U.S. patent application number 11/976382 was filed with the patent office on 2009-10-29 for purified tnfr preparations.
This patent application is currently assigned to IMMUNEX CORPORATION. Invention is credited to M. Patricia Beckmann, Raymond G. Goodwin, Craig A. Smith.
Application Number | 20090270592 11/976382 |
Document ID | / |
Family ID | 27503488 |
Filed Date | 2009-10-29 |
United States Patent
Application |
20090270592 |
Kind Code |
A1 |
Smith; Craig A. ; et
al. |
October 29, 2009 |
Purified TNFR preparations
Abstract
Purified protein preparations comprising tumor necrosis factor
are disclosed.
Inventors: |
Smith; Craig A.; (Seattle,
WA) ; Goodwin; Raymond G.; (Seattle, WA) ;
Beckmann; M. Patricia; (Poulsbo, WA) |
Correspondence
Address: |
Legal Affairs Department;Immunex Corporation
51 University Street
Seattle
WA
98101
US
|
Assignee: |
IMMUNEX CORPORATION
|
Family ID: |
27503488 |
Appl. No.: |
11/976382 |
Filed: |
October 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11260192 |
Oct 28, 2005 |
7459528 |
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11976382 |
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10420785 |
Apr 23, 2003 |
7057022 |
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11260192 |
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09758124 |
Jan 12, 2001 |
6572852 |
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10420785 |
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08953268 |
Oct 17, 1997 |
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09758124 |
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08555629 |
Nov 9, 1995 |
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08953268 |
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08468453 |
Jun 6, 1995 |
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08555629 |
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08038765 |
Mar 19, 1993 |
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08468453 |
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07523635 |
May 10, 1990 |
5395760 |
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08038765 |
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07421417 |
Oct 13, 1989 |
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07523635 |
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07405370 |
Sep 11, 1989 |
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07421417 |
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07403241 |
Sep 5, 1989 |
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07405370 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 9/00 20180101; C07K 14/7151 20130101; G01N 2333/525 20130101;
G01N 2800/26 20130101; A61K 38/00 20130101; G01N 33/6863 20130101;
G01N 2500/02 20130101; A61P 37/00 20180101; A61P 37/02
20180101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/435 20060101
C07K014/435 |
Claims
1. A purified preparation of a protein, wherein the protein
comprises amino acids 1-163 of SEQ ID NO:2, wherein the protein has
been purified by a method comprising the following steps: (a)
concentrating a supernatant from a mammalian cell culture which
contains the protein; (b) purifying the protein in the concentrated
supernatant by subjecting it to affinity chromatography and/or ion
exchange chromatography.
2. The purified preparation of claim 1, wherein the protein
comprises amino acids 1-185 of SEQ ID NO:2.
3. The purified preparation of claim 2, wherein the protein
comprises amino acids 1-235 of SEQ ID NO:2.
4. The purified preparation of claim 1, wherein the mammalian cell
culture is a CHO cell culture.
5. The purified preparation of claim 1, wherein the protein has
been subjected to affinity chromatography and ion exchange
chromatography.
6. The purified preparation of claim 5, wherein ion exchange
chromatography is done after affinity chromatography.
7. The purified preparation of claim 5, wherein the ion exchange
chromatography is anion exchange chromatography.
8. The purified preparation of claim 7, wherein the anion exchange
chromatography is performed on a matrix having pendant
diethylaminoethyl (DEAE) groups.
9. The purified preparation of claim 1, wherein the ion exchange
chromatography is anion exchange chromatography.
10. The purified preparation of claim 9, wherein the anion exchange
chromatography is performed on a matrix having pendant
diethylaminoethyl (DEAE) groups.
11. The purified preparation of claim 1, wherein the ion exchange
chromatography is cation exchange chromatography.
12. The purified preparation of claim 11, wherein the cation
exchange chromatography is performed on a matrix having pendant
carboxymethyl or sulfopropyl groups.
13. The purified preparation of claim 1, wherein the purified
preparation comprises less than about one percent by weight of
non-human cell components.
14. A purified preparation of a protein comprising less than about
one percent by weight of non-human cell components, wherein the
protein comprises amino acids 1-163 of SEQ ID NO:2.
15. The purified preparation of claim 14, wherein the protein has
been purified from a supernatant of a mammalian cell culture.
16. The purified preparation of claim 15, wherein the protein has
been purified by a method comprising the following steps: (a)
concentrating the supernatant; (b) purifying the protein in the
concentrated supernatant by subjecting it to affinity
chromatography.
17. The purified preparation of claim 15, wherein the protein has
been purified by a method comprising the following steps: (a)
concentrating the supernatant; (b) purifying the protein in the
concentrated supernatant by subjecting it to ion exchange
chromatography.
18. The purified preparation of claim 17, wherein the ion exchange
chromatography is anion exchange chromatography.
19. The purified preparation of claim 17, wherein the ion exchange
chromatography is cation exchange chromatography.
20. The purified preparation of claim 15, wherein the mammalian
cell culture is a CHO cell culture.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Continuation of U.S. application Ser.
No. 11/260,192 (Conf. No. 7372), filed Oct. 28, 2005; which is a
Divisional Application of pending prior application Ser. No.
10/420,785 (Conf. No. 4626), filed Apr. 23, 2003 (now U.S. Pat. No.
7,057,022); which is a Divisional of U.S. application Ser. No.
09/758,124, filed Jan. 12, 2001 (now U.S. Pat. No. 6,572,852);
which is a Divisional of U.S. application Ser. No. 08/953,268,
filed Oct. 17, 1997 (now abandoned); which is a Divisional of U.S.
application Ser. No. 08/555,629, filed Nov. 9, 1995 (now
abandoned); which is a Divisional of U.S. application Ser. No.
08/468,453, filed Jun. 6, 1995 (now abandoned); which is a
Continuation of U.S. application Ser. No. 08/038,765, filed Mar.
19, 1993 (now abandoned); which is a Divisional of U.S. application
Ser. No. 07/523,635, filed May 10, 1990 (now U.S. Pat. No.
5,395,760); which is a Continuation-In-Part of U.S. application
Ser. No. 07/421,417, filed Oct. 13, 1989 (now abandoned); which is
a Continuation-In-Part of U.S. application Ser. No. 07/405,370,
filed Sep. 11, 1989 (now abandoned); which is a
Continuation-In-Part of U.S. application Ser. No. 07/403,241, filed
Sep. 5, 1989 (now abandoned). The entire disclosures of each of the
prior applications is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to cytokine
receptors and more specifically to tumor necrosis factor
receptors.
[0003] Tumor necrosis factor-.alpha. (TNF.alpha., also known as
cachectin) and tumor necrosis factor-.beta. (TNF.beta., also known
as lymphotoxin) are homologous mammalian endogenous secretory
proteins capable of inducing a wide variety of effects on a large
number of cell types. The great similarities in the structural and
functional characteristics of these two cytokines have resulted in
their collective description as "TNF." Complementary cDNA clones
encoding TNF.alpha. (Pennica et al., Nature 312: 724, 1984) and
TNF.beta. (Gray et al., Nature 312: 721, 1984) have been isolated,
permitting further structural and biological characterization of
TNF.
[0004] TNF proteins initiate their biological effect on cells by
binding to specific TNF receptor (TNF-R) proteins expressed on the
plasma membrane of a TNF-responsive cell. TNF.alpha. and TNF.beta.
were first shown to bind to a common receptor on the human cervical
carcinoma cell line ME-180 (Aggarwal et al., Nature 318: 665,
1985). Estimates of the size of the TNF-R determined by affinity
labeling studies ranged from 54 to 175 kDa (Creasey et al, Proc.
Natl. Acad Sci. USA 84: 3293, 1987; Stauber et al., J. Biol. Chem.
263: 19098, 1988; Hohmann et al., J. Biol. Chem. 264: 14927, 1989).
Although the relationship between these TNF-Rs of different
molecular mass is unclear, Hohmann et al. (J. Biol. Chem. 264:
14927, 1989) reported that at least two different cell surface
receptors for TNF exist on different cell types. These receptors
have an apparent molecular mass of about 80 kDa and about 55-60
kDa, respectively. None of the above publications, however,
reported the purification to homogeneity of cell surface TNF
receptors.
[0005] In addition to cell surface receptors for TNF, soluble
proteins from human urine capable of binding TNF have also been
identified (Peetre et al., Eur. J. Haematol. 41: 414, 1988;
Seckinger et al., J. Exp. Med. 167: 1511, 1988; Seckinger et al.,
J. Biol. Chem. 264: 11966, 1989; UK Patent Application, Publ. No. 2
218 101 A to Seckinger et al.; Engelmann et al., J. Biol. Chem.
264: 11974, 1989). The soluble urinary TNF binding protein
disclosed by UK 2 218 101 A has a partial N-terminal amino acid
sequence of Asp-Ser-Val-Cys-Pro-, which corresponds to the partial
sequence disclosed later by Engelmann et al. (1989). The
relationship of the above soluble urinary binding proteins was
further elucidated after original parent application (U.S. Ser. No.
403,241) of the present application was filed, when Engelmann et
al. reported the identification and purification of a second
distinct soluble urinary TNF binding protein having an N-terminal
amino acid sequence of Val-Ala-Phe-Thr-Pro- (J. Biol. Chem. 265:
1531, 1990). The two urinary proteins disclosed by the UK 2 218 101
A and the Engelmann et al. publications were shown to be
immunochemically related to two apparently distinct cell surface
proteins by the ability of antiserum against the binding proteins
to inhibit TNF binding to certain cells.
[0006] More recently, two separate groups reported the molecular
cloning and expression of a human 55 kDa TNF-R (Loetscher et al.,
Cell 61: 351, 1990; Schall et al., Cell 61: 361, 1990). The TNF-R
of both groups has an N-terminal amino acid sequence which
corresponds to the partial amino acid sequence of the urinary
binding protein disclosed. by UK 2 218 101 A, Engelmann et al.
(1989) and Englelmann et al. (1990).
[0007] In order to elucidate the relationship of the multiple forms
of TNF-R and soluble urinary TNF binding proteins, or to study the
structural and biological characteristics of TNF-Rs and the role
played by TNF-Rs in the responses of various cell populations to
TNF or other cytokine stimulation, or to use TNF-Rs effectively in
therapy, diagnosis, or assay, purified compositions of TNF-R are
needed. Such compositions, however, are obtainable in practical
yields only by cloning and expressing genes encoding the receptors
using recombinant DNA technology. Efforts to purify the TNF-R
molecule for use in biochemical analysis or to clone and express
mammalian genes encoding TNF-R, however, have been impeded by lack
of a suitable source of receptor protein or mRNA. Prior to the
present invention, no cell lines were known to express high levels
of TNF-R constitutively and continuously, which precluded
purification of receptor for sequencing or construction of genetic
libraries for cDNA cloning.
SUMMARY OF THE INVENTION
[0008] The present invention provides isolated TNF receptors and
DNA sequences encoding mammalian tumor necrosis factor receptors
(TNF-R), in particular, human TNF-Rs. Such DNA sequences include
(a) cDNA clones having a nucleotide sequence derived from the
coding region of a native TNF-R gene; (b) DNA sequences which are
capable of hybridization to the cDNA clones of (a) under moderately
stringent conditions and which encode biologically active TNF-R
molecules; or (c) DNA sequences which are degenerate as a result of
the genetic code to the DNA sequences defined in (a) and (b) and
which encode biologically active TNF-R molecules. In particular,
the present invention provides DNA sequences which encode soluble
TNF receptors.
[0009] The present invention also provides recombinant expression
vectors comprising the DNA sequences defined above, recombinant
TNF-R molecules produced using the recombinant expression vectors,
and processes for producing the recombinant TNF-R molecules using
the expression vectors.
[0010] The present invention also provides isolated or purified
protein compositions comprising TNF-R, and, in particular, soluble
forms of TNF-R.
[0011] The present invention also provides compositions for use in
therapy, diagnosis, assay of TNF-R, or in raising antibodies to
TNF-R, comprising effective quantities of soluble native or
recombinant receptor proteins prepared according to the foregoing
processes.
[0012] Because of the ability of TNF to specifically bind TNF
receptors (TNF-Rs), purified TNF-R compositions will be useful in
diagnostic assays for TNF, as well as in raising antibodies to TNF
receptor for use in diagnosis and therapy. In addition, purified
TNF receptor compositions may be used directly in therapy to bind
or scavenge TNF, thereby providing a means for regulating the
immune activities of this cytokine.
[0013] These and other aspects of the present invention will become
evident upon reference to the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a schematic representation of the coding region of
various cDNAs encoding human and murine TNF-Rs. The leader sequence
is hatched and the transmembrane region is solid.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0015] As used herein, the terms "TNF receptor" and "TNF-R" refer
to proteins having amino acid sequences which are substantially
similar to the native mammalian TNF receptor amino acid sequences,
and which are biologically active, as defined below, in that they
are capable of binding TNF molecules or transducing a biological
signal initiated by a TNF molecule binding to a cell, or
cross-reacting with anti-TNF-R antibodies raised against TNF-R from
natural (i.e., nonrecombinant) sources. The mature full-length
human TNF-R is a glycoprotein having a molecular weight of about 80
kilodaltons (kDa). As used throughout the specification, the term
"mature" means a protein expressed in a form lacking a leader
sequence as may be present in full-length transcripts of a native
gene. Experiments using COS cells transfected with a cDNA encoding
full-length human TNF-R showed that TNF-R bound
.sup.125I-TNF.alpha. with an apparent Ka of about 5.times.10.sup.9
M.sup.-1, and that TNF-R bound .sup.125I-TNF.beta. with an apparent
Ka Of about 2.times.10.sup.9 M.sup.-1. The terms "TNF receptor" or
"TNF-R" include, but are not limited to, analogs or subunits of
native proteins having at least 20 amino acids and which exhibit at
least some biological activity in common with TNF-R, for example,
soluble TNF-R constructs which are devoid of a transmembrane region
(and are secreted from the cell) but retain the ability to bind
TNF. Various bioequivalent protein and amino acid analogs are
described in detail below.
[0016] The nomenclature for TNF-R analogs as used herein follows
the convention of naming the protein (e.g., TNF-R) preceded by
either hu (for human) or mu (for murine) and followed by a .DELTA.
