[0184] In an alternative embodiment, the antibodies are
administered in vivo, in which case blocking antibodies are
desirably employed. Such antibodies may be identified using any
suitable assay procedure, such as by testing antibodies for the
ability to inhibit binding of TRAIL to target cells. Alternatively,
blocking antibodies may be identified in assays for the ability to
inhibit a biological effect of the binding of TRAIL to target
cells. Example 12 illustrates one suitable method of identifying
blocking antibodies, wherein antibodies are assayed for the ability
to inhibit TRAIL-mediated lysis of Jurkat cells.
[0185] The present invention thus provides a method for treating a
thrombotic microangiopathy, involving use of an effective amount of
an antibody directed against TRAIL. Antibodies of the present
invention may be employed in in vivo or ex vivo procedures, to
inhibit TRAIL-mediated damage to (e.g., apoptosis of) microvascular
endothelial cells.
[0186] Anti-TRAIL antibodies may be employed in conjunction with
other agents useful in treating a particular disorder. In an in
vitro study reported by Laurence et al. (Blood 87: 3245, 1996),
some reduction of TTP plasma-mediated apoptosis of microvascular
endothelial cells was achieved by using an anti-Fas blocking
antibody, aurintricarboxylic acid, or normal plasma depleted of
cryoprecipitate.
[0187] Thus, a patient may be treated with an agent that inhibits
Fas-ligand-mediated apoptosis of endothelial cells, in combination
with an agent that inhibits TRAIL-mediated apoptosis of endothelial
cells. In one embodiment, an anti-TRAIL blocking antibody and an
anti-FAS blocking antibody are both administered to a patient
afflicted with a disorder characterized by thrombotic
microangiopathy, such as TTP or HUS. Examples of blocking
monoclonal antibodies directed against Fas antigen (CD95) are
described in PCT application publication number WO 95/10540, hereby
incorporated by reference.
[0188] Pharmaceutical compositions comprising an antibody that is
immunoreactive with TRAIL, and a suitable, diluent, excipient, or
carrier, are provided herein. Suitable components of such
compositions are as described above for the compositions containing
TRAIL proteins.
[0189] The following examples are provided to illustrate particular
embodiments of the present invention, and are not to be construed
as limiting the scope of the invention.
EXAMPLE 1
Isolation of a Human TRAIL DNA
[0190] DNA encoding a human TRAIL protein of the present invention
was isolated by the following procedure. A TBLASTN search of the
dbEST data base at the National Center for Biological Information
(NCBI) was performed, using the query sequence LVVXXXGLYYVYXQVXF
(SEQ ID NO:8). This sequence is based upon the most conserved
region of the TNF ligand family (Smith et al., Cell, 73: 1349,
1993). An expressed sequence tag (EST) file, GenBank accession
number Z36726, was identified using these search parameters. The
GenBank file indicated that this EST was obtained from a human
heart atrium cDNA library.
[0191] Two 30-bp oligonucleotides based upon sequences from the 3'
and 5' ends of this EST file were synthesized. The oligonucleotide
from the 3' end had the sequence TGAAATCGAAAGTATGTGTTGGGAATAGATG
(complement of nucleotides 636 to 665 of SEQ ID NO:1) and the 5'
oligonucleotide was TGACGAAGAGAGTATGA ACAGCCCCTGCTG (nucleotides
291 to 320 of SEQ ID NO:1). The oligonucleotides were 5' end
labeled with .sup.32P .gamma.-ATP and polynucleotide kinase. Two
.lambda.gt10 cDNA libraries were screened by conventional methods
with an equimolar mixture of these labeled oligonucleotides as
probe. One library was a human heart 5' stretch cDNA library
(Stratagene Cloning Systems, La Jolla, Calif.). The other was a
peripheral blood lymphocyte (PBL) library prepared as follows: PBLs
were obtained from normal human volunteers and treated with 10
ng/ml of OKT3 (an anti-CD3 antibody) and 10 ng/ml of human IL-2 for
six days. The PBL cells were washed and stimulated with 500 ng/ml
of ionomycin (Calbiochem) and 10 ng/ml PMA for 4 hours. Messenger
RNA was isolated from the stimulated PBL cells. cDNA synthesized on
the mRNA template was packaged into .lambda.gt10 phage vectors
(Gigapak.RTM., Stratagene Cloning Systems, La Jolla, Calif.).
[0192] Recombinant phages were plated onto E. coli strain C600-HFL
and screened using standard plaque hybridization techniques.
Nitrocellulose filters were lifted from these plates in duplicate,
and hybridized with the .sup.32P-labeled oligonucleotides overnight
at 67.degree. C. in a solution of 60 mM Tris pH 8.0, 2 mM EDTA,
5.times. Denhardt's Solution, 6.times. SSC, 1 mg/ml n-lauroyl
sarcosine, 0.5% NP40, and 4 .mu.g/ml SS salmon sperm DNA. The
filters were then washed in 3.times. SSC at 67.degree. C. for
thirty minutes.
[0193] From the heart 5' stretch cDNA library, one positive plaque
was obtained out of approximately one million plaques. This clone
did not include the 3' end of the gene. Using the PBL library,
approximately 50 positive plaques were obtained out of 500,000
plaques. Fifteen of these first round positive plaques were picked,
and the inserts from the enriched pools were amplified using
oligonucleotide primers designed to amplify phage inserts. The
resulting products were resolved by 1.5% agarose gel
electrophoresis, blotted onto nitrocellulose, and analyzed by
standard Southern blot technique using the .sup.32P-labeled 30-mer
oligonucleotides as probes. The two plaque picks that produced the
largest bands by Southern analysis were purified by secondary
screening, and isolated phage plaques were obtained using the same
procedures described above.
[0194] DNA from the isolated phages was prepared by the plate lysis
method, and the cDNA inserts were excised with EcoRI, purified by
electrophoresis using 1.5% agarose in Tris-Borate-EDTA buffer, and
ligated into the pBluescript.RTM. SK(+) plasmid. These inserts were
then sequenced by conventional methods, and the resulting sequences
were aligned.
[0195] The nucleotide sequence of a human TRAIL DNA is presented in
SEQ ID NO:1 and the amino acid sequence encoded thereby is
presented in SEQ ID NO:2. This human TRAIL protein comprises an
N-terminal cytoplasmic domain (amino acids 1-18), a transmembrane
region (amino acids 19-38), and an extracellular domain (amino
acids 39-281). The calculated molecular weight of this protein is
32,508 daltons.
[0196] E. coli strain DH10B cells transformed with a recombinant
vector containing this TRAIL DNA were deposited with the American
Type Culture Collection on Jun. 14, 1995, and assigned accession
no. 69849. The deposit was made under the terms of the Budapest
Treaty. The recombinant vector in the deposited strain is the
expression vector pDC409 (described in example 5). The vector was
digested with SalI and NotI, and human TRAIL DNA that includes the
entire coding region shown in SEQ ID NO:1 was ligated into the
digested vector.
EXAMPLE 2
Isolation of DNA Encoding a Truncated TRAIL
[0197] DNA encoding a second human TRAIL protein was isolated as
follows. This truncated TRAIL does not exhibit the ability to
induce apoptosis of Jurkat cells.
[0198] PCR analysis, using the 30-mers described in example 1 as
the 5' and 3' primers, indicated that 3 out of 14 of the first
round plaque picks in example 1 contained shorter forms of the
TRAIL DNA. One of the shortened forms of the gene was isolated,
ligated into the pBluescript.RTM. SK(+) cloning vector (Stratagene
Cloning Systems, La Jolla, Calif.) and sequenced.
[0199] The nucleotide sequence of this DNA is presented in SEQ ID
NO:3. The amino acid sequence encoded thereby is presented in SEQ
ID NO:4. The encoded protein comprises an N-terminal cytoplasmic
domain (amino acids 1-18), a transmembrane region (amino acids
19-38), and an extracellular domain (amino acids 39-101).
[0200] The DNA of SEQ ID NO:3 lacks nucleotides 359 through 506 of
the DNA of SEQ ID NO:1, and is thus designated the human TRAIL
deletion variant (huTRAILdv) clone. The deletion causes a shift in
the reading frame, which results in an in-frame stop codon after
amino acid 101 of SEQ ID NO:4. The DNA of SEQ ID NO:3 thus encodes
a truncated protein. Amino acids 1 through 90 of SEQ ID NO:2 are
identical to amino acids 1 through 90 of SEQ ID NO:4. However, due
to the deletion, the C-terminal portion of the huTRAILdv protein
(amino acids 91 through 101 of SEQ ID NO:4) differs from the
residues in the corresponding positions in SEQ ID NO:2.
