U.S. patent application number 12/343429 was filed with the patent office on 2010-01-21 for receptor that binds trail.
Invention is credited to Charles Rauch, Henning Walczak.
Application Number | 20100015137 12/343429 |
Document ID | / |
Family ID | 40584893 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100015137 |
Kind Code |
A1 |
Rauch; Charles ; et
al. |
January 21, 2010 |
RECEPTOR THAT BINDS TRAIL
Abstract
A protein designated TRAIL receptor binds the protein known as
TNF-Related Apoptosis-Inducing Ligand (TRAIL). The TRAIL receptor
finds use in purifying TRAIL or inhibiting activities thereof.
Isolated DNA sequences encoding TRAIL-R polypeptides are provided,
along with expression vectors containing the DNA sequences, and
host cells transformed with such recombinant expression vectors.
Antibodies that are immunoreactive with TRAIL-R are also
provided.
Inventors: |
Rauch; Charles; (Bainbridge
Island, WA) ; Walczak; Henning; (Heidelberg,
DE) |
Correspondence
Address: |
IMMUNEX CORPORATION;LAW DEPARTMENT
1201 AMGEN COURT WEST
SEATTLE
WA
98119
US
|
Family ID: |
40584893 |
Appl. No.: |
12/343429 |
Filed: |
December 23, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09378045 |
Aug 20, 1999 |
7528239 |
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12343429 |
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08883036 |
Jun 26, 1997 |
6072047 |
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09378045 |
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08869852 |
Jun 4, 1997 |
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08883036 |
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08829536 |
Mar 28, 1997 |
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08869852 |
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08815255 |
Mar 12, 1997 |
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08829536 |
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08799861 |
Feb 13, 1997 |
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08815255 |
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Current U.S.
Class: |
424/133.1 ;
424/139.1; 435/375 |
Current CPC
Class: |
C07K 1/22 20130101; A61K
38/00 20130101; C07K 14/715 20130101; C07K 14/70578 20130101 |
Class at
Publication: |
424/133.1 ;
435/375; 424/139.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of inducing apoptosis in mammalian cancel cells
comprising contacting said mammalian cancer cells with an apoptosis
inducing amount of an isolated TRAIL-R agonist monoclonal antibody
which: (a) specifically binds to a TRAIL-R protein, wherein TRAIL-R
is characterized by expression on cell membranes of Jurkat cells,
binds TRAIL, comprises an amino acid sequence VPANEGD (amino acids
327-333 of SEQ ID NO: 2), and has a molecular weight of about 50 to
55 kilodaltons as determined by SDS-polyacrylamide gel
electrophoresis; and, (b) induces apoptosis in said mammalian
cancer cells.
2. The method of claim 1, wherein said TRAIL-R protein consists of
the contiguous amino acid residues 1 to 440 of SEQ ID NO: 2.
3. The method of claim 1, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R protein, said extracellular domain sequence consisting of
the contiguous amino acid residues 52 to 210 of SEQ ID NO: 2.
4. The method of claim 3, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
5. The method of claim 3, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
6. The method of claim l, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R protein, said extracellular domain sequence consisting of
the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2.
7. The method of claim 6, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
8. The method of claim 6, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
9. The method of claim 1, wherein said monoclonal antibody
specifically binds to said TRAIL-R protein having an N-terminal
amino acid residue at either position 52 or 54 of SEQ ID NO: 2.
10. The method of claim 9, wherein said TRAIL-R protein is a
soluble TRAIL-R protein.
11. The method of claim 10, wherein said soluble TRAIL-R protein
lacks a transmembrane region and a cytoplasmic domain.
12. The method of claim 11, wherein said soluble TRAIL-R protein
has an N-terminal amino acid residue at position 54 of SEQ ID NO:
2.
13. The method of claim 1, wherein said TRAIL-R protein is a
soluble TRAIL-R protein and lacks a transmembrane region and a
cytoplasmic domain.
14. A method of inducing apoptosis in mammalian cancer cells
comprising contacting said mammalian cancer cells with an apoptosis
inducing amount of an isolated TRAIL-R agonist monoclonal antibody
which: (a) specifically binds to TRAIL-R, wherein an isolated and
purified human TRAIL-R receptor protein is a product made by the
process comprising the steps of: (i) isolating plasma membranes
from Jurkat cells; (ii) solubilizing and homogenizing said isolated
plasma membranes of step (i); (iii) centrifuging said solubilized
and homogenized isolated plasma membranes of step (ii) to yield a
plasma membrane extract and a pellet; (iv) applying said plasma
membrane extract of step (iii) to an anti-octapeptide monoclonal
antibody affinity chromatography column, whereby said column of
step (iv) adsorbs non-specifically bound material and wherein said
octapeptide has the sequence presented in SEQ ID NO: 5; (v)
applying column flow-through from step (iv) to an octapeptide-TRAIL
ligand affinity chromatography column, whereby said column of step
(v) specifically binds said TRAIL-R receptor protein and wherein
said octapeptide-TRAIL ligand is a fusion protein of said
octapeptide having the sequence presented in SEQ ID NO:5 and TRAIL
ligand; (vi) eluting fractions with TRAIL ligand binding activity
from said column of step (v); and, (vii) purifying said fractions
of step (vi) by reverse-phase HPLC to yield said isolated and
purified TRAIL-R receptor protein, wherein said isolated and
purified TRAIL-R receptor protein has a molecular weight of about
50 to 55 kilodaltons as determined by SDS polyacrylamide gel
electrophoresis, and comprises the amino acid sequence VPANEGD
(amino acids 327-333 of SEQ ID NO: 2); and, (b) induces apoptosis
in said mammalian cancer cells.
15. The method of claim 1 4, wherein said TRAIL-R consists of the
contiguous amino acid residues 1 to 440 of SEQ ID NO: 2.
16. The method of claim 14, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R, said extracellular domain sequence consisting of the
contiguous amino acid residues 52 to 210 of SEQ ID NO: 2.
17. The method of claim 16, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
18. The method of claim 16, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
19. The method of claim 14, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R, said extracellular domain sequence consisting of the
contiguous amino acid residues 54 to 210 of SEQ ID NO: 2.
20. The method of claim 19, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
21. The method of claim 19, wherein said extracellular domain
sequence is a membrane bound extracellular- domain sequence.
22. The method of claim 14, wherein said monoclonal antibody
specifically binds to said TRAIL-R having an N-terminal amino acid
residue at either position 52 or 54 of SEQ ID NO: 2.
23. The method of claim 22, wherein said I-RAIL-R is a soluble
TRAIL-R.
24. The method of claim 23, wherein said soluble TRAIL-R lacks a
transmembrane region and a cytoplasmic domain.
25. The method of claim 24, wherein said soluble TRAIL-R has an
N-terminal amino acid residue at position 54 of SEQ ID NO: 2.
26. The method of claim 14, wherein said TRAIL-R is a soluble
TRAIL-R and lacks a transmembrane region and a cytoplasmic
domain.
27. A method of inducing apoptosis in mammalian cancer cells
comprising contacting said mammalian cancer cells with an apoptosis
inducing amount of an isolated TRAIL-R agonist monoclonal antibody
which: (a) specifically binds to a TRAIL-R protein of SEQ ID NO:2:
and (b) induces apoptosis in said mammalian cancer cells.
28. The method of claim 27, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R protein, said extracellular domain sequence consisting of
the contiguous amino acid residues 52 to 210 of SEQ ID NO: 2.
29. The method of claim 28, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
30. The method of claim 28, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
31. The method of claim 27, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R protein, said extracellular domain sequence consisting of
the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2.
32. The method of claim 31, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
33. The method of claim 31, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
34. The method of claim 27, wherein said monoclonal antibody
specifically binds to said TRAIL-R protein having an N-terminal
amino acid residue at either position 52 or 54 of SEQ ID NO: 2.
35. The method of claim 34, wherein said TRAIL-R protein is a
soluble TRAIL-R receptor protein.
36. The method of claim 35, wherein said soluble TRAIL-R protein
lacks a transmembrane region and a cytoplasmic domain.
37. The method of claim 36, wherein said soluble TRAIL-R protein
has an N-terminal amino acid residue at position 54 of SEQ ID NO:
2.
38. The method of claim 27, wherein said TRAIL-R protein is a
soluble TRAIL-R receptor protein and lacks a transmembrane region
and a cytoplasmic domain.
39. A method of treating cancer comprising contacting mammalian
cancer cells with an apoptosis inducing amount of an isolated
TRAIL-R agonist monoclonal antibody which: (a) specifically binds
to a TRAIL-R protein, wherein TRAIL-R is characterized by
expression on cell membranes of Jurkat cells, binds TRAIL,
comprises an amino acid sequence VPANEGD (amino acids 327-333 of
SEQ ID NO: 2), and has a molecular weight of about 50 to 55
kilodaltons as determined by SDS-polyacrylamide gel
electrophoresis; and, (b) induces apoptosis in said mammalian
cancer cells.
40. The method of claim 39, wherein said TRAIL-R protein consists
of the contiguous amino acid residues 1 to 440 of SEQ ID NO: 2.
41. The method of claim 39, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R protein, said extracellular domain sequence consisting of
the contiguous amino acid residues 52 to 210 of SEQ ID NO: 2.
42. The method of claim 41, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
43. The method of claim 41, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
44. The method of claim 39, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R protein, said extracellular domain sequence consisting of
the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2.
45. The method of claim 44, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
46. The method of claim 44, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
47. The method of claim 39, wherein said monoclonal antibody
specifically binds to said TRAIL-R protein having an N-terminal
amino acid residue at either position 52 or 54 of SEQ ID NO: 2.
48. The method of claim 47, wherein said TRAIL-R protein is a
soluble TRAIL-R receptor protein.
49. The method of claim 48, wherein said soluble TRAIL-R protein
lacks a transmembrane region and a cytoplasmic domain.
50. The method of claim 49, wherein said soluble TRAIL-R protein
has an N-terminal amino acid residue at position 54 of SEQ ID NO:
2.
51. The method of claim 39, wherein said TRAIL-R protein is a
soluble TRAIL-R protein and lacks a transmembrane region and a
cytoplasmic domain.
52. A method of treating cancer comprising contacting mammalian
cancer cells with an apoptosis inducing amount of an isolated
TRAIL-R agonist monoclonal antibody which: (a) specifically binds
to TRAIL-R, wherein an isolated and purified human TRAIL-R receptor
protein is a product made by the process comprising the steps of:
(i) isolating plasma membranes from Jurkat cells; (ii) solubilizing
and homogenizing said isolated plasma membranes of step (i); (iii)
centrifuging said solubilized and homogenized isolated plasma
membranes of step (ii) to yield a plasma membrane extract and a
pellet; (iv) applying said plasma membrane extract of step (iii) to
an anti-octapeptide monoclonal antibody affinity chromatography
column, whereby said column of step (iv) adsorbs non-specifically
bound material and wherein said octapeptide has the sequence
presented in SEQ ID NO: 5; (v) applying column flow-through from
step (iv) to an octapeptide-TRAIL ligand affinity chromatography
column, whereby said column of step (v) specifically binds said
TRAIL-R receptor protein and wherein said octapeptide -TRAIL ligand
is a fusion protein of said octapeptide having the sequence
presented in SEQ ID NO:5 and TRAIL ligand; (vi) eluting fractions
with TRAIL ligand binding activity from said column of step (v);
and, (vii) purifying said fractions of step (vi) by reverse-phase
HPLC to yield said isolated and purified TRAIL-R receptor protein,
wherein said isolated and purified TRAIL-R receptor protein has a
molecular weight of about 50 to 55 kilodaltons as determined by SDS
polyacrylamide gel electrophoresis, and comprises the amino acid
sequence VPANEGD (amino acids 327-333 of SEQ ID NO: 2); and, (b)
induces apoptosis in said mammalian cancer cells.
53. The method of claim 52, wherein said TRAIL-R consists of the
contiguous amino acid residues 1 to 440 of SEQ ID NO: 2.
54. The method of claim 52, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R, said extracellular domain sequence consisting of the
contiguous amino acid residues 52 to 21 0 of SEQ ID NO: 2.
55. The method of claim 54, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
56. The method of claim 54, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
57. The method of claim 52, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R, said extracellular domain sequence consisting of the
contiguous amino acid residues 54 to 210 of SEQ ID NO: 2.
58. The method of claim 57, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
59. The method of claim 57, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
60. The method of claim 52, wherein said monoclonal antibody
specifically binds to said TRAIL-R having an N-terminal amino acid
residue at either position 52 or 54 of SEQ ID NO: 2.
61. The method of claim 60, wherein said TRAIL-R is a soluble
TRAIL-R.
62. The method of claim 61, wherein said soluble TRAIL-R lacks a
transmembrane region and a cytoplasmic domain.
63. The method of claim 62, wherein said soluble TRAIL-R has an
N-terminal amino acid residue at position 54 of SEQ ID NO: 2.
64. The method of claim 52, wherein said TRAIL-R is a soluble
TRAIL-R and lacks a transmembrane region and a cytoplasmic
domain.
65. A method of treating cancer comprising contacting mammalian
cancer cells with an apoptosis inducing amount of an isolated
TRAIL-R agonist monoclonal antibody which: (a) specifically binds
to a TRAIL-R protein of SEQ ID NO:2; and, (b) induces apoptosis in
said mammalian cancer cells.
66. The method of claim 65, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R protein, said extracellular domain sequence consisting of
the contiguous amino acid residues 52 to 210 of SEQ ID NO: 2.
67. The method of claim 66, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
68. The method of claim 66, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
69. The method of claim 65, wherein said monoclonal antibody
specifically binds to an extracellular domain sequence of said
TRAIL-R protein, said extracellular domain sequence consisting of
the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2.
70. The method of claim 69, wherein said extracellular domain
sequence is a soluble extracellular domain sequence.
71. The method of claim 69, wherein said extracellular domain
sequence is a membrane bound extracellular domain sequence.
72. The method of claim 65, wherein said monoclonal antibody
specifically binds to said TRAIL-R protein having an N-terminal
amino acid residue at either position 52 or 54 of SEQ ID NO: 2.
73. The method of claim 72, wherein said TRAIL-R protein is a
soluble TRAIL-R protein.
74. The method of claim 73, wherein said soluble TRAIL-R protein
lacks a transmembrane region and a cytoplasmic domain.
75. The method of claim 74, wherein said soluble TRAIL-R protein
has an N-terminal amino acid residue at position 54 of SEQ ID NO:
2.
76. The method of claim 65, wherein said TRAIL-R protein is a
soluble TRAIL-R protein and lacks a transmembrane region and a
cytoplasmic domain.
77. A method as in one of claims 1-76, wherein said cancer cells
are further contacted with radiation or a chemotherapeutic
agent.
78. A method as in one of claims 1-76, wherein said cancer cells
are lung, breast, ovary, prostate, kidney, liver, bladder,
pancreas, or colon cancer cells.
79. A method as in one of claims 1-76, wherein said cancer cells
are leukemia, lymphoma, or melanoma cancel- cells.
80. A method as in one of claims 1-76, wherein said monoclonal
antibody is a humanized or chimeric monoclonal antibody.
81. A method as in one of claims 1-76, wherein said monoclonal
antibody comprises an Fab or F(ab').sub.2 fragment.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/378,045 filed on Aug. 20, 1999, currently pending, which is a
continuation-in-part of application Ser. No. 08/883,036, filed Jun.
26, 1997, currently pending, which is a continuation-in-part of
application Ser. No. 08/869,852, filed Jun. 4, 1997, now abandoned,
which is a continuation-in-part of application Ser. No. 08/829,536,
filed Mar. 28, 1997, now abandoned, which is a continuation-in-part
of application Ser. No. 08/815,255, filed Mar. 12, 1997, now
abandoned, which is a continuation-in-part of application Ser. No.
08/799,861, filed Feb. 13, 1997, now abandoned.
BACKGROUND OF THE INVENTION
[0002] A protein known as TNF-related apoptosis-inducing ligand
(TRAIL) is a member of the tumor necrosis factor family of ligands
(Wiley et al., Immunity 3:673-682, 1995). TRAIL has demonstrated
the ability to induce apoptosis of certain transformed cells,
including a number of different types of cancer cells as well as
virally infected cells (PCT application WO 97/01633 and Wiley et
al., supra).
[0003] Identification of receptor protein(s) that bind TRAIL would
prove useful in further study of the biological activities of
TRAIL. However, prior to the present invention, no receptor for
TRAIL had been reported.
SUMMARY OF THE INVENTION
[0004] The present invention is directed to a novel protein
designated TRAIL receptor (TRAIL-R), which binds to a protein known
as TNF-related apoptosis-inducing ligand (TRAIL). DNA encoding
TRAIL-R, and expression vectors comprising such DNA, are provided.
A method for producing TRAIL-R polypeptides comprises culturing
host cells transformed with a recombinant expression vector
encoding TRAIL-R, under conditions that promote expression of
TRAIL-R, then recovering the expressed TRAIL-R polypeptides from
the culture. Antibodies that are immunoreactive with TRAIL-R are
also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1 presents the nucleotide sequence of a human TRAIL
receptor DNA fragment (SEQ ID NO:3), as well as the amino acid
sequence encoded thereby (SEQ ID NO:4). This DNA fragment is
described in Example 3.