(to designate a deletion) and the number of the C-terminal amino
acid. For example, huTNF-R.DELTA.235 refers to human TNF-R having
Asp.sup.235 as the C-terminal amino acid (i.e., a polypeptide
having the sequence of amino acids 1-235 of SEQ ID NO:1). In the
absence of any human or murine species designation, TNF-R refers
generically to mammalian TNF-R. Similarly, in the absence of any
specific designation for deletion mutants, the term TNF-R means all
forms of TNF-R, including mutants and analogs which possess TNF-R
biological activity.
[0017] "Soluble TNF-R" or "sTNF-R" as used in the context of the
present invention refer to proteins, or substantially equivalent
analogs, having an amino acid sequence corresponding to all or part
of the extracellular region of a native TNF-R, for example,
huTNF-R.DELTA.235, huTNF-R.DELTA.185 and huTNF-R.DELTA.163, or
amino acid sequences substantially similar to the sequences of
amino acids 1-163, amino acids 1-185, or amino acids 1-235 of SEQ
ID NO:1, and which are biologically active in that they bind to TNF
ligand. Equivalent soluble TNF-Rs include polypeptides which vary
from these sequences by one or more substitutions, deletions, or
additions, and which retain the ability to bind TNF or inhibit TNF
signal transduction activity via cell surface bound TNF receptor
proteins, for example huTNF-R.DELTA.x, wherein x is selected from
the group consisting of any one of amino acids 163-235 of SEQ ID
NO:1. Analogous deletions may be made to muTNF-R. Inhibition of TNF
signal transduction activity can be determined by transfecting
cells with recombinant TNF-R DNAs to obtain recombinant receptor
expression. The cells are then contacted with TNF and the resulting
metabolic effects examined. If an effect results which is
attributable to the action of the ligand, then the recombinant
receptor has signal transduction activity. Exemplary procedures for
determining whether a polypeptide has signal transduction activity
are disclosed by Idzerda et al., J. Exp. Med 171: 861 (1990);
Curtis et al., Proc. Natl. Acad Sci. USA 86: 3045 (1989); Prywes et
al., EMBO J. 5: 2179 (1986) and Chou et al., J. Biol. Chem. 262:
1842 (1987). Alternatively, primary cells or cell lines which
express an endogenous TNF receptor and have a detectable biological
response to TNF could also be utilized.
[0018] The term "isolated" or "purified", as used in the context of
this specification to define the purity of TNF-R protein or protein
compositions, means that the protein or protein composition is
substantially free of other proteins of natural or endogenous
origin and contains less than about 1% by mass of protein
contaminants residual of production processes. Such compositions,
however, can contain other proteins added as stabilizers, carriers,
excipients or co-therapeutics. TNF-R is isolated if it is
detectable as a single protein band in a polyacrylamide gel by
silver staining.
[0019] The term "substantially similar," when used to define either
amino acid or nucleic acid sequences, means that a particular
subject sequence, for example, a mutant sequence, varies from a
reference sequence by one or more substitutions, deletions, or
additions, the net effect of which is to retain biological activity
of the TNF-R protein as may be determined, for example, in one of
the TNF-R binding assays set forth in Example 1 below.
Alternatively, nucleic acid subunits and analogs are "substantially
similar" to the specific DNA sequences disclosed herein if: (a) the
DNA sequence is derived from the coding region of a native
mammalian TNF-R gene; (b) the DNA sequence is capable of
hybridization to DNA sequences of (a) under moderately stringent
conditions (50.degree. C., 2.times.SSC) and which encode
biologically active TNF-R molecules; or DNA sequences which are
degenerate as a result of the genetic code to the DNA sequences
defined in (a) or (b) and which encode biologically active TNF-R
molecules.
[0020] "Recombinant," as used herein, means that a protein is
derived from recombinant (e.g., microbial or mammalian) expression
systems. "Microbial" refers to recombinant proteins made in
bacterial or fungal (e.g., yeast) expression systems. As a product,
"recombinant microbial" defines a protein produced in a microbial
expression system which is essentially free of native endogenous
substances. Protein expressed in most bacterial cultures, e.g., E.
coli, will be free of glycan. Protein expressed in yeast may have a
glycosylation pattern different from that expressed in mammalian
cells.
[0021] "Biologically active," as used throughout the specification
as a characteristic of TNF receptors, means that a particular
molecule shares sufficient amino acid sequence similarity with the
embodiments of the present invention disclosed herein to be capable
of binding detectable quantities of TNF, transmitting a TNF
stimulus to a cell, for example, as a component of a hybrid
receptor construct, or cross-reacting with anti-TNF-R antibodies
raised against TNF-R from natural (i.e., nonrecombinant) sources.
Preferably, biologically active TNF receptors within the scope of
the present invention are capable of binding greater than 0.1
nmoles TNF per nmole receptor, and most preferably, greater than
0.5 nmole TNF per nmole receptor in standard binding assays (see
below).
[0022] "Isolated DNA sequence" refers to a DNA polymer, in the form
of a separate fragment or as a component of a larger DNA construct,
which has been derived from DNA isolated at least once in
substantially pure form, i.e., free of contaminating endogenous
materials and in a quantity or concentration enabling
identification, manipulation, and recovery of the sequence and its
component nucleotide sequences by standard biochemical methods, for
example, using a cloning vector. Such sequences are preferably
provided in the form of an open reading frame uninterrupted by
internal nontranslated sequences, or introns, which are typically
present in eukaryotic genes. Genomic DNA containing the relevant
sequences could also be used as a source of coding sequences.
Sequences of non-translated DNA may be present 5' or 3' from the
open reading frame, where the same do not interfere with
manipulation or expression of the coding regions.
[0023] "Nucleotide sequence" refers to a heteropolymer of
deoxyribonucleotides. DNA sequences encoding the proteins provided
by this invention can be assembled from cDNA fragments and short
oligonucleotide linkers, or from a series of oligonucleotides, to
provide a synthetic gene which is capable of being expressed in a
recombinant transcriptional unit.
Isolation of cDNAs Encoding TNF-R
[0024] The coding sequence of TNF-R is obtained by isolating a
complementary DNA (cDNA) sequence encoding TNF-R from a recombinant
cDNA or genomic DNA library. A cDNA library is preferably
constructed by obtaining polyadenylated mRNA from a particular cell
line which expresses a mammalian TNF-R, for example, the human
fibroblast cell line WI-26 VA4 (ATCC CCL 95.1) and using the mRNA
as a template for synthesizing double stranded cDNA. The double
stranded cDNA is then packaged into a recombinant vector, which is
introduced into an appropriate E. coli strain and propagated.
Murine or other mammalian cell lines which express TNF-R may also
be used. TNF-R sequences contained in the cDNA library can be
readily identified by screening the library with an appropriate
nucleic acid probe which is capable of hybridizing with TNF-R cDNA.
Alternatively, DNAs encoding TNF-R proteins can be assembled by
ligation of synthetic oligonucleotide subunits corresponding to all
or part of the sequence of SEQ ID NO:1 or SEQ ID NO:3 to provide a
complete coding sequence.
[0025] The human TNF receptor cDNAs of the present invention were
isolated by the method of direct expression cloning. A cDNA library
was constructed by first isolating cytoplasmic mRNA from the human
fibroblast cell line WI-26 VA4. Polyadenylated RNA was isolated and
used to prepare double-stranded cDNA. Purified cDNA fragments were
then ligated into pCAV/NOT vector DNA which uses regulatory
sequences derived from pDC201 (a derivative of pMLSV, previously
described by Cosman et al., Nature 312: 768, 1984), SV40 and
cytomegalovirus DNA, described in detail below in Example 2.
pCAV/NOT has been deposited with the American Type Culture
Collection under accession No. ATCC 68014. The pCAV/NOT vectors
containing the W126-VA4 cDNA fragments were transformed into E.
coli strain DH5.alpha.. Transformants were plated to provide
approximately 800 colonies per plate. The resulting colonies were
harvested and each pool used to prepare plasmid DNA for
transfection into COS-7 cells essentially as described by Cosman et
al. (Nature 312: 768, 1984) and Luthman et al. (Nucl. Acid Res. 11:
1295, 1983). Transformants expressing biologically active cell
surface TNF receptors were identified by screening for their
ability to bind .sup.125I-TNF. In this screening approach,
transfected COS-7 cells were incubated with medium containing
.sup.125I-TNF, the cells washed to remove unbound labeled TNF, and
the cell monolayers contacted with X-ray film to detect
concentrations of TNF binding, as disclosed by Sims et al, Science
241: 585 (1988). Transfectants detected in this manner appear as
dark foci against a relatively light background.
[0026] Using this approach, approximately 240,000 cDNAs were
screened in pools of approximately 800 cDNAs until assay of one
transfectant pool indicated positive foci for TNF binding. A frozen
stock of bacteria from this positive pool was grown in culture and
plated to provide individual colonies, which were screened until a
single clone (clone 1) was identified which was capable of
directing synthesis of a surface protein with detectable TNF
binding activity. The sequence of cDNA clone 1 isolated by the
above method is depicted in SEQ ID NO:1.
[0027] Additional cDNA clones can be isolated from cDNA libraries
of other mammalian species by cross-species hybridization. For use
in hybridization, DNA encoding TNF-R may be covalently labeled with
a detectable substance such as a fluorescent group, a radioactive
atom or a chemiluminescent group by methods well known to those
skilled in the art. Such probes could also be used for in vitro
diagnosis of particular conditions.
[0028] Like most mammalian genes, mammalian TNF receptors are
presumably encoded by multi-exon genes. Alternative mRNA constructs
which can be attributed to different mRNA splicing events following
transcription, and which share large regions of identity or
similarity with the cDNAs claimed herein, are considered to be
within the scope of the present invention.
[0029] Other mammalian TNF-R cDNAs are isolated by using an
appropriate human TNF-R DNA sequence as a probe for screening a
particular mammalian cDNA library by cross-species
hybridization.
Proteins and Analogs
[0030] The present invention provides isolated recombinant
mammalian TNF-R polypeptides. Isolated TNF-R polypeptides of this
invention are substantially free of other contaminating materials
of natural or endogenous origin and contain less than about 1% by
mass of protein contaminants residual of production processes. The
native human TNF-R molecules are recovered from cell lysates as
glycoproteins having an apparent molecular weight by SDS-PAGE of
about 80 kilodaltons (kDa). The TNF-R polypeptides of this
invention are optionally without associated native-pattern
glycosylation.
[0031] Mammalian TNF-R of the present invention includes, by way of
example, primate, human, murine, canine, feline, bovine, ovine,
equine and porcine TNF-R. Mammalian TNF-Rs can be obtained by cross
species hybridization, using a single stranded cDNA derived from
the human TNF-R DNA sequence as a hybridization probe to isolate
TNF-R cDNAs from mammalian cDNA libraries.
[0032] Derivatives of TNF-R within the scope of the invention also
include various structural forms of the primary protein which
retain biological activity. Due to the presence of ionizable amino
and carboxyl groups, for example, a TNF-R protein may be in the
form of acidic or basic salts, or may be in neutral form.
Individual amino acid residues may also be modified by oxidation or
reduction.
[0033] The primary amino acid structure may be modified by forming
covalent or aggregative conjugates with other chemical moieties,
such as glycosyl groups, lipids, phosphate, acetyl groups and the
like, or by creating amino acid sequence mutants. Covalent
derivatives are prepared by linking particular functional groups to
TNF-R amino acid side chains or at the N- or C-termini. Other
derivatives of TNF-R within the scope of this invention include
covalent or aggregative conjugates of TNF-R or its fragments with
other proteins or polypeptides, such as by synthesis in recombinant
culture as N-terminal or C-terminal fusions. For example, the
conjugated peptide may be a signal (or leader) polypeptide sequence
at the N-terminal region of the protein which co-translationally or
post-translationally directs transfer of the protein from its site
of synthesis to its site of function inside or outside of the cell
membrane or wall (e.g., the yeast .alpha.-factor leader). TNF-R
protein fusions can comprise peptides added to facilitate
purification or identification of TNF-R (e.g., poly-His). The amino
acid sequence of TNF receptor can also be linked to the peptide
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK (SEQ ID NO:22)) (Hopp et
al., Bio/Technology 6: 1204, 1988.) The latter sequence is highly
antigenic and provides an epitope reversibly bound by a specific
monoclonal antibody, enabling rapid assay and facile purification
of expressed recombinant protein. This sequence is also
specifically cleaved by bovine mucosal enterokinase at the residue
immediately following the Asp-Lys pairing. Fusion proteins capped
with this peptide may also be resistant to intracellular
degradation in E. coli.
[0034] TNF-R derivatives may also be used as immunogens, reagents
in receptor-based immunoassays, or as binding agents for affinity
purification procedures of TNF or other binding ligands. TNF-R
derivatives may also be obtained by cross-linking agents, such as
M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at
cysteine and lysine residues. TNF-R proteins may also be covalently
bound through reactive side groups to various insoluble substrates,
such as cyanogen bromide-activated, bisoxirane-activated,
carbonyldiimidazole-activated or tosyl-activated agarose
structures, or by adsorbing to polyolefin surfaces (with or without
glutaraldehyde cross-linking). Once bound to a substrate, TNF-R may
be used to selectively bind (for purposes of assay or purification)
anti-TNF-R antibodies or TNF.
[0035] The present invention also includes TNF-R with or without
associated native-pattern glycosylation. TNF-R expressed in yeast
or mammalian expression systems, e.g., COS-7 cells, may be similar
or slightly different in molecular weight and glycosylation pattern
than the native molecules, depending upon the expression system.
Expression of TNF-R DNAs in bacteria such as E. coli provides
non-glycosylated molecules. Functional mutant analogs of mammalian
TNF-R having inactivated N-glycosylation sites can be produced by
oligonucleotide synthesis and ligation or by site-specific
mutagenesis techniques. These analog proteins can be produced in a
homogeneous, reduced-carbohydrate form in good yield using yeast
expression systems. N-glycosylation sites in eukaryotic proteins
are characterized by the amino acid triplet Asn-A.sub.1-Z, where
A.sub.1 is any amino acid except Pro, and Z is Ser or Thr. In this
sequence, asparagine provides a side chain amino group for covalent
attachment of carbohydrate. Such a site can be eliminated by
substituting another amino acid for Asn or for residue Z, deleting
Asn or Z, or inserting a non-Z amino acid between A.sub.1 and Z, or
an amino acid other than Asn between Asn and A.sub.1.
