U.S. patent application number 12/498256 was filed with the patent office on 2011-07-14 for ligand for herpes simplex virus entry mediator and methods of use.
This patent application is currently assigned to La Jolla Institute of Allergy & Immunology. Invention is credited to CARL WARE.
Application Number | 20110171242 12/498256 |
Document ID | / |
Family ID | 38861825 |
Filed Date | 2011-07-14 |
United States Patent
Application |
20110171242 |
Kind Code |
A1 |
WARE; CARL |
July 14, 2011 |
LIGAND FOR HERPES SIMPLEX VIRUS ENTRY MEDIATOR AND METHODS OF
USE
Abstract
A novel polypeptide ligand, p30, or LIGHT, for herpes virus
entry mediator, HVEM, is provided. LIGHT is useful for modulating
immune responses and in inhibiting infection and/or subsequent
proliferation by herpesvirus. HVEM fusion proteins are also
provided. Methods for treating subjects with lymphoid cell
disorders, tumors, autoimmune diseases, inflammatory disorders or
those having or suspected of having a herpesvirus infection,
utilizing p30 and the fusion proteins of the invention, are also
provided.
Inventors: |
WARE; CARL; (Solana Beach,
CA) |
Assignee: |
La Jolla Institute of Allergy &
Immunology
La Jolla
CA
|
Family ID: |
38861825 |
Appl. No.: |
12/498256 |
Filed: |
July 6, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11513290 |
Aug 29, 2006 |
7575745 |
|
|
12498256 |
|
|
|
|
09967604 |
Sep 28, 2001 |
7118742 |
|
|
11513290 |
|
|
|
|
09549096 |
Apr 12, 2000 |
|
|
|
09967604 |
|
|
|
|
08898234 |
Jul 30, 1997 |
6140467 |
|
|
09549096 |
|
|
|
|
60051964 |
Jul 7, 1997 |
|
|
|
Current U.S.
Class: |
424/173.1 ;
435/375; 530/350 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2710/16622 20130101; C07K 14/705 20130101; C07K 14/5255
20130101; C07K 14/70578 20130101; A61K 2039/505 20130101; G01N
33/6863 20130101; A61P 31/22 20180101; G01N 2500/00 20130101; A61P
29/00 20180101; A61P 31/12 20180101; C07K 16/2878 20130101; A61K
38/00 20130101; C07K 2319/30 20130101; G01N 33/56983 20130101; C07K
2319/00 20130101; C07K 2317/74 20130101; C07K 14/47 20130101 |
Class at
Publication: |
424/173.1 ;
530/350; 435/375 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 14/435 20060101 C07K014/435; C12N 5/071 20100101
C12N005/071; A61P 31/12 20060101 A61P031/12; A61P 29/00 20060101
A61P029/00 |
Goverment Interests
STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH
[0002] The United States Government has certain rights in this
invention pursuant to grant nos. AI33068 and CA69381 awarded by
National Institutes of Health (NIH), DHHS.
Claims
1. An isolated or recombinant homotrimeric p30 polypeptide
comprising a monomer polypeptide having an apparent molecular
weight of about 30 kDa, wherein the homotrimeric polypeptide binds
to a herpes virus entry mediator (HVEM) polypeptide or a
lymphotoxin .beta. receptor (LT.beta.R) polypeptide under
physiologic conditions.
2. The isolated or recombinant homotrimeric p30 polypeptide of
claim 1, wherein the monomer polypeptide comprises isomers having a
pI from about 7 to about 8.5.
3. A soluble isolated or recombinant homotrimeric p30 polypeptide
lacking a transmembrane domain, wherein the soluble homotrimeric
polypeptide binds to a herpes virus entry mediator (HVEM)
polypeptide or a lymphotoxin .beta. receptor (LT.beta.R)
polypeptide under physiologic conditions.
4.-25. (canceled)
26. A method for inhibiting a p30 polypeptide-mediated cellular
response comprising (a) providing a composition that inhibits
binding of a cell surface expressed p30 polypeptide to a cell
surface expressed HVEM or LT.beta.R, and (b) contacting the cell
expressing the cell surface expressed p30 polypeptide or the cell
surface expressed HVEM or LT.beta.R with an amount of the
composition sufficient to inhibit a p30 polypeptide-mediated
cellular response.
27. The method of claim 26, wherein the cell is contacted with the
composition in vivo.
28. The method of claim 26, wherein the inhibited p30
polypeptide-mediated cellular response comprises inhibition of a
lymphocyte cellular response.
29. The method of claim 28, wherein the inhibited lymphocyte
response is lymphocyte proliferation.
30. The method of claim 28, wherein the inhibited lymphocyte is a
pathogenic effector cell.
31. The method of claim 28, wherein the inhibited lymphocyte
response modulates a T or a B lymphoma or leukemia or an autoimmune
disease.
32.-36. (canceled)
37. The method of claim 26, wherein the contacted cell expresses
p30 polypeptide on its cell surface and the composition is an
anti-p30 antibody.
38. (canceled)
39. A method of modulating a lymphotoxin beta receptor
(LT.beta.R)-mediated cellular response, the method comprising: (a)
providing a composition that inhibits binding of an LT.beta.R to a
p30 polypeptide; and (b) contacting a cell expressing the LT.beta.R
or the p30 polypeptide with an amount of the composition sufficient
to modulate the lymphotoxin .beta. receptor (LT.beta.R)-mediated
cellular response.
40. (canceled)
41. The method of claim 39, wherein the cell expresses a p30
polypeptide and the composition comprises an anti-p30 antibody.
42.-95. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 11/513,290, filed Aug. 29, 2006, which is a divisional
application of U.S. Ser. No. 09/967,604, filed Sep. 28, 2001, which
is a continuation-in-part of U.S. Ser. No. 09/549,096, filed Apr.
12, 2000, which is a continuation-in-part of U.S. Ser. No.
08/898,234, filed Jul. 30, 1997, now U.S. Pat. No. 6,140,467, which
claims benefit of priority to U.S. Ser. No. 60/051,964, filed Jul.
7, 1997, and all of which applications are incorporated herein by
reference in their entirety for all purposes.
FIELD OF THE INVENTION
[0003] The invention relates generally to compounds and methods
useful in regulating immune responses and viral infection.
BACKGROUND OF THE INVENTION
[0004] Herpes simplex virus (HSV), types 1 and 2, causes recurrent
infections that range in severity from benign to serious. HSV
emerges from latency in neurons to infect the skin and other
tissues in the presence of a competent cellular immune system. The
D glycoprotein (gD) of HSV, a transmembrane protein located in the
virion envelope, initiates infection by binding to cellular
receptors (Spear et al. (1993) Viral Fusion Mechanisms. Ed. Bentz.
CRC press, Boca Raton). Recently, a cellular protein used by HSV
for infection was identified and given the term HSV entry mediator
(HVEM) (Montgomery (1996) Cell 87:427). HVEM is a transmembrane
type 1 protein with a cysteine-rich extracellular domain that
exhibits significant homology with receptors for tumor necrosis
factor (TNF)-related cytokines (Smith et al. (1994) Cell 76:959;
Ware et al. (1995) in, Pathways of Cytolysis. Eds. Griffiths and
Tschopp. Springer-Verlag, Basel). Many of the TNF superfamily
members initiate a variety of cellular responses necessary to mount
effective inflammatory and immune responses.
[0005] TNF is a type 2 transmembrane protein (Pennica (1984) Nature
312:724) that is proteolyzed to form the secreted protein (Black
(1997) Nature 385:729), whereas LT.alpha. lacks a transmembrane
domain (Gray (1984) Nature 312:721) and is exclusively secreted as
a homotrimer (in this form it was also known as TNF.beta.). When
expressed as a surface protein, LT.alpha. is associated with a 33
kDa protein (Androlewicz (1992) J. Biol. Chem. 267:2542), termed
LT.beta. (Browning (1993) Cell 72:847), also a type 2 transmembrane
glycoprotein, in heterotrimers of .alpha.1.beta.2 and
.alpha.2.beta.1 subunit ratios (Androlewicz (1992) supra; Browning
(1996) J. Biol. Chem. 271:8618). LT.alpha. and TNF both bind and
signal through two receptors, the 55-60 kDa TNF receptor (TNFR60;
CD120a or type 1) (Schall (1990) Cell 61:361; Loetscher (1990) Cell
61:351) and the 75-80 kDa TNFR (TNFR80; type 2 or CD120b) (Smith
(1990) Science 248:1019). By contrast, the surface
LT.alpha.1.beta.2 complex is recognized specifically by the
LT.beta. receptor (LT.beta.R) (Crowe (1994) Science 264:707), which
does not bind either LT.alpha. or TNF (Crowe (1994) supra) whereas
both TNFRs bind the LT.alpha.2.beta.1 heterotrimer (Crowe (1994)
supra; Browning (1995) J. Immunol. 154:33).
[0006] Genetic deletions of LT.alpha. and LT.beta. genes in mice
have revealed roles for these two genes in the development of lymph
nodes and Peyer's patches (De Togni (1994) Science 264:703; Banks
(1995) J. Immunol. 155:1685), and along with TNF and TNFR60, are
also critical cytokines controlling the formation of germinal
centers and immunoglobulin isotype switching (e.g., IgA production)
during immune responses in adults (Matsumoto (1996) Science
271:1289; Mariathasan (1995) J. Inflammation 45:72). Most studies
have pointed towards the LT.alpha.1.beta.2/LT.beta.R as the
critical cytokine-receptor system controlling these functions
(Crowe (1994) Science 264:707; Koni (1997) Immunity 5:491; Ettinger
(1996) Proc. Natl. Acad. Sci. USA 93:13102; Rennert (1996) J. Exp.
Med. 184:1999).
SUMMARY OF THE INVENTION
[0007] The present invention is based on the identification of an
endogenous polypeptide that functions as a ligand for HVEM, which
previously was known only to bind HSV gD. This HVEM-binding ligand,
also referred to as p30, or LIGHT, is provided, as well as nucleic
acid sequences encoding p30 and antibodies which bind to p30. The
invention also includes methods for identifying compounds that
modulate viral infection, e.g., herpesvirus (e.g., CMV or HSV)
infection, and methods for modulating lymphoid cell responses.
Thus, the methods of the invention are useful for treating subjects
with autoimmune diseases, lymphoid malignancies and viral infection
associated with herpesviridae including, for example, herpesvirus
and cytomegalovirus infection.
[0008] In one embodiment the invention features an assay for
identifying a compound which affects an HVEM-binding agent-mediated
cellular response. Also within the invention is an assay for
identifying a compound which affects an LT.beta.R-p30-mediated
cellular response.
[0009] The present invention provides a method for inhibiting an
inflammatory disorder in a subject, by contacting the subject with
an inhibiting effective amount of an agent which prevents the
interaction of p30 (LIGHT) with its receptor. The subject, tissue
or cell used in the methods of the present invention may be any
subject, tissue or cell, including of a mammalian specie, but is
preferably human. The agents can be administered in vivo, ex vivo,
or in vitro depending upon the source (e.g., whole organism, tissue
or cell). For in vivo administration or contacting, delivery may be
by systemic methods or topical. The agent may be any agent which
prevents interaction of p30 with its receptor. Examples of such
agents include the antibodies (e.g., anti-p30 antibodies),
peptidomimetics, polypeptides, and fragments of HVEM of the
invention.
[0010] In another embodiment, the present invention provides a
fusion polypeptide comprising an HVEM polypeptide or fragment
thereof operatively linked to a polypeptide of interest. The HVEM
polypeptide can be from amino acid 1 to SER205 of HVEM and the
polypeptide of interest an Fc region of an antibody.
[0011] In yet another embodiment, the present invention provides a
polynucleotide sequence encoding the fusion polypeptide comprising
an HVEM polypeptide or fragment thereof operatively linked to a
polypeptide of interest.
[0012] In another embodiment, the present invention provides a
vector containing the polynucleotide sequence encoding the HVEM
fusion polypeptide.
[0013] In yet a further embodiment, the present invention provides
pharmaceutical composition comprising the fusion polypeptide and/or
polynucleotides and a pharmaceutically acceptable carrier.
[0014] The invention provides an isolated or recombinant
homotrimeric p30 polypeptide comprising a monomer polypeptide
having an apparent molecular weight (MW) of about 30 kilodaltons
(kDa), wherein the homotrimeric polypeptide binds to a herpes virus
entry mediator (HVEM) polypeptide or a lymphotoxin receptor (LTR)
polypeptide under physiologic conditions. In alternative
embodiments, the isolated or recombinant homotrimeric p30
polypeptide comprises isomers having a pI from about 7 to about
8.5.
[0015] The invention provides a soluble isolated or recombinant
homotrimeric p30 polypeptide lacking a transmembrane domain,
wherein the homotrimeric polypeptide binds to a herpes virus entry
mediator (HVEM) polypeptide or a lymphotoxin receptor (LT.beta.R)
polypeptide under physiologic conditions.
[0016] The invention provides a fusion protein comprising a p30
polypeptide of the invention and a heterologous sequence. In one
embodiment, the heterologous sequence is a tag or another
detectable moiety.
[0017] The invention provides a liposome comprising a p30
polypeptide of the invention, including a homotrimeric p30
polypeptide of the invention, a soluble homotrimeric p30
polypeptide lacking a transmembrane domain, a fusion protein of the
invention, or a combination thereof.
[0018] The invention provides a pharmaceutical composition
comprising a p30 polypeptide of the invention, including a
homotrimeric p30 polypeptide of the invention, a soluble
homotrimeric p30 polypeptide lacking a transmembrane domain, a
fusion protein of the invention, or a liposome of the invention, or
a combination thereof, and a pharmaceutically acceptable
excipient.
[0019] The invention provides a pharmaceutical composition
comprising a LT.beta.R or TNFR1 agonist. In one embodiment, the
agonist comprises a ligand such as a polypeptide p30 (LIGHT),
LT.alpha., TNF or LT.alpha.1.beta.2, or an antibody, such as a
fully human or humanized form, or a fusion protein or functional
fragment thereof. In a particular aspect, the antibody is a
monoclonal antibody denoted 3C8, 3H4 and 4H8. Pharmaceutical
compositions additionally include those having one or more
antiviral agents (e.g. an agent for treatment of herpesvirus, such
as CMV).
[0020] The invention provides antibodies having a LT.beta.R or
TNFR1 agonist activity. In one embodiment, an antibody has the
binding specificity of a monoclonal antibody denoted 3C8, 3H4 or
4H8. In particular aspects, the antibody having the binding
specificity of a monoclonal antibody denoted 3C8, 3H4 or 4H8 is
humanized or fully human.
[0021] The invention provides a kit comprising a pharmaceutical
composition and printed matter, wherein the pharmaceutical
composition comprises a p30 polypeptide, wherein the p30
polypeptide comprises a homotrimeric p30 polypeptide comprising a
monomer polypeptide having an apparent molecular weight of about 30
kDa, wherein the homotrimeric polypeptide binds to a herpes virus
entry mediator (HVEM) polypeptide or a lymphotoxin receptor (LTR)
polypeptide under physiologic conditions, or, a soluble
homotrimeric p30 polypeptide lacking a transmembrane domain,
wherein the soluble homotrimeric polypeptide binds to a herpes
virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta.
receptor (LT.beta.R) polypeptide under physiologic conditions, and
a pharmaceutically acceptable excipient, wherein the printed matter
comprises instructions for a use of the pharmaceutical composition,
wherein a use comprises inhibiting virus entry into a cell or virus
proliferation in a cell. The instructions can include use of the
pharmaceutical composition for inhibiting virus entry into a cell
or inhibiting virus proliferation in a cell in vivo. In alternative
embodiments, the inhibited virus is a herpesvirus, a herpes simplex
virus (HSV), a cytomegalovirus (CMV), a .gamma.-herpesvirus or an
Epstein Barr virus (EBV). The inhibition of virus entry or virus
proliferation in the cell can be in a mammal, including a
human.
[0022] The invention provides a kit comprising a pharmaceutical
composition and printed matter, wherein the pharmaceutical
composition comprises a p30 polypeptide, wherein the p30
polypeptide comprises a homotrimeric p30 polypeptide comprising a
monomer polypeptide having an apparent molecular weight of about 30
kDa, wherein the homotrimeric polypeptide binds to a herpes virus
entry mediator (HVEM) polypeptide or a lymphotoxin receptor (LTR)
polypeptide under physiologic conditions, or, a soluble
homotrimeric p30 polypeptide lacking a transmembrane domain,
wherein the soluble homotrimeric polypeptide binds to a herpes
virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta.
receptor (LT.beta.R) polypeptide under physiologic conditions, and
a pharmaceutically acceptable excipient, wherein the printed matter
comprises instructions for a use of the pharmaceutical composition,
wherein a use comprises modulating diseases with unwanted
lymphocyte proliferation. In alternative embodiments, the
instructions comprise use of the pharmaceutical composition to
modulate a T or a B lymphoma or leukemia, or an autoimmune disease.
The autoimmune disease can be rheumatoid arthritis,
insulin-dependent diabetes mellitus, multiple sclerosis, systemic
lupus erythematosus or myasthenia gravis.
[0023] The invention provides a pharmaceutical composition
comprising an expression vector encoding a p30 polypeptide having
an apparent molecular weight of about 30 kDa or a p30 polypeptide
lacking a transmembrane domain, wherein the p30 polypeptide forms a
homotrimeric polypeptide that binds to a herpes virus entry
mediator (HVEM) polypeptide or a lymphotoxin .beta. receptor
(LT.beta.R) polypeptide under physiologic conditions.
[0024] The invention provides a kit comprising a pharmaceutical
composition and printed matter, wherein the pharmaceutical
composition comprises an expression vector encoding a p30
polypeptide having an apparent molecular weight of about 30 kDa or
a p30 polypeptide lacking a transmembrane domain, wherein the p30
polypeptide forms a homotrimeric polypeptide that binds to a herpes
virus entry mediator (HVEM) polypeptide or a lymphotoxin .beta.
receptor (LT.beta.R) polypeptide under physiologic conditions, and
a pharmaceutically acceptable excipient, and, wherein the printed
matter comprises instructions for a use of the pharmaceutical
composition, wherein a use comprises targeting of tumor cells or
activated lymphocytes. In one embodiment, the use comprises
treatment of a tumor by direct injection of the pharmaceutical
composition into the tumor.
[0025] The invention provides a kit comprising an LT.beta.R or
TNFR1 agonist, and instructions for use in treating a subject
having or at risk of having a herpesvirus infection. The invention
provides a kit comprising a pharmaceutical composition comprising
an LT.beta.R or TNFR1 agonist and instructions for use in treating
a subject having or at risk of having a herpesvirus infection. The
invention provides a kit comprising an LT.beta.R or TNFR1 agonist,
an antiviral agent, and instructions for use in treating a subject
having or at risk of having a herpesvirus infection. The invention
provides a kit comprising a pharmaceutical composition comprising
an LT.beta.R or TNFR1 agonist, an antiviral agent, and instructions
for use in treating a subject having or at risk of having a
herpesvirus infection.
[0026] The invention provides a method for inducing a
proliferation-inducing signal to a lymphocyte comprising (a)
providing a composition that binds to cell surface expressed HVEM,
and (b) contacting the lymphocyte with a proliferation-inducing
amount of the composition. In alternative embodiments, the
composition comprises an anti-HVEM antibody or a polypeptide
comprising an anti-HVEM antibody binding site. In alternative
embodiments, providing a composition that binds to cell surface
expressed HVEM comprises providing a composition comprising a p30
polypeptide, a soluble p30 polypeptide, a liposome-associated p30
polypeptide, or, a vector encoding a p30 polypeptide or a cell
expressing a recombinant p30 as a cell-associated p30 polypeptide.
In this method, the lymphocyte can be a T cell or a B cell. The
lymphocyte can be contacted in vivo.
[0027] The invention provides a method for inhibiting a p30
polypeptide-mediated cellular response comprising (a) providing an
composition that inhibits binding of a cell surface expressed p30
polypeptide to a cell surface expressed HVEM or LT.beta.R, and, (b)
contacting the cell expressing the cell surface expressed p30
polypeptide or the cell surface expressed HVEM or LT.beta.R with an
amount of the composition sufficient to inhibit a p30
polypeptide-mediated cellular response. In this method, the cell
can be contacted with the composition in vivo. In alternative
embodiments, the inhibited p30 polypeptide-mediated cellular
response comprises inhibition of a lymphocyte cellular response,
the inhibited lymphocyte response is lymphocyte proliferation, and
the inhibited lymphocyte is a pathogenic effector cell. The
inhibited lymphocyte response can comprise modulation of a T or a B
lymphoma or leukemia or an autoimmune disease. The autoimmune
disease can be rheumatoid arthritis, insulin-dependent diabetes
mellitus, multiple sclerosis, systemic lupus erythematosus or
myasthenia gravis. The inhibited lymphocyte response can comprise
modulation of a reaction to a transplant.
[0028] In one embodiment, the contacted cell expresses HVEM and the
composition is a soluble p30 polypeptide. In an alternative
embodiment, the contacted cell expresses LT.beta.R and the
composition is a soluble p30 polypeptide. In other embodiments, the
contacted cell expresses p30 polypeptide on its cell surface and
the composition is a soluble HVEM polypeptide; and, the contacted
cell expresses p30 polypeptide on its cell surface and the
composition is an anti-p30 antibody.
[0029] The invention provides a method for treating tumors
comprising (a) providing a pharmaceutical composition comprising an
expression vector encoding a p30 polypeptide having an apparent
molecular weight of about 30 kDa or a p30 polypeptide lacking a
transmembrane domain, wherein the p30 polypeptide forms a
homotrimeric polypeptide that binds to a herpes virus entry
mediator (HVEM) polypeptide or a lymphotoxin receptor (LT.beta.R)
polypeptide under physiologic conditions, and (b) directly
injecting the pharmaceutical composition into the tumor.
[0030] The invention provides a method of modulating a lymphotoxin
beta receptor (LT.beta.R)-mediated cellular response, the method
comprising: (a) providing a composition that inhibits binding of an
LT.beta.R to a p30 polypeptide; and (b) contacting a cell
expressing the LT.beta.R or the p30 polypeptide with an amount of
the composition sufficient to modulate the lymphotoxin beta
receptor (LT.beta.R)-mediated cellular response. In one embodiment,
the cell expresses LT.beta.R and the composition comprises a
pharmaceutical composition comprising a p30 polypeptide comprising
a homotrimeric p30 polypeptide comprising a monomer polypeptide
having an apparent molecular weight of about 30 kDa, wherein the
homotrimeric polypeptide binds to a herpes virus entry mediator
(HVEM) polypeptide or a lymphotoxin receptor (LTR) polypeptide
under physiologic conditions, or, a soluble homotrimeric p30
polypeptide lacking a transmembrane domain, wherein the soluble
homotrimeric polypeptide binds to a herpes virus entry mediator
(HVEM) polypeptide or a lymphotoxin .beta. receptor (LT.beta.R)
polypeptide under physiologic conditions, and a pharmaceutically
acceptable excipient. In one embodiment, the cell expresses a p30
polypeptide and the composition comprises an anti-p30 antibody. In
another embodiment, the lymphotoxin beta receptor
(LT.beta.R)-mediated cellular response comprises binding of a
herpesvirus to a cell. In one embodiment, the herpesvirus is
blocked from entry into the cell. In another embodiment, the
herpesvirus is inhibited from proliferating in the cell. In
alternative embodiments, the herpesvirus is a herpes simplex virus
(HSV), a cytomegalovirus (CMV), a .gamma.-herpesvirus or an Epstein
Ban virus (EBV).