[0006] FIG. 2 presents the results of the assay described in
example 7. In the assay, a soluble TRAIL-R/Fc fusion protein
blocked TRAIL-induced apoptosis of Jurkat cells.
[0007] FIG. 3 presents the results of the experiment described in
example 8. The indicated compounds were demonstrated to inhibit
apoptosis of cells expressing TRAIL receptor.
[0008] FIGS. 4A to 4C depict targeted insertion of a neo cassette
into the Sma I site of the .mu.1 exon. The construct was employed
in generating transgenic mice, as described in example 10. FIG. 4A
is a schematic diagram of the genomic structure of the .mu. locus.
The filled boxes represent the .mu. exons. FIG. 4B is a schematic
diagram of the CmD targeting vector. The dotted lines denote those
genomic .mu. sequences included in the construct. Plasmid sequences
are not shown. FIG. 4C is a schematic diagram of the targeted .mu.
locus in which the neo cassette has been inserted into .mu.1.
[0009] FIGS. 5A and 5B present the nucleotide sequence (SEQ ID
NO:6) of a vector designated pGP1k, as described in Example 11
below.
DETAILED DESCRIPTION OF THE INVENTION
[0010] A novel protein designated TRAIL receptor (TRAIL-R) is
provided herein. TRAIL-R binds to the cytokine designated
TNF-related apoptosis-inducing ligand (TRAIL). Certain uses of
TRAIL-R flow from this ability to bind TRAIL, as discussed further
below. TRAIL-R finds use in inhibiting biological activities of
TRAIL, or in purifying TRAIL by affinity chromatography, for
example.
[0011] The nucleotide sequence of the coding region of a human
TRAIL receptor DNA is presented in SEQ ID NO:1. The amino acid
sequence encoded by the DNA sequence of SEQ ID NO:1 is shown in SEQ
ID NO:2. This sequence information identifies the TRAIL receptor
protein as a member of the tumor necrosis factor receptor (TNF-R)
family of receptors (reviewed in Smith et al., Cell 76:959-962,
1994) The extracellular domain contains cysteine rich repeats; such
motifs have been reported to be important for ligand binding in
other receptors of this family. TRAIL-R contains a so-called "death
domain" in the cytoplasmic region; such domains in certain other
receptors are associated with transduction of apoptotic signals.
These and other features of the protein are discussed in more
detail below.
[0012] TRAIL-R protein or immunogenic fragments thereof may be
employed as immunogens to generate antibodies that are
immunoreactive therewith. In one embodiment of the invention, the
antibodies are monoclonal antibodies.
[0013] A human TRAIL-R protein was purified as described in example
1. In example 2, amino acid sequence information derived from
fragments of TRAIL-R is presented. One embodiment of the invention
is directed to a purified human TRAIL-R protein that is capable of
binding TRAIL, wherein the TRAIL-R is characterized as comprising
the amino acid sequence VPANEGD (amino acids 327 to 333 of SEQ ID
NO:2). In another embodiment, the TRAIL-R additionally comprises
the sequence ETLRQCFDDFADLVPFDSWEPLMRKLGLMDNEIKVAKAEAAGHRDTLXTML
(amino acids 336 to 386 of SEQ ID NO:2, with one unknown amino acid
indicated as X). Also provided are TRAIL-R fragments comprising
only one of these characterizing amino acid sequences.
[0014] The nucleotide sequence of a TRAIL-R DNA fragment, and the
amino acid sequence encoded thereby, are presented in FIG. 1 (SEQ
ID NO:3 and SEQ ID NO:4); see example 3. The amino acid sequence
presented in FIG. 1 has characteristics of the so-called "death
domains" found in the cytoplasmic region of certain other receptor
proteins. Such domains have been reported to be associated with
transduction of apoptotic signals. Cytoplasmic death domains have
been identified in Fas antigen (Itoh and Nagata, J. Biol. Chem.
268:10932, 1993), TNF-receptor type I (Tartaglia et al. Cell
74:845, 1993), DR3 (Chinnaiyan et al., Science 274:990-992, 1996),
and CAR-1 (Brojatsch et al., Cell 87:845-855, 1996). The role of
these death domains in initiating intracellular apoptotic signaling
cascades is discussed further below.
[0015] SEQ ID NO:1 presents the nucleotide sequence of the coding
region of a human TRAIL receptor DNA, including an initiation codon
(ATG) and a termination codon (TAA). The amino acid sequence
encoded by the DNA of SEQ ID NO:1 is presented in SEQ ID NO:2. The
fragment depicted in FIG. 1 corresponds to the region of TRAIL-R
that is presented as amino acids 336 to 386 in SEQ ID NO:2.
[0016] The TRAIL-R protein of SEQ ID NO:2 includes an N-terminal
hydrophobic region that functions as a signal peptide, followed by
an extracellular domain, a transmembrane region comprising amino
acids 211 through 231, and a C-terminal cytoplasmic domain.
Computer analysis predicts that the signal peptide corresponds to
residues 1 to 51 of SEQ ID NO:2. Cleavage of the signal peptide
thus would yield a mature protein comprising amino acids 52 through
440 of SEQ ID NO:2. The calculated molecular weight for a mature
protein containing residues 52 to 440 of SEQ ID NO:2 is about 43
kilodaltons. The next most likely computer-predicted signal
peptidase cleavage sites (in descending order) occur after amino
acids 50 and 58 of SEQ ID NO:2.
[0017] In another embodiment of the invention, the N-terminal
residue of a mature TRAIL-R protein is the isoleucine residue at
position 56 of SEQ ID NO:2. Sequences of several tryptic digest
peptide fragments of TRAIL-R were determined by a combination of
N-terminal sequencing and Nano-ES MS/MS (nano electrospray tandem
mass spectrometry). The N-terminal amino acid of one of the peptide
fragments was the isoleucine at position 56 of SEQ ID NO:2. Since
this fragment was not preceded by a trypsin cleavage site, the
(Ile)56 residue may correspond to the N-terminal residue resulting
from cleavage of the signal peptide.
[0018] A further embodiment of the invention is directed to mature
TRAIL-R having amino acid 54 as the N-terminal residue. In one
preparation of TRAIL-R (a soluble TRAIL-R/Fc fusion protein
expressed in CV1-EBNA cells), the signal peptide was cleaved after
residue 53 of SEQ ID NO:2.
[0019] The skilled artisan will recognize that the molecular weight
of particular preparations of TRAIL-R protein may differ, according
to such factors as the degree of glycosylation. The glycosylation
pattern of a particular preparation of TRAIL-R may vary according
to the type of cells in which the protein is expressed, for
example. Further, a given preparation may include multiple
differentially glycosylated species of the protein. TRAIL-R
polypeptides with or without associated native-pattern
glycosylation are provided herein. Expression of TRAIL-R
polypeptides in bacterial expression systems, such as E. coli,
provides non-glycosylated molecules.
[0020] In one embodiment, the protein is characterized by a
molecular weight within the range of about 50 to 55 kilodaltons,
which is the molecular weight determined for a preparation of
native, full length, human TRAIL-R. Molecular weight can be
determined by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
[0021] Example 1 presents one method for purifying a TRAIL-R
protein. Jurkat cells are disrupted, and the subsequent
purification process includes affinity chromatography (employing a
chromatography matrix containing TRAIL), and reversed phase
HPLC.
[0022] TRAIL-R polypeptides of the present invention may be
purified by any suitable alternative procedure, using known protein
purification techniques. In one alternative procedure, the
chromatography matrix instead comprises an antibody that binds
TRAIL-R. Other cell types expressing TRAIL-R (e.g., the PS-1 cells
described in example 2) can be substituted for the Jurkat cells.
The cells can be disrupted by any of the numerous known techniques,
including freeze-thaw cycling, sonication, mechanical disruption,
or by use of cell lysing agents.
[0023] The desired degree of purity depends 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-R polypeptides are purified such that no protein bands
corresponding to other (non-TRAIL-R) proteins are detectable upon
analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It
will be recognized by one skilled in the pertinent field that
multiple bands corresponding to TRAIL-R protein may be visualized
by SDS-PAGE, due to differential glycosylation, differential
post-translational processing, and the like. TRAIL-R 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.
[0024] The present invention encompasses TRAIL-R in various forms,
including those that are naturally occurring or produced through
various techniques such as procedures involving recombinant DNA
technology. Such forms of TRAIL-R include, but are not limited to,
fragments, derivatives, variants, and oligomers of TRAIL-R, as well
as fusion proteins containing TRAIL-R or fragments thereof.
[0025] TRAIL-R may be modified to create derivatives thereof 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-R may be prepared by
linking the chemical moieties to functional groups on TRAIL-R amino
acid side chains or at the N-terminus or C-terminus of a TRAIL-R
polypeptide. Conjugates comprising diagnostic (detectable) or
therapeutic agents attached to TRAIL-R are contemplated herein, as
discussed in more detail below.
[0026] Other derivatives of TRAIL-R within the scope of this
invention include covalent or aggregative conjugates of TRAIL-R
polypeptides with other proteins or polypeptides, such as by
synthesis in recombinant culture as N-terminal or C-terminal
fusions. Examples of fusion proteins are discussed below in
connection with TRAIL-R oligomers. Further, TRAIL-R-containing
fusion proteins can comprise peptides added to facilitate
purification and identification of TRAIL-R. 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-Lys, which is highly antigenic
and provides an epitope reversibly bound by a specific monoclonal
antibody, enabling rapid assay and facile purification of expressed
recombinant protein. A murine hybridoma designated 4E11 produces a
monoclonal antibody that binds the Flag.RTM. peptide in the
presence of certain divalent metal cations, as described in U.S.
Pat. No. 5,011,912, hereby incorporated by reference. The 4E11
hybridoma cell line has been deposited with the American Type
Culture Collection under accession no. HB 9259. Monoclonal
antibodies that bind the Flag.RTM. peptide are available from
Eastman Kodak Co., Scientific Imaging Systems Division, New Haven,
Conn.
[0027] Both cell membrane-bound and soluble (secreted) forms of
TRAIL-R are provided herein. Soluble TRAIL-R may be identified (and
distinguished from non-soluble membrane-bound counterparts) by
separating intact cells expressing a TRAIL-R polypeptide from the
culture medium, e.g., by centrifugation, and assaying the medium
(supernatant) for the presence of the desired protein. The presence
of TRAIL-R in the medium indicates that the protein was secreted
from the cells and thus is a soluble form of the desired
protein.
[0028] Soluble forms of receptor proteins typically lack the
transmembrane region that would cause retention of the protein on
the cell surface. In one embodiment of the invention, a soluble
TRAIL-R polypeptide comprises the extracellular domain of the
protein. A soluble TRAIL-R polypeptide may include the cytoplasmic
domain, or a portion thereof, as long as the polypeptide is
secreted from the cell in which it is produced. One example of a
soluble TRAIL-R is a soluble human TRAIL-R comprising amino acids
52 to 210 of SEQ ID NO:2. Other soluble TRAIL-R polypeptides
include, but are not limited to, polypeptides comprising amino
acids x to 210 of SEQ ID NO:2. wherein x is an integer from 51
through 59.
[0029] Soluble forms of TRAIL-R possess certain advantages over the
membrane-bound form of the protein. Purification of the protein
from recombinant host cells is facilitated, since the soluble
proteins are secreted from the cells. Further, soluble proteins are
generally more suitable for certain applications, e.g., for
intravenous administration.
[0030] TRAIL-R fragments are provided herein. Such fragments may be
prepared by any of a number of conventional techniques. Desired
peptide fragments may be chemically synthesized. An alternative
involves generating TRAIL-R fragments by enzymatic digestion. e.g.,
by treating the protein with an enzyme known to cleave proteins at
sites defined by particular amino acid residues. Yet another
suitable technique involves isolating and amplifying a DNA fragment
encoding a desired polypeptide fragment, by polymerase chain
reaction (PCR). Oligonucleotides that define the desired termini of
the DNA fragment are employed as the 5' and 3' primers in the
PCR.
[0031] Examples of fragments are those comprising at least 20, or
at least 30, contiguous amino acids of the sequence of SEQ ID NO:2.
Fragments derived from the cytoplasmic domain find use in studies
of TRAIL-R-mediated signal transduction, and in regulating cellular
processes associated with transduction of biological signals.
TRAIL-R polypeptide fragments also may be employed as immunogens,
in generating antibodies. Particular embodiments are directed to
TRAIL-R polypeptide fragments that retain the ability to bind
TRAIL. Such a fragment may be a soluble TRAIL-R polypeptide, as
described above.
[0032] Naturally occurring variants of the TRAIL-R protein of SEQ
ID NO:2 are provided herein. Such variants include, for example,
proteins that result from alternate mRNA splicing events or from
proteolytic cleavage of the TRAIL-R protein. Alternate splicing of
mRNA may, for example, yield a truncated but biologically active
TRAIL-R protein, such as a naturally occurring soluble form of the
protein. 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-R protein (generally from
1-5 terminal amino acids). TRAIL-R proteins in which differences in
amino acid sequence are attributable to genetic polymorphism
(allelic variation among individuals producing the protein) are
also contemplated herein.
[0033] The skilled artisan will also recognize that the position(s)
at which the signal peptide is cleaved may differ from that
predicted by computer program, and may vary according to such
factors as the type of host cells employed in expressing a
recombinant TRAIL-R polypeptide. A protein preparation may include
a mixture of protein molecules having different N-terminal amino
acids, resulting from cleavage of the signal peptide at more than
one site. As discussed above, particular embodiments of mature
TRAIL-R proteins provided herein include, but are not limited to,
proteins having the residue at position 51, 52, 54, 56, or 59 of
SEQ ID NO:2 as the N-terminal amino acid.
[0034] Regarding the discussion herein of various domains of
TRAIL-R protein the skilled artisan will recognize that the
above-described boundaries of such regions of the protein are
approximate. To illustrate, the boundaries of the transmembrane
region (which may be predicted by using computer programs available
for that purpose) may differ from those described above. Thus,
soluble TRAIL-R polypeptides in which the C-terminus of the
extracellular domain differs from the residue so identified above
are contemplated herein.
[0035] Other naturally occurring TRAIL-R DNAs and polypeptides
include those derived from nonhuman species. Homologs of the human
TRAIL-R of SEQ ID NO:2, from other mammalian species, are
contemplated herein, for example. Probes based on the human DNA
sequence of SEQ ID NO:3 or SEQ ID NO:1 may be used to screen cDNA
libraries derived from other mammalian species, using conventional
cross-species hybridization techniques.
[0036] TRAIL-R DNA sequences may vary from the native sequences
disclosed herein. Due to the known degeneracy of the genetic code,
wherein more than one codon can encode the same amino acid, a DNA
sequence can vary from that shown in SEQ ID NO:1 and still encode a
TRAIL-R protein having the amino acid sequence of SEQ ID NO:2. Such
variant DNA sequences may result from silent mutations (e.g.,
occurring during PCR amplification), or may be the product of
deliberate mutagenesis of a native sequence. Thus, among the DNA
sequences provided herein are native TRAIL-R sequences (e.g., cDNA
comprising the nucleotide sequence presented in SEQ ID NO:1) and
DNA that is degenerate as a result of the genetic code to a native
TRAIL-R DNA sequence.
[0037] Among the TRAIL-R polypeptides provided herein are variants
of native TRAIL-R polypeptides that retain a biological activity of
a native TRAIL-R. Such variants include polypeptides that are
substantially homologous to native TRAIL-R, but which have an amino
acid sequence different from that of a native TRAIL-R because of
one or more deletions, insertions or substitutions. Particular
embodiments include, but are not limited to, TRAIL-R polypeptides
that comprise from one to ten deletions, insertions or
substitutions of amino acid residues, when compared to a native
TRAIL-R sequence. The TRAIL-R-encoding DNAs of the present
invention include variants that differ from a native TRAIL-R DNA
sequence because of one or more deletions, insertions or
substitutions, but that encode a biologically active TRAIL-R
polypeptide. One biological activity of TRAIL-R is the ability to
bind TRAIL.
[0038] Nucleic acid molecules capable of hybridizing to the DNA of
SEQ ID NO:1 or SEQ ID NO:3 under moderately stringent or highly
stringent conditions, and which encode a biologically active
TRAIL-R, are provided herein. Such hybridizing nucleic acids
include, but are not limited to, variant DNA sequences and DNA
derived from non-human species, e.g., non-human mammals.
[0039] Moderately stringent conditions include conditions described
in, for example. Sambrook et al, Molecular Cloning: A Laboratory
Journal, 2nd ed., Vol. 1, pp 1.101-104, Cold Spring Harbor
Laboratory Press. 1989. Conditions of moderate stringency, as
defined by Sambrook et al., include use of a prewashing solution of
5.times.SSC, 0.5% SDS, 1.0 mM EDTA (ply 8.0) and hybridization
conditions of about 55.degree. C., 5.times.SSC, overnight. Highly
stringent conditions include higher temperatures of hybridization
and washing. One embodiment of the invention is directed to DNA
sequences that will hybridize to the DNA of SEQ ID NOS:1 or 3 under
highly stringent conditions, wherein said conditions include
hybridization at 68.degree. C. followed by washing in
0.1.times.SSC/0.1% SDS at 63-68.degree. C.