[0036] TNF-R derivatives may also be obtained by mutations of TNF-R
or its subunits. A TNF-R mutant, as referred to herein, is a
polypeptide homologous to TNF-R but which has an amino acid
sequence different from native TNF-R because of a deletion,
insertion or substitution.
[0037] Bioequivalent analogs of TNF-R proteins may be constructed
by, for example, making various substitutions of residues or
sequences or deleting terminal or internal residues or sequences
not needed for biological activity. For example, cysteine residues
can be deleted (e.g., Cys.sup.178) or replaced with other amino
acids to prevent formation of unnecessary or incorrect
intramolecular disulfide bridges upon renaturation. Other
approaches to mutagenesis involve modification of adjacent dibasic
amino acid residues to enhance expression in yeast systems in which
KEX2 proteasc activity is present. Generally, substitutions should
be made conservatively; i.e., the most preferred substitute amino
acids are those having physiochemical characteristics resembling
those of the residue to be replaced. Similarly, when a deletion or
insertion strategy is adopted, the potential effect of the deletion
or insertion on biological activity should be considered.
Substantially similar polypeptide sequences, as defined above,
generally comprise a like number of amino acids sequences, although
C-terminal truncations for the purpose of constructing soluble
TNF-Rs will contain fewer amino acid sequences. In order to
preserve the biological activity of TNF-Rs, deletions and
substitutions will preferably result in homologous or
conservatively substituted sequences, meaning that a given residue
is replaced by a biologically similar residue. Examples of
conservative substitutions include substitution of one aliphatic
residue for another, such as Ile, Val, Leu, or Ala for one another,
or substitutions of one polar residue for another, such as between
Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative
substitutions, for example, substitutions of entire regions having
similar hydrophobicity characteristics, are well known. Moreover,
particular amino acid differences between human, murine and other
mammalian TNF-Rs is suggestive of additional conservative
substitutions that may be made without altering the essential
biological characteristics of TNF-R.
[0038] Subunits of TNF-R may be constructed by deleting terminal or
internal residues or sequences. Particularly preferred sequences
include those in which the transmembrane region and intracellular
domain of TNF-R are deleted or substituted with hydrophilic
residues to facilitate secretion of the receptor into the cell
culture medium. The resulting protein is referred to as a soluble
TNF-R molecule which retains its ability to bind TNF. A
particularly preferred soluble TNF-R construct is TNF-R.DELTA.235
(the sequence of amino acids 1-235 of SEQ ID NO:1), which comprises
the entire extracellular region of TNF-R, terminating with
Asp.sup.235 immediately adjacent the transmembrane region.
Additional amino acids may be deleted from the extracellular region
while retaining TNF binding activity. For example,
huTNF-R.DELTA.183 which comprises the sequence of amino acids 1-183
of SEQ ID NO:1, and TNF-R.DELTA.163 which comprises the sequence of
amino acids 1-163 of SEQ ID NO:1, retain the ability to bind TNF
ligand as determined using the binding assays described below in
Example 1. TNF-R.DELTA.142, however, does not retain the ability to
bind TNF ligand. This suggests that one or both of Cys.sup.157 and
Cys.sup.163 is required for formation of an intramolecular
disulfide bridge for the proper folding of TNF-R. Cys.sup.178,
which was deleted without any apparent adverse effect on the
ability of the soluble TNF-R to bind TNF, does not appear to be
essential for proper folding of TNF-R. Thus, any deletion
C-terminal to Cys.sup.163 would be expected to result in a
biologically active soluble TNF-R. The present invention
contemplates such soluble TNF-R constructs corresponding to all or
part of the extracellular region of TNF-R terminating with any
amino acid after Cys.sup.163. Other C-terminal deletions, such as
TNF-F.DELTA.157, may be made as a matter of convenience by cutting
TNF-R cDNA with appropriate restriction enzymes and, if necessary,
reconstructing specific sequences with synthetic oligonucleotide
linkers. The resulting soluble TNF-R constructs are then inserted
and expressed in appropriate expression vectors and assayed for the
ability to bind TNF, as described in Example 1. Biologically active
soluble TNF-Rs resulting from such constructions are also
contemplated to be within the scope of the present invention.
[0039] Mutations in nucleotide sequences constructed for expression
of analog TNF-R must, of course, preserve the reading frame phase
of the coding sequences and preferably will not create
complementary regions that could hybridize to produce secondary
mRNA structures such as loops or hairpins which would adversely
affect translation of the receptor mRNA. Although a mutation site
may be predetermined, it is not necessary that the nature of the
mutation per se be predetermined. For example, in order to select
for optimum characteristics of mutants at a given site, random
mutagenesis may be conducted at the target codon and the expressed
TNF-R mutants screened for the desired activity.
[0040] Not all mutations in the nucleotide sequence which encodes
TNF-R will be expressed in the final product, for example,
nucleotide substitutions may be made to enhance expression,
primarily to avoid secondary structure loops in the transcribed
mRNA (see EPA 75,444A, incorporated herein by reference), or to
provide codons that are more readily translated by the selected
host, e.g., the well-known E. coli preference codons for E. coli
expression.
[0041] Mutations can be introduced at particular loci by
synthesizing oligonucleotides containing a mutant sequence, flanked
by restriction sites enabling ligation to fragments of the native
sequence. Following ligation, the resulting reconstructed sequence
encodes an analog having the desired amino acid insertion,
substitution, or deletion.
[0042] Alternatively, oligonucleotide-directed site-specific
mutagenesis procedures can be employed to provide an altered gene
having particular codons altered according to the substitution,
deletion, or insertion required. Exemplary methods of making the
alterations set forth above are disclosed by Walder et al. (Gene
42: 133, 1986); Bauer et al. (Gene 37: 73, 1985); Craik
(BioTechniques, January 1985, 12-19); Smith et al. (Genetic
Engineering: Principles and Methods, Plenum Press, 1981); and U.S.
Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, and
are incorporated by reference herein.
[0043] Both monovalent forms and polyvalent forms of TNF-R are
useful in the compositions and methods of this invention.
Polyvalent forms possess multiple TNF-R binding sites for TNF
ligand. For example, a bivalent soluble TNF-R may consist of two
tandem repeats of amino acids 1-235 of SEQ ID NO: 1, separated by a
linker region. Alternate polyvalent forms may also be constructed,
for example, by chemically coupling TNF-R to any clinically
acceptable carrier molecule, a polymer selected from the group
consisting of Ficoll, polyethylene glycol or dextran using
conventional coupling techniques. Alternatively, TNF-R may be
chemically coupled to biotin, and the biotin-TNF-R conjugate then
allowed to bind to avidin, resulting in tetravalent
avidin/biotin/TNF-R molecules. TNF-R may also be covalently coupled
to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting
conjugate precipitated with anti-DNP or anti-TNP-IgM, to form
decameric conjugates with a valency of 10 for TNF-R binding
sites.
[0044] A recombinant chimeric antibody molecule may also be
produced having TNF-R sequences substituted for the variable
domains of either or both of the immunoglobulin molecule heavy and
light chains and having unmodified constant region domains. For
example, chimeric TNF-R/IgG.sub.1 may be produced from two chimeric
genes--a TNF-R/human .kappa. light chain chimera
(TNF-R/C.sub..kappa.) and a TNF-R/human .gamma..sub.1 heavy chain
chimera (TNF-R/C.sub..gamma.-1). Following transcription and
translation of the two chimeric genes, the gene products assemble
into a single chimeric antibody molecule having TNF-R displayed
bivalently. Such polyvalent forms of TNF-R may have enhanced
binding affinity for TNF ligand. Additional details relating to the
construction of such chimeric antibody molecules are disclosed in
WO 89/09622 and EP 315062.
Expression of Recombinant TNF-R
[0045] The present invention provides recombinant expression
vectors to amplify or express DNA encoding TNF-R. Recombinant
expression vectors are replicable DNA constructs which have
synthetic or cDNA-derived DNA fragments encoding mammalian TNF-R or
bioequivalent analogs operably linked to suitable transcriptional
or translational regulatory elements derived from mammalian,
microbial, viral or insect genes. A transcriptional unit generally
comprises an assembly of (1) a genetic element or elements having a
regulatory role in gene expression, for example, transcriptional
promoters or enhancers, (2) a structural or coding sequence which
is transcribed into mRNA and translated into protein, and (3)
appropriate transcription and translation initiation and
termination sequences, as described in detail below. Such
regulatory elements may include an operator sequence to control
transcription, a sequence encoding suitable mRNA ribosomal binding
sites. The ability to replicate in a host, usually conferred by an
origin of replication, and a selection gene to facilitate
recognition of transformants may additionally be incorporated. DNA
regions are operably linked when they are functionally related to
each other. For example, DNA for a signal peptide (secretory
leader) is operably linked to DNA for a polypeptide if it is
expressed as a precursor which participates in the secretion of the
polypeptide; a promoter is operably linked to a coding sequence if
it controls the transcription of the sequence; or a ribosome
binding site is operably linked to a coding sequence if it is
positioned so as to permit translation. Generally, operably linked
means contiguous and, in the case of secretory leaders, contiguous
and in reading frame. Structural elements intended for use in yeast
expression systems preferably include a leader sequence enabling
extracellular secretion of translated protein by a host cell.
Alternatively, where recombinant protein is expressed without a
leader or transport sequence, it may include an N-terminal
methionine residue. This residue may optionally be subsequently
cleaved from the expressed recombinant protein to provide a final
product.
[0046] DNA sequences encoding mammalian TNF receptors which are to
be expressed in a microorganism will preferably contain no introns
that could prematurely terminate transcription of DNA into mRNA;
however, premature termination of transcription may be desirable,
for example, where it would result in mutants having advantageous
C-terminal truncations, for example, deletion of a transmembrane
region to yield a soluble receptor not bound to the cell membrane.
Due to code degeneracy, there can be considerable variation in
nucleotide sequences encoding the same amino acid sequence. Other
embodiments include sequences capable of hybridizing to the
sequences of the provided cDNA under moderately stringent
conditions (50.degree. C., 2.times.SSC) and other sequences
hybridizing or degenerate to those which encode biologically active
TNF receptor polypeptides.
[0047] Recombinant TNF-R DNA is expressed or amplified in a
recombinant expression system comprising a substantially
homogeneous monoculture of suitable host microorganisms, for
example, bacteria such as E. coli or yeast such as S. cerevisiae,
which have stably integrated (by transformation or transfection) a
recombinant transcriptional unit into chromosomal DNA or carry the
recombinant transcriptional unit as a component of a resident
plasmid. Generally, cells constituting the system are the progeny
of a single ancestral transformant. Recombinant expression systems
as defined herein will express heterologous protein upon induction
of the regulatory elements linked to the DNA sequence or synthetic
gene to be expressed.
[0048] Transformed host cells are cells which have been transformed
or transfected with TNF-R vectors constructed using recombinant DNA
techniques. Transformed host cells ordinarily express TNF-R, but
host cells transformed for purposes of cloning or amplifying TNF-R
DNA do not need to express TNF-R. Expressed TNF-R will be deposited
in the cell membrane or secreted into the culture supernatant,
depending on the TNF-R DNA selected. Suitable host cells for
expression of mammalian TNF-R include prokaryotes, yeast or higher
eukaryotic cells under the control of appropriate promoters.
Prokaryotes include gram negative or gram positive organisms, for
example E. coli or bacilli. Higher eukaryotic cells include
established cell lines of mammalian origin as described below.
Cell-free translation systems could also be employed to produce
mammalian TNF-R using RNAs derived from the DNA constructs of the
present invention. Appropriate cloning and expression vectors for
use with bacterial, fungal, yeast, and mammalian cellular hosts are
described by Pouwels et al. (Cloning Vectors: A Laboratory Manual,
Elsevier, New York, 1985), the relevant disclosure of which is
hereby incorporated by reference.
[0049] Prokaryotic expression hosts may be used for expression of
TNF-R that do not require extensive proteolytic and disulfide
processing. Prokaryotic expression vectors generally comprise one
or more phenotypic selectable markers, for example a gene encoding
proteins conferring antibiotic resistance or supplying an
autotrophic requirement, and an origin of replication recognized by
the host to ensure amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium, and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0050] 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 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed. E. coli is
typically transformed using derivatives of pBR322, a plasmid
derived from an E. coli species (Bolivar et al., Gene 2: 95, 1977).
pBR322 contains genes for ampicillin and tetracycline resistance
and thus provides simple means for identifying transformed
cells.
[0051] Promoters commonly used in recombinant microbial expression
vectors include the .beta.-lactamase (penicillinase) and lactose
promoter system (Chang et al., Nature 275: 615, 1978; and Goeddel
et al., Nature 281: 544, 1979), the tryptophan (trp) promoter
system (Goeddel et al., Nucl. Acids Res. 8: 4057, 1980; and EPA
36,776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A
particularly useful bacterial expression system employs the phage
.lamda. P.sub.L promoter and cI857ts thermolabile repressor.
Plasmid vectors available from the American Type Culture Collection
which incorporate derivatives of the .lamda. P.sub.L promoter
include plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092)
and pPLc28, resident in E. coli RR1 (ATCC 53082).
[0052] Recombinant TNF-R proteins may also be expressed in yeast
hosts, preferably from the Saccharomyces species, such as S.
cerevisiae. Yeast of other genera, such as Pichia or Kluyveromyces
may also be employed. Yeast vectors will generally contain an
origin of replication from the 2.mu. yeast plasmid or an
autonomously replicating sequence (ARS), promoter, DNA encoding
TNF-R, sequences for polyadenylation and transcription termination
and a selection gene. Preferably, yeast vectors will include an
origin of replication and selectable marker permitting
transformation of both yeast and E. coli, e.g., the ampicillin
resistance gene of E. coli and S. cerevisiae TRP1 or URA3 gene,
which provides a selection marker for a mutant strain of yeast
lacking the ability to grow in tryptophan, and a promoter derived
from a highly expressed yeast gene to induce transcription of a
structural sequence downstream. The presence of the TRP1 or URA3
lesion in the yeast host cell genome then provides an effective
environment for detecting transformation by growth in the absence
of tryptophan or uracil.