[0031] The invention provides a method for inhibiting virus
production in a cell, the method comprising (a) providing a p30
polypeptide; and, (b) contacting a cell infected with a herpesvirus
or a cell susceptible to infection by a herpesvirus with an
effective amount of a p30 polypeptide, thereby inhibiting
herpesvirus production in the cell. In one embodiment, the entry of
the herpesvirus into the cell is inhibited. In another embodiment,
the contacting is in vivo and the p30 composition is provided as a
pharmaceutical composition, wherein the pharmaceutical composition
comprises a homotrimeric p30 polypeptide comprising a monomer
polypeptide having an apparent molecular weight of about 30 kDa,
wherein the homotrimeric polypeptide binds to a herpes virus entry
mediator (HVEM) polypeptide or a lymphotoxin receptor (LTR)
polypeptide under physiologic conditions, or, a soluble
homotrimeric p30 polypeptide lacking a transmembrane domain,
wherein the soluble homotrimeric polypeptide binds to a herpes
virus entry mediator (HVEM) polypeptide or a lymphotoxin 13
receptor (LT.beta.R) polypeptide under physiologic conditions, and
a pharmaceutically acceptable excipient. The virus can be a herpes
simplex virus (HSV), a cytomegalovirus (CMV), a .gamma.-herpesvirus
or an Epstein Barr virus (EBV). In one embodiment, the contacting
is in a mammal, such as a human.
[0032] In another embodiment, a herpesvirus infection (e.g., CMV, a
(.beta.-herpesvirus or a .gamma.-herpesvirus) or a disorder
associated with herpesvirus infection is treated. In one aspect, a
method includes contacting a subject having or at risk of having a
herpesvirus infection or a disorder associated with herpesvirus
infection with an amount of a LT.beta.R or TNFR1 agonist sufficient
to treat herpesvirus infection. Agonists include ligands, such as
polypeptides (e.g., p30 (LIGHT), LT.alpha., TNF or
LT.alpha.1.beta.2) and antibodies, such as fully human or humanized
forms, as well as agonists that do not produce substantial
apoptosis in cells infected with the virus. In additional aspects,
virus proliferation, nucleic acid replication or protein
expression, or virus reactivation from latency is reduced in the
subject following treatment. Target subjects include subjects that
are or are at risk of being immunosuppressed, subjects having an
HIV infection or a tumor, subjects having or is at risk of having a
blood or bone marrow, organ, or tissue transplant. Target subjects
further include neonates. Disorders include pneumonia,
arteriosclerosis, CMV hepatitis, CMV retinitis, CMV pneumonitis,
CMV nephritis or CMV mononucleosis, child febrile illness,
cytomegalaic inclusion disease or a demyelinating disease (e.g.,
multiple sclerosis), Kaposis sarcoma, Hodgkins leukemia or
non-Hodgkins leukemia or lymphoma. Various embodiments include
additionally contacting the subject with an antiviral agent or
other form of herpesvirus treatment.
[0033] The invention provides a method for identifying a compound
that inhibits CMV infection, including (a) contacting LT.beta.R or
TNFR1 with a test compound under conditions allowing binding; (b)
measuring LT.beta.R or TNFR1 activity in the presence of the test
compound; and (c) comparing activity in the presence of the test
compound to the absence of the test compound, wherein an increase
in LT.beta.R or TNFR1 activity in the presence of the test compound
identifies the test compound as a compound that inhibits CMV
infection. In one aspect, the test compound is a library of
compounds, for example, a peptide or small molecule library. In
other aspects, the activity comprises activation of an NFkB target
gene (e.g., ICAM1, VCAM1, interleukin-8 (IL-8) or secondary
lymphoid organ chemokine (SLC), for example), or activation of
IFNgamma or IFNbeta gene expression. In yet other aspects, the
identified compound is tested for inhibiting CMV infection in cells
or animals.
[0034] The invention provides a method for identifying a compound
that inhibits CMV infection, including (a) contacting a cell that
expresses LT.beta.R or TNFR1 in the presence of CMV with a test
compound under conditions allowing binding between LT.beta.R or
TNFR1 and the test compound; (b) measuring CMV proliferation,
replication, protein expression or cytopathicity in the presence of
the test compound; and (c) comparing CMV proliferation,
replication, protein expression or cytopathicity in the presence of
the test compound to the absence of the test compound, wherein a
decrease in CMV proliferation, replication, protein expression or
cytopathicity in the presence of the test compound identifies the
test compound as a compound that inhibits CMV infection. In one
aspect, the test compound is a library of compounds, for example, a
peptide or small molecule library.
[0035] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present application, including
definitions, will control. In addition, the materials, methods and
examples described herein are illustrative only and not intended to
be limiting.
[0036] Other features and advantages of the invention, e.g.,
therapy for a variety of human diseases, will be apparent from the
following detailed description, from the drawings and from the
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0037] FIG. 1A is a pair of flow cytometric histograms showing the
binding of HVEM:Fc fusion protein to II-23.D7 cells after
activation with PMA (upper histogram) or PMA and ionomycin (lower
histogram).
[0038] FIG. 1B is a pair of flow cytometric histograms showing the
binding of HVEM:Fc fusion protein to normal human CD4+ (upper
histogram) and CD8+ (lower histogram) T cells.
[0039] FIG. 1C is a line graph showing saturation binding of
HVEM:Fc fusion protein to activated II-23.D7 cells.
[0040] FIG. 2A is a pair of diagrams. The upper diagram is a flow
cytometric histogram showing that HVEM:Fc fusion protein binding to
activated II-23.D7 cells is competed by LT.beta.R:Fc fusion
protein. The lower diagram is a line graph showing dose-dependent
inhibition of HVEM:Fc fusion protein binding by LT.beta.R:Fc fusion
protein.
[0041] FIG. 2B is a pair of diagrams. The upper diagram is a flow
cytometric histogram showing that HVEM:Fc fusion protein binding is
competed by LT.alpha. homotrimer. The
[0042] FIG. 10 shows a graph of the CIA score for collagen-induced
arthritic mice treated with PBS, hIgG or mHVEM:Fc, see Example 8,
below.
[0043] FIG. 11 shows the morphology of dermal fibroblasts infected
with HCMV-F at an MOI of 0.05 and cultured in the present or medium
with 5 nM of A) LT.alpha., B) LT.alpha.+RNFR:Fc, C) LT.alpha.Y108F,
D) LT.alpha.1.beta.2, E) LIGHT, F) LIGHT-G119E, and G) Virus only.
See Example 9.
[0044] FIG. 12 shows antiviral effect of LIGHT and LT.alpha. on
.gamma.-herpesvirus, MHV68, in vivo (see Example 9).
[0045] FIG. 13 shows gel demonstrating the purity of LIGHTt66 (A)
stained by Coomassie blue or (B) western blotted with anti-FLAG
(M2) to detect the recombinant protein. Molecular weight markers
are shown in left lane.
[0046] FIG. 14 shows a number of gels demonstrating the Anti-HCMV
effect of LIGHT and Lymphotoxins are dependent upon activation of
NF.kappa.B.
[0047] FIG. 15 shows the purification and biochemical
characterization of LIGHT t66 and its mutants.
[0048] FIG. 15 A. Purification of LIGHTt66-FLAG from 293 cell
supernatant. Top panel. Coomassie stained SDS PAGE gel (15%). 20
.mu.l was loaded per well. Starting supernatant and ion-exchange
purified LIGHT t66 appeared as multiple bands. Affinity purified
LIGHT t66 appeared as a single band, Mr=27 kDa. Lower Panel.
Western blot stained with anti-FLAG (M2). 20 .mu.l was loaded per
well. LIGHTt66-FLAG was detected with Mr=27 kDa. Relative intensity
of the bands agreed with ELISA data indicating that LIGHT t66-FLAG
was concentrated .times.30 after the ion exchange procedure and
.times.100 after affinity purification. Final yield was 60-80% of
starting material.
[0049] FIG. 15 B. Purification of mutants of LIGHTt66-FLAG from
293T cell supernatants. Top Panel. Silver stained SDS PAGE gel.
Mutants of LIGHTt66-FLAG produced using 293T cells as described.
FLAG-tagged proteins were affinity purified from tissue culture
supernatant using monoclonal M2 anti-FLAG antibody coupled to
Affigel. 100 ng purified protein was loaded per well. All four
mutants appeared as single bands, Mr=27 kDa. In some preparations
an additional faint band Mr=52 kDa was present. No other
contaminating proteins were detected. Lower panel. Western blot
stained with M2 anti-FLAG. LIGHTt66-FLAG and its mutants were
detected with Mr=27 k. In some cases a weakly reactive band with
Mr=52 kDa was also detected, corresponding to the 52 kDa band
detected by silver staining. This band is likely to be a dimer.
[0050] FIG. 15 C. Crosslinking of LIGHTt66-myc. LIGHTt66 was
crosslinked by addition of glutaraldehyde (0.1%) or BSCOES (5 nM)
for 30 min at 4.degree. C.; the reaction was stopped by addition of
TRIS (20 mM, pH 8.0). Samples were analyzed by Western blotting
using 9ElO (anti-myc) antibody. 200 ng was loaded per well. A,
control LIGHTt66-myc, B, crosslinking with glutaraldehyde, C,
crosslinking with BSCOES. In crosslinked samples bands of 52 and 76
kDa were detected, corresponding to expected Mrs for dimer and
trimer.
[0051] FIG. 15D. Gel filtration analysis of LIGHTt66-FLAG. LIGHT
t66-FLAG and mutants were analyzed by FPLC gel filtration on a
Superose 12 column. Upper Panel. Fractions from gel filtration of
LIGHTt66-FLAG were analyzed by ELISA using HVEM:Fc as capture
molecule and M2 anti-FLAG as detecting antibody. Active LIGHT t66
eluted with Mr=76 kDa, indicating a homotrimeric molecule. Lower
Panel. Dot blot stained with M2 anti-FLAG. Fractions from gel
filtration analysis of LIGHTt66-FLAG and mutants were further
analyzed by dot blotting (from above down, t66, G119E, Y 173F,
Q117T, L120Q). Anti-FLAG reactive material was detected only in
those fractions determined by ELISA to contain homotrimeric LIGHT,
demonstrating that the preparations contained no free monomer and
no aggregated material.
[0052] FIG. 16 shows graphs demonstrating that LIGHT t66 is
cytotoxic to HT29 cells.
[0053] FIG. 16A. HT29 cells were incubated with serial dilutions of
LIGHT t66, LT.alpha.1.beta.2, or TNF in the presence of IFNY (80
U/ml) After 72 h cell viability was assessed by MIT assay. LIGHT
t66 inhibited growth of HT29s with comparable efficiency to
LT.alpha.1.beta.2.
[0054] FIG. 16B. LIGHT t66 cytotoxicity is dependent on IFN.gamma..
HT29 cells were incubated with serial dilutions of LIGHT t66 in the
presence or absence of IFN.gamma. (80 U/ml) and MTT assay was
performed after 72 h.
[0055] FIG. 16C. LIGHT t66 cytotoxicity is blocked by co-incubation
with LT.beta.R:Fc and HVEM:Fc. LIGHT t66 (200 pM) was pre-incubated
with varying dilutions of LT.beta.R:Fc, HVEM:Fc or Fas:Fc for 30
min before addition to HT29 cells in the presence of IFN.gamma..
MTT assay was performed after 72 h. LT.beta.R:Fc and HVEM:Fc,
inhibited cytotoxicity of LIGHT t66 in a dose-dependent manner,
whereas Fas:Fc did not affect LIGHT t66-mediated cytotoxicity.
[0056] FIG. 17 are graphs showing the receptor binding
characteristics of LIGHT t66 and its mutants. LIGHTt66-FLAG and
mutants were analyzed by ELISA using the stated receptors as
capture molecule and M2 anti-FLAG as detecting antibodies. L120Q
and Q117T bound human HVEM and LT.beta.R with affinity comparable
to or greater than LIGHT t66. G119E bound human HVEM:Fc with
reduced affinity and showed no detectable binding for human
LT.beta.R:Fc. Y173F bound human LT.beta.R with reduced affinity and
bound G119E weakly. L12OQ showed enhanced affinity for murine
HVEM:Fc and LT.beta.R:Fc. Binding of G119E and Y173F to murine
receptors was not detected in this system.
[0057] FIG. 18 shows overlays of sensorgrams demonstrating surface
plasmon resonance. Overlays of sensorgrams for binding of LIGHT t66
and its mutants to huHVEM:Fc and huLT.beta.R:Fc at ligand
concentration 300 nM. Kinetics of binding were similar for LIGHT
t66 and Q117T. G119E and Y173F dissociated from HVEM:Fc with
increased off rates relative to LIGHT-t66. Y173F dissociated from
huLT.beta.R with an increased off rate, whereas G119E failed to
bind this receptor.
[0058] FIG. 19 shows cell cytotoxicity of LIGHT t66 mutants in HT29
cells. HT29 cells were incubated with serial dilutions of LIGHT t66
and its mutants in the presence of IFN.gamma., an MTT assay was
performed after 72 h. L12OQ and Q117T were toxic to HT29 cells with
comparable efficiency to LIGHT t66 (50% cell death at 10 .mu.M
cytokine). Y173F was weakly cytotoxic (50% cell death at 1 nM
cytokine). G199E showed negligible cytotoxicity to these cells.
[0059] FIG. 20 shows the effect of anti-LT.beta.R and anti-HVEM
antibodies on HT29 cells.
[0060] FIG. 20A. Binding of anti-huHVEM and anti-huLT.beta.R
antibodies to HT29 cells. Cells were incubated with primary
antibody (5 .mu.g/ml) or normal mouse IgG (filled area) for 30 min
at 4'C, followed by goat anti-mouse PE and analyzed by flow
cytometry. Both anti-HVEM antibodies (CW1 and CW8) and both
anti-LT.beta.R antibodies (BDA8 and CDHl0) stained the cells.
[0061] FIG. 20 B. Effect of anti-hu HVEM antibodies on binding of
LIGHT to hu HVEM. Wells of an ELISA plate coated with huHVEM:Fc (3
pg/ml) were incubated with varying concentrations of goat
polyclonal anti-HVEM or the monoclonal anti-HVEM antibodies CW1 or
CW8 for 30 min and then with LIGHT (0.25 nM) for 1 h before
detection of bound LIGHT with monoclonal anti-FLAG (M2) and goat
anti-mouse IgG-HRP. Goat anti-HVEM and CW8 markedly inhibited LIGHT
binding to HVEM whereas CW1 had no effect.
[0062] FIG. 20 C. Effect of anti-LT.beta.R antibodies on binding of
LIGHT to huLT.beta.R. The experiment was conducted as described in
FIG. 20 B. Goat anti-LT.beta.R and monoclonal anti-LT.beta.R BDA8
markedly inhibited LIGHT binding to LT.beta.R whereas CDHl0 had no
effect.
[0063] FIG. 20 D. Effect of antibody crosslinking of huHVEM and
huLT.beta.R on growth of HT29 cells. HT29 cells were incubated with
varying doses of polyclonal anti-HVEM, polyclonal anti-LT.beta.R,
or a mixture of the two. MTT assay was performed after 72 h.
Antibody crosslinking of LT.beta.R significantly reduced cell
number in a dose-dependent manner, whereas antibody crosslinking of
HVEM had no effect. Inclusion of polyclonal anti-HVEM antibody had
no effect on the cytotoxicity of polyclonal anti-LT.beta.R.
[0064] FIG. 20 E. Effect of polyclonal anti-huHVEM and
anti-huLT.beta.R on LIGHT-mediated cytotoxicity. Hi29 cells were
incubated with 10 pg/ml of goat polyclonal anti-huHVEM, goat
polyclonal anti-huLT.beta.R, or the stated monoclonal antibodies
for 10 min before addition of LIGHT (0.25 nM). MTT assay was
performed after 72 h in culture. LIGHT alone resulted in 50% growth
inhibition. Inclusion of goat polyclonal anti-LT.beta.R and the
monoclonal anti-LT.beta.R CDHl0 markedly enhanced LIGHT-mediated
cytotoxicity, whereas monoclonal anti-LT.beta.R BDA8 had a slight
protective effect. Goat polyclonal anti-huHVEM and the monoclonal
anti-HVEM antibodies CW1 and CW8 did not affect LIGHT-mediated
cytotoxicity.
[0065] FIG. 21 shows the effect of LIGHT t66 on the up-regulation
of ICAM expression in NHDF cells. NHDF cells (60,000/well) were
incubated with the stated concentrations of cytokine in 600 .mu.l
of tissue culture medium. After 36 h cells were and analyzed by
FACS with mAb P2A4 (Chemicon International Inc.) to determine the
surface levels of ICAM-1. The fold induction represents the
specific fluorescence of the cytokine-treated wells over an
untreated negative control.
[0066] FIG. 22 shows TRAF recruitment by receptors.
[0067] FIG. 22 A. Co-immunoprecipitation of HVEM and FLAG-tagged
TRAFs from the lysates of transfected 293 T cells. Protein
complexes were precipitated using polyclonal rat anti-HVEM specific
antibodies. Immunoblots were detected using mouse monoclonal
anti-FLAG antibodies.
[0068] FIG. 22 B. Communo-precipitations of LT.beta.R and
FLAG-tagged TRAFs from the lysates of transfected 293 T cells.
Protein complexes were precipitated using goat anti-LT.beta.R
specific antibodies. Immunoblots were detected using mouse
monoclonal anti-FLAG antibodies. Cells were transfected with single
vectors or with vectors in combination as indicated in the figure
and described in experimental procedures. pBABE was included as a
negative control.
[0069] FIG. 23 shows inhibition of HCMV-induced cytopathicity by
lymphotoxins and LIGHT. NHDF were infected with HCMV at an MOI of
0.01, and various purified recombinant cytokines were added to
medium at a final concentration of 5 nM. After culture for 7 days
the cytopathic effect was visualized by light microscopy (20.times.
magnification). (a) NHDF infected with virus in medium or in medium
with, (b) LT.alpha.; (c) LT.alpha. with TNFR1-Fc (25 .mu.g/ml); (d)
mutant LT.alpha.Y108F; (e) LIGHT; f. mutant LIGHTG119E; g.
LT.alpha..beta.2.
[0070] FIG. 24 shows HCMV protein expression in lymphotoxin-treated
cells. a. HCMV infected fibroblasts (MOI=0.01) treated with
indicated purified cytokines, and cells were harvested and analyzed
for the major immediate early I protein (IE1) or late tegument
protein (pp28). M, mock; V, virus with no cytokine added. b. The
percentage of maximal protein expression in the western blot was
calculated as a ratio of the pp28 band density in cytokine treated
cells to cells infected with virus in the absence of cytokine. c.
Infectious virus production (.+-.SD) was measured in supernatants
collected from NHDF infected with HCMV alone, or infected in the
presence of LT.alpha. (0.1 nM), LT.alpha.1.beta.2 (1 nM), or LIGHT
(1 nM), FasL (12 nM) or TRAIL (12 nM). Cytokine dose response
curves and viral titers were performed at least 3 times; data from
a single representative experiment is shown.
[0071] FIG. 25 shows that the anti-viral effect of LT is
reversible. a. NHDF were infected with HCMV at an MOI of 0.01 and
cultured with LT.alpha. (1 nM)(+). After 7 days, the medium was
replaced with fresh medium without LT.alpha.(-), and virus
replication was allowed to proceed for an additional 7 days. Cells
were harvested at the indicated time post infection and virus
protein expression analyzed by Western blot (IE1, gB, gH and pp28).
The "no LT.alpha." lanes represent HCMV proteins at 7 or 14 in the
absence of LT.alpha.. b. HCMV titers (.+-.SD) were measured every
24 hours after infection of NHDF (MOI=0.01) for cells infected in
the absence of LT.alpha. (n), or with LT.alpha. for the initial 7
days and then was removed for days 8-14 (s).
[0072] FIG. 26 shows that NF.kappa.B, but not TRAF3 or FADD, is
critical for anti-HCMV signaling by TNFRI and LT.beta.R. a. NHDF
were transduced with retroviral vectors expressing either a
dominant negative (dn) I.kappa.B.alpha. mutant (IkB.alpha.M),
TRAF3.D11 mutant, FADD-dn (F-dn), or empty vector (LXSN).
NHDF-I.kappa.B.alpha.M cells were treated with TNF or LT.alpha. (1
nM) for 24 hrs and ICAM-1 expression was measured by flow cytometry
(LXSN, upper 2 panels; I.kappa.B.alpha.M, lower 2 panels). Cell
lysates were prepared and analyzed by Western blot for expression
of IkB.alpha., FLAG tagged TRAF3.D11 and FADD-dn mutants. For the
I.kappa.B.alpha. degradation assay, cells were either mock treated
(lanes 1,4) or treated with 1 nM TNF (lanes 2,5) or LT.alpha.
(lanes 3,6) in the presence of 10 .mu.g/ml cycloheximide for 4
hours. C, control LXSN cell lysate. b. NHDF expressing dominant
negative mutants were compared to control cells (LXSN) for the
ability of LT.alpha., LT.alpha.1.beta.2 or LIGHT to inhibit
expression of IE1 and pp28. Blots were reprobed with an
anti-.beta.-actin (.beta.A) antibody. Concentrations of cytokines
or antibodies added to the culture medium were: LIGHT and
LT.alpha., serial 10 fold dilution starting at 10 nM;
LT.alpha.1.beta.2, 5 nM, 1 nM, 0.1 nM and 0.01 nM. c. Supernatants
were collected from HCMV infected NHDF cell lines treated with
cytokine (LT.alpha., 0.1 nM; LT.alpha.1.beta.2, 1 nM; LIGHT, 1 nM)
7 days and then analyzed for plaque forming units (PFU). HCMV PFU
were determined in quadruplicate, and SD are contained within the
symbols. Western blots and titers were performed at least 3
independent times, and representative results are shown.
[0073] FIG. 27 shows that the anti-HCMV activity of LT is mediated
through induction of IFN.beta.. a. Medium from NHDF infected with
HCMV (MOI=0.01)(lanes 1,4,7), treated with 1 nM LT.alpha. (lanes 2,
5, 8), or infected and treated with LT.alpha. (lanes 3, 6, 9) was
collected after 2 days and transferred to NHDF freshly infected
with HCMV (MOI=0.01). The medium from infected cells was untreated
(lanes 1-3), or treated with 25 .mu.g/ml TNFR1-Fc (lanes 4-6), or
25 .mu.g/ml TNFR1-Fc and 500 units anti-IFN.beta. neutralizing
antibody (lanes 7-9). Cells were harvested and analyzed for IE1
expression by Western blot. b. NHDF cells were infected with HCMV
(MOI=0.01) and at the time of infection, treated with LT.alpha. (1
nM) (except for lane 2) with anti-IFN.alpha., or anti-IFN.beta.
neutralizing antibodies. Cells were harvested 7 days after
infection and analyzed for expression of IE1 by western blot. M,
mock infected cells; lane 1, LT.alpha. alone; lane 2, virus only.
Cells were treated with 1 nM LT.alpha. (lanes 3-9). Dose titration
(500 units serial diluted 10 fold) with anti-IFN.alpha. (lanes
3-5), anti-IFN.beta. (lanes 6-8) or anti-IFN.gamma. neutralizing
antibodies (25 mg/ml) (lane 9). c. Supernatants were collected from
NHDF infected with HCMV alone (none), or infected in the presence
of anti-IFN.beta. neutralizing antibody (.alpha.IFN.beta., 500U),
LT.alpha. (1 nM), LT.alpha. plus neutralizing anti-IFN.beta. (500U,
50U, 5U) or anti-IFN.alpha. antibody (500U) as in (b).
[0074] FIG. 28 shows that signaling through TNFR1 and LT.beta.R
induces transcription of IFN.beta. in HCMV infected cells. a. NHDF
were infected with HCMV at indicated MOI in medium with or without
LT.alpha. (1 nM). b. NHDF were infected with HCMV (MOI=1) and
treated with LT.alpha., LT.alpha.1.beta.2 or LIGHT (5 nM and 1 nM
each) or treated with cytokine without virus infection (5 nM). c.