[0040] Certain DNAs and polypeptides provided herein comprise
nucleotide or amino acid sequences, respectively, that are at least
80% identical to a native TRAIL-R sequence. Also contemplated are
embodiments in which a TRAIL-R DNA or polypeptide comprises a
sequence that is at least 90% identical, at least 95% identical, or
at least 98% identical to a native TRAIL-R sequence. The percent
identity may be determined, for example, by comparing sequence
infornationi using 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 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] In particular embodiments of the invention, a variant
TRAIL-R polypeptide differs in amino acid sequence from a native
TRAIL-R, but is substantially equivalent to a native TRAIL-R in a
biological activity. One example is a variant TRAIL-R that binds
TRAIL with essentially the same binding affinity as does a native
TRAIL-R. Binding affinity can be measured by conventional
procedures. e.g., as described in U.S. Pat. No. 5,512,457.
[0042] Variant amino acid sequences may comprise conservative
substitution(s), meaning that one or more amino acid residues of a
native TRAIL-R is replaced by a different residue, but that the
conservatively substituted TRAIL-R polypeptide retains a desired
biological activity of the native protein (e.g., the ability to
bind TRAIL). A given amino acid may be replaced by a residue having
similar physiochemical characteristics. Examples of conservative
substitutions include substitution of one aliphatic residue for
another, such as Ile, Val, Leu, or Ala for one another, or
substitutions of one polar residue for another, such as between Lys
and Arg; Glu and Asp; or Gln and Asn. Other conservative
substitutions, e.g., involving substitutions of entire regions
having similar hydrophobicity characteristics, are well known.
[0043] In another example of variants, 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. Certain receptors of the TNF-R
family contain cysteine-rich repeat motifs in their extracellular
domains (Marsters et al., J. Biol. Chem. 267:5747-5750, 1992).
These repeats are believed to be important for ligand binding. To
illustrate. Marsters et al., supra, reported that soluble TNF-R
type I polypeptides lacking one of the repeats exhibited a ten fold
reduction in binding affinity for TNF.alpha. and TNF.beta.;
deletion of the second repeat resulted in a complete loss of
detectable binding of the ligands. The human TRAIL-R of SEQ ID NO:2
contains two such cysteine rich repeats, the first including
residues 94 through 137, and the second including residues 138
through 178. Cysteine residues within these cysteine rich domains
advantageously remain unaltered in TRAIL-R variants, when retention
of TRAIL-binding activity is desired.
[0044] 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. Mature human TRAIL-R contains such
adjacent basic residue pairs at amino acids 72-73, 154-155,
322-323, 323-324, and 359-360 of SEQ ID NO:2. 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.
[0045] TRAIL-R polypeptides, including variants and fragments
thereof, can be tested for biological activity in any suitable
assay. The ability of a TRAIL-R polypeptide to bind TRAIL can be
confirmed in conventional binding assays, examples of which are
described below.
Expression Systems
[0046] The present invention also provides recombinant cloning and
expression vectors containing TRAIL-R DNA, as well as host cell
containing the recombinant vectors. Expression vectors comprising
TRAIL-R DNA may be used to prepare TRAIL-R polypeptides encoded by
the DNA. A method for producing TRAIL-R polypeptides comprises
culturing host cells transformed with a recombinant expression
vector encoding TRAIL-R, under conditions that promote expression
of TRAIL-R, then recovering the expressed TRAIL-R polypeptides firm
the culture. The skilled artisan will recognize that the procedure
for purifying the expressed TRAIL-R will vary according to such
factors as the type of host cells employed, and whether the TRAIL-R
is membrane-bound or a soluble form that is secreted from the host
cell.
[0047] Any suitable expression system may be employed. The vectors
include a DNA encoding a TRAIL-R 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-R DNA sequence. Thus, a
promoter nucleotide sequence is operably linked to an TRAIL-R DNA
sequence if the promoter nucleotide sequence controls the
transcription of the FRAIL-R 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.
[0048] In addition, a sequence encoding an appropriate signal
peptide (native or heterologous) can be incorporated into
expression vectors. A DNA sequence for a signal peptide (secretory
leader) may be fused in frame to the TRAIL-R sequence so that the
TRAIL-R 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-R
polypeptide. The signal peptide is cleaved from the TRAIL-R
polypeptide upon secretion of TRAIL-R from the cell.
[0049] Suitable host cells for expression of TRAIL-R polypeptides
include prokaryotes, yeast or higher eukaryotic cells. Mammalian or
insect cells are generally preferred for use as host 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, New York. (1 985). Cell-free translation systems could
also be employed to produce TRAIL-R polypeptides using RNAs derived
from DNA constructs disclosed herein.
[0050] 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-R 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-R polypeptide.
[0051] 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-R 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).
[0052] 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 .lamda. P.sub.L promoter and a cI857ts
thermolabile repressor sequence. Plasmid vectors available from the
American Type Culture Collection which incorporate derivatives of
the .lamda. P.sub.L promoter include plasmid pHUB2 (resident in E.
coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1,
ATCC 53082).
[0053] TRAIL-R 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 2.mu. 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-phosphloglycerate 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, pyrvate
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.
[0054] 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., Kur an 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.
[0055] 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.
[0056] 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.
[0057] Mammalian or insect host cell culture systems also may be
employed to express recombinant TRAIL-R 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 CV1/EBNA cell line derived from the African
green monkey kidney cell line CV1 (ATCC CCL 70) as described by
McMahan et al. (EMBO J. 10: 2821, 1991).
[0058] 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.
[0059] 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.
Overexpression of full length TRAIL-R has resulted in membrane
blebbing and nuclear condensation of transfected CV-1/EBNA cells,
indicating that the mechanism of cell death was apoptosis. For host
cells in which such TRAIL-R-mediated apoptosis occurs, a suitable
apoptosis inhibitor may be included in the expression system.
[0060] To inhibit TRAIL-R-induced apoptosis of host cells
expressing recombinant TRAIL-R, the cells may be co-transfected
with an expression vector encoding a polypeptide that functions as
an apoptosis inhibitor. Expression vectors encoding such
polypeptides can be prepared by conventional procedures. Another
approach involves adding an apoptosis inhibitor to the culture
medium. The use of poxvirus CrmA, baculovirus P35, a C-terminal
fragment of FADD, and the tripeptide derivative zVAD-fmk, to reduce
death of host cells is illustrated in examples 6 and 8.
[0061] zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone)
is a tripeptide based compound, available from Enzyme System
Products, Dublin, California. As illustrated in example 8, zVAD-fmk
may be added to the medium in which cells expressing TRAIL-R are
cultured.
[0062] The 38-kilodalton cowpox-derived protein that was
subsequently designated CrmA is described in Pickup et al. (Proc.
Natl. Acad. Sci. USA 83:7698-7702, 1986; hereby incorporated by
reference). Sequence information for CrmA is presented in FIG. 4 of
Pickup et al., supra. One approach to producing and purifying CrmA
protein is described in Ray et al. (Cell. 69:597-604, 1992; hereby
incorporated by reference).
[0063] A 35-kilodalton protein encoded by Autographa californica
nuclear polyhedrosis virus, a baculovirus, is described in Friesen
and Miller (J. Virol. 61:2264-2272, 1987; hereby incorporated by
reference). Sequence information for this protein, designated
baculovirus p35 herein, is presented in FIG. 5 of Friesen and
Miller, supra.
[0064] The death domain-containing cytoplasmic protein FADD (also
known as MORT1) is described in Boldin et al. (J. Biol. Chem.
270:7795-7798, 1995; hereby incorporated by reference). FADD has
been reported to associate, directly or indirectly, with the
cytoplasmic death domain of certain receptors that mediate
apoptosis (Boldin et al., Cell 85:803-815. June 1996; Hsu et al.
(Cell 84:299-308, January 1996).
[0065] In one embodiment of the present invention, truncated FADD
polypeptides that include the death domain (located in the
C-terminal portion of the protein), but lack the N-terminal region
to which apoptosis effector functions have been attributed, are
employed to reduce apoptosis. The use of certain FADD deletion
mutant polypeptides, truncated at the N-terminus, to inhibit death
of cells expressing other apoptosis-inducing receptors, is
described in Hsu et al. (Cell 84:299-308, 1996; hereby incorporated
by reference).
[0066] This approach is illustrated in example 8, which employs one
suitable FADD-dominant negative (FADD-DN) polypeptide, having an
amino acid sequence corresponding to amino acids 117 through 245 of
the MORT1 amino acid sequence presented in Boldin et al. (J. Biol.
Chem. 270:7795-779S, 1995). In example 8, cells were co-transfected
with a TRAIL-R-encoding expression vector, and with an expression
vector encoding the above-described Flag.RTM. peptide, fused to the
N-terminus of the FADD-DN polypeptide.
[0067] While not wishing to be bound by theory, one possible
explanation is that the C-terminal fragments of FADD associate with
the intracellular death domain of the receptor, but lack the
N-terminal portion of the protein that is necessary for effecting
apoptosis (Hsu et al., Cell 84:299-308). January 1996; Boldin et
al., Cell 85:803-815, June 1996). The truncated FADD thereby may
block association of endogenous, full length FADD with the
receptor's death domain; consequently, the apoptosis that would be
initiated by such endogenous FADD is inhibited.
[0068] Other apoptosis inhibitors useful in expression systems of
the present invention can be identified in conventional assay
procedures. One such assay, in which compounds are tested for the
ability to reduce apoptosis of cells expressing TRAIL-R, is
described in example 8.
[0069] Poxvirus CrmA, baculovirus P35, and zVAD-fmk are viral
caspase inhibitors. Other caspase inhibitors may be tested for the
ability to reduce TRAIL-R-mediated cell death.
[0070] The use of CrmA, baculovirus p35, and certain peptide
derivatives (including zVAD-fmk) as inhibitors of apoptosis in
particular cells/systems is discussed in Sarin et al. (J. Exp. Med.
184:2445-2450, December 1996; hereby incorporated by reference).
The role of interleukin-1.beta. converting enzyme (ICE) family
proteases in signal transduction cascades leading to programmed
cell death, and the use of inhibitors of such proteases to block
apoptosis, is discussed in Sarin et al., supra, and Muzio et al.,
Cell 85:817-827, 1996).
[0071] Apoptosis inhibitors generally need not be employed for
expression of TRAIL-R polypeptides lacking the cytoplasmic domain
(i.e., lacking the region of the protein involved in signal
transduction). Thus, expression systems for producing soluble
TRAIL-R polypeptides comprising only the extracellular domain (or a
fragment thereof) need not include one of the above-described
apoptosis inhibitors.
[0072] Regarding signal peptides that may be employed in producing
TRAIL-R, the native signal peptide of TRAIL-R may be replaced by a
heterologous signal peptide or leader sequence, if desired. The
choice of signal peptide or leader may depend on factors such as
the type of host cells in which the recombinant TRAIL-R is to be
produced. To illustrate, examples of heterologous signal peptides
that are functional in mammalian host cells 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-4 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.
Oligomeric Forms of TRAIL-R
[0073] Encompassed by the present invention are oligomer-s that
contain TRAIL-R polypeptides. TRAIL-R oligomers may be in the form
of covalently-linked or non-covalently-linked dimers, trimers, or
higher oligomers.
[0074] One embodiment of the invention is directed to oligomers
comprising multiple TRAIL-R polypeptides joined via covalent or
non-covalent interactions between peptide moieties fused to the
TRAIL-R 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-R polypeptides attached thereto, as
described in more detail below.
[0075] In particular embodiments, the oligomers comprise from two
to four TRAIL-R polypeptides. The TRAIL-R moieties of the oligomer
may be soluble polypeptides, as described above.
[0076] As one alternative, a TRAIL-R oligomer is prepared using
polypeptides derived from immunoglobulins. Preparation oft fusion
proteins comprising certain 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 344:677, 1990); and
Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion
Proteins", in Current Protocol in Immunology, Suppl. 4, pages
10.19.1-10.19.11, 1992).
[0077] One embodiment of the present invention is directed to a
TRAIL-R dimer comprising two fusion proteins created by fusing
TRAIL-R to the Fc region of an antibody. A gene fusion encoding the
TRAIL-R/Fc fusion protein is inserted into an appropriate
expression vector. TRAIL-R/Fc fusion proteins are expressed in host
cells transformed with the recombinant expression vector, and
allowed to assemble much like antibody molecules, whereupon
interchain disulfide bonds form between the Fc moieties to yield
divalent TRAIL-R.
[0078] Provided herein are fusion proteins comprising a TRAIL-R
polypeptide fused to an Fc polypeptide derived from an antibody.
DNA encoding such fusion proteins, as well as dimers containing two
fusion proteins joined via disulfide bonds between the Fc moieties
thereof, are also provided. The term "Fc polypeptide" as used
herein includes native and mutein forms of polypeptides derived
from the Fc region of an antibody. Truncated forms of such
polypeptides containing the hinge region that promotes dimerization
are also included.
[0079] One suitable Fc polypeptide, described in PCT application WO
93/10151 (hereby incorporated by reference), is a single chain
polypeptide extending from the N-terminal hinge region to the
native C-terminus of the Fc region of a human IgG1 antibody.
Another useful Fc polypeptide is the Fc mutein described in U.S.
Pat. No. 5,457,035 and in Baum et al. (EMBO J. 13:3992-4001. 1994).
The amino acid sequence of this 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. The mutein exhibits reduced affinity for Fc receptors.
[0080] In other embodiments, TRAIL-R 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 a TRAIL-R oligomer with as many as four
TRAIL-R extracellular regions.
[0081] Alternatively, the oligomer is a fusion protein comprising
multiple TRAIL-R polypeptides, with or without peptide linkers
(spacer peptides). Among the suitable peptide linkers are those
described in U.S. Pat. Nos. 4,751,180 and 4,935,233, which are
hereby incorporated by reference. A DNA sequence encoding a desired
peptide linker may be inserted between, and in the same reading
frame as, the DNA sequences encoding TRAIL-R, using any suitable
conventional technique. For example, a chemically synthesized
oligonucleotide encoding the linker may be ligated between
sequences encoding TRAIL-R. In particular embodiments, a fusion
protein comprises from two to four soluble TRAIL-R polypeptides,
separated by peptide linkers.
[0082] Another method for preparing oligomeric TRAIL-R 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.
[0083] Examples of leucine zipper domains suitable for producing
soluble oligomeric proteins are described in PCT application WO
94/10308, and the leucine zipper derived from lung surfactant
protein D (SPD) described in Hoppe et al. (FEBS Letters 344:191,
1994), hereby incorporated by reference. The use of a modified
leucine zipper that allows for stable trimerization of a
heterologous protein fused thereto is described in Fanslow et al.
(Semin. Immunol. 6:267-278, 1994). Recombinant fusion proteins
comprising a soluble TRAIL-R polypeptide fused to a leucine zipper
peptide are expressed in suitable host cells, and the soluble
oligomeric TRAIL-R that forms is recovered from the culture
supernatant.
[0084] Oligomeric TRAIL-R has the property of bivalent, trivalent.
etc. binding sites for TRAIL. The above-described fusion proteins
comprising Fc moieties (and oligomers formed therefrom) offer the
advantage of facile purification by affinity chromatography over
Protein A or Protein G columns. DNA sequences encoding oligomeric
TRAIL-R, or encoding fusion proteins useful in preparing TRAIL-R
oligomers, are provided herein.
Assays
[0085] TRAIL-R proteins (including fragments, variants, oligomers,
and other forms of TRAIL-R) may be tested for the ability to bind
TRAIL in any suitable assay, such as a conventional binding assay.
To illustrate, TRAIL-R may be labeled with a detectable reagent
(e.g., a radionuclide, chromophore, enzyme that catalyzes a
calorimetric or fluorometric reaction, and the like). The labeled
TRAIL-R is contacted with cells expressing TRAIL. The cells then
are washed to remove unbound labeled TRAIL-R, and the presence of
cell-bound label is determined by a suitable technique, chosen
according to the nature of the label.
[0086] One example of a binding assay procedure is as follows. A
recombinant expression vector containing TRAIL cDNA is constructed,
e.g., as described in in PCT application WO 97/01633, hereby
incorporated by reference. DNA and amino acid sequence information
for human and mouse TRAIL is presented in WO 97/01633. TRAIL
comprises an N-terminal cytoplasmic domain, a transmembrane region,
and a C-terminal extracellular domain. CV1-EBNA-1 cells in 40
cm.sup.2 dishes are transfected with the recombinant expression
vector. CV-1/EBNA-1 cells (ATCC CRL 10478) constitutively express
EBV nuclear antigen-1 driven from the CMV immediate-early
enhancer/promoter. CV1-EBNA-1 was derived from the African Green
Monkey kidney cell line CV-1 (ATCC CCL 70), as described by McMahan
et al. (EMBO J. 10:2821, 1991).