[0053] Suitable promoter sequences in yeast vectors include the
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, phosphoglucose isomerase, and glucokinase. Suitable
vectors and promoters for use in yeast expression are further
described in R. Hitzeman et al., EPA 73,657.
[0054] Preferred yeast vectors can be assembled using DNA sequences
from pUC18 for selection and replication in E. coli (Amp.sup.r gene
and origin of replication) and yeast DNA sequences including a
glucose-repressible ADH2 promoter and .alpha.-factor secretion
leader. The ADH2 promoter has been described by Russell et al. (J.
Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724,
1982). The yeast .alpha.-factor leader, which directs secretion of
heterologous proteins, can be inserted between the promoter and the
structural gene to be expressed. See, e.g., Kurjan et al., Cell 30:
933, 1982; and Bitter et al., Proc. Natl. Acad Sci. USA 81: 5330,
1984. The leader sequence may be modified to contain, near its 3'
end, one or more useful restriction sites to facilitate fusion of
the leader sequence to foreign genes.
[0055] Suitable yeast transformation protocols are known to those
of skill in the art; an exemplary technique is described by Hinnen
et al., Proc. Natl. Acad Sci. USA 75: 1929, 1978, selecting for
Trp.sup.+ transformants in a selective medium consisting of 0.67%
yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 .mu.g/ml
adenine and 20 .mu.g/ml uracil or URA+ tranformants in medium
consisting of 0.67% YNB, with amino acids and bases as described by
Sherman et al., Laboratory Course Manual for Methods in Yeast
Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1986.
[0056] Host strains transformed by vectors comprising the ADH2
promoter may be grown for expression in a rich medium consisting of
1% yeast extract, 2% peptone, and 1% or 4% glucose supplemented
with 80 .mu.g/ml adenine and 80 .mu.g/ml uracil. Derepression of
the ADH2 promoter occurs upon exhaustion of medium glucose. Crude
yeast supernatants are harvested by filtration and held at
4.degree. C. prior to further purification.
[0057] Various mammalian or insect cell culture systems are also
advantageously employed to express recombinant protein. Expression
of recombinant proteins in mammalian cells is particularly
preferred because such proteins are generally correctly folded,
appropriately modified and completely functional. Examples of
suitable mammalian host cell lines include the COS-7 lines of
monkey kidney cells, described by Gluzman (Cell 23: 175, 1981), and
other cell lines capable of expressing an appropriate vector
including, for example, L cells, C127, 3T3, Chinese hamster ovary
(CHO), HeLa and BHK cell lines. Mammalian expression vectors may
comprise nontranscribed elements such as an origin of replication,
a suitable promoter and enhancer linked to the gene to be
expressed, and other 5' or 3' flanking nontranscribed sequences,
and 5' or 3' nontranslated sequences, such as necessary ribosome
binding sites, a polyadenylation site, splice donor and acceptor
sites, and transcriptional termination sequences. Baculovirus
systems for production of heterologous proteins in insect cells are
reviewed by Luckow and Summers, Bio/Technology 6: 47 (1988).
[0058] The transcriptional and translational control sequences in
expression vectors to be used in transforming vertebrate cells may
be provided by viral sources. For example, commonly used promoters
and enhancers are derived from Polyoma, 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 the other genetic elements required for expression of a
heterologous DNA sequence. The early and late promoters are
particularly useful because both are obtained easily from the virus
as a fragment which also contains the SV40 viral origin of
replication (Fiers et al., Nature 273: 113, 1978). Smaller or
larger SV40 fragments may also be used, provided the approximately
250 bp sequence extending from the Hind 3 site toward the Bgl1 site
located in the viral origin of replication is included. Further,
mammalian genomic TNF-R promoter, control and/or signal sequences
may be utilized, provided such control sequences are compatible
with the host cell chosen. Additional details regarding the use of
a mammalian high expression vector to produce a recombinant
mammalian TNF receptor are provided in Examples 2 and 7 below.
Exemplary vectors can be constructed as disclosed by Okayama and
Berg (Mol. Cell. Biol. 3: 280,1983).
[0059] A useful system for stable high level expression of
mammalian receptor cDNAs in C127 murine mammary epithelial cells
can be constructed substantially as described by Cosman et al.
(Mol. Immunol. 23: 935, 1986).
[0060] In preferred aspects of the present invention, recombinant
expression vectors comprising TNF-R cDNAs are stably integrated
into a host cell's DNA. Elevated levels of expression product is
achieved by selecting for cell lines having amplified numbers of
vector DNA. Cell lines having amplified numbers of vector DNA are
selected, for example, by transforming a host cell with a vector
comprising a DNA sequence which encodes an enzyme which is
inhibited by a known drug. The vector may also comprise a DNA
sequence which encodes a desired protein. Alternatively, the host
cell may be co-transformed with a second vector which comprises the
DNA sequence which encodes the desired protein. The transformed or
co-transformed host cells are then cultured in increasing
concentrations of the known drug, thereby selecting for
drug-resistant cells. Such drug-resistant cells survive in
increased concentrations of the toxic drug by over-production of
the enzyme which is inhibited by the drug, frequently as a result
of amplification of the gene encoding the enzyme. Where drug
resistance is caused by an increase in the copy number of the
vector DNA encoding the inhibitable enzyme, there is a concomitant
co-amplification of the vector DNA encoding the desired protein
(TNF-R) in the host cell's DNA.
[0061] A preferred system for such co-amplification uses the gene
for dihydrofolate reductase (DHFR), which can be inhibited by the
drug methotrexate (MTX). To achieve co-amplification, a host cell
which lacks an active gene encoding DHFR is either transformed with
a vector which comprises DNA sequence encoding DHFR and a desired
protein, or is co-transformed with a vector comprising a DNA
sequence encoding DHFR and a vector comprising a DNA sequence
encoding the desired protein. The transformed or co-transformed
host cells are cultured in media containing increasing levels of
MTX, and those cells lines which survive are selected.
[0062] A particularly preferred co-amplification system uses the
gene for glutamine synthetase (GS), which is responsible for the
synthesis of glutamate and ammonia using the hydrolysis of ATP to
ADP and phosphate to drive the reaction. GS is subject to
inhibition by a variety of inhibitors, for example methionine
sulphoximine (MSX). Thus, TNF-R can be expressed in high
concentrations by co-amplifying cells transformed with a vector
comprising the DNA sequence for GS and a desired protein, or
co-transformed with a vector comprising a DNA sequence encoding GS
and a vector comprising a DNA sequence encoding the desired
protein, culturing the host cells in media containing increasing
levels of MSX and selecting for surviving cells. The GS
co-amplification system, appropriate recombinant expression vectors
and cells lines, are described in the following PCT applications:
WO 87/04462, WO 89/01036, WO 89/10404 and WO 86/05807.
[0063] Recombinant proteins are preferably expressed by
co-amplification of DHFR or GS in a mammalian host cell, such as
Chinese Hamster Ovary (CHO) cells, or alternatively in a murine
myeloma cell line, such as SP2/0-Ag14 or NS0 or a rat myeloma cell
line, such as YB2/3.0-Ag20, disclosed in PCT applications
WO/89/10404 and WO 86/05807.
[0064] A preferred eukaryotic vector for expression of TNF-R DNA is
disclosed below in Example 2. This vector, referred to as pCAV/NOT,
was derived from the mammalian high expression vector pDC201 and
contains regulatory sequences from SV40, adenovirus-2, and human
cytomegalovirus.
Purification of Recombinant TNF-R
[0065] Purified mammalian TNF receptors or analogs are prepared by
culturing suitable host/vector systems to express the recombinant
translation products of the DNAs of the present invention, which
are then purified from culture media or cell extracts.
[0066] For example, supernatants from systems which secrete
recombinant protein into culture media can be first 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 suitable purification matrix. For example, a suitable affinity
matrix can comprise a TNF or lectin or antibody molecule bound to a
suitable support. Alternatively, an anion exchange resin can be
employed, for example, a matrix or substrate having pendant
diethylaminoethyl (DEAE) groups. The matrices can be acrylamide,
agarose, dextran, cellulose or other types commonly employed in
protein purification. Alternatively, a cation exchange step can be
employed. Suitable cation exchangers include various insoluble
matrices comprising sulfopropyl or carboxymethyl groups.
Sulfopropyl groups are preferred.
[0067] Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify a TNF-R composition. Some or all
of the foregoing purification steps, in various combinations, can
also be employed to provide a homogeneous recombinant protein.
[0068] Recombinant protein produced in bacterial culture is usually
isolated by initial extraction from cell pellets, followed by one
or more concentration, salting-out, aqueous ion exchange or size
exclusion chromatography steps. Finally, high performance liquid
chromatography (HPLC) can be employed for final purification steps.
Microbial cells employed in expression of recombinant mammalian
TNF-R can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
[0069] Fermentation of yeast which express mammalian TNF-R as a
secreted protein greatly simplifies purification. Secreted
recombinant protein resulting from a large-scale fermentation can
be purified by methods analogous to those disclosed by Urdal et al.
(J. Chromatog. 296: 171, 1984). This reference describes two
sequential, reversed-phase HPLC steps for purification of
recombinant human GM-CSF on a preparative HPLC column.
[0070] Human TNF-R synthesized in recombinant culture is
characterized by the presence of non-human cell components,
including proteins, in amounts and of a character which depend upon
the purification steps taken to recover human TNF-R from the
culture. These components ordinarily will be of yeast, prokaryotic
or non-human higher eukaryotic origin and preferably are present in
innocuous contaminant quantities, on the order of less than about 1
percent by weight. Further, recombinant cell culture enables the
production of TNF-R free of proteins which may be normally
associated with TNF-R as it is found in nature in its species of
origin, e.g. in cells, cell exudates or body fluids.
Therapeutic Administration of Recombinant Soluble TNF-R
[0071] The present invention provides methods of using therapeutic
compositions comprising an effective amount of soluble TNF-R
proteins and a suitable diluent and carrier, and methods for
suppressing TNF-dependent inflammatory responses in humans
comprising administering an effective amount of soluble TNF-R
protein.
[0072] For therapeutic use, purified soluble TNF-R protein is
administered to a patient, preferably a human, for treatment in a
manner appropriate to the indication. Thus, for example, soluble
TNF-R protein compositions can be administered by bolus injection,
continuous infusion, sustained release from implants, or other
suitable technique. Typically, a soluble TNF-R therapeutic agent
will be administered in the form of a composition comprising
purified protein in conjunction with physiologically acceptable
carriers, excipients or diluents. Such carriers will be nontoxic to
recipients at the dosages and concentrations employed. Ordinarily,
the preparation of such compositions entails combining the TNF-R
with buffers, antioxidants such as ascorbic acid, low molecular
weight (less than about 10 residues) polypeptides, proteins, amino
acids, carbohydrates including glucose, sucrose or dextrins,
chelating agents such as EDTA, glutathione and other stabilizers
and excipients. Neutral buffered saline or saline mixed with
conspecific serum albumin are exemplary appropriate diluents.
Preferably, product is formulated as a lyophilizate using
appropriate excipient solutions (e.g., sucrose) as diluents.
Appropriate dosages can be determined in trials. The amount and
frequency of administration will depend, of course, on such factors
as the nature and severity of the indication being treated, the
desired response, the condition of the patient, and so forth.
[0073] Soluble TNF-R proteins are administered for the purpose of
inhibiting TNF-dependent responses. A variety of diseases or
conditions are believed to be caused by TNF, such as cachexia and
septic shock. In addition, other key cytokines (IL-1, IL-2 and
other colony stimulating factors) can also induce significant host
production of TNF. Soluble TNF-R compositions may therefore be
used, for example, to treat cachexia or septic shock or to treat
side effects associated with cytokine therapy. Because of the
primary roles IL-1 and IL-2 play in the production of TNF,
combination therapy using both IL-1 receptors or IL-2 receptors may
be preferred in the treatment of TNF-associated clinical
indications.
[0074] The following examples are offered by way of illustration,
and not by way of limitation.
EXAMPLES
Example 1
Binding Assays
[0075] A. Radiolabeling of TNF.alpha. and TNF.beta.. Recombinant
human TNF.alpha., in the form of a fusion protein containing a
hydrophilic octapeptide at the N-terminus, was expressed in yeast
as a secreted protein and purified by affinity chromatography (Hopp
et al., Bio/Technology 6: 1204, 1988). Purified recombinant human
TNF.beta. was purchased from R&D Systems (Minneapolis, Minn.).
Both proteins were radiolabeled using the commercially available
solid phase agent, IODO-GEN (Pierce). In this procedure, 5 .mu.g of
IODO-GEN were plated at the bottom of a 10.times.75 mm glass tube
and incubated for 20 minutes at 4.degree. C. with 75 .mu.l of 0.1 M
sodium phosphate, pH 7.4 and 20 .mu.l (2 mCi) Na .sup.125I. This
solution was then transferred to a second glass tube containing 5
.mu.g TNF.alpha. (or TNF.beta.) in 45 .mu.l PBS for 20 minutes at
4.degree. C. The reaction mixture was fractionated by gel
filtration on a 2 ml bed volume of Sephadex G-25 (Sigma)
equilibrated in Roswell Park Memorial Institute (RPMI) 1640 medium
containing 2.5% (w/v) bovine serum albumin (BSA), 0.2% (w/v) sodium
azide and 20 mM Hepes pH 7.4 (binding medium). The final pool of
.sup.125I-TNF was diluted to a working stock solution of
1.times.10.sup.-7 M in binding medium and stored for up to one
month at 4.degree. C. without detectable loss of receptor binding
activity. The specific activity is routinely 1.times.10.sup.6
cpm/mmole TNF.
[0076] B. Binding to Intact Cells. Binding assays with intact cells
were performed by two methods. In the first method, cells were
first grown either in suspension (e.g., U 937) or by adherence on
tissue culture plates (e.g., W126-VA4, COS cells expressing the
recombinant TNF receptor). Adherent cells were subsequently removed
by treatment with 5 mM EDTA treatment for ten minutes at 37 degrees
centigrade. Binding assays were then performed by a phthalate oil
separation method (Dower et al., J. Immunol. 132: 751, 1984)
essentially as described by Park et al. (J. Biol. Chem. 261: 4177,
1986). Non-specific binding of .sup.125I-TNF was measured in the
presence of a 200-fold or greater molar excess of unlabeled TNF.