[0201] The huTRAILdv protein lacks the above-described conserved
regions found at the C-terminus of members of the TNF family of
proteins. The inability of this huTRAILdv protein to cause
apoptotic death of Jurkat cells further confirms the importance of
these conserved regions for biological activity.
EXAMPLE 3
DNA Encoding a Murine TRAIL
[0202] DNA encoding a murine TRAIL was isolated by the following
procedure. A cDNA library comprising cDNA derived from the mouse T
cell line 7B9 in the vector .lambda. ZAP was prepared as described
in Mosley et al. (Cell 59: 335, 1989). DNA from the library was
transferred onto nitrocellulose filters by conventional
techniques.
[0203] Human TRAIL DNA probes were used to identify hybridizing
mouse cDNAs on the filters. Two separate probes were used, in two
rounds of screening. PCR reaction products about 400 bp in length,
isolated and amplified using the human TRAIL DNA as template, were
employed as the probe in the first round of screening. These PCR
products consisted of a fragment of the human TRAIL coding region.
The probe used in the second round of screening consisted of the
entire coding region of the human TRAIL DNA of SEQ ID NO: 1. A
random primed DNA labeling kit (Stratagene, La Jolla, Calif.) was
used to radiolabel the probes.
[0204] Hybridization was conducted at 37.degree. C. in 50%
formamide, followed by washing with 1.times. SSC, 0.1% SDS at
50.degree. C. A mouse cDNA that was positive in both rounds of
screening was isolated.
[0205] The nucleotide sequence of this DNA is presented in SEQ ID
NO:5 and the amino acid sequence encoded thereby is presented in
SEQ ID NO:6. The encoded protein comprises an N-terminal
cytoplasmic domain (amino acids 1-17), a transmembrane region
(amino acids 18-38), and an extracellular domain (amino acids
39-291). This mouse TRAIL is 64% identical to the human TRAIL of
SEQ ID NO:2, at the amino acid level. The coding region of the
mouse TRAIL nucleotide sequence is 75% identical to the coding
region of the human nucleotide sequence of SEQ ID NO:1.
EXAMPLE 4
Antibodies that Bind TRAIL
[0206] This example illustrates the preparation of monoclonal
antibodies that specifically bind TRAIL. Suitable immunogens that
may be employed in generating such antibodies include, but are not
limited to, purified TRAIL protein or an immunogenic fragment
thereof (e.g., the extracellular domain), fusion proteins
containing TRAIL polypeptides (e.g., soluble TRAIL/Fc fusion
proteins), and cells expressing recombinant TRAIL on the cell
surface.
[0207] Known techniques for producing monoclonal antibodies include
those described in U.S. Pat. No. 4,411,993. Briefly, mice are
immunized with TRAIL as an immunogen emulsified in complete
Freund's adjuvant, and injected in amounts ranging from 10-100
.mu.g subcutaneously or intraperitoneally. Ten to twelve days
later, the immunized animals are boosted with additional TRAIL
emulsified in incomplete Freund's adjuvant. Mice are periodically
boosted thereafter on a weekly to bi-weekly immunization schedule.
Serum samples are periodically taken by retro-orbital bleeding or
tail-tip excision for testing by dot blot assay or ELISA
(Enzyme-Linked Immuno-sorbent Assay) for TRAIL antibodies.
[0208] Following detection of an appropriate antibody titer,
positive animals are provided one last intravenous injection of
TRAIL in saline. Three to four days later, the animals are
sacrificed, spleen cells harvested, and spleen cells are fused to a
murine myeloma cell line such as NS1 or, preferably, P3x63Ag 8.653
(ATCC CRL 1580). Fusions generate hybridoma cells, which are plated
in multiple microtiter plates in a HAT (hypoxanthine, aminopterin
and thymidine) selective medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0209] The hybridoma cells are screened by ELISA for reactivity
against purified TRAIL by adaptations of the techniques disclosed
in Engvall et al. (Immunochem. 8: 871, 1971) and in U.S. Pat. No.
4,703,004. Positive hybridoma cells can be injected
intraperitoneally into syngeneic BALB/c mice to produce ascites
containing high concentrations of anti-TRAIL monoclonal antibodies.
Alternatively, hybridoma cells can be grown in vitro in flasks or
roller bottles by various techniques. Monoclonal antibodies
produced in mouse ascites can be purified by ammonium sulfate
precipitation, followed by gel exclusion chromatography.
Alternatively, affinity chromatography based upon binding of
antibody to protein A or protein G can be used, as can affinity
chromatography based upon binding to TRAIL.
EXAMPLE 5
DNA Laddering Apoptosis Assay
[0210] Human TRAIL was expressed and tested for the ability to
induce apoptosis. Oligonucleotides were synthesized that
corresponded to the 3' and 5' ends of the coding region of the
human TRAIL gene, with SalI and NotI restriction sites incorporated
at the ends of the oligonucleotides. The coding region of the human
TRAIL gene was amplified by standard PCR techniques, using these
oligonucleotides as primers. The PCR reaction products were
digested with the restriction endonucleases SalI and NotI, then
inserted into SalI/NotI-digested vector pDC409. pDC409 is an
expression vector for use in mammalian cells, but is also
replicable in E. coli cells.
[0211] pDC409 is derived from an expression vector designated
pDC406 (described in McMahan et al., EMBO J. 10: 2821, 1991, and in
PCT application WO 91/18982, hereby incorporated by reference).
pDC406 contains origins of replication derived from SV40,
Epstein-Barr virus and pBR322 and is a derivative of HAV-EO
described by Dower et al., J. Immunol. 142: 4314 (1989). pDC406
differs from HAV-EO by the deletion of an intron present in the
adenovirus 2 tripartite leader sequence in HAV-EO. DNA inserted
into a multiple cloning site (containing a number of restriction
endonuclease cleavage sites) is transcribed and translated using
regulatory elements derived from HIV and adenovirus. The vector
also contains a gene that confers ampicillin resistance.
[0212] pDC409 differs from pDC406 in that a Bgl II site outside the
mcs has been deleted so that the mcs Bgl II site is unique. Two Pme
1 sites and one Srf 1 site have been added to the mcs, and three
stop codons (TAG) have been positioned downstream of the mcs to
function in all three reading frames. A T7 primer/promoter has been
added to aid in the DNA sequencing process.
[0213] The monkey kidney cell line CV-1/EBNA-1 (ATCC CRL 10478) was
derived by transfection of the CV-1 cell line (ATCC CCL 70) with a
gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1) that
constitutively expresses EBNA-1 driven from the human CMV
intermediate-early enhancer/promoter, as described by McMahan et
al., supra. The EBNA-1 gene allows for episomal replication of
expression vectors, such as pDC409, that contain the EBV origin of
replication.
[0214] CV1/EBNA cells grown in Falcon T175 flasks were transfected
with 15 .mu.g of either "empty" pDC409 or pDC409 containing the
human TRAIL coding region. The transformed cells were cultured for
three days at 37.degree. C. and 10% CO.sub.2. The cells then were
washed with PBS, incubated for 20 minutes at 37.degree. C. in 50 mM
EDTA, scraped off of the flask with a cells scraper, and washed
once in PBS. Next, the cells were fixed in 1% paraformaldehyde PBS
for 10 minutes at 4.degree. C., and washed 3.times. in PBS.
[0215] Jurkat cells were used as the target cells in this assay, to
determine whether the TRAIL-expressing cells could induce apoptosis
thereof. The Jurkat cell line, clone E6-1, is a human acute T cell
leukemia cell line available from the American Type Culture
Collection under accession no. ATCC TIB 152, and described in Weiss
et al. (J. Immunol. 133: 123-128, 1984). The Jurkat cells were
cultured in RPMI media supplemented with 10% fetal bovine serum and
10 .mu.g/ml streptomycin and penicillin to a density of 200,000 to
500,000 cells per ml. Four million of these cells per well were
co-cultured in a 6 well plate with 2.5 mls of media with various
combinations of fixed cells, supernatants from cells transfected
with Fas ligand, and various antibodies, as indicated below.
[0216] After four hours the cells were washed once in PBS and
pelleted at 1200 RPM for 5 minutes in a desktop centrifuge. The
pellets were resuspended and incubated for ten minutes at 4.degree.
C. in 500 .mu.l of buffer consisting of 10 mM Tris-HCl, 10 mM EDTA,
pH 7.5, and 0.2% Triton X-100, which lyses the cells but leaves the
nuclei intact. The lysate was then spun at 4.degree. C. for ten
minutes in a micro-centrifuge at 14,000 RPM. The supernatants were
removed and extracted three times with 1 ml of 25:24:1
phenol-chloroform-isoamyl alcohol, followed by precipitation with
NaOAC and ethanol in the presence of 1 .mu.g of glycogen carrier
(Sigma).
