U.S. patent application number 09/877156 was filed with the patent office on 2002-05-09 for members of tnf and tnfr families.
Invention is credited to Tribouley, Catherine.
Application Number | 20020055625 09/877156 |
Document ID | / |
Family ID | 23099033 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020055625 |
Kind Code |
A1 |
Tribouley, Catherine |
May 9, 2002 |
Members of TNF and TNFR families
Abstract
New members of the TNF and the TNFR superfamily of proteins have
been identified. These proteins are promising targets for
therapeutic intervention and mimesis. TNF-L and TNFR-L proteins can
be used to induce cell death and/or proliferation of cells. Members
of these superfamilies have been implicated in a broad variety of
disease processes, making them central biological and physiological
regulators.
Inventors: |
Tribouley, Catherine; (San
Francisco, CA) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property R338
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Family ID: |
23099033 |
Appl. No.: |
09/877156 |
Filed: |
June 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09877156 |
Jun 8, 2001 |
|
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09286529 |
Apr 5, 1999 |
|
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60068959 |
Dec 30, 1997 |
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Current U.S.
Class: |
536/23.5 ; 435/4;
530/351 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 14/70575 20130101; C07K 14/70578 20130101; C07K 2319/00
20130101; C07K 2319/02 20130101 |
Class at
Publication: |
536/23.5 ;
530/351; 435/4 |
International
Class: |
C07H 021/04; C12Q
001/00; C07K 014/525 |
Claims
1. An isolated human protein having an amino acid sequence which is
at least 85% identical to an amino acid sequence selected from the
group consisting of SEQ ID NOS:1, 2, 17, and 20, wherein percent
identity is determined using a Smith-Waterman homology search
algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 1.
2. The isolated human protein of claim 1 which has an amino acid
sequence selected from the group consisting of SEQ ID NOS:1, 2, 17,
and 20.
3. A fusion protein comprising a first protein segment and a second
protein segment fused together by means of a peptide bond, wherein
the first protein segment consists of a protein having an amino
acid sequence selected from the group consisting of SEQ ID NOS:1,
2, and 17.
4. A preparation of antibodies which specifically bind to a protein
having an amino acid sequence selected from the group consisting of
SEQ ID NOS:1, 2, 17, and 20.
5. A cDNA molecule which encodes a protein having an amino acid
sequence which is at least 85% identical to an amino acid sequence
selected from the group consisting of SEQ ID NOS:1, 2, 17, and 20,
wherein percent identity is determined using a Smith-Waterman
homology search algorithm using an affine gap search with a gap
open penalty of 12 and a gap extension penalty of 1.
6. The cDNA molecule of claim 5 which encodes an amino acid
sequence selected from the group consisting of SEQ ID NOS:1, 2, 17,
and 20.
7. The cDNA molecule of claim 6 which comprises a nucleotide
sequence selected from the group consisting of SEQ ID NOS:6, 7, 18
and 19.
8. A cDNA molecule which is at least 85% identical to a nucleotide
sequence selected from the group consisting of SEQ ID NOS:6, 7, 18,
and 19, wherein percent identity is determined using a
Smith-Waterman homology search algorithm using an affine gap search
with a gap open penalty of 12 and a gap extension penalty of 1.
9. An isolated and purified subgenomic polynucleotide comprising a
nucleotide sequence which hybridizes to a nucleotide sequence
selected from the group consisting of SEQ ID NOS:6, 7, 18, and 19
after washing with 0.2.times.SSC at 65.degree. C., wherein the
nucleotide sequence encodes a protein having an amino acid sequence
selected from the group consisting of SEQ ID NOS:1, 2, and 17.
10. A construct comprising. a promoter; and a polynucleotide
segment encoding an amino acid sequence selected from the group
consisting of SEQ ID NOS:1, 2, 17, and 20, wherein the
polynucleotide segment is located downstream from the promoter,
wherein transcription of the polynucleotide segment initiates at
the promoter.
11. A host cell comprising a construct which comprises: a promoter;
and a polynucleotide segment encoding an amino acid sequence
selected from the group consisting of SEQ ID NOS:1, 2, 17, and
20.
12. A recombinant host cell comprising a new transcription
initiation unit, wherein the new transcription initiation unit
comprises in 5' to 3' order: (a) an exogenous regulatory sequence,
(b) an exogenous exon; and (c) a splice donor site, wherein the new
transcription initiation unit is located upstream of a coding
sequence of a gene, wherein the coding sequence is selected from
the group consisting of SEQ ID NOS:6, 7, 18, and 19, wherein the
exogenous regulatory sequence controls transcription of the coding
sequence of the gene.
13. A method of screening for a compound capable of modulating cell
death inducing activity of a protein, comprising the steps of:
incubating a first population of cells and a protein in the
presence of a test compound, wherein the protein comprises an amino
acid sequence selected from the group of amino acid sequences shown
in SEQ ID NOS:1-5, 17, and 20; incubating a second population of
cells and the protein in the absence of a test compound; and
determining viability of the first and second populations, wherein
a test compound which increases or decreases viability of the first
population relative to the second population is identified as
capable of modulating the cell death inducing activity of the
protein.
14. The method of claim 13 wherein the protein is provided to the
first and second populations of cells by transfecting the first and
second populations of cells with a polynucleotide encoding the
protein.
15. The method of claim 13 wherein the polynucleotide comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NOS:6-10, 18, and 19.
16. A method of identifying a binding partner of a first protein,
comprising the steps of: incubating a first protein with a second
protein, wherein the first protein comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, 17, and 20;
detecting formation of a complex between the first and second
proteins, wherein formation of the complex identifies the second
protein as a binding partner of the first protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of application Ser. No.
60/068,959 filed Dec. 30, 1997, and of application Ser. No.
09/212,270, filed Dec. 16, 1998, both of which are incorporated by
reference herein.
BACKGROUND OF THE INVENTION
[0002] Tumor necrosis factor (TNF) is a pro-inflammatory cytokine
which is produced by a wide spectrum of cells. It has a key role in
host defense and immunosurveillance, mediating complex cellular
responses. In excess, TNF may have detrimental effects.
[0003] Two specific, high affinity cell surface receptors, p55
TNF-R and p75 TNF-R, function as transducing elements, providing
the intracellular signal for cell responses to TNF. While both
types of TNF receptors are expressed by almost all cell types, the
p75 receptor has been shown to be expressed primarily by cells of
the immune system (B and T cells), cells of myeloid origin, and
endothelial cells. Both receptors participate in the induction of
NF.kappa.B and interleukin-6, in the generation of lymphocyte
activated killer (LAK) cells, and in the proliferation of natural
killer (NK) cells, as well as in anti-proliferation, cytotoxicity,
and apoptosis.
[0004] TNF signaling to cells is largely mediated by the p55 TNF-R,
while the main function of the p75 surface receptor is "ligand
passing," i.e., TNF presentation to the p55 TNF-R. Presence of the
cell surface p75 TNF receptor greatly enhances the rate of
association of TNF to the p55 TNF receptor and may reverse the
desensitization of p55 TNF-R to TNF. Pharmaceutical agents which
affect p75 TNF-R may have a general impact on TNF function,
including those activities in which the major signaling receptor is
the p55 TNF-R.
[0005] The TNF-Rs also mediate many non-overlapping functions: the
p55 receptor is involved in interleukin-2 receptor induction,
anti-viral activities, growth stimulation, HLA antigen expression,
and endothelial cell adhesion, while the p75 receptor mediates the
TNF-induced thymocyte proliferation.
[0006] The p55 and p75 TNF-Rs are members of a superfamily which
includes nerve growth factor receptor (NGFR), Fas antigen, CD27,
CD30, CD40, OX40 and 4-1BB. The cysteine-rich domains of the
extracellular part of these receptors are homologous to several
viral proteins produced by cowpox virus, Shope fibroma virus, and
the myxoma virus.
[0007] Because of the central role of TNF and its receptors in host
defense and immunosurveillance, there is a need in the art to
identify new members of the TNF and TNFR superfamilies
SUMMARY OF THE INVENTION
[0008] It is an object of the invention to provide new members of
the TNF and TNFR families, as well as methods of screening for
compounds capable of modifying the activities of these proteins.
This and other objects of the invention are provided by one or more
of the embodiments described below.
[0009] One embodiment of the invention is an isolated human protein
having an amino acid sequence which is at least 85% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOS:1, 2, 17 and 20. Percent identity is determined using a
Smith-Waterman homology search algorithm using an affine gap search
with a gap open penalty of 12 and a gap extension penalty of 1.
[0010] Another embodiment of the invention is a fusion protein
comprising a first protein segment and a second protein segment
fused together by means of a peptide bond. The first protein
segment consists of a protein having an amino acid sequence
selected from the group consisting of SEQ ID NOS:1, 2, 17 and
20.
[0011] Still another embodiment of the invention is a preparation
of antibodies which specifically bind to a protein having an amino
acid sequence selected from the group consisting of SEQ ID NOS:1,
2, 17 and 20.
[0012] Even another embodiment of the invention is a cDNA molecule
which encodes a protein having an amino acid sequence which is at
least 85% identical to an amino acid sequence selected from the
group consisting of SEQ ID NOS:1, 2, 17 and 20. Percent identity is
determined using a Smith-Waterman homology search algorithm using
an affine gap search with a gap open penalty of 12 and a gap
extension penalty of 1.
[0013] Yet another embodiment of the invention is a cDNA molecule
which is at least 85% identical to a nucleotide sequence selected
from the group consisting of SEQ ID NOS:6, 7, 18 and 19. Percent
identity is determined using a Smith-Waterman homology search
algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 1.
[0014] A further embodiment of the invention is an isolated and
purified subgenomic polynucleotide comprising a nucleotide sequence
which hybridizes to a nucleotide sequence selected from the group
consisting of SEQ ID NOS:6, 7, 18 and 19 after washing with
0.2.times.SSC at 65.degree. C. The nucleotide sequence encodes a
protein having an amino acid sequence selected from the group
consisting of SEQ ID NOS:1, 2, 17and 20.
[0015] Another embodiment of the invention is a construct
comprising a promoter and
[0016] a polynucleotide segment encoding an amino acid sequence
selected from the group consisting of SEQ ID NOS:1, 2, 17 and 20
The polynucleotide segment is located downstream from the promoter.
Transcription of the polynucleotide segment initiates at the
promoter.
[0017] Still another embodiment of the invention is a host cell
comprising a construct which comprises a promoter and a
polynucleotide segment encoding an amino acid sequence selected
from the group consisting of SEQ ID NOS:1, 2, 17 and 20.
[0018] Even another embodiment of the invention is a recombinant
host cell comprising a new transcription initiation unit, wherein
the new transcription initiation unit comprises in 5' to 3' order
(a) an exogenous regulatory sequence, (b) an exogenous exon, and
(c) a splice donor site. The new transcription initiation unit is
located upstream of a coding sequence of a gene. The coding
sequence is selected from the group consisting of SEQ ID NOS:6, 7,
18 and 19. The exogenous regulatory sequence controls transcription
of the coding sequence of the gene.
[0019] Yet another embodiment of the invention is a method of
screening for a compound capable of modulating cell death inducing
activity of a protein. A first population of cells and a protein
are incubated in the presence of a test compound. The protein
comprises an amino acid sequence selected from the group of amino
acid sequences shown in SEQ ID NOS:1-5, 17 and 20. A second
population of cells and the protein are incubated in the absence of
a test compound. Viability of the first and second populations is
determined. A test compound which increases or decreases viability
of the first population relative to the second population is
identified as capable of modulating the cell death inducing
activity of the protein.
[0020] Even another embodiment of the invention is a method of
identifying a binding partner of a first protein. A first protein
is incubated with a second protein. The first protein comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOS:1-5, 17 and 20 Formation of a complex between the first and
second proteins is detected. Formation of the complex identifies
the second protein as a binding partner of the first protein.
[0021] The present invention thus provides the art with the amino
acid sequences of proteins which are new members of the TNF and
TNFR families, as well as nucleotide sequences of polynucleotides
which encode the proteins. These proteins and polynucleotides can
be used to enhance or decrease TNF activities thereby providing
therapeutic benefits, such as induction of cell death, lymphoid
organogenesis, or host bacterial resistance, and inhibition of
endotoxic shock, contact hypersensitivity, delayed type
hypersensitivity, or immunocompetence of a transplant recipient.
Methods of diagnosing neoplasia or predisposition to neoplasia are
also provided. Proteins of the present invention are also useful
for identifying compounds which can regulate the TNF-like or
TNFR-like activities of these proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1. FIG. 1A shows the protein sequence of human TNFL1.
The transmembrane domain is underlined. FIG. 1B shows alignments of
the extracellular domains of several members of the TNF family with
TNFL1. The conserved amino acids are boxed.
[0023] FIG. 2. FIG. 2A shows TNFL1 mRNA expression in human tissues
and cell lines. FIG. 2B shows TNFL1 expression in mouse tissues
PBL, peripheral blood lymphocytes; HL60 promyelocytic leukemia
HL60; HeLa, HeLa cell line S3; K562, chromic myelogenous leukemia
K562; MOLT-4, lymphoblastic leukemia MOLT-4; Raji, Burkitt's
lymphoma Raji; SW480, colorectal adenocarcinoma SW480; A549, lung
carcinoma A549; G361, melanoma G361.
[0024] FIG. 3. FIG. 3 shows detection of TNFL1 expression using a
purified polyclonal anti-peptide antibody.
[0025] FIG. 3A shows a Western blot analysis using the
affinity-purified D2710 antibody at a final concentration of 1
.mu.g/ml. The extracts analyzed were total cell extracts of human
monocytes isolated from PBMCs (lanes 1 and 2) and mouse bone
marrow-derived dendritic cells (lanes 3 and 4). Lanes 5 and 6
contained 20 ng of TNFL1 protein purified from E. coli In lanes 2,
4, and 6 the antibody was pre-incubated with a hundred-fold molar
excess of the peptide used to generate the antibody.
[0026] FIG. 3B shows intracellular staining of baculovirus-infected
cells expressing TNFL1, using the D2710 antibody or a control
rabbit antibody at a concentration of 10 .mu.g/ml. The insect cells
were fixed and permeabilized before staining and flow cytometry
analysis.
[0027] FIG. 3C shows the pattern of expression of TNFL1 in mouse
spleen sections. Left panels: rabbit antibody control plus
secondary antibody; right panels, anti-TNFL1 antibody plus
secondary antibody. The magnification is 100x in the top panels and
200x in the bottom panels. RP, red pulp; PALS, periarteriolar lymph
sheath.
[0028] FIG. 3D shows staining of adjacent murine spleen sections
for Thy-1.2, B220, TNFL1, CD11c, and Mac-3.
[0029] FIG. 4. FIG. 4 shows flow cytometric analysis of the
cell-surface expression of TNFL1 on sub-populations of human PBMCs
and cultured cells.
[0030] FIG. 4A shows expression of TNFL1 on CD14+, CD19+, CD4+, and
CD8+ human PBMCs and on mouse bone marrow-derived dendritic cells.
Human PBMCs were stained with affinity purified D2710, followed by
FITC-conjugated anti-rabbit IgG. They were subsequently stained
with PE-conjugated CD14, CD19, CD4, or CD8. The histograms show
TNFL1 expression (FITC) on PE-positive gated cells. The dotted
lines represent the staining in the absence of primary antibody
D2710.
[0031] FIG. 4B shows upregulation of TNFL1 surface expression on T
cells after activation with anti-CD3 and anti-CD28 at 10 .mu.g/ml
in the presence of IL2 at 50 .mu.g/ml for six days.
[0032] FIG. 5. FIG. 5 shows the effect of recombinant TNFL1 on
activated T and B cells.
[0033] FIG. 5A shows the amino acid sequence of a soluble form of
TNFL1 (sequence enclosed in brackets) which was expressed in E.
coli as a fusion protein and used for all biological assays. The
cleavage sites identified after microsequencing of the truncated
form of the protein are represented as vertical bars.
[0034] FIG. 5B shows inhibition of DNA synthesis in activated but
not in resting T cells by TNFL1. T cells enriched from human PBMCs
were either activated with 10 .mu.g/ml of anti-CD3 and 10 .mu.g/ml
of anti-CD28 (left panel) or untreated (right panel) for 24 hours.
TNFL1 was then added at various concentrations, and
.sup.3H-thymidine incorporation over a period of 8 hours was
measured 48 hours after addition of TNFL1.
[0035] FIG. 5C shows inhibition of DNA synthesis by TNFL1 in
activated B cells and activated T cells. B cells were activated
with 10 .mu.g/ml of anti-CD40 antibody. T cells were activated as
described in FIG. 5B. TNFL1 was added 48 hours after activation.
.sup.3H-thymidine incorporation over a period of 8 hours was
measured 48 hours after addition of TNFL1.
[0036] FIG. 5D shows that TNFL1 induces apoptosis of activated T
cells. T cells were activated with anti-CD3 and anti-CD28 for 48
hours. TNFL1 was added at 2 .mu.g/ml and incubated for an
additional 48 hours. Apoptosis was assessed by TUNEL assay. (R1),
blasting cells; (R2), apoptotic cells; (R3), resting non-apoptotic
cells.
[0037] FIG. 5E shows that TNFL1 induces NF.kappa.B activation in
Jurkat cells. An electrophoretic mobility shift assay was performed
with an NF.kappa.B probe on nuclear cell extracts prepared from
Jurkat cells treated with PMA (1 .mu.g/ml) or TNFL1 (3 .mu.g/ml)
for one hour. Wild-type (wt, 20 ng) and mutated (mut, 100 ng)
non-radiolabeled oligonucleotides were used as competitors in the
reaction.
DETAILED DESCRIPTION OF THE INVENTION
[0038] New members of the TNF and TNFR families are a discovery of
the present invention. In particular, the present invention
identifies two cDNA clones which encode new members of TNF ligand
family and three cDNA clones which encode new members of TNFR
family. Proteins and polynucleotides of the invention provide the
art with diagnostic and therapeutic reagents as well as tools for
discovering other therapeutic agents.
[0039] Two cDNA clones have been identified which show significant
homology to members of TNF family. One cDNA clone (SEQ ID NO:6) was
isolated from a human liver cDNA library. It encodes a protein
designated as TNFL1 (SEQ ID NO:1), which has 27%-30% homology with
TNF and 38% homology with lymphotoxin. TNFL1 contains a
transmembrane domain. The TNFL1 cDNA clone encodes a 3 kb mRNA
which can be detected in tissues associated with the immune system,
such as peripheral blood lymphocytes, spleen, and thymus, as well
as small intestine and ovary. TNFL1 protein is constitutively
expressed on monocytes and B cells isolated from human peripheral
blood lymphocytes, as well as on mouse dendritic cells and in mouse
spleen. Expression of the protein can be up-regulated in natural
killer cells. Activation of dendritic cells, for example with
anti-CD4d antibody, can down-regulate expression of TNFL1
protein.
