Note: Descriptions are shown in the official language in which they were submitted.
CA 02211003 1999-07-19
This invention relates to newly identified
polynucleotides, polypeptides encoded by such
polynucleotides, the use of such polynucleotides and
polypeptides, as well as the production of such
polynucleotides and polypeptides. The polypeptide of the
present invention has been putatively identified as a Tumor
Necrosis Factor receptor, and more particularly as a type 2
Tumor Necrosis Factor Receptor. The polypeptide of the
present ivention will hereinafter be referred to as "TNF
receptor". The invention also relates to inhibiting the
receptor.
Human tumor necrosis factors a (TNF-a) and (3 (TNF-/3 or
lymphotoxin) are related members of a broad class of
polypeptide mediators, which includes the interferons,
interleukins and growth factors, collectively called
cytokines (Beutler, B. and Cerami, A., Annu. Rev. Immunol.,
7:625-655 (1989)).
Tumor necrosis factor-(TNF-a and TNF-S) was originally
discovered as a result of its anti-tumor activity, however,
now it is recognized as a DlP;orY~-sic cytokine playing
important roles in a host of biological p~cesses and
pathologies. To date, there are eight known members of the
TNF-related cytokine family, TNF-a, TNF-(3 (lymphotoxin-a),
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CA 02211003 1997-09-19
LT-S, and ligands for the Fas receptor, CD30, CD27, CD40 and ~~
4-1BB receptors. These proteins have conserved C-terminal
sequences and variable N-terminal sequences which are often
used as membrane anchors, with the exception of TNF-~. Both
TNF-a and TNF-~ function as homotrimers when they bind to TNF
receptors.
TNF is produced by a number of cell types, including
monocytes, fibroblasts, T cells, natural killer (NR) cells
and predominately by activated machrophages. TNF-a has been
. reported to have a role in the rapid necrosis of tumors,
immunostimulation, autoimmune disease, graft rejection,
producing an anti-viral response, septic shock, cerebral
malaria, cytotoxicity, protection against deleterious effects
of ionizing radiation produced during a course of
chemotherapy, such as denaturation of enzymes, lipid
peroxidation and DNA damage (Nata et al, J. Immunol.
136(7):2483 (1987)), growth regulation, vascular endothelium
effects. and metabolic effects. TNF-a also triggers
endothelial cells to secrete various factors, including PAI-
1, IL-1, GM-CSF and IL-6 to promote cell proliferation. In
addition, TNF-a up-regulates various cell adhesion molecules .
such as E-Selectin, ICAM-1 and VCAM-1. TNF-a and the Fas
ligand have also been shown to induce programmed cell death.
A related molecule, lymphotoxin (LT, also referred to as
TNF-~B), which is produced by activated lymphocytes shows a
similar but not identical spectrum of biological activities
as TNF. Two different types of LT have been found, LT-a and
LT-~. LT-a has many activities, including tumor necrosis,
induction of an antiviral state, activation of
polymorphonuclear leukocytes, induction of class I major
histocompatibility complex antigens on endothelial cells,
induction of adhesion molecules on endothelium and growth
hormone stimulation (Ruddle, N. and Homer, R. , Prog. Allergy,
40:162-182 (1988)).
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r
The first step in the induction of the various cellular
responses mediated by TNF or LT is their binding to specific
cell surface or soluble receptors. Two distinct TNF
receptors of approximately 55-KDa (TNF-R1) and 75-KDa (TNF-
R2) have been identified (Hohman, H.P. et al., J. Biol.
Chem., 264:14927-14934 (1989)), and human and mouse cDNAs
corresponding to both receptor types have been isolated and
characterized (Loetscher, H. et al., Cell, 61:351 (1990)).
Both TNF-Rs share the typical structure of cell surface
receptors including extracellular, transmembrane and
intracellular regions.
These molecules exist not only in cell bound forms, but
also in soluble forms, consisting of the cleaved extra-
cellular domains of the intact receptors (Nophar et al., EMBO
Journal, 9 (10):3269-76 (1990)). The extracellular domains
of TNF-Rl and TNF-R2 share 28~ identity and are characterized
by four repeated cysteine-rich motifs with significant
intersubunit sequence homology. The majority of cell types
and tissues appear to express both TNF receptors and both
receptors are active in signal transduction, however, they
are able to mediate distinct cellular responses. Further,
TNF-R2 was shown to exclusively mediate human T cell
proliferation by TNF as shown in PCT WO 94/09137.
TNF-R1 dependent responses include accumulation of C-
FOS, IL-6, and manganese superoxide dismutase mRNA,
prostaglandin E2 synthesis, IL-2 receptor and MHC class I and
II cell surface antigen expression, growth inhibition, and
cytotoxicity. TNF-R1 also triggers second messenger systems
such as phospholipase A2, protein kinase C,
phosphatidylcholine-specific phospholipase C and
sphingomyelinase (Pfefferk et al., Cell, 73:457-467 (1993)).
The receptor polypeptide of the present invention binds
TNF, and in particular, TNF-~. Further, the TNF receptor may
also bind other ligands, including but not limifed to Nerve
Growth Factor, due to homology to a family of receptors and
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CA 02211003 1997-09-19 ,
antigens which are involved in other critical biological
processes.. This family shows highly conserved cysteine
residues and includes the low affinity NGF receptor, which
plays an important role in the regulation of growth and
differentiation of nerve cells, the Fas receptor also called
APO, a receptor which is involved is signalling for apoptosis
and which, based on a study with mice deficient in its
function, seems to play an important role in the etiology of
a lupus-like disease, the TNF-R1, the B cell antigen CD40,
and the T cell activation antigen CD27.
In accordance with one aspect of the present invention,
there is provided a novel mature polypeptide which is a
putative TNF receptor, as well as fragments, analogs and
derivatives thereof. The polypeptide of the present
invention is of human origin.
In accordance with another aspect of the present
invention, there are provided isolated nucleic acid molecules
encoding the polypeptide of the present invention, including
mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogs
thereof and biologically active and diagnostically or .
therapeutically useful fragments thereof.
In accordance with yet a further aspect of the present
invention, there is provided a process for producing such
polypeptides by recombinant techniques which comprises
culturing recombinant prokaryotic and/or eukaryotic host
cells, containing a nucleic acid sequence encoding a
polypeptide of the present invention, under conditions
promoting expression of said protein and subsequent recovery
of said protein.
In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such
polypeptide, or polynucleotide encoding such polypeptide to
screen for receptor antagonists and/or agonists and/or
receptor ligands.
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In accordance with yet a further aspect of the present
invention, there are provided nucleic acid probes comprising -
nucleic acid molecules of sufficient length to specifically
hybridize to the polypeptide of the present invention.
In accordance with still another aspect of the present
invention, there is provided a process of using such agonists
for treating conditions related to insufficient TNF receptor
' activity, for example, to inhibit tumor growth, to stimulate
human cellular proliferation, e.g., T-cell proliferation, to
regulate the immune response and antiviral responses, to
protect against the effects of ionizing radiation, to protect
against chlamidiae infection, to regulate growth and to treat
immunodeficiencies such as is. found in HIV.
In accordance with another aspect of the present
invention, there is provided a process of using such
antagonists for treating conditions associated with over-
expression of the TNF receptor, for example, for treating T-
cell mediated autoimmune diseases such as AIDS, septic shock,
cerebral malaria, graft rejection, cytotoxicity, cachexia,
apoptosis and inflammation.
These and other aspects of the present invention should
be apparent to those skilled in the art from the teachings
herein.
The following drawings are illustrative of embodiments
of the invention and are not meant to limit the scope of the
invention as encompassed by the claims.
Figure 1 shows the cDNA sequence and corresponding
deduced amino acid sequence of the polypeptide of the present
invention. The initial 21 amino acids represent the putative
leader sequence and are underlined. The standard one-letter
abbreviations for amino acids are used. Sequencing was
performed using a 373 automated DNA sequencer (Applied
Biosystems, Inc.). Sequencing accuracy is predicted to be
greater than 97~ accurate.
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' CA 02211003 1997-09-19
Figure 2 illustrates an amino acid sequence alignment
of the polypeptide of the present invention (upper line)
and the human type 2 TNF receptor (lower line).
The term "gene" or "cistron" means the segment of DNA
involved in producing a polypeptide chain; it includes
regions preceding and following the coding region (leader
and trailer) as well as intervening sequences (introns)
between individual coding segments (exons).
