Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
PROTEIN THAT BINDS TRAIL
BACKGROUND OF THE INVENTION
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). Identification of cell
surface
proteins) that bind TRAIL would prove useful in further elucidating the
biological
activities of TRAIL.
SUMMARY OF THE INVENTION
The present invention is directed to a novel protein that binds to the protein
known
as TNF-related apoptosis-inducing ligand (TRAIL), and thus is designated a
TRAIL
Binding Protein (TRAIL-BP). DNA encoding TRAIL-BP, and expression vectors
comprising such DNA, are provided. A method for producing TRAIL-BP
polypeptides
comprises culturing host cells transformed with a recombinant expression
vector encoding
TRAIL-BP, under conditions that promote expression of TRAIL-BP, then
recovering the
expressed TRAIL-BP polypeptides from the culture. Antibodies that are
immunoreactive
with TRAIL-BP are also provided.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 presents the nucleotide sequence of a DNA encoding a human TRAIL-
Binding Protein, as well as the amino acid sequence encoded thereby.
DETAILED DESCRIPTION OF THE INVENTION
A novel protein designated TRAIL-Binding Protein (TRAIL-BP) is provided
herein. TRAIL-BP binds to the cytokine designated TNF-related apoptosis-
inducing
ligand (TRAIL). Certain uses of TRAIL-BP flow from this ability to bind TRAIL,
as
discussed further below. TRAIL-BP finds use in inhibiting biological
activities of
TRAIL, or in purifying TRAIL by affinity chromatography, for example.
TRAIL-BP 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.
The nucleotide sequence of a human TRAIL-BP cDNA is presented in Figure 1
(SEQ ID NO: l ), along with the amino acid sequence encoded by the cDNA (SEQ
ID
N0:2). The TRAIL-BP protein of Figure 1 (SEQ ID N0:2) includes an N-terminal
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hydrophobic region that functions as a signal peptide, an extracellular
domain, and a C-
terminal hydrophobic region.
Computer analysis predicts that the signal peptide is cleaved after amino acid
69
of Figure 1 (SEQ ID N0:2). Cleavage of the signal peptide thus would yield a
mature
protein comprising amino acids 70 through 299 of Figure 1 (SEQ ID N0:2). The
next
most likely computer-predicted signal peptidase cleavage sites (in descending
order)
occur after amino acids 63 or 65 of Figure 1 (SEQ ID N0:2).
The coding region of the DNA sequence shown in Figure 1 (SEQ ID NO:1 ) begins
with an initiation codon (ATG). A second potential initation codon is found at
nucleotides 144-146 of Figure 1 (SEQ ID NO:I). Since the second (downstream)
ATG is
found within the region encoding the signal peptide, the mature form of the
protein would
be the same, regardless of which ATG functions as an initiation codon.
The extracellular domain, which follows the signal peptide, terminates at
amino
acid 278 of Figure 1 (SEQ ID N0:2). The amino acid sequence of the TRAIL-BP
extracellular domain shows significant homology to the extracellular domains
of members
of the tumor necrosis factor receptor (TNF-R) family of receptors (reviewed in
Smith et
al., Cell 76:959-962, 1994).
The C-terminal hydrophobic domain comprises amino acids 279 through 299 of
Figure 1 (SEQ ID N0:2). TRAIL-BP proteins containing this hydrophobic domain
are
attached to the cell surface.
The present invention encompasses TRAIL-BP in various forms, which may be
naturally occurring or non-naturally occurring. Forms that are not naturally
occurring
may be produced through various techniques, such as procedures involving
recombinant
DNA technology. The forms of TRAIL-BP provided herein include, but are not
limited
to, fragments, derivatives, variants, and oligomers of TRAIL-BP, as discussed
further
below.
TRAIL-BP 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-BP may be
prepared by linking the chemical moieties to functional groups on TRAIL-BP
amino acid
side chains or at the N-terminus or C-terminus of a TRAIL-BP polypeptide.
Conjugates
comprising diagnostic (detectable) or therapeutic agents attached to TRAIL-BP
are
contemplated herein, as discussed in more detail below.
Other derivatives of TRAIL-BP within the scope of this invention include
covalent or aggregative conjugates of TRAIL-BP 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-BP
oligomers.
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Further, TRAIL-BP-containing fusion proteins can comprise peptides added to
facilitate purification and identification of TRAIL-BP. Such peptides include,
for
example, poly-His or the antigenic identification peptides described in U.S.
Patent No.
5,011,912 and in Hopp et al., BiolTechnology 6:1204, 1988. One such peptide is
the
Flag~ peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID N0:3), 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~
peptide
in the presence of certain divalent metal cations, as described in U.S. Patent
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~ peptide are available from Eastman Kodak Co.,
Scientific
Imaging Systems Division, New Haven, Connecticut.
Both cell membrane-bound and soluble (secreted) forms of TRAIL-BP are
provided herein. Soluble TRAIL-BP may be identified (and distinguished from
non
soluble membrane-bound counterparts) by separating intact cells expressing a
TRAIL-BP
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-
BP in the
medium indicates that the protein was secreted from the cells and thus is a
soluble form of
the desired protein.
TRAIL-BP is believed to be anchored to the cell surface via glycosyl-
phosphatidylinositol (GPI) linkage. GPI membrane anchors, including the
chemical
structure and processing thereof, are described in Ferguson, M. and A.
Williams, Ann.
Rev. Biochem., 57:285, 1988. When initially expressed, certain proteins
comprise a C-
terminal hydrophobic domain that contains signals for GPI anchoring. A
cleavage site is
located upstream, often about 10-12 amino acids upstream of the N-terminus of
the
hydrophobic domain. Post-translational processing includes cleavage of the
protein at
this cleavage site. A GPI anchor attaches to the newly exposed C-terminal
amino acid of
the processed, mature protein.
Soluble forms of TRAIL-BP typically lack the C-terminal hydrophobic region
that
would cause retention of the protein on the cell surface. In one embodiment of
the
invention, a soluble TRAIL-BP polypeptide comprises the extracellular domain
of the
protein. Examples of soluble TRAIL-BP include, but are not limited to, mature
soluble
human TRAIL-BP comprising amino acids x to 278 of the Figure 1 (SEQ ID N0:2}
. 35 sequence, wherein x represents an integer from 64 to 70, inclusive.
Soluble forms of TRAIL-BP possess certain advantages over the membrane-
bound form of the protein. Purification of the protein from recombinant host
cells is
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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.
Naturally occurring variants of the TRAIL-BP protein of Figure 1 are provided
herein. Such variants include, for example, proteins that result from
alternate mRNA
splicing events or from proteolytic cleavage of the TRAIL-BP protein.
Alternate splicing
of mRNA may, for example, yield a truncated but biologically active TRAIL-BP
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-BP protein (generally from 1-5 terminal amino acids).
TRAIL-BP
proteins in which differences in amino acid sequence are attributable to
genetic
polymorphism (allelic variation among individuals producing the protein) are
also
contemplated herein.
Regarding the discussion herein of various domains of TRAIL-BP protein, the
skilled artisan will recognize that the above-described boundaries of such
regions of the
protein are approximate. To illustrate, the N-terminal residue of the C-
terminal
hydrophobic region (which may be predicted by using computer programs
available for
that purpose) may differ from that described above. Thus, soluble TRAIL-BP
polypeptides in which the C-terminus of the extracellular domain differs from
the residue
so identified above are contemplated herein.
The skilled artisan will also recognize that the positions) 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-BP 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-BP proteins provided herein include, but are not limited to, proteins
having the
residue at position 64, 66, or 70 of Figure 1 (SEQ ID N0:2) as the N-terminal
amino acid.
Other naturally occurring TRAIL-BP DNAs and polypeptides include those
derived from non-human species. Homologs of the human TRAIL-BP of Figure 1,
(SEQ
ID NOS:1 and 2) from other mammalian species, are contemplated herein, for
example.
Probes based on the human DNA sequence of Figure 1 (SEQ ID NO:1 ) may be used
to
screen cDNA libraries derived from other mammalian species, using conventional
cross
species hybridization techniques.