[0040] TNFL1 shares some common features with other members of the
TNF family TNFL1 is upregulated on activated T cells, as are TNF,
Fas ligand, and CD30 ligand TNFL1 induces activation of NF.kappa.B,
which is also triggered by every member of the TNF-R family with
the exception of Fas and DR4. TNFL1 also leads to apoptosis of
activated T cells, a well documented effect in the case of Fas
ligand and TNF. TNFL1 differs in its expression pattern, however,
when compared to TNF or Fas ligand. Fas ligand, which is involved
in activation-induced cell death (AICD) of CD4+ T cells and
tolerance to self-antigens, is classically expressed on activated T
cells and the immuno-privileged eye and testis. TNF is
constitutively expressed on both mature and immature thymocytes, is
upregulated on activated T cells, and is induced by LPS on
macrophages. In contrast, TNFL1 is constitutively expressed on
antigen presenting cells, specialized or not, such as monocytes, B
cells from peripheral blood lymphocytes in humans, and likely on
macrophages or dendritic cells in the red pulp and marginal zone of
mouse spleen and cultured dendritic cells.
[0041] This localization in the blood and in the spleen suggests a
possible function for TNFL1 in the recognition process of
blood-borne pathogens, such as bacteria or viruses. Furthermore,
TNFL1 is expressed at the surface of dendritic cells cultured from
bone marrow in the presence of GM-CSF TNFL1 may therefore be
expressed on a subtype of dendritic cells which were recently
individualized, myeloid dendritic cells (MDCs), rather than on
lymphoid dendritic cells (LDCs). MDCs share a precursor with
macrophages, are GM-CSF dependant, and are present in the marginal
zone of secondary lymphoid tissues. LDCs, in contrast, are located
in the T-cell zone of the secondary organs, are IL3-dependent, and
share a precursor with T and B cells.
[0042] A second cDNA clone was isolated from an oligodT-primed
library of a human ovarian tumor. It encodes a protein designated
as TNFL2, which has about 25% homology with TNF. TNFL2 does not
have a transmembrane domain and thus can be secreted. The sequence
of TNFL2 is shown in SEQ ID NO:5 The TNFL2 cDNA clone detects a
major population of mRNA in a range of about 1.5 kb to about 2 kb
in tissues associated with the immune system, e.g., peripheral
blood lymphocytes and spleen. A slightly bigger mRNA is also
expressed in spleen as well as in colon, prostate, and to a lesser
extent in ovary and small intestine. The fill length polynucleotide
sequence of TNFL2 cDNA is shown in SEQ ID NO:10
[0043] TNFL1 might be able to bind to the TNF receptors or to Fas,
as a homotrimer or in association with another member of the TNF
family. TNFL1 and TNFL2 may form heterodimers and work together in
a manner similar to that of lymphotoxin .alpha. and .beta.. The
TNF-like ligands disclosed herein can be used, inter alia, to
induce cell death in tumors, to induce apoptosis of activated T
cells, to induce inflammation, and to rescue resting T cells from
apoptosis.
[0044] Proteins which are members of the TNFR superfamily have also
been identified. These are soluble receptors which have the amino
acid sequences shown in SEQ ID NOS:2 and 3 (human) and SEQ ID NO:4
(mouse). These proteins are encoded by the nucleotide sequences
shown in SEQ ID NOS:7, 8, and 9, respectively. These receptors can
be used, inter alia, to regulate the function of a TNF ligand which
plays a role in apoptosis, inflammation, differentiation, or
proliferation. Expression of the receptors can also be useful as
markers for cancer, especially for colon cancer. Diseases which can
be treated using the ligands and/or receptors of the TNF/TNFR
superfamily include rheumatoid arthritis, cancer, septic shock,
Crohn's disease, and osteoporosis.
[0045] Two forms of the soluble receptor corresponding to SEQ ID
NO:2 have been identified. The first (tnfrGT-1) has 300 amino
acids, as shown in SEQ ID NO:17. The second form (tnfrGT-2) is
shown in SEQ ID NO:20. Polynucleotides encoding these two forms are
shown SEQ ID NOS:18 and 19, respectively.
[0046] The human TNF-like (TNF-L) and mammalian TNF receptor-like
(TNFR-L) proteins or polypeptides, biologically active polypeptides
or protein variants, and fusion proteins disclosed herein can be
used in various therapeutic compositions and methods, as described
below. Any naturally occurring variants of SEQ ID NOS:1-5 which may
occur in human or mammalian tissues and which retain the functional
properties of the TNF-L or TNFR-L proteins disclosed herein are
biologically active TNF-L or TNFR-L variants. Non-naturally
occurring TNF-L or TNFR-L variants which contain conservative amino
acid substitutions relative to SEQ ID NOS:1-5 but which retain
substantially the same ligand or receptor activity as naturally
occurring TNF-L or TNFR-L are also biologically active TNF-L or
TNFR-L variants.
[0047] Naturally or non-naturally occurring TNF-L or TNFR-L
variants preferably are at least 85%, 90%, or 95% identical to SEQ
ID NOS 1-5 and have similar biological functions, which are
described below. More preferably, the molecules are 98% or 99%
identical. Percent identity is determined using the Smith-Waterman
homology search algorithm, using an affine gap search with a gap
open penalty of 12 and a gap extension penalty of 1. The
Smith-Waterman homology search algorithm is taught in Smith and
Waterman, Adv. Appl. Math. (1981) 2:482-489.
[0048] Biologically active TNF-L or TNFR-L variants include
glycosylated forms of the proteins, aggregative conjugates of the
proteins with other molecules, and covalent conjugates of the
proteins with unrelated chemical moieties. Covalent conjugates are
prepared by linkage of functionalities to groups which are found in
the amino acid chain or at the N- or C-terminal residues of the
proteins of the invention by means well known in the art. TNF-L or
TNFR-L variants also include allelic variants, species variants,
and muteins. Truncations or deletions of regions which do not
affect the biological functions of the TNF-L or TNFR-L proteins
disclosed herein are also biologically active TNF-L or TNFR-L
variants.
[0049] A subset of mutants, called muteins, is a group of
polypeptides with the non-disulfide bond participating cysteines
substituted with a neutral amino acid, generally, with serines.
These mutants may be stable over a broader temperature range than
naturally occurring TNF-L or TNFR-L proteins. See Mark et al., U.S.
Pat. No. 4,959,314.
[0050] Biologically active TNF-L or TNFR-L polypeptides can
comprise at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50,
75, 100, 125, 150, 175, 200, 225, 250, or 275 contiguous amino
acids of SEQ ID NO:1, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,
35, 40, 50, 75, 100, 125, or 150 contiguous amino acids of SEQ ID
NO:2, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75,
100, 125, 150, 175, or 200 contiguous amino acids of SEQ ID NO:3,
at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100,
125, 130, or 140 contiguous amino acids of SEQ ID NO:4, or at least
6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125, 150,
175, or 200 contiguous amino acids of SEQ ID NO:5, at least 6, 7,
8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150,
175, 200, 225, 230, 231, 240, 250, 275, or 295 contiguous amino
acids of SEQ ID NO:17, or at least 6, 7, 8, 9, 10, 12, 15, 20, 25,
30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, or 210 contiguous
amino acids of SED ID NO:20. Polypeptide molecules having
substantially the same amino acid sequences as the TNF-L or TNFR-L
proteins disclosed herein but possessing minor amino acid
substitutions which do not substantially affect the ability of the
TNF-L or TNFR-L polypeptides to interact with their respective
receptors or ligands are within the definition of biologically
active TNF-L or TNFR-L polypeptide variants.
[0051] Preferably, biologically active TNF-L or TNFR-L polypeptides
or polypeptide variants are at least 65%, 75%, 85%, 90%, 95%, 98%,
or 99% identical to TNF-L or TNFR-L polypeptide fragments of SEQ ID
NOS:1-5, 17 or 20. Percent identity of potential polypeptides or
polypeptide variants with fragments of SEQ ID NOS:1-5, 17 or 20 is
determined as described above.
[0052] Guidance in determining which amino acid residues can be
substituted, inserted, or deleted without abolishing biological or
immunological activity can be found using computer programs well
known in the art, such as DNASTAR software. Preferably the amino
acid changes in TNF-L or TNFR-L protein or polypeptide variants are
conservative amino acid changes, i.e., changes of similarly charged
or uncharged amino acids. Conservative replacements are those which
take place within a family of amino acids which are related in
their side chains. Genetically encoded amino acids are generally
divided into four families acidic (aspartate, glutamate); basic
(lysine, arginine, histidine); non-polar (alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan); and
uncharged polar (glycine, asparagine, glutamine, cystine, serine,
threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are
sometimes classified jointly as aromatic amino acids.
[0053] It is reasonable to expect that an isolated replacement of a
leucine with an isoleucine or valine, an aspartate with a
glutamate, a threonine with a serine, or a similar replacement of
an amino acid with a structurally related amino acid will not have
a major effect on the binding properties of the resulting TNF-L or
TNFR-L molecule, especially if the replacement does not involve an
amino acid at a binding site involved in an interaction of a TNF-L
protein with its receptor or a TNFR-L protein with its ligand.
Binding between a TNF-L protein and its receptor or a TNFR-L
protein and its ligand can be measured, for example, using a yeast
two-hybrid assay, as is known in the art (Fields & Song, Nature
340:245-46, 1989).
[0054] Alternatively, the amino acid sequence of a TNF-L or TNFR-L
protein can be modified to alter its biological activity. For
example, amino acids 174-193 (the TNF ligand binding domain) can be
deleted in a TNFR-L protein to form an inactive variant of the
TNFR-L protein and thereby inhibit or decrease the function of its
ligand.
[0055] TNF-L or TNFR-L proteins or polypeptides can be isolated
from TNF-L and TNFR-L-producing cells, such as spleen, thymus,
prostate, colon, ovary, small intestine, peripheral blood
lymphocytes, or from cell lines such as K562 (chronic
myeoleukemia), G361 (melanoma), or SW480 (colorectal
adenocarcinoma), using biochemical methods which are standard in
the art. These methods include, but are not limited to, size
exclusion chromatography, ammonium sulfate fractionation, ion
exchange chromatography, affinity chromatography, crystallization,
electrofocusing, and preparative gel electrophoresis. The skilled
artisan can readily select methods which will result in a
preparation of TNF-L or TNFR-L protein which is substantially free
from other proteins and from carbohydrates, lipids, or subcellular
organelles. A preparation of isolated and purified TNF-L or TNFR-L
protein is at least 80% pure; preferably, the preparations are 90%,
95%, or 99% pure Purity of the preparations can be assessed by any
means known in the art, such as SDS-polyacrylamide gel
electrophoresis.
[0056] Human TNF-L and human or mammalian TNFR-L proteins,
polypeptides, or variants can be produced by recombinant DNA
methods or by synthetic chemical methods. For production of
recombinant TNF-L or TNFR-L proteins or polypeptides, coding
sequences selected from the nucleotide sequences shown in SEQ ID
NOS:6-10, 18 and 19 can be expressed in known prokaryotic or
eukaryotic expression systems. Bacterial, yeast, insect, or
mammalian expression systems can be used, as is known in the
art.
[0057] Alternatively, synthetic chemical methods, such as solid
phase peptide synthesis, can be used to synthesize human TNF-L or
human or mammalian TNFR-L protein, polypeptides, or variants.
General means for the production of peptides, analogs or
derivatives are outlined in CHEMISTRY AND BIOCHEMISTRY OF AMINO
ACIDS, PEPTIDES, AND PROTEINS--A SURVEY OF RECENT DEVELOPMENTS, B.
Weinstein, ed. (1983). Substitution of D-amino acids for the normal
L-stereoisomer of a TNF-L or TNFR-L protein of the invention can be
carried out to increase the half-life of the molecule.
[0058] Fusion proteins comprising at least 6, 7, 8, 9, 10, 12, 15,
20, 25, 30, 35, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, or
275 contiguous amino acids of SEQ ID NO:1, at least 6, 7, 8, 9, 10,
12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 125, or 150 contiguous
amino acids of SEQ ID NO:2, at least 6, 7, 8, 9, 10, 12, 15, 20,
25, 30, 35, 40, 50, 75, 100, 125, 150, 175, or 200 contiguous amino
acids of SEQ ID NO:3, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,
35, 40, 50, 75, 100, 125, 130, or 140 contiguous amino acids of SEQ
ID NO:4, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50,
75, 100, 125, 150, 175, or 200 contiguous amino acids of SEQ ID
NO:5, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50,
75, 100, 125, 150, 175, 200, 225, 230, 231, 240, 250, 275, or 295
contiguous amino acids of SEQ ID NO:17, or at least 6, 7, 8, 9, 10,
12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, or
210 contiguous amino acids of SEQ ID NO:20 can also be constructed.
TNF-L and TNFR-L fusion proteins are useful for generating
antibodies against TNF-L and TNFR-L amino acid sequences and for
use in various assay systems For example, TNF-L and TNFR-L fusion
proteins can be used to identify proteins which interact with these
proteins which influence their biological activity and/or ability
to bind to their respective binding partners. Physical methods,
such as protein affinity chromatography, or library-based assays
for protein-protein interactions such as the yeast two-hybrid or
phage display systems, can also be used for this purpose. Such
methods are well known in the art and can also be used as drug
screens.
[0059] A TNF-L or TNFR-L fusion protein comprises two protein
segments fused together by means of a peptide bond. The first
protein segment consists of at least 6, 7, 8, 9, 10, 12, 15, 20,
25, 30, 35, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, or 275
contiguous amino acids of SEQ ID NO:1, at least 6, 7, 8, 9, 10, 12,
15, 20, 25, 30, 35, 40, 50, 75, 100, 125, or 150 contiguous amino
acids of SEQ ID NO:2, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,
35, 40, 50, 75, 100, 125, 150, 175, or 200 contiguous amino acids
of SEQ ID NO:3, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35,
40, 50, 75, 100, 125, 130, or 140 contiguous amino acids of SEQ ID
NO:4, at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75,
100, 125, 150, 175, or 200 contiguous amino acids of SEQ ID NO:5,
at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75,
100, 125, 150, 175, 200, 225, 230, 231, 240, 250, 275, or 295
contiguous amino acids of SEQ ID NO:17, or at least 6, 7, 8, 9, 10,
12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, or
210 contiguous amino acids of SEQ ID NO:20 The amino acids can be
selected from the amino acid sequences shown in SEQ ID NOS:1-5, 17
or 20 or from a biologically active variants of those sequences The
first protein segment can also be a full-length TNF-L or TNFR-L
protein comprising an amino acid sequence as shown in SEQ ID
NOS:1-5, 17 or 20. The first protein segment can be N-terminal or
C-terminal, as is convenient.
[0060] The second protein segment can be a full-length protein or a
protein fragment or polypeptide. Proteins commonly used in fusion
protein construction include .beta.-galactosidase,
.beta.-glucuronidase, green fluorescent protein (GFP),
autofluorescent proteins, including blue fluorescent protein (BFP),
glutathione-S-transferase (GST), luciferase, horseradish peroxidase
(HRP), and chloramphenicol acetyltransferase (CAT). Epitope tags
can be used in fusion protein constructions, including histidine
(His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags,
VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions
can include maltose binding protein (MBP), S-tag, Lex A DNA binding
domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes
simplex virus (HSV) BP 16 protein fusions.
[0061] According to one particularly preferred embodiment, a TNFR-L
protein is fused to the Fc domain of an IgG1 molecule. Such a
fusion protein is be useful for inhibiting the action of a TNF
ligand.
[0062] TNF-L or TNFR-L fusion proteins can be made by covalently
linking the first and second protein segments or by standard
procedures in the art of molecular biology. Recombinant DNA methods
can be used to prepare the fusion proteins, for example, by making
a DNA construct which comprises coding sequences selected from SEQ
ID NOS:6-10, 18, and 19 in proper reading frame with nucleotides
encoding the second protein segment and expressing the DNA
construct in a host cell, as is known in the art. Many kits for
constructing fusion proteins are available from companies which
supply research labs with tools for experiments, including, for
example, Promega Corporation (Madison, Wis.), Stratagene (La Jolla,
Calif.), Clontech (Mountain View, Calif.), Santa Cruz Biotechnology
(Santa Cruz, Calif.), NML International Corporation (MIC;
Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada,
1-888-DNA-KITS).
[0063] Isolated TNF-L or TNFR-L proteins, polypeptides,
biologically active variants, or fusion proteins can be used as
immunogens, to obtain a preparation of antibodies which
specifically bind to epitopes of TNF-L or TNFR-L proteins. The
antibodies can be used, inter alia, to detect TNF-L or TNFR-L
proteins in tissue of humans or other mammals or in fractions
thereof The antibodies can also be used to detect the presence of
mutations in genes which result in under- or over-expression of
TNF-L or TNFR-L proteins or in expression of a TNF-L or TNFR-L
protein with altered size or electrophoretic mobility By binding to
TNF-L or TNFR-L proteins, antibodies can also alter the binding
properties or biological functions of the proteins, for example for
therapeutic use.
[0064] Antibodies which specifically bind to epitopes of TNF-L or
TNFR-L proteins, polypeptides, fusion proteins, or biologically
active variants can be used in immunochemical assays, including but
not limited to Western blots, ELISAs, radioimmunoassay,
immunohistochemical assays, immunoprecipitations, or other
immunochemical assays known in the art. Typically, antibodies
provide a detection signal at least 5-, 10-, or 20-fold higher than
a detection signal provided with other proteins when used in such
immunochemical assays. Preferably, antibodies which specifically
bind to TNF-L or TNFR-L protein epitopes do not detect other
proteins in immunochemical assays and can immunoprecipitate TNF-L
or TNFR-L proteins or polypeptides from solution.
[0065] TNF-L- or TNFR-L-specific antibodies specifically bind to
epitopes present in a protein having the amino acid sequence shown
in SEQ ID NOS:1-5, 17 and 20 or to biologically active variants of
those sequences. Typically, at least 6, 8, 10, or 12 contiguous
amino acids are required to form an epitope. However, epitopes
which involve non-contiguous amino acids may require more, e.g., at
least 15, 25, or 50 amino acids. Preferably, TNF-L or TNFR-L
epitopes are not present in other proteins. A preferred epitope
comprises amino acids 208-211 of SEQ ID NO:20. Antibodies capable
of specifically binding to a protein comprising this epitope are
useful for identifying a protein expressed by the polynucleotide of
SEQ ID NO:19.