In accordance with an aspect of the present invention,
there is provided an isolated nucleic acid (polynucleotide)
which encodes for the mature polypeptide having the deduced
amino acid sequence of Figure 1 (SEQ ID No. 2) or for the
mature polypeptide encoded by the cDNA of the clone
deposited as ATCC Deposit No. 75899 on September 28, 1994.
A polynucleotide encoding a polypeptide of. the present
invention may be obtained from human pulmonary tissue,
hippocampus and adult heart. The polynucleotide of this
invention was discovered in a cDNA library derived from
human early passage fibroblasts (HSA 172 cells). It is
structurally related to the human TNF-R2 receptor. It
contains an open reading frame encoding a protein of 401
amino acid residues of which approximately the first 21
amino acid residues are the putative leader sequence such
that the mature protein comprises 380 amino acids. The
protein exhibits the highest degree of homology to a human
type 2 TNF receptor with 39% identity and 46% similarity
over an 88 amino acid stretch. Six conserved cyteines
present in modules of 40 residues in all TNF receptors are
conserved in this receptor.
The TNF receptor of the present invention is a soluble
receptor and is secreted, however, it may also exist as a
membrane bound receptor having a transmembrane region and
an intra- and extracellular region. The polypeptide of the
present invention may bind TNF and lymphotoxin ligands.
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CA 02211003 1997-09-19 ,:
In accordance with an aspect of the present invention
there is provided a polynucleotide which may be in the .form
of RNA or in the form of DNA, which DNA includes cDNA,
genomic DNA, and synthetic DNA. The DNA may be double-
stranded or single-stranded, and if single stranded may be
the coding strand or non-coding (anti-sense) strand. The
coding sequence which encodes the mature polypeptide may be
identical to the coding sequence shown in Figure 1 (SEQ ID
No. 1) or that of the deposited clone or may be a different
coding sequence which coding sequence, as a result of the
redundancy or degeneracy of the genetic code, encodes the
same mature polypeptide as the DNA of Figure 1 (SEQ ID No. 1)
or the deposited cDNA.
The polynucleotide which encodes for the mature
polypeptide of Figure 1 (SEQ ID No. 2) or for the mature
polypeptide encoded by the deposited cDNA may include: only
the coding sequence for the mature polypeptide; the coding
sequence for the mature polypeptide and additional coding
sequence such as a leader or secretory sequence or a
proprotein sequence; the coding sequence for the mature
polypeptide (and optionally additional coding sequence) and .
non-coding sequence, such as introns or non-coding sequence
5' and/or 3' of the coding sequence for the mature
polypeptide.
Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding
sequence for the polypeptide as well as a polynucleotide
which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the
hereinabove described polynucleotides which encode for
fragments, analogs and derivatives of the polypeptide having
the deduced amino acid sequence of Figure 1 (SEQ ID No. 2) or
the polypeptide encoded by the cDNA of the deposited clone.
The variant of the polynucleotide may be a naturally
CA 02211003 1997-09-19 ,.
occurring allelic variant of the polynucleotide or a non-
naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as shown in Figure 1
(SEQ ID No. 2) or the same mature polypeptide encoded by the
cDNA of the deposited clone as well as-variants of such
polynucleotides which variants encode for a fragment,
derivative or analog of the polypeptide of Figure 1 (SEQ ID
No. 2) or the polypeptide encoded by the cDNA of the
deposited clone. Such nucleotide variants include deletion
variants, substitution variants and addition or insertion
variants.
As hereinabove indicated, the polynucleotide may have a
coding sequence which is a naturally occurring allelic
variant of the coding sequence shown in Figure 1 (SEQ ID No.
1) or of the coding sequence of the deposited clone. As
known in the art, an allelic variant is an alternate form of
a polynucleotide sequence which may have a substitution,
deletion or addition of one or more nucleotides, which does
not substantially alter the function of the encoded
polypeptide.
The present invention also includes polynucleotides,
wherein the coding sequence for the mature polypeptide may be
fused in the same reading frame to a.polynucleotide sequence
which aids in expression and secretion of a polypeptide from
a host cell, for example, a leader sequence which functions
as a secretory sequence for controlling transport of a
polypeptide from the cell. The polypeptide having a leader
sequence is a preprotein and may have the leader sequence
cleaved by the host cell to form the mature form of the
polypeptide. The polynucleotides may also encode for a
proprotein which is the mature protein plus additional 5'
amino acid~residues. A mature protein having a prosequence
is a proprotein and is an inactive form of the protein. Once
the prosequence is cleaved an active mature protein remains.
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CA 02211003 1997-09-19
Thus, for example, the polynucleotide of the present
invention may encode for a mature protein, or for a protein -
having a prosequence or for a protein having both a
prosequence and a presequence (leader sequence).
The polynucleotides of the present invention may also
' have the coding sequence fused in frame to~a marker sequence
which allows for purification of the polypeptide of the
' present invention. The marker sequence may be a hexa-
histidine tag supplied by a pQE-9 vector to provide for
purification of the mature polypeptide fused to the marker in
the case of a bacterial host, or, for example, the marker
sequence may be a hemagglutinin (HA) tag when a mammalian
host, e.g. COS-7 cells, is used. The HA tag corresponds to
an epitope derived from the influenza hemagglutinin protein
(Wilson, I., et al., Cell, 37:767 (1984)). The coding
sequence may also be fused to a sequence which codes for a
fusion protein such as an IgG Fc fusion protein.
The present invention further relates to
polynucleotides which hybridize to the hereinabove-described
sequences if there is at least 50$ and preferably 70$
identity between the sequences. The present invention
particularly relates to polynucleotides which hybridize under
stringent conditions to the hereinabove-described
polynucleotides. As herein used, the term "stringent
conditions" means hybridization will occur only if there is
at least 95~ and preferably at least 97~ identity between the
sequences. The polynucleotides which hybridize to the
hereinabove described polynucleotides in a preferred
embodiment encode polypeptides which retain substantially the
same biological function or activity as the mature
polypeptide encoded by the cDNA of Figure 1 (SEQ ID No. 1) or
the deposited cDNA.
The deposits) referred to herein will be maintained
under the terms of the Budapest Treaty on the Irnternational
Recognition of the Deposit of Micro-organisms for purposes of
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CA 02211003 1997-09-19 ..
Patent Procedure. These deposits are provided merely as.~
convenience to. those of skill in the art and are not an
admission that a deposit is required under 35 U.S.C. ~112.
The sequence of the polynucleotides contained in the
deposited materials, as well as the amino acid sequence of
the polypeptides encoded thereby, are inco=porated herein by
reference and are controlling in the event of any conflict
with any description of sequences herein. A license may be
required to make, use or sell the deposited materials, and
no such license is hereby granted.
The present invention further relates to a polypeptide
which has the deduced amino acid sequence of Figure 1 (SEQ ID
No. 2) or which has the amino acid sequence encoded by the
deposited cDNA, as well as fragments, analogs and derivatives
of such polypeptide.
The terms "fragment," "derivative" and "analog" when
referring to the polypeptide of Figure 1 (SEQ ID No. 2) or
that encoded by the deposited cDNA, means a polypeptide which
retains essentially the same biological function or activity
as such polypeptide. Thus, an analog includes a proprotein
which can be activated by cleavage of the proprotein portion
to produce an active mature polypeptide.
The polypeptide of the present invention may be a
recombinant polypeptide, a natural polypeptide or a synthetic
polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide
of Figure 1 (SEQ ID No. 2) or that encoded by the deposited
cDNA may be (i) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved
amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may
not be one encoded by the genetic code, or (ii) one in which
one or more of the amino acid residues includes a substituent
group, or (iii) one in which the mature polypeptide is fused
with another compound, such as a compound to increase the
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CA 02211003 1997-09-19
half-life of the polypeptide (for example, polyethylene
glycol), or (iv) one in which the additional amino acids are
fused to the mature polypeptide, such as an IbG Fc fusion
region peptide or leader or secretory sequence or a sequence
which is employed for purification of the mature polypeptide
or a proprotein sequence. Such fragments-, derivatives and
analogs are deemed to be within the scope of those skilled in
the art from the teachings herein.
The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
The term "isolated" means that the material is removed
from its original environment (e. g., the natural environment
if it is naturally occurring). For example, a naturally-
occurring polynucleotide or polypeptide present in a living
animal is not isolated, but the same polynucleotide or
polypeptide, separated from some or all of the coexisting
materials in the natural system, is isolated. Such
polynucleotides could be part of a vector and/or such
polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or
composition is not part of its natural environment.