Sodium azide (0.2%) was included in a binding assay to inhibit
internalization of .sup.125I-TNF by cells. In the second method,
COS cells transfected with the TNF-R-containing plasmid, and
expressing TNF receptors on the surface, were tested for the
ability to bind .sup.125I-TNF by the plate binding assay described
by Sims et al. (Science 241: 585, 1988).
[0077] C. Solid Phase Binding Assays. The ability of TNF-R to be
stably adsorbed to nitrocellulose from detergent extracts of human
cells yet retain TNF-binding activity provided a means of detecting
TNF-R. Cell extracts were prepared by mixing a cell pellet with a
2.times.volume of PBS containing 1% Triton X-100 and a cocktail of
protease inhibitors (2 mM phenylmethyl sulfonyl fluoride, 10 .mu.M
pepstatin, 10 .mu.M leupeptin, 2 mM o-phenanthroline and 2 mM EGTA)
by vigorous vortexing. The mixture was incubated on ice for 30
minutes after which it was centrifuged at 12,000.times.g for 15
minutes at 8.degree. C. to remove nuclei and other debris. Two
microliter aliquots of cell extracts were placed on dry BA85/21
nitrocellulose membranes (Schleicher and Schuell, Keene, N H) and
allowed to dry. The membranes were incubated in tissue culture
dishes for 30 minutes in Tris (0.05 M) buffered saline (0.15 M) pH
7.5 containing 3% w/v BSA to block nonspecific binding sites. The
membrane was then covered with 5.times.10.sup.-11 M .sup.125I-TNF
in PBS+3% BSA and incubated for 2 hr at 4.degree. C. with shaking.
At the end of this time, the membranes were washed 3 times in PBS,
dried and placed on Kodak X-Omat AR film for 18 hr at -70.degree.
C.
Example 2
Isolation of Human TNF-R cDNA by Direct Expression of Active
Protein in COS-7 Cells
[0078] Various human cell lines were screened for expression of
TNF-R based on their ability to bind .sup.125I-labeled TNF. The
human fibroblast cell line WI-26 VA4 was found to express a
reasonable number of receptors per cell. Equilibrium binding
studies showed that the cell line exhibited biphasic binding of
.sup.125I-TNF with approximately 4,000 high affinity sites
(K.sub.a=1.times.10.sup.10 M.sup.-1) and 15,00 low affinity sites
(K.sub.a=1.times.10.sup.8 M.sup.-1) per cell.
[0079] An unsized cDNA library was constructed by reverse
transcription of polyadenylated mRNA isolated from total RNA
extracted from human fibroblast WI-26 VA4 cells grown in the
presence of pokeweed mitogen using standard techniques (Gubler, et
al., Gene 25: 263, 1983; Ausubel et al., eds., Current Protocols in
Molecular Biology, Vol. 1, 1987). The cells were harvested by
lysing the cells in a guanidine hydrochloride solution and total
RNA isolated as previously described (March et al., Nature 315:
641, 1985).
[0080] Poly A.sup.+ RNA was isolated by oligo dT cellulose
chromatography and double-stranded cDNA was prepared by a method
similar to that of Gubler and Hoffman (Gene 25: 263, 1983).
Briefly, the poly A.sup.+ RNA was converted to an RNA-cDNA hybrid
by reverse transcriptase using oligo dT as a primer. The RNA-cDNA
hybrid was then converted into double-stranded cDNA using RNAase H
in combination with DNA polymerase I. The resulting double stranded
cDNA was blunt-ended with T4 DNA polymerase. To the blunt-ended
cDNA is added EcoRI linker-adapters (having internal Not1 sites)
which were phosphorylated on only one end (Invitrogen). The
linker-adaptered cDNA was treated with T4 polynucleotide kinase to
phosphorylate the 5' overhanging region of the linker-adapter and
unligated linkers were removed by running the cDNA over a Sepharose
CL4B column. The linker-adaptered cDNA was ligated to an equimolar
concentration of EcoR1 cut and dephosphorylated arms of
bacteriophage .lamda.kgt10 (Huynh et al, DNA Cloning: A Practical
Approach, Glover, ed., IRL Press, pp. 49-78). The ligated DNA was
packaged into phage particles using a commercially available kit to
generate a library of recombinants (Stratagene Cloning Systems, San
Diego, Calif., USA). Recombinants were further amplified by plating
phage on a bacterial lawn of E. coli strain c600(hfl.sup.-).
[0081] Phage DNA was purified from the resulting .lamda.gt10 cDNA
library and the cDNA inserts excised by digestion with the
restriction enzyme Not1. Following electrophoresis of the digest
through an agarose gel, cDNAs greater than 2,000 bp were
isolated.
[0082] The resulting cDNAs were ligated into the eukaryotic
expression vector pCAV/NOT, which was designed to express cDNA
sequences inserted at its multiple cloning site when transfected
into mammalian cells. pCAV/NOT was assembled from pDC201 (a
derivative of pMLSV, previously described by Cosman et al., Nature
312: 768, 1984), SV40 and cytomegalovirus DNA and comprises, in
sequence with the direction of transcription from the origin of
replication: (1) SV40 sequences from coordinates 5171-270 including
the origin of replication, enhancer sequences and early and late
promoters; (2) cytomegalovirus sequences including the promoter and
enhancer regions (nucleotides 671 to +63 from the sequence
published by Boechart et al. (Cell 41: 521, 1985); (3) adenovirus-2
sequences containing the first exon and part of the intron between
the first and second exons of the tripartite leader, the second
exon and part of the third exon of the tripartite leader and a
multiple cloning site (MCS) containing sites for Xho1, Kpn1, Sma1,
Not1 and Bgl1; (4) SV40 sequences from coordinates 4127-4100 and
2770-2533 that include the polyadenylation and termination signals
for early transcription; (5) sequences derived from pBR322 and
virus-associated sequences VAI and VAII of pDC201, with adenovirus
sequences 10532-11156 containing the VAI and VAII genes, followed
by pBR322 sequences from 4363-2486 and 1094-375 containing the
ampicillin resistance gene and origin of replication.
[0083] The resulting WI-26 VA4 cDNA library in pCAV/NOT was used to
transform E. coli strain DH5.alpha., and recombinants were plated
to provide approximately 800 colonies per plate and sufficient
plates to provide approximately 50,000 total colonies per screen.
Colonies were scraped from each plate, pooled, and plasmid DNA
prepared from each pool. The pooled DNA was then used to transfect
a sub-confluent layer of monkey COS-7 cells using DEAE-dextran
followed by chloroquine treatment, as described by Luthman et al.
(Nucl. Acids Res. 11: 1295, 1983) and McCutchan et al. (J. Natl.
Cancer Inst. 41: 351, 1986). The cells were then grown in culture
for three days to permit transient expression of the inserted
sequences. After three days, cell culture supernatants were
discarded and the cell monolayers in each plate assayed for TNF
binding as follows. Three ml of binding medium containing
1.2.times.10.sup.-11 M .sup.125I-labeled FLAG.RTM.-TNF was added to
each plate and the plates incubated at 4.degree. C. for 120
minutes. This medium was then discarded, and each plate was washed
once with cold binding medium (containing no labeled TNF) and twice
with cold PBS. The edges of each plate were then broken off,
leaving a flat disk which was contacted with X-ray film for 72
hours at -70.degree. C. using an intensifying screen. TNF binding
activity was visualized on the exposed films as a dark focus
against a relatively uniform background.
[0084] After approximately 240,000 recombinants from the library
had been screened in this manner, one transfectant pool was
observed to provide TNF binding foci which were clearly apparent
against the background exposure.
[0085] A frozen stock of bacteria from the positive pool was then
used to obtain plates of approximately 150 colonies. Replicas of
these plates were made on nitrocellulose filters, and the plates
were then scraped and plasmid DNA prepared and transfected as
described above to identify a positive plate. Bacteria from
individual colonies from the nitrocellulose replica of this plate
were grown in 0.2 ml cultures, which were used to obtain plasmid
DNA, which was transfected into COS-7 cells as described above. In
this manner, a single clone, clone 1, was isolated which was
capable of inducing expression of human TNF-R in COS cells. The
expression vector pCAV/NOT containing the TNF-R cDNA clone 1 has
been deposited with the American Type Culture Collection, 12301
Parklawn Drive, Rockville, Md. 20852, USA (Accession No. 68088)
under the name pCAV/NOT-TNF-R.
Example 3
Construction of cDNAs Encoding Soluble huTNF-R.DELTA.235
[0086] A cDNA encoding a soluble huTNF-R.DELTA.235 (having the
sequence of amino acids 1-235 of SEQ ID NO:1) was constructed by
excising an 840 bp fragment from pCAV/NOT-TNF-R with the
restriction enzymes Not1 and Pvu2. Not1 cuts at the multiple
cloning site of pCAV/NOT-TNF-R and Pvu2 cuts within the TNF-R
coding region 20 nucleotides 5' of the transmembrane region. In
order to reconstruct the 3' end of the TNF-R sequences, two
oligonucleotides (encoding amino acids corresponding to
Ala.sup.229-Ala.sup.235 of SEQ ID NO:1) were synthesized and
annealed to create the following oligonucleotide linker:
TABLE-US-00001 Pvu2 BamH1 Bgl2 CTGAAGGGAGCACTGGCGACTAAGGATCCA (SEQ
ID NO: 5 GACTTCCCTCGTGACCGCTGATTCCTAGGTCTAG (SEQ ID NO: 6)
AlaGluGlySerThrGlyAspEnd (SEQ ID NO: 7)
This oligonucleotide linker has terminal Pvu2 and Bgl2 restriction
sites, regenerates 20 nucleotides of the TNF-R, followed by a
termination codon (underlined) and a BamH1 restriction site (for
convenience in isolating the entire soluble TNF-R by Not1/BamH1
digestion). This oligonucleotide was then ligated with the 840 bp
Not1/Pvu2 TNF-R insert into Bgl2/Notl cut pCAV/NOT to yield
psolhuTNF-R.DELTA.235/CAVNOT, which was transfected into COS-7
cells as described above. This expression vector induced expression
of soluble human TNF-R which was capable of binding TNF.
Example 4
Construction of cDNAs Encoding Soluble huTNF-R.DELTA.185
[0087] A cDNA encoding a soluble huTNF-R.DELTA.185 (having the
sequence of amino acids 1-185 of SEQ ID NO:1) was constructed by
excising a 640 bp fragment from pCAV/NOT-TNF-R with the restriction
enzymes Not1 and Bgl2. Notl cuts at the multiple cloning site of
pCAV/NO-TNF-R and Bgl2 cuts within the TNF-R coding region at
nucleotide 637, which is 237 nucleotides 5' of the transmembrane
region. The following oligonucleotide linkers (encoding amino acids
corresponding to Ile.sup.162-Ala.sup.176 and
Val.sup.177-Arg.sup.185 of SEQ ID NO:1) were synthesized:
TABLE-US-00002 Bg12 (SEQ ID NO: 8)
5'-GATCTGTAACGTGGTGGCCATCCCTGGGAATGCAAGCATGGATGC- 3' (SEQ ID NO: 9)
ACATTGCACCACCGGTAGGGACCCTTACGTTCG (SEQ ID NO: 10)
IleCysAsnValValAlaIleProGlyAsnAlaSerMetAspAla Not 1 (SEQ ID NO: 11)
5'-AGTCTGCACGTCCACGTCCCCCACCCGGTGAGC-3' (SEQ ID NO: 12)
TACCTACGTCAGACGTGCAGGTGCAGGGGGTGGGCCACTCGCCGG (SEQ ID NO: 13)
ValCysThrSerThrSerProThrArgEnd
The above oligonucleotide linkers reconstruct the 3' end of the
receptor molecule up to nucleotide 708, followed by a termination
codon (underlined). These oligonucleotides were then ligated with
the 640 bp Not1 TNF-R insert into Notl cut pCAV/NOT to yield the
expression vector psolTNFR.DELTA.185/CAVNOT, which was transfected
into COS-7 cells as described above. This expression vector induced
expression of soluble human TNF-R which was capable of binding
TNF.
Example 5
Construction of cDNAs Encoding Soluble huTNF-RA163
[0088] A cDNA encoding a soluble huTNF-R.DELTA.163 (having the
sequence of amino acids 1-163 of SEQ ID NO:1) was constructed by
excising a 640 bp fragment from from pCAV/NOT-TNF-R with the
restriction enzymes Not1 and Bgl2 as described in Example 4. The
following oligonucleotide linkers (encoding amino acids
corresponding to Ile.sup.165-Cys.sup.163 of SEQ ID NO:1) were
synthesized:
TABLE-US-00003 Bgl2 Not1 5'-GATCTGTTGAGC-3' (SEQ ID NO: 14)
ACAACTCGCCGG (SEQ ID NO: 15) IleCysEnd
This above oligonucleotide linker reconstructs the 3' end of the
receptor molecule up to nucleotide 642 (amino acid 163), followed
by a termination codon (underlined). This oligonucleotide was then
ligated with the 640 bp Not1 TNF-R insert into Notl cut pCAV/NOT to
yield the expression vector psolTNFR.DELTA.163/CAVNOT, which was
transfected into COS-7 cells as described above. This expression
vector induced expression of soluble human TNF-R which was capable
of binding TNF in the binding assay described in Example 1.