[0217] The resulting pellets were resuspended in 10 mM Tris-HCl, 10
mM EDTA, pH 7.5, and incubated with 10 .mu.g/ml RNase A at
37.degree. C. for 20 minutes. The DNA solutions were then resolved
by 1.5% agarose gel electrophoresis in Tris-Borate EDTA buffer,
stained with ethidium bromide and photographed while
trans-illuminated with UV light.
[0218] The results were as follows. Fixed CV1/EBNA cells
transfected with either pDC409 or pDC409-TRAIL produced no
detectable DNA laddering. pDC409-TRAIL fixed cells co-cultured with
Jurkat cells produced DNA laddering, but pDC409 fixed cells
co-cultured with Jurkat cells did not.
[0219] DNA laddering was also seen when Jurkat cells were
co-cultured with concentrated supernatants from COS cells
transfected with DNA encoding human Fas ligand in pDC409. The
supernatants are believed to contain soluble Fas ligand that is
proteolytically released from the cell surface. The Fas
ligand-induced DNA laddering could be blocked by adding 10 .mu.g/ml
of a soluble blocking monoclonal antibody directed against Fas.
This same antibody could not inhibit laddering of Jurkat DNA by the
pDC409-TRAIL cells, which indicates that TRAIL does not induce
apoptosis through Fas.
[0220] In the same assay procedure, fixed CV1/EBNA cells
transfected with pDC409-TRAIL induced DNA laddering in U937 cells.
U937 (ATCC CRL 1593) is a human histiocytic lymphoma cell line. The
ratio of effector to target cells was 1:4 (the same as in the assay
on Jurkat target cells).
[0221] The fragmentation of cellular DNA into a pattern known as
DNA laddering is a hallmark of apoptosis. In the foregoing assay,
TRAIL induced apoptosis of a leukemia cell line and a lymphoma cell
line.
EXAMPLE 6
Northern Blot Analysis
[0222] Expression of TRAIL in a number of different tissue types
was analysed in a conventional northern blot procedure. Northern
blots containing poly A.sup.+ RNA from a variety of adult human
tissues (multiple tissue northern blots I and II) were obtained
from Clonetech (Palo Alto, Calif.). Other blots were prepared by
resolving RNA samples on a 1.1% agarose-formaldehyde gel, blotting
onto Hybond-N as recommended by the manufacturer (Amersham
Corporation), and staining with methylene blue to monitor RNA
concentrations. The blots were probed with an antisense RNA
riboprobe corresponding to the entire coding region of human
TRAIL.
[0223] Human TRAIL mRNA was detected in peripheral blood
lymphocytes, colon, small intestine, ovary, prostate, thymus,
spleen, placenta, lung, kidney, heart, pancreas, and skeletal
muscle. TRAIL transcripts were found to be abundant in the large
cell anaplastic lymphoma cell line Karpas 299 (Fischer et al.,
Blood, 72: 234, 1988) and in tonsilar T cells. TRAIL message was
present to a lesser degree in the Burkitt lymphoma cell line
designated Raji.
[0224] TRAIL mRNA was not detected in testis, brain, or liver, or
in several T cell lines. Little or no TRAIL transcripts were
detected in freshly isolated peripheral blood T cells (PBT), either
unstimulated or stimulated with PMA and calcium ionophore for 20
hours.
EXAMPLE 7
Production of a Soluble TRAIL Polypeptide
[0225] A soluble human TRAIL polypeptide comprising amino acids 95
to 281 of SEQ ID NO:2 was prepared as follows. This polypeptide is
a fragment of the extracellular domain, lacking the spacer region
discussed above.
[0226] An expression vector encoding soluble human TRAIL was
constructed by fusing in-frame DNA encoding the following amino
acid sequences (listed from N- to C-terminus): a leader sequence
derived from human cytomegalovirus (CMV), a synthetic epitope
designated Flag.RTM., and amino acids 95-281 of human TRAIL. The
Flag.RTM. octapeptide (SEQ ID NO:7) facilitates purification of
proteins fused thereto, as described above and in Hopp et al.
(Biotechnology 6: 1204-1210, 1988).
[0227] The TRAIL-encoding DNA fragment was isolated and amplified
by polymerase chain reaction (PCR), using oligonucleotide primers
that defined the termini of a DNA fragment encoding amino acids
95-281 of SEQ ID NO:2. The 3' primer was a 31-mer that additionally
added a NotI site downstream of the TRAIL-encoding sequence. The 5'
primer added an SpeI site and a Flag.RTM. epitope encoding sequence
upstream of the TRAIL-encoding sequence. PCR was conducted by
conventional procedures, using the above-described human TRAIL cDNA
as the template.
[0228] The reaction products were digested with SpeI and NotI, and
inserted into the expression vector pDC409 (described in example
5), which had been cleaved with SalI and NotI. Annealed
oligonucleotides that form a SalI-SpeI fragment encoding a CMV open
reading frame leader were also ligated into the vector. The amino
acid sequence of the CMV-derived leader is presented as SEQ ID
NO:9. Amino acids 1 to 29 of SEQ ID NO:9 are encoded by CMV DNA,
whereas amino acids 30 to 32 are encoded by oligonucleotides
employed in constructing the vector. E. coli cells were transfected
with the ligation mixture, and the desired recombinant expression
vector was isolated therefrom.
[0229] CV1-EBNA cells (ATCC CRL 10478; described in example 5) were
transfected with the recombinant vector, which is designated
pDC409-Flag-shTRAIL, and cultured to allow expression and secretion
of the soluble Flag.RTM.-TRAIL polypeptide. Culture supernatants
were harvested 3 days after transfection and applied to a column
containing an anti-Flag.RTM. antibody designated M2 immobilized on
a solid support. The column then was washed with PBS. The
monoclonal antibody M2 is described in Hopp et al., supra, and
available from Kodak Scientific Imaging Systems, New Haven, Conn.
800 .mu.l fractions were eluted from the column with 50 mM citrate,
and immediately neutralized in 0.45 ml 1M Tris (pH 8). Fractions
were adjusted to 10% glycerol and stored at -20.degree. C. until
needed.
[0230] This soluble recombinant Flag.RTM./human TRAIL expressed in
CV1/EBNA cells has an apparent molecular weight of 28 ld) when
analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The
Flag.RTM. moiety contributes an estimated 880 daltons to the total
molecular weight. Gel filtration analysis of purified soluble
Flag.RTM./TRAIL suggests that the molecule is multimeric in
solution with a size of .about.80 kD. While not wishing to be bound
by theory, the gel filtration analysis suggests that the soluble
recombinant Flag.RTM./human TRAIL naturally formed a combination of
dimers and trimers, with trimers predominating.
[0231] An expression vector designated pDC409-Flag-smTRAIL, which
encodes a CMV leader-Flag.RTM.-soluble murine TRAIL protein, was
constructed by analogous procedures. A DNA fragment encoding a
soluble murine TRAIL polypeptide was isolated and amplified by PCR.
Oligonucleotides that defined the termini of DNA encoding amino
acids 99 to 291 of the murine TRAIL sequence of SEQ ID NO:6 were
employed as the 5' and 3' primers in the PCR.
EXAMPLE 8
Lysis of Leukemia Cells by Soluble TRAIL
[0232] In example 5, cells expressing human TRAIL induced apoptosis
of Jurkat cells (a leukemia cell line). In the following study, a
soluble human TRAIL polypeptide killed Jurkat cells.
[0233] Jurkat cells were cultured to a density of 200,000 to
500,000 cells per ml in RPMI medium supplemented with 10% fetal
bovine serum, 100 .mu.g/ml streptomycin, and 100 .mu.g/ml
penicillin. The cells (in 96-well plates at 50,000 cells per well
in a volume of 100 .mu.l) were incubated for twenty hours with the
reagents indicated in FIG. 1. "TRAIL supe." refers to conditioned
supernatant (10 .mu.l per well) from CV1/EBNA cells transfected
with pDC409-Flag-shTRAIL (see example 7). "Control supe." refers to
supernatant from CV1/EBNA cells transfected with empty vector.
Where indicated, immobilized anti-Flag.RTM. antibody M2 ("Imm. M2")
was added at a concentration of 10 .mu.g/ml in a volume of 100
.mu.l per well and allowed to adhere either overnight at 4.degree.
C. or for 2 hours at 37.degree. C., after which wells were
aspirated and washed twice with PBS to remove unbound antibody.