[0066] Protein epitopes which are particularly antigenic can be
selected, for example, by routine screening of polypeptides for
antigenicity or by applying a theoretical method for selecting
antigenic regions of a protein to the amino acid sequences shown in
SEQ ID NOS:1-5. Such methods are taught, for example, in Hopp and
Wood, Proc. Nat. Acad. Sci. U.S.A. 78, 3824-28 (1981), Hopp and
Wood, Mol. Immunol. 20, 483-89 (1983), and Sutcliffe et al.,
Science 219, 660-66 (1983).
[0067] Any type of antibody known in the art can be generated to
bind specifically to TNF-L or TNFR-L epitopes. For example,
preparations of polyclonal and monoclonal antibodies can be made
using standard methods which are well known in the art Similarly,
single-chain antibodies can also be prepared. Single-chain
antibodies which specifically bind to TNF-L or TNFR-L epitopes can
be isolated, for example, from a single-chain immunoglobulin
display library, as is known in the art. The library is "panned"
against the amino acid sequences disclosed herein, and a number of
single chain antibodies which bind with high-affinity to different
epitopes of proteins of the invention can be isolated. Hayashi et
al., 1995, Gene 160:129-30. Single-chain antibodies can also be
constructed using a DNA amplification method, such as the
polymerase chain reaction (PCR), using hybridoma cDNA as a
template. Thirion et al., 1996, Eur. J. Cancer Prev. 5.507-11.
[0068] Single-chain antibodies can be mono- or bispecific, and can
be bivalent or tetravalent. Construction of tetravalent, bispecific
single-chain antibodies is taught, for example, in Coloma and
Morrison, 1997, Nat. Biotechnol. 15: 159-63. Construction of
bivalent, bispecific single-chain antibodies is taught inter alia
in Mallender and Voss, 1994, J. Biol. Chem. 269:199-206.
[0069] A nucleotide sequence encoding a single-chain antibody can
be constructed using manual or automated nucleotide synthesis,
cloned into an expression construct using standard recombinant DNA
methods, and introduced into a cell to express the coding sequence,
as described below. Alternatively, single-chain antibodies can be
produced directly using, for example, filamentous phage technology.
Verhaar et al., 1995, Int. J. Cancer 61:497-501, Nicholls et al.,
1993, J. Immunol. Meth. 165:81-91.
[0070] Monoclonal and other antibodies can also be "humanized" in
order to prevent a patient from mounting an immune response against
the antibody when it is used therapeutically. Such antibodies may
be sufficiently similar in sequence to human antibodies to be used
directly in therapy or may require alteration of a few key
residues. Sequence differences between, for example, rodent
antibodies and human sequences can be minimized by replacing
residues which differ from those in the human sequences, for
example, by site directed mutagenesis of individual residues, or by
grafting of entire complementarity determining regions
Alternatively, one can produce humanized antibodies using
recombinant methods, as described in GB2188638B. Antibodies which
specifically bind to TNF-L or TNFR-L epitopes can contain antigen
binding sites which are either partially or fully humanized, as
disclosed in U.S. Pat. No. 5,565,332.
[0071] Other types of antibodies can be constructed and used in
methods of the invention. For example, chimeric antibodies can be
constructed as disclosed, for example, in WO 93/03151. Binding
proteins which are derived from immunoglobulins and which are
multivalent and multispecific, such as the "diabodies" described in
WO 94/13804, can also be prepared
[0072] Antibodies can be purified by methods well known in the art.
For example, antibodies can be affinity purified by passing the
antibodies over a column to which a TNF-L or TNFR-L protein,
polypeptide, biologically active variant, or fusion protein is
bound. The bound antibodies can then be eluted from the column,
using a buffer with a high salt concentration.
[0073] TNF-L- or TNFR-L-specific binding polypeptides other than
antibodies can also be identified. These polypeptides include
ligands of TNFR-L proteins and receptors of TNF-L proteins. TNF-L-
or TNFR-L-specific binding polypeptides are polypeptides which bind
with TNF-L or TNFR-L proteins or their variants and which have a
measurably higher binding affinity for TNF-L or TNFR-L and
polypeptide variants of these proteins than for other polypeptides
tested for binding. Higher affinity by a factor of 10 is preferred,
more preferably a factor of 100. Such polypeptides can be found,
for example, using the yeast two-hybrid system.
[0074] Nucleotide sequences which encode TNF-L or TNFR-L proteins
are shown in SEQ ID NOS:6-10, 18 and 19 Isolated and purified
polynucleotides according to the invention can be single- or
double-stranded, are subgenomic, and contain less than a whole
chromosome. Preferably, the subgenomic polynucleotides are
intron-free.
[0075] Isolated and purified subgenomic polynucleotides according
to the invention can comprise at least 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 75, 100,
150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000,
1100, or 1200 contiguous nucleotides of SEQ ID NO:6, at least 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,
40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, or 450
contiguous nucleotides of SEQ ID NO:7, at least 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 75,
100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 575, 600, 650,
700, 750, 800, or 850 contiguous nucleotides of SEQ ID NO:8, at
least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,
500, 550, 575, or 600 contiguous nucleotides of SEQ ID NO:9, or at
least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,
500, 600, 700, 800, 900, 1000, 1100, or 1200 contiguous nucleotides
of SEQ ID NO:10, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250,
300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200,
1250, 1300, 1400, 1500, 1600, 1700, 1800, or 1830 contiguous
nucleotides of SEQ ID NO:19, or can comprise SEQ ID NOS:6, 7, 8, 9,
10, 18, or 19. Such polynucleotides can be used, for example, as
primers or probes or for expression of TNF-L or TNFR-L proteins or
polypeptides.
[0076] The complements of the nucleotide sequences shown in SEQ ID
NOS:6-10, 18, and 19 are contiguous nucleotide sequences which form
Watson-Crick base pairs with a contiguous nucleotide sequence as
shown in SEQ ID NOS:6-10, 18, and 19. The complements of SEQ ID
NOS:6-10, 18, and 19 are polynucleotides of the invention and can
be used, for example, to provide antisense oligonucleotides,
primers, and probes.
[0077] Antisense oligonucleotides, primers, and probes of the
invention can consist of at least 11, 12, 15, 20, 25, 30, 50, or
100 contiguous nucleotides which are complementary to the coding
sequences shown in SEQ ID NOS:6-10, 18, and 19. A complement of the
entire coding sequence can also be used. Double-stranded subgenomic
polynucleotides which comprise all or a portion of the nucleotide
sequences shown in SEQ ID NOS:6-10, 18, and 19, as well as
polynucleotides which encode TNF-L- or TNFR-L-specific antibodies
or ribozymes, are also subgenomic polynucleotides according to the
invention.
[0078] Degenerate nucleotide sequences encoding amino acid
sequences of proteins or biologically active protein variants as
well as homologous nucleotide sequences which are at least 65%,
75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the
nucleotide sequences shown in SEQ ID NOS.6-10, 18, or 19 are also
subgenomic polynucleotides according to the invention and can be
used in the methods disclosed herein. Percent sequence identity
between a nucleotide sequence of SEQ ID NOS:6-10, 18 or 19 and a
putative homologous or degenerate nucleotide sequence is determined
using computer programs which employ the Smith-Waterman algorithm,
for example as implemented in the MPSRCH program (Oxford
Molecular), using an affine gap search with the following
parameters: a gap open penalty of 12 and a gap extension penalty of
1.
[0079] Nucleotide sequences which hybridize to the coding sequences
shown in SEQ ID NOS:6-10, 18 or 19, or their complements with at
most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35% basepair mismatches
are also TNF-L or TNFR-L subgenomic polynucleotides. For example,
using the following wash conditions--2.times.SSC (0.3 M sodium
chloride, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room
temperature twice, 30 minutes each; then 2.times.SSC, 0.1% SDS,
50.degree. C. once, 30 minutes; then 2.times.SSC, room temperature
twice, 10 minutes each--homologous TNF-L or TNFR-L sequences can be
identified which contain at most about 25-30% basepair mismatches
with SEQ ID NOS:6-10, 18 or 19, or their complements More
preferably, homologous nucleic acid strands contain 15-25% basepair
mismatches, even more preferably 5-15% basepair mismatches.
[0080] Species homologs of TNF-L or TNFR-L subgenomic
polynucleotides of the invention can also be identified by making
suitable probes or primers and screening cDNA expression libraries
from other species, such as mice, monkeys, yeast, or bacteria. It
is well known that the T.sub.m of a double-stranded DNA decreases
by 1-1.5.degree. C. with every 1% decrease in homology (Bonner et
al, J. Mol. Biol. 81, 123 (1973). Homologous TNF-L or TNFR-L human
polynucleotides or TNF-L or TNFR-L polynucleotides of other species
can therefore be identified, for example, by hybridizing a putative
homologous TNF-L or TNFR-L polynucleotide with a polynucleotide
having a nucleotide sequence of SEQ ID NO:6, 7, 8, 9, 10, 18, or
19, comparing the melting temperature of the test hybrid with the
melting temperature of a hybrid comprising a polynucleotide having
a nucleotide sequence of SEQ ID NOS:6, 7, 8, 9, 10, 18, or 19 and a
polynucleotide which is perfectly complementary to SEQ ID NO:6, 7,
8, 9, 10, 18, or 19, and calculating the number of basepair
mismatches within the test hybrid.
[0081] Nucleotide sequences which hybridize to the coding sequences
shown in SEQ ID NOS:6-10, 18, or 19, or their complements following
stringent hybridization and/or wash conditions are also TNF-L or
TNFR-L subgenomic polynucleotides. Stringent wash conditions are
well known and understood in the art and are disclosed, for
example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY
MANUAL, 2d ed, 1989, at pages 9.50-9.51.
[0082] Typically, for stringent hybridization conditions a
combination of temperature and salt concentration should be chosen
that is approximately 12-20.degree. C. below the calculated T.sub.m
of the hybrid under study. The T.sub.m of a hybrid between a
sequence shown in SEQ ID NO:6, 7, 8, 9, 10, 18 or 19, and a
polynucleotide sequence which is 65%, 75%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% identical to SEQ ID NO:6, 7, 8, 9, 10, 18, or 19 can be
calculated, for example, using the equation of Bolton and McCarthy,
Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
[0083] T.sub.m=81.5.degree. C.-16 6(log.sub.10[Na.sup.30
])+0.41(%G+C)-0.63(%formamide)-600/l), where l=the length of the
hybrid in basepairs.
[0084] Stringent wash conditions include, for example, 4.times.SSC
at 65.degree. C., or 50% formamide, 4.times.SSC at 42.degree. C.,
or 05.times.SSC, 0.1% SDS at 65.degree. C. Highly stringent wash
conditions include, for example, 0.2.times.SSC at 65.degree. C.
[0085] Subgenomic polynucleotides can be isolated and purified free
from other nucleotide sequences using standard nucleic acid
purification techniques. For example, restriction enzymes and
probes can be used to isolate subgenomic polynucleotide fragments
which comprise TNF-L or TNFR-L coding sequences. Isolated and
purified subgenomic polynucleotides are in preparations which are
free or at least 90% free of other molecules.
[0086] Complementary DNA (cDNA) molecules which encode TNF-L or
TNFR-L proteins are also TNF-L or TNFR-L subgenomic
polynucleotides. cDNA molecules can be made with standard molecular
biology techniques, using TNF-L or TNFR-L mRNA as a template. cDNA
molecules can thereafter be replicated using molecular biology
techniques known in the art and disclosed in manuals such as
Sambrook et al., 1989. An amplification technique, such as the
polymerase chain reaction (PCR), can be used to obtain additional
copies of subgenomic polynucleotides, using either human or
mammalian genomic DNA or cDNA as a template.
[0087] Alternatively, synthetic chemistry techniques can be used to
synthesize subgenomic polynucleotide molecules The degeneracy of
the genetic code allows alternate nucleotide sequences to be
synthesized which will encode a TNF-L protein comprising an amino
acid sequence shown in SEQ ID NOS:1, 2, 3, 4, 5, 17, 20 or a
biologically active variant of one of those sequences.
[0088] The invention also provides polynucleotide probes which can
be used to detect TNF-L or TNFR-L sequences, for example, in
hybridization protocols such as Northern or Southern blotting or in
situ hybridizations. TNF-L or TNFR-L polynucleotide probes of the
invention comprise at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 30,
or 40 or more contiguous nucleotides selected from SEQ ID NOS:6-10,
18, or 19. Polynucleotide probes can comprise a detectable label,
such as a radioisotopic, fluorescent, enzymatic, or
chemiluminescent label.
[0089] TNF-L or TNFR-L subgenomic polynucleotides can be propagated
in vectors and cell lines using techniques well known in the art.
Expression systems in bacteria include those described in Chang et
al, Nature (1978) 275 615, Goeddel et al, Nature (1979) 281: 544,
Goeddel et al., Nucleic Acids Res. (1980) 8: 4057, EP 36,776, U.S.
Pat. No. 4,551,433, deBoer et al, Proc. Natl. Acad. Sci. USA (1983)
80: 21-25, and Siebenlist et al, Cell (1980) 20: 269.
[0090] Expression systems in yeast include those described in
Hinnen et al., Proc. Natl Acad. Sci. USA (1978) 75. 1929; Ito et
al., J. Bacteriol (1983) 153: 163; Kurtz et al., Mol. Cell. Biol
(1986) 6 142; Kunze et al., J. Basic Microbiol (1985) 25: 14 1;
Gleeson et al., J. Gen. Microbiol (1986) 132: 3459, Roggenkamp et
al., Mol. Gen. Genet. (1986) 202 :302) Das et al., J. Bacteriol.
(1984) 158. 1165, De Louvencourt et al., J. Bacteriol (1983) 154:
737, Van den Berg et al., Bio/Technology (1990) 8 135; Kunze et
al., J. Basic Microbiol (1985) 25 141; Cregg et al., Mol. Cell.
Biol. (1985) 5. 3376, U.S. Pat. Nos. 4,837,148, 4,929,555, Beach
and Nurse, Nature (1981) 300: 706, Davidow et al., Curr. Genet.
(1985) 10 380, Gaillardin et al., Curr. Genet. (1985) 10: 49,
Ballance et al., Biochem. Biophy. Res. Commun. (1983) 112 284-289;
Tilburn et al, Gene (1983) 26: 205-221, Yelton et al., Proc. Natl
Acad. Sci. USA (1984) 81: 1470-1474, Kelly and Hynes, EMBO J (1985)
4: 475479, EP 244,234, and WO 91/00357.
[0091] Expression of TNF-L or TNFR-L subgenomic polynucleotides in
insects can be accomplished as described in U.S. Pat. No.
4,745,051, Friesen et al (1986) "The Regulation of Baculovirus Gene
Expression" in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W.
Doerfler, ed ), EP 127,839, EP 155,476, and Viak et al., J. Gen.
Virol. (1988) 69: 765-776, Miller et al., Ann. Rev. Microbiol.
(1988) 42: 177, Carbonell et al., Gene (1988) 73: 409, Maeda et
al., Nature (1985) 315: 592-594, Lebacq-Verheyden et al., Mol.
Cell. Biol. (1988) 8: 3129; Smith et al., Proc. Natl. Acad. Sci.
USA (1985) 82: 8404, Miyajima et al., Gene (1987) 58: 273; and
Martin et al., DNA (1988) 7:99. Numerous baculoviral strains and
variants and corresponding permissive insect host cells from hosts
are described in Luckow et al, Bio/Technology (1988) 6: 47-55,
Miller et al., in GENETIC ENGINEERING (Setlow, J. K. et al. eds.),
Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al.,
Nature, (1985) 315: 592-594.
[0092] Mammalian expression of TNF-L or TNFR-L subgenomic
polynucleotides can be accomplished as described in Dijkema et al.,
EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad. Sci. USA
(1982b) 79. 6777, Boshart et al., Cell (1985) 41: 521 and U.S. Pat.
No. 4,399,216. Other features of mammalian expression can be
facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:
44, Barnes and Sato, Anal. Biochem. (1980) 102: 255, U.S. Pat. Nos.
4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO
87/00195, and U.S. Pat. No. RE 30,985.
[0093] TNF-L or TNFR-L subgenomic polynucleotides can be on linear
or circular molecules. They can be on autonomously replicating
molecules or on molecules without replication sequences They can be
regulated by their own or by other regulatory sequences, as is
known in the art TNF-L or TNFR-L subgenomic polynucleotides can be
introduced into suitable host cells using a variety of techniques
which are available in the art, such as
transferrin-polycation-mediated DNA transfer, transfection with
naked or encapsulated nucleic acids, liposome-mediated DNA
transfer, intracellular transportation of DNA-coated latex beads,
protoplast fusion, viral infection, electroporation, and calcium
phosphate-mediated transfection.
[0094] Polynucleotides of the invention can also be used in gene
delivery vehicles, for the purpose of delivering an mRNA or
oligonucleotide (either with the sequence of a native mRNA or its
complement), full-length protein, fusion protein, polypeptide, or
ribozyme, or single-chain antibody, into a cell, preferably a
eukaryotic cell. According to the present invention, a gene
delivery vehicle can be, for example, naked plasmid DNA, a viral
expression vector comprising a polynucleotide of the invention, or
a polynucleotide of the invention in conjunction with a liposome or
a condensing agent.
[0095] In one embodiment of the invention, the gene delivery
vehicle comprises a promoter and one of the polynucleotides
disclosed herein. Preferred promoters are tissue-specific promoters
and promoters which are activated by cellular proliferation, such
as the thymidine kinase and thymidylate synthase promoters. Other
preferred promoters include promoters which are activatable by
infection with a virus, such as the .alpha.- and .beta.-interferon
promoters, and promoters which are activatable by a hormone, such
as estrogen. Other promoters which can be used include the Moloney
virus LTR, the CMV promoter, and the mouse albumin promoter.
[0096] A gene delivery vehicle can comprise viral sequences such as
a viral origin of replication or packaging signal. These viral
sequences can be selected from viruses such as astrovirus,
coronavirus, orthomyxovirus, papovavirus, paramyxovirus,
parvovirus, picornavirus, poxvirus, retrovirus, togavirus or
adenovirus. In a preferred embodiment, the gene delivery vehicle is
a recombinant retroviral vector. Recombinant retroviruses and
various uses thereof have been described in numerous references
including, for example, Mann et al., Cell 33:153, 1983, Cane and
Mulligan, Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al,
Human Gene Therapy 1:5-14, 1990, U.S. Pat. Nos. 4,405,712,
4,861,719, and 4,980,289, and PCT Application Nos WO 89/02,468, WO
89/05,349, and WO 90/02,806 Numerous retroviral gene delivery
vehicles can be utilized in the present invention, including for
example those described in EP 0,415,731; WO 90/07936; WO 94/03622;
WO 93/25698; WO 93/25234, U.S. Pat. No. 5,219,740; WO 9311230; WO
9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and
Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res.