Example 6
Construction of cDNAs Encoding Soluble huTNF-RA142
[0089] A cDNA encoding a soluble huTNF-R.DELTA.142 (having the
sequence of amino acids 1-142 of SEQ ID NO:1) was constructed by
excising a 550 bp fragment from from pCAV/NOT-TNF-R with the
restriction enzymes Not1 and AlwN1. AlwN1 cuts within the TNF-R
coding region at nucleotide 549. The following oligonucleotide
linker (encoding amino acids corresponding to
Thr.sup.132-Cys.sup.142 of SEQ ID NO:1) was synthesized:
TABLE-US-00004 Bgl2 Not1 (SEQ ID NO: 16)
5'-CTGAAACATCAGACGTGGTGTGCAAGCCCTGTTAAA-3' (SEQ ID NO: 17)
CTTGACTTTGTAGTCTGCACCACACGTTCGGGACAATTTCTAGA End
This above oligonucleotide linker reconstructs the 3' end of the
receptor molecule up to nucleotide 579 (amino acid 142), followed
by a termination codon (underlined). This oligonucleotide was then
ligated with the 550 bp Not1/AlwN1 TNF-R insert into Not1/Bgl2 cut
pCAV/NOT to yield the expression vector psolTNFR.DELTA.142/CAVNOT,
which was transfected into COS-7 cells as described above. This
expression vector did not induce expression of soluble human TNF-R
which was capable of binding TNF. It is believed that this
particular construct failed to express biologically active TNF-R
because one or more essential cysteine residue (e.g., Cys.sup.157
or Cys.sup.163) required for intramolecular bonding (for formation
of the proper tertiary structure of the TNF-R molecule) was
eliminated.
Example 7
Expression of Soluble TNF Receptors in CHO Cells
[0090] Soluble TNF receptor was expressed in Chinese Hamster Ovary
(CHO) cells using the glutamine-synthetase (GS) gene amplification
system, substantially as described in PCT patent application Nos.
WO87/04462 and WO89/01036. Briefly, CHO cells are transfected with
an expression vector containing genes for both TNF-R and GS. CHO
cells are selected for GS gene expression based on the ability of
the transfected DNA to confer resistance to low levels of
methionine sulphoximine (MSX). GS sequence amplification events in
such cells are selected using elevated MSX concentrations. In this
way, contiguous TNF-R sequences are also amplified and enhanced
TNF-R expression is achieved.
[0091] The vector used in the GS expression system was
psolTNFR/P6/PSVLGS, which was constructed as follows. First, the
vector pSVLGS.1 (described in PCT Application Nos. WO87/04462 and
WO89/01036, and available from Celltech, Ltd., Berkshire, UK) was
cut with the BamH1 restriction enzyme and dephosphorylated with
calf intestinal alkaline phosphatase (CIAP) to prevent the vector
from religating to itself. The BamH1 cut pSVLGS.1 fragment was then
ligated to a 2.4 kb BamH1 to Bgl2 fragment of pEE6hCMV (described
in PCT Application No. WO89/01036, also available from Celltech)
which was cut with Bgl2, BamH1 and Fsp1 to avoid two fragments of
similar size, to yield an 11.2 kb vector designated p6/PSVLGS.1.
pSVLGS.1 contains the glutamine synthetase selectable marker gene
under control of the SV40 later promoter. The BamH1 to Bgl2
fragment of pEE6hCMV contains the human cytomegalovirus major
immediate early promoter (hCMV), a polylinker, and the SV40 early
polyadenylation signal. The coding sequences for soluble TNF-R were
added to p6/PSVLGS.1 by excising a Not1 to BamH1 fragment from the
expression vector psolTNFR/CAVNOT (made according to Example 3
above), blunt ending with Klenow and ligating with Sma1 cut
dephosphorylated p6/PSVLGS.1, thereby placing the solTNF-R coding
sequences under the control of the hCMV promoter. This resulted in
a single plasmid vector in which the SV40/GS and hCMB/solTNF-R
transcription units are transcribed in opposite directions. This
vector was designated psolTNFR/P6/PSVLGS.
[0092] psolTNFR/P6/PSVLGS was used to transfect CHO-K1 cells
(available from ATCC, Rochville, Md., under accession number CCL
61) as follows. A monolayer of CHO-K1 cells were grown to
subconfluency in Minimum Essential Medium (MEM) 10.times. (Gibco:
330-1581AJ) without glutamine and supplemented with 10% dialysed
fetal bovine serum (Gibco: 220-6300AJ), 1 mM sodium pyruvate
(Sigma), MEM non-essential amino acids (Gibco: 320-1140AG), 500
.mu.M asparagine and glutamate (Sigma) and nucleosides (30 .mu.M
adenosine, guanosine, cytidine and uridine and 10 .mu.M
thymidine)(Sigma).
[0093] Approximately 1.times.10.sup.6 cells per 10 cm petri dish
were transfected with 10 .mu.g of psolTNFR/P6/PSVLGS by standard
calcium phosphate precipitation, substantially as described by
Graham & van der Eb, Virology 52: 456 (1983). Cells were
subjected to glycerol shock (15% glycerol in serum-free culture
medium for approximately 1.5 minutes) approximately 4 hours after
transfection, substantially as described by Frost & Williams,
Virology 91: 39 (1978), and then washed with serum-free medium. One
day later, transfected cells were fed with fresh selective medium
containing MSX at a final concentration of 25 .mu.M. Colonies of
MSX-resistant surviving cells were visible within 3-4 weeks.
Surviving colonies were transferred to 24-well plates and allowed
to grow to confluency in selective medium. Conditioned medium from
confluent wells were then assayed for soluble TNF-R activity using
the binding assay described in Example 1 above. These assays
indicated that the colonies expressed biologically active soluble
TNF-R.
[0094] In order to select for GS gene amplification, several
MSX-resistant cell lines are transfected with psolTNFR/P6/PSVLGS
and grown in various concentrations of MSX. For each cell line,
approximately 1.times.10.sup.6 cells are plated in gradually
increasing concentrations of 100 .mu.M, 250 .mu.M, 500 .mu.M and 1
mM MSX and incubated for 10-14 days. After 12 days, colonies
resistant to the higher levels of MSX appear. The surviving
colonies are assayed for TNF-R activity using the binding assay
described above in Example 1. Each of these highly resistant cell
lines contains cells which arise from multiple independent
amplification events. From these cells lines, one or more of the
most highly resistant cells lines are isolated. The amplified cells
with high production rates are then cloned by limiting dilution
cloning. Mass cell cultures of the transfectants secrete active
soluble TNF-R.
Example 8
Expression of Soluble Human TNF-R in Yeast
[0095] Soluble human TNF-R was expressed in yeast with the
expression vector pIXY432, which was derived from the yeast
expression vector pIXY120 and plasmid pYEP352. pIXY120 is identical
to pY.alpha.HuGM (ATCC 53157), except that it contains no cDNA
insert and includes a polylinker/multiple cloning site with a Nco1
restriction site.
[0096] A DNA fragment encoding TNF receptor and suitable for
cloning into the yeast expression vector pIXY120 was first
generated by polymerase chain reaction (PCR) amplification of the
extracellular portion of the full length receptor from
pCAV/NOT-TNF-R (ATCC 68088). The following primers (encoding amino
acids corresponding in part to Leu.sup.1-Thr.sup.8 and
Pro.sup.225-Asp.sup.235 of SEQ ID NO:1) were used in this PCR
amplification:
TABLE-US-00005 5' End Primer 5'-TTCCGGTACCTTTGGATAAAAGAGACTACAAGGAC
Asp718- >ProLeuAspLysArgAspTyrLysAsp (SEQ ID NO: 18)
GACGATGACAAGTTGCCCGCCCAGGTGGCATTTACA-3' (SEQ ID NO: 19)
AspAspAspLys<--------TNF-R----------> 3' End Primer
(antisense) (SEQ ID NO 20)
5'-CCCGGGATCCTTAGTCGCCAGTGCTCCCTTCAGCTGGG-3'
BamH1>End<-------------TNF-R------->
The 5' end oligonucleotide primer used in the amplification
included an Asp718 restriction site at its 5' end, followed by
nucleotides encoding the 3' end of the yeast .alpha.-factor leader
sequence (Pro-Leu-Asp-Lys-Arg (SEQ ID NO:21)) and those encoding
the 8 amino acids of the FLAG.RTM. peptide
(AspTyrLysAspAspAspAspLys (SEQ ID NO:22)) fused to a sequence
encoding the 5' end of the mature receptor. The FLAG.RTM. peptide
(Hopp et al., Bio/Technology 6: 1204, 1988) is a highly antigenic
sequence which reversibly binds the monoclonal antibody M1 (ATCC HB
9259). The oligonucleotide used to generate the 3' end of the
PCR-derived fragment is the antisense strand of DNA encoding
sequences which terminate the open reading frame of the receptor
after nucleotide 704 of the mature coding region (following the Asp
residue preceding the transmembrane domain) by introducing a TAA
stop codon (underlined). The stop codon is then followed by a BamH1
restriction site. The DNA sequences encoding TNF-R are then
amplified by PCR, substantially as described by Innis et al., eds.,
PCR Protocols: A Guide to Methods and Applications (Academic Press,
1990).
[0097] The PCR-derived DNA fragment encoding soluble human TNF-R
was subcloned into the yeast expression vector pIXY120 by digesting
the PCR-derived DNA fragment with BamH1 and Asp718 restriction
enzymes, digesting pIXY120 with BamH1 and Asp718, and ligating the
PCR fragment into the cut vector in vitro with T4 DNA ligase. The
resulting construction (pIXY424) fused the open reading frame of
the FLAG.RTM.-soluble TNF receptor in-frame to the complete
.alpha.-factor leader sequence and placed expression in yeast under
the aegis of the regulated yeast alcohol dehydrogenase (ADH2)
promoter. Identity of the nucleotide sequence of the soluble TNF
receptor carried in pIXY424 with those in cDNA clone 1 were
verified by DNA sequencing using the dideoxynucleotide chain
termination method. pIXY424 was then transformed into E. coli
strain RR1.
[0098] Soluble human TNF receptor was also expressed and secreted
in yeast in a second vector. This second vector was generated by
recovering the pIXY424 plasmid from E. coli and digesting with
EcoR1 and BamH1 restriction enzymes to isolate the fragment
spanning the region encoding the ADH2 promoter, the .alpha.-factor
leader, the FLAG.RTM.-soluble TNF receptor and the stop codon. This
fragment was ligated in vitro into EcoR1 and BamH1 cut plasmid
pYEP352 (Hill et al., Yeast 2: 163 (1986)), to yield the expression
plasmid pIXY432, which was transformed into E. coli strain RR1.
[0099] To assess secretion of the soluble human TNF receptor from
yeast, pIXY424 was purified and introduced into a diploid yeast
strain of S. cerevisiae (XV2181) by electroporation and selection
for acquisition of the plasmid-borne yeast TRP1.sup.+ gene on media
lacking tryptophan. To assess secretion of the receptor directed by
pIXY432, the plasmid was introduced into the yeast strain PB149-6b
by electroporation followed by selection for the plasmid-borne
URA3.sup.+ gene with growth on media lacking uracil. Overnight
cultures were grown at 30.degree. C. in the appropriate selective
media. The PB149-6b/pIXY434 transformants were diluted into YEP-1%
glucose media and grown at 30.degree. C. for 38-40 hours.
Supernatants were prepared by removal of cells by centrifugation,
and filtration of supernatants through 0.45.mu. filters.
[0100] The level of secreted receptor in the supernatants was
determined by immuno-dotblot. Briefly, 1 ul of supernatants, and
dilutions of the supernatants, were spotted onto nitrocellulose
filters and allowed to dry. After blocking non-specific protein
binding with a 3% BSA solution, the filters were incubated with
diluted M1 anti-FLAG.RTM. antibody, excess antibody was removed by
washing and then dilutions of horseradish peroxidase conjugated
anti-mouse IgG antibodies were incubated with the filters. After
removal of excess secondary antibodies, peroxidase substrates were
added and color development was allowed to proceed for
approximately 10 minutes prior to removal of the substrate
solution.
[0101] The anti-FLAG.RTM. reactive material found in the
supernatants demonstrated that significant levels of receptor were
secreted by both expression systems. Comparisons demonstrated that
the pIXY432 system secreted approximately 8-16 times more soluble
human TNF receptor than the pIXY424 system. The supernatants were
assayed for soluble TNF-R activity, as described in Example 1, by
their ability to bind .sup.125I-TNF.alpha. and block TNF.alpha.
binding. The pIXY432 supernatants were found to contain significant
levels of active soluble TNF-R.
Example 9
Isolation of Murine TNF-R cDNAs
[0102] Murine TNF-R cDNAs were isolated from a cDNA library made
from murine 7B9 cells, an antigen-dependent helper T cell line
derived from C57BL/6 mice, by cross-species hybridization with a
human TNF-R probe. The cDNA library was constructed in .lamda.ZAP
(Stratagene, San Diego), substantially as described above in
Example 2, by isolating polyadenylated RNA from the 7B9 cells.
[0103] A double-stranded human TNF-R cDNA probe was produced by
excising an approximately 3.5 kb Not1 fragment of the human TNF-R
clone 1 and .sup.32P-labeling the cDNA using random primers
(Boehringer-Mannheim).
[0104] The murine cDNA library was amplified once and a total of
900,000 plaques were screened, substantially as described in
Example 2, with the human TNF-R cDNA probe. Approximately 21
positive plaques were purified, and the Bluescript plasmids
containing EcoR1-linkered inserts were excised (Stratagene, San
Diego). Nucleic acid sequencing of a portion of murine TNF-R clone
11 indicated that the coding sequence of the murine TNF-R was
approximately 88% homologous to the corresponding nucleotide
sequence of human TNF-R. A partial nucleotide sequence of murine
TNF-R cDNA clone 11 is set forth in SEQ ID NO:3.
Example 10
Preparation of Monoclonal Antibodies to TNF-R
[0105] Preparations of purified recombinant TNF-R, for example,
human TNF-R, or transfected COS cells expressing high levels of
TNF-R are employed to generate monoclonal antibodies against TNF-R
using conventional techniques, for example, those disclosed in U.S.
Pat. No. 4,411,993. Such antibodies are likely to be useful in
interfering with TNF binding to TNF receptors, for example, in
ameliorating toxic or other undesired effects of TNF, or as
components of diagnostic or research assays for TNF or soluble TNF
receptor.