The present invention also relates to vectors which
include polynucleotides of the present invention, host cells
which are genetically engineered with vectors of the
invention and the production of polypeptides of the invention
by recombinant techniques.
Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this
invention which may be, for example, a cloning vector or an
expression vector. The vector may be, for example, in the
form of a plasmid, a viral particle, a phage, etc. The
engineered host cells can be cultured in conventional
nutrient media modified as appropriate for activating
promoters, selecting transformants or amplifying the nucleic
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CA 02211003 1997-09-19
acid sequences of the present invention. The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be
employed for producing polypeptides-- by recombinant
techniques. Thus, for example, the polynucleotide may be
included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g.,
derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from
combinations of plasmids and phage DNA, viral DNA such as
vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is
replicable and viable in the host.
The appropriate DNA sequence may be inserted into the
vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction
endonuclease sites) by procedures known in the art. Such
procedures and others are deemed to be within the scope of
those skilled in the art.
The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequences)
(promoter) to direct mRNA synthesis. As representative
examples of such promoters, there may be mentioned: LTR or
SV40 promoter, the E. coli. lac or try, the phage lambda PL
promoter and other promoters known to control expression of
genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also contains a ribosome binding site
for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for
amplifying expression.
In addition, the expression vectors preferably contain
one or more selectable marker genes to provide a phenotypic
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trait for selection of transformed host cells such as
dihydrofolate reductase or neomycin resistance for eukaryotic -
cell culture, or such as tetracycline or ampicillin
resistance in E. coli.
The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate
host to permit the host to express the protein.
As representative examples of appropriate hosts, there
may be mentioned: bacterial cells, such as E. coli,
Streptomyces, Salmonella typhimurium; fungal cells, such as
yeast; insect cells such as Drosophila S2 and Spodoptera Sf9;
animal cells such as CHO, COS or Bowes melanoma;
adenoviruses; plant cells, etc. The selection of an
appropriate host is deemed to be within the scope of those
skilled in the art from the teachings herein.
More particularly, the present invention also includes
recombinant constructs comprising one or more of the
sequences as broadly described above. The constructs
comprise a vector, such as a plasmid or viral vector, into
which a sequence of the invention has been inserted, in a
forward or reverse orientation. In a preferred aspect of
this embodiment, the construct further comprises regulatory
sequences, including, for example, a promoter, operably
linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided
by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),
pBS, pDlO, phagescript, psiX174, pbluescript SK, pbsks,
pNHBA, pNHl6a, pNHl8A, pNH46A (Stratagene); pTRC99a, pKK223-
3, pKK233-3, pDR540, pRITS (Pharmacia). Eukaryotic: pWLNEO,
pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG,
pSVL (Pharmacia). However, any other plasmid or vector may
be used as long as they are replicable and viable in the
host.
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CA 02211003 1997-09-19 ,.
Promoter regions can be selected from any desired gene
using CAT (chloramphenicol transferase) vectors or other
vectors with selectable markers. Two appropriate vectors are
pKK232-8 and pCM7. Particular named bacterial promoters
include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp.
Eukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus,
and mouse metallothionein-I. Selection of the appropriate
vector and promoter is well within the level of ordinary
skill in the art. .
In a further embodiment, the present invention relates
to host cells containing the above-described constructs. The
host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the construct into the host
cell can be effected by calcium phosphate transfection, DEAF-
Dextranznediated transfection, or electroporation (Davis, L.,
Dibner, M., Battey, I., Basic Methods in Molecular Biology,
(1986)).
The constructs in host cells can be used in a
conventional manner to produce the gene product encoded by
the recombinant sequence. Alternatively, the polypeptides of
the invention can be synthetically produced by conventional
peptide synthesizers.
Mature proteins can be expressed in mammalian cells,
yeast, bacteria, or other cells under the control of
appropriate promoters. Cell-free translation systems can
also be employed to produce such proteins using RNAs derived
from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with
prokaryotic and eukaryotic hosts are described by Sambrook,
et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of
which is hereby incorporated by reference.
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Transcription of the DNA encoding the polypeptides of
the present invention by higher eukaryotes is increased by -
inserting an enhancer sequence into the vector. Enhancers
are cis-acting elements of DNA, usually about from 10 to 300
by that act on a promoter to increase its transcription.
Examples including the SV40 enhancer on the late side of the
replication origin by 100 to 270, a cytomegalovirus early
promoter enhancer, the polyoma enhancer on the late side of
the replication origin, and adenovirus enhancers.
Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin
resistance gene of E. coli and S. cerevisiae TR.P1 gene, and
a promoter derived from a highly-expressed gene to direct
transcription of a downstream structural sequence. Such
promoters can be derived from operons encoding glycolytic
enzymes such as 3-phosphoglycerate kinase (PGK), a-factor,
acid phQSphatase, or heat shock proteins, among others. The
heterologous structural sequence is assembled in appropriate
phase with translation initiation and termination sequences,
and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence
can encode a fusion protein including an N-terminal
identification peptide imparting desired characteristics,
e.g., stabilization or simplified purification of expressed
recombinant product.
Useful expression vectors for bacterial use are
constructed by inserting a structural DNA sequence encoding
a desired protein together with suitable translation
initiation and termination signals in operable reading phase
with a functional promoter. The vector will comprise one or
more phenotypic selectable markers and an origin of
replication to ensure maintenance of the vector and to, if
desirable, provide amplification within the host. Suitable
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CA 02211003 1997-09-19 ,,
prokaryotic hosts for transformation include E. coli, -
Bacillus subtilis a Salmonella typhimurium and various species
within the genera Pseudomonas, Streptomyces, and
Staphylococcus, although others may also be employed as a
matter of choice.
As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a
selectable marker and bacterial origin of replication derived
from commercially available plasmids comprising genetic
elements of the well known cloning vector pBR322 (ATCC
3701?). Such commercial vectors include, for example,
pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1
(Promega Biotec, Madison, WI, USA). These pBR322 "backbone"
sections are combined with an appropriate promoter and the
structural sequence to be expressed.
Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e. g.,
temperature shift or chemical induction) and cells are
cultured for an additional period.
Cells are typically harvested by centrifugation,
disrupted by physical or chemical means, and the resulting
crude extract retained for further purification.
Microbial cells employed in expression of proteins can
be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell
lysing agents, such methods are well know to those skilled in
the art.
Various mammalian cell culture systems can also be
employed to express recombinant protein. Examples of
mammalian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described by Gluzman, Cell, 23:175
(1981), and other cell lines capable of expressing a
compatible vector, for example, the C127, 3T3, CHO, HeLa and
BHR cell lines. Mammalian expression vectors will comprise
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CA 02211003 1997-09-19
an origin of replication, a suitable promoter and enhancer,
and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the
SV40 splice, and polyadenylation sites may be used to provide
the required nontranscribed genetic elements.
The polypeptide of the present invention can be
recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation,
acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding
steps can be used, as necessary, in completing configuration
of the mature protein. Finally, high performance liquid
chromatography (HPLC) can be employed for final purification
steps. ,
The polypeptides of the present invention may be a
naturally purified product, or a product of chemical
synthetic procedures, or produced by recombinant techniques
from a prokaryotic or eukaryotic host (for example, by
bacterial, yeast, higher plant, insect and mammalian cells in
culture). Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present
invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue.
The TNF receptor of the present invention was assayed
for the ability to bind TNF-a and TNF-~, however, the present
invention also contemplates the ability of the receptor to
bind other TNF-like proteins. Monoclonal antibodies specific
to TNF-a and TNF-S were prepared. These monoclonal
antibodies were bound to TNF-a and TNF-~i and a control ELISA
assay was performed to quantify the amount of monoclonal
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CA 02211003 1997-09-19
antibody present. The TNF receptor was then bound to TNF-a -
and TNF-~ in the same way in which the monoclonal antibody -
was bound and another ELISA assay was performed. The TNF
receptor was found to bind to TNF-S just as strongly as the
monoclonal antibody, while it only bound TNF-a two-thirds as
strongly.
Fragments of the full length polynucleotide seqeunces of
the present invention may be used as a hybridization probe
for a cDNA library to isolate other genes which have a high
sequence similarity to the polynucleotide sequence of the
present invention or similar biological activity. Probes of
this type generally have at least 50 bases, although they may
have a greater number of bases. The probe may also be used
as markers to identify a cDNA clone corresponding to a full
length transcript and a genomic clone or clones that contain
the complete polynucleotide sequence of the present invention
including regulatory and promotor regions, exons, and
introns. An example of a screen comprises isolating the
coding region of the gene of the present invention by using
the known DNA sequence to synthesize an oligonucleotide
probe. Labeled oligonucleotides having a sequence .
complementary to that of the gene of the present invention
are used to screen a library of human cDNA, genomic DNA or
mRNA to determine which members of the library the probe
hybridizes to.