Jurkat cells treated with Fas ligand or M3, a blocking monoclonal
antibody directed against Fas, (Alderson et al., J. Exp. Med. 181:
71, 1995; and PCT application WO 95/10540) were included in the
assay as indicated.
[0234] Metabolic activity of the thus-treated cells was assayed by
metabolic conversion of alamar Blue dye, in the following
procedure. Alamar Blue conversion was measured by adding 10 .mu.l
of alamar Blue dye (Biosource International, Camarillo, Calif.) per
well, and subtracting the optical density (OD) at 550-600 nm at the
time the dye was added from the OD 550-600 nm after four hours. No
conversion of dye is plotted as 0 percent viability, and the level
of dye conversion in the absence of TRAIL is plotted as 100 percent
viability. Percent viability was calculated by multiplying the
ratio of staining of experimental versus control cultures by
100.
[0235] The results are presented in FIG. 1. Error bars represent
the standard deviation of measurements from four independent wells,
and the values are the average of these measurements.
[0236] The TRAIL-containing supernatant caused a significant
reduction in viability of Jurkat cells. A greater reduction of cell
viability resulted from contact with a combination of
TRAIL-containing supernatant and immobilized anti-Flag.RTM.
antibody M2. One possible explanation is that M2 facilitates
cross-linking of the Flag.RTM./TRAIL-receptor complexes, thereby
increasing the strength of signaling.
[0237] Fas ligand demonstrated the ability to kill Jurkat cells.
The anti-Fas antibody M3 inhibited the activity of Fas ligand, but
not the activity of TRAIL.
[0238] In order to confirm that the changes in dye conversion in
the alamar Blue assay were due to cell death, the decrease in cell
viability induced by TRAIL was confirmed by staining the cells with
trypan blue.
EXAMPLE 9
Lysis of Leukemia and Lymphoma Cells
[0239] In examples 5 and 8, TRAIL induced apoptosis of a leukemia
cell line (Jurkat) and a lymphoma cell line (U937). The following
study further demonstrates the ability of TRAIL to kill leukemia
and lymphoma cells.
[0240] The human cell lines indicated in Table I were cultured to a
density of 200,000 to 500,000 cells per ml in RPMI medium
supplemented with 10% fetal bovine serum, 100 .mu.g/ml
streptomycin, and 100 .mu.g/ml penicillin. The cells (in 96-well
plates at 50,000 cells per well in a volume of 100 .mu.l) were
incubated for twenty hours with conditioned supernatants (10 .mu.l
per well) from pDC409-Flag-shTRAIL transfected CV1/EBNA cells.
[0241] Metabolic activity was assayed by conversion of alamar Blue
dye, in the assay procedure described in example 8. The results are
presented in Table I.
[0242] In order to confirm that the changes in dye conversion in
the alamar Blue assay were due to cell death, the decrease in cell
viability induced by TRAIL was confirmed by staining the cells with
trypan blue. Crystal violet staining, performed as described by
Flick and Gifford (J. Immunol. Methods 68: 167-175, 1984), also
confirmed the results seen in the alamar Blue assay. The apoptotic
nature of the cell death was confirmed by trypan blue staining and
visualization of apoptotic fragmentation by microscopy.
[0243] As shown in Table I, many cancer cell lines were sensitive
to TRAIL mediated killing. The susceptibility of additional cell
types to TRAIL mediated apoptosis can be determined using the assay
procedures described in this examples section.
[0244] TRAIL exhibited no significant cytotoxic effect on the cell
lines THP-1, K562, Karpas 299, and MP-1. K299, also known as Karpas
299, (DSM-ACC31) was established from peripheral blood of a male
diagnosed with high grade large cell anaplastic lymphoma (Fischer
et al., Blood, 72: 234, 1988). MP-1 is a spontaneously derived
EBV-transformed B cell line (Goodwin et al., Cell 73: 447, 1993).
While not wishing to be bound by theory, it is possible that these
four cell lines do not express a receptor for TRAIL, or are
characterized by upregulation of a gene that inhibits
apoptosis.
2TABLE 1 Effect of soluble TRAIL on cell line viability Cell Line
Description Percent Viability.sup.a Bjab Burkitt lymphoma 0.5 .+-.
3.8 Ramos Burkitt lymphoma 12.1 .+-. 2.1 U937 histiocytic lymphoma
25.2 .+-. 8.2 HL60 promyelocytic leukemia 59.5 .+-. 3.2 Raji
Burkitt lymphoma 64.9 .+-. 4.5 Daudi Burkitt lymphoma 70.2 .+-. 4.2
THP-1 monocytic cell line 92.3 .+-. 6.8 K562 chronic myelogenous
leukemia 97.1 .+-. 4.8 K 299 large cell anaplastic lymphoma 99.0
.+-. 4.3 MP-1 spontaneous B cell line 104.9 .+-. 11.7 .sup.aResults
are means .+-. SEMs of 4 wells for each data point
EXAMPLE 10
Cross-Species Activity of TRAIL
[0245] Interspecies cross-reactivity of human and murine TRAIL was
tested as follows. Murine and human TRAIL were incubated with the
human melanoma cell line A375 (ATCC CRL 1619). Since this is an
adherent cell line, a crystal violet assay, rather than alamar
Blue, was used to determine cell viability. A375 cells were
cultured in DMEM supplemented with 10% fetal bovine serum, 100
.mu.g/ml streptomycin, and 100 .mu.g/ml penicillin. The cells (in
96-well plates at 10,000 cells per well in a volume of 100 .mu.l)
were incubated for 72 hours with the soluble murine TRAIL described
in example 7. Crystal violet staining was performed as described by
(Flick and Gifford (J. Immunol. Methods 68: 167-175, 1984). The
results demonstrated that both human and murine TRAIL are active on
these human cells, in that murine and human TRAIL killed A375
cells.
[0246] The ability of human TRAIL to act on murine cells was
tested, using the immortalized murine fibroblast cell line L929.
Incubation of L929 cells with either human or murine TRAIL resulted
in a decrease in crystal violet staining, thus demonstrating that
human and murine TRAIL are active on (induced apoptosis of) murine
cells. In addition to crystal violet, cell death was confirmed by
trypan-blue staining.
EXAMPLE 11
Lysis of CMV-Infected Cells
[0247] The following experiment demonstrates that the soluble
Flag.RTM.-human TRAIL protein prepared in example 7 has a cytotoxic
effect on virally infected cells.
[0248] Normal human gingival fibroblasts were grown to confluency
on 24 well plates in 10% CO.sub.2 and DMEM medium supplemented with
10% fetal bovine serum, 100 .mu.g/ml streptomycin, and 100 .mu.g/ml
penicillin. Samples of the fibroblasts were treated as indicated in
FIG. 2. Concentrations of cytokines were 10 ng/ml for
.gamma.-interferon and 30 ng/ml of soluble Flag.RTM.-human TRAIL.
All samples receiving TRAIL also received a two-fold excess by
weight of anti-Flag.RTM. antibody M2 (described above), which
enhances TRAIL activity (presumably by crosslinking).
[0249] Pretreatment of cells with the indicated cytokines was for
20 hours. To infect cells with cytomegalovirus (CMV), culture media
were aspirated and the cells were infected with CMV in DMEM with an
approximate MOI (multiplicity of infection) of 5. After two hours
the virus containing media was replaced with DMEM and cytokines
added as indicated. After 24 hours the cells were stained with
crystal violet dye as described (Flick and Gifford, 1984, supra).
Stained cells were washed twice with water, disrupted in 200 .mu.l
of 2% sodium deoxycholate, diluted 5 fold in water, and the OD
taken at 570 nm. Percent maximal staining was calculated by
normalizing ODs to the sample that showed the greatest staining.
Similar results were obtained from several independent
experiments.
[0250] The results presented in FIG. 2 demonstrate that TRAIL
specifically killed CMV infected fibroblasts. This cell death was
enhanced by pretreatment of the cells with .gamma.-interferon. No
significant death of non-virally infected fibroblasts resulted from
contact with TRAIL.
EXAMPLE 12
Assay to Identify Blocking Antibodies
[0251] Blocking antibodies directed against TRAIL may be identified
by testing antibodies for the ability to inhibit a particular
biological activity of TRAIL. In the following assay, a monoclonal
antibody was tested for the ability to inhibit TRAIL-mediated
apoptosis of Jurkat cells. The Jurkat cell line is described in
example 5.
[0252] A hybridoma cell line producing a monoclonal antibody raised
against a Flag.RTM./soluble human TRAIL fusion protein was employed
in the assay. Supernatants from the hybridoma cultures were
incubated with 20 ng/ml Flag.RTM./soluble human TRAIL crosslinked
with 40 ng/ml anti-Flag.RTM. monoclonal antibody M2, in RPMI
complete media in a 96 well microtiter plate. An equivalent amount
of fresh hybridoma culture medium was added to control cultures.