TRAIL-BP fragments are provided herein. Such fragments may be truncated at
the N-terminus or C-terminus, or may lack internal residues, for example, when
compared
with a full length native protein. Certain fragments lack amino acid residues
that are not
essential for a desired biological activity, such as TRAIL binding.
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TRAIL-BP fragments may be prepared by any of a number of conventional
techniques. Desired peptide fragments may be chemically synthesized. An
alternative
approach involves generating TRAIL-BP 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, or by digesting the DNA with suitable restriction enzymes
and
isolating the desired fragment. 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.
TRAIL-BP polypeptide fragments may be employed as immunogens, in
generating antibodies. Certain embodiments are directed to TRAIL-BP
polypeptide
fragments that possess a desired biological activity, e.g., the ability to
bind TRAIL. Such
a fragment may be a soluble TRAIL-BP polypeptide, as described above.
In particular embodiments, the TRAIL-BP fragments include cysteine-rich repeat
motifs found within the extracellular domain. A number of 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 1
polypeptides lacking one of the repeats exhibited a ten fold reduction in
binding affinity
for TNFa and TNF13; deletion of the second repeat resulted in a complete loss
of
detectable binding of the ligands.
The human TRAIL-BP of Figure 1 (SEQ ID N0:2) contains two such cysteine
rich repeats, the first including residues 108 through 149, and the second
including
residues 150 through 190 of Figure 1 (SEQ ID N0:2}. TRAIL-BP fragments
provided
herein include, but are not limited to, polypeptides that are truncated at the
N-terminus
and/or the C-terminus, but include the cysteine residues found within the
cysteine rich
repeats. Examples of such TRAIL-BP fragments include, but are not limited to,
polypeptides comprising amino acids y to z of Figure 1 (SEQ ID N0:2), wherein
y
represents an integer from b4 to 109, and z represents an integer from 189 to
299. In
particular embodiments, y is 64, b6, 70, 108, or 109, and z is 189, 190, or
278. Soluble
TRAIL-BP polypeptides provided herein include, but are not limited to,
fragments of the
extracellular domain, wherein the fragments comprise the cysteine residues in
the cysteine
rich repeats.
Two expressed sequence tags (ESTs) contain regions of identity with the DNA
sequence of Figure 1 (SEQ ID NO:1 ). The computer databank record for an EST
having
accession no. T71406 presents a DNA sequence 352 nucleotides in length. When
the
EST T71406 sequence is aligned with the TRAIL-BP DNA sequence of Figure 1 (SEQ
ID NO:1 ), regions of identity are found between nucleotides 9 and 358 of
Figure 1 (SEQ
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ID NO:1 ). Certain of the nucleotides in EST T71406 are unidentified {i.e.,
are designated
"N" in the databank record because their identity was unknown). The EST T71406
databank sequence also includes insertions, mismatches, and a deletion, when
compared
to the corresponding region of the nucleotide sequence of Figure 1 (SEQ ID
NO:1 ).
A DNA sequence 398 nucleotides in length is presented in a computer databank
for an EST having accession no. AAI50849. When the EST AA150849 sequence is
aligned with the TRAIL-BP DNA sequence of Figure 1 (SEQ ID NO:1 ), regions of
identity are found between nucleotides 10 and 409 of Figure 1 (SEQ ID NO: l).
However,
the EST AA 150849 sequence contains deletions and mismatches when compared to
the
corresponding nucleotide sequence of Figure 1 (SEQ ID NO:1 ).
The EST T71406 sequence is not identical to the overlapping region of EST
AA150849. Alignment of the databank sequences of these two ESTs reveals
insertions
and mismatches. No reading frame is identified in the databank file for either
of the two
ESTs. However, even if the DNA sequences set forth in the computer databank
file were
translated in accordance with the reading frame elucidated herein, neither EST
T71406
nor AA150849 would encode a TRAIL-BP that is expected to bind TRAIL. The
translates lack most of the conserved cysteine residues discussed above.
TRAIL-BP 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 Figure
1 {SEQ
ID NO:1) and still encode a TRAIL-BP protein having the amino acid sequence of
Figure
1 (SEQ ID N0:2). 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-
BP sequences (e.g., cDNA comprising the nucleotide sequence presented in
Figure 1
(SEQ ID NO:1 ) and DNA that is degenerate as a result of the genetic code to a
native
TRAIL-BP DNA sequence.
Among the TRAIL-BP polypeptides provided herein are variants of native
TRAIL-BP polypeptides that retain a biological activity of a native TRAIL-BP.
Such
variants include polypeptides that are substantially homologous to native
TRAIL-BP, but
which have an amino acid sequence different from that of a native TRAIL-BP
because of
one or more deletions, insertions or substitution Particular embodiments
include, but
are ~:ot limited to, TRAIL-BP polypeptides tl..° <.omprise from one to
ten deletions,
insertions or substitutions of amino acid residues, when compared to a native
TRAIL-BP
sequence. The TRAIL-BP-encoding DNAs of the present invention include variants
that
differ from a native TRAIL-BP DNA sequence because of one or more deletions,
insertions or substitutions, but that encode a biologically active TRAIL-BP
polypeptide.
One biological activity of TRAIL-BP is the ability to bind TRAIL.
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Nucleic acid molecules capable of hybridizing to the DNA of Figure 1 (SEQ ID
NO: I ) under moderately stringent or highly stringent conditions, and which
encode a
biologically active TRAIL-BP, 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.
. Moderately stringent conditions include conditions described in, for
example,
Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. l, 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 SX SSC,
0.5% SDS,
1.0 mM EDTA (pH 8.0) and hybridization conditions of about 55 C, 5 X 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 Figure 1 (SEQ ID NO: l ) under highly stringent
conditions,
wherein said conditions include hybridization at 68°C followed by
washing in 0.1X
SSC/0.1% SDS at 63-68°C.
Certain DNAs and polypeptides provided herein comprise nucleotide or amino
acid sequences, respectively, that are at least 80% identical to a native
TRAIL-BP
sequence. Also contemplated are embodiments in which a TRAIL-BP 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-BP sequence. The percent identity may
be
determined, for example, by comparing sequence information 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. For fragments, the percent identity is calculated by
comparing the
sequence of the fragment with the corresponding portion of a native TRAIL-BP.
In particular embodiments of the invention, a variant TRAIL-BP polypeptide
, differs in amino acid sequence from a native TRAIL-BP, but is substantially
equivalent to
a native TRAIL-BP in a biological activity. One example is a variant TRAIL-BP
that
binds TRAIL with essentially the same binding affinity as does a native TRAIL-
BP.
Binding affinity can be measured by conventional procedures, e.g., as
described in U.S.
Patent no. 5,512,457.
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Variant amino acid sequences may comprise conservative substitution(s),
meaning
that one or more amino acid residues of a native TRAIL-BP is replaced by a
different
residue, but that the conservatively substituted TRAIL-BP 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 G1n and Asn. Other
conservative substitutions, e.g., involving substitutions of entire regions
having similar
hydrophobicity characteristics, are well known.
In further examples of variants, sequences are altered so that cysteine
residues that
are not essential for biological activity are deleted or replaced with other
amino acids.
Such deletion or substitution of Cys residues may reduce formation of
incorrect
intramolecular disulfide bridges during renaturation of the expressed protein.
In one
embodiment, Cys residues within the above-described cysteine rich domains
remain
unaltered in the TRAIL-BP variants.
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. The human TRAIL-BP
of
Figure 1 (SEQ ID N0:2) contains one such adjacent basic residue pair, at amino
acids
166-167. 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.
In still other variants, N-glycosylation sites in a native TRAIL-BP are
inactivated.
N-glycosylation sites can be modified to preclude glycosylation, allowing
expression of a
more homogeneous, reduced carbohydrate analog in mammalian and yeast
expression
systems. N-glycosylation sites in eukaryotic polypeptides are characterized by
an amino
acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or
Thr. The
mature form of the human TRAIL-BP protein of Figure 1 (SEQ ID N0:2) comprises
five
such triplets, at amino acids I17-I 19, 180-182, I96-198, 209-211, and 224-226
of Figure
1 (SEQ ID N0:2). Appropriate substitutions, additions or deletions to the
nucleotide
sequence encoding these triplets will result in prevention of attachment of
carbohydrate
residues to the Asn side chain. Alteration of a single nucleotide, chosen so
that Asn is
replaced by a different amino acid, for example, is sufficient to inactivate
an N-
glycosylation site. Known procedures for inactivating N-glycosylation sites in
proteins
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include those described in U.S. Patent 5,071,972 and EP 276,846, hereby
incorporated by
reference.