[0087] The transfected cells are cultured for 24 hours, and the
cells in each dish then are split into a 24-well plate. After
culturing an additional 48 hours, the transfected cells (about
4.times.10.sup.4 cells/well) are washed with BM-NFDM, which is
binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin,
2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg/ml nonfat
dry milk has been added. The cells then are incubated for 1 hour at
37.degree. C. with various concentrations of a soluble TRAIL-R/Fc
fusion protein. Cells then are washed and incubated with a constant
saturating concentration of a .sup.125I-mouse anti-human IgG in
binding medium, with gentle agitation for 1 hour at 37.degree. C.
After extensive washing, cells are released via trypsinization.
[0088] The mouse anti-human IgG employed above is directed against
the Fc region of human IgG and can be obtained from Jackson
Immunoresearch Laboratories, Inc., West Grove, Pa. The antibody is
radioiodinated using the standard chloramine-T method. The antibody
will bind to the Fc portion of any I 0 TRAIL-R/Fc protein that has
bound to the cells. In all assays, non-specific binding of
.sup.125I-antibody is assayed in the absence of TRAIL-R Fc, as well
as in the presence of TRAIL-R/Fc and a 200-fold molar excess of
unlabeled mouse anti-human IgG antibody.
[0089] Cell-bound .sup.125I-antibody is quantified on a Packard
Autogamma counter. Affinity calculations (Scatchard, Ann. N.Y.
Acad. Sci. 51:660, 1949) are generated on RS/1 (BBN Software,
Boston, Mass.) run on a Microvax computer.
[0090] Another type of suitable binding assay is a competitive
binding assay. To illustrate, biological activity of a TRAIL-R
variant may be determined by assaying for the variant's ability to
compete with a native TRAIL-R for binding to TRAIL.
[0091] Competitive binding assays can be performed by conventional
methodology. Reagents that may be employed in competitive binding
assays include radiolabeled TRAIL-R and intact cells expressing
TRAIL (endogenous or recombinant) on the cell surface. For example,
a radiolabeled soluble TRAIL-R fragment can be used to compete with
a soluble TRAIL-R variant for binding to cell surface TRAIL.
Instead of intact cells, one could substitute a soluble TRAIL/Fc
fusion protein bound to a solid phase through the interaction of
Protein A or Protein G (on the solid phase) with the Fc moiety.
Chromatography columns that contain Protein A and Protein G include
those available from Pharmacia Biotech, Inc., Piscataway, N.J.
Another type of competitive binding assay utilizes radiolabeled
soluble TRAIL, such as a soluble TRAIL/Fc fusion protein, and
intact cells expressing TRAIL-R. Qualitative results can be
obtained by competitive autoradiographic plate binding assays,
while Scatchard plots (Scatchard, Ann. N.Y. Acad. Sci. 51:660,
1949) may be utilized to generate quantitative results.
[0092] Another type of assay for biological activity involves
testing a TRAIL-R polypeptide for the ability to block
TRAIL-mediated apoptosis of target cells, such as the human
leukemic T-cell line known as Jurkat cells, for example.
TRAIL-mediated apoptosis of the cell line designated Jurkat clone
E6-1 (ATCC TIB 152) is demonstrated in assay procedures described
in PCT application WO 97/01633, hereby incorporated by
reference.
Uses of TRAIL-R
[0093] Uses of TRAIL-R include, but are not limited to, the
following. Certain of these uses of TRAIL-R flow from its ability
to bind TRAIL.
[0094] TRAIL-R finds use as a protein purification reagent. TRAIL-R
polypeptides may be attached to a solid support material and used
to purify TRAIL proteins by affinity chromatography. In particular
embodiments, a TRAIL-R polypeptide (in any form described herein
that is capable of binding TRAIL) is attached to a solid support by
conventional procedures. As one example, chromatography columns
containing functional groups that will react with functional groups
on amino acid side chains of proteins are available (Pharmacia
Biotech, Inc., Piscataway, N.J.). In an alternative, a TRAIL-R/Fc
protein is attached to Protein A- or Protein G-containing
chromatography columns through interaction with the Fc moiety.
[0095] TRAIL-R proteins also find use in measuring the biological
activity of TRAIL proteins in teems of their binding affinity for
TRAIL-R. TRAIL-R proteins thus may be employed by those conducting
"quality assurance" studies, e.g., to monitor shelf life and
stability of TRAIL protein under different conditions. To
illustrate, TRAIL-R may be employed in a binding affinity study to
measure the biological activity of a TRAIL protein that has been
stored at different temperatures, or produced in different cell
types. TRAIL-R also may be used to determine whether biological
activity is retained after modification of a TRAIL protein (e.g.,
chemical modification, truncation, mutation, etc.). The binding
affinity of the modified TRAIL protein for TRAIL-R is compared to
that of an unmodified TRAIL protein to detect any adverse impact of
the modifications on biological activity of TRAIL. The biological
activity of a TRAIL protein thus can be ascertained before it is
used in a research study, for example.
[0096] TRAIL-R also finds use in purifying or identifying cells
that express TRAIL on the cell surface. TRAIL-R polypeptides are
bound to a solid phase such as a column chromatography matrix or a
similar suitable substrate. For example, magnetic microspheres can
be coated with TRAIL-R and held in an incubation vessel through a
magnetic field. Suspensions of cell mixtures containing
TRAIL-expressing cells are contacted with the solid phase having
TRAIL-R thereon. Cells expressing TRAIL on the cell surface bind to
the fixed TRAIL-R, and unbound cells then are washed away.
[0097] Alternatively, TRAIL-R can be conjugated to a detectable
moiety, then incubated with cells to be tested for TRAIL
expression. After incubation, unbound labeled TRAIL-R is removed
and the presence or absence of the detectable moiety on the cells
is determined.
[0098] In a further alternative, mixtures of cells suspected of
containing TRAIL cells are incubated with biotinylated TRAIL-R.
Incubation periods are typically at least one hour in duration to
ensure sufficient binding. The resulting mixture then is passed
through a column packed with avidin-coated beads, whereby the high
affinity of biotin for avidin provides binding of the desired cells
to the beads. Procedures for using avidin-coated beads are known
(see Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing to
remove unbound material, and the release of the bound cells, are
performed using conventional methods.
[0099] TRAIL-R polypeptides also find use as carriers for
delivering agents attached thereto to cells bearing TRAIL. Cells
expressing TRAIL include those identified in Wiley et al.
(Immunity, 3:673-682, 1995). TRAIL-R proteins thus can be used to
deliver diagnostic or therapeutic agents to such cells (or to other
cell types found to express TRAIL on the cell surface) in in vitro
or in vivo procedures.
[0100] Detectable (diagnostic) and therapeutic agents that may be
attached to a TRAIL-R polypeptide include, but are not limited to,
toxins, other cytotoxic agents, drugs, radionuclides, chromophores,
enzymes that catalyze a colorimetric or fluorometric reaction, and
the like, with the particular agent being chosen according to the
intended application. Among the toxins are ricin, abrin, diphtheria
toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating
proteins, mycotoxins such as trichothecenes, and derivatives and
fragments (e.g., single chains) thereof. Radionuclides suitable for
diagnostic use include, but are not limited to, 123I1. .sup.131i.
.sup.99mTc, .sup.111In, and .sup.76Br. Examples of radionuclides
suitable for therapeutic use are .sup.131I, .sup.211At, .sup.77Br,
.sup.186Re, .sup.188Re, .sup.212Pb, .sup.212Bi, .sup.109Pd,
.sup.64Cu, and .sup.67Cu.
[0101] Such agents may be attached to the TRAIL-R by any suitable
conventional procedure. TRAIL-R, being a protein, comprises
functional groups on amino acid side chains that can be reacted
with functional groups on a desired agent to form covalent bonds,
for example. Alternatively, the protein or agent may be derivatized
to generate or attach a desired reactive functional group. The
derivatization may involve attachment of one of the bifunctional
coupling reagents available for attaching various molecules to
proteins (Pierce Chemical Company, Rockford, Ill.). A number of
techniques for radiolabeling proteins are known. Radionuclide
metals may be attached to TRAIL-R by using a suitable bifunctional
chelating agent, for example.
[0102] Conjugates comprising TRAIL-R and a suitable diagnostic or
therapeutic agent (preferably covalently linked) are thus prepared.
The conjugates are administered or otherwise employed in an amount
appropriate for the particular application.
[0103] TRAIL-R DNA and polypeptides of the present invention may be
used in developing treatments for any disorder mediated (directly
or indirectly) by defective, or insufficient amounts of, TRAIL-R.
TRAIL-R polypeptides may be administered to a mammal afflicted with
such a disorder. Alternatively, a gene therapy approach may be
taken. Disclosure herein of native TRAIL-R nucleotide sequences
permits the detection of defective TRAIL-R genes, and the
replacement thereof with normal TRAIL-R-encoding genes. Defective
genes may be detected in in vitro diagnostic assays, and by
comparison of a native TRAIL-R nucleotide sequence disclosed herein
with that of a TRAIL-R gene derived from a person suspected of
harboring a defect in this gene.
[0104] Another use of the protein of the present invention is as a
research tool for studying the biological effects that result from
inhibiting TRAIL/TRAIL-R interactions on different cell types.
TRAIL-R polypeptides also may be employed in in vitro assays for
detecting TRAIL or TRAIL-R or the interactions thereof.
[0105] TRAIL-R may be employed in inhibiting a biological activity
of TRAIL, in in vitro or in vivo procedures. A purified TRAIL-R
polypeptide may be used to inhibit binding of TRAIL to endogenous
cell surface TRAIL-R. Biological effects that result from the
binding of TRAIL to endogenous receptors thus are inhibited.
Various forms of TRAIL-R may be employed, including, for example,
the above-described TRAIL-R fragments, oligomers, derivatives, and
variants that are capable of binding TRAIL. In one embodiment, a
soluble TRAIL-R is employed to inhibit a biological activity of
TRAIL, e.g., to inhibit TRAIL-mediated apoptosis of particular
cells.
[0106] TRAIL-R may be administered to a mammal to treat a
TRAIL-mediated disorder. Such TRAIL-mediated disorders include
conditions caused (directly or indirectly) or exacerbated by
TRAIL.
[0107] TRAIL-R 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).
[0108] 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. As described in PCT
application WO 97/01633 (hereby incorporated by reference), TRAIL
is present in the serum of TTP patients, and may play a role in
inducing apoptosis of microvascular endothelial cells.
[0109] 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 TRAIL-R 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-associated HUS differs in etiology from adult
BUS.
[0110] Other conditions characterized by clotting of small blood
vessels may be treated using TRAIL-R. 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.
[0111] In one embodiment, a patient's blood or plasma is contacted
with TRAIL-R ex vivo. The TRAIL-R may be bound to a suitable
chromatography matrix by conventional procedures. The patient's
blood or plasma flows through a chromatography column containing
TRAIL-R bound to the matrix, before being returned to the patient.
The immobilized receptor binds TRAIL, thus removing TRAIL protein
from the patient's blood.
[0112] Alternatively, TRAIL-R may be administered in vivo to a
patient afflicted with a thrombotic microangiopathy. In one
embodiment, a soluble form of TRAIL-R is administered to the
patient.
[0113] The present invention thus provides a method for treating a
thrombotic microangiopathy, involving use of an effective amount of
TRAIL-R. A TRAIL-R polypeptide may be employed in in vivo or ex
vivo procedures, to inhibit TRAIL-mediated damage to (e.g.,
apoptosis of) microvascular endothelial cells.
[0114] TRAIL-R 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.
[0115] 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, TRAIL-R 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.
[0116] Another embodiment of the present invention is directed to
the use of TRAIL-R to reduce TRAIL-mediated death of T cells in
HIV-infected patients. The role of T cell apoptosis in the
development of AIDS has been the subject of a number of studies
(see, for example, Meyaard et al., Science 257:217-219, 1992; Groux
et al., J. Exp. Med., 175:331, 1992; and Oyaizu et al., in Cell
Activation and Apoptosis in HIV Infection, Andrieu and Lu, Eds.,
Plenum Press, New York, 1995, pp. 101-114). Certain investigators
have studied the role of Fas-mediated apoptosis: the involvement of
interleukin-1I3-converting enzyme (ICE) also has been explored
(Estaquier et al. Blood 87:4959-4966, 1996; Mitra et al.,
Immunology 87:581-585, 1996; Katsikis et al., J. Exp. Med.
181:2029-2036, 1995). It is possible that T cell apoptosis occurs
through multiple mechanisms.
[0117] At least some of the T cell death seen in HIV.sup.+ patients
is believed to be mediated by TRAIL. While not wishing to be bound
by theory, such TRAIL-mediated T cell death is believed to occur
through the mechanism known as activation-induced cell death
(AICD).
[0118] Activated human T cells are induced to undergo programmed
cell death (apoptosis) upon triggering through the CD3/T cell
receptor complex, a process termed activated-induced cell death
(AICD). AICD of CD4.sup.+ T cells isolated from HIV-infected
aymptomatic individuals has been reported (Groux et al., supra).
Thus, AICD may play a role in the depletion of CD4.sup.+ cells and
the progression to AIDS in HIV-infected individuals.
[0119] The present invention provides a method of inhibiting
TRAIL-mediated T cell death in HIV.sup.+ patients, comprising
administering TRAIL-R (preferably, a soluble TRAIL-R polypeptide)
to the patients. In one embodiment, the patient is asymptomatic
when treatment with TRAIL-R commences. If desired, prior to
treatment, peripheral blood T cells may be extracted from an
HIV.sup.+ patient, and tested for susceptibility to TRAIL-mediated
cell death by conventional procedures.
[0120] In one embodiment, a patient's blood or plasma is contacted
with TRAIL-R flex vivo. The TRAIL-R may be bound to a suitable
chromatography matrix by conventional procedures. The patient's
blood or plasma lows through a chromatography column containing
TRAIL-R bound to the matrix, before being returned to the patient.
The immobilized TRAIL-R binds TRAIL, thus removing TRAIL protein
from the patient's blood.
[0121] In treating HIV- patients, TRAIL-R may be employed in
combination with other inhibitors of T cell apoptosis. Fas-mediated
apoptosis also has been implicated in loss of T cells in HIV.sup.+
individuals (Katsikis et al., J. Exp. Med. 181:2029-2036. 1995).
Thus, a patient susceptible to both Fas ligand (Fas-L)-mediated and
TRAIL-mediated T cell death may be treated with both an agent that
blocks TRAIL/TRAIL-R interactions and an agent that blocks
Fas-L/Fas interactions. Suitable agents for blocking binding of
Fas-L to Fas include, but are not limited to, soluble Fas
polypeptides; oligomeric forms of soluble Fas polypeptides (e.g.,
dimers of sFas/Fc); anti-Fas antibodies that bind Fas without
transducing the biological signal that results in apoptosis;
anti-Fas-L antibodies that block binding of Las-L to Fas; and
muteins of Fas-L that bind Fas but don't transduce the biological
signal that results in apoptosis. Preferably, the antibodies
employed in the method are monoclonal antibodies. Examples of
suitable agents for blocking Fas-L/Fas interactions, including
blocking anti-Fas monoclonal antibodies, are described in WO
95/10540, hereby incorporated by reference.
[0122] Compositions comprising an effective amount of a TRAIL-R
polypeptide of the present invention, in combination with other
components such as a physiologically acceptable diluent, carrier,
or excipient, are provided herein. TRAIL-R can be formulated
according to known methods used to prepare pharmaceutically useful
compositions. TRAIL-R can be combined in admixture, either as the
sole active material or with other known active materials suitable
for a given indication, with pharmaceutically acceptable diluents
(e.g., saline, Tris-HCl, acetate, and phosphate buffered
solutions), preservatives (e.g., thimerosal, benzyl alcohol,
parabens), emulsifiers, solubilizers, adjuvants and/or carriers.
Suitable formulations for pharmaceutical compositions include those
described in Remington's Pharmaceutical Sciences, 16th ed. 1980,
Mack Publishing Company, Easton, Pa.
[0123] In addition, such compositions can contain TRAIL-R complexed
with polyethylene glycol (PEG), metal ions, or incorporated into
polymeric compounds such as polyacetic acid, polyglycolic acid,
hydrogels, dextran, etc., or incorporated into liposomes,
microemulsions, micelles, unilamellar or multilamellar vesicles,
erythrocyte ghosts or spheroblasts. Such compositions will
influence the physical state, solubility, stability, rate of in
vivo release, and rate of in vivo clearance of TRAIL-R, and are
thus chosen according to the intended application. TRAIL-R
expressed on the surface of a cell may find use, as well.
[0124] Compositions of the present invention may contain a TRAIL-R
polypeptide in any form described herein, such as native proteins,
variants, derivatives, oligomers, and biologically active
fragments. In particular embodiments, the composition comprises a
soluble TRAIL-R polypeptide or an oligomer comprising soluble
TRAIL-R polypeptides.
[0125] TRAIL-R can be administered in any suitable manner, e.g.,
topically, parenterally, or by inhalation. The term "parenteral"
includes injection, e.g., by subcutaneous, intravenous, or
intramuscular routes, also including localized administration,
e.g., at a site of disease or injury. Sustained release from
implants is also contemplated. One skilled in the pertinent art
will recognize that suitable dosages will vary, depending upon such
factors as the nature of the disorder to be treated, the patient's
body weight, age, and general condition, and the route of
administration. Preliminary doses can be determined according to
animal tests, and the scaling of dosages for human administration
are performed according to art- accepted practices.