53:83-88, 1993; Takamiya et al., J. Neurosci. Res. 33:493-503,
1992; Baba et al, J. Neurosurg. 79:729-735, 1993 (U.S. Pat. No.
4,777,127, GB 2,200,651, EP 0,345,242 and WO91/02805).
[0097] Particularly preferred retroviruses are derived from
retroviruses which include avian leukosis virus (ATCC Nos VR-535
and VR-247), bovine leukemia virus (VR-1315), murine leukemia virus
(MLV), mink-cell focus-inducing virus (Koch et al, J. Vir. 49:828,
1984; and Oliff et al, J. Vir. 48:542, 1983), murine sarcoma virus
(ATCC Nos. VR-844, 45010 and 45016), reticuloendotheliosis virus
(ATCC Nos VR-994, VR-770 and 45011), Rous sarcoma virus,
Mason-Pfizer monkey virus, baboon endogenous virus, endogenous
feline retrovirus (e.g., RD114), and mouse or rat gL30 sequences
used as a retroviral vector. Particularly preferred strains of MLV
from which recombinant retroviruses can be generated include 4070A
and 1504A (Hartley and Rowe, J. Vir. 19:19, 1976), Abelson (ATCC
No. VR-999), Friend (ATCC No. VR-245), Graffi (Ru et al., J. Vir.
67:4722, 1993; and Yantchev Neoplasma 26:397, 1979), Gross (ATCC
No. VR-590), Kirsten (Albino et al, J. Exp. Med. 164:1710, 1986),
Harvey sarcoma virus (Manly et al., J. Vir. 62:3540, 1988; and
Albino et al., J. Exp. Med. 164:1710, 1986) and Rauscher (ATCC No.
VR-998), and Moloney MLV (ATCC No. VR-190). A particularly
preferred non-mouse retrovirus is Rous sarcoma virus. Preferred
Rous sarcoma viruses include Bratislava (Manly et al., J. Vir.
62:3540, 1988; and Albino et al, J. Exp. Med. 164:1710, 1986),
Bryan high titer (e.g. ATCC Nos. VR-334, VR-657, VR-726, VR-659,
and VR-728), Bryan standard (ATCC No. VR-140), Carr-Zilber
(Adgighitov et al., Neoplasma 27 159, 1980), Engelbreth-Holm
(Laurent et al, Biochem Biophys Acta 908 241, 1987), Harris, Prague
(e.g., ATCC Nos VR-772, and 45033), and Schmidt-Ruppin (e.g. ATCC
Nos. VR-724, VR-725, VR-354) viruses.
[0098] Any of the above retroviruses can be readily utilized in
order to assemble or construct retroviral gene delivery vehicles
given the disclosure provided herein and standard recombinant
techniques (e.g., Sambrook et al., 1989, and Kunkle, Proc. Natl
Acad. Sci. U.S.A. 82.488, 1985) known in the art. Portions of
retroviral expression vectors can be derived from different
retroviruses. For example, retrovector LTRs can be derived from a
murine sarcoma virus, a tRNA binding site from a Rous sarcoma
virus, a packaging signal from a murine leukemia virus, and an
origin of second strand synthesis from an avian leukosis virus
These recombinant retroviral vectors can be used to generate
transduction competent retroviral vector particles by introducing
them into appropriate packaging cell lines (see Ser. No.
07/800,921, filed Nov. 29, 1991). Recombinant retroviruses can be
produced which direct the site-specific integration of the
recombinant retroviral genome into specific regions of the host
cell DNA. Such site-specific integration can be mediated by a
chimeric integrase incorporated into the retroviral particle (see
Ser. No. 08/445,466 filed May 22, 1995). It is preferable that the
recombinant viral gene delivery vehicle is a replication-defective
recombinant virus.
[0099] Packaging cell lines suitable for use with the
above-described retroviral gene delivery vehicles can be readily
prepared (see Ser. No. 08/240,030, filed May 9, 1994; see also WO
92/05266) and used to create producer cell lines (also termed
vector cell lines or "VCLs") for production of recombinant viral
particles. In particularly preferred embodiments of the present
invention, packaging cell lines are made from human (e.g., HT1080
cells) or mink parent cell lines, thereby allowing production of
recombinant retroviral gene delivery vehicles which are capable of
surviving inactivation in human serum. The construction of
recombinant retroviral gene delivery vehicles is described in
detail in WO 91/02805 These recombinant retroviral gene delivery
vehicles can be used to generate transduction competent retroviral
particles by introducing them into appropriate packaging cell lines
(see Ser. No. 07/800,921). Similarly, adenovirus gene delivery
vehicles can also be readily prepared and utilized given the
disclosure provided herein (see also Berkner, Biotechniques
6:616-627, 1988, and Rosenfeld et al, Science 252-431-434, 1991, WO
93/07283, WO 93/06223, and WO 93/07282)
[0100] A gene delivery vehicle can also be a recombinant adenoviral
gene delivery vehicle. Such vehicles can be readily prepared and
utilized given the disclosure provided herein (see Berkner,
Biotechniques 6.616, 1988, and Rosenfeld et al., Science 252:431,
1991, WO 93/07283, WO 93/06223, and WO 93/07282). Adeno-associated
viral gene delivery vehicles can also be constructed and used to
deliver proteins or polynucleotides of the invention to cells in
vitro or in vivo The use of adeno-associated viral gene delivery
vehicles in vitro is described in Chatterjee et al., Science 258:
1485-1488 (1992), Walsh et al., Proc. Nat'l. Acad. Sci. 89
7257-7261 (1992), Walsh et al., J. Clin. Invest. 94: 1440-1448
(1994), Flotte et al, J. Biol. Chem. 268 3781-3790 (1993),
Ponnazhagan et al., J. Exp. Med. 179 733-738 (1994), Miller et al.,
Proc. Nat'l Acad. Sci. 91: 10183-10187 (1994), Einerhand et al.,
Gene Ther. 2 336-343 (1995), Luo et al, Exp. Hematol. 23: 1261-1267
(1995), and Zhou et al., Gene Therapy 3: 223-229 (1996). In vivo
use of these vehicles is described in Flotte et al., Proc. Nat'l
Acad. Sci. 90 10613-10617 (1993), and Kaplitt et al., Nature Genet.
8: 148-153 (1994).
[0101] In another embodiment of the invention, a gene delivery
vehicle is derived from a togavirus. Preferred togaviruses include
alphaviruses, in particular those described in U.S. Ser. No.
08/405,627, filed Mar. 15, 1995, WO 95/07994. Alpha viruses,
including Sindbis and ELVS viruses can be gene delivery vehicles
for polynucleotides of the invention. Alpha viruses are described
in WO 94/21792, WO 92/10578 and WO 95/07994. Several different
alphavirus gene delivery vehicle systems can be constructed and
used to deliver polynucleotides to a cell according to the present
invention. Representative examples of such systems include those
described in U.S. Pat. Nos. 5,091,309 and 5,217,879 Particularly
preferred alphavirus gene delivery vehicles for use in the present
invention include those which are described in WO 95/07994, and
U.S. Ser. No. 08/405,627.
[0102] Preferably, the recombinant viral vehicle is a recombinant
alphavirus viral vehicle based on a Sindbis virus. Sindbis
constructs, as well as numerous similar constructs, can be readily
prepared essentially as described in U.S. Ser. No. 08/198,450.
Sindbis viral gene delivery vehicles typically comprise a 5'
sequence capable of initiating Sindbis virus transcription, a
nucleotide sequence encoding Sindbis non-structural proteins, a
viral junction region inactivated so as to prevent fragment
transcription, and a Sindbis RNA polymerase recognition sequence
Optionally, the viral junction region can be modified so that
polynucleotide transcription is reduced, increased, or maintained
As will be appreciated by those in the art, corresponding regions
from other alphaviruses can be used in place of those described
above.
[0103] The viral junction region of an alphavirus-derived gene
delivery vehicle can comprise a first viral junction region which
has been inactivated in order to prevent transcription of the
polynucleotide and a second viral junction region which has been
modified such that polynucleotide transcription is reduced. An
alphavirus-derived vehicle can also include a 5' promoter capable
of initiating synthesis of viral RNA from cDNA and a 3' sequence
which controls transcription termination.
[0104] Other recombinant togaviral gene delivery vehicles which can
be utilized in the present invention include those derived from
Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus
(ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246),
Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250;
ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat. Nos.
5,091,309 and 5,217,879 and in WO 92/10578. The Sindbis vehicles
described above, as well as numerous similar constructs, can be
readily prepared essentially as described in U.S. Ser. No.
08/198,450.
[0105] Other viral gene delivery vehicles suitable for use in the
present invention include, for example, those derived from
poliovirus (Evans et al., Nature 339:385, 1989, and Sabin et al.,
J. Biol. Standardization 1:115, 1973) (ATCC VR-58); rhinovirus
(Arnold et al., J. Cell. Biochem. L401, 1990) (ATCC VR- 1110); pox
viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et
al., PROC. NATL. ACAD. SCI. U.S.A. 86:317, 1989; Flexner et al.,
Ann. N.Y. Acad. Sci. 569:86, 1989; Flexner et al, Vaccine 8:17,
1990; U.S. Pat. Nos. 4,603,112 and 4,769,330; WO 89/01973) (ATCC
VR-111, ATCC VR-2010); SV40 (Mulligan et al., Nature 277:108, 1979)
(ATCC VR-305), (Madzak et al., J. Gen. Vir. 73-1533, 1992);
influenza virus (Luytjes et al., Cell 59:1107, 1989; McMicheal et
al, The New England Journal of Medicine 309:13, 1983; and Yap et
a., Nature 273 238, 1978) (ATCC VR-797); parvovirus such as
adeno-associated virus (Samulski et al., J. Vir. 63:3822, 1989, and
Mendelson et al, Virology 166.154, 1988) (ATCC VR-645); herpes
simplex virus (Kit et al., Adv. Exp. Med. Biol. 215:219, 1989)
(ATCC VR-977, ATCC VR-260); Nature 277: 108, 1979); human
immunodeficiency virus (EPO 386,882, Buchschacher et al, J. Vir.
66:2731, 1992); measles virus (EPO 440,219) (ATCC VR-24); A (ATCC
VR-67; ATCC VR-1247), Aura (ATCC VR-368), Bebaru virus (ATCC
VR-600; ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus
(ATCC VR-64; ATCC VR-1241), Fort Morgan (ATCC VR-924), Getah virus
(ATCC VR-369; ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC
VR-66), Mucambo virus (ATCC VR-580; ATCC VR-1244), Ndumu (ATCC
VR-371), Pixuna virus (ATCC VR-372; ATCC VR-1245), Tonate (ATCC
VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Whataroa (ATCC
VR-926), Y-62-33 (ATCC VR-375), O'Nyong virus, Eastern encephalitis
virus (ATCC VR-65; ATCC VR-1242), Western encephalitis virus (ATCC
VR-70; ATCC VR-1251; ATCC VR-622; ATCC VR-1252), and coronavirus
(Hamre et al., Proc. Soc. Exp. Biol. Med. 121:190, 1966) (ATCC
VR-740).
[0106] A polynucleotide of the invention can also be combined with
a condensing agent to form a gene delivery vehicle. In a preferred
embodiment, the condensing agent is a polycation, such as
polylysine, polyarginine, polyornithine; protamine, spermine,
spermidine, and putrescine. Many suitable methods for making such
linkages are known in the art (see, for example, Ser. No.
08/366,787, filed Dec. 30, 1994).
[0107] In an alternative embodiment, a polynucleotide is associated
with a liposome to form a gene delivery vehicle. Liposomes are
small, lipid vesicles comprised of an aqueous compartment enclosed
by a lipid bilayer, typically spherical or slightly elongated
structures several hundred Angstroms in diameter. Under appropriate
conditions, a liposome can fuse with the plasma membrane of a cell
or with the membrane of an endocytic vesicle within a cell which
has internalized the liposome, thereby releasing its contents into
the cytoplasm Prior to interaction with the surface of a cell,
however, the liposome membrane acts as a relatively impermeable
barrier which sequesters and protects its contents, for example,
from degradative enzymes. Additionally, because a liposome is a
synthetic structure, specially designed liposomes can be produced
which incorporate desirable features See Stryer, Biochemistry, pp.
236-240, 1975 (W. H. Freeman, San Francisco, Calif.); Szoka et al.,
Biochim. Biophys. Acta 600-1, 1980; Bayer et al., Biochim. Biophys.
Acta. 550:464, 1979; Rivnay et al, Meth. Enzymol. 149:119, 1987;
Wang et al, PROC NATL. ACAD. SCI. U.S.A. 84: 7851, 1987, Plant et
al., Anal Biochem. 176:420, 1989, and U.S. Pat. No. 4,762,915.
Liposomes can encapsulate a variety of nucleic acid molecules
including DNA, RNA, plasmids, and expression constructs comprising
polynucleotides such those disclosed in the present invention.
[0108] Liposomal preparations for use in the present invention
include cationic (positively charged), anionic (negatively charged)
and neutral preparations. Cationic liposomes have been shown to
mediate intracellular delivery of plasmid DNA (Felgner et al.,
Proc. Natl. Acad. Sci. USA 84:7413-7416, 1987), mRNA (Malone et
al., Proc. Natl. Acad. Sci. USA 86:6077-6081, 1989), and purified
transcription factors (Debs et al., J. Biol. Chem. 265:10189-10192,
1990), in functional form. Cationic liposomes are readily
available. For example, N[1 -2,3-dioleyloxy)propyl]-
-N,N,N-triethylammonium (DOTMA) liposomes are available under the
trademark Lipofectin, from GEBCO BRL, Grand Island, N.Y. See also
Feigner et al., Proc. Natl. Acad. Sci. USA 91: 5148-5152.87, 1994.
Other commercially available liposomes include Transfectace
(DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes
can be prepared from readily available materials using techniques
well known in the art. See, e.g., Szoka et al., Proc. Natl. Acad.
Sct USA 75:4194-4198, 1978; and WO 90/11092 for descriptions of the
synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)
liposomes.
[0109] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios Methods for making liposomes using these materials are well
known in the art.
[0110] The liposomes can comprise multilammelar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs) The various liposome-nucleic acid complexes are prepared
using methods known in the art. See, e.g., Straubinger et al.,
METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al.,
Proc. Natl. Acad. Sci. USA 87:3410-3414, 1990; Papahadjopoulos et
al., Biochim. Biophys. Acta 394:483, 1975, Wilson et al., Cell
17:77, 1979, Deamer and Bangham, Biochim. Biophys. Acta 443 629,
1976; Ostro et al., Biochem. Biophys. Res. Commun. 76:836 , 1977;
Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348, 1979; Enoch and
Strittmatter, Proc. Natl. Acad. Sci. USA 76:145, 1979; Fraley et
al., J. Biol. Chem. 255:10431, 1980; Szoka and Papahadjopoulos,
Proc. Natl. Acad. Sci. USA 75:145, 1979; and Schaefer-Ridder et
al., Science 215:166, 1982.
[0111] In addition, lipoproteins can be included with a
polynucleotide of the invention for delivery to a cell. Examples of
such lipoproteins include chylomicrons, HDL, IDL, LDL, and VLDL.
Mutants, fragments, or fusions of these proteins can also be used.
Modifications of naturally occurring lipoproteins can also be used,
such as acetylated LDL. These lipoproteins can target the delivery
of polynucleotides to cells expressing lipoprotein receptors.
Preferably, if lipoproteins are included with a polynucleotide, no
other targeting ligand is included in the composition.
[0112] In another embodiment, naked polynucleotide molecules are
used as gene delivery vehicles, as described in WO 90/11092 and
U.S. Pat. No. 5,580,859. Such gene delivery vehicles can be either
DNA or RNA and, in certain embodiments, are linked to killed
adenovirus. Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other
suitable vehicles include DNA-ligand (Wu et al., J. Biol. Chem. 264
16985-16987, 1989), lipid-DNA combinations (Feigner et al., Proc.
Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et al.,
Proc. Natl. Acad. Sci. 84:7851-7855, 1987) and microprojectiles
(Williams et al., Proc. Natl. Acad. Sci. 88 2726-2730, 1991).
[0113] One can increase the efficiency of naked polynucleotide
uptake into cells by coating the polynucleotides onto biodegradable
latex beads. This approach takes advantage of the observation that
latex beads, when incubated with cells in culture, are efficiently
transported and concentrated in the perinuclear region of the
cells. The beads will then be transported into cells when injected
into muscle Polynucleotide-coated latex beads will be efficiently
transported into cells after endocytosis is initiated by the latex
beads and thus increase gene transfer and expression efficiency.
This method can be improved further by treating the beads to
increase their hydrophobicity, thereby facilitating the disruption
of the endosome and release of polynucleotides into the
cytoplasm.
[0114] The newly identified receptor proteins play regulatory roles
in cell proliferation and/or differentiation For example, they can
induce the production of cytokines, immunoglobulins, and cell
surface antigens The receptors can also play a role in the negative
regulation of osteoclastogenesis Soluble TNFR-like receptors can be
useful in the neutralization of TNF or TNF-like ligands for the
treatment of rheumatoid arthritis and Crohn's disease. Similarly,
restoring normal apoptosis to a cell via these receptors can be
used to treat viral diseases.
[0115] A variety of diseases and conditions can be treated by
modulating the activity of TNF-L or TNFR-L proteins of the
invention. For example, TNFL proteins induce apoptosis of activated
T cells, but rescue resting T cells from apoptosis. A TNF-L protein
can therefore be used to treat autoimmune diseases, such as
myasthenia gravis, insulin-dependent diabetes mellitus, rheumatoid
arthritis, multiple sclerosis, and systemic lupus erythematosus.
TNF-L proteins also have tumor stimulating properties Tumors can
therefore be treated by inhibiting expression or activity of a
TNF-L protein. Similarly, reducing expression of a TNFR-L protein
or blocking its ligand binding site can be used to treat tumors,
whereas increasing expression of a TNFR-L protein can be used to
treat autoimmune diseases such as those disclosed above.
[0116] In one embodiment of the invention, expression of a TNF-L or
TNFR-L gene is decreased using a ribozyme, an RNA molecule with
catalytic activity. See, e.g., Cech, 1987, Science 236: 1532-1539;
Cech. 1990, Ann. Rev. Biochem. 59:543-568, Cech, 1992, Curr. Opin.