The calculated molecular weight for a mature protein containing residues 70 to
299 of Figure 1 (SEQ ID N0:2) is about 24.3 kilodaltons. The skilled artisan
will
recognize that the molecular weight of particular preparations of TRAIL-BP
protein may
differ, according to such factors as the degree of glycosylation. The
glycosylation pattern
of a particular preparation of TRAIL-BP may vary according to the type of
cells in which
the protein is expressed, for example, and a given preparation may include
multiple
differentially glycosylated species of the protein. TRAIL-BP polypeptides with
or
I0 without associated native-pattern glycosylation are provided herein.
Expression of
TRAIL-BP polypeptides in bacterial expression systems, such as E. coli,
provides non-
glycosylated molecules. Further, N-glycosylation sites in the native protein
may be
inactivated, as discussed above.
TRAIL-BP polypeptides, including variants and fragments thereof, can be tested
for biological activity in any suitable assay. The ability of a TRAIL-BP
polypeptide to
bind TRAIL can be confirmed in conventional binding assays, examples of which
are
described below.
Expression S, sty ems
The present invention also provides recombinant cloning and expression vectors
containing TRAIL-BP DNA, as well as host cells containing the recombinant
vectors.
Expression vectors comprising TRAIL-BP DNA may be used to prepare TRAIL-BP
polypeptides encoded by the DNA. A method for producing TRAIL-BP polypeptides
comprises culturing host cells containing a recombinant expression vector
encoding
TRAIL-BP, under conditions that allow expression of TRAIL-BP, then recovering
the
expressed TRAIL-BP polypeptides from the culture. The skilled artisan will
recognize
that the procedure for purifying the expressed TRAIL-BP will vary according to
such
factors as the type of host cells employed, and whether the TRAIL-BP is cell
membrane-
bound or a soluble form that is secreted from the host cell.
Any suitable expression system may be employed. The vectors include a DNA
encoding a TRAIL-BP 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
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relates to the TRAIL-BP DNA sequence. Thus, a promoter nucleotide sequence is
operably linked to an TRAIL-BP DNA sequence if the promoter nucleotide
sequence
controls the transcription of the TRAIL-BP 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.
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-BP sequence so
that the
TRAIL-BP 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-BP polypeptide. The signal peptide is cleaved from the TRAIL-BP
polypeptide upon secretion of TRAIL-BP from the cell.
Suitable host cells for expression of TRAIL-BP 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, (1985). Cell-free
translation
systems could also be employed to produce TRAIL-BP polypeptides using RNAs
derived
from DNA constructs disclosed herein.
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 typhinaurium, and various other species
within the
genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host
cell, such
as E. coli, a TRAIL-BP 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-BP
polypeptide.
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-BP DNA sequence are inserted into the pBR322
CA 02294704 1999-12-16
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vector. Other commercially available vectors include, for example, pKK223-3
(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMI (Promega Biotec,
Madison,
WI, USA).
Promoter sequences commonly used for recombinant prokaryotic host cell
expression vectors include p-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. 4I2, 1982). A particularly useful prokaryotic host cell
expression system
employs a phage ~ PL promoter and a cI857ts thermolabile repressor sequence.
Plasmid
vectors available from the American Type Culture Collection which incorporate
derivatives of the ~ PL promoter include plasmid pHUB2 (resident in E. toll
strain JMB9,
ATCC 37092) and pPLc28 (resident in E. toll RR1, ATCC 53082).
TRAIL-BP alternatively may be expressed in yeast host cells, preferably from
the
I5 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~ yeast plasmid, an autonomously replicating
sequence
(ARS), a promoter region, sequences for polyadenylation, sequences for
transcription
termination, and a selectable marker gene. Suitable promoter sequences for
yeast vectors
include, among others, promoters for metallothionein, 3-phosphoglycerate
kinase
(Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes
(Hess et al.,
J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),
such as
enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-
phosphoglycerate
mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase,
and
glucokinase. Other suitable vectors and promoters for use in yeast expression
are further
described in Hitzeman, EPA-73,657. Another alternative is the glucose-
repressible
ADH2 promoter described by Russell et al. (J. Biol. CJ~em. 258:2674, 1982) and
Beier et
al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and E.
toll may be
constructed by inserting DNA sequences from pBR322 for selection and
replication in E.
toll (Ampr gene and origin of replication) into the above-described yeast
vectors.
The yeast a-factor leader sequence may be employed to direct secretion of the
TRAIL polypeptide. The a-factor leader sequence is often inserted between the
promoter
I1
CA 02294704 1999-12-16
WO 99100423 PCT/US98I13491
sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell
30:933, 1982 and
Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984. Other leader
sequences suitable
for facilitating secretion of recombinant polypeptides from yeast hosts are
known to those
of skill in the art. A leader sequence may be modified near its 3' end to
contain one or
more restriction sites. This will facilitate fusion of the leader sequence to
the structural
gene.
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+ transformants in a selective medium,
wherein the
selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids,
2%
glucose, 10 ~g/ml adenine and 20 ~g/ml uracil.
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
pg/ml adenine and 80 ~g/ml uracil. Derepression of the ADH2 promoter occurs
when
glucose is exhausted from the medium.
Mammalian or insect host cell culture systems also may be employed to express
recombinant TRAIL-BP polypeptides. Bacculovirus systems for production of
heterologous proteins in insect cells are reviewed by Luckow and Summers,
BiolTechnology 6:47 ( 1988). Established cell Iines 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,
C 127 cells, 3T3 cells (ATCC CCL 163}, Chinese hamster ovary (CHO) cells, HeLa
cells,
BHK (ATCC CRL 10) cells, and the CV IIEBNA cell line (ATCC CRL 10478} that was
derived from the African green monkey kidney cell line CV 1 (ATCC CCL 70) as
described by MeMahan et al. (EMBO J. 10: 2821, 1991 ).
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
12
CA 02294704 1999-12-16
WO 99100423 PCT/US98/13491
particularly useful because both are easily obtained from a viral genome as a
fragment
which may also contain a viral origin of replication (Hers et al., Nature
273:113, 1978).
Smaller or larger SV40 fragments may also be used, provided the approximately
250 by
sequence extending from the Hifzd III site toward the Bgl I site located in
the SV40 viral
origin of replication site is included.
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 C 127 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
91118982. As
one alternative, the vector may be derived from a retrovirus.
Regarding signal peptides that may be employed in producing TRAIL-BP, the
native signal peptide of TRAIL-BP 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-BP 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 United
States Patent
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. Patent
4,968,607; and the
type II interleukin-1 receptor signal peptide described in EP 460,846.
Purified Protein
TRAIL-BP polypeptides of the present invention may be produced by
recombinant expression systems as described above, or purified from naturally
occurring
cells. TRAIL-BP may be purified by any of a number of suitable methods, which
may
employ conventional protein purification techniques. As is known to the
skilled artisan,
procedures for purifying a given protein are chosen according to such factors
as the types
of contaminants that are to be removed, which may vary according to the
particular cells
in which the TRAIL-BP is expressed. For recombinant proteins, other
considerations
include the particular expression systems employed and whether or not the
desired protein
is secreted into the culture medium.
13
CA 02294704 1999-12-16
WO 99100423 PCT/US98/13491
In one method, cells expressing the protein are disrupted by any of the
numerous
known techniques, including freeze-thaw cycling, sonication, mechanical
disruption, or
by use of cell lysing agents. Alternatively, a soluble TRAIL-BP may be
expressed and
secreted from the cell. The subsequent purification process may include
affinity
chromatography, e.g., employing a chromatography matrix containing TRAIL. The
chromatography matrix may instead comprise an antibody that binds TRAIL-BP.
The
TRAIL-BP polypeptides can be recovered from an affinity chromatography column
using
conventional techniques (e.g., elution in a high salt buffer), then dialyzed
into a lower salt
buffer for use.