[0126] Compositions comprising TRAIL-R nucleic acids in
physiologically acceptable formulations are also contemplated.
TRAIL-R DNA may be formulated for injection, for example.
Antibodies
[0127] Antibodies that are immunoreactive with TRAIL-R polypeptides
are provided herein. Such antibodies specifically bind TRAIL-R, in
that the antibodies bind to TRAIL-R via the antigen-binding sites
of the antibody (as opposed to non- specific binding).
[0128] The TRAIL-R protein prepared as described in example 1 may
be employed as an immunogen in producing antibodies immunoreactive
therewith. Alternatively, another form of TRAIL-R, such as a
fragment or fusion protein, is employed as the immunogen.
[0129] Polyclonal and monoclonal antibodies may be prepared by
conventional techniques. See, for example, Monoclonal Antibodies,
Hybridomas: A New Dimension 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 directed against TRAIL-R is further
illustrated in example 4.
[0130] 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 and F(ab').sub.2 fragments. Antibody fragments and
derivatives produced by genetic engineering techniques are also
provided.
[0131] 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).
[0132] Procedures that have been developed for generating human
antibodies in non-human animals may be employed in producing
antibodies of the present invention. The antibodies may be
partially human, or preferably completely human. For example,
transgenic mice into which genetic material encoding one or more
human immunoglobulin chains has been introduced may be employed.
Such mice may be genetically altered in a variety of ways. The
genetic manipulation may result in human immunoglobulin polypeptide
chains replacing endogenous immunoglobulin chains in at least some
(preferably virtually all) antibodies produced by the animal upon
immunization.
[0133] Mice in which one or more endogenous immunoglobulin genes
have been inactivated by various means have been prepared. Human
immunoglobulin genes have been introduced into the mice to replace
the inactivated mouse genes. Antibodies produced in the animals
incorporate human immunoglobulin polypeptide chains encoded by the
human genetic material introduced into the animal.
[0134] Examples of techniques for production and use of such
transgenic animals are described in U.S. Pat. Nos. 5,814,31 ,
5,569,825, and 5,545,806, which are incorporated by reference
herein. See examples 10-12 below for further description of the
preparation of transgenic mice useful for generating human
antibodies directed against an antigen of interest.
[0135] Antibodies produced by procedures that comprise immunizing
transgenic animals with a TRAIL-R polypeptide are provided herein.
Transgenic mice into which genetic material encoding human
immunoglobulin polypeptide chain(s) has been introduced are among
the suitable transgenic animals. Examples of such mice include, but
are not limited to, those containing the genetic alterations
described in the examples below.
[0136] Monoclonal antibodies may be produced by conventional
procedures. e.g., by immortalizing spleen cells harvested from the
transgenic animal after completion of the immunization schedule.
The spleen cells may be fused with myeloma cells to produce
hybridomas, by conventional procedures (see example 4 for an
illustration of such techniques).
[0137] A method for producing a hybridoma cell line comprises
immunizing such a transgenic animal with a TRAIL-R immunogen;
harvesting spleen cells from the immunized animal; fusing the
harvested spleen cells to a myeloma cell line, thereby generating
hybridoma cells; and identifying a hybridoma cell line that
produces a monoclonal antibody that binds TRAIL-R. Such hybridoma
cell lines, and monoclonal antibodies produced therefrom, are
encompassed by the present invention.
[0138] Among the uses of antibodies of the present invention, which
are directed against TRAIL-R, is use in assays to detect the
presence of TRAIL-R polypeptides, either in vitro or in vivo. The
antibodies also may be employed in purifying TRAIL-R proteins by
immunoaffinity chromatography.
[0139] Those antibodies that additionally can block binding of
TRAIL-R to TRAIL may be used to inhibit a biological activity that
results from such binding. Such blocking antibodies may be
identified using any suitable assay procedure, such as by testing
antibodies for the ability to inhibit binding of TRAIL to cells
expressing TRAIL-R. Examples of such cells are the Jurkat cells and
PSI cells described in example 2 below. Alternatively, blocking
antibodies may be identified in assays for the ability to inhibit a
biological effect that results from binding of TRAIL to target
cells. Antibodies may be assayed for the ability to inhibit
TRAIL-mediated lysis of Jurkat cells, for example.
[0140] Such an antibody may be employed in an in vitro procedure,
or administered in vivo to inhibit a TRAIL-R-mediated biological
activity. Disorders caused or exacerbated (directly or indirectly)
by the interaction of TRAIL, with cell surface TRAIL receptor thus
may be treated. A therapeutic method involves in vivo
administration of a blocking antibody to a mammal in an amount
effective in inhibiting a TRAIL-mediated biological activity.
Disorders caused or exacerbated by TRAIL, directly or indirectly,
are thus treated. Monoclonal antibodies are generally preferred for
use in such therapeutic methods. In one embodiment, an
antigen-binding antibody fragment is employed.
[0141] A blocking antibody directed against TRAIL-R may be
substituted for TRAIL-R in the above-described method of treating
thrombotic microangiopathy. e.g., in treating TTP or HUS. The
antibody is administered in vivo, to inhibit TRAIL-mediated damage
to (e.g., apoptosis of) microvascular endothelial cells.
[0142] Antibodies raised against TRAIL-R may be screened for
agonistic (i.e., ligand-mimicking) properties. Such antibodies,
upon binding to cell surface TRAIL-R, induce biological effects
(e.g., transduction of biological signals) similar to the
biological effects induced when TRAIL binds to cell surface
TRAIL-R. Agonistic antibodies may be used to induce apoptosis of
certain cancer cells or virally infected cells, as has been
reported for TRAIL. The ability of TRAIL to kill cancer cells
(including but not limited to leukemia, lymphoma, and melanoma
cells) and virally infected cells is described in Wiley et al.
(Immunity, 3:673-682, 1995); and in PCT application WO
97/01633.
[0143] Conventional techniques may be employed to confirm the
susceptibility of various cancer cell types and virally infected
cells to cell death induced by agonistic antibodies of the present
invention. Use of agonistic TRAIL-R2 antibodies for treating
cancers that include, but are not limited to, carcinomas, sarcomas,
lymphomas, leukemia, melanoma, cancers of the lung, breast, ovary,
prostate, kidney, liver, bladder, pancreas, and colon (including
colorectal cancer) is contemplated herein.
[0144] Viral infections and associated conditions include, but are
not limited to, cytomegalovirus, encephalomyocarditis, influenza,
Newcastle disease virus, vesicular stomatitus virus, herpes simplex
virus, hepatitis, adenovirus-2, bovine viral diarrhea virus, HIV,
and Epstein-Barr virus. Agonistic antibodies of the present
invention may be administered alone or in combination with other
agents useful for combatting a particular virus. As one example,
the TRAIL-R antibody may be administered with an interferon such as
.gamma.-interferon, to treat a viral infection.
[0145] Agonistic TRAIL-R antibodies may be employed in conjunction
with other agent(s) useful in treating cancer. Examples of such
agents include both proteinaceous and non-proteinaceous drugs, and
radiation therapy. A wide variety of drugs have been employed in
chemotherapy of cancer. Examples include, but are not limited to,
cisplatin, taxol etoposide, Novantrone.RTM. (mitoxantrone),
actinomycin D, camptothecin (or water soluble derivatives thereof
such as irinotecan or topotecan), methotrexate, mitomycin (e.g.,
mitomycin C), dacarbazine (DTIC), 5-fluorouracil and
anti-neoplastic antibiotics such as doxorubicin and daunomycin.
[0146] The TRAIL-R antibody may be co-administered with other
proteinaceous agents for cancer therapy. Examples include various
cytokines that induce a desired immune or other biological
response, interferons such as .gamma.-interferon. TRAIL, and other
antibodies. One such antibody is an agonistic antibody directed
against DR4. which is a receptor protein that binds TRAIL, but is
distinct from the TRAIL-R of the present invention. (See PCT
application WO 98/32856, hereby incorporated by reference and
further discussion of DR4 below.)
[0147] Drugs employed in cancer therapy may have a cytotoxic or
cytostatic effect on cancer cells, or may reduce proliferation of
the malignant cells. 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 such factors as the particular type of cancer and the general
condition of the patient, as is recognized in the pertinent
field.
[0148] An agonistic TRAIL-R antibody may administered alone, or may
be co-administered with one or more anti-cancer agents to a
patient. Co-administration is not limited to simultaneous
administrations but includes treatment regimens in which such an
antibody is administered at least one during a course of treatment
that involves administering at least one other agent to the
patient.
[0149] Agonistic TRAIL-R antibodies may be added to a standard
chemotherapy regimen, in treating a cancer patient. For those
combinations in which the antibody and 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. The antibody may be
co-administered with an amount of an anti-cancer drug that is
effective in enhancing sensitivity of cancer cells to the
antibody.
[0150] One embodiment of the invention is directed to agonistic
TRAIL-R antibodies that are more effective than TRAIL in killing
cancer cells in vivo. Such an antibody exhibits greater anti-cancer
activity, compared to TRAIL, on at least one cancer- cell type. One
example is an antibody that reduces the tumor burden (size or
number of tumors) in a mammal to a greater degree than does an
equivalent dosage of TRAIL. One method for identifying such
antibodies involves administering equal amounts (by weight) of the
antibody and TRAIL to mice harboring tumors arising from human
tumor cells introduced into the mice. Methods for generating tumors
by implanting human cancer cells in mice are well known. Reduction
in tumor size is measured and compared, for mice treated with the
antibody or with TRAIL.
[0151] Especially preferred for in vivo use are antibodies that
effectively induce cell death in vivo without being immobilized or
administered in conjunction with a cross-linking reagent. Neither a
reagent for cross-linking the antibody, nor a reagent for
cross-linking cell surface TRAIL-R need be employed with such
preferred antibodies.
[0152] In one embodiment, an antibody is specific for the TRAIL-R
of the present invention and does not cross-react with other
protein(s). Such an antibody may lack cross-reactivity with other
proteins that bind TRAIL, for example. Several such TRAIL-binding
proteins, which are distinct from the TRAIL-R of the present
invention, have been identified.
[0153] One such TRAIL receptor is the protein designated DR4,
described in Pan et al. (Science 276:111-113, 1997) and PCT
application WO 98/32856. Another TRAIL receptor is described in Pan
et al., supra, Sheridan et al., supra, and Degli-Esposti et al. (J.
Exp. Med. 186:1165, 1997), wherein the receptor is designated TRID,
DcR1, or TRAIL-R3, respectively. A TRAIL receptor designated
TRAIL-R4 is described in Degli-Esposti et al. (Immunity 7:813,
1997). Osteoprotegerin (OPG) also binds TRAIL. Secreted (i.e.,
naturally occurring soluble) human, mouse, and rat osteoprotegerin
are described, and amino acid sequences presented, in Simonet et
al. (Cell 89:309, 1997). OPG in both monomeric and dimeric form, as
well as certain mutants and variants thereof, are disclosed in EP
816,380. Each of the foregoing references describing TRAIL-binding
proteins is incorporated by reference herein.
[0154] As discussed above, the cytoplasmic domain of the TRAIL-R of
the present invention contains a so-called "death domain". Such
domains, found in the cytoplasmic region of certain other receptor
proteins, are associated with transduction of apoptotic signals,
i.e., play a role in initiating intracellular apoptotic signaling
cascades. DR4 likewise contains a functional death domain.
[0155] TRAIL-R3, TRAIL-R4, and OPG lack the functional cytoplasmic
death domains that are associated with the ability to transduce an
apoptotic signal. TRAIL-R3 lacks a cytoplasmic domain, and is
attached to the cell surface by glycosylphosphatidyl-inositol (GPI)
linkage. TRAIL-R4 contains only a partial death domain. OPG lacks a
cytoplasmic domain, and is secreted rather than being attached to
the surface of cells in which it is expressed.
[0156] While not wishing to be bound by theory, one advantage that
a non-cross-reactive antibody specific for TRAIL-R of the present
invention may offer is that it does not bind to "non-signaling"
receptors. Thus, the administered dosage of such an antibody binds
to a receptor capable of mediating cell death. In contrast, TRAIL
is capable of binding to both the signaling and non-signaling
receptors described above. Thus, a portion of a dosage of TRAIL may
bind to proteins that do not transduce apoptotic signals in the
target cells. Non-cross-reactive antibodies would be expected to
have the same advantage over any antibody that is raised against a
signaling receptor but that cross-reacts with a non-signaling
receptor.
[0157] Examples of monoclonal antibodies of the present invention
are described in example 9. Humanized derivatives of the antibodies
described herein, including but not limited to humanized M412 or
M413, are provided,in accordance with the present invention. A
number of procedures for generating such humanized antibodies are
known, including those discussed above. Antibodies that exhibit a
biological activity of M412 or M413 are provided, as are antibodies
that bind to the same epitope as M412 or M4413.
[0158] Whole antibodies may be administered in vivo. Alternatively,
antigen-binding antibody fragments may be employed. Whole
antibodies may be advantageous when an effector function or other
property conferred by the Fc moiety is desired. If prolonging the
half life of the antibody in vivo is desired, a whole antibody may
be chosen.
[0159] Compositions comprising an antibody that is directed against
TRAIL-R, and a physiologically acceptable diluent, excipient, or
carrier, are provided herein. Suitable components of such
compositions are as described above for compositions containing
TRAIL-R proteins.
[0160] Also provided herein are conjugates comprising a detectable
(e.g., diagnostic) or therapeutic agent, attached to an antibody
directed against TRAIL-R. Examples of such agents are presented
above. The conjugates find use in in vitro or in vivo
procedures.
Nucleic Acids
[0161] The present invention provides TRAIL-R nucleic acids. Such
nucleic acids include, but are not limited to, DNA encoding the
peptide described in example 2. Such DNAs can be identified from
knowledge of the genetic code. Other nucleic acids of the present
invention include isolated DNAs comprising the nucleotide sequence
presented in SEQ ID NO:1 or SEQ ID NO:3.
[0162] The present invention provides isolated nucleic acids useful
in the production of TRAIL-R polypeptides, e.g., in the recombinant
expression systems discussed above. Such nucleic acids include, but
are not limited to, the human TRAIL-R DNA of SEQ ID NO:1. Nucleic
acid molecules of the present invention include TRAIL-R DNA in both
single-stranded and double-stranded form, as well as the RNA
complement thereof. TRAIL-R DNA includes, for example, cDNA,
genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and
combinations thereof. Genomic DNA may be isolated by conventional
techniques, e.g., using the cDNA of SEQ ID NO:1 or 3, or a suitable
fragment thereof, as a probe.
[0163] DNAs encoding TRAIL-R in any of the forms contemplated
herein (e.g., full length TRAIL-R or fragments thereof) are
provided. Particular embodiments of TRAIL-R-encoding DNAs include a
DNA encoding the full length human TRAIL-R of SEQ ID NO:2
(including the N-terminal signal peptide), and a DNA encoding a
full length mature human TRAIL-R. Other embodiments include DNA
encoding a soluble TRAIL-R (e.g., encoding the extracellular domain
of the protein of SEQ ID NO:2, either with or without the signal
peptide).
[0164] One embodiment of the invention is directed to -fragments of
TRAIL-R nucleotide sequences comprising at least about 17
contiguous nucleotides of a TRAIL-R DNA sequence. In other
embodiments, a DNA fragment comprises at least 30, or at least 60,
contiguous nucleotides of a TRAIL-R DNA sequence. Nucleic acids
provided herein include DNA and RNA complements of said fragments,
along with both single-stranded and double-stranded forms of the
TRAIL-R DNA.
[0165] Among the uses of TRAIL-R nucleic acid fragments is use as
probes or primers. Using knowledge of the genetic code in
combination with the amino acid sequences set forth in example 2,
sets of degenerate oligonucleotides can be prepared.
[0166] Such oligonucleotides find use as primers, e.g., in
polymerase chain reactions (PCR), whereby TRAIL-R DNA fragments are
isolated and amplified.
[0167] Other useful fragments of the TRAIL-R nucleic acids include
antisense or sense oligonucleotides comprising a single-stranded
nucleic acid sequence (either RNA or DNA) capable of binding to
target TRAIL-R mRNA (sense) or TRAIL-R DNA (antisense) sequences.
Antisense or sense oligonucleotides, according to the present
invention, comprise a fragment of the coding region of TRAIL-R DNA.
Such a fragment generally comprises at least about 14 nucleotides,
preferably from about 14 to about 30 nucleotides. The ability to
derive an antisense or a sense oligonucleotide, based upon a cDNA
sequence encoding 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).
[0168] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block transcription or translation of the target sequence 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-R proteins. 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.
[0169] 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 oligonucleotide for the target nucleotide sequence.
[0170] 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 by using gene transfer vectors
such as Epstein-Barr virus. In a preferred procedure, an antisense
or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic acid sequence is
contacted with the recombinant retroviral vector, either in vivo or
ex vivo. Suitable retroviral vectors include, but are not limited
to, those derived from the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
[0171] Sense or antisense oligonucleotides also may 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.
[0172] 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.