This invention also provides a method of screening
compounds to identify compounds which interact with the
polypeptide of the present invention which comprises
contacting a mammalian cell comprising an isolated DNA
molecule encoding and expressing a the polypeptide of the
present invention with a plurality of compounds, determining
those which activate or block the activation of the receptor,
and thereby identifying compounds which specifically interact
with, and activate or block the activation of the polypeptide
of the present invention.
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CA 02211003 1997-09-19
This invention also contemplates the use of the
polynucleotide of the present invention as a diagnostic. For
example, if a mutation is present, c..::~ditions would result
from a lack of TNF receptor activity. Further, mutations
which enhance TNF receptor activity would lead to diseases
associated with an over-expression of the receptor, e.g.,
endotoxic shock. Mutated genes can be detected by comparing
the sequence of the defective gene with that of a normal one.
Subsequently one can verify that a mutant gene is associated
with a disease condition or the susceptibility to a disease
condition. That is, a mutant gene which leads to the
underexpression of the TNF receptor would be associated with
an inability of TNF to inhibit tumor growth.
Individuals carrying mutations in the polynucleotide of
the present invention may be detected at the DNA level by a
variety of techniques. Nucleic acids used for diagnosis may
be obtained from a patient's cells which include, but are not
limited~to, blood, urine, saliva and tissue biopsy. The
genomic DNA may be used directly for detection or may be
amplified enzymatically by using PCR (Saiki et al., Nature,
324:163-166_(1986)) prior to analysis. RNA or cDNA may also
be used for the same purpose. As an example, PCR primers
complementary to the nucleic acid of the instant invention
can be used to identify and analyze gene mutations. For
example, deletions and insertions can be detected by a change
in the size of the amplified product in comparison to the
normal genotype. Point mutations can be identified by
hybridizing amplified DNA to radiolabeled RNA or
alternatively, radiolabeled TNF receptor antisense DNA
sequences. Perfectly matched sequences can be distinguished
from mismatched duplexes by RNase A digestion or by
differences in melting temperatures. Such a diagnostic would
be particularly useful for prenatal or even neonatal testing.
Sequence differences between the reference gene and
"mutants" may be revealed by the direct DNA sequencing
-19-
CA 02211003 1997-09-19 -
method. In addition, cloned DNA segments may be used as
probes to detect specific DNA segments. The sensitivity of -
this method is greatly enhanced when combined with PCR. For
example, a sequencing primary used with double stranded PCR
product or a single stranded template molecule generated by
a modified PCR product. The sequence--determination is
performed by conventional procedures with radiolabeled
nucleotides or by automatic sequencing procedures with
fluorescent tags.
Sequence changes at the specific locations may be
revealed by nuclease protection assays, such as RNase and S1
protection or the chemical cleavage method (for example,
Cotton et al., PNAS, 85:4397-4401 (1985)).
The present invention further relates to a diagnostic
assay which detects an altered level of a soluble form of the
polypeptide of the present invention where an elevated level
in a sample derived from a host is indicative of certain
diseases. Assays available to detect levels of soluble
receptors are well known to those of skill in the art, for
example, radioimmunoassays, competitive-binding assays,
Western blot analysis, and preferably an ELISA assay may be
employed.
An ELISA assay initially comprises preparing an antibody
specific to an antigen to the polypeptide of the present
invention, preferably a monoclonal antibody. In addition a
reporter antibody is prepared against the monoclonal
antibody. To the reporter antibody is attached a detectable
reagent such as radioactivity, fluorescence or in this
example a horseradish peroxidase enzyme. A sample is now
removed from a host and incubated on a solid support, e.g, a
polystyrene dish, that binds the proteins in the sample. Any
free protein binding sites on the dish are then covered by
incubating~with a non-specific protein such as bovine serum
albumen. Next, the monoclonal antibody is incubated in the
dish during which time the monoclonal antibodies attach to
-20-
CA 02211003 1997-09-19
any proteins of the present invention which are attached to
the polystyrene dish. All unbound monoclonal antibody is -
washed out with buffer. The reporter antibody linked to
horseradish peroxidase is now placed in the dish resulting in
binding of the reporter antibody to any monoclonal antibody
bound to the polypeptide of the present invention.
Unattached reporter antibody is then washed out. Peroxidase
substrates are then added to the dish and the amount of color
developed in a given time period is a measurement of the
amount of the protein of interest present in a given volume
of patient sample when compared against a standard curve.
A competition assay may be employed wherein antibodies
specific to the polypeptides of the present invention are
attached to a solid support. Labeled TNF receptor
polypeptides, and a sample derived from the host are passed
over the solid support and the amount of label detected
attached to the solid support can be correlated to a quantity
in the~sample. The soluble form of the receptor may also be
employed to identify agonists and antagonists.
A thymocyte proliferation assay may be employed to
identify both ligands and potential agonists and antagonists
to the polypeptide of the present invention. For example,
thymus cells are disaggregated from tissue and grown in
culture medium. Incorporation of DNA prescursors such as 3H-
thymidine or 5-bromo-2'-deoxyuridine (HrdU) is monitored as
a parameter for DNA synthesis and cellular proliferation.
Cells which have incorporated BrdU into DNA can be detected
using a monoclonal antibody against BrdU and measured by an
enzyme or fluorochrome-conjugated second antibody. The
reaction is quantitated by fluorimetry or by
spectrophotometry. Two control wells and an experimental
well are set up. TNF-~3 is added to all wells, while soluble
receptors of the present invention are added to the
experimental well. Also added to the experimental well is a
compound to be screened. The ability of the compound to be
-21-
CA 02211003 1997-09-19 ~:
3
screened to inhibit the interaction of TNF-~ with the -
receptor polypeptides of the present invention may then be
quantified. In the case of the agonists, the ability of the
compound to enhance this interaction is quantified.
A determination may be made whether a ligand not known
to be capable of binding to the polypeptide of the present
invention can bind thereto comprising contacting a mammalian
cell comprising an isolated molecule encoding a polypeptide
of the present invention with a ligand under conditions
permitting binding of ligands known to bind thereto,
detecting the presence of any bound ligand, and thereby
determining whether such ligands bind to a polypeptide of the
present invention. Also, a soluble form of the receptor may
utilized in the above assay where it is secreted in to the
extra-cellular medium and contacted with ligands to determine
which will bind to the soluble form of the receptor.
Other agonist and antagonist screening procedures
involve, providing appropriate cells which express the
receptor on the surface thereof. In particular, a
polynucleotide encoding a polypeptide of the present
invention is employed to transfect cells to thereby express
the polypeptide. Such transfection may be accomplished by
procedures as hereinabove described.
Thus, for example, such assay may be employed for
screening for a receptor antagonist by contacting the cells
which encode the polypeptide of the present invention with
both the receptor ligand and a compound to be screened.
Inhibition of the signal generated by the ligand indicates
that a compound is a potential antagonist for the receptor,
i.e., inhibits activation of the receptor.
The screening may be employed for determining an agonist
by contacting such cells with compounds to be screened and
determining whether such compounds generate a signal, i.e.,
activates the receptor.
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f.
CA 02211003 1997-09-19
Other screening techniques include the use of cells
which express the polypeptide of the present invention (for -
example, transfected CHO cells ) in a system which measures
extracellular pH changes caused by receptor activation, for
example, as described in Science, Volume 246, pages 181-296
(1989). In another example, potential agonists or
antagonists may be contacted with a cell which expresses the
polypeptide of the present invention and a second messenger
response, e.g., signal transduction may be measured to
determine whether the potential antagonist or agonist is
effective.
Another screening technique involves expressing the
receptor polypeptide wherein it is linked to phospholipase C
or D. As representative examples of such cells, there may be
mentioned endothelial cells, smooth muscle cells, embryonic
kidney cells and the like. The screening for an antagonist
or agonist may be accomplished as hereinabove described by
detecting activation of the receptor or inhibition of
activation of the receptor from the phospholipase second
signal.
Antibodies may be utilized as both an agonist and
antagonist depending on which part of the polypeptide of the
present invention the antibody binds to. The antibody in one
instance can bind to the active site and block ligand access.
However, it has been observed that monoclonal antibodies
directed against certain TNF receptors can act as specific
agonists when binding to the extra-cellular domain of the
receptor.