In one approach, when an expression system that secretes the recombinant
protein
is employed, the culture medium first may be concentrated using a commercially
available protein concentration filter, for example, an Amicon or Millipore
Pellicon
ultrafiltration unit. Following the concentration step, the concentrate can be
applied to a
purification matrix such as a gel filtration matrix. Alternatively, an anion
exchange resin
can be employed, for example, a matrix or substrate having pendant
diethylaminoethyl
(DEAE) groups. The matrices can be acrylamide, agarose, dexiran, cellulose or
other
support materials commonly employed in protein purification. Alternatively, a
cation
exchange step can be employed. Suitable cation exchangers include various
insoluble
matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups
are
preferred. In addition, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g.,
silica
gel having pendant methyl or other aliphatic groups) can be employed. Some or
all of the
foregoing purification steps, in various combinations, may be employed.
Recombinant protein produced in bacterial culture can be isolated by initial
disruption of the host cells, centrifugation, extraction from cell pellets if
an insoluble
polypeptide, or from the supernatant fluid if a soluble polypeptide, followed
by one or
more concentration, salting-out, ion exchange, affinity purification or size
exclusion
chromatography steps. Finally, RP-HPLC can be employed for final purification
steps.
Microbial cells can be disrupted by any convenient method, including freeze-
thaw
cycling, sonication, mechanical disruption, or use of cell lysing agents.
In yeast host cells, TRAIL-BP is preferably expressed as a secreted
polypeptide, to
simplify purification. Recombinant polypeptides secreted from a yeast host
cell
fermentation can be purified by methods analogous to those disclosed by Urdal
et al. (J.
Chromatog. 29b:171, 1984). Urdal et al. describe two sequential, reversed-
phase HPLC
steps for purification of recombinant human IL-2 on a preparative HPLC column.
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-BP polypeptides are purified such that no
protein
14
CA 02294704 1999-12-16
WO 99/00423 PCT/US98/13491
bands corresponding to other (non-TRAIL-BP) proteins are detectable upon
analysis by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). One skilled in the
pertinent field
will recognize that multiple bands corresponding to TRAIL-BP protein may be
visualized
by SDS-PAGE, due to differential glycosylation, differential post-
translational
processing, and the like. TRAIL-BP 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.
Oligomeric Forms of TRAIL-BP
Encompassed by the present invention are oligomers that contain TRAIL-BP
polypeptides. TRAIL-BP oligomers may be in the form of covalently-linked or
non-
covalently-linked dimers, trimers, or higher oligomers.
One embodiment of the invention is directed to oligomers comprising multiple
TRAIL-BP polypeptides joined via covalent or non-covalent interactions between
peptide
moieties fused to the TRAIL-BP 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-BP polypeptides attached thereto, as
described in
more detail below.
In particular embodiments, the oligomers comprise from two to four TRAIL-BP
polypeptides. The TRAIL-BP moieties of the oligomer may be soluble
polypeptides, as
described above.
As one alternative, a TRAIL-BP oligomer is prepared using polypeptides derived
from immunoglobulins. Preparation of 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 Protocols in Immunology, Suppl. 4,
pages
10.19.1 - 10.19.11, 1992).
One embodiment of the present invention is directed to a TRAIL-BP dimer
comprising two fusion proteins created by fusing TRAIL-BP to the Fc region of
an
antibody. The TRAIL-BP moiety preferably is a soluble polypeptide. A gene
fusion
encoding the TRAIL-BP/Fc fusion protein is inserted into an appropriate
expression
vector. TRAIL-BPIFc 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-BP.
CA 02294704 1999-12-16
WO 99/04423 PCT/US98/13491
Provided herein are fusion proteins comprising a TRAIL-BP 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.
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 IgG 1
antibody.
Another useful Fc polypeptide is the Fc mutein described in U.S. Patent
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.
In other embodiments, TRAIL-BP is 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-BP oligomer with as many
as four
TRAIL-BP extracellular regions.
Alternatively, the oligomer is a fusion protein comprising multiple TRAIL-BP
polypeptides, with or without peptide linkers (spacer peptides). Among the
suitable
peptide linkers are those described in U.S. Patents 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-BP, using any suitable conventional technique. In one approach, a
chemically
synthesized oligonucleotide encoding the linker is ligated between sequences
encoding
TRAIL-BP. In particular embodiments, a fusion protein comprises from two to
four
soluble TRAIL-BP polypeptides, separated by peptide linkers.
Another method for preparing oligomeric TRAIL-BP 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), at~d 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.
Examples of
leucine zipper domains suitable for producing soluble oligomeric proteins are
described in
PCT application WO 94/10308 (hereby incorporated by reference), and the
leucine zipper
derived from lung surfactant protein D (SPD) described in Hoppe et al. (FEES
Letters
16
CA 02294704 1999-12-16
WO 99/00423 PCT/US98/13491
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-BP polypeptide fused to a leucine zipper peptide
are
expressed in suitable host cells, and the soluble oligomeric TRAIL-BP that
forms is
recovered from the culture supernatant.
Oligomeric TRAIL-BP 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-
BP,
or encoding fusion proteins useful in preparing TRAIL-BP oligomers, are
provided
herein.
Assavs
IS TRAIL-BP proteins (including fragments, variants, oligomers, and other
forms of
TRAIL-BP) may be tested for the ability to bind TRAIL in any suitable assay,
such as a
conventional binding assay. To illustrate, a soluble TRAIL-BP may be labeled
with a
detectable reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a
colorimetric or fluorometric reaction, and the like). The labeled TRAIL-BP is
contacted
with cells expressing TRAIL. The cells then are washed to remove unbound
labeled
TRAIL-BP, and the presence of cell-bound label is determined by a suitable
technique,
chosen according to the nature of the label.
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. CV 1-EBNA-1 cells in 10 cm2 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. CV 1-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).
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 x 104 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
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CA 02294704 1999-12-16
WO 99/00423 PCT/US98/13491
for i hour at 37 ° C with various concentrations of a soluble TRAIL-
BP/Fc fusion protein.
Cells then are washed and incubated with a constant saturating concentration
of a 125/_
mouse anti-human IgG in binding medium, with gentle agitation for I hour at
37°C.
After extensive washing, cells are released via trypsinization.
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 TRAIL-BP/Fc protein that has bound
to the
cells. In all assays, non-specific binding of t25I-antibody is assayed in the
absence of
TRAIL-BP/Fc, as well as in the presence of TRAIL-BP/Fc and a 200-fold molar
excess of
unlabeled mouse anti-human IgG antibody.
Cell-bound t25I_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, MA) run on a Microvax computer.
Another type of suitable binding assay is a competitive binding assay. To
illustrate, biological activity of a TRAIL-BP variant may be determined by
assaying for
the variant's ability to compete with a native TRAIL-BP for binding to TRAIL.
Competitive binding assays can be performed by conventional methodology.
Reagents that may be employed in competitive binding assays include
radiolabeled
TRAIL-BP and intact cells expressing TRAIL (endogenous or recombinant) on the
cell
surface. For example, a radiolabeled soluble TRAIL-BP fragment can be used to
compete
with a soluble TRAIL-BP 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, NJ. In one alternative, LZ-TRAIL (a
fusion
protein comprising a leucine zipper peptide fused to a soluble TRAIL
polypeptide; see
example I ) is employed instead of TRAIL/Fc. The LZ-TRAIL may be attached to a
Protein A or Protein G column via a monoclonal antibody specific for the
leucine zipper
peptide.
Another type of competitive binding assay utilizes radiolabeled soluble TRAIL,
such as a soluble TRAIL/Fc fusion protein, and intact cells expressing TRAIL-
BP. A
soluble leucine zipper/TRAIL fusion protein may be employed in place of a
TRAIL/Fc
fusion protein. Qualitative results can be obtained by competitive
autoradiographic plate
binding assays, while Scatchard plots (Scatchard, Ann. N. Y. Acad. Sei.
51:660, 1949) may
be utilized to generate quantitative results.
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CA 02294704 1999-12-16
WO 99/00423 PCT/US98/13491
Another type of assay for biological activity involves testing a TRAIL-BP
polypeptide (preferably a soluble TRAIL-BP) 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
97J01633, hereby incorporated by reference.
Uses of TRAIL-BP
Uses of TRAIL-BP include, but are not limited to, the following. Certain of
these
uses of TRAIL-BP flow from its ability to bind TRAIL.