[0173] The following examples are provided to further illustrate
particular embodiments of the invention, and are not to be
construed as limiting the scope of the present invention.
Example 1
Purification of TRAIL-R Protein
[0174] A human TRAIL receptor (TRAIL-R) protein was prepared by the
following procedure. Trail-R was isolated from the cell membranes
of Jurkat cells, a human acute T leukemia cell line. Jurkat cells
were chosen because a specific band can be affinity precipitated
from surface-biotinylated Jurkat cells, using Flag.RTM.-TRAIL
covalently coupled to affi-gel beads (Biorad Laboratories,
Richmond, Calif.). The precipitated band has a molecular weight of
about 52 kD. A minor specific band of about 42 kD also was present,
possibly accounting for a proteolytic breakdown product or a less
glycosylated form of TRAIL-R.
[0175] Approximately 50 billion Jurkat cells were harvested by
centrifugation (80 ml of cell pellet), washed once with PBS, then
shock frozen on liquid nitrogen. Plasma membranes were isolated
according to method number three described in Maeda et al.,
Biochim. et Biophys. Acta, 731:115, 1983; hereby incorporated by
reference) with five modifications: [0176] 1. The following
protease inhibitors were included in all solutions at the indicated
concentrations: Aprotinin, 150 nM; EDTA, 5 mM; Leupeptin, 1 .mu.M;
pA-PMSF, 20 .mu.M; Pefabloc, 500 .mu.M; Pepstatin A, 1 .mu.M; PMSF,
500 .mu.M. [0177] 2. Dithiothreitol was not used. [0178] 3. DNAase
was not used in the homogenization solution. [0179] 4. 1.25 ml of
homogenization buffer was used per ml of cell pellet. [0180] 5. The
homogenization was accomplished by five passages through a ground
glass dounce homogenizer.
[0181] After isolation of the cell membranes, proteins were
solubilize(d by resuspending the isolated membranes in 50 ml PBS
containing 1% octylglucoside and all of the above mentioned
protease inhibitors at the above indicated concentrations. The
resulting solution was then repeatedly vortexed during a
thirty-minute incubation at 4.degree. C. The solution was then
centrifuged at 20,000 rpm in an SW28 rotor in an LE-80 Beckman
ultracentrifuge (Beckman Instruments. Inc., Palo Alto, Calif.) at
4.degree. C. for 30 minutes to obtain the supernatant (the membrane
extract).
Chromatography
[0182] The first step of purification of TRAIL-R out of the
membrane extract prepared above was affinity chromatography. The
membrane extract was first applied to an anti-Flag.RTM. M2 affi-gel
column (10 in of monoclonal antibody M2 coupled to 2 ml of Affi-gel
beads) to adsorb any nonspecifically binding material. The
flow-through was then applied to a Flag.RTM.-TRAIL affi-gel column
(10 mg of recombinant protein coupled to 1 ml of affi-gel
beads).
[0183] The Affi-gel support is an N-hydroxysuccinimide ester of a
derivatized, crosslinked agarose gel bead (available from Biorad
Laboratories, Richmond, Calif.).
[0184] As discussed above, the Flag.RTM. peptide,
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, provides an epitope reversibly
bound by specific monoclonal antibodies, enabling rapid assay and
facile purification of expressed recombinant protein. M2 is a
monoclonal antibody that binds Flag.RTM.. Monoclonal antibodies
that bind the Flag.RTM. peptide, as well as other reagents for
preparing and using Flag.RTM. fusion proteins, are available from
Eastman Kodak Co., Scientific Imaging Systems Division, New Haven,
Conn. Preparation of Flag.RTM.-TRAIL fusion proteins (comprising
Flag.RTM. fused to a soluble TRAIL polypeptide) is further
described in PCT application WO 97/01633, hereby incorporated by
reference.
[0185] The column was washed with 25 ml of each of the following
buffers, in the order indicated: [0186] 1. PBS containing 1%
octylglucoside [0187] 2. PBS [0188] 3. PBS containing an additional
200 mM NaCl [0189] 4. PBS
[0190] The bound material was eluted with 50 mM Na Citrate (pH 3)
in 1 ml fractions and immediately neutralized with 300 .mu.l of 1 M
Tris-HCl (pH 8.5) per fraction. The TRAIL-binding activity of each
fraction was determined by a TRAIL-R-specific ELISA as described
below. Fractions with high TRAIL-binding activity were pooled,
brought to 0.1% Trifluoroacetic acid (TFA), and subsequently
chromatographed on a capillary reversed-phase HPLC column [500
.mu.m internal diameter.times.25 cm fused silicone capillary column
packed with 5 .mu.m Vydac C.sub.4 material (Vydac, Hesperia,
Calif.)] using a linear gradient (2% per minute) from 0% to 100%
acetonitrile in water containing 0.1% TFA. Fractions containing
high TRAIL-binding activity are then determined as above. pooled,
and, if desired lyophilized.
TRAIL-R-Specific ELISA:
[0191] Serial dilutions of TRAIL-R-containing samples (in 50 mN/m
NaHCO.sub.3, brought to pH 9 with NaOH) were coated onto
Linbro/Titertek 96 well flat bottom E.I.A. microtitration plates
(ICN Biomedicals Inc., Aurora, Ohio) at 100 .mu.l/well. After
incubation at 4.degree. C. for 16 hours, the wells were washed six
times with 200 .mu.l PBS containing 0.05% Tween-20 (PBS-Tween). The
wells were then incubated with Flag.RTM.-TRAIL at 1 .mu.g/ml in
PBS-Tween with 5% fetal calf serum (FCS) for 90 minutes (100 .mu.l
per well), followed by washing as above. Next, each well was
incubated with the anti-Flag.RTM.; monoclonal antibody M2 at 1
.mu.g/ml in PBS-Tween containing 5% FCS for 90 minutes (100 .mu.l
per well), followed by washing as above. Subsequently, wells were
incubated with a polyclonal goat anti-mIgG1-specific horseradish
peroxidase-conjugated antibody (a 1:5000 dilution of the commercial
stock in PBS-Tween containing 5% FCS) for 90 minutes (100 .mu.l per
well). The HRP-conjugated antibody was obtained from Southern
Biotechnology Associates, Inc., Birmingham, Ala. Wells then were
washed six times, as above.
[0192] For development of the ELISA, a substrate mix [100 .mu.l per
well of a 1:1 premix of the TMB Peroxidase Substrate and Peroxidase
Solution B (Kirkegaard Perry Laboratories, Gaithersburg, Md.)] was
added to the wells. After sufficient color reaction, the enzymatic
reaction was terminated by addition of 2 N H.sub.2SO.sub.4 (50
.mu.l per well). Color intensity (indicating TRAIL-binding
activity) was determined by measuring extinction at 450 nm on a V
Max plate reader (Molecular Devices. Sunnyvale, Calif.).
Example 2
Amino Acid Sequence
[0193] (a) TRAIL-R Purified from Jurkat Cells
[0194] TRAIL-R protein isolated from Jurkat cells was digested with
trypsin, using conventional procedures. Amino acid sequence
analysis was conducted on one of the peptide fragments produced by
the tryptic digest. The fragment was found to contain the following
sequence which corresponds to amino acids 327 to 333 of the
sequence presented in SEQ ID NO:2: VPANEGD.
[0195] (b) TRAIL-R Purified from PS-1 Cells
[0196] TRAIL-R protein was also isolated from PS-1 cells. PS-1 is a
human B cell line that spontaneously arose after lethal irradiation
of human peripheral blood lymphocytes (PBLs). The TRAIL-R protein
was digested with trypsin, using conventional procedures. Amino
acid sequence analysis was conducted on peptide fragments that
resulted from the tryptic digest. One of the fragments was found to
contain the following sequence, which, like the fragment presented
in (a) corresponds to amino acids 327 to 333 of the sequence
presented in SEQ ID NO:2: VPANEGD.
Example 3
DNA and Amino Acid Sequences
[0197] The amino acid sequence of additional tryptic digest peptide
fragments of TRAIL-R was determined. Degenerate oligonucleotides
based upon the amino acid sequence of two of the peptides, were
prepared. A TRAIL-R DNA fragment was isolated and amplified by
polymerase chain reaction (PCR), using the degenerate
oligonucleotides as 5' and 3' primers. The PCR was conducted
according to conventional procedures, using cDNA derived from the
PS-1 cell line described in example 2 as the template. The
nucleotide sequence of the isolated TRAIL-R DNA fragment (excluding
portions corresponding to part of the primers), and the amino acid
sequence encoded thereby, are presented in FIG. 1 (SEQ ID NOS:3 and
4). The sequence of the entire TRAIL-R DNA fragment isolated by PCR
corresponds to nucleotides 988 to 1164 of SEQ ID NO:1. which encode
amino acids 330 to 388 of SEQ ID NO:2.
[0198] The amino acid sequence in SEQ ID NO:4 bears significant
homology to the so-called death domains found in certain other
receptors. The cytoplasmic region of Fas and TNF receptor type I
each contain a death domain, which is associated with transduction
of an apoptotic signal (Tartaglia et al. Cell 74:845, 1993; Itoh
and Nagata, J. Biol. Chem. 268:10932, 1993). Thus, the sequence
presented in SEQ ID NO:4 is believed to be found within the
cytoplasmic domain of TRAIL-R.
[0199] A probe derived from the fragment isolated above was used to
screen a cDNA library (human foreskin fibroblast-derived cDNA in
.lamda.gt10 vector), and a human TRAIL-R cDNA was isolated. The
nucleotide sequence of the coding region of this cDNA is presented
in SEQ ID NO:1, and the amino acid sequence encoded thereby is
shown in SEQ ID NO:2.
Example 4
Monoclonal Antibodies that Bind TRAIL-R
[0200] This example illustrates a method for preparing monoclonal
antibodies that bind TRAIL-R. Suitable immunogens that may be
employed in generating such antibodies include, but are not limited
to, purified TRAIL-R protein or an immunogenic fragment thereof
such as the extracellular domain, or fusion proteins containing
TRAIL-R (e.g., a soluble TRAIL-R/Fc fusion protein).
[0201] Purified TRAIL-R can be used to generate monoclonal
antibodies immunoreactive therewith, using conventional techniques
such as those described in U.S. Pat. No. 4,411,993. Briefly, mice
are immunized with TRAIL-R 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-R
emulsified in incomplete Freund's adjuvant. Mice are periodically
boosted thereafter on a weekly to biweekly immunization schedule.
Serum samples are periodically taken by retro-orbital bleeding or
tail-tip excision to test for TRAIL-R antibodies by dot blot assay,
ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition of TRAIL
binding.
[0202] Following detection of an appropriate antibody titer,
positive animals are provided one last intravenous injection of
TRAIL-R 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, e.g., NS1 or preferably P3x63Ag8.653
(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.
[0203] The hybridoma cells are screened by ELISA for reactivity
against purified TRAIL-R by adaptations of the techniques disclosed
in Engvall et al., Immunochem. 8:871, 1971 and in U.S. Pat. No.
4,703,004. A preferred screening technique is the antibody capture
technique described in Beckmann et al., (J. Immunol. 144:4212,
1990) Positive hybridoma cells can be injected intraperitoneally
into syngeneic BALB/c mice to produce ascites containing high
concentrations of anti-TRAIL-R 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 C can also be used, as can
affinity chromatography based upon binding to TRAIL-R.
Example 5
Northern Blot Analysis
[0204] The tissue distribution of TRAIL-R mRNA was investigated by
Northern blot analysis, as follows. An aliquot of a radiolabeled
probe (the same radiolabeled probe used to screen the cDNA library
in example 3) was added to two different human multiple tissue
Northern blots (Clontech, Palo Alto, Calif.; Biochain, Palo Alto,
Calif.). Hybridization was conducted overnight at 63.degree. C in
50% formamide as previously described (March et al. Nature
315:641-647, 1985). The blots then were washed with 2.times.SSC.
0.1% SDS at 68.degree. C. for 30 minutes. A
glycerol-aldehyde-phosphate dehydrogenase (GAPDII) specific probe
was used to standardize for RNA loadings.
[0205] A single transcript of 4.4 kilobases (kb) was present in all
tissues examined, including spleen, thymus, peripheral blood
lymphocytes (PBLs), prostate, testis, ovary, uterus, placenta, and
multiple tissues along the gastro-intestinal tract (including
esophagus, stomach, duodenum, jejunum/ileum, colon, rectum, and
small intestine). The cells and tissues with the highest levels of
TRAIL-R mRNA are PBLs, spleen, and ovary, as shown by comparison to
control hybridizations with a GAPDH-specific probe.
Example 6
Binding Assay
[0206] Full length human TRAIL-R was expressed and tested for the
ability to bind TRAIL. The binding assay was conducted as
follows.
[0207] A fusion protein comprising a leucine zipper peptide fused
to the N-terminus of a soluble TRAIL, polypeptide (LZ-TRAIL) was
employed in the assay. An expression construct was prepared,
essentially as described for preparation of the Flag.RTM.-TRAIL
expression construct in Wiley et al. (i Immunity, 3:673-682, 1995;
hereby incorporated by reference), except that DNA encoding the
Flag.RTM. peptide was replaced with a sequence encoding a modified
leucine zipper that allows for trimerization. The construct, in
expression vector pDC409. encoded a leader sequence derived from
human cytomegalovirus, followed by the leucine zipper moiety fused
to the N-terminus of a soluble TRAIL, polypeptide. The TRAIL
polypeptide comprised amino acids 95-281 of human TRAIL (a fragment
of the extracellular domain), as described in Wiley et al. (supra).
The LZ-TRAIL was expressed in CHO cells, and purified from the
culture supernatant.
[0208] The expression vector designated pDC409 is a mammalian
expression vector derived from the pDC406 vector described in
McMahan et al. (EMBO J. 10:2821-2832, 1991; hereby incorporated by
reference). Features added to pDC409 (compared to pDC406) include
additional unique restriction sites in the multiple cloning site
(mcs); three stop codons (one in each reading frame) positioned
downstream of the mcs; and a T7 polymerase promoter, downstream of
the mcs, that faciliates sequencing of DNA inserted into the
mcs.
[0209] For expression of full length human TRAIL-R protein, the
entire coding region (i.e., the DNA sequence presented in SEQ ID
NO:1) was amplified by polymerase chain reaction (PCR). The
template employed in the PCR was the cDNA clone isolated from a
human foreskin fibroblast cDNA library, as described in example 3.
The isolated and amplified DNA was inserted into the expression
vector pDC409, to yield a construct designated pDC409-TRAIL-R.
[0210] CrmA protein was employed to inhibit apoptosis of host cells
expressing recombinant TRAIL-R, as discussed above and in example
8. An expression vector designated pDC409-CrmA contained DNA
encoding poxvirus CrmA in pDC409. The 38-kilodalton cowpox-derived
protein that was subsequently designated CrmA is described in
Pickup et al. (Proc. Natl. Acad. Sci. USA 83:7698-7702, 1986;
hereby incorporated by reference).
[0211] CV-1/EBNA cells were co-transfected with pDC409-TRAIL-R
together with pDC409-CrmA, or with pDC409-CrmA alone. The cells
were cultured in DMEM supplemented with 10% fetal bovine serum,
penicillin, streptomycin, and glutamine. 48 hours after
transfection, cells were detached non-enzymatically and incubated
with LZ-TRAIL (5 .mu.g/ml), a biotinylated anti-L/Z monoclonal
antibody (5 .mu.g/ml), and phycoerythrin-conjugated streptavidin
1:400)), before analysis by fluorescence-activated cell scanning
(FACS). The cytometric analysis was conducted on a FACscan (Beckton
Dickinson, San Jose, Calif.).
[0212] The CV-1/EBNA cells co-transfected with vectors encoding
TRAIL-R and CrmA showed significantly enhanced binding of LZ-TRAIL,
compared to the cells transfected with the CrmA-encoding vector
alone.
Example 7
TRAIL-R Blocks TRAIL-Induced Apoptosis of Target Cells
[0213] TRAIL-R was tested for the ability to block TRAIL-induced
apoptosis of Jurkat cells. The TRAIL-R employed in the assay was in
the form of a fusion protein designated sTRAIL-R/Fc, which
comprised the extracellular domain of human TRAIL-R, fused to the
N-terminus of an Fc polypeptide derived from human IgG1.
[0214] CV1-EBNA cells were transfected with a recombinant
expression vector comprising a gene fusion encoding the sTRAIL-R/Fc
protein, in the pDC409 vector described in example 6, and cultured
to allow expression of the fusion protein. The sTRAIL-R/Fc fusion
protein was recovered from the culture supernatant. Procedures for
fusing DNA encoding an IgG1 Fc polypeptide to DNA encoding a
heterologous protein are described in Smith et al., (Cell
73:1349-1360, 1993); analogous procedures were employed herein.
[0215] A fusion protein designated TNF-R2-Fc-employed as a control,
comprised the extracellular domain of the TNF receptor protein
known as p75 or p80 TNF-R (Smith et al., Science 248:1019-1023,
1990: Smith et al. Cell 76:959-962 1994), fused to an Fc
polypeptide. A mouse monoclonal antibody that is a blocking
antibody directed against human TRAIL, was employed in the assay as
well.