The Flag.RTM./soluble human TRAIL fusion protein and the monoclonal
antibody designated M2 are described in example 7.
[0253] The hybridoma supernatant was employed at a 1:50 (v/v)
dilution (starting concentration), and at two fold serial dilutions
thereof. After incubation at 37.degree. C., 10% CO.sub.2, for 30
minutes, 50,000 Jurkat cells were added per well, and incubation
was continued for 20 hours.
[0254] Cell viability was then assessed measuring metabolic
conversion of alamar blue dye. An alamar blue conversion assay
procedure is described in example 8. The monoclonal antibody was
found to inhibit the apoptosis of Jurkat cells induced by
Flag.RTM./soluble human TRAIL.
EXAMPLE 13
TRAIL Blocking Study
[0255] Human microvascular endothelial cells of dermal origin were
treated for 16-18 hours with plasma from patients with thrombotic
thrombocytopenic purpura (TTP) or with control plasma, either alone
or in the presence of anti-TRAIL polyclonal antiserum. A 1:2000
dilution of the antiserum was employed. The plasma was from two TTP
patients, designated #1 and #2 below, The cells employed in the
assays were MVEC-1 (HMVEC 2753, purchased from Clonetics, San
Diego, Calif.) and MVEC-2 (DHMVEC 30282, purchased from Cell
Systems, Kirkland, Wash.). Cultures of these cells can be
maintained as described in Laurence et al. (Blood, 87: 3245,
1996).
[0256] The results were as follows. The data shown are from DNA
histograms of cells stained with propidium iodide, and "A.sub.0
peak" represents the apoptotic peak (see Oyaizu et al., Blood, 82:
3392, 1993; Nicoletti et al., J. Immunol. Methods, 139: 271, 1991;
and Laurence et al., Blood, 75: 696, 1990).
3 Microvascular EC Plasma (1%) Antibody % A.sub.0 peak Experiment 1
Dermal MVEC-1 control - 0 Dermal MVEC-1 TTP (#1) - 19.5 Dermal
MVEC-1 TTP (#1) + 0.3 Experiment 2 Dermal MVEC-2 control - 0 Dermal
MVEC-2 TTP (#2) - 20.0 Dermal MVEC-2 TTP (#2) control Ab 13.1
Dermal MVEC-2 TTP (#2) + 0.2 Experiment 3 Dermal MVEC-1 TTP (#1) -
50.1 Dermal MVEC-1 TTP (#1) + 10.6 Experiment 4 Dermal MVEC-2
control - 0 Dermal MVEC-2 TTP (#1) - 13.9 Dermal MVEC-2 TTP (#1)
control Ab 14.1 Dermal MVEC-2 TTP (#1) + 0.6
[0257] The data reveal that plasma derived from TTP patients
induces apoptosis of microvascular endothelial cells of dermal
origin. This apoptosis was inhibited by polyclonal antibodies
directed against TRAIL.
EXAMPLE 14
Expression of LZ-TRAIL
[0258] Examples of fusion proteins comprising leucine zipper (LZ)
peptides fused to the N-terminus of a soluble TRAIL polypeptide are
as follows. The leucine zipper moieties promote oligomerization of
the TRAIL polypeptides fused thereto, as described above.
[0259] An expression vector is constructed, containing DNA encoding
(from N- to C-terminus) a human growth hormone signal peptide, a
leucine zipper peptide, and a soluble human TRAIL polypeptide. The
TRAIL polypeptide comprises amino acids 95 to 281 of SEQ ID NO:2.
This TRAIL polypeptide is a fragment of the extracellular domain of
human TRAIL, lacking the spacer region, as described in example
7.
[0260] The growth hormone signal peptide comprises the amino acid
sequence: Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly
Leu Leu Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala (SEQ ID NO:19). The
leucine zipper peptide is selected from peptides comprising the
amino acid sequence:
[0261] (Asp).sub.n (Arg).sub.n Met Lys Gln Ile Glu Asp Lys Ile Glu
Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys
Lys Leu Ile Gly Glu Arg, wherein each n independently represents 1
or 0 (SEQ ID NO:15);
[0262] Ser Leu Ala Ser Leu Arg Gln Gln Leu Glu Ala Leu Gln Gly Gln
Leu Gln His Leu Gln Ala Ala Leu Ser Gln Leu Gly Glu (SEQ ID NO:16);
or
[0263] Ser Ile Ala Ser Ile Arg Gln Gln Ile Glu Ala Ile Gln Gly Gln
Ile Gln His Ile Gln Ala Ala Ile Ser Gln Ile Gly Glu (SEQ ID
NO:17).
[0264] The fusion protein may comprise additional amino acid
residue(s), encoded by DNA segments that result from construction
of the vector, or that are added to facilitate vector construction.
In one particular embodiment, the tripeptide Thr-Ser-Ser is
positioned between the growth hormone signal peptide and the
leucine zipper peptide. This tripeptide is encoded by a DNA segment
that comprises an Spe I restriction endonuclease recognition site.
The tripeptide Thr-Arg-Ser, encoded by a DNA segment that comprises
a Bgl II restriction site, may be positioned between the leucine
zipper and the TRAIL polypeptide.
[0265] DNA encoding the desired fusion protein is inserted into a
suitable expression vector, such as the pDC409 vector described in
example 7. CV1-EBNA cells are transformed with the recombinant
expression vector, then cultured to allow expression of the fusion
protein, and oligomerization thereof.
Sequence CWU 1
1
25 1 1751 DNA human CDS (88)..(933) 1 cctcactgac tataaaagaa
tagagaagga agggcttcag tgaccggctg cctggctgac 60 ttacagcagt
cagactctga caggatc atg gct atg atg gag gtc cag ggg gga 114 Met Ala
Met Met Glu Val Gln Gly Gly 1 5 ccc agc ctg gga cag acc tgc gtg ctg
atc gtg atc ttc aca gtg ctc 162 Pro Ser Leu Gly Gln Thr Cys Val Leu
Ile Val Ile Phe Thr Val Leu 10 15 20 25 ctg cag tct ctc tgt gtg gct
gta act tac gtg tac ttt acc aac gag 210 Leu Gln Ser Leu Cys Val Ala
Val Thr Tyr Val Tyr Phe Thr Asn Glu 30 35 40 ctg aag cag atg cag
gac aag tac tcc aaa agt ggc att gct tgt ttc 258 Leu Lys Gln Met Gln
Asp Lys Tyr Ser Lys Ser Gly Ile Ala Cys Phe 45 50 55 tta aaa gaa
gat gac agt tat tgg gac ccc aat gac gaa gag agt atg 306 Leu Lys Glu
Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu Glu Ser Met 60 65 70 aac
agc ccc tgc tgg caa gtc aag tgg caa ctc cgt cag ctc gtt aga 354 Asn
Ser Pro Cys Trp Gln Val Lys Trp Gln Leu Arg Gln Leu Val Arg 75 80
85 aag atg att ttg aga acc tct gag gaa acc att tct aca gtt caa gaa
402 Lys Met Ile Leu Arg Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu
90 95 100 105 aag caa caa aat att tct ccc cta gtg aga gaa aga ggt
cct cag aga 450 Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly
Pro Gln Arg 110 115 120 gta gca gct cac ata act ggg acc aga gga aga
agc aac aca ttg tct 498 Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg
Ser Asn Thr Leu Ser 125 130 135 tct cca aac tcc aag aat gaa aag gct
ctg ggc cgc aaa ata aac tcc 546 Ser Pro Asn Ser Lys Asn Glu Lys Ala
Leu Gly Arg Lys Ile Asn Ser 140 145 150 tgg gaa tca tca agg agt ggg
cat tca ttc ctg agc aac ttg cac ttg 594 Trp Glu Ser Ser Arg Ser Gly
His Ser Phe Leu Ser Asn Leu His Leu 155 160 165 agg aat ggt gaa ctg
gtc atc cat gaa aaa ggg ttt tac tac atc tat 642 Arg Asn Gly Glu Leu
Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr 170 175 180 185 tcc caa