Stryct. Biol. 2 605-609; Couture and Stinchcomb, 1996, Trends
Genet. 12: 510-515. Ribozymes can be used to inhibit gene function
by cleaving an RNA sequence, as is known in the art (e.g., Haseloff
et al., U.S. Pat. No. 5,641,673)
[0117] The coding sequences shown in SEQ ID NOS:6-10, 18, and 19
can be used to generate ribozymes which will specifically bind to
mRNA transcribed from a TNF-L or TNFR-L gene. Methods of designing
and constructing ribozymes which can cleave other RNA molecules in
trans in a highly sequence specific manner have been developed and
described in the art (see Haseloff et al (1988), Nature
334:585-591). For example, the cleavage activity of ribozymes can
be targeted to specific RNAs by engineering a discrete
"hybridization" region into the ribozyme. The hybridization region
contains a sequence complementary to the target RNA and thus
specifically hybridizes with the target (see, for example, Gerlach
et al., EP 321,201). Longer complementary sequences can be used to
increase the affinity of the hybridization sequence for the target.
The hybridizing and cleavage regions of the ribozyme can be
integrally related; thus, upon hybridizing to the target RNA
through the complementary regions, the catalytic region of the
ribozyme can cleave the target.
[0118] TNF-L and TNFR-L-specific ribozymes can be introduced into
cells, such as neoplastic cells, as part of a DNA construct, as is
known in the art. The DNA construct can also include
transcriptional regulatory elements, such as a promoter element, an
enhancer or UAS element, and a transcriptional terminator signal,
for controlling transcription of the ribozyme in the cells.
[0119] Mechanical methods, such as microinjection,
liposome-mediated transfection, electroporation, or calcium
phosphate precipitation, can be used to introduce the
ribozyme-containing DNA construct into cells whose division it is
desired to decrease, as described above. Alternatively, if it is
desired that the cells stably retain the DNA construct, the DNA
construct can be supplied on a plasmid and maintained as a separate
element or integrated into the genome of the cells, as is known in
the art.
[0120] Expression of a TNF-L or TNFR-L gene can also be altered
using an antisense oligonucleotide sequence The antisense sequence
is complementary to at least a portion of the coding sequence of a
gene having a coding sequence shown in SEQ ID NO:6-10, 18, or 19
Preferably, the antisense oligonucleotide sequence is at least six
nucleotides in length, but can be about 8, 12, 15, 20, 25, 30, 35,
40, 45, or 50 nucleotides long. Longer sequences can also be used.
TNF-L or TNFR-L antisense oligonucleotide molecules can be provided
in a DNA construct and introduced into cells whose division is to
be decreased, as described above.
[0121] Antisense oligonucleotides can be composed of
deoxyribonucleotides, ribonucleotides, or a combination of both.
Oligonucleotides can be synthesized manually or by an automated
synthesizer, by covalently linking the 5' end of one nucleotide
with the 3' end of another nucleotide with non-phosphodiester
internucleotide linkages such alkylphosphonates, phosphorothioates,
phosphorodithioates, alkylphosphonothioates, alkylphosphonates,
phosphoramidates, phosphate esters, carbamates, acetamidate,
carboxymethyl esters, carbonates, and phosphate triesters See
Brown, 1994, Meth. Mol. Biol 20-1-8; Sonveaux, 1994, Meth. Mol.
Biol. 26 1-72; Uhlmann et al., 1990, Chem. Rev. 90:543-583
[0122] Precise complementarity is not required for successful
duplex formation between an antisense molecule and the
complementary coding sequence of a TNF-L or TNFR-L gene. Antisense
molecules which comprise, for example, 2, 3, 4, or 5 or more
stretches of contiguous nucleotides which are precisely
complementary to a TNF-L or TNFR-L coding sequence, each separated
by a stretch of contiguous nucleotides which are not complementary
to adjacent TNF-L or TNFR-L coding sequences, can provide targeting
specificity for TNF-L or TNFR-L mRNA. Preferably, each stretch of
contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more
nucleotides in length. Non-complementary intervening sequences are
preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the
art can easily use the calculated melting point of an
antisense-sense pair to determine the degree of mismatching which
will be tolerated between a particular antisense oligonucleotide
and a particular TNF-L or TNFR-L coding sequence.
[0123] TNF-L or TNFR-L antisense oligonucleotides can be modified
without affecting their ability to hybridize to a TNF-L or TNFR-L
coding sequence. These modifications can be internal or at one or
both ends of the antisense molecule. For example, internucleoside
phosphate linkages can be modified by adding cholesteryl or diamine
moieties with varying numbers of carbon residues between the amino
groups and terminal ribose. Modified bases and/or sugars, such as
arabinose instead of ribose, or a 3', 5'-substituted
oligonucleotide in which the 3' hydroxyl group or the 5' phosphate
group are substituted, can also be employed in a modified antisense
oligonucleotide. These modified oligonucleotides can be prepared by
methods well known in the art. Agrawal et al., 1992, Trends
Biotechnol. 10:152-158; Uhlmann et al., 1990, Chem. Rev.
90:543-584; Uhlmann et al., 1987, Tetrahedron. Lett.
215:3539-3542.
[0124] Antibodies which specifically bind to a TNF-L and TNFR-L
protein can also be used to alter effective levels of TNF-L or
TNFR-L gene expression. TNF-L and TNFR-L-specific antibodies bind
to TNF-L and TNFR-L proteins and prevent the proteins from
functioning in the cell Construction of such antibodies is
disclosed above.
[0125] Expression of an endogenous TNF-L or TNFR-L gene in a cell
can also be altered by introducing in frame with the endogenous
7NF-L or TNFR-L gene a DNA construct comprising a TNF-L or TNFR-L
targeting sequence, a regulatory sequence, an exon, and an unpaired
splice donor site by homologous recombination, such that a
homologously recombinant cell comprising the DNA construct is
formed. The new transcription unit can be used to turn the TNF-L or
TNFR-L gene on or off as desired. This method of affecting
endogenous gene expression is taught in U.S. Pat. No.
5,641,670.
[0126] The targeting sequence is a segment of at least 10, 12, 15,
20, or 50 contiguous nucleotides selected from a nucleotide
sequence shown in SEQ ID NO:6-10, 18, or 19. The transcription unit
is located upstream of a coding sequence of the endogenous TNF-L or
TNFR-L gene. The exogenous regulatory sequence directs
transcription of the coding sequence of the TNF-L or TNFR-L
gene.
[0127] Preferably, the mechanism used to decrease expression of the
TNF-L or TNFR-L gene, whether ribozyme, antisense nucleotide
sequence, or antibody, decreases expression of the gene by 50%,
60%, 70%, or 80% Most preferably, expression of the gene is
decreased by 90%, 95%, 99%, or 100%. The effectiveness of the
mechanism chosen to alter expression of the gene can be assessed
using methods well known in the art, such as hybridization of
nucleotide probes to mRNA of the gene, quantitative RT-PCR, or
detection of a TNF-L and TNFR-L protein using specific antibodies
of the invention.
[0128] TNF-L and TNFR-L proteins or subgenomic polynucleotides can
be used in therapeutic compositions for treating a variety of
TNF-mediated disorders. Therapeutic compositions of the invention
which comprise TNF-L protein or TNF-L protein encoding
polynucleotides can be used, for example, to treat disorders in
which abnormal numbers of T cells become activated. Activated
T-lymphocytes are associated with disease in graft versus host
reactions (e.g., bone marrow transplantation) and most forms of
autoimmunity, including but not restricted to, multiple sclerosis,
rheumatoid arthritis, lupus, and myasthenia gravis.
T-lymphocyte-mediated primary diseases, such as juvenile diabetes,
can also be treated using TNF-L protein or protein encoding
polynucleotides.
[0129] TNF-L and TNFR-L therapeutic compositions of the invention
can comprise a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are well known to those in the
art. Such carriers include, but are not limited to, large, slowly
metabolized macromolecules, such as proteins, polysaccharides,
polylactic acids, polyglycolic acids, polymeric amino acids, amino
acid copolymers, and inactive virus particles. Pharmaceutically
acceptable salts can also be used in the composition, for example,
mineral salts such as hydrochlorides, hydrobromides, phosphates, or
sulfates, as well as the salts of organic acids such as acetates,
proprionates, malonates, or benzoates.
[0130] TNF-L or TNFR-L therapeutic compositions can also contain
liquids, such as water, saline, glycerol, and ethanol, as well as
substances such as wetting agents, emulsifying agents, or pH
buffering agents. Liposomes, such as those described in U.S. Pat.
No. 5,422,120, WO 95/13796, WO 91/14445, or EP 524,968 B1, can also
be used as a carrier for a therapeutic TNF-L or TNFR-L
composition.
[0131] Typically, a therapeutic TNF-L or TNFR-L composition is
prepared as an injectable, either as a liquid solution or
suspension; however, solid forms suitable for solution in, or
suspension in, liquid vehicles prior to injection can also be
prepared. A TNF-L or TNFR-L composition can also be formulated into
an enteric coated tablet or gel capsule according to known methods
in the art, such as those described in U.S. Pat. No. 4,853,230, EP
225,189, AU 9,224,296, and AU 9,230,801.
[0132] Proliferative disorders, such as neoplasias, dysplasias, and
hyperplasias, can be treated by administration of a therapeutic
TNF-L and TNFR-L composition which will inhibit TNF-L activity or
expression Neoplasias which can be treated with the therapeutic
composition include, but are not limited to, melanomas, squamous
cell carcinomas, adenocarcinomas, hepatocellular carcinomas, renal
cell carcinomas, sarcomas, myosarcomas, non-small cell lung
carcinomas, leukemias, lymphomas, osteosarcomas, central nervous
system tumors such as gliomas, astrocytomas, oligodendrogliomas,
and neuroblastomas, tumors of mixed origin, such as Wilms' tumor
and teratocarcinomas, and metastatic tumors.
[0133] Proliferative disorders which can be treated with a
therapeutic TNF-L composition include disorders such as anhydric
hereditary ectodermal dysplasia, congenital alveolar dysplasia,
epithelial dysplasia of the cervix, fibrous dysplasia of bone, and
mammary dysplasia Hyperplasias, for example, endometrial, adrenal,
breast, prostate, or thyroid hyperplasias, or pseudoepitheliomatous
hyperplasia of the skin can be treated with TNF-L or TNFR-L
therapeutic compositions.
[0134] Even in disorders in which TNF-L or TNFR-L mutations are not
implicated, decreasing expression of a TNF-L gene or a TNFR-L gene
or decreasing a TNF-L or TNFR-L protein function can have a
therapeutic application. In these disorders, decreasing TNF-L or
TNFR-L expression or function can help to suppress tumors.
Similarly, in tumors in which TNF-L or TNFR-L expression is not
aberrant, effecting TNF-L or TNFR-L downregulation or decrease of
TNF-L or TNFR-L activity can suppress metastases.
[0135] Administration of therapeutic compositions of the invention
can include local or systemic administration, including injection,
oral administration, particle gun, or catheterized administration,
and topical administration. Various methods can be used to
administer a therapeutic composition directly to a specific site in
the body. For example, a small metastatic lesion can be located and
a therapeutic composition injected several times in several
different locations within the body of tumor Alternatively,
arteries which serve a tumor can be identified, and a therapeutic
composition injected into such an artery, in order to deliver the
composition directly into the tumor.
[0136] A tumor which has a necrotic center can be aspirated and the
composition injected directly into the now empty center of the
tumor A therapeutic composition can be directly administered to the
surface of a tumor, for example, by topical application of the
composition X-ray imaging can be used to assist in certain of the
above delivery methods. Combination therapeutic agents, such as an
anti-TNF-L neutralizing antibody and another therapeutic agent, can
be administered simultaneously or sequentially.
[0137] Alternatively, a therapeutic composition can be introduced
into human cells ex vivo, and the cells then replaced into the
human. Cells can be removed from a variety of locations including,
for example, from a selected tumor or from an affected organ. In
addition, the therapeutic composition can be inserted into
non-tumorigenic cells, for example, dermal fibroblasts or
peripheral blood leukocytes. If desired, particular fractions of
cells such as a T cell subset or stem cells can also be
specifically removed from the blood (see, for example, PCT WO
91/16116). The removed cells can then be contacted with the
therapeutic composition utilizing any of the above-described
techniques, followed by the return of the cells to the human,
preferably to or within the vicinity of a tumor. The
above-described methods can additionally comprise the steps of
depleting fibroblasts or other non-contaminating tumor cells
subsequent to removing tumor cells from a human, and/or the step of
inactivating the cells, for example, by irradiation.
[0138] Receptor-mediated targeted delivery of therapeutic
compositions containing TNF-L or TNFR-L subgenomic polynucleotides
to specific tissues can also be used. Receptor-mediated DNA
delivery techniques are described in, for example, Findeis et al.
(1993), Trends in Biotechnol. 11, 202-05; Chiou et al (1994), GENE
THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.
A. Wolff, ed.); Wu & Wu (1988), J. Biol. Chem. 263, 621-24; Wu
et al. (1994), J. Biol Chem. 269, 542-46; Zenke et al. (1990),
Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59; Wu et al. (1991), J.
Biol. Chem. 266, 338-42.
[0139] Both the dose of the TNF-L or TNFR-L composition and the
means of administration can be determined based on the specific
qualities of the therapeutic composition, the condition, age, and
weight of the patient, the progression of the disease, and other
relevant factors If the composition contains TNF-L or TNFR-L
protein, polypeptide, or antibody, effective dosages of the
composition are in the range of about 5 .mu.g to about 50 .mu.g/kg
of patient body weight, about 50 .mu.g to about 5 .mu.g/kg, about
100 .mu.g to about 500 .mu.g/kg of patient body weight, and about
200 to about 250 .mu.g/kg.
[0140] Therapeutic compositions containing TNF-L or TNFR-L
subgenomic polynucleotides, ribozymes, or antisense
oligonucleotides can be administered in a range of about 100 ng to
about 200 mg of DNA for local administration in a gene therapy
protocol. Concentration ranges of about 500 ng to about 50 mg,
about 1 .mu.g to about 2 .mu.g, about 5 .mu.g to about 500 .mu.g,
and about 20 .mu.g to about 100 .mu.g of DNA can also be used
during a gene therapy protocol. Factors such as method of action
and efficacy of transformation and expression are considerations
that will effect the dosage required for ultimate efficacy of the
TNF-L or TNFR-L subgenomic polynucleotides.
[0141] Where greater expression is desired over a larger area of
tissue, larger amounts of a TNF-L or TNFR-L therapeutic composition
or the same amount readministered in a successive protocol of
administrations, or several administrations to different adjacent
or close tissue portions of for example, a tumor site, may be
required to effect a positive therapeutic outcome. In all cases,
routine experimentation in clinical trials will determine specific
ranges for optimal therapeutic effect.
[0142] The invention provides knock-out mammals whose endogenous
TNF-L or TNFR-L gene is not expressed Methods of making knock-out
mammals are well known in the art. The mammal can be any
experimental mammal, such as a mouse, rat, or rabbit; however, a
mouse is preferred The endogenous wild-type TNF-L or TNFR-L gene of
the mammal can be deleted entirely, resulting in an absence of
TNF-L or TNFR-L protein in the mammal. Alternatively, mutations
such as deletions, insertions, missense substitutions, or
inversions, can be introduced into a TNF-L or TNFR-L gene. Such
mutations result in expression of truncated or otherwise aberrant
forms of TNF-L or TNFR-L protein in the knock-out mammal Mammalian
cell lines which do not express an endogenous TNF-L or TNFR-L gene
can also be constructed, as is known in the art.
[0143] Knock-out mammals and cells of the invention are useful as
model systems for studying the effects of drugs in the absence of
wild-type TNF-L or TNFR-L protein or in the presence of altered
forms of the TNF-L or TNFR-L protein in the mammal or cell.
Knock-out mammals can also be used to develop therapeutic
treatments for diseases associated with alterations in TNF-L or
TNFR-L gene expression, such as neoplasia or various autoimmune
diseases.
[0144] The invention also provides screening methods which can be
used to identify chemical agents which may have use in therapy, for
example, regulators of the disclosed genes and proteins can be
screened using a variety of methods. These include ligand binding
(Zhang et al., J. Biol. Chem 267:24069-24075), cytotoxicity
(Creasey, Cancer Res. 47:145-149, 1987, Geigert, Develop. Biol.
Standard 69.129; Tsujimoto, J. Biochem. 101:919-925, 1987; Kamijo,
Biochem. Biophys. Res. Commun. 160:830-825, 1989; Sidhu, Anticancer
Res. 9:1569-1576, 1989), differentiation (Kamijo, 1989), maturation
of osteoclasts from hematopoietic precursors (Lacey, Endocrinology
136:2367-2376, 1995), and proliferation (Tsujimoto, 1989).
[0145] The ability of a test compound or a potential therapeutic
agent to stimulate or inhibit activity of a TNF-L or TNFR-L protein
can be assessed by determining or measuring the viability of the
population of cells A test compound which increases or decreases
cell lysis or cell death is a modulator of the TNF-L or TNF-LR
protein and can be used as a therapeutic agent to regulate TNF
activities, such as cell lysis or cell death. A test compound which
increases cell lysis or cell death may be particularly useful in
treatment of neoplastic growth. The polypeptide of the invention
can be applied to the cell exogenously, or it can be expressed by a
cell which has been transfected with a subgenomic polynucleotide
encoding the polypeptide.
[0146] Methods for measuring the viability of cells can be any
which are known in the art. Cell death can be determined by
contacting the cell with a dye and viewing it under a microscope
Viable cells can be observed to have an intact membrane and do not
stain, whereas dying or dead cells having "leaky" membranes do
stain. Incorporation of the dye by the cell indicates the death of
the cell The most common dye used in the art for this purpose is
trypan blue. Viability of cells can also be determined by detecting
DNA synthesis. Cells can be cultured in cell medium with labeled
nucleotides, such as [.sup.3H]-thymidine. The uptake or
incorporation of the labeled nucleotides by cells indicates DNA
synthesis and cell viability. Death of tumor cells in vivo can be
monitored by observing regression or shrinkage of a tumor. Any
suitable diagnostic technique can be applied.
[0147] Other cellular proteins which are involved in the same
biological pathways can be identified by looking for proteins which
interact with the disclosed polypeptides. Natural ligands can
therefore be identified for the receptor proteins, and natural
receptor proteins can be identified for the ligands. Complex
formation can be detected in vitro or in vivo. Many methods for
detecting formation of protein complexes are known in the art, and
any such methods can be used. For example, the yeast two-hybrid
system can be used in cells to detect proteins which interact with
the disclosed ligands and receptors. Alternatively, protein complex
formation can be tested in vitro and complexes detected by altered
mobility on non-denaturing gels, or by co-immunoprecipitation.
[0148] Expression of TNFR-L proteins can serve as a marker of
neoplasia. TNFR-L proteins can be detected in body samples,
including tissues, serum, urine, sputum, and feces, using
immunological techniques. Expression can also be observed by
measuring or detecting mRNA encoding the receptors. Any suitable
technique can be used including but not limited to Northern
blotting and RT-PCR.