[0216] Jurkat cells were incubated with varying or constant
concentrations of LZ-TRAIL (the LZ-TRAIL fusion protein described
in example 6), in the absence or presence of varying concentrations
of sTRAIL-R-Fc. TNF-R2-1-c, or the TRAIL-specific monoclonal
antibody. Cell death was quantitated using an MTT cell viability
assay (MTT is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide), as previously described (Mosmann, J. Immunol. Methods
65:55-63, 1983). The results are shown in FIG. 2, which presents
the percent cell death for Jurkat cells that were untreated
(.DELTA.) or were treated with varying (.tangle-solidup.) or
constant (.smallcircle. .quadrature..box-solid.) concentrations of
LZ-TRAIL (13 ng/ml) in the absence ( ) or presence of varying
concentrations of TRAIL-R2-Fc (.box-solid.), TNF-R2-Fc
(.quadrature.), or the anti-TRAIL antibody (.smallcircle.). Varying
concentrations for all substances started at 10 .mu.g/ml and were
serially diluted.
[0217] The anti-TRAIL monoclonal antibody and sTRAIL-R/Fc each
blocked TRAIL-induced apoptosis in a dose dependent fashion,
whereas TNFR2-Fc did not. Thus, the extracellular domain of TRAIL-R
is capable of binding to TRAIL and inhibiting TRAIL-mediated
apoptosis of target cells.
Example 8
TRAIL-R-Induced Apoptosis is Blocked by Caspase Inhibitors and
FADD-DN
[0218] CV-1/EBNA cells were transfected, by the DEAE-dextran
method, with expression plasmids for TRAIL-R (pDC409-TRAIL-R),
together with a threefold excess of empty expression vector
(pDC409) in the presence or absence of z-VAD-fink (10 .mu.M; in the
culture medium), or together with a threefold excess of expression
vector pDC409-CrmA, pDC409-p35, or pDC409-FADD-DN. In addition, 400
ng/slide of an expression vector for the E. coli lacz gene was
co-transfected together with all DNA mixes. The transfected cells
were cultured in chambers mounted on slides.
[0219] The mammalian expression vector pDC409, and the
pDC409-TRAIL-R vector encoding full length human TRAIL-R, are
described in example 6. The tripeptide derivative zVAD-fmk
(benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone) is available
from Enzyme System Products, Dublin, Calif.
[0220] The 38-kilodalton cowpox-derived protein that was
subsequently designated CrmA is described in Pickup et al. (Proc.
Natl. Acad. Sci. USA 83:7698-7702, 1986; hereby incorporated by
reference). Sequence information for CrmA is presented in FIG. 4 of
Pickup et al., supra.
[0221] A 35-kilodalton protein encoded by Autographa californica
nuclear polyhedrosis virus, a baculovirus, is described in Friesen
and Miller (J. Virol. 61:2264-2272, 1987; hereby incorporated by
reference). Sequence information for this protein, designated
baculovirus p35 herein, is presented in FIG. 5 of Friesen and
Miller, supra.
[0222] FADD (also designated MORT1) is described in Boldin et al.
(J. Biol. Chem. 270:7795-7798, 1995; hereby incorporated by
reference). The protein referred to as FADD-DN (FADD dominant
negative) is a C-terminal fragment of FADD that includes the death
domain. DNA encoding FADD-DN, fused to an N-terminal Flag.RTM.
epitope tag (described above), was inserted into the pDC409
expression vector described in example 6, to form pDC409-FADD-DN.
The FADD-DN polypeptide corresponds to amino acids 117 through 245
of the MORT1 amino acid sequence presented in Boldin et al.,
supra.
[0223] 48 hours after transfection, cells were washed with PBS,
fixed with glutaraldehyde and incubated with X-gal
(5-bromo-4-chloro-3-indoxyl-.beta.-D-galactopyranoside). Cells
expressing .beta.-galactosidase stain blue. A decrease in the
percentage of stained cells indicates loss of .beta.-galactosidase
expression, and correlates with death of cells that express the
protein(s) co-transfected with the lacz gene.
[0224] The results are presented in FIG. 3, wherein the values
plotted represent the mean and standard deviation of at least three
separate experiments. Poxvirus CrmA, baculovirus p35. FADD-DN, and
z-VAD-fmk each effectively reduced death of transfected cells
expressing TRAIL-R.
Example 9
Agonistic Monoclonal Antibodies Specific for TRAIL-R
[0225] BALB/c mice (The Jackson Laboratory, Bar Harbor, Me.) were
immunized with a TRAIL-R immunogen in Titermax (Cytrx Corporation,
Norcross, Ga.). The immunogen was a purified fusion protein
consisting of the extracellular domain of human TRAIL-R fused to
the constant region of a human IgG1 (huTRAIL-R:Fc). Mice were
boosted three times, and spleen cells were fused with the murine
myeloma cell line NS1 in the presence of 50% polyethylene glycol in
PBS followed by culture in DMEM/HAT and DMEM/HT selective
media.
[0226] Supernatants from positive wells were tested for the ability
to bind TRAIL-R in an ELISA (cell-based ELISA using CV1 cells
transfected with TRAIL-R cDNA) and for reactivity to huTRAIL-R:Fc
in Western blots. Hybridomas that produced antibodies that bound to
huTRAIL-R:Fc but not human IgG1 were cloned by three rounds of
limiting dilution. Monoclonal antibodies (MAbs) were purified by
protein A affinity chromatography.
[0227] Resulting MAbs were tested in a slide binding assay for
cross-reactivity with other TRAIL receptors (DR4, TRAIL-R3. and
TRAIL-R4, described above). MAbs specific for TRAIL-R, in that
binding to the other three receptors was not detected, were
identified. Two such antibodies are designated M412 and M413; both
are isotype IgG1 MAbs.
[0228] M412 and M413 exhibited agonistic properties in vivo. Both
MAbs induced death of cancer cells, when administered to mice
implanted with human tumor cells (including but not limited to the
colon carcinoma cells designated COLO 205, available from American
Type Culture Collection).
[0229] M412 is a preferred agonistic anti-TRAIL-R monoclonal
antibody. M412 has exhibited cancer cell-killing activity exceeding
that of TRAIL in vivo, in mice bearing human tumor cells. The human
colon carcinoma cells COLO 205 are an example of cancer cells on
which M412 exhibited cell killing activity superior to that of
TRAIL, in mice bearing the COLO 205 cells. Whole M412 MAb was
administered by IP injection to the mice: no cross-linking reagent
was co-administered. The TRAIL employed in the experiment was a
soluble human TRAIL with a leucine zipper peptide fused to the
N-terminus thereof (LZ-huTRAIL).
[0230] M412 and M413 were tested for ability to inhibit TRAIL
binding to TRAIL-R. One experiment employed a cell line that
expresses TRAIL-R but not DR4 or TRAIL-R3. Another assay was a
modified ELISA that employed a soluble human TRAIL-R/Fc fusion
protein. M413 inhibited LZ-huTRAIL binding in both experiments. In
contrast. M412 did not inhibit binding of LZ-huTRAIL to
TRAIL-R.
Example 10
Generation of Cmu Targeted Mice
[0231] Construction of a CMD targeting vector. The plasmid pICEmu
contains an EcoRI/XhoI fragment of the murine Ig heavy chain locus,
spanning the mu gene, that was obtained from a Balb/C genomic
lambda phage library (Marcu et al. (Cell 22: 187. 1980). This
genomic fragment was subcloned into the XhoI/EcoRI sites of the
plasmid pICEMI9H (Marsh et al; Gene 32, 481-485, 1984). The heavy
chain sequences included in pICEmu extend downstream of the EcoRI
site located just 3 of the mu intronic enhancer, to the XhoI site
located approximately 1 kb downstream of the last transmembrane
exon of the mu gene; however, much of the mu switch repeat region
has been deleted by passage in E. coli.
[0232] The targeting vector was constructed as follows (see FIG.
4). A 1.3 kb HindIII/SmaI fragment was excised from pICEmu and
subcloned into HindIII/SmaI digested pBluescript (Stratagene, La
Jolla, Calif.). This pICEmu fragment extends from the HindIII site
located approximately 1 kb 5' of Cmu1 to the SmaI site located
within Cmu1. The resulting plasmid was digested with SmaI/SpeI and
the approximately 4 kb SmaI/XbaI fragment from pICEmu, extending
from the Sma I site in Cmu1 3' to the XbaI site located just
downstream of the last Cmu exon, was inserted. The resulting
plasmid, pTAR1, was linearized at the SmaI site, and a neo
expression cassette inserted. This cassette consists of the neo
gene under the transcriptional control of the mouse
phosphoglycerate kinase (pgk) promoter (XbaI/TaqI fragment; Adra et
al. (1987) Gene 60: 65-74) and containing the pgk polyadenylation
site (PvuII/HindIII fragment; Boer et al. (1990) Biochemical
Genetics 28: 299-308). This cassette was obtained from the plasmid
pKJ1 (described by Tybulewicz et al. (1991) Cell 65: 1153-1163)
from which the neo cassette was excised as an EcoRI/HindIII
fragment and subcloned into EcoRI/HindIII digested pGEM-7Zf (+) to
generate pGEM-7 (KJ1). The neo cassette was excised from pGEM-7
(KJ1) by EcoRI/SalI digestion, blunt ended and subcloned into the
SmaI site of the plasmid pTAR1, in the opposite orientation of the
genomic Cmu sequences.
[0233] The resulting plasmid was linearized with Not I, and a
herpes simplex virus thymidine kinase (tk) cassette was inserted to
allow for enrichment of ES clones bearing homologous recombinants,
as described by Mansour et al. (1988) Nature 336: 348-352. This
cassette consists of the coding sequences of the tk gene bracketed
by the mouse pgk promoter and polyadenylation site, as described by
Tybulewicz et al. (1991) Cell 65: 1153-1163.
[0234] The resulting CMD targeting vector contains a total of
approximately 5.3 kb of homology to the heavy chain locus and is
designed to generate a mutant mu gene into which has been inserted
a neo expression cassette in the unique SmaI site of the first Cmu
exon. The targeting vector was linearized with PvuI, which cuts
within plasmid sequences, prior to electroporation into ES
cells.
[0235] Generation and analysis of targeted ES cells. AB-1 ES cells
(McMahon, A. P. and Bradley. A. (1990) Cell 62: 1073-1085) were
grown on mitotically inactive SNL76/7 cell feeder layers (ibid.),
essentially as described in Teratocarcinomas and Embryonic Stem
Cells: a Practical Approach, E. J. Robertson, Ed., Oxford: IRE
Press. 1987, pp. 71-112. The linearized CMD targeting vector was
electroporated into AB-1 cells by the methods described in Hasty et
al. (1I991) Nature 350: 243-246. Electroporated cells were plated
into 100 mm dishes at a density of 1-2.times.10.sup.6 cells/dish.
After 24 hours, G4 18 (200 micrograms/ml of active component) and
FIAU (5.times.10.sup.-7 M) were added to the medium, and
drug-resistant clones were allowed to develop over 8-9 days. Clones
were picked, trypsinized, divided into two portions, and further
expanded. Half of the cells derived from each clone were then
frozen and the other half analyzed for homologous recombination
between vector and target sequences.
[0236] DNA analysis was carried out by Southern blot hybridization.
DNA was isolated from the clones as described by Laird et al.
(1991) Nucleic Acids Res. 19:4293). Isolated genomic DNA was
digested with SpeI and probed with a 915 bp Sac fragment, probe A
(FIG. 4C), which hybridizes to a sequence between the mu intronic
enhancer and the mu switch region. Probe A detects a 9.9 kb SpeI
fragment from the wild type locus, and a diagnostic 7.6 kb band
from a mu locus which has homologously recombined with the CMD
targeting vector (the neo expression cassette contains a SpeI
site).
[0237] Of 1132 G418 and FIAU resistant clones screened by Southern
blot analysis, 3 displayed the 7.6 kb Spe I band indicative of
homologous recombination at the mu locus. These 3 clones were
further digested with the enzymes BglI, BstXI, and EcoRI to verify
that the vector integrated homologously into the mu gene. When
hybridized with probe A. Southern blots of wild type DNA digested
with BglI, BstXI, or EcoRI produce fragments of 15.7, 7.3, and 12.5
kb, respectively, whereas the presence of a targeted mu allele is
indicated by fragments of 7.7, 6.6, and 14.3 kb, respectively. All
3 positive clones detected by the SpeI digest showed the expected
BglI, BstXI, and EcoRI restriction fragments diagnostic of
insertion of the neo cassette into the Cmu1 exon.
[0238] Generation of mice bearing the mutated mu gene. The three
targeted ES clones, designated number 264, 272, and 408, were
thawed and injected into C57BL/6J blastocysts as described by A.
Bradley in Teratocarcinomas and Embryonic Stem Cells: a Practical
Approach, E. J. Robertson. Ed., Oxford: IRL Press, 987, pp.
113-151. Injected blastocysts were transferred into the uteri of
pseudopregnant females to generate chimeric mice representing a
mixture of cells derived from the input ES cells and the host
blastocyst. The extent of ES cell contribution to the chimera can
be visually estimated by the amount of agouti coat coloration,
derived from the ES cell line, on the black C57BL/6J background.
Clones 272 and 408 produced only low percentage chimeras (i.e. low
percentage of agouti pigmentation) but clone 264 produced high
percentage male chimeras. These chimeras were bred with C57BL/6J
females and agouti offspring were generated, indicative of germline
transmission of the ES cell genome. Screening for the targeted mu
gene was carried out by Southern blot analysis of BglI digested DNA
from tail biopsies (as described above for analysis of ES cell
DNA). Approximately 50% of the agouti offspring showed a
hybridizing BglI band of 7.7 kb in addition to the wild type band
of 1 5.7 kb, demonstrating a germline transmission of the targeted
mu gene.
[0239] Analysis of transgenic mice for functional inactivation of
mu gene. To determine whether the insertion of the neo cassette
into Cmu1 has inactivated the Ig heavy chain gene, a clone 264
chimera was bred with a mouse homozygous for the JHD mutation,
which inactivates heavy chain expression as a result of deletion of
the JH gene segments (Chen et al, (1993) Immunol. 5: 647-656). Four
agouti offspring were generated. Serum was obtained firm these
animals at the age of 1 month and assayed by ELISA for the presence
of murine IgM. Two of the four offspring were completely lacking
IgM (Table 1). Genotyping of the four animals by Southern blot
analysis of DNA from tail biopsies by BglI digestion and
hybridization with probe A (FIG. 1), and by StuI digestion and
hybridization with a 475 bp EcoRI/Stul fragment (ibid.)
demonstrated that the animals which fail to express serum IgM are
those in which one allele of the heavy chain locus carries the JHD
mutation, the other allele the Cmu1 mutation. Mice heterozygous for
the JHD mutation display wild type levels of serum Ig. These data
demonstrate that the Cmu1 mutation inactivates expression of the mu
gene.
[0240] Table 1 presents the level of serum IgM, detected by ELISA,
for mice candying both the CMD and JHD mutations (CMD/JHD), for
mice heterozygous for the JHD mutation (+/JHD), for wild type
(129Sv.times.C57BL/6J)F1 mice (+/+), and for B cell deficient mice
homozygous for the JHD mutation (JHD/JHD).
TABLE-US-00001 TABLE 1 Serum IgM Mouse (micrograms/ml) Ig H chain
genotype 42 <0.002 CMD/JHD 43 196 +/JHD 44 <0.002 CMD/JHD 45
174 +/JHD 129 .times. BL6 F1 153 +/+ JHD <0.002 JHD/JHD
Example 11
Generation of HCo12 Transgenic Mice
[0241] The HCo12 human heavy chain transgene. The HCo12 transgene
was generated by coinjection of the 80 kb insert of pHC2 (Taylor et
al., 1994, Int. Immunol., 6: 579-591) and the 25 kb insert of pVx6.
The plasmid pVx6 was constructed as described below.
[0242] An 8.5 kb HindIII/SalI DNA fragment, comprising the germline
human VH1-18 (DP-14) gene together with approximately 2.5 kb of 5'
flanking, and 5 kb of 3' flanking genomic sequence was subcloned
into the plasmid vector pSP72 (Promega, Madison, Wis.) to generate
the plasmid p343.7.16. A 7 kb BamHI/HindIII DNA fragment,
comprising the germline human VH5-51 (DP-73) gene together with
approximately 5 kb of 5' flanking and 1 kb of 3' flanking genomic
sequence, was cloned into the pBR322 based plasmid cloning vector
pGP1f (Taylor et al. 1992, Nucleic Acids Res. 20: 6287-6295), to
generate the plasmid p251f
[0243] A new cloning vector derived from pGP1f, pGP1k (the sequence
of which is presented in FIGS. 5A and 5B), was digested with
EcoRV/BamHI, and ligated to a 10 kb EcoRV/BamHI DNA fragment,
comprising the germline human VH3-23 (DP47) gene together with
approximately 4 kb of 5 flanking and 5 kb of 3' flanking genomic
sequence. The resulting plasmid, p112.2RR.7, was digested with
BamHI/SalI and ligated with the the 7 kb purified BamHI/SalI insert
of p251f. The resulting plasmid, pVx4. was digested with XhoI and
ligated with the 8.5 kb XhoI/SalI insert of p343.7.16. A clone was
obtained with the VH1-18 gene in the same orientation as the other
two V genes. This clone, designated pVx6, was then digested with
NotI and the purified 26 kb insert coinjected, together with the
purified 50 kb NotI insert of pHC2 at a 1:1 molar ratio, into the
pronuclei of one-half day (C57BL/6J.times.DBA/2J)F2 embryos as
described by Hogan et al. (B. Hogan et al., Manipulating the Mouse
Embryo, A Laboratory Manual, 2.sup.nd edition, 1994, Cold Spring
Harbor Laboratory Press, Plainview N.Y.).