aca tac ttt cga ttt cag gag gaa ata aaa gaa aac aca aag 690 Ser Gln
Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys 190 195 200
aac gac aaa caa atg gtc caa tat att tac aaa tac aca agt tat cct 738
Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro 205
210 215 gac cct ata ttg ttg atg aaa agt gct aga aat agt tgt tgg tct
aaa 786 Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser
Lys 220 225 230 gat gca gaa tat gga ctc tat tcc atc tat caa ggg gga
ata ttt gag 834 Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly
Ile Phe Glu 235 240 245 ctt aag gaa aat gac aga att ttt gtt tct gta
aca aat gag cac ttg 882 Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val
Thr Asn Glu His Leu 250 255 260 265 ata gac atg gac cat gaa gcc agt
ttt ttc ggg gcc ttt tta gtt ggc 930 Ile Asp Met Asp His Glu Ala Ser
Phe Phe Gly Ala Phe Leu Val Gly 270 275 280 taa ctgacctgga
aagaaaaagc aataacctca aagtgactat tcagttttca 983 ggatgataca
ctatgaagat gtttcaaaaa atctgaccaa aacaaacaaa cagaaaacag 1043
aaaacaaaaa aacctctatg caatctgagt agagcagcca caaccaaaaa attctacaac
1103 acacactgtt ctgaaagtga ctcacttatc ccaagaaaat gaaattgctg
aaagatcttt 1163 caggactcta cctcatatca gtttgctagc agaaatctag
aagactgtca gcttccaaac 1223 attaatgcaa tggttaacat cttctgtctt
tataatctac tccttgtaaa gactgtagaa 1283 gaaagcgcaa caatccatct
ctcaagtagt gtatcacagt agtagcctcc aggtttcctt 1343 aagggacaac
atccttaagt caaaagagag aagaggcacc actaaaagat cgcagtttgc 1403
ctggtgcagt ggctcacacc tgtaatccca acattttggg aacccaaggt gggtagatca
1463 cgagatcaag agatcaagac catagtgacc aacatagtga aaccccatct
ctactgaaag 1523 tgcaaaaatt agctgggtgt gttggcacat gcctgtagtc
ccagctactt gagaggctga 1583 ggcaggagaa tcgtttgaac ccgggaggca
gaggttgcag tgtggtgaga tcatgccact 1643 acactccagc ctggcgacag
agcgagactt ggtttcaaaa aaaaaaaaaa aaaaaaactt 1703 cagtaagtac
gtgttatttt tttcaataaa attctattac agtatgtc 1751 2 281 PRT human 2
Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys 1 5
10 15 Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val
Ala 20 25 30 Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met
Gln Asp Lys 35 40 45 Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys
Glu Asp Asp Ser Tyr 50 55 60 Trp Asp Pro Asn Asp Glu Glu Ser Met
Asn Ser Pro Cys Trp Gln Val 65 70 75 80 Lys Trp Gln Leu Arg Gln Leu
Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95 Glu Glu Thr Ile Ser
Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105 110 Leu Val Arg
Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly 115 120 125 Thr
Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu 130 135
140 Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly
145 150 155 160 His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu
Leu Val Ile 165 170 175 His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln
Thr Tyr Phe Arg Phe 180 185 190 Gln Glu Glu Ile Lys Glu Asn Thr Lys
Asn Asp Lys Gln Met Val Gln 195 200 205 Tyr Ile Tyr Lys Tyr Thr Ser
Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220 Ser Ala Arg Asn Ser
Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr 225 230 235 240 Ser Ile
Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile 245 250 255
Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala 260
265 270 Ser Phe Phe Gly Ala Phe Leu Val Gly 275 280 3 1521 DNA
human CDS (78)..(383) 3 aattccggaa tagagaagga agggcttcag tgaccggctg
cctggctgac ttacagcagt 60 cagactctga caggatc atg gct atg atg gag gtc
cag ggg gga ccc agc 110 Met Ala Met Met Glu Val Gln Gly Gly Pro Ser
1 5 10 ctg gga cag acc tgc gtg ctg atc gtg atc ttc aca gtg ctc ctg
cag 158 Leu Gly Gln Thr Cys Val Leu Ile Val Ile Phe Thr Val Leu Leu
Gln 15 20 25 tct ctc tgt gtg gct gta act tac gtg tac ttt acc aac
gag ctg aag 206 Ser Leu Cys Val Ala Val Thr Tyr Val Tyr Phe Thr Asn
Glu Leu Lys 30 35 40 cag atg cag gac aag tac tcc aaa agt ggc att
gct tgt ttc tta aaa 254 Gln Met Gln Asp Lys Tyr Ser Lys Ser Gly Ile
Ala Cys Phe Leu Lys 45 50 55 gaa gat gac agt tat tgg gac ccc aat
gac gaa gag agt atg aac agc 302 Glu Asp Asp Ser Tyr Trp Asp Pro Asn
Asp Glu Glu Ser Met Asn Ser 60 65 70 75 ccc tgc tgg caa gtc aag tgg
caa ctc cgt cag ctc gtt aga aag act 350 Pro Cys Trp Gln Val Lys Trp
Gln Leu Arg Gln Leu Val Arg Lys Thr 80 85 90 cca aga atg aaa agg
ctc tgg gcc gca aaa taa actcctggga atcatcaagg 403 Pro Arg Met Lys
Arg Leu Trp Ala Ala Lys 95 100 agtgggcatt cattcctgag caacttgcac
ttgaggaatg gtgaactggt catccatgaa 463 aaagggtttt actacatcta
ttcccaaaca tactttcgat ttcaggagga aataaaagaa 523 aacacaaaga
acgacaaaca aatggtccaa tatatttaca aatacacaag ttatcctgac 583
cctatattgt tgatgaaaag tgctagaaat agttgttggt ctaaagatgc agaatatgga
643 ctctattcca tctatcaagg gggaatattt gagcttaagg aaaatgacag
aatttttgtt 703 tctgtaacaa atgagcactt gatagacatg gaccatgaag
ccagtttttt cggggccttt 763 ttagttggct aactgacctg gaaagaaaaa
gcaataacct caaagtgact attcagtttt 823 caggatgata cactatgaag
atgtttcaaa aaatctgacc aaaacaaaca aacagaaaac 883 agaaaacaaa
aaaacctcta tgcaatctga gtagagcagc cacaaccaaa aaattctaca 943
acacacactg ttctgaaagt gactcactta tcccaagaga atgaaattgc tgaaagatct
1003 ttcaggactc tacctcatat cagtttgcta gcagaaatct agaagactgt
cagcttccaa 1063 acattaatgc agtggttaac atcttctgtc tttataatct
actccttgta aagactgtag 1123 aagaaagcgc aacaatccat ctctcaagta
gtgtatcaca gtagtagcct ccaggtttcc 1183 ttaagggaca acatccttaa
gtcaaaagag agaagaggca ccactaaaag atcgcagttt 1243 gcctggtgca
gtggctcaca cctgtaatcc caacattttg ggaacccaag gtgggtagat 1303
cacgagatca agagatcaag accatagtga ccaacatagt gaaaccccat ctctactgaa
1363 agtgcaaaaa ttagctgggt gtgttggcac atgcctgtag tcccagctac
ttgagaggct 1423 gaggcaggag aatcgtttga acccgggagg cagaggttgc
agtgtggtga gatcatgcca 1483 ctacactcca gcctggcgac agagcgagac
ttggtttc 1521 4 101 PRT human 4 Met Ala Met Met Glu Val Gln Gly Gly
Pro Ser Leu Gly Gln Thr Cys 1 5 10 15 Val Leu Ile Val Ile Phe Thr
Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30 Val Thr Tyr Val Tyr
Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45 Tyr Ser Lys
Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60 Trp
Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val 65 70
75 80 Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Thr Pro Arg Met Lys
Arg 85 90 95 Leu Trp Ala Ala Lys 100 5 1366 DNA murine CDS
(47)..(919) 5 tgctgggctg caagtctgca ttgggaagtc agacctggac agcagt
atg cct tcc 55 Met Pro Ser 1 tca ggg gcc ctg aag gac ctc agc ttc
agt cag cac ttc agg atg atg 103 Ser Gly Ala Leu Lys Asp Leu Ser Phe
Ser Gln His Phe Arg Met Met 5 10 15 gtg att tgc ata gtg ctc ctg cag
gtg ctc ctg cag gct gtg tct gtg 151 Val Ile Cys Ile Val Leu Leu Gln
Val Leu Leu Gln Ala Val Ser Val 20 25 30 35 gct gtg act tac atg tac
ttc acc aac gag atg aag cag ctg cag gac 199 Ala Val Thr Tyr Met Tyr
Phe Thr Asn Glu Met Lys Gln Leu Gln Asp 40 45 50 aat tac tcc aaa