[0149] A TNF-R or TNFR-L subgenomic polynucleotide can also be
delivered to subjects for the purpose of screening test compounds
for those which are useful for enhancing transfer of TNF-L or
TNFR-L subgenomic polynucleotides to the cell or for enhancing
subsequent biological effects of TNF-L or TNFR-L subgenomic
polynucleotides within the cell Such biological effects include
hybridization to complementary TNF-L or TNFR-L mRNA and inhibition
of its translation, expression of a TNF-L or TNFR-L subgenomic
polynucleotide to form a TNF-L or TNFR-L mRNA, single-chain
antibody, ribozyme, oligonucleotide, or protein and/or replication
and integration of a TNF-L or TNFR-L subgenomic polynucleotide. The
subject can be a cell culture or an animal, preferably a mammal
more preferably a human.
[0150] Test compounds which can be screened include any substances,
whether natural products or synthetic, which can be administered to
the subject in vitro or in vivo Libraries or mixtures of test
compounds can be tested The test compound can be a pharmacologic
agent already known in the art or can be a compound previously
unknown to have any pharmacological activity. The test compound can
be naturally occurring or designed in the laboratory. It can be
isolated from microorganisms, animals, or plants, and can be
produced recombinantly, or synthesized by chemical methods known in
the art. Test compounds or substances can be delivered before,
after, or concomitantly with a TNF-L or TNFR-L subgenomic
polynucleotide. They can be administered separately or in admixture
with a TNF-L or TNFR-L subgenomic polynucleotide.
[0151] Integration of a delivered TNF-L or TNFR-L subgenomic
polynucleotide can be monitored by any means known in the art. For
example, Southern blotting of the delivered TNF-L or TNFR-L
subgenomic polynucleotide can be performed. A change in the size of
the fragments of a delivered polynucleotide indicates integration.
Replication of a delivered polynucleotide can be monitored inter
alia by detecting incorporation of labeled nucleotides combined
with hybridization to a TNF-L or TNFR-L probe. Expression of a
TNF-L or TNFR-L subgenomic polynucleotide can be monitored by
detecting production of TNF-L or TNFR-L mRNA which hybridizes to
the delivered polynucleotide or by detecting TNF-L or TNFR-L
protein. TNF-L or TNFR-L protein can be detected immunologically.
Thus, the delivery of TNF-L or TNFR-L subgenomic polynucleotides
according to the present invention provides an excellent system for
screening test compounds for their ability to enhance transfer of
TNF-L or TNFR-L polynucleotides to a cell, by enhancing delivery,
integration, hybridization, expression, replication or integration
in a cell vitro or in vivo in an animal, preferably a mammal, more
preferably a human.
[0152] The TNFL1 gene (SEQ ID NO:6) maps to human chromosome 13q34.
Polynucleotide probes of TNFL1 can therefore be used to identify
this region of chromosome 13 in metaphase spreads of human
chromosomes. Preparations of human metaphase chromosomes can be
prepared using standard cytogenetic techniques from human primary
tissues or cell lines. Polynucleotide probes comprising at least 12
contiguous nucleotides selected from the nucleotide sequence shown
in SEQ ID NO:6 are used to identify the human chromosome The
polynucleotide probes can be labeled, for example, with a
radioactive, fluorescent, biotinylated, or chemiluminescent label,
and detected by well known methods appropriate for the particular
label selected. Protocols for hybridizing polynucleotide probes to
preparations of metaphase chromosomes are also well known in the
art. A polynucleotide probe will hybridize specifically to
nucleotide sequences in the chromosome preparations which are
complementary to the nucleotide sequence of the probe.
[0153] A polynucleotide probe which hybridizes specifically to
human chromosome region 13q34 hybridizes to nucleotide sequences
present in the TNFL1 gene and not to nucleotide sequences present
in other human genes. A polynucleotide probe which hybridizes
specifically to an TNFL1 gene provides a detection signal at least
5-, 10-, or 20-fold higher than the background hybridization
provided with non-TNFL1 coding sequences.
[0154] A human chromosome which specifically hybridizes to an TNFL1
polynucleotide probe is identified as a human chromosome 13.
Preferably, the polynucleotide probe identifies the long arm of
human chromosome 13. More preferably, the polynucleotide probe
identifies a q34 region of human chromosome 13.
[0155] The complete contents of the references cited in this
disclosure are expressly incorporated by reference herein The
following examples are illustrative and are not meant to limit the
scope of the invention disclosed herein.
EXAMPLE 1
[0156] This example describes cloning of the fill-length cDNA for
TNFL1.
[0157] TNFL1 was first identified from a database of expressed
sequence tags (ESTs) by its homology to other members of the TNF
family. The full-length cDNA was isolated by screening a liver cDNA
library applying the genetrapper technique (Gibco). A liver library
from Gibco BRL was screened using the Genetrapper cDNA positive
selection system (catalog no 10356-020) and two oligonucleotide
primers. The sequence of the biotinylated primer is:
5'AGGTCCATGTCTTTGGG3' (SEQ ID NO:11) the sequence of the non
biotinylated primer is: 5'GGGGATGAATTGAGTCTG3' (SEQ ID NO:12). The
product of the repair reaction was transformed, plated on LB+ Amp
(100 .mu.g/ml) plates. The colonies were analyzed by colony
hybridization with a radioactive fragment prepared by PCR using the
primers 5'GTGCCCTCGAAGAAAAAG3' (SEQ ID NO:13) and
5'GCAAGTTGGAGTTCATC3' (SEQ ID NO:14).
[0158] The longest open reading frame was 1280 bp long and
contained a poly A tail as well as an in-frame stop codon at
position -117 upstream of the ATG at position +1, suggesting that
this clone was full-length. The nucleotide sequence surrounding
this ATG also matched the Kozak consensus sequence. The open
reading frame encodes a protein of 285 amino acids which we named
Tumor Necrosis Factor Like 1 (TNFL1 ) (FIG. 1A).
[0159] The lack of a signal sequence at the N-terminus and the
presence of an internal hydrophobic domain are indicative of a type
II transmembrane structure, which is similar to the structure of
most of the other members of the TNF family with the exception of
lymphotoxin a. Two potential N-glycosylation sites were also
identified in the extracellular region of the protein. When aligned
with the extra-cellular domains from other members of the TNF
family, the extracellular domain of the TNFL1 protein showed an
overall homology of 28% to the proteolytically cleaved form of TNF
(FIG. 1B).
EXAMPLE 2
[0160] This example shows the tissue distribution of TNFL1
mRNA.
[0161] Northern blots showing mRNAs from different tissues and
cancerous cell lines were purchased from Clontech. A Northern blot
with mRNAs from hematopoietic cell lines and various cell types of
the immune system was prepared with 2 .mu.g of poly A mRNA.
[0162] A probe prepared by digestion of the TNFL1 cDNA with EcoRI
and XhoI was labeled by random priming with .sup.35S and Klenow
enzyme (Rediprime kit from Amersham). The hybridization was
performed in the Expresshyb buffer purchased from Clontech.
[0163] A 3 kb messenger mRNA corresponding to TNFL1 mRNA was
detected mainly in the organs of the immune system, such as
peripheral blood lymphocytes, spleen, and thymus, as well as in the
small intestine and ovary (FIG. 2A) Human TNFL1 mRNA was also
detected in a few human cancer cell lines such as the chronic
myelogenous leukemia cell line K562 and the melanoma cell line G361
(FIG. 2A). Mouse mRNA was detected in heart, spleen, and lung using
as a probe a mouse EST sequence which is homologous to the human
TNFL1 sequence (Accession No. AA254417) (FIG. 2B).
[0164] Because TNFL1 mRNA was expressed in the spleen of both mouse
and human samples as well as in peripheral blood leukocytes, a more
precise analysis of the protein expression levels was carried out
in the same tissues.
EXAMPLE 3
[0165] This example demonstrates expression of protein levels in
mouse and human tissues.
[0166] A polyclonal antibody (D2710) was raised against amino acids
234-248 of TNFL1 and purified on a protein G column followed by a
peptide affinity column. Amino acids 234-248 are highly conserved
between the human and the mouse protein and differ by only 4 amino
acids.
[0167] This antibody was able to recognize a purified 30 kDa TNFL1
protein by Western blot analysis (FIG. 3A, lane 5). A single band
corresponding to a 45 kDa protein was detected in cytoplasmic
extracts from mouse bone marrow-derived dendritic cells and human
monocytes. Both the 30 kDa and the 45 kDa bands were absent after
incubation of D2710 with an excess of competitor peptide (FIG. 3A,
lanes 2, 4, and 6), suggesting that the 45 kDa protein corresponds
to the full-length TNFL1 protein.
[0168] The affinity purified antibody was also able to detect TNFL1
expressed in insect cells. Insect cells were infected with a
recombinant baculovirus expressing the TNFL1 protein and analyzed
by flow cytometry. The protein TNFL1 was detected by intracellular
staining with D2710 after fixation and permeabilization of the
cells infected with the recombinant virus but not the wild type
virus (FIG. 3B).
[0169] Immunohistochemistry experiments were performed on sections
from mouse spleen and lymphoid organs using the polyclonal antibody
D2710. The spleen was isolated from an animal perfused with 4%
paraformaldehyde in PBS, incubated in the same solution for one
additional hour, and incubated overnight at 4.degree. C. in a 10%
sucrose solution. The spleen was then embedded in OCT prior to
cryo-sectioning. The sections were stored at -80.degree. C.
Immunostaining was carried out using the following protocol. The
sections were blocked in normal donkey IgG (whole molecule H+L;
Jackson 017 000 003; lot 39113 at 25 5 g/l) diluted 1.multidot.100
in 1.times.PBS and Fc block diluted 1:50 (Pharmingen, catalog no.
0124A). The sections were then incubated with primary antibodies
D2710, anti-mouse CD11c (HL3, Pharmingen, catalog no 09702D),
anti-mouse Th1.2 CD90.2 (53-2.1, Pharmingen, catalog no. 01122A),
or anti-mouse CD45R/B220 (RA3-6B2, Pharmingen, catalog no. 01122A)
diluted 1:50 in blocking reagent. The sections were washed three
times for 3 minutes each in PBS.
[0170] The sections were then incubated in secondary antibody
(biotin-labeled donkey anti-rabbit F(ab).sub.2 (Jackson
711-066-152) or biotin-labeled donkey anti-rat F(ab').sub.2(Jackson
721-066-153) diluted 1:100 in PBS, washed 3 times for 3 min in PBS,
and incubated for 30 minutes at room temperature with
pre-equilibrated ABC-Alkaline Phosphatase reagent (Vector) or
ABC-peroxidase (Vector). The sections were again washed 3 times for
3 minutes in PBS and incubated in a color developing reaction with
levamisole (Vector; SK 5000) using a Vector black AP substrate kit
(SK5200), Vector red AP substrate kit (SK 5100), or Vector AEC
peroxidase substrate kit (SK4200). After washing again in PBS,
alkaline phosphatase stained sections were counterstained with
hematoxylin nuclear counterstain (Vector; H3401) and methyl green
(Vector; H3402). Other sections were mounted in fluoromount
(Southern Biotechnology Associates, catalog no 100-01, Fisher
OB100-01).
[0171] Normal rabbit IgG (R& D Systems, catalog no. AB-105C) at
a 1:50 dilution, secondary antibody at a dilution of 1.100, and
secondary antibody alone at a dilution of 1.100 were used as
negative controls.
[0172] TNFL1 was constitutively and specifically expressed as a
cell surface-bound protein in normal spleen (FIG. 3C), but was
weakly expressed in lymph nodes, mesenteric lymph nodes, and
Peyer's patches. In the spleen, the pattern of expression was
restricted to the marginal zone and the red pulp. The region
stained with monoclonal antibodies directed against markers of the
T cell population and of the B cell zone (Th1-2 and B220
respectively) did not overlap with the region stained with the
antibody specific for TNFL1 (FIG. 3D) A monoclonal antibody
directed against the dendritic cell marker CD11c stained the T cell
area and the marginal zone, as well as some isolated cell in the
red pulp (FIG. 3D)
[0173] Although it is possible that some cell sub-types present
both the CD11c antigen and the TNFL1 protein at their surface,
TNFL1 does not seem to be an exclusive marker of dendritic cells in
the spleen The Mac-3 antigen, a marker for macrophages and
monocytes mainly localized in the red pulp, showed a pattern of
expression very similar to the one observed with TNFL1 (FIG. 3D).
Overall, these results suggest that TNFL1 is expressed at the
surface of splenic macrophages, monocytes, or dendritic cells
usually present in the marginal zone and the red pulp.
[0174] Flow cytometry experiments were performed on human PBMCs
isolated from whole blood by Ficoll gradient centrifugation and the
PBMCs analyzed. TNFL1 was found to be constitutively expressed on
monocytes and B cells, but not on resting CD4+ and CD8+ T cells.
Mouse bone marrow-derived dendritic cells cultivated for 10 days in
the presence of GM-CSF also showed some surface staining with the
TNFL1 antibody (FIG. 4A). After incubation of PBMCs with anti-CD3
and anti-CD28 antibodies for 6 days in the presence of IL2, TNFL1
was shown to be upregulated at the surface of T cells (FIG.
4B).
EXAMPLE 4
[0175] This example demonstrates expression of a soluble form of
TNFL1 in the periplasm of E. coli.
[0176] A chimeric soluble version of TNFL1 was expressed in E. coli
as a fusion protein comprising the extracellular portion of TNFL1
(amino acids 113-285; FIG. 5A) and the pelB signal sequence for
periplasmic localization. The EYMPMD peptide (SEQ ID NO:15) was
inserted between the signal sequence and the TNFL1 sequence for
convenient affinity purification. The cDNA for TNFL1 was cloned
into the vector pET-22b(+) from Invitrogen, which contains the pelB
signal sequence for periplasmic localization. A 100 ml culture was
grown at 37.degree. C. until it reached an OD of 0.7-0.9 and then
grown at 25.degree. C. for 24 hours after induction by 1 mM IPTG.
The pellet was centrifuged at 4000.times.g for 10 minutes and
resuspended in 10 ml 30 mM Tris HCl, 20% sucrose, pH 8.0. After
addition of 1 mM EDTA, the sample was incubated at room temperature
for 5-10 min. The sample was then centrifuged at 8000.times.g at
4.degree. C. for 10 min. The supernatant was removed, and the
pellet was resuspended in 10 ml ice-cold 5 mM MgSO.sub.4. After a
10 minute incubation in an ice/water bath, the sample was
centrifuged at 8000.times.g at 4.degree. C. for 10 min. The
supernatant containing the periplasmic fraction was stored at
-80.degree. C. in 15% glycerol. A control sample was processed in a
similar way with an empty vector construct.
[0177] A similar fusion construct was also designed for expression
and purification from COS cells using the pSecTag vector
(Invitrogen) with a signal sequence from the mouse Ig e chain.
EXAMPLE 5
[0178] This example demonstrates purification of the TNFL1 fusion
protein from E. coli.
[0179] BL21 (DE3) transformed E. coli were grown in a 10-1
fermenter to an OD of 29-31 before induction with IPTG The cells
were harvested in a Beckman J-6B centrifuge. The wet cell paste was
subjected to osmotic shock treatment for periplasmic extraction
(lot 10229-142) or lysozyme/EDTA spheroplast formation with
retention of the spheroplast supernatant as the periplasmic
fraction (lot 981001-M8). Using a Pall Filtron Centrasette
tangential flow apparatus and 2 Centrasette 10 kD NMWCO membranes,
the resulting periplasmic fraction was concentrated to 1 liter,
then buffer-exchanged by constant-flow diafiltration in the same
apparatus against at least 6 volumes of PBS. The resulting solution
was centrifuged at 10,000 rpm at 4.degree. C. for 50 minutes in a
Beckman J2-21 centrifuge with a JA-10 rotor
[0180] The resulting supernatant was precipitated with 50% ammonium
sulfate using an equal volume of saturated ammonium sulfate. The
resulting pellet was resuspended in 1/4 the original volume of PBS
and loaded onto a glu-tag monoclonal antibody affinity column at a
flow rate of 30 cm/hr. Following the load, a wash of 5-10 CV of PBS
+0.2% Tween 20 was performed, followed by 2 CV of PBS. Elution was
effected by 5 CV of 0.1 mg/ml EYMPTD peptide (SEQ ID NO. 16)
(Research Genetics) followed by PBS. A strip of 1.5 CV 0.1 M
glycine pH 2.7 was also collected into one-tenth volume of 1 M Tris
pH 8, and the column was neutralized with 1 CV of the same.
[0181] The peptide elution was concentrated in an Amicon 8400
stirred-cell with a YM-10 membrane. Peptide removal was effected by
overnight dialysis of the concentrate against PBS (except for the
samples treated to remove residual detergent). Endotoxin removal
was by Triton X-114 phase separation. One-ninth volume of 10%
Triton X-114 protein grade (Calbiochem) was added to samples
chilled on ice. Constant agitation for 30 min. at 4.degree. C. was
followed by 15 min. incubation in a 37.degree. C. water bath.
Centrifugation at 10,000.times.g recovered the supernatant as the
detergent-poor aqueous fraction. The cycle was repeated 3-5 times,
resulting in endotoxin levels of less than or equal to 0.05
EU/ml.
[0182] Detergent was removed by an anion-exchange chromatography
step in which the protein was bound and eluted while the detergent
and elution peptide flowed through. A Pharmacia HR 10/5 column
packed with 1.7 ml TMAE-Fractogel (EM Merck) was equilibrated in 25
mM NaPi, pH 7.4, at 1 ml/minute on an Akta Explorer FPLC system.
Fractions of 1 ml were collected across a 15 CV gradient from 0-0 6
M NaCl in 25 mM NaPi, pH 7 4. The protein eluted at a conductivity
of about 22 mS/cm. The peak fractions were pooled and concentrated,
if necessary, in a Amicon Centriplus-10 centrifugal concentration
device.
[0183] The purified protein was detected in a Western blot with
both the tag antibody and the D2710 antibody as a 30 kD protein
(FIG. 3A). A minor additional band at 21 kD was also observed. This
band was identified by microsequencing as a degradation product of
the 30 kD protein (FIG. 5A)
EXAMPLE 6
[0184] This example demonstrates the effects of TNFL1 on T and B
cells.
[0185] T cells isolated from healthy donors were activated for two
days in the presence of anti-CD3 and anti-CD28 antibodies and
incubated for an additional two days in the presence of different
concentrations of purified TNFL1. Mouse and human T cells were
purified by negative selection from PBMCs on a mouse or human T
cell enrichment column (R&D Systems), and B cells were isolated
by positive selection on Dynabeads M-450 Pan-B and detached from
the beads with the polyclonal antibody Detachabead from Dynal. The
T cells were activated for 2 days with anti-CD3 and anti-CD28
antibodies at a concentration of 10 ig/ml.