In addition to the antagonists identified above,
oligonucleotides which bind to the TNF receptor may also act
as TNF receptor antagonists. Alternatively, a potential TNF
receptor antagonist may be a soluble form of the TNF receptor
which contains the complete extra-cellular region of the TNF
receptor and which binds to ligands to inhibit their
biological activity.
-23-
CA 02211003 1997-09-19 ,;
Another potential TNF receptor antagonist is an
antisense construct prepared using antisense technology. -
Antisense technology can be used to control gene expression
through triple-helix formation or antisense DNA or RNA, both
of which methods are based on binding of a polynucleotide to
DNA or RNA. For example, the 5' coding portion of the
polynucleotide sequence, which encodes for the mature
polypeptides of the present invention, is used to design an
antisense RNA oligonucleotide of from about 10 to 40 base
pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in
transcription (triple helix -see Lee et al., Nucl. Acids
Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988);
and Dervan et al., Science, 251: 1360 (1991)), thereby
preventing transcription and the production of TNF receptors.
The antisense RNA oligonucleotide hybridizes to the mRNA in
vivo and blocks translation of the mRNA molecule into the TNF
receptor- polypeptide (antisense - Okano, ,7. Neurochem.,
56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors
of Gene Expression, CRC Press, Boca Raton, FL (1988)). The
oligonucleotides described above can also be delivered to
cells such that the antisense RNA or DNA may be expressed in
vivo to inhibit production of TNF receptors.
TNF receptor antagonists also include a small molecule
which binds to and occupies the TNF receptor thereby making
the receptor inaccessible to ligands which bind thereto such
that normal biological activity is prevented. Examples of
small molecules include but are not limited to small peptides
or peptide-like molecules.
The TNF receptor agonists may be employed to stimulate
ligand activities, such as inhibition of tumor growth and
necrosis of certain transplantable tumors. The agonists may
also be employed to stimulate cellular differentiation, for
example, T-cell, fibroblasts and haemopoietic cell
differentiation. Agonists to the TNF receptor may also
-24-
CA 02211003 1997-09-19
augment TNF's role in the host's defense against
microorganisms and prevent related diseases (infections such
as that from L. monocytogenes) and chlamidiae. The agonists
may also be employed to protect against the deleterious
effects of ionizing radiation produced during a course of
radiotherapy, such as denaturation of enzymes, lipid
peroxidation, and DNA damage.
The agonists may also be employed to mediate an anti-
viral response, to regulate growth, to mediate the immune
response and to treat immunodeficiencies related to diseases
such as HIV.
Antagonists to the TNF receptor may be employed to treat
autoimmune diseases, for example, graft versus host rejection
and allograft rejection, and T-cell mediated autoimmune
diseases such as AIDS. It has been shown that T-cell
proliferation is stimulated via a type 2 TNF receptor.
Accordingly, antagonizing the receptor may prevent the
prolifer-ation of T-cells and treat T-cell mediated autoimmune
diseases.
The antagonists may also be employed to prevent
apoptosis, which is the basis for diseases such as viral
infection, rheumatoid arthritis, systemic lupus
erythematosus, insulin-dependent diabetes mellitus, and graft
rejection. Similarly, the antagonists may be employed to
prevent cytotoxicity.
The antagonists to the TNF receptor may also be employed
to treat B cell cancers which are stimulated by TNF.
Antagonists to the TNF receptor may also be employed to
treat and/or prevent septic shock, which remains a critical
clinical condition. Septic shock results from an exaggerated
host response, mediated by protein factors such as TNF and
IL-1, rather than from a pathogen directly. For example,
lipopolysaccharides have been shown to elicit the release of
TNF leading to a strong and transient increase of its serum
concentration. TNF causes shock and tissue injury when
-25-
CA 02211003 1997-09-19 ,:
administered in excessive amounts. Accordingly, antagonists
to the TNF receptor will block the actions of TNF and
treat/prevent septic shock. These antagonists may also be
employed to treat meningococcemia in children which
correlates with high serum levels of TNF.
Among other disorders which may be- treated by the
antagonists to TNF receptors, there are included,
inflammation which is mediated by TNF receptor ligands, and
the bacterial infections cachexia and cerebral malaria.
TNF receptor antagonists may also be employed to treat
inf lammation mediated by ligands to the receptor such as TNF .
The soluble TNF receptor and agonists and antagonists
may be employed in combination with a suitable pharmaceutical
carrier. Such compositions comprise a therapeutically
effective amount of the soluble receptor or agonist or
antagonist, and a pharmaceutically acceptable carrier or
excipient. Such a carrier includes but is not limited to
saline,.buffered saline, dextrose, water, glycerol, ethanol,
and combinations thereof. The formulation should suit the
mode of administration.
The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of
the ingredients of the pharmaceutical compositions of the
invention. Associated with such containers) can be a notice
in the form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals or biological
products, which notice reflects approval by the agency of
manufacture, use or sale for human administration. In
addition, the soluble form of the receptor and agonists and
antagonists of the present invention may also be employed in
conjunction with other therapeutic compounds.
The pharmaceutical compositions may be administered in
a convenient manner such as by the oral, topical,
intravenous, intraperitoneal, intramuscular, subcutaneous,
intranasal or intradermal routes. The pharmaceutical
-26-
CA 02211003 1997-09-19
m
compositions are administered in an amount which is effective
for treating and/or prophylaxis of the specific indication.
In general, they are administered in an amount of at least
about 10 ~,g/kg body weight and in most cases they will be
administered in an amount not in excess of about 8 mg/Kg body
weight per day. In most cases, the dosage is from about 10
~cg/kg to about 1 mg/kg body weight daily, taking into account
the routes of administration, symptoms, etc.
The TNF receptor and agonists and antagonists which are
polypeptides may also be employed in accordance with the
present invention by expression of such polypeptides in vivo,
which is often referred to as "gene therapy."
Thus, for example, cells from a patient may be
engineered with a polynucleotide (DNA or RNA) encoding a
polypeptide ex vivo, with the engineered cells then being
provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells
may be engineered by procedures known in the art by use~of a
retroviral particle containing RNA encoding a polypeptide of
the present invention.
Similarly, cells may be engineered in vivo for
expression of a polypeptide in vivo by, for example,
procedures known in the art. As known in the art, a producer
cell for producing a retroviral particle containing RNA
encoding the polypeptide of the present invention may be
administered to a patient for engineering cells in vivo and
expression of the polypeptide in vivo. These and other
methods for administering a polypeptide of the present
invention by such method should be apparent to those skilled
in the art from the teachings of the present invention. For
example, the expression vehicle for engineering cells may be
other than a retrovirus, for example, an adenovirus which may
be used to engineer cells in vivo after combination with a
suitable delivery vehicle.
_27_
CA 02211003 1997-09-19 .:
The sequences of the present invention are also valuable
for chromosome identification. The sequence is specifically
targeted to and can hybridize with a particular location on
an individual human chromosome. Moreover, there is a current
need for identifying particular sites on the chromosome. Few
chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes
according to the present invention is an important first step
in correlating those sequences with genes associated with
disease.
Briefly, sequences can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the 3' untranslated region is used to
rapidly select primers that do not span more than one exon in
the genomic DNA, thus complicating the amplification process .
These primers are then used for PCR screening of somatic cell
hybrids.containing individual human chromosomes. Only those
hybrids containing the human gene corresponding to the primer
will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure
for assigning a particular DNA to a particular chromosome.
Using the present invention with the same oligonucleotide
primers, sublocalization can be achieved with panels of
fragments from specific chromosomes or pools of large genomic
clones in an analogous manner. Other mapping strategies that
can similarly be used to map to its chromosome include in
situ hybridization, prescreening with labeled flow-sorted
chromosomes and preselection by hybridization to construct
chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA
clone to a metaphase chromosomal spread can be used to
provide a precise chromosomal location in one step. This
technique can be used with cDNA as short as 500 or 600 bases;
however, clones larger than 2, 000 by have a higher likelihood
-28-
CA 02211003 1997-09-19
of binding to a unique chromosomal location with sufficient
signal intensity for simple detection. For example, 2,000 by -
is good, 4, 000 is better, and more than 4, 000 is probably not
necessary to get good results a reasonable percentage of the
time. For a review of this technique, see Verma et al.,
Human Chromosomes: a Manual of Basic Techniques, Pergamon
Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the
chromosome can be correlated with genetic map data. Such
data are found, for example, in V. McKusick, Mendelian
Inheritance in Man (available on line through Johns Hopkins
University Welch Medical Library). The relationship between
genes and diseases that have been mapped to the same
chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in
the cDNP or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then
the mutation is likely to be the causative agent of the
disease.