TRAIL-BP finds use as a protein purification reagent. TRAIL-BP polypeptides
may be attached to a suitable support material (generally an insoluble matrix)
and used to
purify TRAIL proteins by affinity chromatography. In particular embodiments, a
TRAIL-
BP 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,
NJ). In an
alternative, a TRAIL-BP/Fc protein is attached to Protein A- or Protein G-
containing
chromatography columns through interaction with the Fc moiety.
TRAIL-BP proteins also find use in measuring the biological activity of TRAIL
proteins in terms of their binding affinity for TRAIL-BP. TRAIL-BP 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-BP 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-BP 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-BP 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
preparation thus
can be ascertained before it is used in a research study, for example.
TRAIL-BP also finds use in purifying or identifying cells that express TRAIL
on
the cell surface. TRAIL-BP 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-BP 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-BP thereon. Cells expressing TRAIL
on
the cell surface bind to the fixed TRAIL-BP, and unbound cells then are washed
away.
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WO 99/00423 PCT/US98/13491
Alternatively, TRAIL-BP can be conjugated to a detectable moiety, then
incubated
with cells to be tested for TRAIL expression. After incubation, unbound
labeled TRAIL-
BP is removed and the presence or absence of the detectable moiety on the
cells is
determined.
In a further alternative, mixtures of cells suspected of containing TRAIL
cells are
incubated with biotinylated TRAIL-BP. 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
promotes binding of the desired cells to the beads. Procedures for using
avidin-coated
beads are known (see Berenson, et al. J. Cell. Biochem., lOD:239, 1986).
Washing to
remove unbound material, and the release of the bound cells, are performed
using
conventional methods.
TRAIL-BP 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-BP 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.
Detectable (diagnostic) and therapeutic agents that may be attached to a TRAIL
BP 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, 1231, 1311, 99mTc, 111In, and
~6Br.
Examples of radionuclides suitable for therapeutic use are ~ 3 t I, 2 t ~ At,
~~Br, ~ g6Re, 1 ggRe,
212pb, 212Bi, 109pd, 64Cu, and 6~Cu.
Such agents may be attached to the TRAIL-BP by any suitable conventional
procedure. TRAIL-BP, 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 o~
one of the bifunctional coupling reagents available for attaching various
molecules
proteins (Pierce Chemical Company, Rockford, Illinois). A number of techniques
foc°
radiolabeling proteins are known. Radionuclide metals may be attached to TRAIL-
BP by
using a suitable bifunctional chelating agent, for example.
CA 02294704 1999-12-16
WO 99100423 PCT/US98/13491
Conjugates comprising TRAIL-BP 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.
TRAIL-BP 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-BP. TRAIL-BP 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-BP nucleotide sequences permits the
detection
of defective TRAIL-BP genes, and the replacement thereof with normal TRAIL-BP
encoding genes. Defective genes may be detected in in vitro diagnostic assays,
and by
comparision of a native TRAIL-BP nucleotide sequence disclosed herein with
that of a
TRAIL-BP gene derived from a person suspected of harboring a defect in this
gene.
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-BP
interactions
on different cell types. TRAIL-BP polypeptides also may be employed in in
vitro assays
for detecting TRAIL or TRAIL-BP or the interactions thereof.
A purified TRAIL-BP polypeptide may be used to inhibit binding of TRAIL to
endogenous cell surface TRAIL receptors. Certain ligands of the TNF family (of
which
TRAIL is a member) have been reported to bind to more than one distinct cell
surface
receptor protein. TRAIL likewise may bind to multiple cell surface proteins. A
receptor
protein designated DR4 that reportedly binds TRAIL, but is distinct from the
TRAIL-BP
of the present invention, is described in Pan et al. (Science 276:111-113,
1997; hereby
incorporated by reference). By binding TRAIL, soluble TRAIL-BP polypeptides of
the
present invention may be employed to inhibit the binding of TRAIL not only to
cell
surface TRAIL-BP, but also to TRAIL receptor proteins that are distinct from
TRAIL-BP.
TRAIL-BP may be used to inhibit a biological activity of TRAIL, in in vitro or
in
vivo procedures. By inhibiting binding of TRAIL to cell surface receptors,
TRAIL-BP
also inhibits biological effects that result from the binding of TRAIL to
endogenous
receptors. Various forms of TRAIL-BP may be employed, including, for example,
the
above-described TRAIL-BP fragments, oligomers, derivatives, and variants that
are
capable of binding TRAIL. In a preferred embodiment, a soluble TRAIL-BP is
employed
to inhibit a biological activity of TRAIL, e.g., to inhibit TRAIL-mediated
apoptosis of
cells susceptible to such apoptosis.
TRAIL-BP 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.
TRAIL-BP may be useful for treating thrombotic microangiopathies. One such
disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, H.C., Semin.
Hematol.,
21
CA 02294704 1999-12-16
WO 99/00423 PCT/US98/13491
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).
Plasma from patients afflicted with TTP (including HIV and HIV patients)
induces apoptosis of human endothelial cells of dermal microvascular origin,
but not large
vessel origin (Lawrence et al., Blood, 87:3245, April 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.
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-
BP 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 HUS.
Other conditions characterized by clotting of small blood vessels may be
treated
using TRAIL-BP. Such conditions include but are not limited to the following.
Cardiac
problems seen in about 5-10% of pediatric AIDS patients are believed to
invoive 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.
In one embodiment, a patient's blood or plasma is contacted with TRAIL-BP ex
vivo. The TRAIL-BP may be bound to a suitable chromatography matrix by
conventional
procedures. The patient's blood or plasma flows through a chromatography
column
containing TRAIL-BP bound to the matrix, before being returned to the patient.
The
immobilized TRAIL-BP binds TRAIL, thus removing TRAIL protein from the
patient's
blood.
Alternatively, TRAIL-BP may be administered in vivo to a patient afflicted
with a
thrombotic microangiopathy. In one embodiment, a soluble form of TRAIL-BP is
administered to the patient.
The present invention thus provides a method for treating a thrombotic
microangiopathy> involving use of an effec ~.ve amount of TRAIL-BP. A TRAIL-BP
polypeptide may be employed in in vivo or ex vivo procedures, to inhibit TRAIL-
mediated
damage to (e.g., apoptosis of) microvascular endothelial cells.
TRAIL-BP may be employed in conjunction with other agents useful in treating a
particular disorder. In an in vitro study reported by Lawrence et al. (Blood
87:3245,
1996), some reduction of TTP plasma-mediated apoptosis of microvascular
endothelial
22
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WO 99100423 PCT/US98/13491
cells was achieved by using an anti-Fas blocking antibody, aurintricarboxylic
acid, or
normal plasma depleted of cryoprecipitate.
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-BP 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.
Another embodiment of the present invention is directed to the use of TRAIL-BP
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 Apoprosis 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-II3-convening 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.
At least some of the T cell death seen in HIV' 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).
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' 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+ T cells and the progression to AIDS in HIV-
infected
individuals.
The present invention provides a method of inhibiting TRAIL-mediated T cell
death in HIV' patients, comprising administering TRAIL-BP (preferably, a
soluble
TRAIL-BP polypeptide) to the patients. In one embodiment, the patient is
asymptomatic
when treatment with TRAIL-BP commences. If desired, prior to treatment,
peripheral
blood T cells may be extracted from an HIV' patient, and tested for
susceptibility to
TRAIL-mediated cell death by conventional procedures.
In one embodiment, a patient's blood or plasma is contacted with TRAIL-BP ex
vivo. The TRAIL-BP may be bound to a suitable chromatography matrix by
conventional
23
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WO 9910Q423 PCT/US98113491
procedures. The patient's blood or plasma flows through a chromatography
column
containing TRAIL-BP bound to the matrix, before being returned to the patient.
The
immobilized TRAIL-BP binds TRAIL, thus removing TRAIL protein from the
patient's
blood.
In treating HIV' patients, a TRAIL-BP 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' 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 sFasIFc); anti-Fas antibodies that
bind Fas
without transducing the biological signal that results in apoptosis; anti-Fas-
L antibodies
that block binding of Fas-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.