NHDF infected with HCMV (MOI=1) plus or minus LT.alpha. (1 nM) were
harvested at various hours post infection (hpi) for analysis of
IFN.alpha., IFN.beta. and HCMV IE1 expression levels. +,
amplification of IFN.alpha. from NHDF genomic DNA. d. Real time PCR
was performed on NHDF-LXSN and NHDF-I.kappa.B.alpha.M cells
infected with HCMV (MOI=1) with or without LT.alpha. (1 nM) in
order to quantify induction of IFN.beta. mRNA. Quantification of
.beta.actin (.beta.A) mRNA was performed in parallel to allow for
normalization and calculation of fold difference in IFN.beta.
induction. Four independent experiments were performed, and the
level of IFN.beta. in infected cells treated with LT.alpha. was
increased by 48-103 fold (mean=77+/-26). e. NHDF-LXSN or
NHDF-I.kappa.B.alpha.M cells were infected with HCMV at MOI (1,
0.5, 0.25, 0.125 from left to right) and incubated in medium with
or without LT.alpha. (1 nM). Right panel, NHDF-LXSN (lanes 1,3,5)
and NHDF-I.kappa.B.alpha.M (lanes 2,4,6) cells were treated with
LT.alpha. (lanes 1,2), polyI:C (lanes 3,4) or mock infected (M)
(lanes 5,6). For all panels except c, cells were harvested 4 hours
post infection for isolation of total cell RNA; M, mock infected.
NHDF were treated with polyI:C (100 mg/ml) with (panel a,c) or
without (panel e) cycloheximide (10 mg/ml) for 4 or 6 (panel c)
hours. All PCR was performed in the linear range excepting panels
(a) and (c) (IFN.alpha. only) to allow for detection of low level
induction.
[0075] FIG. 29 shows the increased susceptibility of LT.alpha.-/-
and LT.beta.R-Fc Tg mice to MCMV infection. Upper panel. Groups of
LT.alpha. deficient mice (n=4 to 6) were infected with MCMV Smith
strain at a dose of MCMV, 5.times.10.sup.3(n), 4.times.10.sup.4(l),
8.times.10.sup.4(t), 2.times.10.sup.5(u), or 5.times.10.sup.5(s)
PFU per mouse. Control wild type mice (C57/BL6; n=4 per group) were
infected with 1.times.10.sub.6 (c) PFU/mouse and the viability of
all mice was monitored daily for 2 weeks. Lower panel. Groups of
LT.beta.R-Fc transgenic mice (filled symbol) or age/sex matched
littermate control mice (open symbol)(n=4 to 6) were infected with
MCMV at 8.times.10.sup.4 (l) or 2.times.10.sup.5 (s) PFU per
animal. Both LT.beta.R-Fc(+) and control mice infected with doses
at or below 4.times.10.sup.4 PFU/mouse demonstrated 100% survival
at 14 days post infection. These studies were repeated on three
separate occasions for the LT.alpha.-/- and twice for the
LT.beta.R-Fc(+) mice.
[0076] FIG. 30 shows the agonist activity of the LT.beta.R antibody
(denoted anti-LT.beta.R). Fluorescence intensity indicates the
amount of VCAM-1.
DETAILED DESCRIPTION OF THE INVENTION
[0077] The invention provides a novel ligand for HVEM, or p30 and
functional variations and fragments thereof. This novel ligand,
which can be found as a membrane protein and can function as a
cytokine, is also called LIGHT, because this polypeptide is
homologous to Lymphotoxins, exhibits Inducible expression, and
competes with HSV Glycoprotein D for HVEM, a receptor expressed by
T lymphocytes. Because LIGHT can compete with HSV glycoprotein D
for HVEM, homotrimeric soluble forms of this polypeptide can be
used to block the entry of herpesvirus into cells. Thus, this novel
HVEM ligand can be used to treat or prevent herpesvirus infections,
such as .beta.-herpesvirus and cytomegalovirus.
[0078] LIGHT also bind to the lymphotoxin beta receptor
(LT.beta.R).
[0079] Experiments involving inhibition of binding of a fusion
protein containing the extracellular domain of the TNF receptor
(TNFR) related polypeptide, HVEM, showed that the both malignant
and normal human T-cells expressed LIGHT, the cell surface ligand
for HVEM.
[0080] The present invention is also based upon the discovery that
HVEM polypeptides have an antagonistic effect on inflammation. In
particular, HVEM fusion proteins are capable of inhibiting
inflammation when administered to a subject.
DEFINITIONS
[0081] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include the plural
unless the context clearly dictates otherwise. Thus, for example,
reference to "a cell" includes a plurality of such cells.
[0082] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below.
[0083] As used herein, to "inhibit" or "inhibiting" activity is to
reduce that activity a measurable amount, such as a reduction of at
least 30% or more. Where there are multiple different activities
that may be inhibited (for example, preventing cell recruitment,
production of pro-inflammatory mediators, cell or viral entry,
viral activation, viral replication, or viral progression), the
reduction of any single activity (with or without the other
activities) is sufficient to fall within the scope of this
definition. In addition, where a single or multiple agents are
administered to inhibit activity, the reduction by a single agent
of any single activity or the reduction by a combination of agents
of any single activity is sufficient to fall within the scope of
this definition. An "inflammation inhibiting amount" means that
amount of an inflammatory agent necessary to modulate, inhibit, or
suppress inflammatory responses or symptoms.
[0084] Inflammation
[0085] Inflammation results from a number of individual and related
cascades or reactions caused by pro-inflammatory mediators
including cytokines, prostaglandins, leukotrienes, chemokines,
adhesion molecules (e.g., LFA-1) and others known to those of skill
in the art. For example, receptors play a pivotal role in
permitting viral entry into cells. The ligands for these receptors
are also important. The ligands along with pro-inflammatory
mediators such as cytokines are the main stimulators of cells but
also play another role in amplification of the inflammatory
cascade. These soluble inflammatory mediators are derived mainly
from T cells. Once produced they can act in a paracrine and
autocrine fashion to further activate cells in their vicinity and
recruit additional T cells to the site of inflammation. These
additional lymphocytes are themselves activated, contributing to
the amplifying inflammatory cascade. Immunosuppressive activity, as
used herein, refers to inhibiting or decreasing the ability of B
and T cells to react or to be recruited or become activated to a
site of inflammation.
[0086] Other signals which activate inflammatory cells include
binding of an adhesion receptor, for example, LFA-1 (CD11a and
CD18), to one of its counter-receptors such as ICAM-1 (CD54)
(Staunton et al. (1990) Cell 61:243-254). If the second signal is
blocked, the antigen-specific T-cells are induced to die by
apoptosis or to enter a state of cellular anergy. Blockage of this
interaction by monoclonal antibodies to LFA-1 and ICAM-1 results in
increased survival time for mice receiving a heart allograft (Isobe
(1992) Science 255: I 125-I 127). Accordingly, the compositions and
methods of the invention may be used alone or in combination with
other anti-inflammatory agents including non-steroidal
anti-inflammatory drugs, steroids, antibodies, receptor antagonists
and others easily identifiable in the art.
[0087] As used herein "inflammation" means the process or series of
events that are elicited by numerous stimuli (e.g., infectious
agents, ischemia, antigen-antibody interactions, and thermal or
other physical injury). Inflammation is characterized by an
increase in erythema, edema, tenderness (hyperalgesia), and pain at
the inflamed site. An inflammatory reaction or cascade is generally
recognized as having a number of distinct stages, for example,
vasodilation and increased capillary permeability; an infiltration
of leukocytes and phagocytes; and a stage of tissue degeneration
and fibrosis. As used herein an "inflammatory disorder" means any
number of diseases characterized as having part of its pathogenesis
and inflammatory cascade or reaction resulting in inflammation.
Such disorders include, for example, arthritis, psoriasis,
inflammatory bowel disease, infectious agents such as herpes, and
others known to those of skill in the art.
[0088] Experiments involving inhibition of binding of a fusion
protein containing the extracellular domain of the TNF receptor
(TNFR) related polypeptide, HVEM, showed that both malignant and
normal human T-cells expressed a cell surface ligand for HVEM.
Competitive inhibition experiments showed that the HVEM ligand has
characteristics in common with LT.alpha..beta. heterotrimers and
LT.alpha., but also has features that distinguish it from
LT.alpha.1.beta.2 and TNF. Thus, LT.alpha.2.beta.1 could be a
putative surface ligand recognized by HVEM, with the caveat that
the HVEM binding site on LT.alpha.2.beta.1 is not the same as
TNFR60. Alternatively, HVEM might recognize a novel ligand. A
biochemical approach was used to distinguish between these
possibilities.
[0089] Immunoprecipitation and sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) studies demonstrated
the presence of a novel 30 kDa polypeptide ligand (p30) for HVEM on
the surface of T cells that was antigenically distinct from both
LT.beta. and LT.alpha.. Affinity chromatography purification and
two-dimensional electrophoresis showed that p30 is also physically
distinct from LT.alpha. and LT.beta. in that it has a molecular
weight of 30 kDa and a pI of about 7-8.5 (FIG. 4C). In addition,
these studies showed that active p30 (e.g. its trimeric form) is
also recognized by LT.beta.R but not by TNFR. The p30 polypeptide
is also referred to as LIGHT in the recent literature (see for
example, Mauri et al., Immunity, 8:21 (1998)).
[0090] Binding inhibition experiments demonstrated that soluble
gD-1 (gD from HSV-1) and a mutant of gD-1, gD-1 .DELTA.290-299t)
bind to HVEM but not to LT.beta.R or TNFR60. This result suggests
that gD-1 has co-evolved specifically for binding to HVEM, even
though HVEM binds to ligands that are recognized by TNFR60 and
LT.beta.R. Furthermore, the findings indicate that gD-1 is a
membrane-anchored virokine of the lymphotoxins and may modulate
HVEM signaling activities during entry or egress of HSV from the
infected cell.
[0091] In vitro cell culture studies showed that anti-HVEM antibody
enhanced proliferation of both naive and memory T cells. Similar
experiments indicated that signaling through HVEM provided an
activating stimulus to B cells and that a positive stimulus,
without a counterbalancing negative stimulus via the TNFR, may be a
unique property of the p30 HVEM ligand. These results indicate that
the physiologic functions of the HVEM ligand is likely to be
distinct from TNF and LT.alpha.1.beta.2. The identification of a
novel 30 kDa ligand for HVEM raises the possibility that this
ligand, may be responsible for physiological responses previously
ascribed to LT.alpha. or LT.beta.. The discoveries presented here
provide a deeper understanding of the LT/TNF cytokine system and
herpes virus that suggest new approaches for controlling these
cytokines in disease processes as well as affecting inflammation
due to infection and injury.
[0092] Together, the results indicate that antagonist and binding
agents such as, for example, Fc fusion proteins containing HVEM or
LT.beta.R will modulate the action of LT.alpha. and the 30 kDa HVEM
ligand, p30. The results also indicate that antagonists and binding
agents (e.g., antibodies and the fusion protein of the invention
(e.g., HVEM:Fc)) are also useful in modulating inflammation and
inflammatory responses. As discussed above, HVEM binding activates
B cells and thus a modulation in the interaction of HVEM with its
ligand reduces the activation of inflammatory cells and activation
of the inflammatory cascade. Similarly, fusion protein HVEM could
be used to identify specific inhibitors of the ligand
receptor-complexes, such as monoclonal antibodies or peptides or
small organic compounds. Inhibitors of p30 or LT.alpha.
interactions with HVEM, or p30 interactions with LT.beta.R, could
be used to modulate diseases where unwanted lymphocyte
proliferation occurs, including T and B lymphomas or leukemias, or
in autoimmune diseases, such as rheumatoid arthritis,
insulin-dependent diabetes mellitus, multiple sclerosis, systemic
lupus erythematosus, myasthenia gravis, and inflammation, for
example, arthritis, psoriasis, inflammatory bowel disease, asthma,
and other known in the art.
[0093] Similarly, herpesvirus gD-1 could be used to inhibit immune
reactions where LT.alpha., p30 and HVEM signaling are implicated as
effector molecules. LT.alpha. or soluble forms of the 30 kDa HVEM
ligand (generated by deletion of its predicted cytoplasmic and
transmembrane domains e.g. LIGHT-t66 described below) may function
as inhibitors of viral infection including, for example, infection
by Herpes viridae (e.g., alphaherpes virinae, betaherpes virinae
and gammaherpes virinae) and recrudesces by blocking the ability of
virus to enter a cellular target.
[0094] Like TNFRs, HVEM has a dual ligand specificity, binding to
LT.alpha. and the trimeric form (e.g., 90 kD form) of LIGHT, a
membrane bound form of the ligand, p30. The LT.alpha. Tyr108Phe
mutation destroys HVEM binding as it does for TNFR60 and TNFR80.
The inability of TNFR60 to block HVEM binding to the surface 30 kDa
form indicates that surface LT.alpha.2.beta.1 is not an HVEM
ligand.
[0095] Furthermore, LIGHT (p30) differs from LT.alpha. because it
is antigenically distinct and remains cell-associated, unlike
LT.alpha. which is exclusively secreted. Thus, the HVEM binding
protein (p30) is predicted to contain a stretch of hydrophobic
residues forming a transmembrane domain arranged as a type-II
transmembrane configuration similar to other proteins related to
TNF. This does not exclude the possibility that p30 might also be
modified in other ways (e.g., lipid modification) to allow
attachment to the cell surface. Furthermore, this protein should
share regions of sequence homology with LT.alpha. and LT.beta. and
related cytokines that define this superfamily and contain a
C-terminal extracellular domain of approximately 150-160
residues.
[0096] The inventors' findings also indicate that HVEM is a
specific receptor for LT.alpha., a property that clearly
distinguishes it from the TNF binding receptors, TNFR60, also
referred to as TNFR1, and TNFR80, also referred to as TNFR2. This
property will allow an HVEM fusion protein or similar protein to
antagonize LT.alpha. specifically without inhibiting TNF or
LT.alpha.1.beta.2 functions.
[0097] The present invention provides antagonists and binding
agents, such as, e.g., a soluble, homotrimeric LIGHT or an HVEM
fusion polypeptide. The HVEM fusion polypeptide can be
characterized as having a molecular weight of about 58 kDa as
determined by reducing SDS-PAGE, as demonstrated in FIG. 8, see
Example 6.
[0098] The present invention also provides a substantially pure
LIGHT, or p30 polypeptide. The p30 polypeptide is characterized as
having a predicted molecular weight of 30 kDa as determined by
reducing SDS-PAGE and a pI in the range of about 7-8.5 (FIG. 4C).
p30 exists in less than ten, such as less than eight, particularly
less than six, (e.g., three, four or five) isomeric forms. The
invention also provides LIGHT, or p30, as a homotrimer. As
demonstrated in the examples, below, p30 can be expressed as a
homotrimer, and, when expressed as a recombinant, truncated,
soluble form, it is secreted exclusively as a homotrimer. The LIGHT
polypeptide can be cell bound, i.e., is not secreted. In its cell
surface form, p30 binds HVEM and LT.beta.R.
[0099] The term "substantially pure" as used herein refers to
polypeptide which is substantially free of other proteins, lipids,
carbohydrates or other materials with which it is naturally
associated. One skilled in the art can purify such polypeptides and
fusion proteins using standard techniques for protein purification
(see, e.g., Protein Purification, Principles and Practice, second
edition (1987) Scopes, Springer Verlag, N.Y.). For example, a
substantially pure HVEM:Fc polypeptide will yield a single major
band of about 58 kDa on a reducing SDS-PAGE gel. The LIGHT p30
polypeptide will yield a single major band of about 30 kDa on a
reducing SDS-PAGE gel; in its homotrimeric form, it forms a single
major band of about 90 kDa on a non-reducing gel (which does not
disturb the trimeric tertiary structure).
[0100] The invention includes a functional fusion polypeptide and
functional fragments thereof. As used herein, the term "functional
polypeptide" refers to a polypeptide which possesses a biological
function or other activity, such as the ability to bind to a
receptor, which can be identified through routing and defined
functional and receptor binding assays (see, e.g., the Examples
below), and which, e.g., are associated with particular biologic,
morphologic, or phenotypic alterations in the cell, or binding
events, such as the ability of a soluble LIGHT polypeptide of the
invention to block entry of a herpesvirus.
[0101] "Functional fragments" as used herein include subsequences
of the polypeptides of the invention which a biological function,
as described above. For example, fusion polypeptides of the
invention can include fragments of, e.g., LIGHT or HVEM. For
example, the invention provides fragments of HVEM and a second
polypeptide sequence so long as an activity of substantially the
same as that of HVEM:Fc remains, e.g., modulation of cellular
responses by inhibiting binding of HSV to HVEM, antigenicity, or
inhibition of inflammation. Smaller peptides containing the
biological activity of HVEM or HVEM fusion polypeptides are
included in the invention. One of skill in the art can assay for
functional activity of such polypeptides by standard methods, e.g.,
viral plaque reduction assay or cell activation assays including
cytokine production assays and measurement of inflammatory
responses as described in the Examples. Similarly, functional
fragments of p30 may be determined through a defined functional
assay and which is associated with a particular biologic,
morphologic, or phenotypic alteration in the cell.
[0102] The invention provides polypeptides with minor modifications
of the LIGHT (p30), HVEM or fusion proteins (e.g., p30 of HVEM
fusion protein) primary amino acid sequences. This can result in
proteins which have substantially equivalent activity, e.g., as
compared to the p30, the LIGHT-t66, and other mutants described
below; and, HVEM:Fc polypeptide, as described herein. Such
modifications can be specifically engineered, e.g., as generated by
site-directed mutagenesis. Alternatively, they can be spontaneous
mutations. All of the polypeptides produced by these modifications
are within the scope of the invention so long as the binding or
biological activity of LIGHT (p30) or HVEM:Fc is present, e.g.,
modulation of cellular responses by binding to HVEM and LT.beta.R
or inhibiting binding of HSV to HVEM or modulation of inflammation.
Further, deletion of one or more amino acids can also result in a
modification of the structure of the resultant molecule without
significantly altering its activity (e.g. LIGHT-t66, see
below).
[0103] Thus, the invention includes smaller, active (i.e.,
"functional") LIGHT (p30), including truncated, soluble
homotrimeric forms, and HVEM molecules having alternative
utilities. For example, amino or carboxyl terminal amino acids
which are not required for activity can be removed. For example,
the HVEM:Fc fusion polypeptide (described below) is characterized
as having amino acids 1 through 205 of HVEM. Another example
includes a truncated soluble homotrimeric p30 protein, the
LIGHT-t66, described in detail in Example 12, below.
[0104] The polypeptides of the invention also include conservative
variations, mimetics and peptidomimetics of the polypeptide
sequence. The term "conservative variation" as used herein denotes
the replacement of an amino acid residue by another, biologically
similar residue. Examples of conservative variations include the
substitution of one hydrophobic residue such as isoleucine, valine,
leucine or methionine for another, or the substitution of one polar
residue for another, such as the substitution of arginine for
lysine, glutamic for aspartic acids, or glutamine for asparagine,
and the like. The term "conservative variation" also includes the
use of a substituted amino acid in place of an unsubstituted parent
amino acid provided that antibodies raised to the substituted
polypeptide also immunoreact with the unsubstituted
polypeptide.
[0105] The terms "mimetic" and "peptidomimetic" refer to a
synthetic chemical compound that has substantially the same
structural and/or functional characteristics of the polypeptides,
e.g., translocation domains or odorant-ligand binding domains or
chimeric receptors of the invention. The mimetic can be either
entirely composed of synthetic, non-natural analogues of amino
acids, or, is a chimeric molecule of partly natural peptide amino
acids and partly non-natural analogs of amino acids. The mimetic
can also incorporate any amount of natural amino acid conservative
substitutions as long as such substitutions also do not
substantially alter the mimetic's structure and/or activity. As
with polypeptides of the invention which are conservative variants,
routine experimentation will determine whether a mimetic is within
the scope of the invention, i.e., that its structure and/or
function is not substantially altered. Polypeptide mimetic
compositions can contain any combination of non-natural structural
components, which are typically from three structural groups: a)
residue linkage groups other than the natural amide bond ("peptide
bond") linkages; b) non-natural residues in place of naturally
occurring amino acid residues; or c) residues which induce
secondary structural mimicry, i.e., to induce or stabilize a
secondary structure, e.g., a beta turn, gamma turn, beta sheet,
alpha helix conformation, and the like. A polypeptide can be
characterized as a mimetic when all or some of its residues are
joined by chemical means other than natural peptide bonds.
Individual peptidomimetic residues can be joined by peptide bonds,
other chemical bonds or coupling means, such as, e.g.,
glutaraldehyde, N-hydroxysuccinimide esters, bifunctional
maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or
N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be an
alternative to the traditional amide bond ("peptide bond") linkages
include, e.g., ketomethylene (e.g., --C(.dbd.O)--CH.sub.2-- for
--C(.dbd.O)--NH--), aminomethylene (CH.sub.2--NH), ethylene, olefin
(CH.dbd.CH), ether (CH.sub.2-O), thioether (CH.sub.2-S), tetrazole
(CN.sub.4-), thiazole, retroamide, thioamide, or ester (see, e.g.,
Spatola (1983) In: Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone
Modifications," Marcell Dekker, NY). A polypeptide can also be
characterized as a mimetic by containing all or some non-natural
residues in place of naturally occurring amino acid residues;
non-natural residues are well described in the scientific and
patent literature.
[0106] Antagonists and binding agents of the invention can also
include antibodies to p30 (LIGHT), antibodies and polypeptides
which bind to or act as antagonist of HVEM (as discussed more fully
below) and antibodies which act as agonists of LT.beta.R or TNFR1
(see, e.g., Examples 21 and 22), as well as the fusion proteins
describe herein.
[0107] The fusion protein of the invention includes the full length
HVEM polypeptide sequence as well as fragments of the sequence
which may exclude certain peptides sequences. Similarly, the LIGHT
(p30) polypeptide of the invention includes the full length p30,
homotrimer forms, fusion proteins comprising p30 sequence, as well
as fragments of the sequence which may exclude certain amino acids,
peptides or sequences, and homo- or hetero-trimeric forms of these
fragments, such as, e.g., LIGHT-t66, as described below. Such
fragments can be useful in the creation of the antibodies of the
invention, including polyclonal and monoclonal antibodies.
[0108] For example, such excluded sequences may include fragments
lacking the carboxyl terminal region of the full length HVEM
polypeptide. In addition, the fusion polypeptides of the invention
can include an HVEM polypeptide sequence or a fragment thereof.
[0109] The "second" polypeptide sequence of the fusion protein can
be any polypeptide desired to be linked to a polypeptide sequence
of the invention (e.g., p30 or HVEM sequence) so long as the
p30/HVEM:second polypeptide fusion protein retains a binding (e.g.,
virus blocking or receptor binding) or biological activity that is
substantially similar to the binding (e.g., to receptors) or
biological activity of a p30:Fc or HVEM:Fc (e.g., inhibits or
modulates inflammation). The identification and determination of
second polypeptide sequences useful in the present invention are
readily identifiable to one skilled in the art. For example, one
skilled in the art can determine whether a fusion construct retains
the desired receptor binding or biological activity by using the
methods and techniques described in Examples below.
[0110] The invention also provides isolated nucleic acid sequences
or polynucleotides encoding the polypeptide of the invention.