[0244] Three independent lines of transgenic mice comprising
sequences from both Vx6 and HC2 were established from mice that
developed from the injected embryos. These lines are designated
(HCo12)14881, (HCo12)15053, and (HCo12)15087. Each of the three
lines were then bred with mice comprising the CMD mutation
described in Example 10, the JKD mutation (Chen et al. 1993, EMBO
J. 12: 811-820), and the (KCo5)9272 transgene (Fishwild et al.
1996, Nature Biotechnology 14: 845-851). The resulting mice express
human heavy and kappa light chain transgenes in a background
homozygous for disruption of the endogenous mouse heavy and kappa
light chain loci.
Example 12
Generation of Human IgG Kappa Anti-TRAIL-R Monoclonal
Antibodies
[0245] Transgenic mice. Two different strains of mice are used to
generate Trail-R-reactive monoclonal antibodies. Strain ((CMD)++;
(JKD)++; (HCo7)11952+/++; (KCo5)9272+/++), and strain ((CMD)++;
(JKD)++; (HCo12)15087+/++; (KCo5)9272+/++). Each of these strains
are homozygous for disruptions of the endogenous heavy chain (CMD)
and kappa light chain (JKD) loci. Both strains also comprise a
human kappa light chain transgene (HCo7), with individual animals
either hemizygous or homozygous for insertion #11952. The two
strains differ in the human heavy chain transgene used. Mice were
hemizygous or homozygous for either the HCo7 or the HCo12
transgene. The CMD mutation is described above in Example 10. The
generation of (HCo12)15087 mice is described in Example 11. The JKD
mutation (Chen et al. 1993, EMBO J. 12: 811-820) and the (KCo5)9272
(Fishwild et al. 1996, Nature Biotechnology 14: 845-851) and
(HCo7)11952 mice, are described in U.S. Pat. No. 5,770,429, which
is hereby incorporated by reference.
[0246] Immunization. Transgenic mice are initially immunized i.p.
with 10-50 ug TRAIL-R protein, such as a soluble TRAIL-R fragment,
in adjuvant (either complete Freund's or Ribi). Immunized mice are
subsequently boosted every 2 to 4 weeks i.p. with TRAIL-R in
incomplete Freund's adjuvant. Animals are kept on protocol for 2 to
5 months. Prior to fusion, animals are boosted i.v. on days -3 and
-2 with 10 to 20 ug TRAIL-R immunogen. Some animals also receive
antigen i.v. on day -4.
[0247] Fusions. Spleen cells harvested from the immunized mice are
fused to mouse myeloma cells (line P3 X63 Ag8.6.53, ATCC CRL 1580,
or SP2/0-Ag14, ATCC CRL 1581) by standard procedures (Harlow and
Lane, 1988. Antibodies A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor N.Y.; Kennett et al. 1980,
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analysis. Plenum, New York: Oi and Hertzenberg, 1980,
Immunoglobulin Producing Hybrid Cell Lines, in Selected Methods In
Cellular Immunology, ed. Mishell and Shiigi, pp. 357-372. Freeman,
San Francisco). Cells are cultured in DMEM, 10% FBS, OPI (Sigma
O-5003), BME (Gibco 21985-023), 3% Origen Hybridoma Cloning Factor
(Igen IG50-0615), and 5% P388d1 (ATCC TIB 63) conditioned media.
HAT or HT supplement is added to the medium durin(g initial growth
and selection.
[0248] Hybridoma Screening. To identify hybridomas secreting human
IgG kappa antibodies, ELISA plates (Nunc MaxiSorp) are coated
overnight at 4.degree. C. with 100 ul/well goat anti-human Fcgamma
specific antibody (Jackson Immuno Research #109-006-098) at 1 ug/ml
in PBS. Plates are washed and blocked with 100 ul/well PBS-Tween
containing 1% BSA. Fifty ul cell culture supernatent is added
followed by a 1-2 hour incubation. Plates are washed and then
incubated for one hour with 100 ul/well goat anti-Kappa light chain
conjugated to alkaline phosphatase or horseradish peroxidase (Sigma
#A-3813, or #A-7164). Plates are washed three times in PBS-Tween
between each step. An analogous assay was used to identify
hybridomas that secrete human antibodies reactive with TRAIL-R.
This assay is identical except that the ELISA plates were coated
with recombinant TRAIL-R protein instead of goat anti-human Fcgamma
antibody.
Sequence CWU 1
1
611323DNAHomo sapiensCDS(1)..(1323) 1atg gaa caa cgg gga cag aac
gcc ccg gcc gct tcg ggg gcc cgg aaa 48Met Glu Gln Arg Gly Gln Asn
Ala Pro Ala Ala Ser Gly Ala Arg Lys1 5 10 15agg cac ggc cca gga ccc
agg gag gcg cgg gga gcc agg cct ggg ccc 96Arg His Gly Pro Gly Pro
Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro 20 25 30cgg gtc ccc aag acc
ctt gtg ctc gtt gtc gcc gcg gtc ctg ctg ttg 144Arg Val Pro Lys Thr
Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45gtc tca gct gag
tct gct ctg atc acc caa caa gac cta gct ccc cag 192Val Ser Ala Glu
Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60cag aga gcg
gcc cca caa caa aag agg tcc agc ccc tca gag gga ttg 240Gln Arg Ala
Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu65 70 75 80tgt
cca cct gga cac cat atc tca gaa gac ggt aga gat tgc atc tcc 288Cys
Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser 85 90
95tgc aaa tat gga cag gac tat agc act cac tgg aat gac ctc ctt ttc
336Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe
100 105 110tgc ttg cgc tgc acc agg tgt gat tca ggt gaa gtg gag cta
agt ccg 384Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu
Ser Pro 115 120 125tgc acc acg acc aga aac aca gtg tgt cag tgc gaa
gaa ggc acc ttc 432Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu
Glu Gly Thr Phe 130 135 140cgg gaa gaa gat tct cct gag atg tgc cgg
aag tgc cgc aca ggg tgt 480Arg Glu Glu Asp Ser Pro Glu Met Cys Arg
Lys Cys Arg Thr Gly Cys145 150 155 160ccc aga ggg atg gtc aag gtc
ggt gat tgt aca ccc tgg agt gac atc 528Pro Arg Gly Met Val Lys Val
Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175gaa tgt gtc cac aaa
gaa tca ggt aca aag cac agt ggg gaa gcc cca 576Glu Cys Val His Lys
Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro 180 185 190gct gtg gag
gag acg gtg acc tcc agc cca ggg act cct gcc tct ccc 624Ala Val Glu
Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205tgt
tct ctc tca ggc atc atc ata gga gtc aca gtt gca gcc gta gtc 672Cys
Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val 210 215
220ttg att gtg gct gtg ttt gtt tgc aag tct tta ctg tgg aag aaa gtc
720Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys
Val225 230 235 240ctt cct tac ctg aaa ggc atc tgc tca ggt ggt ggt
ggg gac cct gag 768Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly
Gly Asp Pro Glu 245 250 255cgt gtg gac aga agc tca caa cga cct ggg
gct gag gac aat gtc ctc 816Arg Val Asp Arg Ser Ser Gln Arg Pro Gly
Ala Glu Asp Asn Val Leu 260 265 270aat gag atc gtg agt atc ttg cag
ccc acc cag gtc cct gag cag gaa 864Asn Glu Ile Val Ser Ile Leu Gln
Pro Thr Gln Val Pro Glu Gln Glu 275 280 285atg gaa gtc cag gag cca
gca gag cca aca ggt gtc aac atg ttg tcc 912Met Glu Val Gln Glu Pro
Ala Glu Pro Thr Gly Val Asn Met Leu Ser 290 295 300ccc ggg gag tca
gag cat ctg ctg gaa ccg gca gaa gct gaa agg tct 960Pro Gly Glu Ser
Glu His Leu Leu Glu Pro Ala Glu Ala Glu Arg Ser305 310 315 320cag
agg agg agg ctg ctg gtt cca gca aat gaa ggt gat ccc act gag 1008Gln
Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp Pro Thr Glu 325 330
335act ctg aga cag tgc ttc gat gac ttt gca gac ttg gtg ccc ttt gac
1056Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro Phe Asp
340 345 350tcc tgg gag ccg ctc atg agg aag ttg ggc ctc atg gac aat
gag ata 1104Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn
Glu Ile 355 360 365aag gtg gct aaa gct gag gca gcg ggc cac agg gac
acc ttg tac acg 1152Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp
Thr Leu Tyr Thr 370 375 380atg ctg ata aag tgg gtc aac aaa acc ggg
cga gat gcc tct gtc cac 1200Met Leu Ile Lys Trp Val Asn Lys Thr Gly
Arg Asp Ala Ser Val His385 390 395 400acc ctg ctg gat gcc ttg gag
acg ctg gga gag aga ctt gcc aag cag 1248Thr Leu Leu Asp Ala Leu Glu
Thr Leu Gly Glu Arg Leu Ala Lys Gln 405 410 415aag att gag gac cac
ttg ttg agc tct gga aag ttc atg tat cta gaa 1296Lys Ile Glu Asp His
Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu 420 425 430ggt aat gca
gac tct gcc atg tcc taa 1323Gly Asn Ala Asp Ser Ala Met Ser 435
4402440PRTHomo sapiens 2Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala
Ser Gly Ala Arg Lys1 5 10 15Arg His Gly Pro Gly Pro Arg Glu Ala Arg
Gly Ala Arg Pro Gly Pro 20 25 30Arg Val Pro Lys Thr Leu Val Leu Val
Val Ala Ala Val Leu Leu Leu 35 40 45Val Ser Ala Glu Ser Ala Leu Ile
Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60Gln Arg Ala Ala Pro Gln Gln
Lys Arg Ser Ser Pro Ser Glu Gly Leu65 70 75 80Cys Pro Pro Gly His
His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser 85 90 95Cys Lys Tyr Gly
Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100 105 110Cys Leu
Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120
125Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe
130 135 140Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr
Gly Cys145 150 155 160Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr
Pro Trp Ser Asp Ile 165 170 175Glu Cys Val His Lys Glu Ser Gly Thr
Lys His Ser Gly Glu Ala Pro 180 185 190Ala Val Glu Glu Thr Val Thr
Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205Cys Ser Leu Ser Gly
Ile Ile Ile Gly Val Thr Val Ala Ala Val Val 210 215 220Leu Ile Val
Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val225 230 235
240Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro Glu
245 250 255Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn
Val Leu 260 265 270Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val
Pro Glu Gln Glu 275 280 285Met Glu Val Gln Glu Pro Ala Glu Pro Thr
Gly Val Asn Met Leu Ser 290 295 300Pro Gly Glu Ser Glu His Leu Leu
Glu Pro Ala Glu Ala Glu Arg Ser305 310 315 320Gln Arg Arg Arg Leu
Leu Val Pro Ala Asn Glu Gly Asp Pro Thr Glu 325 330 335Thr Leu Arg
Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro Phe Asp 340 345 350Ser
Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn Glu Ile 355 360
365Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu Tyr Thr
370 375 380Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala Ser
Val His385 390 395 400Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu
Arg Leu Ala Lys Gln 405 410 415Lys Ile Glu Asp His Leu Leu Ser Ser
Gly Lys Phe Met Tyr Leu Glu 420 425 430Gly Asn Ala Asp Ser Ala Met
Ser 435 4403157DNAHomo
sapiensCDS(3)..(155)misc_feature(145)..(145)n is a, c, g, or t 3ct
gag act ctg aga cag tgc ttc gat gac ttt gca gac ttg gtg ccc 47Glu
Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro1 5 10 15ttt
gac tcc tgg gag ccg ctc atg agg aag ttg ggc ctc atg gac aat 95Phe
Asp Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn 20 25
30gag ata aag gtg gct aaa gct gag gca gcg ggc cac agg gac acc ttg
143Glu Ile Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu
35 40 45tnc acn atg ctg at 157Xaa Thr Met Leu 50451PRTHomo
sapiensmisc_feature(48)..(48)The 'Xaa' at location 48 stands for
Tyr, Cys, Ser, or Phe. 4Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala
Asp Leu Val Pro Phe1 5 10 15Asp Ser Trp Glu Pro Leu Met Arg Lys Leu
Gly Leu Met Asp Asn Glu 20 25 30Ile Lys Val Ala Lys Ala Glu Ala Ala
Gly His Arg Asp Thr Leu Xaa 35 40 45Thr Met Leu 5058PRTArtificial
SequenceFLAG peptide 5Asp Tyr Lys Asp Asp Asp Asp Lys1
563159DNAArtificial SequenceCloning Vector pGP1k 6aattagcggc
cgctgtcgac aagcttcgaa ttcagtatcg atgtggggta cctactgtcc 60cgggattgcg
gatccgcgat gatatcgttg atcctcgagt gcggccgcag tatgcaaaaa
120aaagcccgct cattaggcgg gctcttggca gaacatatcc atcgcgtccg
ccatctccag 180cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg
ccacgggtgc gcatgatcgt 240gctcctgtcg ttgaggaccc ggctaggctg
gcggggttgc cttactggtt agcagaatga 300atcaccgata cgcgagcgaa
cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc 360aacaacatga
atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
420gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
cctgtggaac 480acctacatct gtattaacga agcgctggca ttgaccctga
gtgatttttc tctggtcccg 540ccgcatccat accgccagtt gtttaccctc
acaacgttcc agtaaccggg catgttcatc 600atcagtaacc cgtatcgtga
gcatcctctc tcgtttcatc ggtatcatta cccccatgaa 660cagaaattcc
cccttacacg gaggcatcaa gtgaccaaac aggaaaaaac cgcccttaac
720atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
cgagctggac 780gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
acgctgatga gctttaccgc 840agctgcctcg cgcgtttcgg tgatgacggt
gaaaacctct gacacatgca gctcccggag 900acggtcacag cttgtctgta
agcggatgcc gggagcagac aagcccgtca gggcgcgtca 960gcgggtgttg
gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
1020tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
catatgcggt 1080gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
caggcgctct tccgcttcct 1140cgctcactga ctcgctgcgc tcggtcgttc
ggctgcggcg agcggtatca gctcactcaa 1200aggcggtaat acggttatcc
acagaatcag gggataacgc aggaaagaac atgtgagcaa 1260aaggccagca
aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
1320tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
cgaaacccga 1380caggactata aagataccag gcgtttcccc ctggaagctc
cctcgtgcgc tctcctgttc 1440cgaccctgcc gcttaccgga tacctgtccg
cctttctccc ttcgggaagc gtggcgcttt 1500ctcatagctc acgctgtagg
tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 1560gtgtgcacga
accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
1620agtccaaccc ggtaagacac gacttatcgc cactggcagc agccaggcgc
gccttggcct 1680aagaggccac tggtaacagg attagcagag cgaggtatgt
aggcggtgct acagagttct 1740tgaagtggtg gcctaactac ggctacacta
gaaggacagt atttggtatc tgcgctctgc 1800tgaagccagt taccttcgga
aaaagagttg gtagctcttg atccggcaaa caaaccaccg 1860ctggtagcgg
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
1920aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
aactcacgtt 1980aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
ctagatcctt ttaaattaaa 2040aatgaagttt taaatcaatc taaagtatat
atgagtaaac ttggtctgac agttaccaat 2100gcttaatcag tgaggcacct
atctcagcga tctgtctatt tcgttcatcc atagttgcct 2160gactccccgt
cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
2220caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
aaccagccag 2280ccggaagggc cgagcgcaga agtggtcctg caactttatc
cgcctccatc cagtctatta 2340attgttgccg ggaagctaga gtaagtagtt
cgccagttaa tagtttgcgc aacgttgttg 2400ccattgctgc aggcatcgtg
gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 2460gttcccaacg
atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
2520ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
ctcatggtta 2580tggcagcact gcataattct cttactgtca tgccatccgt
aagatgcttt tctgtgactg 2640gtgagtactc aaccaagtca ttctgagaat
agtgtatgcg gcgaccgagt tgctcttgcc 2700cggcgtcaac acgggataat
accgcgccac atagcagaac tttaaaagtg ctcatcattg 2760gaaaacgttc
ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
2820tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
agcgtttctg 2880ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
aataagggcg acacggaaat 2940gttgaatact catactcttc ctttttcaat
attattgaag catttatcag ggttattgtc 3000tcatgagcgg atacatattt
gaatgtattt agaaaaataa acaaataggg gttccgcgca 3060catttccccg
aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
3120ataaaaatag gcgtatcacg aggccctttc gtcttcaag 3159
* * * * *