[0106] To immunize mice, TNF-R immunogen is emulsified in complete
Freund's adjuvant and injected in amounts ranging from 10-100 .mu.g
subcutaneously into Balb/c mice. Ten to twelve days later, the
immunized animals are boosted with additional immunogen emulsified
in incomplete Freund's adjuvant and periodically boosted thereafter
on a weekly to biweekly immunization schedule. Serum samples are
periodically taken by retro-orbital bleeding or tail-tip excision
for testing by dot-blot assay (antibody sandwich) or ELISA
(enzyme-linked immunosorbent assay). Other assay procedures are
also suitable. Following detection of an appropriate antibody
titer, positive animals are given an intravenous injection of
antigen in saline. Three to four days later, the animals are
sacrificed, splenocytes harvested, and fused to the murine myeloma
cell line NS1. Hybridoma cell lines generated by this procedure are
plated in multiple microtiter plates in a HAT selective medium
(hypoxanthine, aminopterin, and thymidine) to inhibit proliferation
of non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0107] Hybridoma clones thus generated can be screened by ELISA for
reactivity with TNF R, for example, by adaptations of the
techniques disclosed by Engvall et al., Immunochem. 8: 871 (1971)
and in U.S. Pat. No. 4,703,004. Positive clones are then injected
into the peritoneal cavities of syngeneic Balb/c mice to produce
ascites containing high concentrations (>1 mg/ml) of anti-TNF-R
monoclonal antibody. The resulting monoclonal antibody can be
purified by ammonium sulfate precipitation followed by gel
exclusion chromatography, and/or affinity chromatography based on
binding of antibody to Protein A of Staphylococcus aureus.
DETAILED DESCRIPTION OF THE SEQUENCE LISTING
[0108] SEQ ID NO:1 and SEQ ID NO:2 show the partial cDNA sequence
and derived amino acid sequence of the human TNF-R clone 1.
Nucleotides are numbered from the beginning of the 5' untranslated
region. Amino acids are numbered from the beginning of the signal
peptide sequence. The putative signal sequence is represented by
amino acids -22 to -1. The N-terminus of the mature TNF-R begins
with amino acid 1. The predicted transmembrane region extends from
amino acids 236-265.
[0109] SEQ ID NO:3 and SEQ ID NO:4 show the cDNA sequence and
derived amino acid sequence of the murine TNF-R clone 11. The
putative signal peptide sequence is represented by amino acids -22
to -1. The N-terminus of the mature TNF-R begins with amino acid 1.
The predicted transmembrane region extends from amino acids
234-265.
Sequence CWU 1
1
2211641DNAHomo
sapiensCDS(88)..(1470)sig_peptide(88)..(153)mat_peptide(154)..(1470)
1gcgaggcagg cagcctggag agaaggcgct gggctgcgag ggcgcgaggg cgcgagggca
60gggggcaacc ggaccccgcc cgcatcc atg gcg ccc gtc gcc gtc tgg gcc gcg
114 Met Ala Pro Val Ala Val Trp Ala Ala -20 -15ctg gcc gtc gga ctg
gag ctc tgg gct gcg gcg cac gcc ttg ccc gcc 162Leu Ala Val Gly Leu
Glu Leu Trp Ala Ala Ala His Ala Leu Pro Ala -10 -5 -1 1cag gtg gca
ttt aca ccc tac gcc ccg gag ccc ggg agc aca tgc cgg 210Gln Val Ala
Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser Thr Cys Arg 5 10 15ctc aga
gaa tac tat gac cag aca gct cag atg tgc tgc agc aaa tgc 258Leu Arg
Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys Ser Lys Cys20 25 30
35tcg ccg ggc caa cat gca aaa gtc ttc tgt acc aag acc tcg gac acc
306Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr Ser Asp Thr
40 45 50gtg tgt gac tcc tgt gag gac agc aca tac acc cag ctc tgg aac
tgg 354Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp Asn
Trp 55 60 65gtt ccc gag tgc ttg agc tgt ggc tcc cgc tgt agc tct gac
cag gtg 402Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp
Gln Val 70 75 80gaa act caa gcc tgc act cgg gaa cag aac cgc atc tgc
acc tgc agg 450Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys
Thr Cys Arg 85 90 95ccc ggc tgg tac tgc gcg ctg agc aag cag gag ggg
tgc cgg ctg tgc 498Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly
Cys Arg Leu Cys100 105 110 115gcg ccg ctg cgc aag tgc cgc ccg ggc
ttc ggc gtg gcc aga cca gga 546Ala Pro Leu Arg Lys Cys Arg Pro Gly
Phe Gly Val Ala Arg Pro Gly 120 125 130act gaa aca tca gac gtg gtg
tgc aag ccc tgt gcc ccg ggg acg ttc 594Thr Glu Thr Ser Asp Val Val
Cys Lys Pro Cys Ala Pro Gly Thr Phe 135 140 145tcc aac acg act tca
tcc acg gat att tgc agg ccc cac cag atc tgt 642Ser Asn Thr Thr Ser
Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys 150 155 160aac gtg gtg
gcc atc cct ggg aat gca agc atg gat gca gtc tgc acg 690Asn Val Val
Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr 165 170 175tcc
acg tcc ccc acc cgg agt atg gcc cca ggg gca gta cac tta ccc 738Ser
Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu Pro180 185
190 195cag cca gtg tcc aca cga tcc caa cac acg cag cca act cca gaa
ccc 786Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu
Pro 200 205 210agc act gct cca agc acc tcc ttc ctg ctc cca atg ggc
ccc agc ccc 834Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly
Pro Ser Pro 215 220 225cca gct gaa ggg agc act ggc gac ttc gct ctt
cca gtt gga ctg att 882Pro Ala Glu Gly Ser Thr Gly Asp Phe Ala Leu
Pro Val Gly Leu Ile 230 235 240gtg ggt gtg aca gcc ttg ggt cta cta
ata ata gga gtg gtg aac tgt 930Val Gly Val Thr Ala Leu Gly Leu Leu
Ile Ile Gly Val Val Asn Cys 245 250 255gtc atc atg acc cag gtg aaa
aag aag ccc ttg tgc ctg cag aga gaa 978Val Ile Met Thr Gln Val Lys
Lys Lys Pro Leu Cys Leu Gln Arg Glu260 265 270 275gcc aag gtg cct
cac ttg cct gcc gat aag gcc cgg ggt aca cag ggc 1026Ala Lys Val Pro
His Leu Pro Ala Asp Lys Ala Arg Gly Thr Gln Gly 280 285 290ccc gag
cag cag cac ctg ctg atc aca gcg ccg agc tcc agc agc agc 1074Pro Glu
Gln Gln His Leu Leu Ile Thr Ala Pro Ser Ser Ser Ser Ser 295 300
305tcc ctg gag agc tcg gcc agt gcg ttg gac aga agg gcg ccc act cgg
1122Ser Leu Glu Ser Ser Ala Ser Ala Leu Asp Arg Arg Ala Pro Thr Arg
310 315 320aac cag cca cag gca cca ggc gtg gag gcc agt ggg gcc ggg
gag gcc 1170Asn Gln Pro Gln Ala Pro Gly Val Glu Ala Ser Gly Ala Gly
Glu Ala 325 330 335cgg gcc agc acc ggg agc tca gat tct tcc cct ggt
ggc cat ggg acc 1218Arg Ala Ser Thr Gly Ser Ser Asp Ser Ser Pro Gly
Gly His Gly Thr340 345 350 355cag gtc aat gtc acc tgc atc gtg aac
gtc tgt agc agc tct gac cac 1266Gln Val Asn Val Thr Cys Ile Val Asn
Val Cys Ser Ser Ser Asp His 360 365 370agc tca cag tgc tcc tcc caa
gcc agc tcc aca atg gga gac aca gat 1314Ser Ser Gln Cys Ser Ser Gln
Ala Ser Ser Thr Met Gly Asp Thr Asp 375 380 385tcc agc ccc tcg gag
tcc ccg aag gac gag cag gtc ccc ttc tcc aag 1362Ser Ser Pro Ser Glu
Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Lys 390 395 400gag gaa tgt
gcc ttt cgg tca cag ctg gag acg cca gag acc ctg ctg 1410Glu Glu Cys
Ala Phe Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu Leu 405 410 415ggg
agc acc gaa gag aag ccc ctg ccc ctt gga gtg cct gat gct ggg 1458Gly
Ser Thr Glu Glu Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly420 425
430 435atg aag ccc agt taaccaggcc ggtgtgggct gtgtcgtagc caaggtgggc
1510Met Lys Pro Sertgagccctgg caggatgacc ctgcgaaggg gccctggtcc
ttccaggccc ccaccactag 1570gactctgagg ctctttctgg gccaagttcc
tctagtgccc tccacagccg cagcctccct 1630ctgacctgca g 16412461PRTHomo
sapiens 2Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu
Glu Leu -20 -15 -10Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala
Phe Thr Pro Tyr -5 -1 1 5 10Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu
Arg Glu Tyr Tyr Asp Gln 15 20 25Thr Ala Gln Met Cys Cys Ser Lys Cys
Ser Pro Gly Gln His Ala Lys 30 35 40Val Phe Cys Thr Lys Thr Ser Asp
Thr Val Cys Asp Ser Cys Glu Asp 45 50 55Ser Thr Tyr Thr Gln Leu Trp
Asn Trp Val Pro Glu Cys Leu Ser Cys 60 65 70Gly Ser Arg Cys Ser Ser
Asp Gln Val Glu Thr Gln Ala Cys Thr Arg75 80 85 90Glu Gln Asn Arg
Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 95 100 105Ser Lys
Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg 110 115
120Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val
125 130 135Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser
Ser Thr 140 145 150Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val
Ala Ile Pro Gly155 160 165 170Asn Ala Ser Met Asp Ala Val Cys Thr
Ser Thr Ser Pro Thr Arg Ser 175 180 185Met Ala Pro Gly Ala Val His
Leu Pro Gln Pro Val Ser Thr Arg Ser 190 195 200Gln His Thr Gln Pro
Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser 205 210 215Phe Leu Leu
Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly 220 225 230Asp
Phe Ala Leu Pro Val Gly Leu Ile Val Gly Val Thr Ala Leu Gly235 240
245 250Leu Leu Ile Ile Gly Val Val Asn Cys Val Ile Met Thr Gln Val
Lys 255 260 265Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val Pro
His Leu Pro 270 275 280Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu
Gln Gln His Leu Leu 285 290 295Ile Thr Ala Pro Ser Ser Ser Ser Ser
Ser Leu Glu Ser Ser Ala Ser 300 305 310Ala Leu Asp Arg Arg Ala Pro
Thr Arg Asn Gln Pro Gln Ala Pro Gly315 320 325 330Val Glu Ala Ser
Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser 335 340 345Asp Ser
Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile 350 355
360Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln
365 370 375Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu
Ser Pro 380 385 390Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys
Ala Phe Arg Ser395 400 405 410Gln Leu Glu Thr Pro Glu Thr Leu Leu
Gly Ser Thr Glu Glu Lys Pro 415 420 425Leu Pro Leu Gly Val Pro Asp
Ala Gly Met Lys Pro Ser 430 43533813DNAMus
musculusCDS(55)..(1476)sig_peptide(55)..(120)mat_peptide(121)..(1476)
3cgcagctgag gcactagagc tccaggcaca agggcgggag ccaccgctgc ccct atg 57
Metgcg ccc gcc gcc ctc tgg gtc gcg ctg gtc ttc gaa ctg cag ctg tgg
105Ala Pro Ala Ala Leu Trp Val Ala Leu Val Phe Glu Leu Gln Leu Trp
-20 -15 -10gcc acc ggg cac aca gtg ccc gcc cag gtt gtc ttg aca ccc
tac aaa 153Ala Thr Gly His Thr Val Pro Ala Gln Val Val Leu Thr Pro
Tyr Lys-5 -1 1 5 10ccg gaa cct ggg tac gag tgc cag atc tca cag gaa
tac tat gac agg 201Pro Glu Pro Gly Tyr Glu Cys Gln Ile Ser Gln Glu
Tyr Tyr Asp Arg 15 20 25aag gct cag atg tgc tgt gct aag tgt cct cct
ggc caa tat gtg aaa 249Lys Ala Gln Met Cys Cys Ala Lys Cys Pro Pro
Gly Gln Tyr Val Lys 30 35 40cat ttc tgc aac aag acc tcg gac acc gtg
tgt gcg gac tgt gag gca 297His Phe Cys Asn Lys Thr Ser Asp Thr Val
Cys Ala Asp Cys Glu Ala 45 50 55agc atg tat acc cag gtc tgg aac cag
ttt cgt aca tgt ttg agc tgc 345Ser Met Tyr Thr Gln Val Trp Asn Gln
Phe Arg Thr Cys Leu Ser Cys60 65 70 75agt tct tcc tgt acc act gac
cag gtg gag atc cgc gcc tgc act aaa 393Ser Ser Ser Cys Thr Thr Asp
Gln Val Glu Ile Arg Ala Cys Thr Lys 80 85 90cag cag aac cga gtg tgt