Nucleic acids encoding any of the above polypeptides, fragments,
modifications and fusion proteins are also encompassed by the
present invention. Thus, the term "isolated" as used herein
includes polynucleotides substantially free of other nucleic acids,
proteins, lipids, carbohydrates or other materials with which it is
naturally associated. Polynucleotide sequences of the invention
include DNA, cDNA and RNA sequences which encode antagonists,
binding agents or a fusion polypeptide of the invention. For
example, it is understood that all polynucleotides encoding all or
a portion of LIGHT or HVEM or fusion polypeptides thereof are also
included herein, so long as they encode a polypeptide with LIGHT:Fc
or HVEM:Fc activity, as described herein. Such polynucleotides
include (isolated) naturally occurring, recombinant, synthetic, and
intentionally manipulated polynucleotides. For example, portions of
the mRNA sequence may be altered due to alternate RNA splicing
patterns or the use of alternate promoters for RNA transcription.
As another example, the polynucleotide may be subjected to
site-directed mutagenesis. The polynucleotides of the invention
include sequences that are degenerate as a result of the genetic
code. There are 20 natural amino acids, most of which are specified
by more than one codon. Therefore, all degenerate nucleotide
sequences are included in the invention so long as the amino acid
sequence of LIGHT or HVEM and (in the case of a fusion protein) a
second polypeptide encoded by the nucleotide sequence are
functionally unchanged.
[0111] The nucleic acids of the invention comprise sequences that
encode an polypeptide of the invention, an antagonist, a binding
agent or a fusion protein (e.g., LIGHT-FLAG or HVEM:Fc), as well as
fragments of naturally occurring LIGHT or HVEM and any nucleotide
sequence that can hybridize to the complement of these sequences
under stringent conditions. The invention also includes degenerate
variants of these sequences for both p30 and the fusion
polypeptides of the invention. For example, the LIGHT p30 nucleic
acids of the invention include sequences that encode naturally
occurring p30 and any nucleotide sequences that hybridize to the
complement of the sequences under stringent conditions as described
herein.
[0112] The phrase "stringent conditions" refers to hybridization or
wash conditions under which a nucleic acid, e.g., a sample nucleic
acid or a probe will primarily hybridize to its target subsequence,
typically in a complex mixture of nucleic acid, but to no other
sequences in significant amounts. A positive signal (e.g.,
identification of a nucleic acid of the invention) is about 10
times background hybridization. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Probes, "Overview of principles
of hybridization and the strategy of nucleic acid assays" (1993).
Generally, stringent conditions are selected to be about
5-10.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength, pH, and nucleic acid
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium).
[0113] Stringent conditions will be those in which the salt
concentration is less than about 1.0 M sodium ion, typically about
0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0
to 8.3 and the temperature is at least about 30.degree. C. for
short probes (e.g., 10 to 50 nucleotides) and at least about
60.degree. C. for long probes (e.g., greater than 50 nucleotides).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide.
[0114] Stringent hybridization conditions that can be used to
identify nucleic acids within the scope of the invention can
include hybridization in a buffer comprising 50% formamide,
5.times.SSC, and 1% SDS at 42.degree. C., or hybridization in a
buffer comprising 5.times.SSC and 1% SDS at 65.degree. C., both
with a wash of 0.2.times.SSC and 0.1% SDS at 65.degree. C.
Exemplary stringent hybridization conditions can also include a
hybridization in a buffer of 40% formamide, 1 M NaCl, and 1% SDS at
37.degree. C., and a wash in 1.times.SSC at 45.degree. C.
Alternatively, hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. can be used to identify and isolate nucleic acids within the
scope of the invention. Those of ordinary skill will readily
recognize that alternative but comparable hybridization and wash
conditions can be utilized to provide conditions of similar
stringency.
[0115] However, the selection of a hybridization format is not
critical, as is known in the art, it is the stringency of the wash
conditions that set forth the conditions which determine whether a
nucleic acid is within the scope of the invention. Wash conditions
used to identify nucleic acids within the scope of the invention
include, e.g.: a salt concentration of about 0.02 molar at pH 7 and
a temperature of at least about 50.degree. C. or about 55.degree.
C. to about 60.degree. C.; or, a salt concentration of about 0.15 M
NaCl at 72.degree. C. for about 15 minutes; or, a salt
concentration of about 0.2.times.SSC at a temperature of at least
about 50.degree. C. or about 55.degree. C. to about 60.degree. C.
for about 15 to about 20 minutes; or, the hybridization complex is
washed twice with a solution with a salt concentration of about
2.times.SSC containing 0.1% SDS at room temperature for 15 minutes
and then washed twice by 0.1.times.SSC containing 0.1% SDS at
68.degree. C. for 15 minutes; or, equivalent conditions. Stringent
conditions for washing can also be 0.2.times.SSC/0.1% SDS at
42.degree. C. In instances wherein the nucleic acid molecules are
deoxyoligonucleotides ("oligos"), stringent conditions can include
washing in 6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C.
(for 14_base oligos), 48.degree. C. (for 17-base oligos),
55.degree. C. (for 20-base oligos), and 60.degree. C. (for 23-base
oligos). See Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL
(2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989);
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley
& Sons, Inc., New York (1997), or Tijssen (1993) supra, for
detailed descriptions of equivalent hybridization and wash
conditions and for reagents and buffers, e.g., SSC buffers and
equivalent reagents and conditions.
[0116] These nucleic acid molecules may encode or act as p30
antisense molecules, useful, for example, in p30 regulation (for
and/or as antisense primers in amplification reactions of p30
nucleic acid sequences). Still further, such molecules may be used
as components of screening methods whereby, for example, the
presence of a p30 gene may be detected.
[0117] In addition to the nucleotide sequences described above,
full length cDNA or genomic sequences can be identified and readily
isolated, without undue experimentation, by molecular biological
techniques well known in the art. The invention encompasses these
nucleic acid molecules.
[0118] DNA sequences of the invention can be obtained by several
methods. For example, the DNA can be isolated using hybridization
or computer-based techniques which are well known in the art. These
include, but are not limited to: (a) hybridization of genomic or
cDNA libraries with probes to detect homologous nucleotide
sequences; (b) antibody screening of expression libraries to detect
cloned DNA fragments with shared structural features; (c)
polymerase chain reaction (PCR) on genomic DNA or cDNA using
primers capable of annealing to the DNA sequence of interest; (d)
computer searches of sequence databases for similar sequences; (e)
differential screening of a subtracted DNA library; and (f) large
scale genomic sequencing by expressed sequence tags (EST) of a T
cell cDNA library. Preferably the polynucleotides (e.g., p30
polynucleotide and HVEM:fusion protein polynucleotide) of the
invention are derived from a mammalian organism.
[0119] Screening procedures which rely on nucleic acid
hybridization make it possible to isolate any gene sequence from
any organism, provided the appropriate probe is available.
Oligonucleotide probes, which correspond to a part of the sequence
encoding the protein in question, can be synthesized chemically.
This requires that short, oligopeptide stretches of amino acid
sequence must be known. The DNA sequence encoding the protein can
be deduced from the genetic code, however, the degeneracy of the
code must be taken into account. It is possible to perform a mixed
addition reaction when the sequence is degenerate. This includes a
heterogeneous mixture of denatured double-stranded DNA. For such
screening, hybridization is preferably performed on either
single-stranded DNA or denatured double-stranded DNA. Hybridization
is particularly useful in the detection of cDNA clones derived from
sources where an extremely low amount of mRNA sequences relating to
the polypeptide of interest are present. In other words, by using
stringent hybridization conditions directed to avoid non-specific
binding, it is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement (Wallace et al. (1981) Nucl. Acid Res. 9:879).
Alternatively, a subtractive library, is useful for elimination of
non-specific cDNA clones.
[0120] When the entire sequence of amino acid residues of the
desired polypeptide is not known, the direct synthesis of DNA
sequences is not possible and the method of choice is the synthesis
of cDNA sequences. Among the standard procedures for isolating cDNA
sequences of interest is the formation of plasmid- or
phage-carrying cDNA libraries which are derived from reverse
transcription of mRNA which is abundant in donor cells that have a
high level of genetic expression. When used in combination with
polymerase chain reaction technology, even rare expression products
can be cloned. In those cases where significant portions of the
amino acid sequence of the polypeptide are known, the production of
labeled single or double-stranded DNA or RNA probe sequences
duplicating a sequence putatively present in the target cDNA may be
employed in DNA/DNA hybridization procedures which are carried out
on cloned copies of the cDNA which have been denatured into a
single-stranded form (Jay et al., (1983) Nucl. Acid Res. 11:2325).
Appropriate oligonucleotide probes and primers can be constructed
by "back-translating" the amino acid sequence of the p30
polypeptide obtained by N-terminal amino acid sequencing.
[0121] A cDNA expression library, such as lambda gt11, can be
screened indirectly for p30 peptides having at least one epitope,
using antibodies specific for p30. Such antibodies can be either
polyclonally or monoclonally derived and used to detect expression
product indicative of the presence of p30 cDNA.
[0122] Alterations in p30 nucleic acid include intragenic mutations
(e.g., point mutation, nonsense (stop), missense, splice site and
frameshift) and heterozygous or homozygous deletions. Detection of
such alterations can be done by standard methods known to those of
skill in the art including sequence analysis, Southern blot
analysis, PCR based analyses (e.g., multiplex PCR, sequence tagged
sites (STSs)) and in situ hybridization. Such proteins can be
analyzed by standard SDS-PAGE and/or immunoprecipitation analysis
and/or Western blot analysis, for example.
[0123] The invention also encompasses DNA vectors that contain any
of the foregoing p30 coding sequences and/or their complements
(i.e., antisense) and expression vectors that contain any of the
foregoing p30 coding sequences.
[0124] The invention also encompasses DNA vectors that contain any
of the foregoing fusion polypeptide coding sequences and/or their
complements and expression vectors that contain any of the
foregoing coding sequences. An expression vector is composed of or
contains a nucleic acid in which a polynucleotide sequence encoding
a peptide or polypeptide of the invention is operatively linked to
a promoter or enhancer-promoter combination. A promoter is a
transcriptional regulatory element composed of a region of a DNA
molecule typically within 100 nucleotide pairs in front (upstream
of) of the point at which transcription starts. Another
transcriptional regulatory element is an enhancer. An enhancer
provides specificity in terms of time, location and expression
level. Unlike a promoter, an enhancer can function when located at
variable distances from the transcription site, provided a promoter
is present. An enhancer can also be located downstream of the
transcription initiation site. A coding sequence of an expression
vector is operatively linked to a transcription terminating region.
To bring a coding sequence under control of a promoter, it is
necessary to position the translation initiation site of the
translational reading frame of the peptide or polypeptide between
one and about fifty nucleotides downstream (3') of the promoter.
Such regulatory elements include but are not limited to the
cytomegalovirus hCMV immediate early gene, the early or late
promoters of SV40 adenovirus, the lac system, the trp system, the
TAC system, the TRC system, the major operator and promoter regions
of phage A, the control regions of fd coat protein, the promoter
for 3 phosphoglycerate kinase, the promoters of acid phosphatase,
and the promoters of the yeast a mating factors.
[0125] Expression vectors and methods for their construction are
known to those familiar with the art. Suitable vectors include
plasmids, and viral vectors such as herpes viruses, retroviruses,
canary pox viruses, adenoviruses and adeno-associated viruses,
among others.
[0126] The invention includes suitable host cell lines transfected
with expression vectors containing the nucleic acid sequences
described. Cells to be used for transfection include, but are not
restricted to HEK293 cells of mammalian origin or Sf9 and TN5
insect cells, for example, for expression of a fusion polypeptide
of the invention in its various natural or engineered forms. Cells
are transfected by a variety of methods commonly used in the art,
for example, electroporation or calcium phosphate precipitation.
Genes can also be introduced into the cells by transduction with
viral vectors, e.g., retroviruses. Successfully transfected cell
lines are selected by appropriate means familiar to those of
average skill in the art, e.g., using tissue culture medium
supplemented with a drug such as Geneticin.TM. (G418) or puromycin,
for example, for which the relevant expression vector contains a
resistance gene. Successfully transfected cell lines are screened
expression of the fusion molecules by a variety of possible
methods, e.g., flow cytometry analysis.
[0127] "Host cells" are cells in which a vector can be propagated
and its DNA expressed. The term also includes any progeny of the
subject host cell. It is understood that all progeny may not be
identical to the parental cell since there may be mutations that
occur during replication. However, such progeny are included when
the term "host cell" is used.
[0128] Antibodies that specifically recognize antigenic epitopes
within an amino acid sequence of p30 or HVEM or LT.beta.R or TNFR1
are also encompassed by the invention. Such antibodies include but
are not limited to non-human, humanized and fully human antibody
sequences. For example, humanized and fully human antibodies having
the binding specificity of a monoclonal antibody denoted 3C8, 3H4
or 4H8. Antibodies include polyclonal antibodies (IgG, IgM, IgA,
IgE and IgD), monoclonal antibodies, chimeric antibodies, single
chain antibodies, Fab fragments, F(ab').sub.2 fragments, Fv, and
epitope-binding fragments of any of the above. The term "antibody"
therefore includes intact antibody molecules as well as fragments
thereof. Exemplary fragments include, for example, those capable of
binding to an antigenic determinant present in p30 or HVEM or
LT.beta.R or TNFR1 or a functional subsequence thereof, and having
agonist or antagonist activity. Generally, antigenic determinants
have at least 3-5 contiguous amino acids.
[0129] Such antibodies can act as antagonists or binding agents in
modulation an inflammatory reaction. For example, antibodies to p30
can act by interaction with p30 and preventing p30's binding to its
receptor. Similarly, antibodies that bind to or interact with HVEM
can also act to prevent binding of HVEM with its ligand (e.g.,
p30). Such antibodies can also act in modulation of viral
infection. For example, antibodies that bind LT.beta.R or TNFR1 and
which have agonist activity (e.g., antibody having the binding
specificity of a monoclonal antibody denoted 3C8, 3H4 or 4H8) can
be used to activate LT.beta.R or TNFR1 activity which, in turn
inhibits or prevents herpesvirus (e.g., CMV) infection,
reactivation from latency or disorders associated with herpesvirus
(e.g., CMV) infection.
[0130] The antibodies of the invention can be used, for example, in
the treatment of autoimmune diseases and lymphocytic malignancies.
They can also be used to test for expression of p30 on a cell and
may thus be utilized as part of a screening procedure to select an
appropriate treatment for a particular subject. For example, if the
tumor cells of a lymphoma or leukemia patient express p30, anti-p30
antibody or immunotoxin conjugates of anti-p30 antibody or
immunotoxin conjugates of anti-p30 antibody may be used as therapy
in that patient. Such antibodies may also be utilized in the
screening assays of the invention. Antibodies that bind LT.beta.R
or TNFR1 and which have agonist activity can be used to treat,
inhibit or prevent herpesvirus (e.g., CMV) infection.
[0131] Protein suitable for generating antibodies can be produced
by any of a variety of standard synthetic or recombinant expression
techniques known in the art. For example, protein can be produced
by standard peptide synthesis techniques, such as solid-phase
synthesis. Nucleic acid encoding protein may be expressed in a cell
and the protein produced by the cells may be purified. For example,
using a baculovirus expression construct and isolating the produced
protein from infected Tn5 insect cells. The protein may be
expressed as a part of a larger protein by recombinant methods. The
protein may then be purified with a protein A column or affinity
column. Alternatively, the protein may contain an amino acid
sequence such as a T7 tag or polyhistidine sequence to facilitate
purification of the expressed or synthesized protein.
[0132] Antibodies may be produced using intact polypeptide,
functional subsequences thereof or small peptide fragments, or
fusion protein as the immunizing antigen. To produce antibodies
that specifically bind to the N- or C-terminal domains of a
protein, or internal amino acid sequences, appropriate regions can
be used as immunizing antigen.
[0133] For the production of antibodies of the invention, a host
animal is immunized by injection with either the fusion
polypeptide, the individual polypeptides comprising the fusion
polypeptide (e.g., HVEM), with cells expressing the fusion
polypeptide, or the p30 polypeptide. Alternatively, peptides
corresponding to specific regions (i.e., antigenic epitopes) of
these polypeptides may be used as immunogens. Such host animals may
include but are not limited to rabbits, mice, and rats. Various
adjuvants may be used to increase the immunological response,
depending on the host species, including but not restricted to
Freund's (complete and incomplete) adjuvant, mineral gels such as
aluminum hydroxide, lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, BCG (bacille Calmette-Guerin) and
Corynebacterium parvum. Polyclonal antibodies are heterogeneous
populations of antibody molecules derived from the sera of the
immunized animals.
[0134] In order to further enhance immunogenicity, the immunogen
may be coupled to a carrier. Examples of such carriers are keyhole
limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other
albumins such as ovalbumin, mouse serum albumin or rabbit serum
albumin can also be used as carriers. Methods of coupling a peptide
to a carrier are well known in the art and include the use of
glutaraldehyde, carbodiimide and
m-maleimidobenzoyl-N-hydroxysuccinimide ester.
[0135] The amount of antigen to be used can be determined readily
by those with average skill in the art without undue
experimentation. The antigen can be administered by a number of
routes (e.g., subcutaneous, intramuscular, intradermal, intravenous
and intraperitoneal). The production of polyclonal antibodies is
monitored by sampling blood of the immunized animal at various time
points after administration. When the desired level of antibody is
obtained, the animal is bled and the serum is stored.
[0136] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, may be obtained by any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique (Kohler and Milstein (1975)
Nature 256:495-497; U.S. Pat. No. 4,376,110; Howell and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Press, N.Y.),
the human B-cell hybridoma technique (Kosbor (1983) Immunology
Today 4:72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026),
and the EBV-hybridoma technique (Cole et al. (1985), Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc.). Monoclonal
antibodies can be isolated and purified from hybridoma cultures by
a variety of well-established techniques which include, for
example, affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography
(see, e.g., Coligan et al. (1994) In: Current Protocols in
Immunology, Wiley; and Barnes et al. (1992) In: Methods in
Molecular Biology, Vol. 10, pages 79-104, Humana Press). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof.
[0137] In addition, techniques developed for the production of
"chimeric antibodies" can be used (Morrison et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851; Neuberger (1984) Nature 312:604;
Takeda (1985) Nature 314:452). These involve, for example, splicing
a portion of a gene encoding a mouse antibody of appropriate
antigen specificity to a portion of a gene encoding a human
antibody of appropriate biological activity. A chimeric antibody is
a molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine monoclonal antibody and a human immunoglobulin constant
region. Such chimeric antibodies could also be generated, for
example, by immunizing mice containing the human genetic loci
encoding IgH and .kappa. and .lamda. light chain loci.
[0138] Chimeric antibodies can be produced using recombinant DNA
technology and expression of the recombinant construct in cells to
produce the antibody chimera. Alternatively, techniques described
for the production of single chain antibodies (U.S. Pat. No.
4,946,778; Bird (1988) Science 242:423; Huston et al. (1988) Proc.
Natl. Acad. Sci. USA 85:5879; and Ward et al. (1989) Nature
334:544) can be adapted to produce single chain antibodies against
epitopes of the fusion polypeptide of the invention. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. They are conveniently produced by
recombinant DNA techniques. The antibody may be purified as
discussed herein, for example, protein A or G purification or
affinity purification, or using other methods known in the art.
[0139] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, such fragments include
but are not limited to the F(ab')2 fragments which can be produced
by pepsin digestion of the antibody molecule, and the Fab fragments
which can be generated by reducing the disulfide bridges of the
F(ab').sub.2 fragments. Alternatively, Fab expression libraries may
be constructed (Huse (1989) Science 246:1275) to allow rapid and
easy identification of monoclonal Fab fragments with the desired
specificity. Methods for screening antibodies for binding
specificity are well known in the art.
[0140] Humanized antibodies can be produced by transferring
non-human complementarity determining regions (CDR's) from heavy
and light variable chains into a human variable domain, and then
substituting human residues in the framework regions of the
non-human counterparts. Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.
(1986) Nature 321:522; Riechmann et al. (1988) Nature 332:323;
Verhoeyen et al. (1988) Science 239:1534; Carter et al. (1992)
Proc. Natl. Acad. Sci. USA 89:4285; Sandhu (1992) Crit. Rev.
Biotech. 12:437; and Singer et al. (1993) J. Immunol. 150:2844. The
use of fully human and humanized antibodies obviates potential
problems associated with immunogenicity of non-human sequences.
[0141] In order to produce fully human antibodies, a protein
antigen can be produced using techniques known in the art and
subsequently used to immunize human transchromosomic (Tc) mice,
which contain kappa or lambda human IgG chains in their chromosomes
(Tomizuka et al., (2000) Proc. Natl. Acad. Sci. USA 97:722 and
Tomizuka et al., (1997) Nat. Genet. 16:133). Mice exhibiting
positive antibody titer can be used for spleen cell fusions and
monoclonal antibodies prepared using the general method of Kohler
and Milstein (Nature 256:495 (1975)). The resulting hybridomas are
assayed for the production of heavy or kappa chains and then for
antibody production. Cells may be cloned and rescreened for
antibody production. The selected or cloned hybridomas are then
cultured either in vitro (e.g., in tissue culture), or in vivo (as
ascites in mice) and human antibodies purified. Suitable
purification techniques include affinity purification,
non-denaturing gel purification, HPLC or RP-HPLC, purification on
protein A column, or any combination of these techniques. The
antibodies are confirmed to be human Ig by using mouse Ig-absorbed
anti-human Ig in an ELISA assay.
[0142] Thus, in another embodiment, the invention provides methods
of producing human antibodies that modulate activity of LT.beta.R
or TNFR1. In one embodiment, a method includes administering
LT.beta.R or TNFR1 or an immunogenic fragment thereof to an animal
(e.g., a mouse) capable of expressing human immunoglobulin;
screening the animal for expression of human LT.beta.R or TNFR1
antibody; selecting an animal that produces a human LT.beta.R or
TNFR1 antibody; isolating the antibody from the animal; and
determining whether the human LT.beta.R or TNFR1 antibody modulates
an activity of LT.beta.R or TNFR1 thereby identifying a human
LT.beta.R or TNFR1 antibody that modulates an activity of LT.beta.R
or TNFR1. In yet another embodiment, a method includes
administering human LT.beta.R or TNFR1 or an immunogenic fragment
thereof to an animal (e.g., a mouse) capable of expressing human
immunoglobulin; isolating spleen cells from the animal that
produces human LT.beta.R or TNFR1 antibody; fusing the spleen cells
with a myeloma cell to produce a hybridoma; and screening the
hybridoma for expression of a human LT.beta.R or TNFR1 antibody
that modulates an activity of LT.beta.R or TNFR1 thereby
identifying a hybridoma that produces a human LT.beta.R or TNFR1
antibody.
[0143] The invention features in vitro systems designed to identify
compounds capable of modulating cellular responses mediated via
either the HVEM or LT.beta.R receptor polypeptides. "Cellular
responses" refers herein to cell activation, cell internalization
of HSV or changes in activation or production of inflammatory
mediators such as inflammatory cells, cytokines, prostaglandins and
leukotrienes. These cellular responses are elicited by an
interaction of (a) HVEM with p30, gD or LT.alpha.; or (b) LT.beta.R
with p30.
[0144] The term "ligand" refers to a polypeptide or a compound that
binds to a receptor protein in a high affinity and specific manner
to elicit a functional response. For example ligands of the
invention include p30, gD or LT.alpha.. The term "receptor" refers
herein to a polypeptide which, when bound by a ligand, induces a
cellular response. Receptors of the invention include HVEM or
LT.beta.R or TNFR1. The term "binding agent" refers to a
polypeptide or a compound that binds to a receptor or a ligand in a
high affinity and specific manner and may or may not elicit a
functional response.