att gga cta gct tgc ttc tca aag acg gat gag gat 247 Asn Tyr Ser Lys
Ile Gly Leu Ala Cys Phe Ser Lys Thr Asp Glu Asp 55 60 65 ttc tgg
gac tcc act gat gga gag atc ttg aac aga ccc tgc ttg cag 295 Phe Trp
Asp Ser Thr Asp Gly Glu Ile Leu Asn Arg Pro Cys Leu Gln 70 75 80
gtt aag agg caa ctg tat cag ctc att gaa gag gtg act ttg aga acc 343
Val Lys Arg Gln Leu Tyr Gln Leu Ile Glu Glu Val Thr Leu Arg Thr 85
90 95 ttt cag gac acc att tct aca gtt cca gaa aag cag cta agt act
cct 391 Phe Gln Asp Thr Ile Ser Thr Val Pro Glu Lys Gln Leu Ser Thr
Pro 100 105 110 115 ccc ttg ccc aga ggt gga aga cct cag aaa gtg gca
gct cac att act 439 Pro Leu Pro Arg Gly Gly Arg Pro Gln Lys Val Ala
Ala His Ile Thr 120 125 130 ggg atc act cgg aga agc aac tca gct tta
att cca atc tcc aag gat 487 Gly Ile Thr Arg Arg Ser Asn Ser Ala Leu
Ile Pro Ile Ser Lys Asp 135 140 145 gga aag acc tta ggc cag aag att
gaa tcc tgg gag tcc tct cgg aaa 535 Gly Lys Thr Leu Gly Gln Lys Ile
Glu Ser Trp Glu Ser Ser Arg Lys 150 155 160 ggg cat tca ttt ctc aac
cac gtg ctc ttt agg aat gga gag ctg gtc 583 Gly His Ser Phe Leu Asn
His Val Leu Phe Arg Asn Gly Glu Leu Val 165 170 175 atc gag cag gag
ggc ctg tat tac atc tat tcc caa aca tac ttc cga 631 Ile Glu Gln Glu
Gly Leu Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg 180 185 190 195 ttt
cag gaa gct gaa gac gct tcc aag atg gtc tca aag gac aag gtg 679 Phe
Gln Glu Ala Glu Asp Ala Ser Lys Met Val Ser Lys Asp Lys Val 200 205
210 aga acc aaa cag ctg gtg cag tac atc tac aag tac acc agc tat ccg
727 Arg Thr Lys Gln Leu Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro
215 220 225 gat ccc ata gtg ctc atg aag agc gcc aga aac agc tgt tgg
tcc aga 775 Asp Pro Ile Val Leu Met Lys Ser Ala Arg Asn Ser Cys Trp
Ser Arg 230 235 240 gat gcc gag tac gga ctg tac tcc atc tat cag gga
gga ttg ttc gag 823 Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly
Gly Leu Phe Glu 245 250 255 cta aaa aaa aat gac agg att ttt gtt tct
gtg aca aat gaa cat ttg 871 Leu Lys Lys Asn Asp Arg Ile Phe Val Ser
Val Thr Asn Glu His Leu 260 265 270 275 atg gac ctg gat caa gaa gcc
agc ttc ttt gga gcc ttt tta att aac 919 Met Asp Leu Asp Gln Glu Ala
Ser Phe Phe Gly Ala Phe Leu Ile Asn 280 285 290 taaatgacca
gtaaagatca aacacagccc taaagtaccc agtaatcttc taggttgaag 979
gcatgcctgg aaagcgactg aactggttag gatatggcct ggctgtagaa acctcaggac
1039 agatgtgaca gaaaggcagc tggaactcag cagcgacagg ccaacagtcc
agccacagac 1099 actttcggtg tttcatcgag agacttgctt tctttccgca
aaatgagatc actgtagcct 1159 ttcaatgatc tacctggtat cagtttgcag
agatctagaa gacgtccagt ttctaaatat 1219 ttatgcaaca attgacaatt
ttcacctttg ttatctggtc caggggtgta aagccaagtg 1279 ctcacaagct
gtgtgcagac caggatagct atgaatgcag gtcagcataa aaatcacaga 1339
atatctcacc tactaaaaaa aaaaaaa 1366 6 291 PRT murine 6 Met Pro Ser
Ser Gly Ala Leu Lys Asp Leu Ser Phe Ser Gln His Phe 1 5 10 15 Arg
Met Met Val Ile Cys Ile Val Leu Leu Gln Val Leu Leu Gln Ala 20 25
30 Val Ser Val Ala Val Thr Tyr Met Tyr Phe Thr Asn Glu Met Lys Gln
35 40 45 Leu Gln Asp Asn Tyr Ser Lys Ile Gly Leu Ala Cys Phe Ser
Lys Thr 50 55 60 Asp Glu Asp Phe Trp Asp Ser Thr Asp Gly Glu Ile
Leu Asn Arg Pro 65 70 75 80 Cys Leu Gln Val Lys Arg Gln Leu Tyr Gln
Leu Ile Glu Glu Val Thr 85 90 95 Leu Arg Thr Phe Gln Asp Thr Ile
Ser Thr Val Pro Glu Lys Gln Leu 100 105 110 Ser Thr Pro Pro Leu Pro
Arg Gly Gly Arg Pro Gln Lys Val Ala Ala 115 120 125 His Ile Thr Gly
Ile Thr Arg Arg Ser Asn Ser Ala Leu Ile Pro Ile 130 135 140 Ser Lys
Asp Gly Lys Thr Leu Gly Gln Lys Ile Glu Ser Trp Glu Ser 145 150 155
160 Ser Arg Lys Gly His Ser Phe Leu Asn His Val Leu Phe Arg Asn Gly
165 170 175 Glu Leu Val Ile Glu Gln Glu Gly Leu Tyr Tyr Ile Tyr Ser
Gln Thr 180 185 190 Tyr Phe Arg Phe Gln Glu Ala Glu Asp Ala Ser Lys
Met Val Ser Lys 195 200 205 Asp Lys Val Arg Thr Lys Gln Leu Val Gln
Tyr Ile Tyr Lys Tyr Thr 210 215 220 Ser Tyr Pro Asp Pro Ile Val Leu
Met Lys Ser Ala Arg Asn Ser Cys 225 230 235 240 Trp Ser Arg Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly 245 250 255 Leu Phe Glu
Leu Lys Lys Asn Asp Arg Ile Phe Val Ser Val Thr Asn 260 265 270 Glu
His Leu Met Asp Leu Asp Gln Glu Ala Ser Phe Phe Gly Ala Phe 275 280
285 Leu Ile Asn 290 7 8 PRT synthetic 7 Asp Tyr Lys Asp Asp Asp Asp
Lys 1 5 8 17 PRT conserved peptide 8 Leu Val Val Xaa Xaa Xaa Gly
Leu Tyr Tyr Val Tyr Xaa Gln Val Xaa 1 5 10 15 Phe 9 32 PRT CMV
leader 9 Met Ala Arg Arg Leu Trp Ile Leu Ser Leu Leu Ala Val Thr
Leu Thr 1 5 10 15 Val Ala Leu Ala Ala Pro Ser Gln Lys Ser Lys Arg
Arg Thr Ser Ser 20 25 30 10 759 DNA synthetic fusion CDS (1)..(759)
10 atg gct aca ggc tcc cgg acg tcc ctg ctc ctg gct ttt ggc ctg ctc
48 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu
1 5 10 15 tgc ctg ccc tgg ctt caa gag ggc agt gca act agt tct gac
cgt atg 96 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Thr Ser Ser Asp
Arg Met 20 25 30 aaa cag ata gag gat aag atc gaa gag atc cta agt
aag att tat cat 144 Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser
Lys Ile Tyr His 35 40 45 ata gag aat gaa atc gcc cgt atc aaa aag
ctg att ggc gag cgg act 192 Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys
Leu Ile Gly Glu Arg Thr 50 55 60 aga tct acc tct gag gaa acc att
tct aca gtt caa gaa aag caa caa 240 Arg Ser Thr Ser Glu Glu Thr Ile
Ser Thr Val Gln Glu Lys Gln Gln 65 70 75 80 aat att tct ccc cta
gtg aga gaa aga ggt cct cag aga gta gca gct 288 Asn Ile Ser Pro Leu
Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala 85 90 95 cac ata act
ggg acc aga gga aga agc aac aca ttg tct tct cca aac 336 His Ile Thr
Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn 100 105 110 tcc
aag aat gaa aag gct ctg ggc cgc aaa ata aac tcc tgg gaa tca 384 Ser
Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser 115 120
125 tca agg agt ggg cat tca ttc ctg agc aac ttg cac ttg agg aat ggt
432 Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly
130 135 140 gaa ctg gtc atc cat gaa aaa ggg ttt tac tac atc tat tcc
caa aca 480 Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser
Gln Thr 145 150 155 160 tac ttt cga ttt cag gag gaa ata aaa gaa aac
aca aag aac gac aaa 528 Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn
Thr Lys Asn Asp Lys 165 170 175 caa atg gtc caa tat att tac aaa tac
aca agt tat cct gac cct ata 576 Gln Met Val Gln Tyr Ile Tyr Lys Tyr
Thr Ser Tyr Pro Asp Pro Ile 180 185 190 ttg ttg atg aaa agt gct aga
aat agt tgt tgg tct aaa gat gca gaa 624 Leu Leu Met Lys Ser Ala Arg
Asn Ser Cys Trp Ser Lys Asp Ala Glu 195 200 205 tat gga ctc tat tcc
atc tat caa ggg gga ata ttt gag ctt aag gaa 672 Tyr Gly Leu Tyr Ser
Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu 210 215 220 aat gac aga
att ttt gtt tct gta aca aat gag cac ttg ata gac atg 720 Asn Asp Arg
Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met 225 230 235 240