With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a
chromosomal region associated with the disease could be one
of between 50 and 500 potential causative genes.. (This
assumes 1 megabase mapping resolution and one gene per 20
kb).
The polypeptides, their fragments or other derivatives,
or analogs thereof, or cells expressing them can be used as
an immunogen to produce antibodies thereto . These antibodies
can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chimeric, single chain,
and humanized antibodies, as well as Fab fragments, or the
product of an Fab expression library. Various procedures
-29-
;x
CA 02211003 1997-09-19
,..
known in the art may be used for the production of such
antibodies and fragments.
Antibodies generated against the polypeptides
corresponding to a sequence of the present invention can be
obtained by direct injection of the polypeptides into an
animal or by administering the polypeptides to an animal,
preferably a nonhuman. The antibody so obtained will then
bind the polypeptides itself. In this manner, even a
sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native
polypeptides. Such antibodies can then be used to isolate
the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line
cultures can be used. Examples include the hybridoma
technique (Kohler and Milstein, 1975, Nature, 256:495-497),
the trioma technique, the human B-cell hybridoma technique
(Rozbor~et al., 1983, Immunology Today 4:72), and the EBV-
hybridoma technique to produce human monoclonal antibodies
(Cole, et al., 1985, in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain
antibodies (U. S. Patent 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products
of this invention. Also, transgenic mice may be used to
express humanized antibodies to immunogenic polypeptide
products of this invention.
The present invention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified,
are by weight.
In order to facilitate understanding of the following
examples certain frequently occurring methods and~or terms
will be described.
-30-
CA 02211003 1997-09-19 ..
"Plasmids" are designated by a lower case p preceded
and/or followed by capital letters and/or numbers. The
starting plasmids herein are either commercially available,
publicly available on an unrestricted basis, or can be
constructed from available plasmids in accord with published
procedures. In addition, equivalent plasmids to those
described are known in the art and will be apparent to the
ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the
DNA with a restriction enzyme that acts only at certain
sequences in the DNA. The various restriction enzymes used
herein are commercially available and their reaction
conditions, cofactors and other requirements were used as
would be known to the ordinarily skilled artisan. For
analytical purposes, typically 1 ug of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ul
of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 erg of
DNA are digested with 20 to 250 units of enzyme in a larger
volume. Appropriate buffers and substrate amounts for
particular restriction enzymes are specified by the
manufacturer . Incubation times of about 1 hour at 37 ' C are
ordinarily used, but may vary in accordance with the
supplier's instructions. After digestion the reaction is
electrophoresed.directly on a polyacrylamide gel to isolate
the desired fragment.
Size separation of the cleaved fragments is performed
using 8 percent polyacrylamide gel described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not
ligate to another oligonucleotide without adding a phosphate
with an ATP in the presence of a kinase. A synthetic
-31-
CA 02211003 1997-09-19 ,:
F _ .,,
oligonucleotide will ligate to a fragment that has not been
dephosphorylated. -
"Ligation" refers to the process of forming
phosphodiester bonds between two double stranded nucleic acid
fragments (Maniatis, T., et al., Id., p. 146). Unless
otherwise provided, ligation may be accomplished using known
buffers and conditions with 10 units of T4 DNA ligase
("ligase") per 0.5 Ng of approximately equimolar amounts of
the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as
described in the method of Graham, F. and Van der Eb, A.,
Virology, 52:456-457 (1973).
Example 1
Bacterial Expression and Purification of the TNF receptor
The DNA sequence encoding TNF receptor, ATCC # 75899, is
initially amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' end sequences of the processed
TNF receptor nucleic acid sequence (minus the signal peptide
sequence). Additional nucleotides corresponding to TNF
receptor gene are added to the 5' and 3' end sequences
respectively. The 5' oligonucleotide primer has the sequence ,
5' GCCAGAGGATCCGAAACGTTTCCTCCAAAGTAC 3' (SEQ ID No. 3)
contains a BamHI restriction enzyme site (bold) followed by
21 nucleotides of TNF receptor coding sequence starting from
the presumed initiation codon. The 3' sequence 5'
CGGCTTCTAGAATTACCTATCATTTCTAAAAAT 3' (SEQ ID No. 4) contains
complementary sequences to a Hind III site (bold) and is
followed by 18 nucleotides of TNF receptor. The restriction
enzyme sites correspond to the restriction enzyme sites on
the bacterial expression vector pQE-9 (Qiagen, Inc.
Chatsworth, CA). pQE-9 encodes antibiotic resistance (Amp'),
a bacterial origin of replication (ori), an IPTG-regulatable
promoter operator (P~O), a ribosome binding site (RBS), a 6-
His tag and restriction enzyme sites. pQE-9 is then digested
with BamHI and XbaI. The amplified sequences are ligated
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CA 02211003 1997-09-19
into pQE-9 and are inserted in frame with the sequence
encoding for the histidine tag and the RBS. The ligation
mixture is then used to transform E. coli strain M15/rep 4
(Qiagen, Inc.) by the procedure described in Sambrook, J. et
al., Molecular Cloning: A Laboratory Manual, Cold Spring
Laboratory Press, (1989). M15/rep4 contains multiple copies
of the plasmid pREP4, which expresses the lacI repressor and
also confers kanamycin resistance (Kan'). Transformants are
identified by their ability to grow on LB plates and
ampicillin/kanamycin resistant colonies are selected.
Plasmid DNA is isolated and confirmed by restriction
analysis. Clones containing the desired constructs are grown
overnight (O/N) in liquid culture in LB media supplemented
with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N
culture is used to inoculate a large culture at a ratio of
1:100 to 1:250. The cells are grown to an optical density
600 (O.D.6°°) of between 0.4 and 0.6. IPTG ("Isopropyl-B- D-
thiogalacto pyranoside") is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene
expression. Cells are grown an extra 3 to 4 hours. Cells .
are then harvested by centrifugation. The cell pellet is
solubilized in the chaotropic agent 6 Molar Guanidine HC1.
After clarification, solubilized TNF receptor is purified
from this solution by chromatography on a Nickel-Chelate
column under conditions that allow for tight binding by
proteins containing the 6-His tag (Hochuli, E. et al., J.
Chromatography 411:177-184 (1984)). TNF receptor (90$ pure)
is eluted from the column in 6 molar guanidine HC1 pH 5.0 and
for the purpose of renaturation adjusted to 3 molar guanidine
HC1, 100mM sodium phosphate, 10 mmolar glutathione (reduced)
and 2 mmolar glutathione (oxidized). After incubation in
this solution for 12 hours the protein is dialyzed to 10
mmolar sodium phosphate.
-33-
CA 02211003 1997-09-19 , .
,, . .
Exa~le 2 ~'
CloninQ and expression of TNF receptor and extracellular
Ssoluble) TNF' receptor using the baculovirus expression
system
The DNA sequence encoding the full length TNF receptor
protein, ATCC # 75899, was amplified using PCR
oligonucleotide primers corresponding to the 5' and 3'
sequences of the gene. The 5' primer has the sequence 5'
GCGCGGATCCATGAACAAGTTGCTGTGCTGC 3' (SEQ ID No. 5) and
contains a BamHI restriction enzyme site (in bold) and which
is just behind the first 21 nucleotides of the TNF receptor
gene (the initiation codon for translation "ATG" is
underlined).
The 3' primer has the sequence 5' GCGCTCTAGATTA
CCTATCATTTCTAAAAATAAC 3' (SEQ ID No. 6) and 5'
GCGCGGTACCTCAGTGGTTTGGGCTCCTCCC 3' (SEQ ID No. 7) and
contains the cleavage site for the restriction endonuclease
XbaI and 21 nucleotides complementary to the 3' non-
translated sequence of the TNF receptor gene. The amplified
sequences were isolated from a 1~ agarose gel using a
commercially available kit ("Geneclean", BIO 101 Inc., La
Jolla, Ca.). The fragments were then digested with the
endonucleases BamHI and XbaI and then purified again on a 1~
agarose gel. This fragment is designated F2.