[0145] In one embodiment the invention features an assay for
identifying a compound which affects an HVEM-binding agent-mediated
cellular response. This assay involves: (a) incubating the compound
with an HVEM polypeptide or a cell expressing an HVEM polypeptide,
and an HVEM-binding agent, under conditions which allow the
components to interact; and (b) determining the effect of the
compound on the HVEM-binding agent-mediated cellular response. Also
within the invention is an assay for identifying a compound which
affects an LT.beta.R-p30-mediated cellular response. This assay
involves: a) incubating the compound with an LT.beta.R polypeptide
or a cell expressing an LT.beta.R polypeptide, and with p30, under
conditions which allow the components to interact; and (b)
determining the effect of the compound on the
LT.beta.R-p30-mediated cellular response. In the assays of the
invention compounds are screened for their ability to either
modulate a cell activation mediated by interaction HVEM or
LT.beta.R with a ligand or to inhibit infection of susceptible
cells by HSV.
[0146] In another embodiment the invention features an assay for
identifying a compound that inhibits herpesvirus (e.g., CMV)
infection. This assay involves: (a) contacting LT.beta.R or TNFR1
with a test compound under conditions allowing binding; (b)
measuring LTR or TNFR1 activity in the presence of the test
compound; and (c) comparing activity in the presence of the test
compound to the absence of the test compound. An increase in
LT.beta.R or TNFR1 activity in the presence of the test compound
identifies the test compound as a compound that inhibits
herpesvirus (e.g., CMV) infection. The identified compound may
further be tested for inhibiting herpesvirus (e.g., CMV) infection
in cells or in vivo (e.g., in animals).
[0147] In yet another embodiment the invention features an assay
for identifying a compound that inhibits herpesvirus (e.g., CMV)
infection. This assay involves: (a) contacting a cell that
expresses LT.beta.R or TNFR1 in the presence of herpesvirus (e.g.,
CMV) with a test compound under conditions allowing binding between
LT.beta.R or TNFR1 and the test compound; (b) measuring herpesvirus
(e.g., CMV) proliferation, replication, protein expression or
cytopathicity in the presence of the test compound; and (c)
comparing herpesvirus (e.g., CMV) proliferation, replication,
protein expression or cytopathicity in the presence of the test
compound to the absence of the test compound. A decrease in
herpesvirus (e.g., CMV) proliferation, replication, protein
expression or cytopathicity in the presence of the test compound
identifies the test compound as a compound that inhibits
herpesvirus (e.g., CMV) infection.
[0148] The invention features cellular response assays. These
cellular response assays measure either protein activity, changes
in gene expression (e.g., nuclear factor .kappa.B(NF.kappa.B)
responsive genes such as intercellular adhesion molecule-1 (ICAM1)
and vascular adhesion molecule 1 (VCAM1) and chemokines such as
interleukin-8 and secondary lymphoid organ chemokine), cell
activation, cell infection by herpesvirus or modulation of
inflammation via affecting inflammatory mediators such as
inflammatory cell recruitment, activation, and production of
pro-inflammatory cytokines (e.g., interleukins (IL) and interferons
(IFN), prostaglandins and leukotrienes.
[0149] Test compounds can be tested for their ability to modulate a
response of cells expressing receptors (e.g., HVEM or LT.beta.R or
TNFR1) stimulated by ligands (e.g., p30, LT.alpha. or gD) and a
suboptimal dose of a stimulus appropriate for the cells. The
"responder" receptor expressing cells can be freshly obtained from
a subject or they can be a cultured cell line. The cells can
express endogenously encoded receptor or a receptor encoded by a
transfected gene. The ligand may be added to the cellular response
cultures in the form of an isolated polypeptide or by addition to
the cultures of cells expressing the ligands. The ligand expressing
cells may express an endogenous gene encoding the ligand or may
express a transfected gene encoding the ligand. Furthermore the
ligand may be expressed on the cell surface (p30 or gD) or be may
be secreted (p30, gD or LT.alpha.). In order for p30 or gD to be
secreted, the gene encoding it would need to have the region
encoding the transmembrane domain deleted. Cellular activation can
be measured by, for example, cell proliferation, de novo expression
of cell-surface activation markers, or soluble factor
production.
[0150] In a preferred embodiment, the cells are lymphocytes. In the
case of T cells, the receptor (HVEM) expressing responder T cells
can be cultured in the presence of the test compound, the ligand
and a suboptimal dose of a T cell activator, e.g., anti-CD3
antibody, a lectin such as phytohemoglutinin (PHA) or a
superantigen such as staphylococcal enterotoxin C (SEC). Controls
will be cultures containing: (a) T cells alone; (b) T cells with T
cell activator, with ligand and without test compound; (c) T cells
with T cell activator, without ligand and without test compound;
(d) T cells with T cell activator, without ligand and with test
compound, (e) T cells without T cell activator, with ligand and
without test compound; (f) T cells without T cell activator,
without ligand and without test compound and (g) T cells without T
cell activator, without ligand and with test compound. T-cell
activation can be measured in terms of T cell proliferation by
incorporation of .sup.3H-thymidine (see Example 5), induction of
activation markers such as CD69 or CD25, or production of cytokines
such as interleukin-2 (IL-2), interleukin-4 (IL-4) or
interferon-.gamma. (IFN.gamma.).
[0151] In the case of B lymphocytes similar response assays can be
carried out. The B cell activators may be mitogens such as poke
weed mitogen, staphylococcal protein A or anti-immunoglobulin. Cell
activation cell can be measured by cell proliferation (again by
.sup.3H-thymidine incorporation) or Ig secretion. Alternatively,
the survival of B cells in nutritionally suboptimal medium may be
measured (see Example 5).
[0152] The ability of a test compound to inhibit lymphocyte
activation would be an indication that such a compound may be
useful in the treatment of an autoimmune disease largely involving
T-cells (rheumatoid arthritis, insulin dependent diabetes mellitus
and multiple sclerosis, for example) or T and B cells (systemic
lupus erythematosus and myasthenia gravis, for example) as well as
inflammatory disorders, for example, arthritis, psoriasis, and
inflammatory bowel disease. The ability of a test compound to
stimulate lymphocyte activation would be an indication that such a
compound may be useful in stimulating immune responses in subjects
with infectious diseases, or in which the subject is
immunosuppressed as, for example, in patients undergoing
chemotherapy or radiation therapy for cancer or in patients with
AIDS.
[0153] In assays for test compounds that prevent herpesvirus
infection, the test compounds can be added to cultures of
herpesvirus susceptible cells and herpesvirus. Permissive cell
lines for virus infection include human dermal fibroblasts,
peripheral blood lymphocytes treated with agents that cause
activation (e.g., anti-CD3 antibody, or phytohemagglutinin), and
transformed cell lines (e.g., Hela cells). Virus production can be
measured by any number of methods known by those skilled in the art
including viral plaque assays, production of specific virus
proteins measured by an ELISA or use of recombinant virus that
contains an indicator gene product like .beta.-galactosidase, an
enzyme easily detectable by colorimetric assays (Montgomery (1996)
supra).
[0154] The ability of a test compound to inhibit cell infection by
HSV would be an indication that such a compound may be useful in
the treatment of a subject with an HSV infection. In order to test
whether compounds which affect cellular responses function by
binding either member of the relevant receptor-ligand pair, they
can be tested for their ability to bind to soluble forms of the
receptor or ligand by assays well known in the art, for example,
ELISAs, Western blotting or radioimmunoassays. Furthermore, to test
whether binding of a test compound to either the receptor or the
ligand results in inhibition of their binding to each other, the
test compound can be tested for its capacity to inhibit binding of
soluble forms of the receptor and the ligand. Examples of these
assays are competitive ELISAs, competitive Western blotting and
competitive radioimmunoassays.
[0155] Test compounds include purified and unpurified compounds,
e.g. extracts from living organisms. Natural compounds in the form
of bacterial, fungal, plant and animal cell or tissue extracts are
available or can be readily produced.
[0156] Generally test compounds will be found among biomolecules
including, but not limited to: polypeptides, peptidomimetics,
saccharides, fatty acids, steroids, purines, pyrimidines,
derivatives, structural analogs or combinations thereof. Test
compounds further include chemical compounds (e.g., small organic
molecules having a molecular weight of more than 50 and less than
5,000 Daltons, such as hormones). Candidate organic test compounds
comprise functional groups for interaction with proteins,
particularly hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, preferably at least
two of the functional chemical groups. Candidate organic compounds
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Known pharmacological agents are
considered test compounds and may further include agents subjected
to directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification, etc., to produce
structural analogs, for example.
[0157] Test compounds include libraries of compounds, for example,
synthetic or natural compounds in a combinatorial library such as
peptides (e.g., antibodies, or other peptides having common
functional or structural features, such as nucleic acid binding,
intracellular signaling, binding activity, etc.), and small organic
molecules, such as drugs. Numerous libraries are commercially
available or can be readily produced; means for random and directed
synthesis of a wide variety of organic compounds and biomolecules,
including expression of randomized oligonucleotides and
oligopeptides, are known. Additionally, natural or synthetically
produced libraries and compounds are readily modified through
conventional chemical, physical and biochemical means, and may be
used to produce combinatorial libraries. Such libraries are useful
for the screening of a large number of different test compounds.
Moreover, such test compounds additionally can be modified so as to
facilitate their identification or purification. Such modifications
are well known to the skilled artisan (e.g., biotin and
streptavidin conjugated compounds, polyhistidine and T7 tags). The
test compounds may therefore be a defined, isolated and purified
candidate compound (e.g., a synthetic small molecule), a member of
a combinatorial library or may be present in a biological sample
such as a biological fluid, tissue extract, subcellular fraction or
cellular lysate.
[0158] Peptides and polypeptides used in the screening assays of
the invention may be obtained by a variety of means. Smaller
peptides (less than 50 amino acids long) may be conveniently
synthesized by standard chemical methods. Some polypeptides (e.g.
antibodies) may be purchased from commercial sources. Where
otherwise unavailable, antibodies can be generated as described
supra. Detectably labeled antibodies either can be purchased from
commercial sources or are readily prepared by those of ordinary
skill in the art.
[0159] Polypeptides such as HVEM, LT.beta.R, p30 (LIGHT), gD or
LT.alpha. may be purified from biological sources by methods
well-known to those skilled in the art (Protein Purification,
Principles and Practice, second edition (1987) Scopes, Springer
Verlag, N.Y.). They may also be produced in their naturally
occurring, truncated, fusion or chimeric protein forms by
recombinant DNA technology using techniques well known in the art.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. See, for example, the techniques described in
Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Press, N.Y.; and Ausubel et al., cited supra.
Alternatively, RNA encoding the proteins may be chemically
synthesized. See, for example, the techniques described in
Oligonucleotide Synthesis, (1984) Gait, M. J. ed., IRL Press,
Oxford, which is incorporated by reference herein in its
entirety.
[0160] A variety of host-expression vector systems may be utilized
to express the nucleotide sequences. Where the peptide or
polypeptide is soluble, it can be recovered from: (a) the culture,
i.e., from the host cell in cases where the peptide or polypeptide
is not secreted or (b) from the culture medium in cases where the
peptide or polypeptide is secreted by the cells. The expression
systems also encompass engineered host cells that express the
polypeptide in situ, e.g., anchored in the cell membrane.
Purification or enrichment of the polypeptide from such an
expression system can be accomplished using appropriate detergents,
lipid micelles and other methods well known to those skilled in the
art. Alternatively, such engineered host cells themselves may be
used in situations where it is important not only to retain the
structural and functional characteristics of the protein, but also
to assess biological activity.
[0161] The expression systems that may be used for purposes of the
invention include but are not limited to microorganisms such as
bacteria (for example, E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing the nucleotide sequences; yeast transformed with
recombinant yeast expression vectors; insect cells infected with
recombinant viral expression vectors (baculovirus); plant cell
systems infected with recombinant viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors; or
mammalian cells (e.g., COS, CHO, BHK, 293, 3T3) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g. metallothionein promoter) or
from mammalian viruses.
[0162] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
gene product being expressed. For example, when a large quantity of
such a protein is to be produced, e.g. for raising antibodies to
the protein, vectors which direct the expression of high levels of
fusion protein products that are readily purified may be desirable.
Such vectors include, but are not limited to, the E. coli
expression vector pUR278 (Ruther et al. (1983) EMBO J. 2:1791), in
which the coding sequence may be ligated individually into the
vector in frame with the lacZ coding region so that a fusion
protein is produced; pIN vectors (Inouye (1985) Nucleic Acids Res.
13:3101; Van Heeke (1989) J. Biol. Chem. 264:5503); and the like.
pGEX vectors may also be used to express foreign polypeptides as
fusion proteins with glutathione S transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. The pGEX vectors are
designed to include thrombin or factor Xa protease cleavage sites
so that the cloned target gene product can be released from the GST
moiety. It is understood that the polypeptides used for the
screening assays can be either the naturally occurring forms of the
polypeptides or fusion proteins containing the polypeptides. The
irrelevant part of the fusion protein can be, for example, the Fc
portion of immunoglobulin G, hexahistidine or GST.
[0163] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the nucleotide sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the gene
product in infected hosts (e.g., See Logan & Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655). Specific initiation signals may also
be required for efficient translation of inserted nucleotide
sequences. These signals include the ATG initiation codon and
adjacent sequences. In cases where an entire gene or cDNA,
including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only a portion of the coding sequence is inserted, exogenous
translational control signals, including, perhaps, the ATG
initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (Bittner (1987) Methods in Enzymol. 153:516).
[0164] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Appropriate cell lines or host systems can be chosen to
ensure the correct modification and processing of the foreign
protein expressed. Mammalian host cells include but are not limited
to CHO, VERO, BHK, Held, COS, MDCK, 293, 3T3, and WI38.
[0165] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the sequences described above may be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1 to 2 days in an enriched medium, and then are
switched to a selective medium. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the gene product. Such engineered cell lines
may be particularly useful in screening and evaluation of compounds
that affect the endogenous activity of the gene product.
[0166] A fusion protein may be readily purified by utilizing an
antibody or a moiety that specifically binds to the fusion protein
being expressed. For example, a system described by Janknecht
(1991) Proc. Natl. Acad. Sci. USA 88:8972, allows for the ready
purification of non-denatured fusion proteins expressed in human
cell lines. In this system, the gene of interest is subcloned into
a vaccinia recombination plasmid such that the gene's open reading
frame is translationally fused to an amino-terminal tag consisting
of six histidine residues. Extracts from cells infected with
recombinant vaccinia virus are loaded onto Ni.sup.2+ nitriloacetic
acid-agarose columns and histidine-tagged proteins are selectively
eluted with imidazole-containing buffers. If desired, the histidine
tag can be selectively cleaved with an appropriate enzyme.
[0167] Chimeric proteins may also be derived by methods known to
those in the art. These involve splicing a portion of a gene
encoding a given protein to one or more portions derived from one
or more genes encoding different proteins. A chimeric polypeptide
is a molecule in which different portions are derived from
different proteins. For example, a chimeric protein may contain a
domain of HVEM and another domain of LT.beta.R or a domain of HVEM
and a domain of an Fc region of an antibody.
[0168] The invention provides methods for modulating an
HVEM-mediated cellular response by contacting a cell expressing the
receptor polypeptide, HVEM, with an HVEM binding agent.
Alternatively, an HVEM-mediated cellular response is modulated by
contacting a ligand for HVEM with a ligand binding agent. Such
ligands include LIGHT (p30), LT.alpha. or gD. LIGHT (p30) can be
expressed on the surface of a cell or it can be soluble, e.g., in
homotrimeric form. LT.alpha. can be secreted and gD can be
expressed on an HSV virion or on the surface of an HSV-infected
cell.
[0169] The phrase "cellular responses" refers again herein to cell
activation (e.g., an increase in production or inflammatory
mediators) or to internalization of HSV by the cell. The invention
also features methods for modulating LT.beta.R-mediated cellular
responses by contacting a cell expressing LT.beta.R or a cell
expressing the LT.beta.R ligand, p30, with a binding agent that
binds either to HVEM or the p30.
[0170] As used herein, the term "contacting" means exposing the
receptor or the ligand to the binding agent, in a
receptor-modulating effective amount, so that the binding agent can
effectively modulate the cellular response initiated by interaction
of the ligand with the receptor. Modulation can result in
inhibition or activation of the cellular response. These
alternative properties of a particular binding agent for a
particular receptor-ligand pair can be tested for in advance using
the screening assays described (see, e.g., Examples 6-8).
[0171] With respect to receptor binding agents, HVEM binding agents
include soluble gD, soluble p30 (e.g. LIGHT-t66) or a peptide
fragment of LT.alpha., preferably a peptide fragment that contains
the amino acid Tyr at a position corresponding to position 108 from
the N-terminus of naturally occurring LT.alpha.. An LT.beta.R
binding agent is soluble p30.
[0172] With respect to ligand binding agents, p30 binding agents
include soluble HVEM, soluble LT.beta.R, chimeric constructs (e.g.,
fusion proteins) such as for example, HVEM:Fc or antibody that
binds specifically to p30. An LT.alpha. binding agent is soluble
HVEM, for example, an HVEM:Fc fusion protein or chimeric
construct.
[0173] Contacting may be in vitro, for example, by adding a binding
agent or antagonist to a culture of cells expressing HVEM or
LT.beta.R, e.g., lymphocytes, undergoing activation by HVEM ligands
(p30, LT.alpha. or gD) or the LT.beta.R ligand, p30. Binding agents
may also be added, for example, to a culture of HVEM expressing
cells exposed to gD on the surface of HSV virions or on the surface
of HSV infected cells. The ability of the binding agent to modulate
these cellular responses could be tested for in advance using the
screening methods described. The binding agent may be added as an
isolated polypeptide or as cells transfected with an expressing
vector containing a binding agent encoding nucleic acid molecule.
In these in vitro methods, a "receptor-modulating effective amount"
of binding agent, is the amount required to modulate cell
activation or HSV infection by greater than 20%, preferably greater
than 50%, more preferably greater than 80% and most preferably
greater than 95%.
[0174] Contacting may be in vivo in a subject. The subject may be a
mammal, preferably a human, with an autoimmune disease such as
rheumatoid arthritis, insulin dependent diabetes mellitus, multiple
sclerosis, systemic lupus erythematosus or myasthenia gravis, a
lymphoid (T or B cell) malignancy, an HSV infection, an infection
with an organism other than HSV, an inflammatory disorder, or for
immunosuppression. Inhibition of a HVEM-p30 or a LT.beta.R-p30
mediated cellular response could be advantageous in patients with
autoimmune diseases or lymphoid malignancies in that it could
prevent T cell proliferation or activation (as in rheumatoid
arthritis, insulin dependent diabetes mellitus, multiple sclerosis,
systemic lupus erythematosus, myasthenia gravis and T cell
malignancies) and B cell proliferation (as in systemic lupus
erythematosus, myasthenia gravis and B cell malignancies).
Inhibition of an HVEM-gD mediated cellular response (i.e., HSV
internalization) could be therapeutic for subjects with an HSV
infection in that it would prevent viral spread mediated by
internalization of gD-expressing HSV virions present in the
extracellular space or from HSV-infected cells expressing gD on
their surface. Stimulation of an HVEM-p30 or a LT.beta.R-p30
mediated cellular response would be useful in treating subjects
with an infection other than HSV or immunosuppressed subjects
(e.g., patients undergoing radiation and/or chemotherapy for
cancer, other than lymphoid malignancies) or AIDS patients in that
both T and B cell proliferation would be stimulated. Naturally, one
would avoid using binding agents that stimulate an HVEM-p30
mediated cellular response in a subject with an HSV infection in
that such an agent might also enhance an HVEM-gD cellular response
and, thereby, the spread of HSV virus. However, this activity in
the relevant binding agent could be tested for in advance using the
screening assays described supra. Similarly, these stimulatory
binding agents would not be used in lymphoid malignancies as they
could promote growth of the tumor cells.
[0175] The binding agents to be used for in vivo modulation of
cellular responses include the naturally occurring forms of HVEM,
LT.beta.R, p30, antibodies, gD and LT.alpha. as well as engineered
forms such as HVEM:Fc constructs. These will be produced by the
methods described supra. Peptides derived from LT.alpha. and which
modulate the HVEM-LT.alpha. interaction will also be used. The
peptides will contain about 205 amino acids or less. For example,
they may contain five, eight, twelve, fifteen or eighteen amino
acids. The peptides will preferably contain the residue Tyr, or a
conservative replacement thereof, at a position corresponding to
amino acid residue 108 from the N-terminus of naturally occurring
LT.alpha..
[0176] Also included as binding agents are peptidomimetics of the
peptides described supra. Peptidomimetic compounds are synthetic
compounds having a three-dimensional structure (i.e. a "peptide
motif") based upon the three-dimensional structure of a selected
peptide. The peptide motif provides the peptidomimetic compound
with the activity of modulating cellular responses that is the same
or greater than the activity of the peptide from which the
peptidomimetic was derived. Peptidomimetic compounds can have
additional characteristics that enhance their therapeutic
application such as greater affinity and/or avidity and prolonged
biological half-life. The peptidomimetics of the invention
typically have a backbone that is partially or completely
non-peptide, but with side groups identical to the side groups of
the amino acid residues that occur in the peptide on which the
peptidomimetic is based. Several types of chemical bonds, e.g.
ester, thioester, thioamide, retroamide, reduced carbonyl,
dimethylene and ketomethylene bonds, are known in the art to be
generally useful substitutes for peptide bonds in the construction
of protease-resistant peptidomimetics.
[0177] Polypeptide and peptide binding agents may be modified by
the addition at either or both the amino- and carboxyl-terminal
ends, of a blocking agent in order to facilitate survival of the
relevant polypeptide or peptide in vivo. This can be useful in
those situations in which the peptide termini tend to be degraded
("nibbled") by proteases. Such blocking agents can include, without
limitation, additional related or unrelated peptide sequences that
can be attached to the amino and/or carboxyl terminal residues of
the polypeptide or peptide to be administered. This can be done
either chemically during the synthesis of the peptide or
polypeptide or by recombinant DNA technology. Alternatively,
blocking agents such as pyroglutamic acid or other molecules known
to those of average skill in the art may be attached to the amino
and/or carboxyl terminal residues, or the amino group at the amino
terminus or carboxyl group at the carboxyl terminus replaced with a
different moiety. Likewise, the binding agents can be covalently or
noncovalently coupled to pharmaceutically acceptable "carrier"
proteins prior to administration.
[0178] In vivo delivery involves administering to a subject either
the binding agent itself, a nucleic acid encoding the binding
agent, an expression vector encoding the binding agent, or cells
transfected or transduced with the vector. Expression systems
therefore further include vectors specifically designed for in vivo
or ex vivo applications including adenoviral vectors (U.S. Pat.
Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Pat.
No. 5,604,090), herpes simplex virus vectors (U.S. Pat. No.
5,501,979) and retroviral vectors (U.S. Pat. Nos. 5,624,820,
5,693,508 and 5,674,703 and WIPO publications WO92/05266 and
WO92/14829). Bovine papilloma virus (BPV) and CMV based vectors
have also been employed for in vivo uses (U.S. Pat. Nos. 5,719,054
and 5,561,063).
[0179] Binding agents may be delivered to a cell of a mammal using
techniques substantially the same as those described infra for
delivery to human subjects. Examples of appropriate mammals include
but are not restricted to humans, non-human primates, horses,
cattle, sheep, dogs, cats, mice, rats, guinea pigs, hamsters,
rabbits and goats.
[0180] A binding agent may be delivered to cells of a patient in
its unmodified state, dissolved in an appropriate physiological
solution, e.g. physiological saline. Naturally, it is desirable
that these peptides be selectively targeted to relevant tissues and
cell types. This can be achieved by contacting the peptides
directly with the affected organ or tissue, e.g., by localized
injection or implantation. Thus, in autoimmune diseases such as
rheumatoid arthritis or insulin-dependent diabetes mellitus, the
peptides could be introduced directly into affected joints or the
pancreas, respectively, or, preferably, into draining lymphoid
tissue in which the active autoimmune response occurs.