gct tgc gaa gct ggc agg tac tgc gcc ttg 441Gln Gln Asn Arg Val Cys
Ala Cys Glu Ala Gly Arg Tyr Cys Ala Leu 95 100 105aaa acc cat tct
ggc agc tgt cga cag tgc atg agg ctg agc aag tgc 489Lys Thr His Ser
Gly Ser Cys Arg Gln Cys Met Arg Leu Ser Lys Cys 110 115 120ggc cct
ggc ttc gga gtg gcc agt tca aga gcc cca aat gga aat gtg 537Gly Pro
Gly Phe Gly Val Ala Ser Ser Arg Ala Pro Asn Gly Asn Val 125 130
135cta tgc aag gcc tgt gcc cca ggg acg ttc tct gac acc aca tca tcc
585Leu Cys Lys Ala Cys Ala Pro Gly Thr Phe Ser Asp Thr Thr Ser
Ser140 145 150 155act gat gtg tgc agg ccc cac cgc atc tgt agc atc
ctg gct att ccc 633Thr Asp Val Cys Arg Pro His Arg Ile Cys Ser Ile
Leu Ala Ile Pro 160 165 170gga aat gca agc aca gat gca gtc tgt gcg
ccc gag tcc cca act cta 681Gly Asn Ala Ser Thr Asp Ala Val Cys Ala
Pro Glu Ser Pro Thr Leu 175 180 185agt gcc atc cca agg aca ctc tac
gta tct cag cca gag ccc aca aga 729Ser Ala Ile Pro Arg Thr Leu Tyr
Val Ser Gln Pro Glu Pro Thr Arg 190 195 200tcc caa ccc ctg gat caa
gag cca ggg ccc agc caa act cca agc atc 777Ser Gln Pro Leu Asp Gln
Glu Pro Gly Pro Ser Gln Thr Pro Ser Ile 205 210 215ctt aca tcg ttg
ggt tca acc ccc att att gaa caa agt acc aag ggt 825Leu Thr Ser Leu
Gly Ser Thr Pro Ile Ile Glu Gln Ser Thr Lys Gly220 225 230 235ggc
atc tct ctt cca att ggt ctg att gtt gga gtg aca tca ctg ggt 873Gly
Ile Ser Leu Pro Ile Gly Leu Ile Val Gly Val Thr Ser Leu Gly 240 245
250ctg ctg atg tta gga ctg gtg aac tgc atc atc ctg gtg cag agg aaa
921Leu Leu Met Leu Gly Leu Val Asn Cys Ile Ile Leu Val Gln Arg Lys
255 260 265aag aag ccc tcc tgc cta caa aga gat gcc aag gtg cct cat
gtg cct 969Lys Lys Pro Ser Cys Leu Gln Arg Asp Ala Lys Val Pro His
Val Pro 270 275 280gat gag aaa tcc cag gat gca gta ggc ctt gag cag
cag cac ctg ttg 1017Asp Glu Lys Ser Gln Asp Ala Val Gly Leu Glu Gln
Gln His Leu Leu 285 290 295acc aca gca ccc agt tcc agc agc agc tcc
cta gag agc tca gcc agc 1065Thr Thr Ala Pro Ser Ser Ser Ser Ser Ser
Leu Glu Ser Ser Ala Ser300 305 310 315gct ggg gac cga agg gcg ccc
cct ggg ggc cat ccc caa gca aga gtc 1113Ala Gly Asp Arg Arg Ala Pro
Pro Gly Gly His Pro Gln Ala Arg Val 320 325 330atg gcg gag gcc caa
ggg ttt cag gag gcc cgt gcc agc tcc agg att 1161Met Ala Glu Ala Gln
Gly Phe Gln Glu Ala Arg Ala Ser Ser Arg Ile 335 340 345tca gat tct
tcc cac gga agc cac ggg acc cac gtc aac gtc acc tgc 1209Ser Asp Ser
Ser His Gly Ser His Gly Thr His Val Asn Val Thr Cys 350 355 360atc
gtg aac gtc tgt agc agc tct gac cac agt tct cag tgc tct tcc 1257Ile
Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser 365 370
375caa gcc agc gcc aca gtg gga gac cca gat gcc aag ccc tca gcg tcc
1305Gln Ala Ser Ala Thr Val Gly Asp Pro Asp Ala Lys Pro Ser Ala
Ser380 385 390 395cca aag gat gag cag gtc ccc ttc tct cag gag gag
tgt ccg tct cag 1353Pro Lys Asp Glu Gln Val Pro Phe Ser Gln Glu Glu
Cys Pro Ser Gln 400 405 410tcc ccg tgt gag act aca gag aca ctg cag
agc cat gag aag ccc ttg 1401Ser Pro Cys Glu Thr Thr Glu Thr Leu Gln
Ser His Glu Lys Pro Leu 415 420 425ccc ctt ggt gtg ccg gat atg ggc
atg aag ccc agc caa gct ggc tgg 1449Pro Leu Gly Val Pro Asp Met Gly
Met Lys Pro Ser Gln Ala Gly Trp 430 435 440ttt gat cag att gca gtc
aaa gtg gcc tgacccctga caggggtaac 1496Phe Asp Gln Ile Ala Val Lys
Val Ala 445 450accctgcaaa gggacccccg agaccctgaa cccatggaac
ttcatgactt ttgctggatc 1556catttccctt agtggcttcc agagccccag
ttgcaggtca agtgagggct gagacagcta 1616gagtggtcaa aaactgccat
ggtgttttat gggggcagtc ccaggaagtt gttgctcttc 1676catgacccct
ctggatctcc tgggctcttg cctgattctt gcttctgaga ggccccagta
1736ttttttcctt ctaaggagct aacatcctct tccatgaata gcacagctct
tcagcctgaa 1796tgctgacact gcagggcggt tccagcaagt aggagcaagt
ggtggcctgg tagggcacag 1856aggcccttca ggttagtgct aaactcttag
gaagtaccct ctccaagccc accgaaattc 1916ttttgatgca agaatcagag
gccccatcag gcagagttgc tctgttatag gatggtaggg 1976ctgtaactca
gtggtccagt gtgcttttag catgccctgg gtttgatcct cagcaacaca
2036tgcaaaacgt aagtagacag cagacagcag acagcacagc cagccccctg
tgtggtttgc 2096agcctctgcc tttgactttt actctggtgg gcacacagag
ggctggagct cctcctcctg 2156accttctaat gagcccttcc aaggccacgc
cttccttcag ggaatctcag ggactgtaga 2216gttcccaggc ccctgcagcc
acctgtctct tcctacctca gcctggagca ctccctctaa 2276ctccccaacg
gcttggtact gtacttgctg tgaccccaac gtgcattgtc cgggttaggc
2336actgtgagtt ggaacagctc atgacatcgg ttgaaaggcc cacccggaaa
cagctaagcc 2396agctcttttg ccaaaggatt catgccggtt ttctaatcaa
cctgctccct agcattgcct 2456ggaaggaaag ggttcaggag actcctcaag
aagcaagttc agtctcaggt gcttggatgc 2516catgctcacc gattccactg
gatatgaact tggcagagga gcctagttgt tgccatggag 2576acttaaagag
ctcagcactc tggaatcaag atactggaca cttggggccg acttgttaag
2636gctctgcagc atcagactgt agaggggaag gaacacgtct gccccctggt
ggcccgtcct 2696gggatgacct cgggcctcct aggcaacaaa agaatgaatt
ggaaaggatg ttcctgggtg 2756tggcctagct cctgtgcttg tgtggatccc
taaagggtgt gctaaggagc aattgcactg 2816tgtgctggac agaattcctg
cttataaatg ctttttgttg ttgttttgta cactgagccc 2876tggctgagcc
accccacccc acctcccatc ccacctttac acgccactct tgcatgagaa
2936cctggctgtc tcccacttgt agcctgtgga tgctgaggaa acacccagcc
aagtagactc 2996caggcttgcc cctatctcct gctatgagtc tggcctcctc
attgtgttgt gggaaggaga 3056cgggttctgt catctcggaa cgcccacacc
gtggatgtga acaatggctg tactagctta 3116gaccagctta gggctctgca
tatcacagga gggggagcag ggaacaattt gagtgctgac 3176ctataacaca
gttcctaaag gatcgggcag tccagaatct cctccttcag tgtgtgtgtg
3236tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtccatgtt tgcatgtatg
tgtgtgccag 3296tgtgtggagg cccgaggttg gctttgggtg tgtttgatca
ctctccagtt actgaggcgg 3356gctctcatct gtacccagag cttgcacatt
ttctagtcta acttgattca gggatctctg 3416tctgcctatg gaggtgctca
ggttacaggc aggctgccat acctgcccga catttacatg 3476aatactagag
atctgaattc tggtcctcac acttgtatac ctgcatttta tccactaaga
3536catctctcca agggctcccc cttcctattt aataagttag ttttgaactg
gcaagatggc 3596tcagtgggta aggcagtttg cggacaaacc tgatgacctg
agttggatcc ctgaccataa 3656ggtagaagag acctgattcc tgcaagttgt
cctctgacca ccaccccata catgcttctg 3716catatgtgca cacatcacat
tcttgcacac acactcacat accataaatg taataaattt 3776ttttaaataa
attgatttta tcttttaaaa aaaaaaa 38134474PRTMus musculus 4Met Ala Pro
Ala Ala Leu Trp Val Ala Leu Val Phe
Glu Leu Gln Leu -20 -15 -10Trp Ala Thr Gly His Thr Val Pro Ala Gln
Val Val Leu Thr Pro Tyr -5 -1 1 5 10Lys Pro Glu Pro Gly Tyr Glu Cys
Gln Ile Ser Gln Glu Tyr Tyr Asp 15 20 25Arg Lys Ala Gln Met Cys Cys
Ala Lys Cys Pro Pro Gly Gln Tyr Val 30 35 40Lys His Phe Cys Asn Lys
Thr Ser Asp Thr Val Cys Ala Asp Cys Glu 45 50 55Ala Ser Met Tyr Thr
Gln Val Trp Asn Gln Phe Arg Thr Cys Leu Ser 60 65 70Cys Ser Ser Ser
Cys Thr Thr Asp Gln Val Glu Ile Arg Ala Cys Thr75 80 85 90Lys Gln
Gln Asn Arg Val Cys Ala Cys Glu Ala Gly Arg Tyr Cys Ala 95 100
105Leu Lys Thr His Ser Gly Ser Cys Arg Gln Cys Met Arg Leu Ser Lys
110 115 120Cys Gly Pro Gly Phe Gly Val Ala Ser Ser Arg Ala Pro Asn
Gly Asn 125 130 135Val Leu Cys Lys Ala Cys Ala Pro Gly Thr Phe Ser
Asp Thr Thr Ser 140 145 150Ser Thr Asp Val Cys Arg Pro His Arg Ile
Cys Ser Ile Leu Ala Ile155 160 165 170Pro Gly Asn Ala Ser Thr Asp
Ala Val Cys Ala Pro Glu Ser Pro Thr 175 180 185Leu Ser Ala Ile Pro
Arg Thr Leu Tyr Val Ser Gln Pro Glu Pro Thr 190 195 200Arg Ser Gln
Pro Leu Asp Gln Glu Pro Gly Pro Ser Gln Thr Pro Ser 205 210 215Ile
Leu Thr Ser Leu Gly Ser Thr Pro Ile Ile Glu Gln Ser Thr Lys 220 225
230Gly Gly Ile Ser Leu Pro Ile Gly Leu Ile Val Gly Val Thr Ser
Leu235 240 245 250Gly Leu Leu Met Leu Gly Leu Val Asn Cys Ile Ile
Leu Val Gln Arg 255 260 265Lys Lys Lys Pro Ser Cys Leu Gln Arg Asp
Ala Lys Val Pro His Val 270 275 280Pro Asp Glu Lys Ser Gln Asp Ala
Val Gly Leu Glu Gln Gln His Leu 285 290 295Leu Thr Thr Ala Pro Ser
Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala 300 305 310Ser Ala Gly Asp
Arg Arg Ala Pro Pro Gly Gly His Pro Gln Ala Arg315 320 325 330Val
Met Ala Glu Ala Gln Gly Phe Gln Glu Ala Arg Ala Ser Ser Arg 335 340
345Ile Ser Asp Ser Ser His Gly Ser His Gly Thr His Val Asn Val Thr
350 355 360Cys Ile Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln
Cys Ser 365 370 375Ser Gln Ala Ser Ala Thr Val Gly Asp Pro Asp Ala
Lys Pro Ser Ala 380 385 390Ser Pro Lys Asp Glu Gln Val Pro Phe Ser
Gln Glu Glu Cys Pro Ser395 400 405 410Gln Ser Pro Cys Glu Thr Thr
Glu Thr Leu Gln Ser His Glu Lys Pro 415 420 425Leu Pro Leu Gly Val
Pro Asp Met Gly Met Lys Pro Ser Gln Ala Gly 430 435 440Trp Phe Asp
Gln Ile Ala Val Lys Val Ala 445 450530DNAArtificial
Sequence3'-Terminus of human TNF receptor including restriction
endonuclease cleavage sites 5ctgaagggag cactggcgac taaggatcca
30634DNAArtificial Sequence3'-Terminus of human TNF receptor
including restriction endonuclease cleavage sites 6gacttccctc
gtgaccgctg attcctaggt ctag 3477PRTArtificial
SequenceCarboxy-Terminus of human TNF receptor 7Ala Glu Gly Ser Thr
Gly Asp1 5845DNAArtificial Sequence3'-Terminus of human TNF
receptor including restriction endonuclease cleavage sites
8gatctgtaac gtggtggcca tccctgggaa tgcaagcatg gatgc
45933DNAArtificial Sequence3'-Terminus of human TNF receptor
including restriction endonuclease cleavage sites 9acattgcacc
accggtaggg acccttacgt tcg 331015PRTArtificial
SequenceCarboxy-Terminus of human TNF receptor 10Ile Cys Asn Val
Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala1 5 10
151133DNAArtificial Sequence3'-Terminus of human TNF receptor
including restriction endonuclease cleavage sites 11agtctgcacg
tccacgtccc ccacccggtg agc 331245DNAArtificial Sequence3'-Terminus
of human TNF receptor including restriction endonuclease cleavage
sites 12tacctacgtc agacgtgcag gtgcaggggg tgggccactc gccgg
45139PRTArtificial SequenceCarboxy-Terminus of human TNF receptor
13Val Cys Thr Ser Thr Ser Pro Thr Arg1 51412DNAArtificial
Sequence3'-Terminus of human TNF receptor including restriction
endonuclease cleavage sites 14gatctgttga gc 121512DNAArtificial
Sequence3'-Terminus of human TNF receptor including restriction
endonuclease cleavage sites 15acaactcgcc gg 121636DNAArtificial
Sequence3'-Terminus of human TNF receptor including restriction
endonuclease cleavage sites 16ctgaaacatc agacgtggtg tgcaagccct
gttaaa 361744DNAArtificial Sequence3'-Terminus of human TNF
receptor including restriction endonuclease cleavage sites
17cttgactttg tagtctgcac cacacgttcg ggacaatttc taga
441871DNAArtificial SequencePCR Primer 18ttccggtacc tttggataaa
agagactaca aggacgacga tgacaagttg cccgcccagg 60tggcatttac a
711913PRTArtificial SequencePCR Primer 19Pro Leu Asp Lys Arg Asp
Tyr Lys Asp Asp Asp Asp Lys1 5 102038DNAArtificial SequencePCR
Primer 20cccgggatcc ttagtcgcca gtgctccctt cagctggg
38215PRTArtificial SequenceYeast alpha-factor leader sequence 21Pro
Leu Asp Lys Arg1 5228PRTArtificial SequenceFLAG peptide 22Asp Thr
Lys Asp Asp Asp Asp Lys1 5
* * * * *