Compositions comprising an effective amount of a TRAIL-BP polypeptide of the
present invention, in combination with other components such as a
physiologically
acceptable diluent, carrier, or excipient, are provided herein. TRAIL-BP can
be
formulated according to known methods used to prepare pharmaceutically useful
compositions. TRAIL-BP 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-HCI, 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.
In addition, such compositions can contain TRAIL-BP 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-BP,
and are thus
chosen according to the intended application. TRAIL-BP expressed on the
surface of a
cell may find use, as well.
24
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WO 99/00423 PCT/US98/13491
Compositions of the present invention may contain a TRAIL-BP 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-BP polypeptide or an oligomer comprising soluble TRAIL-BP
polypeptides. The additional blocking agents described above may be included
in the
TRAIL-BP composition, or may be formulated separately.
TRAIL-BP 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.
Compositions comprising TRAIL-BP nucleic acids in physiologically acceptable
formulations are also contemplated. TRAIL-BP DNA may be formulated for
injection,
for example.
Antibodies
Antibodies that are immunoreactive with TRAIL-BP polypeptides are provided
herein. Such antibodies specifically bind TRAIL-BP, in that the antibodies
bind to
TRAIL-BP via the antigen-binding sites of the antibody (as opposed to non-
specific
binding).
The TRAIL-BP protein of Figure 1 (SEQ ID N0:2) may be employed as an
immunogen in producing antibodies immunoreactive therewith. Alternatively,
another
form of TRAIL-BP, such as a fragment or fusion protein, may be employed as the
immunogen. The present invention thus provides antibodies obtained by
immunizing an
animal with the TRAIL-BP of Figure 1, or an immunogenic fragment thereof. A
method
for producing antibodies comprises immunizing an animal with a TRAIL-BP
polypeptide,
whereby antibodies directed against the TRAIL-BP are generated in said animal.
The
desired antibodies may be purified, e.g., from the animal's serum, by
conventional
techniques.
Among the procedures for preparing polyclonal and monoclonal antibodies are
those described in Monoclonal Antibodies, Hybridomas: A New Dimen.rion in
Biological
Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies:
A
Laboratory Manual , Harlow and Land (eds.), Cold Spring Harbor Laboratory
Press, Cold
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WO 99/00423 PCT/US98I13491
Spring Harbor, NY, ( 1988). Production of monoclonal antibodies directed
against
TRAIL-BP is further illustrated in example 4.
Antigen-binding fragments of antibodies directed against TRAIL-BP may be
produced by well known procedures, and are encompassed by the present
invention.
Examples of such fragments include, but are not limited to, Fab and F(ab')2
fragments.
Antibody fragments and derivatives produced by genetic engineering techniques
are also
provided.
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.
(Biol1'echnology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993).
In one embodiment, the antibodies are specific for TRAIL-BP, and do not cross-
react with other (non-TRAIL-BP) proteins. Screening procedures by which such
antibodies may be identified are well known, and may involve immunoaffinity
chromatography, for example.
Hybridoma cell lines that produce monoclonal antibodies specific for TRAIL-BP
are also contemplated herein. Such hybridomas may be produced and identified
by
conventional techniques. One method for producing such a hybridoma cell line
comprises immunizing an animal with a TRAIL-BP; harvesting spleen cells from
the
immunized animal; fusing said 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-BP. The monoclonal antibodies may be recovered by
conventional techniques.
Among the uses of the antibodies is use in assays to detect the presence of
TRAIL-BP pr peptides, either in vitro or in vivo. The antibodies also may be
employed
in purifying ~ IL-BP pr~~teins by imrrmnoaffinity chromatography.
In one embodiment, the antibodies additionally can block binding of TRAIL to
TRAIL-BP. Such antibodies may be employed to inhibit binding of TRAIL to cell
surface TRAIL-BP, for example. Blocking antibodies may be identified using
conventional assay procedures.
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WO 99/00423 PCT/US98/13491
Such an antibody may be employed in an in vitro procedure, or administered in
vivo to inhibit a TRAIL-BP-mediated biological activity. Disorders caused or
exacerbated (directly or indirectly) by the interaction of TRAIL with cell
surface TRAIL-
BP 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, directly or indirectly,
by the
interaction of TRAIL with TRAIL-BP are thus treated. Monoclonal antibodies are
generally preferred for use in such therapeutic methods. In one embodiment, an
antigen-
binding antibody fragment is employed.
Antibodies raised against TRAIL-BP may be screened for agonistic (i.e., ligand-
mimicking) properties. Such antibodies may be tested for the ability to induce
particular
biological effects, upon binding to cell surface TRAIL-BP. Agonistic
antibodies may be
screened for activities reported for TRAIL, such as the ability to induce
apoptosis of
certain cancer cells (e.g., leukemia, lymphoma, and melanoma cells) or virally
infected
cells. (See Wiley et al., Immunity 3:673-682, 1995; and PCT application WO
97/01633.)
Compositions comprising an antibody that is directed against TRAIL-BP, and a
physiologically acceptable diluent, excipient, or carrier, are provided
herein. Suitable
components of such compositions are as described above for compositions
containing
TRAIL-BP proteins.
Also provided herein are conjugates comprising a detectable (e.g., diagnostic)
or
therapeutic agent, attached to an antibody directed against TRAIL-BP. Examples
of such
agents are presented above. The conjugates find use in in vitro or ira vivo
procedures.
Nucleic Acids
The present invention provides TRAIL-BP nucleic acids. Examples of such
nucleic acids include, but are not limited to, isolated DNAs comprising the
nucleotide
sequence presented in Figure 1 (SEQ ID NO:1 ), the coding region thereof, or
fragments
thereof.
The present invention provides isolated nucleic acids useful in the production
of
TRAIL-BP polypeptides, e.g., in the recombinant expression systems discussed
above.
Such nucleic acids include, but are not limited to, the human TRAIL-BP DNA of
Figure 1
(SEQ ID NO:1). Nucleic acid molecules of the present invention include TRAIL-
BP
DNA in both single-stranded and double-stranded form, as well as the RNA
complement
thereof. TRAIL-BP 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 DNA of Figure 1
(SEQ ID
NO:1), or a suitable fragment thereof, as a probe.
27
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WO 99/00423 PCT/US98/13491
DNAs encoding TRAIL-BP in any of the forms contemplated herein (e.g., full
length TRAIL-BP or fragments thereof) are provided. Particular embodiments of
TRAIL-
BP-encoding DNAs include a DNA encoding the full length human TRAIL-BP of
Figure
1 and SEQ ID N0:2 (including the N-terminal signal peptide), and a DNA
encoding a full
length mature human TRAIL-BP. Other embodiments include DNA encoding a soluble
TRAIL-BP (e.g., encoding the extracellular domain of the protein of Figure 1
and SEQ ID
N0:2, either with or without the signal peptide).
Particular embodiments include TRAIL-BP-encoding DNAs comprising either of
the above-discussed initiation codons. Thus, examples of such DNAs include
those in
which the coding region begins with the initiation codon (ATG) presented as
nucleotides
24-26 of Figure 1 (SEQ ID NO:1); alternatively, the DNA may be truncated at
the 5' end,
such that the coding region begins with the ATG presented as nucleotides 144-
146 of
Figure 1 (SEQ ID NO: I ).
Fragments of TRAIL-BP nucleotide sequences comprising at least about 17
nucleotides find use as probes or primers, for example. Such oligonucleotides
may be
employed as primers in polymerase chain reactions (PCR), whereby TRAIL-BP DNA
fragments are isolated and amplified. Alternatively, a DNA fragment may
comprise at
least 30, or at least 60, contiguous nucleotides of a TRAIL-BP DNA sequence.
To
illustrate, a probe derived from a fragment of the DNA of Figure 1 (SEQ ID
NO:1 ) may
be used to screen a suitable cDNA library to identify TRAIL-BP clones.
Examples of
human cDNA libraries that may be employed include libraries derived from fetal
Iiver and
spleen, pregnant uterine tissue, foreskin fibroblasts, and peripheral blood
leukocytes.
Other useful fragments of the TRAIL-BP nucleic acids include antisense or
sense
oligonucleotides comprising a single-stranded nucleic acid sequence (either
RNA or
DNA) capable of binding to target TRAIL-BP mRNA (sense) or TRAIL-BP DNA
(antisense) sequences. Antisense or sense oligonucleotides, according to the
present
invention, comprise a fragment of the coding region of TRAIL-BP 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).