[0181] Alternatively, the binding agents may be delivered in
liposomes into which have been incorporated ligands for receptors
on relevant cells (e.g., T cells or B cells) or antibodies to
cell-surface markers expressed by these cells. Thus an antibody
specific for the CD4 T cell surface marker may direct liposomes
containing both the anti-CD4 antibody and the relevant binding
agent to a CD4.sup.+ T cell. This approach could be used in both
autoimmune diseases and HSV infection. In autoimmune diseases in
which the T cell receptor (TCR) expressed by a dominant pathogenic
T-cell clone has been defined, an antibody specific for the
relevant TCR component (e.g. V.beta.) may be used. The latter
methodology would represent an ideal form of immunotherapy in which
pathogenic effector cells are specifically targeted for inhibition
while the immune system as a whole and the cells of the target
organ remain uncompromised.
[0182] In lymphoma or leukemia patients, anti-proliferative binding
agents are preferably directed to cancer cells. The peptides could,
for example, be injected directly into the tissues surrounding the
lymphoma tumor site after surgery to remove the tumor, in order to
inhibit growth of residual tumor cells. Instead of surgery, the
tumor could be treated by in situ injection of the binding agent
into the tumor. The liposome methodology described supra, could
also be exploited. In this case antibodies specific for
tumor-specific antigens (TSA) or tumor-associated antigens (TAA)
would be exploited.
[0183] It is well known in the medical arts that dosages for any
one patient depend on many factors, as well as the particular
compound to be administered, the time and route of administration
and other drugs being administered concurrently. Dosages for the
binding agents of the invention will vary, but can be, when
administered intravenously, approximately 0.01 mg to 10 mg/ml blood
volume. Routes and doses of administration are well known to
skilled pharmacologists and physicians. Routes, in addition to
those described supra, include, but are not restricted to:
intraperitoneal, intramuscular, intrapulmonary, transmucosal,
subcutaneous and intravenous.
[0184] An in vivo gene therapy approach requires delivery of a
genetic construct directly into the patient, preferably targeting
it to the cells or tissue of interest. Targeting of tumor cells or
activated lymphocytes, for example, can be accomplished by the use
of a retrovirus, which stably transfects primarily proliferating
cells. In another embodiment, the inflammatory disorder may be
arthritis, and the tissue target the cartilage in the joint of a
subject. In such an instance the fusion construct of the invention
may be attached to a gene activated matrix (i.e., coated on a
polymer material) so as to provide a slow release material or
injected directly into the joint.
[0185] Tissue specific targeting may also be achieved by the use of
a molecular conjugate composed of a plasmid or other vector
attached to poly-L-lysine by electrostatic or covalent forces.
Poly-L-lysine binds to a ligand that can bind to a receptor on
tumor cells (Cristiano (1995) J. Mol. Med. 73:479). Similarly,
tumor and cell specific antibodies of the type described supra can
be bound to vectors and thereby target them to lymphoid tumors or
cells such as T-lymphocytes. The latter would be useful in
autoimmune diseases and HSV infection. A promoter inducing
relatively tumor-specific expression can be used to achieve a
further level of targeting. Tissue-specific promoters for use in
autoimmune or transplant patients include, for example, the
inducible IL-2 (Thompson (1992) Mol. Cell. Biol. 12:1043), IL-4
(Todd (1993) J. Exp. Med. 177:1663) and gamma-interferon (Penix
(1993) J. Exp. Med. 178:483) T-cell targeting promoters. Such
inducible promoters would have an invaluable additional advantage
in that expression would occur selectively in activated T-cells.
Included in this population of activated T-cells are the effector
cells that an ideal immuno-therapeutic modality would selectively
inhibit in autoimmune patients.
[0186] Vectors can also be delivered by incorporation into
liposomes or other delivery vehicles either alone or
co-incorporated with cell specific antibodies, as described
supra.
[0187] Where the relevant binding agent is normally bound to the
cell membrane (HVEM, LT.beta.R, p30 or gD), the region of the
nucleic acid encoding the transmembrane domain of binding agent
will be deleted from the nucleic acid contained in the expression
vector.
[0188] DNA or transfected cells may be administered in a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are biologically compatible vehicles which are suitable
for administration to a human, e.g., physiological saline. A
therapeutically effective amount is an amount of the DNA of the
invention which is capable of producing a medically desirable
result in a treated animal. As is well known in the medical arts,
the dosage for any one patient depends upon many factors, including
the patient's size, body surface area, age, the particular compound
to be administered, sex, time and route of administration, general
health, and other drugs being administered concurrently. Dosages
will vary, but a preferred dosage for intravenous administration of
DNA is from approximately 10.sup.6 to 10.sup.12 copies of the DNA
molecule. This dose can be repeatedly administered, as needed.
[0189] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0190] Pharmaceutical compositions comprising pharmaceutically
acceptable carriers including invention compositions useful in
practicing the methods of the invention are provided. Exemplary
compositions include, for example, suppositories, pills, capsules,
syrups, elixirs, drops, creams, ointments, lotions, salves, sprays,
and other formulations known by those of skill in the art.
Additional formulations include, for example, polymeric substances,
such as polyesters, polyamine acids, hydrogel, polyvinyl
pyrrolidone, ethylene-vinylacetate, methylcellulose,
carboxymethylcellulose, protamine sulfate, or lactide/glycolide
copolymers, polylactide/glycolide copolymers, or
ethylenevinylacetate copolymers. Microcapsules prepared by
coacervation techniques or by interfacial polymerization, for
example, by the use of hydroxymethylcellulose or
gelatin-microcapsules, or poly (methylmethacrolate) microcapsules,
respectively, or in a colloid drug delivery system can be used for
delivery of invention compositions and useful in practicing the
methods of the invention. Colloidal dispersion systems include
macromolecule complexes, nano-capsules, microspheres, beads, and
lipid-based systems, including oil-in-water emulsions, micelles,
mixed micelles, and liposomes.
[0191] The use of liposomes for introducing various compositions
into cells, including protein and nucleic acid, is known to those
skilled in the art (see, e.g., U.S. Pat. Nos. 4,844,904, 5,000,959,
4,863,740, and 4,975,282). piperazine based amphilic cationic
lipids useful for gene therapy also are known (see, e.g., U.S. Pat.
No. 5,861,397). Cationic lipid systems also are known (see, e.g.,
U.S. Pat. No. 5,459,127).
[0192] Pharmaceutical formulations include carriers compatible with
particular routes of administration. Solutions or suspensions used
for parenteral, intradermal, or subcutaneous administration can
include: water, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose.
[0193] Pharmaceutical compositions for injection include sterile
aqueous solutions (where water soluble) or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water,
Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered
saline (PBS). The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. Fluidity can be
maintained, for example, by the use of surfactants. Antibacterial
and antifungal agents include, for example, parabens,
chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic
agents, for example, sugars, polyalcohols such as manitol,
sorbitol, sodium chloride can be included in the composition.
Including an agent which delays absorption, for example, aluminum
monostearate and gelatin can prolong absorption of injectable
compositions.
[0194] For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, for example, for transmucosal administration, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or aerosols, inhalation devices (e.g., aspirators) or
suppositories. For transdermal administration, compositions may be
formulated into ointments, salves, gels, or creams as generally
known in the art.
[0195] Carriers that protect against rapid elimination from the
body, such as a controlled release formulation or a time delay
material such as glyceryl monostearate or glyceryl stearate may be
used. The compositions can also be delivered using implants or
microencapsulated delivery systems to achieve local or systemic
sustained delivery or controlled release.
[0196] Biodegradable, biocompatable polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations are known to those skilled in the
art. The materials can also be obtained commercially from Alza
Corporation and Nova Pharmaceuticals, Inc.
[0197] Additional pharmaceutical formulations appropriate for the
compositions for administration in the methods of the invention are
known in the art (see, e.g., Remington's Pharmaceutical Sciences
(1990) 18.sup.th ed., Mack Publishing Co., Easton, Pa.; The Merck
Index (1996) 12.sup.th ed., Merck Publishing Group, Whitehouse,
N.J.; and Pharmaceutical Principles of Solid Dosage Forms,
Technonic Publishing Co., Inc., Lancaster, Pa., (1993)).
[0198] The optimal concentration of the peptide, antibodies, fusion
proteins, agonists, antagonists, nucleic acids or a derivative
thereof in a pharmaceutically acceptable composition may vary,
depending on a number of factors, including the preferred dosage of
the compound to be administered, the chemical characteristics of
the compounds employed, the formulation of the compound excipients
and the route of administration. The optimal dosage of a
pharmaceutical composition to be administered may also depend on
such variables as the type and extent of, for example, an
inflammatory disorder, or disease to be treated, the overall health
status of the particular subject, the relative biological efficacy
of the compound selected, the clinical endpoint desired, previous
or simultaneous treatments, the general health, age, sex or race of
the subject and other factors. For example, the compositions of the
invention may be used for the treatment of inflammatory disorders
including, but not limited to, arthritis, psoriasis, and
inflammatory bowel disease. An "effective amount" or "inflammation
inhibiting amount" means that amount of the compound necessary to
modulate, inhibit, or suppress inflammatory responses or symptoms.
In the methods of the invention, including prophylactic and
treatment methods, dosages and protocols may be tailored or
modified based on pharmacogenomic data. The skilled artisan will
appreciate the factors that may influence the dosage and timing
required to provide an amount sufficient for therapeutic
benefit.
[0199] One of one of ordinary skill in the art can use the
following teachings describing the methods and techniques for
determining clinical dosages (Spilker B., Guide to Clinical Studies
and Developing Protocols, Raven Press Books, Ltd., New York, 1984,
pp. 7-13, 54-60; Spilker B., Guide to Clinical Trials, Raven Press,
Ltd., New York, 1991, pp. 93-101; Craig C., and R. Stitzel, eds.,
Modern Pharmacology, 2d ed., Little, Brown and Co., Boston, 1986,
pp. 127-33; T. Speight, ed., Avery's Drug Treatment: Principles and
Practice of Clinical Pharmacology and Therapeutics, 3d ed.,
Williams and Wilkins, Baltimore, 1987, pp. 50-56; R. Tallarida, R.
Raffa and P. McGonigle, Principles in General Pharmacology,
Springer-Verlag, New York, 1988, pp. 18-20) to determine the
appropriate dosage to use.
[0200] Combination compositions and treatment methods using the
combinations and treatments in the methods of the invention are
also included. For example, compositions described herein can be
combined with compositions known in the art that improve or lessen
side effects of treatment or in order to treat a p30 polypeptide
mediated cellular response, or a herpesvirus (e.g. CMV) infection.
Thus, for treatment of inflammation or risk of inflammation, for
example, a steroid (e.g., dexamethasone, triamcinalone, prednisone,
cortisone, hydrocortisone) or a non-steroid anti-inflammatory drug
(e.g., aspirin) may be used in combination with an invention
composition. For treatment of a tumor, for example, an antitumor
drug or treatment (e.g., methotrexate, tamoxifen, 5-Fu,
radiotherapy, surgical resection) may be used in combination with
an invention composition. For a herpesvirus (e.g., CMV) infection,
an antiviral agent, for example, inhibitor of virus fusion to a
cell or entry into a cell, viral RNA or DNA replication, e.g.,
ganciclovir, foscarnet, and ciofovir; protein synthesis or virus
assembly or budding; or an immune response stimulator such as an
interleukine or interferon may be used in combination with an
invention composition. Such combinations can additionally be
formulated into pharmaceutical compositions and kits as set forth
herein.
[0201] The invention further provides kits comprising one or more
compositions of the invention, including pharmaceutical
formulations, packaged into suitable packaging material. In one
embodiment, a kit includes p30. In another embodiment, a kit
includes a nucleic acid encoding p30. In additional embodiments, a
kit includes an LT.beta.R or TNFR1 agonist, and instructions for
use in treating a subject having or at risk of having a herpesvirus
(e.g., CMV) infection. In a particular embodiment, an LT.beta.R or
TNFR1 agonist comprises one or more ligands, for example, p30
(LIGHT), LT.alpha., TNF or LT.alpha.1.beta.2, or an antibody (e.g.,
human or humanized). In still another embodiment, a kit includes an
antiviral agent (e.g., in a pharmaceutical carrier).
[0202] In other embodiments, a kit includes a label or packaging
insert including instructions for identifying a compound that
inhibits herpesvirus (e.g., CMV) infection. The instructions may
comprise, for example, contacting LT.beta.R or TNFR1 with a test
compound under conditions allowing binding; measuring LT.beta.R or
TNFR1 activity in the presence of the test compound; and comparing
activity in the presence of the test compound to the absence of the
test compound in vitro (in solid phase, in solution or in cells),
in vivo, or ex vivo.
[0203] As used herein, the term "packaging material" refers to a
physical structure housing the components of the kit. The packaging
material can maintain the components sterilely, and can be made of
material commonly used for such purposes (e.g., paper, corrugated
fiber, glass, plastic, foil, ampules, etc.). The label or packaging
insert can include appropriate written instructions, for example,
practicing a method of the invention, e.g., treating CMV infection.
Kits of the invention therefore specifically include kits that
include instructions for using the kit components in any of the
methods of the invention.
[0204] Thus, invention pharmaceutical compositions can be included
in a container, pack, or dispenser together with instructions for
administration to a subject, e.g. a subject having or at risk of
having herpesvirus (e.g., CMV). Instructions may additionally
include indications of a satisfactory clinical endpoint or of any
adverse symptoms that may occur, or additional information required
by the Food and Drug Administration for use on a human subject.
[0205] The instructions may be on "printed matter," e.g., on paper
or cardboard within the kit, on a label affixed to the kit or
packaging material, or attached to a vial or tube containing a
component of the kit. Instructions may comprise voice or video tape
and additionally be included on a computer readable medium, such as
a disk (floppy diskette or hard disk), optical CD such as CD- or
DVD-ROM/RAM, magnetic tape, electrical storage media such as RAM
and ROM and hybrids of these such as magnetic/optical storage
media.
[0206] Invention kits can additionally include a buffering agent, a
preservative, or a protein/nucleic acid stabilizing agent. The kit
can also include control components for assaying for activity,
e.g., a control sample or a standard. Each component of the kit can
be enclosed within an individual container or in a mixture and all
of the various containers can be within single or multiple
packages. For example, a composition can be packaged into an ampule
or other dispenser having a label attached thereto including
ingredients, or instructions or dosages for administering the
composition locally or systemically to a subject having or at risk
of having herpesvirus (e.g., CMV) infection.
[0207] As described in further detail below, the polypeptides of
the invention (e.g., HVEM:Fc) have been demonstrated to have
anti-inflammatory properties. Accordingly, using the methods and
compositions described herein, the present invention provides a
method of treating or inhibiting and inflammatory reaction or
disorder in a cell, tissue or subject. The method includes
contacting the cell, tissue or subject having an inflammatory
reaction or disorder with an inhibiting effective amount of the
fusion polypeptide or a composition containing the fusion
polypeptide of the invention. As described herein, the formulation
and compositions can be readily identified by one skilled in the
art using teaching described herein or readily available to a
person skilled in the art. For example, a fusion polypeptide of the
invention may be administered topically to an inflamed site on a
subject. Such topical administration includes administering the
polypeptide of the invention in, for example, a lotion or salve.
Alternatively, the polypeptides of the invention may be
administered systemically. Such systemic administration includes,
for example, intraperitoneal injections, subcutaneous injections,
intravenous injections or orally.
[0208] The invention also provides methods of treating a subject
having or at risk of having herpesvirus (e.g., CMV) infection. A
method includes contacting the subject with an amount of a
LT.beta.R or TNFR1 agonist sufficient to treat herpesvirus (e.g.,
CMV) infection. In one embodiment, the agonist comprises a ligand
(e.g., polypeptide such as p30 (LIGHT), LT.alpha., TNF or
LT.alpha.1.beta.2) or an antibody (e.g., fully human or humanized
antibody having the binding specificity of a monoclonal antibody
denoted 3C8, 3H4 or 4H8). In one aspect, the amount is sufficient
to reduce herpesvirus (e.g., CMV) proliferation, replication,
protein expression, or apoptosis in cells infected with herpesvirus
(e.g., CMV). In another aspect, the amount is sufficient to reduce
one or more symptoms (e.g., cytopathicity) associated with CMV
infection in the subject. In yet another aspect, the subject is
further contacted with an antiviral agent. In still another aspect,
the subject is a neonate.
[0209] Although infection with human cytomegalovirus (HCMV), a
.beta.-herpesvirus, is widespread and acquired early in life, this
virus also causes disease in immune compromised individuals.
Following primary infection, HCMV persists in a latent form, a
characteristic of all herpesviruses. Disease results from
reactivation of latent virus following immune system suppression,
for example, due to immunosppressive drug use in organ or tissue
transplantation, or chemotherapy/radiation for tumors, as well as
disease or pathogens that cause immune suppression, such as HIV
(AIDS).
[0210] The invention therefore further provides methods of treating
a subject having or at risk of reactivating herpesvirus (e.g.,
CMV). A method includes contacting the subject with an amount of a
LT.beta.R or TNFR1 agonist sufficient to inhibit HSV (e.g., CMV)
reactivation. In one embodiment, the subject is immune compromised,
for example, suffers from cancer or HIV infection. In another
embodiment, the subject has or is at risk of having a blood
transfusion or bone marrow transplant, organ transplant (e.g.,
liver, kidney, heart) or tissue transplant (e.g., skin, cornea). In
yet another embodiment, the subject has or is at risk of having
arteriosclerosis or multiple sclerosis.
[0211] Primary CMV infection can cause disease. For example,
infection of neonates when transmitted from mothers to the fetus is
the leading cause of acquired deafness and mental retardation in
children. Thus, in yet another embodiment, the subject is a female
who is pregnant or is at risk of pregnancy.
[0212] The invention additionally provides methods of treating a
subject having or at risk of having a disorder associated with
herpesvirus (e.g., CMV) infection. A method includes contacting the
subject with an amount of a LT.beta.R or TNFR1 agonist sufficient
to inhibit herpesvirus (e.g., CMV) thereby treating a subject
having or at risk of having a disorder associated with herpesvirus
(e.g., CMV) infection. In one embodiment, the disorder comprises
pneumonia, vascular pathology (e.g., arteriosclerosis), CMV
hepatitis, CMV retinitis, CMV pneumonitis, CMV nephritis or CMV
mononucleosis. In another embodiment, the disorder comprises a
demyelinating disease (e.g., multiple sclerosis). In still another
embodiment, the disorder comprises congenital cytomegalaic
inclusion disease.
[0213] The methods of the invention are also applicable to
herpesvirus in general. herpesvirus infection and associated
disorders therefore include .beta.- and .gamma.-herpesvirus
infections and associated disorders. (3-herpesvirus include, for
example, HHV6 and HHV7, which are associated with febrile illness
in children and demyelinating diseases, such as multiple sclerosis
in adults. .gamma.-herpesvirus include, for example, HHV8 and EBV,
which are associated with Kaposis sarcoma, Hodgkins leukemia or
non-Hodgkins leukemia or lymphoma.
[0214] The methods of the invention, including treating a p30
polypeptide-mediated disorder, an inflammatory reaction or
disorder, a tumor or herpesvirus infection or reactivation in a
subject, likely results in an improvement in the subjects'
condition, a reduction of one or more symptoms or decreasing the
subject's risk for developing one or more symptoms associated with
the disorder. Improvements therefore include one or more decreased
symptoms associated with autoimmunity, hypersensitivity, graft vs.
host disease, inflammation (e.g., pain or swelling or immune cell
infiltration into the affected tissue or organ, tissue damage
caused by such inflammation, etc.), tumor size or tumor growth rate
or tumor metastasis, herpesvirus (e.g., CMV, EBV) proliferation,
cytopathicity (cell toxicity), viral titre, susceptibility to
opportunistic infection, reduction of artherosclerotic lesions or
plaque formation, symptoms of multiple sclerosis, etc.
[0215] An improvement may also mean a reduction in the frequency or
amount of a drug used for treating a subject having or at risk of
having the disorder. For example, autoimmune patients treated with
steroids may require less steroid when additionally treated with an
invention composition or method. Similarly, herpesvirus infected
patients treated, for example, with ganciclovir, or other DNA
polymerase inhibiting drug (e.g., foscarnet, cidofovir) may require
less of the drug when additionally treated with an invention
composition or method. An improvement therefore includes reducing
the dosage frequency or dosage amount of steroid that the subject
was administered in comparison to the dosage frequency or amount
administered without treatment with the invention composition or
method.
[0216] An improvement may be relatively short in duration, e.g.,
several hours, days or weeks, or extend over a longer period of
time, e.g., months or years. The improvement need not be a complete
ablation of any or all symptoms of the disorder. For example,
reducing a symptom of inflammation, such as the severity of pain
for several hours is an improvement. Thus, a satisfactory clinical
endpoint for a method of treatment is achieved when there is an
incremental improvement in the subjects' condition or a partial
reduction of associated symptoms, over a short or long
duration.
[0217] Candidate subjects for the methods of the invention include
those having an infection or a disorder as described herein or
known in the art as well as subjects at risk of developing
infection, reactivation or a disorder. The invention methods are
therefore applicable to prophylactic treatment of a subject who is
at risk of reactivation or developing a disorder associated with
infection but either does not have or has not yet exhibited
detectable symptoms of the disorder. A particular example of an at
risk subject would be one with a family history or other genetic
characteristic indicating predisposition to a cancer. Such at risk
subjects can be identified using genetic screening for the presence
of the genetic lesion or inquiry into the subjects' family history
to establish that they are at risk of the disorder. Another example
of an at risk subject would be one susceptible to HSV infection,
for example, an immunocompromised subject or a subject exposed to
HSV from contact with another person. Yet another example of an at
risk subject would be a neonate at risk of HSV transmitted from the
mother to the fetus. Subjects also include those who have had an
initial HSV infection followed by a period of latency in which the
virus is still present but infectious virus is not recovered.
Latency can be reversed, for example, by immunosuppression which
causes virus reactivation. Thus, subjects in which virus is latent
are included in the methods of the invention, particularly at risk
of immunosuppression.
[0218] The term "subject" refers to animals, typically mammalian
animals, such as humans, non human primates (apes, gibbons,
chimpanzees, orangutans, macaques), domestic animals (dogs and
cats), farm animals (horses, cows, goats, sheep, pigs),
experimental animals (mouse, rat, rabbit, guinea pig) etc. Subjects
include neonates. Subjects also include animal disease models
(e.g., inflammatory disease animals, animals susceptible to HSV
infection, etc.).
[0219] The following examples are meant to illustrate the invention
and not to limit it.
EXAMPLES
Materials for the Examples
[0220] Construction, expression and purification of the bivalent
chimeric proteins formed with the Fc region of human IgG1 and the
ligand binding domains of Fas:Fc (Brunner (1995) Nature 373:441),
TNFR60 (Crowe (1994) J. Immunol. Methods 168:79) and human
LT.beta.R:Fc (Crowe (1994) Science 264:707) have been previously
described. The extracellular region of HVEM was generated by PCR
using Taq DNA polymerase amplified sequences from pBEC10 DNA
encoding 1 to K184 using the forward primer 5'
CGGAGATCTGAGTTCATCCTGCTAGCTGG-3' (SEQ ID NO:1) and reverse primer
5' ATAGGATCCCTTGGTCTGGTGCTGACATTCC-3' (SEQ ID NO:2). The amplified
HVEM product was ligated in-frame into the baculovirus vector
pVL1392 (Pharmingen) containing the human Fc IgG1. A similar
construct of HVEM:Fc with Fc region from rabbit IgG1 was produced
in CHO cells, purified and used as an immunogen to produce rabbit
anti-HVEM antibody. LT.beta.R:Fc was constructed from a mouse LTJ3R
(mLT.beta.R) DNA fragment that encodes amino acid residues 1 to
Met221 of the extracellular domain (Force et al. (1996) J. Immunol.