The vector pRGl (modification of pVL941 vector,
discussed below) was used for the expression of the TNF
receptor proteins using the baculovirus expression system
(for review see: Summers, M.D. and Smith, G.E. 1987, A manual
of methods for baculovirus vectors and insect cell culture
procedures, Texas Agricultural Experimental Station Bulletin
No. 1555). This expression vector contains the strong
polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by the recognition sites
for the restriction endonucleases BamHI and XbaI. The
polyadenylation site of the simian virus (SV)40 was used for
-34-
CA 02211003 1997-09-19
efficient polyadenylation. For an easy selection of
recombinant viruses the beta-galactosidase gene from E.coli w
was inserted in the same orientation as the polyhedrin
promoter followed by the polyadenylation signal of the
polyhedrin gene. The polyhedrin sequences were flanked at
both sides by viral sequences for the cell-mediated
homologous recombination of cotransfected wild-type viral
DNA. Many other baculovirus vectors could be used in place
of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and
Summers, M.D., Virology, 170:31-39).
The plasmid was digested with the restriction enzymes
BamHI and XbaI. The DNA was then isolated from a 1~ agarose
gel using the commercially available kit ( "Geneclean" BIO 101
Inc., La Jolla, Ca.). This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were
ligated with T4 DNA ligase. E. coli HB101 cells were then
transformed and cells identified that contained the plasmid
(pBac TP1F receptor) with the TNF receptor genes using the
enzymes BamHI and XbaI. The sequence of the cloned fragment
was confirmed by DNA sequencing. .
5 ug of the plasmid pBac TNF receptor was cotransfected
with 1.0 pg of a commercially available linearized
baculovirus ("BaculoGold~" baculovirus DNA", Pharmingen, San
Diego, CA.) using the lipofection method (Felgner et al.
Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
lug of BaculoGold'" virus DNA and 5 ug of the plasmid
pBac TNF receptors were mixed in a sterile well of a
microtiter plate containing 50 girl of serum free Grace's
medium (Life Technologies Inc., Gaithersburg, MD).
Afterwards 10 ul Lipofectin plus 90 ul Grace's medium were
added, mixed and incubated for 15 minutes at room
temperature. Then the transfection mixture was added
dropwise to the Sf9 insect cells (ATCC CRL 1711) seeded in'a
35 mm tissue culture plate with lml Grace' medium without
serum. The plate was rocked back and forth to mix the newly
-35-
CA 02211003 1997-09-19
added solution. The plate was then incubated for 5 hours at~- -
27°C. After 5 hours the transfection solution was removed -
from the plate and 1 ml of Grace's insect medium supplemented
with 10~ fetal calf serum was added. The plate was put back
into an incubator and cultivation continued at 27°C for four
days.
After four days the supernatant was collected and a
plaque assay performed similar as described by Summers and
Smith (supra). As a modification an agarose gel with "Blue
Gal" (Life Technologies Inc., Gaithersburg) was used which
allows an easy isolation of blue stained plaques. (A
detailed description of a "plaque assay" can also be found in
the user's guide for insect cell culture and baculovirology
distributed by Life Technologies Inc., Gaithersburg, page 9-
10).
Four days after the serial dilution, the viruses were
added to the cells and blue stained plaques were picked with
the tip~of an Eppendorf pipette. The agar containing the
recombinant viruses were then resuspended in an Eppendorf
tube containing 200 ul of Grace's medium. The agar was
removed by a brief centrifugation and the supernatant
containing the recombinant baculoviruses was used to infect
Sf9 cells seeded in 35 mm dishes. Four days later the
supernatants of these culture dishes were harvested and then
stored at 4°C.
Sf9 cells were grown in Grace's medium supplemented with
10~ heat-inactivated FBS. The cells were infected with the
recombinant baculovirus V-TNF receptor at a multiplicity of
infection (MOI) of 2. Six hours later the medium was removed
and replaced with SF900 II medium minus methionine and
cysteine (Life Technologies Inc., Gaithersburg). 42 hours
later 5 ~rCi of 35S-methionine and 5 ~rCi 35S cysteine (Amersham)
were added. The cells are further incubated for 16 hours
before they are harvested by centrifugation and the labelled
proteins visualized by SDS-PAGE and autoradiography.
-36-
CA 02211003 1997-09-19
Example 3
Expression of Recombinant TNF receptor in COS cells
The expression of plasmid, TNF receptor HA is derived
from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40
origin of replication, 2) ampicillin resistance gene, 3)
E.coli replication origin, 4) CMV promoter followed by a
polylinker region, a SV40 intron and polyadenylation site.
A DNA fragment encoding the entire TNF receptor precursor and
a HA tag fused in frame to its 3' end is cloned into the
polylinker region of the vector, therefore, the recombinant
protein expression is directed under the CMV promoter. The
HA tag correspond to an epitope derived from the influenza
hemagglutinin protein as previously described (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner,
1984, Cell 37, 767). The infusion of HA tag to the target
protein allows easy detection of the recombinant protein with
an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as
follows
The DNA sequence encoding TNF receptor, ATCC # 75899, is
constructed by PCR using two primers: the 5' primer 5'
GCCAGAGGATCCGCCACCATGAACAAGTTGCTGTGCTGC 3' (SEQ ID No. 8)
contains a BamFiI site (bold) followed by 21 nucleotides of
TNF receptor coding sequence starting from the initiation
codon; the 3' sequence 5' CGGCTTCTAGAATCAAGCGTAGTCTGGGACG
TCGTATGGGTACCTATCATTTCTAAAAAT 3'(SEQ ID No. 9) contains
complementary sequences to an XbaI site (bold), translation
stop codon, HA tag and the last 18 nucleotides of the TNF
receptor coding sequence (not including the stop codon).
Therefore, the PCR product contains a BamHI site, TNF
receptor coding sequence followed by HA tag fused in frame,
a translation termination stop codon next to the HA tag, and
an XbaI site. The PCR amplified DNA fragment and the vectcr,
pcDNAI/Amp, are digested with BamHI and XbaI restriction
enzymes and ligated. The ligation mixture is transformed into
-37-
CA 02211003 1997-09-19
E. coli strain SURE (Stratagene Cloning Systems, La Jolla,
CA) the transformed culture is plated on ampicillin media
plates and resistant colonies are selected. Plasmid DNA is
isolated from transformants and examined by restriction
analysis for the presence of the correct fragment. For
expression of the recombinant TNF receptor, COS cells are
transfected with the expression vector by DEAE-DEXTRAN method
(J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A
Laboratory Manual, Cold Spring Laboratory Press, (1989)).
The expression of the TNF receptor HA protein is detected by
radiolabelling and immunoprecipitation method (E. Harlow, D.
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, (1988)). Cells are labelled for 8 hours
with 35S-cysteine two days post transfection. Culture media
are then collected and cells are lysed with detergent (RIPA
buffer (150 mM NaCl, 1$ NP-40, 0.1$ SDS, 1$ NP-40, 0.5$ DOC,
50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)).
Both cerl lysate and culture media are precipitated with a HA
specific monoclonal antibody. Proteins precipitated are
analyzed on 15$ SDS-PAGE gels. .
Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, within the scope of the appended claims, the
invention may be practiced otherwise than as particularly
described.
-38-
CA 02211003 1997-09-19
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: HUMAN GENOME SCIENCES, INC.
9410 KEY WEST AVENUE
ROCKVILLE, MD 20850
APPLICANTS/INVENTOR: GREENS, JOHN M .
FLEISCHMANN, ROBERT D
(ii) TITLE OF INVENTION: HUMAN TUMOR NECROSIS FACTOR RECEPTOR
(iii) NUMBER OF SEQUENCES: 9
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: STERNS, KESSLER, GOLDSTEIN & FOX, P.L.L.C.
(B) STREET: 1100 NEW YORK AVENUE, NW, SUITE 600
(C) CITY: WASHINGTON
(D) STATE: DC
(E) COUNTRY: US
(F) ZIP: 20005-3934
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS _
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US95/03216
(B) FILING DATE: 15-MAR-1995
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: GOLDSTEIN, JORGE A.