[0186] T cell proliferation was assessed by thymidine incorporation
at day 5. The assays were performed in a 96 well plate format with
100,000 cells/well. The thymidine incorporation assay was performed
by addition of .sup.3H-thymidine at a concentration of 1 mCi/well.
After 8 hours, the cells were harvested onto a filter-paper and
.sup.3H-thymidine uptake was measured by liquid scintillation
counting.
[0187] The dose-response curve shows a 30-fold decrease in
thymidine incorporation at the highest concentrations of TNFL1
(FIG. 5B, left). Resting T cells incubated with the ligand for two
days did not show any decrease in thymidine incorporation (FIG. 5B,
right).
[0188] In a separate experiment, the effect of TNFL1 was tested on
anti-CD3- and anti-CD28-activated T cells and on
anti-CD40-activated B cells. The T cells were activated for 2 days
with anti-CD3 and anti-CD28 antibodies at a concentration of 10
.mu.g/ml, and the B cells were activated for 2 days in the presence
of anti-CD40 antibody at a concentration of 10 .mu.g/ml. Both cell
types showed a strong and comparable decrease in thymidine
incorporation after addition of TNFL1 (FIG. 5C) TNFL1 also
inhibited the proliferation of murine T cells activated by
allogeneic bone marrow-derived DCs by 90%.
[0189] We also tested the ability of TNFL1 to decrease the
proliferation rate of mouse T cells activated by allogeneic bone
marrow-derived dendritic cells in a mixed lymphocyte reaction (MLR)
For the MLR reaction, 100,000 mouse T cells were mixed with 10,000
mouse dendritic cells Dendritic cells were prepared from red blood
cell-depleted bone marrow cells. The cells were resuspended in
culture medium in a bacterial Petri dish at a concentration of
0.2.times.10.sup.6 cells per ml. Recombinant mouse GM-CSF was added
to the cells at a final concentration of 200 U/ml on days 1, 2, 3,
6 and 8. On day 10, LPS at 1 .mu.g/ml or TNF-.alpha. at 500 U/ml
was added to the cells The cells were harvested on day 11.
[0190] In the absence of TNFL1, the cultured DCs triggered a
100-fold increase in thymidine incorporation in the responding T
cells. When the assay was performed in the presence of TNFL1, the
increase in thymidine incorporation was only 10-fold, showing that
TNFL1 is able to reduce the T cell stimulation induced by
allogeneic antigen presenting cells.
[0191] Overall, these results indicate that TNFL1 is able to induce
a decrease in thymidine incorporation in activated B and T cells.
This effect could be due to a direct inhibition of proliferation or
to the induction of apoptosis of B and T cells.
[0192] In order to elucidate the exact mechanism of action of TNFL1
on activated T cells, a TUNEL assay (TdT-mediated dUTP-FITC
nick-end labeling; Boehringer Mannheim) was performed according to
the manufacturer's specifications. Briefly, treated cells were
washed twice in PBS supplemented with 1% bovine serum albumin,
fixed with 0.4% paraformaldehyde, and permeabilized in 0.1% Triton
X-100 in 0.1% sodium citrate. TUNEL staining was performed with 50
.mu.l of TUNEL mix per sample at 37.degree. C. for 1 hour. Negative
controls were treated similarly but were not exposed to the
enzyme.
[0193] After incubation of the T cells with TNFL1, the amount of
apoptotic cells measured by flow cytometry analysis (FIG. 5D). The
blasted cells, visualized by forward size scattering (region R1),
almost totally disappeared after addition of TNFL1. At the same
time, the amount of apoptotic, FITC-positive cells (region R2)
dramatically increased. No apoptotic cells were detected in the
absence of dUTP-FITC, whether or not TNFL1 was added.
[0194] These results strongly suggest that TNFL1 is able to induce
apoptotic cell death of blasting as well as of slightly activated T
cells.
EXAMPLE 7
[0195] This example demonstrates that TNFL1 activates NF.kappa.B
and leads to its translocation to the nucleus.
[0196] TNF activates the transcription factor NF.kappa.B.
NF.kappa.B represents a family of related proteins involved in the
transcriptional control of numerous cellular genes such as
interleukin-2, interleukin-2 receptor, .beta.-interferon,
granulocyte macrophage colony-stimulating factor,
histocompatibility antigens, TNF, and lymphotoxin a. We therefore
decided to test whether TNFL1 could induce a similar intracellular
response in Jurkat cells.
[0197] Jurkat cells (10.sup.7) were incubated for one hour with PMA
at a concentration of 1 .mu.g/ml or TNFL1 at a concentration of 3
.mu.g/ml in 1 ml of RPMI and 10% FBS. Nuclear extracts were
prepared by centrifuging the cells at 2000 rpm and resuspending the
pellet in 1 ml of cold buffer A (10 mM Hepes, 1.5 mM MgCl.sub.2, 10
mM KCl, 0.5 mM DTT, 0.5 mM PMSF). After a second centrifugation at
2000 rpm, the pellet was resuspended in 20 .mu.l of buffer A and
0.1% NP40, incubated for 10 minutes on ice, and then centrifuged.
The pellet was resuspended in 15 .mu.l of buffer C (20 mM Hepes, pH
7.9, 0.42 M NaCl, 1.5 MM MgCl.sub.2, 0.2 mM EDTA, pH 7.4, 25%
glycerol, 0.5 mM PMSF, 0.5 mM DTT, 50 .mu.g/ml leupeptin, 50
.mu.g/ml pepstatin, and 78 .mu.g/ml benzamidin). The mixture was
incubated for 15 minutes on ice, then centrifuged for 10 minutes at
14,000 rpm at 4.degree. C. The supernatant was diluted in 75 .mu.l
of buffer D (20 mM Hepes, pH 7.9, 20% glycerol, 0.2 mM EDTA, pH
7.4, 50 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF).
[0198] Five .mu.g of nuclear extract was incubated for 10 minutes
at 4.degree. C. in the presence of 5 .mu.g of polydIdc and
wild-type or mutated competitor oligonucleotide (2 ng to 200 ng).
Two-tenths of a nanogram of polynucleotide kinase-radiolabeled
probe was added to the reaction, and the mixture was incubated at
room temperature for 30 min.
[0199] The mixture was loaded on a 5% acrylamide 60:1.times.linked
gel in Tris-glycine buffer, pH 8.3, and analyzed in an
electrophoretic mobility shift assay. Specific bands, identified by
competition with wild-type or mutated NF.kappa.B binding sites,
were observed with both PMA- and TNFL 1-treated extracts (FIG. 5E),
indicating that TNFL1 is indeed able to activate NF.kappa.B and
leads to its translocation to the nucleus.
[0200] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
25 1 285 PRT human 1 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg
Leu Thr Ser Cys Leu 1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys
Glu Cys Val Ser Ile Leu Pro 20 25 30 Arg Lys Glu Ser Pro Ser Val
Arg Ser Ser Lys Asp Gly Lys Leu Leu 35 40 45 Ala Ala Thr Leu Leu
Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val 50 55 60 Ser Phe Tyr
Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg 65 70 75 80 Ala
Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90
95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu
100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser
Gln Asn 115 120 125 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu
Thr Val Thr Gln 130 135 140 Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu
Thr Pro Thr Ile Gln Lys 145 150 155 160 Gly Ser Tyr Thr Phe Val Pro
Trp Leu Leu Ser Phe Lys Arg Gly Ser 165 170 175 Ala Leu Glu Glu Lys
Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr 180 185 190 Phe Phe Ile
Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met 195 200 205 Gly
His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu 210 215
220 Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu
225 230 235 240 Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu
Glu Glu Gly 245 250 255 Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn
Ala Gln Ile Ser Leu 260 265 270 Asp Gly Asp Val Thr Phe Phe Gly Ala
Leu Lys Leu Leu 275 280 285 2 153 PRT human 2 Leu Glu Arg Cys Arg
Tyr Cys Asn Val Leu Cys Gly Glu Arg Glu Glu 1 5 10 15 Glu Ala Arg
Ala Cys His Ala Thr His Asn Arg Ala Cys Arg Cys Arg 20 25 30 Thr
Gly Phe Phe Ala His Ala Gly Phe Cys Leu Glu His Ala Ser Cys 35 40
45 Pro Pro Gly Ala Gly Val Ile Ala Pro Gly Thr Pro Ser Gln Asn Thr
50 55 60 Gln Cys Gln Pro Cys Pro Pro Gly Thr Phe Ser Ala Ser Ser
Ser Ser 65 70 75 80 Ser Glu Gln Cys Gln Pro His Arg Asn Cys Thr Ala
Leu Gly Leu Ala 85 90 95 Leu Asn Val Pro Gly Ser Ser Ser His Asp
Thr Leu Cys Thr Ser Cys 100 105 110 Thr Gly Phe Pro Leu Ser Thr Arg
Val Pro Gly Ala Glu Glu Cys Glu 115 120 125 Arg Ala Val Ile Asp Phe
Val Ala Phe Gln Asp Ile Ser Ile Lys Arg 130 135 140 Leu Gln Arg Leu
Leu Gln Ala Leu Glu 145 150 3 210 PRT human 3 Met Ala Leu Lys Val
Leu Pro Leu His Arg Thr Val Leu Phe Ala Ala 1 5 10 15 Ile Leu Phe
Leu Leu His Leu Ala Cys Lys Val Ser Cys Glu Thr Gly 20 25 30 Asp
Cys Arg Gln Gln Glu Phe Lys Asp Arg Ser Gly Asn Cys Val Leu 35 40
45 Cys Lys Gln Cys Gly Pro Gly Met Glu Leu Ser Lys Glu Cys Gly Phe
50 55 60 Gly Tyr Gly Glu Asp Ala Gln Cys Val Pro Cys Arg Pro His
Arg Phe 65 70 75 80 Lys Glu Asp Trp Gly Phe Gln Lys Cys Lys Pro Cys
Ala Asp Cys Ala 85 90 95 Leu Val Asn Arg Phe Gln Arg Ala Asn Cys
Ser His Thr Ser Asp Ala 100 105 110 Val Cys Gly Asp Cys Leu Pro Gly
Phe Tyr Arg Lys Thr Lys Leu Val 115 120 125 Gly Phe Gln Asp Met Glu
Cys Val Pro Cys Gly Asp Pro Pro Pro Pro 130 135 140 Tyr Glu Pro His
Cys Thr Ser Lys Val Asn Leu Val Lys Ile Ser Ser 145 150 155 160 Thr
Val Ser Ser Pro Arg Asp Thr Ala Val Ala Ala Val Ile Cys Ser 165 170
175 Ala Leu Ala Thr Val Leu Leu Ala Cys Ser Ser Cys Val Ser Ser Thr
180 185 190 Ala Arg Gly Ser Ser Trp Arg Arg Asn Pro Ala Val Ser Ser
His Pro 195 200 205 Ser Val 210 4 151 PRT human 4 Met Ala Leu Lys
Val Leu Pro Leu His Arg Thr Val Leu Phe Ala Ala 1 5 10 15 Ile Leu
Phe Leu Leu His Leu Ala Cys Lys Val Ser Cys Glu Thr Gly 20 25 30
Asp Cys Ser Arg Gln Gln Glu Phe Lys Asp Arg Ser Gly Asn Cys Val 35
40 45 Leu Cys Lys Gln Cys Gly Pro Gly Met Glu Leu Ser Lys Glu Cys
Gly 50 55 60 Phe Gly Tyr Gly Glu Asp Ala Gln Cys Val Pro Cys Arg
Pro His Arg 65 70 75 80 Phe Lys Glu Asp Trp Gly Phe Gln Lys Cys Lys
Pro Cys Ala Asp Cys 85 90 95 Ala Leu Val Asn Arg Phe Gln Arg Ala
Asn Cys Ser His Thr Ser Asp 100 105 110 Ala Val Cys Gly Asp Cys Leu
Pro Gly Phe Tyr Arg Lys Thr Lys Leu 115 120 125 Val Gly Phe Gln Asp
Met Glu Cys Val Pro Cys Gly Asp Pro Pro Pro 130 135 140 Pro Tyr Glu
Pro His Cys Glu 145 150 5 205 PRT human 5 Met Val Gln Leu Thr Gln
Gln Thr Glu Leu Gln Ser Leu Arg Arg Glu 1 5 10 15 Val Ser Arg Leu
Gln Gly Thr Gly Gly Pro Ser Gln Asn Gly Glu Gly 20 25 30 Tyr Pro
Trp Gln Ser Leu Pro Glu Gln Ser Ser Asp Ala Leu Glu Ala 35 40 45
Trp Glu Asn Gly Glu Arg Ser Arg Lys Arg Arg Ala Val Leu Thr Gln 50
55 60 Lys Gln Lys Lys Gln His Ser Val Leu His Leu Val Pro Ile Asn
Ala 65 70 75 80 Thr Ser Lys Asp Asp Ser Asp Val Thr Glu Val Met Trp
Gln Pro Ala 85 90 95 Leu Arg Arg Gly Arg Gly Leu Gln Ala Gln Gly
Tyr Gly Val Arg Ile 100 105 110 Gln Asp Ala Gly Val Tyr Leu Leu Tyr
Ser Gln Val Leu Phe Gln Asp 115 120 125 Val Thr Phe Thr Met Gly Gln
Val Val Ser Arg Glu Gly Gln Gly Arg 130 135 140 Gln Glu Thr Leu Phe
Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp 145 150 155 160 Arg Ala
Tyr Asn Ser Gln Tyr Ser Ala Gly Val Pro His Leu His Gln 165 170 175
Gly Asp Ile Leu Ser Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn 180
185 190 Leu Ser Pro His Gly Thr Phe Leu Gly Phe Val Lys Leu 195 200
205 6 1284 DNA human 6 accggtccgg aattcccggg tcgacccacg cgtccgccca
cgcgtccgag aagactttga 60 aattcttaca aaaactgaaa gtgaaatgag
gaagacagat tgagcaatcc aatcggaggg 120 taaatgccag caaacctact
gtacagtagg ggtagagatg cagaaaggca gaaaggagaa 180 aattcaggat
aactctcctg aggggtgagc caagccctgc catgtagtgc acgcaggaca 240
tcaacaaaca cagataacag gaaatgatcc attccctgtg gtcacttatt ctaaaggccc
300 caaccttcaa agttcaagta gtgatatgga tgactccaca gaaagggagc
agtcacgcct 360 tacttcttgc cttaagaaaa gagaagaaat gaaactgaag
gagtgtgttt ccatcctccc 420 acggaaggaa agcccctctg tccgatcctc
caaagacgga aagctgctgg ctgcaacctt 480 gctgctggca ctgctgtctt
gctgcctcac ggtggtgtct ttctaccagg tggccgccct 540 gcaaggggac
ctggccagcc tccgggcaga gctgcagggc caccacgcgg agaagctgcc 600
agcaggagca ggagccccca aggccggcct ggaggaagct ccagctgtca ccgcgggact
660 gaaaatcttt gaaccaccag ctccaggaga aggcaactcc agtcagaaca
gcagaaataa 720 gcgtgccgtt cagggtccag aagaaacagt cactcaagac
tgcttgcaac tgattgcaga 780 cagtgaaaca ccaactatac aaaaaggatc
ttacacattt gttccatggc ttctcagctt 840 taaaagggga agtgccctag
aagaaaaaga gaataaaata ttggtcaaag aaactggtta 900 cttttttata
tatggtcagg ttttatatac tgataagacc tacgccatgg gacatctaat 960
tcagaggaag aaggtccatg tctttgggga tgaattgagt ctggtgactt tgtttcgatg
1020 tattcaaaat atgcctgaaa cactacccaa taattcctgc tattcagctg
gcattgcaaa 1080 actggaagaa ggagatgaac tccaacttgc aataccaaga
gaaaatgcac aaatatcact 1140 ggatggagat gtcacatttt ttggtgcatt
gaaactgctg tgacctactt acaccatgtc 1200 tgtagctatt ttcctccctt
tctctgtacc tctaagaaga aagaatctaa ctgaaaatac 1260 aaaaaaaaaa
aaaaaaaaaa aaaa 1284 7 459 DNA human 7 ctggagcgct gccgctactg
caacgtcctc tgcggggagc gtgaggagga ggcacgggct 60 tgccacgcca
cccacaaccg tgcctgccgc tgccgcaccg gcttcttcgc gcacgctggt 120
ttctgcttgg agcacgcatc gtgtccacct ggtgccggcg tgattgcccc gggcaccccc
180 agccagaaca cgcagtgcca gccgtgcccc ccaggcacct tctcagccag
cagctccagc 240 tcagagcagt gccagcccca ccgcaactgc acggccctgg
gcctggccct caatgtgcca 300 ggctcttcct cccatgacac cctgtgcacc
agctgcactg gcttccccct cagcaccagg 360 gtaccaggag ctgaggagtg
tgagcgtgcc gtcatcgact ttgtggcttt ccaggacatc 420 tccatcaaga
ggctgcagcg gctgctgcag gccctcgag 459 8 893 DNA human 8 tccggcgccg
cggggcagga caaggggaag gaataaacac gtttggtgag agccatggca 60
ctcaaggtcc tacctctaca caggacggtg ctcttcgctg ccattctctt cctactccac
120 ctggcatgta aagtgagttg cgaaaccgga gattgcaggc agcaggaatt