Binding of antisense or sense oligonucleotides to target nucleic acid
sequences
results in the formation of duplexes that bl~o~k transcription or translation
of the tar:v.,t
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-BP proteins.
Antisense
or sense oligonucleotides further comprise oligonucleotides having modified
sugar-
phosphodiester backbones (or other sugar linkages, such as those described in
28
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WO 99/00423 PCT/US98I13491
W091/06629) 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) hut retain sequence specificity to be able to
bind to
target nucleotide sequences.
S 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.
Antisense or sense oligonucleotides may be introduced into a cell containing
the
target nucleic acid sequence by any gene transfer method, including, for
example, CaP04-
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 DCTSA, DCTSB and DCTSC (see WO 90/13641).
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.
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.
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.
29
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WO 99/00423 PCT/US98113491
EXAMPLE 1 ~ Expression of TRAIL-BP and Binding Assav
A human TRAIL-BP was expressed by the following procedures. The expressed
protein was tested for the ability to bind TRAIL.
DNA comprising nucleotides 144 to 923 of Figure 1 (SEQ ID NO:1 ) (encoding
amino acids 41 to 299 of SEQ ID N0:2) was amplified by polymerise chain
reaction
(PCR). The coding region of this DNA begins with the second (downstream)
initiation
codon of the Figure 1 (SEQ ID NO:l) sequence, as discussed above. The isolated
and
amplified DNA was inserted into expression vector pDC409, to yield a construct
designated pDC409-TRAIL-BP.
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 polymerise promoter, downstream of the mcs, that faciliates sequencing of
DNA
inserted into the mcs.
TRAIL-BP was tested for the ability to bind TRAIL, in a slide binding assay.
Such assays are described in Gearing et al. (EMBO J. 8:3667, 1989); McMahan et
al.
(EMBO J. 10:2821, 1991) and Goodwin et al. (Eur. J. Immunol. 23:2631, 1993),
hereby
incorporated by reference.
The assay was conducted as follows. CV-1/EBNA cells were transfected with
pDC409-TRAIL-BP. The transfected cells were cultured on glass slides in DMEM
supplemented with 10% fetal bovine serum, penicillin, streptomycin, and
glutamine. 48
hours after transfection, cells were incubated with the LZ-TRAIL fusion
protein described
below (1 ~g/ml) in 1 ml of binding media (RPMI 1640 containing 25 mg/mI bovine
serum
albumin, 2 mg/ml sodium azide, 20mM Hepes (pH 7.2), and 50 mg/ml nonfat dry
milk).
After 30 minutes' incubation, the slides were washed once with binding media.
A '~SI-
labeled antibody specific for the leucine zipper (LZ) moiety of the fusion
protein was then
added ( 1 nM in 1 ml binding media). After 30 minutes' incubation, the slides
were
washed, fixed, end dipped in photographic emulsion.
The CV-1/EBNA cells transfected with the pDC409-TRAIL-BP expression vector
showed significantly enhanced binding of LZ-TRAIL, compared to cells
transfected with
the empty pDC409 vector alone (a control).
CA 02294704 1999-12-16
WO 99!00423 PCT/US98/13491
The LZ-TRAIL protein employed in the foregoing binding assay is a fusion
protein comprising a leucine zipper peptide fused to the N-terminus of a
soluble TRAIL
polypeptide. An expression construct was prepared, essentially as described
for
preparation of the Flag~-TRAIL expression construct in Wiley et al. (Immunity,
3:673-
682, 1995; hereby incorporated by reference), except that DNA encoding the
Flag~
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.
EXAMPLE 2: Monoclonal Antibodies That Bind TRAIL-BP
This example illustrates a method for preparing monoclonal antibodies that
bind
TRAIL-BP. Suitable immunogens that may be employed in generating such
antibodies
include, but are not limited to, purified TRAIL-BP protein or an immunogenic
fragment
thereof such as the extracellular domain, or fusion proteins containing TRAIL-
BP (e.g., a
soluble TRAIL-BP/Fc fusion protein).
Purified TRAIL-BP can be used to generate monoclonal antibodies
immunoreactive therewith, using conventional techniques such as those
described in U.S.
Patent 4,411,993. Briefly, mice are immunized with TRAIL-BP immunogen
emulsified
in complete Freund's adjuvant, and injected in amounts ranging from 10-100 Ng
subcutaneously or intraperitoneally. Ten to twelve days later, the immunized
animals are
boosted with additional TRAIL-BP emulsified in incomplete Freund's adjuvant.
Mice are
periodically boosted thereafter on a weekly to bi-weekly immunization
schedule. Serum
samples are periodically taken by retro-orbital bleeding or tail-tip excision
to test for
TRAIL-BP antibodies by dot blot assay, ELISA (Enzyme-Linked Immunosorbent
Assay)
or inhibition of TRAIL binding.
Following detection of an appropriate antibody titer, positive animals are
provided
one last intravenous injection of TRAIL-BP 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., NS 1 or preferably P3x63Ag8.653 (ATCC CRL 1580).
Fusions
generate hybridoma cells, which are plated in multiple microtiter plates in a
HAT
3I
CA 02294704 1999-12-16
WO 99/00423 PCTNS98I13491
(hypoxanthine, aminopterin and thymidine) selective medium to inhibit
proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells are screened by ELISA for reactivity against purified
TRAIL-BP by adaptations of the techniques disclosed in Engvall et al.,
Immunochem.
8:871, 1971 and in U.S. Patent 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-BP monoclonal
antibodies.
Alternatively, hybridoma cells can be grown in vitro in flasks or roller
bottles by various
techniques. Monoclonal antibodies produced in mouse ascites can be purified by
ammonium sulfate precipitation, followed by gel exclusion chromatography.
Alternatively, affinity chromatography based upon binding of antibody to
Protein A or
Protein G can also be used, as can affinity chromatography based upon binding
to
TRAIL-BP.
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CA 02294704 1999-12-16
WO 99/00423 PCT/US98/13491
SEQUENCE LISTING
S (1) GENERAL INFORMATION:
(i) APPLICANT: IMMUNEX CORPORATION
IO (ii) TITLE OF INVENTION: PROTEIN THAT BINDS TRAIL
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
IS (A} ADDRESSEE: Kathryn A. Anderson, Immunex Corporation
(B) STREET: 52 University Street
(C) CITY: Seattle
(D) STATE: WA
(E) COUNTRY: :US
ZO (F) ZIP: 98101
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
2$ (C) OPERATING SYSTEM: MS-DOS/Windows 95
(D) SOFTWARE: Word for Windows 95, 7.Oa
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: --to be assigned--
30 (B} FILING DATE: 25-JUN-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
3S (A) APPLICATION NUMBER: US 08/883,529
(B) FILING DATE: 26-JUN-1997
(C} CLASSIFICATION:
40 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Anderson, Kathryn A.