gac cat gaa gcc agt ttt ttc ggg gcc ttt tta gtt ggc 759 Asp His Glu
Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 245 250 11 253 PRT
synthetic fusion 11 Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala
Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala
Thr Ser Ser Asp Arg Met 20 25 30 Lys Gln Ile Glu Asp Lys Ile Glu
Glu Ile Leu Ser Lys Ile Tyr His 35 40 45 Ile Glu Asn Glu Ile Ala
Arg Ile Lys Lys Leu Ile Gly Glu Arg Thr 50 55 60 Arg Ser Thr Ser
Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln 65 70 75 80 Asn Ile
Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala 85 90 95
His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn 100
105 110 Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu
Ser 115 120 125 Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu
Arg Asn Gly 130 135 140 Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr
Ile Tyr Ser Gln Thr 145 150 155 160 Tyr Phe Arg Phe Gln Glu Glu Ile
Lys Glu Asn Thr Lys Asn Asp Lys 165 170 175 Gln Met Val Gln Tyr Ile
Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile 180 185 190 Leu Leu Met Lys
Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu 195 200 205 Tyr Gly
Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu 210 215 220
Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met 225
230 235 240 Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 245
250 12 768 DNA synthetic fusion CDS (1)..(768) 12 atg gct cgg agg
cta tgg atc ttg agc tta tta gcc gtg acc ttg acg 48 Met Ala Arg Arg
Leu Trp Ile Leu Ser Leu Leu Ala Val Thr Leu Thr 1 5 10 15 gtg gct
ttg gcg gca cct tct cag aaa tcg aag cgc agg act agt tct 96 Val Ala
Leu Ala Ala Pro Ser Gln Lys Ser Lys Arg Arg Thr Ser Ser 20 25 30
gac cgt atg aaa cag ata gag gat aag atc gaa gag atc cta agt aag 144
Asp Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 35
40 45 att tat cat ata gag aat gaa atc gcc cgt atc aaa aag ctg att
ggc 192 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile
Gly 50 55 60 gag cgg act aga tct acc tct gag gaa acc att tct aca
gtt caa gaa 240 Glu Arg Thr Arg Ser Thr Ser Glu Glu Thr Ile Ser Thr
Val Gln Glu 65 70 75 80 aag caa caa aat att tct ccc cta gtg aga gaa
aga ggt cct cag aga 288 Lys Gln Gln Asn Ile Ser Pro Leu Val Arg Glu
Arg Gly Pro Gln Arg 85 90 95 gta gca gct cac ata act ggg acc aga
gga aga agc aac aca ttg tct 336 Val Ala Ala His Ile Thr Gly Thr Arg
Gly Arg Ser Asn Thr Leu Ser 100 105 110 tct cca aac tcc aag aat gaa
aag gct ctg ggc cgc aaa ata aac tcc 384 Ser Pro Asn Ser Lys Asn Glu
Lys Ala Leu Gly Arg Lys Ile Asn Ser 115 120 125 tgg gaa tca tca agg
agt ggg cat tca ttc ctg agc aac ttg cac ttg 432 Trp Glu Ser Ser Arg
Ser Gly His Ser Phe Leu Ser Asn Leu His Leu 130 135 140 agg aat ggt
gaa ctg gtc atc cat gaa aaa ggg ttt tac tac atc tat 480 Arg Asn Gly
Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr 145 150 155 160
tcc caa aca tac ttt cga ttt cag gag gaa ata aaa gaa aac aca aag 528
Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys 165
170 175 aac gac aaa caa atg gtc caa tat att tac aaa tac aca agt tat
cct 576 Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr
Pro 180 185 190 gac cct ata ttg ttg atg aaa agt gct aga aat agt tgt
tgg tct aaa 624 Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys
Trp Ser Lys 195 200 205 gat gca gaa tat gga ctc tat tcc atc tat caa
ggg gga ata ttt gag 672 Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln
Gly Gly Ile Phe Glu 210 215 220 ctt aag gaa aat gac aga att ttt gtt
tct gta aca aat gag cac ttg 720 Leu Lys Glu Asn Asp Arg Ile Phe Val
Ser Val Thr Asn Glu His Leu 225 230 235 240 ata gac atg gac cat gaa
gcc agt ttt ttc ggg gcc ttt tta gtt ggc 768 Ile Asp Met Asp His Glu
Ala Ser Phe Phe Gly Ala Phe Leu Val Gly 245 250 255 13 256 PRT
synthetic fusion 13 Met Ala Arg Arg Leu Trp Ile Leu Ser Leu Leu Ala
Val Thr Leu Thr 1 5 10 15 Val Ala Leu Ala Ala Pro Ser Gln Lys Ser
Lys Arg Arg Thr Ser Ser 20 25 30 Asp Arg Met Lys Gln Ile Glu Asp
Lys Ile Glu Glu Ile Leu Ser Lys 35 40 45 Ile Tyr His Ile Glu Asn
Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 50 55 60 Glu Arg Thr Arg
Ser Thr Ser Glu Glu Thr Ile Ser Thr Val Gln Glu 65 70 75 80 Lys Gln
Gln Asn Ile Ser Pro Leu Val Arg Glu Arg Gly Pro Gln Arg 85 90 95
Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser 100
105 110 Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn
Ser 115 120 125 Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn
Leu His Leu 130 135 140 Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly
Phe Tyr Tyr Ile Tyr 145 150 155 160 Ser Gln Thr Tyr Phe Arg Phe Gln
Glu Glu Ile Lys Glu Asn Thr Lys 165 170 175 Asn Asp Lys Gln Met Val
Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro 180 185 190 Asp Pro Ile Leu
Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys 195 200 205 Asp Ala
Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu 210 215 220
Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu 225
230 235 240 Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu
Val Gly 245 250 255 14 27 PRT LZ peptide 14 Pro Asp Val Ala Ser Leu
Arg Gln Gln Val Glu Ala Leu Gln Gly Gln 1 5 10 15 Val Gln His Leu
Gln Ala Ala Phe Ser Gln Tyr 20 25 15 34 PRT LZ peptide 15 Asp Arg
Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 1 5 10 15
Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 20
25 30 Glu Arg 16 28 PRT LZ peptide 16 Ser Leu Ala Ser Leu Arg Gln
Gln Leu Glu Ala Leu Gln Gly Gln Leu 1 5 10 15 Gln His Leu Gln Ala
Ala Leu Ser Gln Leu Gly Glu 20 25 17 28 PRT LZ peptide 17 Ser Ile
Ala Ser Ile Arg Gln Gln Ile Glu Ala Ile Gln Gly Gln Ile 1 5 10 15
Gln His Ile Gln Ala Ala Ile Ser Gln Ile Gly Glu 20 25 18 77 DNA GH
Leader 18 atggctacag gctcccggac gtccctgtcc tggcttttgg cctgctctgc
ctgccctggc 60 ttcaagaggg cagtgca 77 19 26 PRT GH Leader 19 Met Ala
Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15
Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala 20 25 20 9 PRT Artificial
Sequence Description of Artificial Sequence artificial peptide 20
Cys Asp Cys Arg Gly Asp Cys Phe Cys 1 5 21 13 PRT Artificial
Sequence Description of Artificial Sequence artificial peptide 21
Cys Asn Gly Arg Cys Val Ser Gly Cys Ala Gly Arg Cys 1 5 10 22 6 PRT
Artificial Sequence Description of Artificial Sequence artificial
peptide 22 Asn Gly Arg Ala His Ala 1 5 23 9 PRT Artificial Sequence
Description of Artificial Sequence artificial peptide 23 Cys Val
Leu Asn Gly Arg Met Glu Cys 1 5 24 5 PRT Artificial Sequence
Description of Artificial Sequence artificial peptide 24 Cys Asn
Gly Arg Cys 1 5 25 20 PRT Homo sapiens 25 Met Gly Thr Asp Thr Leu
Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly
20
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