1995.1 155:5280) by PCR using Taq DNA polymerase with forward
primer 5' GACGTCAGATCTTCCCACCTTTCCTCCTA 3' (SEQ ID NO:3) and
reverse primer 5' GAACAGAGATCTCATTGCTCCTGGCTCTG 3' (SEQ ID
NO:4).
[0221] LT.alpha. and LT.alpha.Tyr108Phe were produced in insect
cells using recombinant baculovirus as described (Crowe et al.
(1994) J. Immunol. Methods 168:79). Recombinant soluble
LT.alpha.1.beta.2 (Browning et al. (1996), cited supra), TNF
(Browning and Ribolini (1989) J. Immunol. 143:1859), and monoclonal
antibodies to LT.alpha. (BF7), and LT.beta. (C37, B9 and B27)
(Browning et al. (1995), cited supra) were gifts from Jeffrey
Browning (Biogen, Inc.). The immunoprecipitating anti-LT.alpha.
antibody (clone 9B9) was from Boehringer Mannheim. Anti-CD3 (OKT3)
was produced in ascites in BALB/c mice and used at 1 .mu.g/ml
protein. Purified recombinant HSV gD.sub.--1 proteins and mutants
were produced in baculovirus as previously described in detail
(Nicola et al. (1996) J. Virol. 6:3815). FITC-anti-CD4 and CD8
antibodies were obtained from Becton-Dickenson.
[0222] Normal human dermal fibroblasts (NHDF) (Clonetics, San
Diego, Calif.), were cultured in DMEM (Gibco/BRL) supplemented with
10% fetal bovine serum. HCMV strain AD169 (American type culture
collection) stocks were prepared and quantified by limiting
dilution plaque formation assay on NHDF. Purified recombinant
soluble LIGHT, (Rooney et al. (2000) J. Biol. Chem. 275:14307),
mutant LIGHTG119E (Rooney et al. (2000) J. Biol. Chem. 275:14307),
LT.alpha.Y108F (Williams-Abbott et al. (1997) J. Biol. Chem.
272:19451) were purified as described. Anti-LT.beta.R (goat)
antibodies and decoy receptors TNFR1-Fc and LT.beta.R-Fc were
purified as described (Rooney et al. (2000a) Methods Enzymol.
322:345). Interferon-.alpha., IFN.beta., and their specific
antibodies (sheep polyclonal) were from Research Diagnostics
(Flanders, N.J.), and Fas ligand and TRAIL were from Alexis
Biochemicals (San Diego, Calif.). ICAM-1 was detected with mAB 2146
(Chemicon) by flow cytometry (FACS Caliber, Becton-Dickinson) and
staining with goat anti-mouse IgG conjugated to R-phycoerythrin.
Anti-I.kappa.B.alpha. (rabbit polyclonal Ab, Upstate Biotech) and
anti-FLAG epitope (Sigma) were used in western blotting as
described (Benedict et al., 1999). Anti-HCMV monoclonal antibodies
IE1, (clone 63 and 27); pp28, gB and gH were prepared as described
(Sanchez et al. (1998) J. Virol. 72:3321).
Example 1
Expression of an HVEM Ligand on T Cells
[0223] This example demonstrates that both malignant and normal
human T-cells express a cell surface ligand for HVEM. A fusion
protein containing the extracellular domain of HVEM and the Fc
region of human IgG (HVEM:Fc) was constructed. Experiments
demonstrated that HVEM (as a soluble, recombinant HVEM:Fc) bound to
normal (freshly isolated) human T cells.
[0224] Peripheral blood mononuclear cells were obtained from normal
donors by Ficoll-Hypaque. They were activated with anti-CD3
antibody for 5 days in medium with IL-2. Cells were re-stimulated
with PMA or PMA and ionomycin for 4 hours and then dual stained
with FITC-CD4 or FITC-CD8 and HVEM:Fc detected with anti-huIgG-PE
as described above. FITC fluorescence with compensation was used to
gate on CD4 and CD8 T cell subpopulations.
[0225] Receptor binding was determined by incubating graded
concentrations of HVEM:Fc or control IgG with activated II-23.D7
cells. The II-23.D7 cell line is a human CD4+ T cell hybridoma
(Ware (1986) Lymphokine Res. 5:313) and is maintained in RPMI1640
medium with 10% fetal bovine serum (FBS) and antibiotics. II-23.D7
cells were activated for 4 hours at 37.degree. C. with phorbol
myristate acetate (PMA) (100 ng/ml), or PMA (100 ng/ml) and
ionomycin (1 .mu.g/ml). The cells were washed and incubated for 30
minutes at 4.degree. C. in Hanks Balance Salt Solution (HBSS)
(supplemented with 10% bovine calf serum and 0.1% NaN.sub.3)
containing HVEM:Fc, LT.beta.R:Fc or human IgG at 5 .mu.g/ml, and
then stained with goat anti-human IgG conjugated with phycoerythrin
(anti-huIg-PE). Stained cells were analyzed by flow cytometry
(FACSCaliber, Becton-Dickenson). Receptor binding was determined by
calculating the fluorescence intensity=(mean fluorescent channel)
(% positive fluorescent events), where a positive event has a
fluorescence value >98% of the value for normal IgG. Specific
fluorescence intensity represents the fluorescence intensity after
subtraction of the value for control IgG. Each histogram represents
10.sub.4 events.
[0226] These experiments demonstrated that HVEM:Fc bound
specifically to the human CD4+ T cell hybridoma, II.23.D7 (Ware et
al. (1986) see infra) after activation with the calcium ionophore,
ionomycin, and PMA, but not PMA alone was detected by flow
cytometry (FIG. 1A). Specific HVEM:Fc binding was also detected on
T lymphocytes derived from human peripheral blood (FIG. 1B). These
findings indicated that the both malignant and normal human T-cells
expressed a cell surface ligand for HVEM. Half-maximal binding of
HVEM:Fc to II.23.D7 cells was achieved at .about.20 nM (FIG. 1C).
The II.23.D7 cell line is also induced by PMA to express LT.alpha.
and .beta. and TNF (see, e.g., Ware (1992) J. Immunol.
149:3881).
Example 2
Binding Characteristics of HVEM and its Ligands; HVEM Binds
LT.alpha.
[0227] This example demonstrates the binding characteristics of
HVEM using the soluble, recombinant HVEM:Fc. To determine whether
HVEM might bind to TNF or LT.alpha..beta. complexes, LT.beta.R:Fc
and TNFR:Fc were used as competitive inhibitors of HVEM:Fc binding
to activated II-23.D7 cells. These experiments found that the
LT.alpha. homotrimer, but not TNF or LT.alpha.1.beta.2 competed for
HVEM:Fc (soluble HVEM) binding.
[0228] Competition of binding by LT.beta.R:Fc. Activated II-23.D7
cells (PMA and ionomycin as described in Example 1) were
pre-incubated with LT.beta.R:Fc or TNFR60:Fc (100 .mu.g/ml) for 30
minutes at 4.degree. C. HVEM:Fc-rabbit (2 .mu.g/ml) was then added,
incubated for 30 minutes and the cells stained with goat
anti-rabbit IgG-PE to detect HVEM:Fc-rabbit. Rabbit IgG was used to
determine background staining. Binding of HVEM:Fc to activated
II-23.D7 cells was competed with graded concentrations of
LT.beta.R:Fc, TNFR60, Fas:Fc, or IgG as described above.
[0229] Competition of binding by LT.alpha. homotritner. II-23.D7
cells were activated and HVEM:Fc was preincubated with recombinant
LT.alpha. or LT.alpha.1.beta.2 for 30 minutes at 4.degree. C. The
mixture was added to activated II-23.D7 cells and then stained with
anti-huIgG-PE. Fluorescence staining with HVEM:Fc+LT.alpha. was
equal to background with normal IgG.
[0230] To determine whether HVEM might bind to TNF or
LT.alpha..beta. complexes, LT.beta.R:Fc and TNFR:Fc were used as
competitive inhibitors of HVEM:Fc binding to II-23.D7 cells
activated with PMA and ionomycin utilizing an HVEM:Fc construct
with rabbit IgG Fc (Montgomery (1996) supra). The LT.beta.R:Fc and
HVEM:Fc (Fc of human IgG1), but not TNFR60:Fc competed for binding
of HVEM:Fc (rabbit) (FIG. 2A). In addition, neither of the related
receptor fusion proteins, Fas:Fc and TNFR80:Fc competed for binding
of HVEM:Fc. However, surprisingly, the LT.alpha. homotrimer, but
not TNF or LT.alpha.1.beta.2 competed for HVEM:Fc binding (FIG.
2B). A TNFR60 binding mutant of LT.alpha. in which tyrosine (Tyr)
at position 108 is replaced with phenylalanine (Phe) (Tyr108Phe)
(Goh (1991) Protein Eng. 4:785) did not compete (FIG. 3). These
results indicated that the putative HVEM ligand has characteristics
in common with LT.alpha..beta. heterotrimers and LT.alpha., but
also has features that distinguish it from LT.alpha.1.beta.2 and
TNF. Thus LT.alpha.2.beta.1 could be a putative surface ligand
recognized by HVEM:Fc, with the caveat that the HVEM binding
site(s) on LT.alpha.2.beta.1 is not the same as TNFR60.
Alternatively, HVEM:Fc might recognize a novel ligand. A
biochemical approach was used to distinguish between these
possibilities.
Example 3
Biochemical Characterization of the HVEM Ligand (Light)
[0231] This example demonstrates the purification of LIGHT (p30) by
affinity chromatography using soluble, recombinant HVEM:Fc.
Analysis of proteins that bind HVEM:Fc by Two-dimensional (2D)
electrophoresis showed that p30 polypeptides migrated as a broad
band (pI 7 to 8.5) and that under lower intensity p30 resolves into
three bands.
[0232] SDS-PAGE analysis. II-23.D7 cells were activated for 2.5
hours with PMA or PMA and ionomycin (as in Example 1); washed twice
with phosphate buffered saline (PBS), once with cysteine-methionine
deficient RPMI; and then resuspended in this medium containing 10%
dialyzed FBS, 250 .mu.Ci each of .sup.35S-methionine and
.sup.35S-cysteine, and activating agents for 1.5 hours. The culture
supernatants were harvested and the cells lysed in buffer
containing 2% NP40, HEPES pH7.0, 20 mM EDTA, 150 mM NaCl with
leupeptin and aprotinin (at 10 .mu.g/ml), PMSF (1 mM) and
iodoacetamide (20 mM). The extract was precleared with human IgG
(10 .mu.g), where indicated, anti-LT antibodies and protein G
beads. The receptor:Fc fusion proteins (10 .mu.g/ml) were then
added to the samples and precipitated with protein G beads. Labeled
proteins were analyzed by reducing SDS-PAGE and phosphoimage (pixel
range 6-200).
[0233] Cellular extracts prepared as in the above paragraph were
first pre-cleared with 10 .mu.g of mouse IgG or monoclonal
antibodies to LT.alpha. or LT.beta. and then HVEM:Fc was added to
precipitate ligands. The proteins bound to HVEM:Fc were then
resolved by reducing SDS-PAGE and detected by phosphoimage.
[0234] Purification of HVEM ligand, p30. II-23.D7 cells were
activated with PMA (100 ng/ml) or PMA and ionomycin (1 .mu.g/ml)
for 2.5 hours, followed by labeling with .sup.35S-methionine and
-cysteine as in the above two paragraphs. Cell extracts were
pre-cleared with human IgG (5 .mu.g) and protein G beads to remove
nonspecifically binding proteins. The extract was then depleted of
LT.alpha. by treatment of the extract with TNFR60:Fc and protein G
beads. HVEM:Fc and protein G beads were then added to the extract
and incubated. In each case, the beads were washed three times to
remove the contaminating proteins in the non-bound fraction. The
beads were eluted in buffer containing 8M urea and analyzed in the
first dimension by isoelectric focusing (gradient formed with an
ampholine mixture of pI of 5-7 (50%0, 3-10 (40%), 2-11 (10%) and
reducing SDS-PAGE (15% gel) in the second dimension.
[0235] The purification of p30 by HVEM:Fc was monitored by
comparison to samples purified by LT.beta.R:Fc or TNFR60:Fc.
LT.beta.R:Fc purified proteins, LT.alpha.1.beta.2, were isolated
from II-23.D7 cells stimulated with PMA is shown in FIG. 4A and
proteins bound to TNFR60:Fc that was used to deplete LT.alpha. from
the extract is shown in FIG. 4B. p30 purified by HVEM:Fc as
described above is shown in FIG. 4C. Shown in the first lane of
each gel are .sup.14C-labeled molecular weight markers and in the
second lane are the receptor:Fc bound proteins run in the second
dimension only.
[0236] LT.alpha. is secreted by 1123.D7 cells after activation with
PMA (Ware et al. (1992), cited supra; Crowe et al. (1994) Science
264:707). HVEM:Fc and TNFR:Fc precipitated secreted LT.alpha. from
II-23.D7 cells stimulated with PMA and ionomycin as indicated by
SDS-PAGE. LT.alpha. migrates as a range of molecular weights due to
heterogeneity in glycosylation (Browning et al. (1991), cited
supra). TNFR60:Fc, but not HVEM:Fc also precipitated TNF (17 kDa,
thereby confirming the results of the competition studies described
supra. LT.beta.R:Fc, as expected, did not bind any secreted
proteins, but precipitated the LT.beta. (33 kDa) and LT.alpha.
(23-25 kDa) complex from detergent extracts of PMA activated
II-23.D7 cells. However, when the stimulus included ionomycin and
PMA, LT.beta.R:Fc precipitated a major band at 30 kDa, as well as a
small amount of LT.beta. at 33 kDa and LT.alpha. at 23-25 kDa.
TNFR60:Fc precipitated a 23 kDa protein identical in size to the
LT.alpha. precursor. By contrast, HVEM:Fc precipitated both the 30
kDa and 23 kDa proteins. Three different receptor blocking
monoclonal antibodies to LT.beta. failed to remove the 30 kDa
protein from the extract prior to the addition of HVEM:Fc
indicating that the p30 protein is antigenically unrelated to
LT.beta.. However, anti-LT.alpha. antibodies removed the 23 kDa
band from the extracts indicating relatedness of it to LT.alpha..
The inability of LT.alpha. antibodies to preclear both the 30 kDa
and 23 kDa bands demonstrate that these proteins are not associated
with each other, unlike LT.alpha. and LT.beta. which form
heterotrimers (Androlewicz et al., cited supra).
[0237] LIGHT (p30) was purified from II-23.D7 cells by affinity
chromatography. Successive TNFR60:Fc and HVEM:Fc steps were used,
such that LT.alpha. is removed from the extracts by TNFR60 and thus
does not interfere with p30 binding to HVEM:Fc.
[0238] Two-dimensional (2D) electrophoresis of proteins that bind
HVEM:Fc, TNFR60:Fc or murine LT.beta.R:Fc revealed that p30 has a
distinct charge-to-mass ratio when compared to LT.alpha. and
LT.beta.. LT.beta. in the LT.alpha.1.beta.2 complex precipitated by
LT.beta.R:Fc is acidic with four distinct charge isomers ranging in
pI from 5-6.5 with a detectable increase in mass of the acidic
forms (FIG. 4A). LT.alpha., as a complex with LT.beta. or the
LT.alpha. homotrimer bound to TNFR60 (FIG. 4B), has seven distinct
isomers ranging in pI from 7 to 8.5; the 23 kDa LT.alpha. precursor
has the most basic pI (> or =9). The pI of LT.alpha. without
signal sequence is 8.9. These results are characteristic of
glycosylation adducts and agree fully with previously published
studies for LT.alpha. and LT.beta. (Browning et al. (1991), cited
supra). By contrast, p30 migrated as a broad band (pI 7-8.5) that
under lower intensity resolves into three bands (FIG. 4C). The
charge heterogeneity with no discernable change in mass of p30 is
possibly the result of post-translational modification such as
addition of phosphates or phospholipids.
[0239] These results clearly demonstrate that HVEM binds a novel
cell surface protein of 30 kDa (isolated from the human CD4+ T cell
hybridoma II.23.D7) with isomers of pI 7 to 8.5, which is referred
to as p30 or HVEM ligand (or LIGHT). p30 is antigenically and
physically distinct from LT.beta.. The HVEM ligand is also
recognized by LT.beta.R:Fc, but not TNFR.
Example 4
HSV gD Envelope Glycoprotein Competes with the Endogenous HVEM
Ligand (p30) for Binding to HVEM:Fc
[0240] This example demonstrates the binding of herpesvirus HSV
gD-1 protein to HVEM and that soluble gD-1 competes with
HVEM-ligand, or p30 (LIGHT) for binding to HVEM. Data showing that
HSV gD-1 protein is an antagonist of p30 (LIGHT) binding to HVEM
also demonstrates that p30 can act as an antagonist of herpesvirus
gD-1 protein to HVEM.
[0241] HVEM:Fc (2 .mu.g/ml) was pre-incubated for 30 minutes at
4.degree. C. with gD-1 (250 .mu.g/ml) or gD-1 (.DELTA.290-299) (100
.mu.g/ml), and then added to PMA and ionomycin activated II-23.D7
cells (as in Example 1). Background staining was determined with
huIgG and is equal to HVEM:Fc+gD-1 (.DELTA.290-299). Binding of
HVEM:Fc to activated II-23.D7 cells was competed with graded
concentrations of gD-1 or gD-1 (.DELTA.290-299) (for protocol, see
Example 1).
[0242] The possibility that HSV gD might function as an antagonist
of HVEM-ligand (cellular ligands, e.g., p30) binding to HVEM was
suggested by the binding of HSV gD-1 protein to HVEM. Soluble gD-1
and a mutant of gD, gD-1 (.DELTA.290-299t) with enhanced binding
for HVEM, were both effective at blocking HVEM binding to the
surface of activated II-23.D7 cells (FIG. 5A) (expressing p30). The
effective inhibitory concentration of the gD-1 proteins correlated
with their affinity for HVEM (FIG. 5B). The binding of LT.beta.R:Fc
or TNFR60:Fc to PMA or PMA/ionomycin-activated II-23.D7 cells was
not inhibited by gD-1 (.DELTA.290-299t), indicating that the
HVEM:gD-1 interaction is highly specific. This result suggests that
gD-1 has co-evolved specifically for binding to HVEM, even though
HVEM binds to ligands that are recognized by TNFR60 and LT.beta.R.
These results indicate that gD-1 is a membrane-anchored virokine
and may modulate HVEM signaling activities during entry or egress
of HSV from the infected cell.
Example 5
Crosslinking of Cell Surface HVEM Results in Lymphocyte
Activation
[0243] This example demonstrates that anti-HVEM antibody promoted
the enhanced proliferation of peripheral blood lymphocytes cells
(PBLs), including T cells and B cells. Data showing that anti-HVEM
antibody can promote proliferation of PBLs (which also express
HVEM-ligand, p30, or LIGHT) also demonstrates that cell-associated
p30 (LIGHT) can function as a proliferation-inducing signal for
PBLs. Furthermore, the finding that anti-HVEM antibody added to B
cell lines cultured in low serum medium stimulated their growth in
a dose-dependent fashion also demonstrated that HVEM signaling,
e.g., LIGHT binding to HVEM, can stimulate B cell
proliferation.
[0244] T cell activation. Freshly isolated peripheral blood
lymphocytes were incubated in medium containing graded dilutions of
rabbit anti-HVEM or pre-immune sera (Montgomery (1996) supra) and
PMA at a sub-mitogenic dose (1 .mu.g/ml). Proliferation was
measured after 3 days by incorporation of 3H-thymidine into DNA as
assessed by .beta.-scintillation counting.
[0245] Freshly isolated peripheral blood lymphocytes were activated
with phytohemagglutinin (PHA) at 5 .mu.g/ml and cultured in medium
with IL-2. After 17 days the cells were re-stimulated with graded
dilutions of anti-HVEM antiserum and anti-CD3 (OKT3) antibody at a
sub-mitogenic concentration (1.5 .mu.g/ml). Proliferation was
measured after 3 days as above.
[0246] B cell flow cytometric analysis. Human lymphoblastoid RAJI
cells were subjected to flow cytometric analysis by incubation with
anti-HVEM antiserum (1:100 dilution) or control rabbit IgG at
4.degree. C. and the stained with goat anti-rabbit IgG conjugated
with phycoerythrin. 10.sup.4 cells were analyzed for each
histogram.
[0247] B cell activation. RAJI was transferred into medium
containing 2% FBS for 24 hours and then incubated for 3 days in the
presence of the indicated dilutions of rabbit anti-HVEM antibody or
medium alone. Cell proliferation was assessed as described
above.
[0248] HVEM is expressed on resting CD4+ T cells suggesting that it
could function as a co-stimulatory molecule for cellular
proliferation during the initial phase of an immune response. At
suboptimal concentrations of PMA, anti-HVEM antibody promoted the
enhanced proliferation of peripheral blood lymphocytes indicated by
an increase in the uptake of .sup.3H-thymidine measured after 3
days in culture (FIG. 6A). Memory lymphocytes, generated by
continued culture for 10 to 17 days after activation with PHA, were
also reactivated with anti-HVEM antibody at suboptimal
concentrations of anti-CD3 antibody (FIG. 6B). This result
indicated that HVEM functions in the effector phase of the immune
response. Because antibodies can mimic the action of TNF-related
ligands (Engelmann (1990) J. Biol. Chem. 265:14497), these results
indicate that the cell-associated 30 kDa HVEM ligand may function
as a proliferation-inducing signal for T cells.
[0249] LT.alpha. has previously been shown to stimulate growth
enhancing activities for B lymphocytes, including Epstein-Barr
virus transformed cell lines (Abken (1992) J. Immunol. 149:2785;
Estrov (1993) J. Exp. Med. 177:76; Kehrl (1987) Science 238:1144;
Gibbons (1994) Eur. J. Immunol. 24:1879). HVEM is also expressed on
B lymphoblastoid lines (FIG. 7A). Anti-HVEM antibody, when added to
cultures of RAJI B cell lines in medium with 2% serum, stimulated
the uptake of .sup.3H-thymidine in a dose-dependent fashion,
indicating that HVEM can signal maintenance of B cell viability in
low serum (FIG. 7B). LT.alpha. exhibited a 2 to 3 fold stimulatory
effect in this assay. The presence of TNFR60 and TNFR80 as negative
growth factors may contribute a low response to LT.alpha.. The
positive effect of anti-HVEM antibody may be a property unique to
p30 (HVEM-ligand, LIGHT).
Example 6
Production of Mouse HVEM:Fc
[0250] This example demonstrates the construction of a mouse
HVEM:Fc recombinant construct.
[0251] The extracellular region of mouse HVEM was amplified by PCR
from the mHVEM cDNA (Hsu et al., 1997) starting with Met1 and
ending at Ser205 (forward primer=5' tatGGATTCatggaacctctcccaggat-3%
and reverse primer=5'-tatGGATTCggaggagcaggt ggtgtctgt-3'; both
primers contain a BamHI site. The 550 bpPCR product was purified by
Wizard PCR Preps (Promega), digested with BamHI and then ligated
in-frame into BgIII cut Baculovirus vector pVL1392 (Pharmingen)
containing the Fc region of human IgC1 at the 3' end of the HVEM
insert (pVL1392-mHVEM:Fc). The ligation reaction mixture was used
to transform XL-1 blue competent cells (Stratagene) for a plasmid
preparation.