(B) REGISTRATION NUMBER: 29,021
(C) REFERENCE/DOCKET NUMBER: 1488.071PC00
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 371-2600
(B) TELEFAX: (202) 371 2540
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1527 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
39
' CA 02211003 1997-09-19
(B) LOCATION:46..1248
(ix) '
FEATURE:
(A) NAME/KEY:sig~eptide
(B) LOCATION:46..106
(ix ) FEATURE:
(A) NAME/KEY:mat~eptide
(B) LOCATION:109..1248
(xi ) SEQUENCE IPTION: ID
DESCR SEQ N0:1:
CGCCGAGCCG CCACA 54
CCGCCTCCAA ATG
GCCCCTGAGG AAC
TTTCCGGGGA AAG
Met
Asn
Lys
-21
-20
TTG CTG TGC TGC GCG GTG TTT GAC ATCTCCATTAAG TGG ACC 102
CTC CTG
Leu Leu Cys Cys Ala Val Phe Asp IleSerIleLys Trp Thr
Leu Leu
-15 -10 -5
ACC CAG GAA ACG TTT CCA AAG CTT CATTATGACGAA GAA ACC 150
CCT TAC
Thr Gln Glu Thr Phe Pro Lys Leu HisTyrAspGlu Glu Thr
Pro Tyr
1 5 10
TCT CAT CAG CTG TTG GAC AAA CCT CCTGGTACCTAC CTA AAA 198
TGT TGT
Ser His Gln Leu Leu Asp Lys Pro ProGlyThrTyr Leu Lys
Cys Cys
15 20 25 30
CAA CAC TGT ACA GCA TGG AAG GTG TGCGCCCCTTGC CCT GAC 246 .
AAG ACC
Gln His Cys Thr Ala Trp Lys Val CysAlaProCys Pro Asp _
Lys Thr
35 40 45
CAC TAC TAC ACA GAC TGG CAC AGT GACGAGTGTCTA TAC TGC 294
AGC ACC
His Tyr Tyr Thr Asp Trp His Ser AspGluCysLeu Tyr Cys
Ser Thr
50 55 60
AGC CCC GTG TGC AAG CTG CAG GTC AAGCAGGAGTGC AAT CGC 342
GAG TAC
Ser Pro Val Cys Lys Leu Gln Val LysGlnGluCys Asn Arg
Glu Tyr
65 70 75
ACC CAC AAC CGC GTG GAA TGC GAA GGGCGCTACCTT GAG ATA 390
TGC AAG
Thr His Asn Arg Val Glu Cys Glu GlyArgTyrLeu Glu Ile
Cys Lys
80 85 90
GAG TTC TGC TTG AAA CAT AGG AGC TGC CCT CCT GGA TTT GGA GTG GTG 438
G1u Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val
95 100 105 110
CAA GCT GGA ACC CCA GAG CGA AAT ACA GTT TGC AAA AGA TGT CCA GAT 486
Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp
115 120 125
GGG TTC TTC TCA AAT GAG ACG TCA TCT AAA GCA CCC TGT AGA AAA CAC 534
Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala-Pro Cys Arg Lys His
130 135 140
CA 02211003 1997-09-19
ACA AAT TGC AGT GTC CTC CTG CTA ACT CAG AAA GGA AAT 582
TTT GGT GCA
Thr Asn Cys Ser Val Leu Leu Leu Thr Gln Lys Gly Asn
Phe Gly Ala
145 150 155 '
ACA CAC GAC AAC ATA GGA AAC AGT GAA TCA ACT CAA AAA 630
TGT TCC TGT
Thr His Asp Asn Ile Gly Asn Ser Glu Ser Thr Gln Lys
Cys Ser Cys
160 165 170
GGA ATA GAT GTT ACC GAG GAG GCA TTC TTC AGG TTT GCT 678
CTG TGT GTT
Gly Ile Asp Val Thr Glu Glu Ala Phe Phe Arg Phe Ala
Leu Cys Val
175 i80 185 190
CCT ACA AAG TTT ACG TGG CTT AGT GTC TTG GTA GAC AAT 726
CCT AAC TTG
Pro Thr Lys Phe Thr Trp Leu Ser Val Leu Val Asp Asn
Pro Asn Leu
195 200 205
CCT GGC ACC AAA GTA GAG AGT GTA GAG AGG ATA AAA CGG 774
AAC GCA CAA
Pro Gly Thr Lys Val Glu Ser Val Glu Arg Ile Lys Arg
Asn Ala Gln
210 215 220
CAC AGC TCA CAA GAA TTC CAG CTG CTG AAG TTA TGG AAA 822
CAG ACT CAT
His Ser Ser Gln Glu Phe Gln Leu Leu Lys Leu Trp Lys
Gln Thr His
225 230 235
CAA AAC AAA GAC CAA GTC AAG AAG ATC ATC CAA GAT ATT 870
GAT ATA GAC
Gln Asn Lys Asp Gln Val Lys Lys Ile Ile Gln Asp Ile
Asp Ile Asp
240 245 250
CTCTGTGAA GTGCAGCGG CAC ATT CAT GCT AAC CTC 918
AAC GGA ACC
AGC
LeuCysGlu SerValGlnArg His Ile His Ala Asn Leu
Asn Gly Thr
255 260 265 270
TTCGAGCAG CGTAGCTTGATG GAA AGC CCG GGA AAG AAA 966
CTT TTA GTG
PheGluGln ArgSerLeuMet Glu Ser Pro Gly Lys Lys
Leu Leu Val
275 280 285
GGAGCAGAA ATTGAAAAAACA ATA AAG TGC AAA CCC AGT 1014
GAC GCA GAC
GlyAlaGlu IleGluLysThr Ile Lys Cys Lys Pro Ser
Asp Ala Asp
290 295 300
CAGATCCTG CTGCTCAGTTTG TGG CGA AAA AAT GGC GAC 1062
AAG ATA CAA
GlnIleLeu LeuLeuSerLeu Trp Arg Lys Asn Gly Asp
Lys Ile Gln
305 310 315
GACACCTTG GGCCTAATGCAC GCA CTA CAC TCA AAG ACG 1110'
AAG AAG TAC
AspThrLeu GlyLeuMetHis Ala Leu His Ser Lys Thr
Lys Lys Tyr
320 325 330
CACTTTCCC ACTGTCACTCAG AGT CTA AAG ACC ATC AGG 1158
AAA AAG TTC
HisPhePro ThrValThrGln Ser Leu Lys Thr Ile Arg
Lys Lys Phe
335 340 345 350
CTTCACAGC ACAATGTACAAA TTG TAT AAG TTA TTT TTA 1206
TTC CAG GAA
LeuHisSer ThrMetTyrLys Leu Tyr Lys Leu Phe Leu
Phe Gln Glu
355 360 365
ATGATAGGT CAGGTCCAATCA GTA AAA AGC TGC TTA 1248
AAC ATA
41
' CA 02211003 1997-09-19
Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys Leu
370 375 380
TAACTGGAAA TGGCCATTGA GCTGTTTCCT CACAATTGGC GAGATCCCAT GGATGAGTAA 1308
ACTGTTTCTC AGGCACTTGA GGCTTTCAGT GATATCTTTC TCATTACCAG TGACTAATTT 1368
TGCCACAGGG TACTAAAAGA AACTATGATG TGGAGAAAGG ACTAACATCT CCTCCAATAA 1428
ACCCCAAATG GTTAATCCAA CTGTCAGATC TGGATCGTTA TCTACTGACT ATATTTTCCC I 1488
TTATTACTGC TTGCAGTAAT TCAACTGGAA AAAAAAAAA 1527
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile
-21 -20 -15 -10
Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp
1 5 10
Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr
20 25
Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro
30 35 40
Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys
45 50 55
Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu
60 65 70 75
Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr
80 85 90
Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe
95 100 105
Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg
110 115 120
Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys
125 130 135
Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys
140 145 150 155
42
~CA 02211003 1997-09-19
Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr
160 165 170
Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg
175 180 185
Phe Ala Val Fro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val
190 195 200
Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile '
205 210 213
Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu
220 225 230 235
Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln
240 245 250
Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile Gly His Ala
255 260 265
Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly
270 275 280
Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys
285 290 295
Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn
300 305 310 315
Gly Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser _
320 325 330
Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr
335 340 345
Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu
350 355 360
Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys
365 370 375
Leu
380 '
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
43
ACA 02211003 1997-09-19
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GCCAGAGGAT CCGAAACGTT TCCTCCAAAG TAC 33
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
CGGCTTCTAG AATTACCTAT CATTTCTAAA AAT 33
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear '
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GCGCGGATCC ATGAACAAGT TGCTGTGCTG C 31
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
GCGCTCTAGA TTACCTATCA TTTCTAAAAA TAAC 34
(2) INFORMATION FOR SEQ ID N0:7:
44
'CA 02211003 1997-09-19 .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GCGCGGTACC TCAGTGGTTT GGGCTCCTCC C 31
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
GCCAGAGGAT CCGCCACCAT GAACAAGTTG CTGTGCTGC 3g
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CGGCTTCTAG AATCAAGCGT AGTCTGGGAC GTCGTATGGG TACCTATCAT TTCTAAAAAT 60