caaggatcga 180 tctggaaact gtgtcctctg caaacagtgc ggacctggca
tggagttgtc caaggaatgt 240 ggcttcggct atggggagga tgcacagtgt
gtgccctgca ggccgcaccg gttcaaggaa 300 gactggggtt tccagaagtg
taagccatgt gcggactgtg cgctggtgaa ccgctttcag 360 agggccaact
gctcacacac cagtgatgct gtctgcgggg actgcctgcc aggattttac 420
cggaagacca aactggttgg ttttcaagac atggagtgtg tgccctgcgg agacccacct
480 cctccctacg aaccacactg taccagcaag gtgaaccttg tgaagatctc
ctccaccgtc 540 tccagccctc gggacacggc ggtggctgcc gtcatctgca
gtgctctggc cacggtgctg 600 ctcgcctgct catcctgtgt gtcatctact
gcaagaggca gttcatggag aagaaaccca 660 gctgtaagct cccatccctc
tgtctcactg tgaagtgagc ttgttagcat tgtcacccaa 720 gagttctcaa
gacacctggc tgagacctaa gacctttaga gcatcaacag ctacttagaa 780
tacaagatgc aggaaaacga gcctcttcag gaatctcagg gcctcctagg gatgctggca
840 aggctgtgat gtctcaaggc taccaggaaa aaataaaagt tgtctatacc cta 893
9 623 DNA human 9 gaggcaagat tcggcacgag ggcgtttggc gcggaagtgc
taccaagctg cggaaagcgt 60 gagtctggag cacagcactg gcgagtagca
ggaataaaca cgtttggtga gagccatggc 120 actcaaggtc ctacctctac
acaggacggt gctcttcgct gccattctct tcctactcca 180 cctggcatgt
aaagtgagtt gcgaaaccgg agattgcagg cagcaggaat tcaaggatcg 240
atctggaaac tgtgtcctct gcaaacagtg cggacctggc atggagttgt ccaaggaatg
300 tggcttcggc tatggggagg atgcacagtg tgtgccctgc aggccgcacc
ggttcaagga 360 agactggggt ttccagaagt gtaagccatg tgcggactgt
gcgctggtga accgctttca 420 gagggccaac tgctcacaca ccagtgatgc
tgtctgcggg gactgcctgc caggatttta 480 ccggaagacc aaactggttg
gttttcaaga catggagtgt gtgccctgcg gagacccacc 540 tcctccctac
gaaccacact gtgagtgatg tgccaagtgg cagcagacct ttaaaaaaaa 600
aagaaaaaaa aacaaacaaa aac 623 10 1260 DNA human 10 cttcctagag
ggactggaac ctaattctcc tgaggctgag ggagggtgga gggtctcaag 60
gcaacgctgg ccccacgacg gagtgccagg agcactaaca gtacccttag cttgctttcc
120 tcctccctcc tttttatttt caagttcctt tttatttctc cttgcgtaac
aaccttcttc 180 ccttctgcac cactgcccgt acccttaccc gccccgccac
ctccttgcta ccccactctt 240 gaaaccacag ctgttggcag ggtccccagc
tcatgccagc ctcatctcct ttcttgctag 300 cccccaaagg cctccaggca
acatgggggg cccagtcaga gagccggcac tctcagttgc 360 cctctggttg
agttgggggg cagctctggg ggccgtggtt tgtgcatggt tcagctgacc 420
caacaaacag agctgcagag cctcaggaga gaggtgagcc ggctgcaggg gacaggaggc
480 ccctcccaga atggggaagg gtatccctgg cagagtctcc cggagcagag
ttccgatgcc 540 ctggaagcct gggagaatgg ggagagatcc cggaaaagga
gagcagtgct cacccaaaaa 600 cagaagaagc agcactctgt cctgcacctg
gttcccatta acgccacctc caaggatgac 660 tccgatgtga cagaggtgat
gtggcaacca gctcttaggc gtgggagagg cctacaggcc 720 caaggatatg
gtgtccgaat ccaggatgct ggagtttatc tgctgtatag ccaggtcctg 780
tttcaagacg tgactttcac catgggtcag gtggtgtctc gagaaggcca aggaaggcag
840 gagactctat tccgatgtat aagaagtatg ccctcccacc cggaccgggc
ctacaacagc 900 tgctatagcg caggtgtctt ccatttacac caaggggata
ttctgagtgt cataattccc 960 cgggcaaggg cgaaacttaa cctctctcca
catggaacct tcctggggtt tgtgaaactg 1020 tgattgtgtt ataaaaagtg
gctcccagct tggaagacca gggtgggtac atactggaga 1080 cagccaagag
ctgagtatat aaaggagagg gaatgtgcag gaacagaggc atcttcctgg 1140
gtttggctcc ccgttcctca cttttccctt ttcattccca ccccctagac tttgatttta
1200 cggatatctt gcttctgttc cccatggagc tccgaattct tgcgtgtgtg
tagatgaggg 1260 11 17 DNA human 11 aggtccatgt ctttggg 17 12 18 DNA
human 12 ggggatgaat tgagtctg 18 13 18 DNA human 13 gtgccctcga
agaaaaag 18 14 17 DNA human 14 gcaagttgga gttcatc 17 15 6 PRT human
15 Glu Tyr Met Pro Thr Asp 1 5 16 6 PRT human 16 Glu Tyr Met Pro
Thr Glu 1 5 17 299 PRT Homo sapien 17 Met Arg Ala Leu Glu Gly Pro
Gly Leu Ser Leu Leu Cys Leu Val Leu 1 5 10 15 Ala Leu Pro Ala Leu
Leu Pro Val Pro Ala Val Arg Gly Val Ala Glu 20 25 30 Thr Pro Thr
Tyr Pro Trp Arg Asp Ala Glu Thr Gly Glu Arg Leu Val 35 40 45 Cys
Ala Gln Cys Pro Pro Gly Thr Phe Val Gln Arg Pro Cys Arg Arg 50 55
60 Asp Ser Pro Thr Thr Cys Gly Pro Cys Pro Pro Arg His Tyr Thr Gln
65 70 75 80 Phe Trp Asn Tyr Leu Glu Arg Cys Arg Tyr Cys Asn Val Leu
Cys Gly 85 90 95 Glu Arg Glu Glu Glu Ala Arg Ala Cys His Ala Thr
His Asn Arg Ala 100 105 110 Cys Arg Cys Arg Thr Gly Phe Phe Ala His
Ala Gly Phe Cys Leu Glu 115 120 125 His Ala Ser Cys Pro Pro Gly Ala
Gly Val Ile Ala Pro Gly Thr Pro 130 135 140 Ser Gln Asn Thr Gln Cys
Gln Pro Cys Pro Pro Gly Thr Phe Ser Ala 145 150 155 160 Ser Ser Ser
Ser Glu Gln Cys Gln Pro His Arg Asn Cys Thr Ala Leu 165 170 175 Gly
Leu Ala Leu Asn Val Pro Gly Ser Ser Ser His Asp Thr Leu Cys 180 185
190 Thr Ser Cys Thr Gly Phe Pro Leu Ser Thr Arg Val Pro Gly Ala Glu
195 200 205 Glu Cys Glu Arg Ala Val Ile Asp Phe Val Ala Phe Gln Asp
Ile Ser 210 215 220 Ile Lys Arg Leu Gln Arg Leu Leu Gln Ala Leu Glu
Ala Pro Glu Gly 225 230 235 240 Trp Gly Pro Thr Pro Arg Ala Gly Arg
Ala Ala Leu Gln Leu Lys Leu 245 250 255 Arg Arg Arg Leu Thr Glu Leu
Leu Gly Ala Gln Asp Gly Ala Leu Leu 260 265 270 Val Arg Leu Leu Gln
Ala Leu Arg Val Ala Arg Met Pro Gly Leu Glu 275 280 285 Arg Ser Val
Arg Glu Arg Phe Leu Pro Val His 290 295 18 1347 DNA Homo sapien 18
ccgacacacc aggctgcctg ggctggtccc tggctggtga ggcccctccc agaaccaccc
60 ttggactgag ctctggggag ggatggtacc aggtgggtga ggggggctgc
ctggggaggg 120 aggggttcct atggggcgtg gcgaggctgg cccagccctc
tccccgccca tatatgtagg 180 gcagcagcag gatgggcttc tggacttggg
cggcccctcc gcaggcggac cgggggcaaa 240 ggaggtggca tgtcggtcag
gcacagcagg gtcctgtgtc cgcgctgagc cgcgctctcc 300 ctgctccagc
aaggaccatg agggcgctgg aggggccagg cctgtcgctg ctgtgcctgg 360
tgttggcgct gcctgccctg ctgccggtgc cggctgtacg cggagtggca gaaacaccca
420 cctacccctg gcgggacgca gagacagggg agcggctggt gtgcgcccag
tgccccccag 480 gcacctttgt gcagcggccg tgccgccgag acagccccac
gacgtgtggc ccgtgtccac 540 cgcgccacta cacgcagttc tggaactacc
tggagcgctg ccgctactgc aacgtcctct 600 gcggggagcg tgaggaggag
gcacgggctt gccacgccac ccacaaccgt gcctgccgct 660 gccgcaccgg
cttcttcgcg cacgctggtt tctgcttgga gcacgcatcg tgtccacctg 720
gtgccggcgt gattgccccg ggcaccccca gccagaacac gcagtgccag ccgtgccccc
780 caggcacctt ctcagccagc agctccagct cagagcagtg ccagccccac
cgcaactgca 840 cggccctggg cctggccctc aatgtgccag gctcttcctc
ccatgacacc ctgtgcacca 900 gctgcactgg cttccccctc agcaccaggg
taccaggagc tgaggagtgt gagcgtgccg 960 tcatcgactt tgtggctttc
caggacatct ccatcaagag gctgcagcgg ctgctgcagg 1020 ccctcgaggc
cccggagggc tggggtccga caccaagggc gggccgcgcg gccttgcagc 1080
tgaagctgcg tcggcggctc acggagctcc tgggggcgca ggacggggcg ctgctggtgc
1140 ggctgctgca ggcgctgcgc gtggccagga tgcccgggct ggagcggagc
gtccgtgagc 1200 gcttcctccc tgtgcactga tcctggcccc ctcttattta
ttctacatcc ttggcacccc 1260 acttgcactg aaagaggctt ttttttaaat
agaagaaatg aggtttctta aagcttaaaa 1320 aaaaaaaaaa aaaaaaaaaa aaaaaaa
1347 19 1859 DNA Homo sapien 19 ggaggtggca tgtcggtcag gcacagcagg
gtcctgtgtc cgcgctgagc cgcgctctcc 60 ctgctccagc aaggaccatg
agggcgctgg aggggccagg cctgtcgctg ctgtgcctgg 120 tgttggcgct
gcctgccctg ctgccggtgc cggctgtacg cggagtggca gaaacaccca 180
cctacccctg gcgggacgca gagacagggg agcggctggt gtgcgcccag tgccccccag
240 gcacctttgt gcagcggccg tgccgccgag acagccccac gacgtgtggc
ccgtgtccac 300 cgcgccacta cacgcagttc tggaactacc tggagcgctg
ccgctactgc aacgtcctct 360 gcggggagcg tgaggaggag gcacgggctt
gccacgccac ccacaaccgt gcctgccgct 420
gccgcaccgg cttcttcgcg cacgctggtt tctgcttgga gcacgcatcg tgtccacctg
480 gtgccggcgt gattgccccg ggcaccccca gccagaacac gcagtgccag
ccgtgccccc 540 caggcacctt ctcagccagc agctccagct cagagcagtg
ccagccccac cgcaactgca 600 cggccctggg cctggccctc aatgtgccag
gctcttcctc ccatgacacc ctgtgcacca 660 gctgcactgg cttccccctc
agcaccaggg taccaggtga gccagaggcc tgagggggca 720 gcacactgca
ggccaggccc acttgtgccc tcactcctgc ccctgcacgt gcatctagcc 780
tgaggcatgc cagctggctc tgggaagggg ccacagtgga tttgaggggt caggggtccc
840 tccactagat ccccaccaag tctgccctct caggggtggc tgagaatttg
gatctgagcc 900 agggcacagc ctcccctgga gagctctggg aaagtgggca
gcaatctcct aactgcccga 960 ggggaaggtg gctggctcct ctgacacggg
gaaaccgagg cctgatggta attctcctaa 1020 ctgcctgaga ggaaggtggc
tgcctcctct gacatgggga aaccgaggcc caatgttaac 1080 cactgttgag
aagtcacagg gggaagtgac ccccttaaca tcaagtcagg tccggtccat 1140
ctgcaggtcc caactcgccc cttccgatgg cccaggagcc ccaagccctt gcctgggccc
1200 ccttgcctct tgcagccaag gtccgagtgg ccgctcctgc cccctaggcc
tttgctccag 1260 ctctctgacc gaaggctcct gccccttctc cagtccccat
cgttgcactg ccctctccag 1320 cacggctcac tgcacaggga tttctctctc
ctgcaaaccc cccgagtggg gcccagaaag 1380 cagggtacct ggcagccccc
gccagtgtgt gtgggtgaaa tgatcggacc gctgcctccc 1440 caccccactg
caggagctga ggagtgtgag cgtgccgtca tcgactttgt ggctttccag 1500
gacatctcca tcaagaggct gcagcggctg ctgcaggccc tcgaggcccc ggagggctgg
1560 ggtccgacac caagggcggg ccgcgcggcc ttgcagctga agctgcgtcg
gcggctcacg 1620 gagctcctgg gggcgcagga cggggcgctg ctggtgcggc
tgctgcaggc gctgcgcgtg 1680 gccaggatgc ccgggctgga gcggagcgtc
cgtgagcgct tcctccctgt gcactgatcc 1740 tggccccctc ttatttattc
tacatccttg gcaccccact tgcactgaaa gaggcttttt 1800 tttaaataga
agaaatgagg tttcttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1859 20 211
PRT Homo sapien 20 Met Arg Ala Leu Glu Gly Pro Gly Leu Ser Leu Leu
Cys Leu Val Leu 1 5 10 15 Ala Leu Pro Ala Leu Leu Pro Val Pro Ala
Val Arg Gly Val Ala Glu 20 25 30 Thr Pro Thr Tyr Pro Trp Arg Asp
Ala Glu Thr Gly Glu Arg Leu Val 35 40 45 Cys Ala Gln Cys Pro Pro
Gly Thr Phe Val Gln Arg Pro Cys Arg Arg 50 55 60 Asp Ser Pro Thr
Thr Cys Gly Pro Cys Pro Pro Arg His Tyr Thr Gln 65 70 75 80 Phe Trp
Asn Tyr Leu Glu Arg Cys Arg Tyr Cys Asn Val Leu Cys Gly 85 90 95
Glu Arg Glu Glu Glu Ala Arg Ala Cys His Ala Thr His Asn Arg Ala 100
105 110 Cys Arg Cys Arg Thr Gly Phe Phe Ala His Ala Gly Phe Cys Leu
Glu 115 120 125 His Ala Ser Cys Pro Pro Gly Ala Gly Val Ile Ala Pro
Gly Thr Pro 130 135 140 Ser Gln Asn Thr Gln Cys Gln Pro Cys Pro Pro
Gly Thr Phe Ser Ala 145 150 155 160 Ser Ser Ser Ser Ser Glu Gln Cys
Gln Pro His Arg Asn Cys Thr Ala 165 170 175 Leu Gly Leu Ala Leu Asn
Val Pro Gly Ser Ser Ser His Asp Thr Leu 180 185 190 Cys Thr Ser Cys
Thr Gly Phe Pro Leu Ser Thr Arg Val Pro Gly Glu 195 200 205 Pro Glu
Ala 210 21 145 PRT Homo sapien 21 Thr Val Thr Gln Asp Cys Leu Gln
Leu Ile Ala Asp Ser Glu Thr Pro 1 5 10 15 Thr Ile Gln Lys Gly Ser
Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe 20 25 30 Lys Arg Gly Ser
Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys 35 40 45 Glu Thr
Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys 50 55 60
Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe 65
70 75 80 Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln
Asn Met 85 90 95 Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala
Gly Ile Ala Lys 100 105 110 Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala
Ile Pro Arg Glu Asn Ala 115 120 125 Gln Ile Ser Leu Asp Gly Asp Val
Thr Phe Phe Gly Ala Leu Lys Leu 130 135 140 Leu 145 22 141 PRT Homo
sapien 22 Lys Glu Leu Arg Lys Val Ala His Leu Thr Gly Lys Ser Asn
Ser Arg 1 5 10 15 Ser Met Pro Leu Glu Trp Glu Asp Thr Tyr Gly Ile
Val Leu Leu Ser 20 25 30 Gly Val Lys Tyr Lys Lys Gly Gly Leu Val
Ile Asn Glu Thr Gly Leu 35 40 45 Tyr Phe Val Tyr Ser Lys Val Tyr
Phe Arg Gly Gln Ser Cys Asn Asn 50 55 60 Leu Pro Leu Ser His Lys
Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln 65 70 75 80 Asp Leu Val Met
Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly 85 90 95 Gln Met
Trp Ala Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr 100 105 110
Ser Ala Asp His Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn 115
120 125 Phe Glu Glu Ser Gln Thr Phe Phe Gly Leu Tyr Lys Leu 130 135
140 23 147 PRT Homo sapien 23 Ser Thr Leu Lys Pro Ala Ala His Leu
Ile Gly Asp Pro Ser Lys Gln 1 5 10 15 Asn Ser Leu Leu Trp Arg Ala
Asn Thr Asp Arg Ala Phe Leu Gln Asp 20 25 30 Gly Phe Ser Leu Ser
Asn Asn Ser Leu Leu Val Pro Thr Ser Gly Ile 35 40 45 Tyr Phe Val
Tyr Ser Gln Val Val Phe Ser Gly Lys Ala Tyr Ser Pro 50 55 60 Lys
Ala Thr Ser Ser Pro Leu Tyr Leu Ala His Glu Val Gln Leu Phe 65 70
75 80 Ser Ser Gln Tyr Pro Phe His Val Pro Leu Leu Ser Ser Gln Lys
Met 85 90 95 Val Tyr Pro Gly Leu Gln Glu Pro Trp Leu His Ser Met
Tyr His Gly 100 105 110 Ala Ala Phe Gln Leu Thr Gln Gly Asp Gln Leu
Ser Thr His Thr Asp 115 120 125 Gly Ile Pro His Leu Val Leu Ser Pro
Ser Thr Val Phe Phe Gly Ala 130 135 140 Phe Ala Leu 145 24 161 PRT
Homo sapien 24 Ser Pro Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro
Leu Lys Gly 1 5 10 15 Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln
Ala Phe Leu Thr Ser 20 25 30 Gly Thr Gln Phe Ser Asp Ala Glu Gly
Leu Ala Leu Pro Gln Asp Gly 35 40 45 Leu Tyr Tyr Leu Tyr Cys Leu
Val Gly Tyr Arg Gly Arg Ala Pro Pro 50 55 60 Gly Gly Gly Asp Pro
Gln Gly Arg Ser Val Thr Leu Arg Ser Ser Leu 65 70 75 80 Tyr Arg Ala
Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu Leu Leu Leu 85 90 95 Glu
Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro Ala Arg Arg Gln 100 105
110 Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly Gly Leu Val
115 120 125 Gln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser His
Pro Asp 130 135 140 Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly
Ala Val Met Val 145 150 155 160 Gly 25 150 PRT Homo sapien 25 Pro
Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro Gln Ala Glu 1 5 10
15 Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn
20 25 30 Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu
Gly Leu 35 40 45 Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln
Gly Cys Pro Ser 50 55 60 Thr His Val Leu Leu Thr His Thr Ile Ser
Arg Ile Ala Val Ser Tyr 65 70 75 80 Gln Thr Lys Val Asn Leu Leu Ser
Ala Ile Lys Ser Pro Cys Gln Arg 85 90 95 Glu Thr Pro Glu Gly Ala
Glu Ala Lys Pro Trp Tyr Glu Pro Ile Tyr 100 105 110 Leu Gly Gly Val
Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu 115 120 125 Ile Asn
Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr 130 135 140
Phe Gly Ile Ile Ala Leu 145 150
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