(B} REGISTRATION NUMBER: 32,172
(C) REFERENCE/DOCKET NUMBER: 2629-WO
4S (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 206-587-0430
(B) TELEFAX: 206-233-0644
SO (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1347 base pairs
(B) TYPE: nucleic acid
SS (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vii) IMMEDIATE SOURCE:
(B) CLONE: TRAIL-BP
33
CA 02294704 1999-12-16
WO 99/00423 PCT/US98/13491
(ix)FEATURE:
(A ) EY: CDS
NAME/K
(B ) ON: 24..920
LOCATI
(xi)SEQ UENCE SCRIPTION: D :1:
DE SEQ N0
I
GGCACGAGGG GACCA G G C C 50
AGTTT GAG CAA GTG TT CTA
AT GG AAG
GAG
CG
Me t n y l u g e
Gl Gl Va Lys Ar Ph Leu
Gl
2 5
CCG TTAGGG AACTCTGGG GACAGAGCG CCCCGGCCG CCTGAT GGCCGA 98
Pro LeuGly AsnSerGly AspArgAla ProArgPro ProAsp GlyArg
10 15 20 25
IS
GGC AGGGTG CGACCCAGG ACCCAGGAC GGCGTCGGG AACCAT ACCATG 146
Gly ArgVal ArgProArg ThrGlnAsp GlyValGly AsnHis ThrMet
30 35 40
ZO GCC CGGATC CCCAAGACC CTAAAGTTC GTCGTCGTC ATCGTC GCGGTC 194
Ala ArgIle ProLysThr LeuLysPhe ValValVal IleVal AlaVal
45 50 55
CTG CTGCCA GTCCTAGCT TACTCTGCC ACCACTGCC CGGCAG GAGGAA 242
25 Leu LeuPro ValLeuAla TyrSerAla ThrThrAla ArgG1n GluGlu
60 65 70
GTT CCCCAG CAGACAGTG GCCCCACAG CAACAGAGG CACAGC TTCAAG 290
Val ProGln GlnThrVal AIaProGln GlnGlnArg HisSer PheLys
3~ 75 80 85
GGG GAGGAG TGTCCAGCA GGATCTCAT AGATCAGAA CATACT GGAGCC 338
Gly GluGlu CysProAla GlySerHis ArgSerGlu HisThr GlyAla
g0 95 100 105
3S
TGT AACCCG TGCACAGAG GGTGTGGAT TACACCAAC GCTTCC AACAAT 386
Cys AsnPro CysThrGlu GlyValAsp TyrThrAsn AlaSer AsnAsn
110 115 120
4O GAA CCTTCT TGCTTCCCA TGTACAGTT TGTAAATCA GATCAA AAACAT 434
Glu ProSer CysPhePro CysThrVal CysLysSer AspGln LysHis
125 130 135
AAA AGTTCC TGCACCATG ACCAGAGAC ACAGTGTGT CAGTGT AAAGAA 482
45 Lys SerSer CysThrMet ThrArgAsp ThrValCys GlnCys LysGlu
140 145 150
GGC ACCTTC CGGAATGAA AACTCCCCA GAGATGTGC CGGAAG TGTAGC 530
Gly ThrPhe ArgAsnGlu AsnSerPro GluMetCys ArgLys CysSer
SD 155 160 165
AGG TGCCCT AGTGGGGAA GTCCAAGTC AGTAATTGT ACGTCC TGGGAT 578
Arg CysPro SerGlyGlu ValGlnVa1 SerAsnCys ThrSer TrpAsp
170 175 180 185
SS
GAT ATCCAG TGTGTTGAA GAATTTGGT GCCAATGCC ACTGTG GAAACC 626
Asp IleGln CysValGlu GluPheGly AlaAsnAla ThrVal GluThr
190 195 200
C7O CCA GCTGCT GAAGAGACA ATGAACACC AGCCCGGGG ACTCCT GCCCCA 674
Pro AlaAla GluGluThr MetAsnThr SerProGly ThrPro AlaPro
205 210 215
GCT GCTGAA GAGACAATG AACACCAGC CCAGGGACT CCTGCC CCAGCT 722
34
CA 02294704 1999-12-16
WO 99100423 PCT/US98/13491
Ala Ala Glu Glu Thr Met Asn Thr Ser Pro Gly Thr Pro Ala Pro Ala
220 225 230
GCT GAA GAG ACA ATG ACC ACC AGC CCG GGG ACT CCT GCC CCA GCT GCT 770
$ Ala Glu Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala
235 240 245
GAA GAG ACA ATG ACC ACC AGC CCG GGG ACT CCT GCC CCA GCT GCT GAA 818
Glu Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu
250 255 260 265
GAG ACA ATG ACC ACC AGC CCG GGG ACT CCT GCC TCT TCT CAT TAC CTC 866
Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Ser Ser His Tyr Leu
270 275 280
TCA TGC ACC ATC GTA GGG ATC ATA GTT CTA ATT GTG CTT CTG ATT GTG 914
Ser Cys Thr Ile Val Gly Ile Ile Val Leu Ile Val Leu Leu Ile Val
285 290 295
2O TTT GTT TGAAAGACTT CACTGTGGAA GAAATTCCTT CCTTACCTGA AAGGTTCAGG 970
Phe Val
TAGGCGCTGG CTGAGGGCGG GGGGCGCTGG ACACTCTCTG CCCTGCCTCC CTCTGCTGTG 1030
TTCCCACAGA CAGAAACGCC TGCCCCTGCC CCAAGTCCTG GTGTCTCCAG CCTGGCTCTA 1090
TCTTCCTCCT TGTGATCGTC CCATCCCCAC ATCCCGTGCA CCCCCCAGGA CCCTGGTCTC 1150
3O ATCAGTCCCT CTCCTGGAGC TGGGGGTCCA CACATCTCCC AGCCAAGTCC AAGAGGGCAG 1210
GGCCAGTTCC TCCCATCTTC AGGCCCAGCC AGGCAGGGGG CAGTCGGCTC CTCAACTGGG 1270
TGACAAGGGT GAGGATGAGA AGTGGTCACG GGATTTATTC AGCCTTGGTC AGAGCAGAAA 1330
P,AAAAA.AAAA AAAAAAA 13 4 7
(2) INFORMATION FORSEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:
299
amino
acids
(B) TYPE:
amino
acid
(D) TOPOLOGY:
linear
(ii) TYPE: protein
MOLECULE
(xi) SEQUENCE DESCRIPTION: ID N0:2:
SEQ
Met Gln Val LysGlu Arg Phe ProLeuGlyAsn SerGly Asp
Gly Leu
1 5 10 15
Arg Ala Arg ProPro Asp Gly GlyArgValArg ProArg Thr
Pro Arg
20 25 30
Gln Asp Val GlyAsn His Thr AlaArgIlePro LysThr Leu
Gly Met
35 40 45
Lys Phe Val ValIle Val Ala LeuLeuProVal LeuAla Tyr
Val Val
50 55 60
Ser Ala Thr AlaArg Gln Glu ValProGlnGln ThrVal Ala
Thr Glu
70 75 80
CA 02294704 1999-12-16
WO 99/00423 PCT/US98/13491
Pro Gln Gln Gln Arg His Ser Phe Lys Gly Glu Glu Cys Pro Ala Gly
85 90 95
Ser His Arg Ser Glu Thr Gly CysAsnPro CysThrGlu Gly
His Ala
100 105 110
Val Asp Tyr Thr Asn Ser Asn GluProSer CysPhePro Cys
Ala Asn
115 120 125
Thr Val Cys Lys Ser Gln Lys LysSerSer CysThrMet Thr
Asp His
130 135 140
Arg Asp Thr Val Cys Cys Lys GlyThrPhe ArgAsnGlu Asn
Gln G1u
145 150 155 160
Ser Pro Glu Met Cys Lys Cys ArgCysPro SerGlyGlu Val
Arg Ser
165 170 175
Gln Val Ser Asn Cys Ser Trp AspIleGln CysValGlu Glu
Thr Asp
180 185 190
Phe Gly Ala Asn A1a Val Glu ProAlaAla GluGluThr Met
Thr Thr
195 200 205
Asn Thr Ser Pro Gly Pro Ala AlaAlaGlu GluThrMet Asn
Thr Pro
210 215 220
Thr Ser Pro Gly Thr Ala Pro AlaGluGlu ThrMetThr Thr
Pro Ala
225 230 235 240
Ser Pro Gly Thr Pro Pro Ala GluGluThr MetThrThr Ser
Ala Ala
245 250 255
Pro Gly Thr Pro Ala Ala Ala GluThrMet ThrThrSer Pro
Pro Glu
3S 260 265 270
Gly Thr Pro Ala Ser His Tyr SerCysThr IleValGly Ile
Ser Leu
275 280 285
Ile Val Leu Ile Val Leu Ile PheVal
Leu Val
290 295
(2) INFORMATION FOR ID N0:3:
SEQ
4S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: amino acids
8
(B) TYPE:
amino acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY:linear
(ii)MOLECULE TYPE:peptide
(iii)HYPOTHETICAL:
No
SS (iw ANTI-SENSE:
No
(vi.7.MMEDIATE
SOURCE:
(B) CLONE:
FLAG peptide
GO (xi)SEQUENCE DESCRIPTION:
SEQ ID N0:3:
Asp Tyr Lys Asp
Asp Asp Asp
Lys
1 5
36