[0252] TN5 insect cells (1.25.times.10.sup.6) were plated on a T25
flask in 4 mL Excell 401 Medium (JRH Biosciences) and allowed to
attach for 2 hours. TN5 cells were co-transfected with 1 .mu.g
pVL1392-HVEMFc plasmid and 250 ng Baculogold.TM. DNA (Pharmingen)
using 14 .mu.g Lipofectin.TM. (Gibco BRL). The following day the
medium was exchanged and the supernatant containing virus was
collected after 4 days. The virus was amplified to make a stock for
protein production.
[0253] Mouse HVEM:Fc was produced in TN5 insect cells and purified
to homogeneity as described (Crowe et al., 1994). Briefly, TN5
cells were infected with recombinant baculovirus containing
mHVEM:Fc at an MOI of 10. After 3 days the supernatant was
harvested, clarified of cells and debris and treated with protease
inhibitors. Purified mHVEM:Fc protein was obtained by protein A
affinity chromatography with an acidic elution (pH 2.5). Analysis
of mHVEM:Fc by SDS-PAGE showed a single band of protein at 58 kDa
under reducing conditions (see FIG. 8). The preparation was judged
by the Limulus lysate test to be free of detectable endotoxin.
Example 7
Inhibitory Effect of Mouse HVEM:Fc on Inflammation (Delayed-Type
Hypersensitivity) in Mice
[0254] This example demonstrates that the soluble, recombinant
HVEM:Fc fusion protein of the invention has an inhibitory effect on
inflammation in vivo using a art-recognized animal model.
[0255] Eight week old female BDF1 mice (Japan SLC Inc., Shizuoka,
Japan) (N=8) were immunized with 50 .mu.g OVA adsorbed to 1 mg alum
by subcutaneous injection. After 7 days mHVEM:Fc or control IgG was
administered by intraperitoneal injection and the mice were
challenged on footpad with 50 .mu.g alum-absorbed 10 .mu.g OVA.
Measurements of the thickness of right footpads were performed just
before and 24 hours after the antigen challenging, and swelling
(percent increase) of footpad thickness was calculated. Suppression
of 300 .mu.g of mHVEM:Fc was statistically significant (P<0.05),
as shown by the data summarized in FIG. 9.
Example 8
Inhibitory Effect of Mouse HVEM:Fc on Collagen-Induced Arthritis in
Mice
[0256] This example demonstrates that the HVEM:Fc fusion protein of
the invention has an inhibitory effect on inflammation in vivo
using a art-recognized animal model for arthritis.
[0257] Six-week-old DBA/1 mice (Seatec Yoshitomi, Hukuoka, Japan)
(N=10) were immunized with emulsions of 100 .mu.g of bovine type II
collagen and 100 mg of Mycobacterium tuberculosis (H37Ra) in
incomplete Freund's adjuvant at the base of tail by subcutaneous
injection, and boosted 28 days later with the same emulsion. Sixty
.mu.g of mHVEM:Fc or control IgG was administered intrapertoneally
twice a week starting at the second day of immunization. Clinical
scoring for each paw was assessed by reference to the following
scale: 0=normal, 1=swelling and/or erythema of one toe, 2=swelling
and/or erythema of two or more toes, 3=swelling and erythema of the
entire paw, 4=complete swelling and erythema of the entire paw and
incapacity to bend the ankle. CIA score was expressed as the
cumulative value for all paws, with a maximum of 16 (FIG. 10). The
data presented in FIG. 10, demonstrate that HVEM:Fc had a
significant effect on inflammation of the footpad and ankle.
Example 9
Inhibition of Herpesvirus Infection by Soluble Homotrimeric
Light
[0258] This example demonstrates that soluble homotrimeric LIGHT
polypeptides of the invention can block the entry of herpesvirus
into cells in vivo. It is also demonstrated that the anti-viral
activity of LIGHT is by its binding to LT.beta.R and not HVEM. This
example also demonstrates that both LIGHT and LT.alpha. can prevent
virus infection of cells in a dose-dependent manner (FIG. 12).
[0259] Herpesviruses have genetic mechanisms that specifically
target cytokine systems of the TNF superfamily, suggesting that the
immune system has evolved specific counter measures to suppress
herpesvirus reactivation or spread in the host.
[0260] LIGHT was tested along with other cytokines to determine
their ability to inhibit cytomegalovirus (CMV) infection. To
investigate the anti-viral and biological properties of LIGHT, a
soluble form was made by genetic engineering that deleted the
cytoplasmic tail and transmembrane domains. A recombinant soluble
form of LIGHT lacking the N-terminal 66 amino acids was produced,
as described in detail in Example 12, below, and designated
"LIGHTt66." The recombinantly produced soluble LIGHT t66 and its
mutants were secreted exclusively as homotrimers (wild type LIGHT
of expressed as a homotrimer).
[0261] Normal human dermal fibroblasts were infected with the
clinical isolate of human CMV (HCMV) Fiala (HCMV-F) or the lab
strain AD169 of HCMV at low multiplicity of infection (MOI) of
0.05. Cytokines that signal through either the TNFR1 (Lt.alpha.) or
the LT.beta.R (LT.alpha.1.beta.2, LIGHT) were added to the culture
supernatant and incubated for 7 days.
[0262] Addition of LT.alpha. completely blocked viral cytopathic
effect (CPE), This inhibition was specific because soluble type I
TNFR protein (TNFR:Fc) neutralized the anti-viral effect (FIGS. 11A
and 11B). Additionally, a point mutant of LT.alpha. (Y108F) which
can no longer bind to TNFR but retains conformation integrity, was
incapable of blocking HCMV spread (FIG. 11C).
[0263] LT.alpha.1.beta.2 and LIGHT, which both bind to the
LT.beta.R, also were able to block viral CPE (FIG. 11D, 11E). A
point mutant of LIGHT (G119E), which is incapable of binding to the
LT.beta.R, but retains binding to HVEM, was incapable of inhibiting
HCMV spread (FIG. 11F). This indicates that the anti-viral activity
of LIGHT is likely to be through binding to the LT.beta.R, and not
HVEM.
[0264] Quantitative analysis of the anti-HCMV activity of
LT.alpha., LT.alpha.1.beta.2 and LIGHT was accomplished by
measuring expression of both the major immediate early protein
(IE1) and the late tegument protein pp28 by Western Blot. LT.alpha.
and LIGHT showed similar relative anti-viral activities, being
capable to completely inhibit cytopathic effect of HCMV at a
concentration of 100 pM, while LT.alpha..beta.2 was approximately
10 fold less effective. Monoclonal antibodies specific for the
lymphotoxin receptor (LT.beta.R) were also potent inhibitors of
HCMV spread.
[0265] In addition, studies using mouse .gamma.-herpesvirus showed
a reduction in plaque forming units (FIG. 12). Owl monkey kidney
cells in 12 well microplates were infected with 500 plaque forming
units (PFU) per well for 60 minutes at 37.degree. C. and overlayed
with carboxymethycellulose (0.75%) in medium containing fetal
bovine serum (FBS) with or without LIGHT or LT.alpha. at the
indicated concentrations. After 7 days, the cells were fixed with
methanol and stained with Gemisa. Each data point in FIG. 12 is the
average number of plaques in two wells. The data clearly
demonstrates that both LIGHT and LT.alpha. were able to
significantly decrease the number of infected cells in a
dose-dependent manner.
[0266] Mouse MHV-68 is similar to the human .gamma.-herpesviruses
EBV and HHV-8. EBV is implicated as the oncogenic factor in certain
human cancers including EBV-associated lymphoma and nasopharyngeal
carcinoma. HHVE is linked to AIDS-associated Kaposi's sarcoma.
Example 10
Biological Activities of Soluble Homotrimeric Light
[0267] This example demonstrates that a recombinant, soluble
homotrimeric LIGHT polypeptide of the invention is active in
several cellular response assays, including apoptosis of
adenocarcinomal HT29 cell lines and the induction of ICAM-1 on
fibroblasts. As discussed in Example 9, above, to investigate the
biological properties of LIGHT, a soluble form, the recombinantly
produced soluble LIGHT t66 (described in detail in Example 12), was
made. While this soluble polypeptide lacks the cytoplasmic tail and
transmembrane domains of wild-type p30, it retains the homotrimeric
wild-type tertiary structure.
[0268] HT29 cells and 293 cell lines were obtained from ATCC; HT29
cells were derived from human adenocarcinoma, ATCC number HTB-38
(see, e.g., Chen (1987) Cancer Genet. Cytogenet. 27: 125-134). Both
cell lines were cultured in DMEM containing 10% FBS with glutamine
and penicillin/streptomycin.
[0269] 293 cells were stably transfected with cDNA encoding human
soluble LIGHT polypeptide (see Example 12). Clones with high
expression soluble polypeptide were selected for large scale
culture. Spent medium from 293-LIGHT cells was harvested for
purification by a combination of ion-exchange and affinity
chromatography. LIGHT was purified to homogeneity (see FIG. 13),
with a yield of 10 to 15 mg per liter and with levels of endotoxin
less than 0.1 endotoxin units/mg.
[0270] The activity of the purified LIGHT protein was measured by a
specific ELISA assay that utilizes soluble forms of LT.beta.R or
HVEM in a plate bound form. Recombinant soluble LIGHT (homotrimeric
LIGHT t66) binds as efficiently to both mouse HVEM and LT.beta.R as
the homologous human forms.
[0271] LIGHT is active in several cellular response assays,
including apoptosis of HT29 cells and the induction of ICAM-1 on
fibroblasts (ICAM is a cell adhesion marker of inflammation). LIGHT
is as efficient as LT.alpha.1.beta.2 in inducing the death of HT29
cells, but weak compared to the ability of TNF or LT.alpha. to
induce ICAM-1 expression. The latter result suggests that LIGHT may
not be pro-inflammatory. In vivo, LIGHT does not appear to be
pro-inflammatory or toxic because mice injected with purified LIGHT
(maximum dose tested of 200 .mu.g per mouse) failed to display the
symptoms of shock observed when TNF or LT.alpha. are administered
at this dose.
Example 11
NF-.kappa.B, but not TRAF, Necessary for LIGHT-Mediated Anti-Viral
Effect
[0272] This example demonstrates that the anti-viral activity of
soluble, homotrimeric LIGHT and lymphotoxin (LT) requires the
activity of NF-.kappa.B (cells lacking NF-.kappa.B activity were
refractory to the effects of LIGHT) (see FIG. 14). Thus, this
example demonstrates that activation of NF-.kappa.B-activity
dependent apoptotic pathways is involved in the anti-CMV activity
of LIGHT.
[0273] No cell death was observed in normal human dermal
fibroblasts (NHDF) treated with LT.alpha., LT.alpha.1.beta.2 or
LIGHT. This demonstrates that the anti-viral activity of these
cytokines may be independent of the apoptotic death pathway that
can be triggered by the TNFR. This suggested that an NF.kappa.B
dependent mechanism may be operative, as this is the other major
pathway activated by this receptor. NF.kappa.B also controls
transcription of several anti-apoptotic proteins in fibroblasts
thus counteracting the apoptosis pathway.
[0274] To test this hypothesis, a dominant negative mutant of the
inhibitor of NF-.kappa.B.alpha.subunit (I.kappa.B.alpha.M) was
introduced into NHDF by transduction with retroviral vector. This
mutant cannot be phosphorylated and thus remains in a stable
complex with NFB. This prevents transcription of .kappa.B dependent
genes. TNF and LT.alpha. both failed to induce ICAM-1 expression in
NHDF-I.kappa.B.alpha.M, demonstrating the efficiency (of inhibiting
NF.kappa.B) of the I.kappa.B.alpha.M dominant negative mutant.
[0275] NHDF-I.kappa.B.alpha.M cells were then compared to cells
transduced with vector alone (NHDF-LXSN) or the ability of the
various cytokines to inhibit HCMV replication. It was found that
NHDF-I.kappa.B.alpha.M cells were refractory to the effects of
lymphotoxins and LIGHT (FIG. 14). Significantly higher levels of
IE1 and pp28 were seen for a given concentration of cytokine in
NHDF-I.kappa.B.alpha.M as compared to NHDF-LXSN cells.
[0276] Additionally, a 10 fold increase in infectious HCMV was
produced from NHDF-I.kappa.B.alpha.M cells in the presence of
cytokine. Interestingly, HCMV replicated equally well in
NHDF-I.kappa.B.alpha.M as in NHDF-LXSN cells in the absence of
cytokine, as assayed by WI and pp28 expression and viral titer.
This result indicates NF-.kappa.B is not essential for efficient
viral replication in fibroblasts, although the IE promoter contain
multiple NF-.kappa.B response elements.
[0277] TRAF3 is recruited directly to the LT.beta.R where it is
involved in signaling apoptosis, but not in the activation of
NF.kappa.B. This was demonstrated by dominant negative mutants of
TRAF3. TRAF3 dominant negative mutants (TRAF3.DELTA.7 and
TRAF3.DELTA.11) introduced into NHDF cells by retrovirus
transduction similar to I.kappa.B.alpha.M, remained sensitive to
the anti-virus activity of LIGHT and lymphotoxins. This indicates
activation of NF.kappa.B-activity dependent apoptotic pathway is
not involved in the anti-viral (anti-CMV) activity of soluble
LIGHT.
Example 12
Production and Characterization of Soluble (Homotrimeric) LIGHTt66
and LIGHTt66 Point Mutants
[0278] This example demonstrates the construction and isolation of
soluble, recombinant LIGHT polypeptides (used in Examples 9 to 11,
13 and 14), including those with single residue changes (i.e.,
point mutants), which, as demonstrated below, are secreted as
homotrimers.
[0279] A recombinant soluble form of LIGHT lacking the N-terminal
66 amino acids was produced (designated LIGHTt66). The truncated,
soluble LIGHT was further engineered to include the "FLAG" tag for
immuno-affinity purification by anti-FLAG antibody (Sigma, St.
Louis, Mo.). The truncated, FLAG-labeled construct was designated
LIGHTt66-FLAG.
[0280] HT29, and 293 cells were obtained from the American Type
Culture Collection (ATCC, Rockville, Md.) and cultured in DMEM
containing 10% fetal bovine serum with glutamine and
penicillin/streptomycin. Neonatal Normal Human dermal fibroblast
(NHDF cells) were purchased from Clonetics, San Diego, Calif. and
grown in DMEM supplemented with 10% fetal bovine serum, insulin (5
pg/ml) and fibroblast growth factor (1 pg/ml) (Sigma, St. Louis,
Mo.).
[0281] LIGHTt66-FLAG was produced in stably transfected mammalian
(293) cells. Stably transfected 293 cells were grown in roller
bottle culture in DMEM containing 0.5% FBS. Concentration of LIGHT
t66 reached 10 mg/l in 7 day cultures. LIGHT t66 was partially
purified from 7-day supernatants by an ion-exchange procedure.
Supernatant diluted 1:2 in 20 mM Tris, pH 7.0, so that initial NaCl
concentration was 50 mM, was loaded on a SP Hitrap.TM. column
(Pharmacia). After washing, LIGHT t66 was eluted using 20 mM
Tris/500 mM NaCl, pH 7.0. After dialysis into PBS, LIGHT t66 was
further purified by affinity chromatography on a column of
monoclonal anti-FLAG (M2) coupled to Affigel.TM. (Biorad). LIGHT
t66 was eluted from the column using 20 mM glycine, 150 mM NaCl, pH
3.0, and neutralized immediately by collection into 50 mM Tris pH
7.4.
[0282] A soluble form of LIGHT (LIGHT t66) with the addition of an
N-terminal FLAG epitope was produced in stably transfected 293
cells and purified to homogeneity by ion exchange followed by
immuno-affinity purification on an affinity matrix of monoclonal
anti-FLAG (M2). Final yield of the protein was 80% and purity
>95% (FIG. 5A).
[0283] Primer-introduced sequence modification was used to generate
soluble LIGHT with the following single amino acid substitutions:
G119E, L120Q Q11TT, and Y173F. Briefly, internal primers were
designed to introduce a restriction site at the mutation location.
Forward and reverse primers containing the mutations were used in
separate PCR reactions to amplify two regions of soluble LIGHT.
Primers were as follows:
TABLE-US-00001 Q117T: 5'_ACGCTGGGCCTGGCCTXCTGA_3', (SEQ ID NO.: 9)
5'_ACTCTCCCATAACAGCGGCC_3'. (SEQ ID NO.: 10) G119E:
5'_GAGCTGGCC_ITGCTGAGGGGCCT_3' (SEQ ID NO.: 11)
5'_CAGCTGAGTCTCCCATAACA_3'. (SEQ ID NO.: 12) L120Q:
5'_CAGGCC_ITCCTGAGGGGCCTCA_3, (SEQ ID NO.: 13)
5'_GCCCAGCTGAGTCTCCCATAA_3'. (SEQ ID NO.: 14) Y 173F:
5'_TTCCCCGAGGAGCTGGAGCT_3, (SEQ ID NO.: 15)
5'_GCGGGGTGTGCGCTTGTAGA_3'. (SEQ ID NO.: 16)
[0284] The PCR products were ligated at the primer-introduced
restriction enzyme site to create soluble LIGHT starting at amino
acid t66 and containing one of the 4 amino acid substitutions. The
LIGHT t66 mutants were ligated into the FLAG-tagged cassette,
pBABE-FLAG which contains the V-cam signal sequence fused to the
FLAG epitope. The V-cam FLAG-LIGHT mutant inserts were cloned into
pcDNA3.1 (+) (Invitrogen). All mutants were sequenced (ABI310
automated sequencer) for unambiguous verification. For protein
production, 293T cells (1.5.times.10.sup.6 cells/10 cm dish) were
transfected with 5 pg DNA. Medium containing soluble protein was
collected after 24 h in culture.
[0285] LIGHTt66-FLAG mutants were purified from 24 h culture
supernatant in a one step immunoaffinity procedure using an
affinity matrix of monoclonal anti-FLAG antibody (M2) coupled to
Affigel.TM. (5 mg antibody per ml of gel). Culture supernatant (50
to 100 ml) was passed over 0.5 ml of affinity matrix. The gel was
washed with 10 volumes PBS and bound protein eluted with 0.5 ml
aliquots of 10 mM glycine/HCI, pH 3.0 and neutralized immediately
by collection into 50 mM TRIS pH 7.4. Protein-containing fractions
were dialyzed against PBS.
[0286] Four single amino acid change mutants of FLAG-tagged LIGHT
t66-Y173F, G119E, L120Q and Q117T were generated in transiently
transfected 293T cells, as described herein. Y173F is the analog of
the Y108F mutant of LT.alpha., which lies in the D-E loop. The LTY
108F homotrimer fails to bind receptors. Q117T, G119E and L120Q are
three adjacent mutations in the A-A loops, conserved between
LT.beta. and LIGHT, which molecular modeling predicts are involved
in receptor binding.
[0287] Protein was immuno-affinity purified from culture
supernatants in a one-step procedure using monoclonal anti-FLAG
(M2) antibody coupled to Affigel (FIG. 15B). Purity for all of the
proteins was >95%.
[0288] It was next determined that the point mutants trimerized.
Analysis by crosslinking and by FPLC gel filtration followed by
ELISA and dot blotting of fractions demonstrated that LIGHT t66 and
its mutants were secreted exclusively as homotrimers, with no
detectable contaminating monomeric or aggregated material (FIGS.
15C and D).
[0289] ELISAs were used to measure recombinant LIGHTt66
polypeptides (including the point mutations). The capture molecule,
murine HVEM:Fc, human HVEM:Fc, murine LT.beta.R:Fc or human
LT.beta.R:Fc, was immobilized in wells of a microtiter plate (150
ng/well in 50 .mu.l 20 mM Tris/150 mM NaCl, pH 9.6) for 16 h at
4.degree. C. After washing with PBS/0.5% Tween, samples were
applied diluted in PBS/3% BSA and incubated for 1 h at RT. After
washing wells were incubated with monoclonal anti-FLAG (M2) (10
.mu.g/ml in PBS/BSA) for 1 h at RT, washed and incubated with goat
anti-mouse HRP (1:5000) for 1 hr at RT. After final washing, color
was developed with 2,2'-AZINO-bis(3-ETHYLBENZ-THIAZOLINE-6-SULFONIC
ACID) (Sigma). The OD was measured at 415 nm in a SpectraMax plate
reader (Molecular Devices Corp., Sunnyvale, Calif.).
[0290] Partially purified LIGHT was cross-linked by the addition of
bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) (Pierce
Chemical Co, Rockford, Ill.) at final concentrations of 0.1 and 1
mM, or by addition of glutaraldehyde (0.1% and 1%) for 30 min at
4.degree. C. with rotation. The reaction was stopped by addition of
TRIS (20 mM, pH 8.0). Crosslinked and control samples were analyzed
by Western blotting using monoclonal M2 (anti-FLAG) antibody.
[0291] In order to determine the molecular weight of native LIGHT
t66, purified protein was analyzed by gel filtration on a Superose
12.TM. column using a FPLC 500 system (both from Pharmacia,
Piscataway, N.J.). Flow rate was 0.5 ml/min, 0.5 ml fractions were
collected, and PBS was the mobile phase. LIGHT in eluted fractions
was detected by ELISA. Relative molecular weight of LIGHT t66 was
determined by comparison with the elution profiles of calibration
proteins.
Example 13
Cytotoxicity Assays Demonstrate that Soluble LIGHT can Trigger
Apoptosis on Cells Expressing Lymphotoxin Receptor
[0292] This example demonstrates that the homotrimeric LIGHT t66
can inhibit growth and cause apoptosis in cells expressing the
human LT.beta.R receptor. These data also indicated that
crosslinking of HVEM does not induce apoptosis.
[0293] HT29 cells (see above), at 5,000/well, were placed in wells
of a microtiter plate in DMEM (50 .mu.l) in the presence or absence
of IFN.gamma. (80 U/ml). Serial dilutions of LIGHT t66 and other
cytokines were added in 50 .mu.l medium, again in the presence or
absence of IFN.gamma., and the cells were incubated at 37.degree.
C. After 24 to 72 h, 20 .mu.l of 5 mg/ml MTT
[3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide] was
added and the plate incubated for 4 h at 37.degree. C. The medium
was then aspirated and 100 .mu.l acidified 70% isopropanol added to
dissolve the formazin. The OD was quantified at 570 nm.
[0294] LIGHT t66 inhibited growth and caused apoptosis of HT29
cells with comparable efficiency to LT.alpha.1.beta.2 (FIG. 16A).
Cytotoxicity was dependent on IFN.gamma. (FIG. 16B) and was maximal
after 72 h. Over a range of experiments, 50% cytotoxicity was
achieved with doses of 10 to 100 pM. LIGHT t66 cytotoxicity could
be blocked by pre-incubation of LIGHT t66 with human LT.beta.R:Fc
or HVEM:Fc in a dose-dependent manner; whereas Fas:Fc, which does
not bind LIGHT, had no effect (FIG. 16C).
[0295] In the presence of IFN.gamma., L120Q and Q117T were
cytotoxic to HT29 cells with comparable efficiency to LIGHT t66
(FIG. 19). Y173F, which binds both hu LT.beta.R and hu HVEM, showed
weaker, but significant cytotoxicity to these cells, whereas G119E,
which has higher affinity for HVEM than Y173F, but fails to bind
LT.beta.R, showed no significant cytotoxicity.
[0296] The data obtained with the LIGHT t66 mutants strongly
suggested that LIGHT-mediated apoptosis of HT29 cells is mediated
via LT.beta.R. To confirm this hypothesis, and to determine whether
LIGHT-mediated cross-linking of HVEM potentiates or inhibits
LT.beta.R-mediated apoptosis, we conducted experiments using a
panel of monoclonal and polyclonal anti-HVEM and anti-LT.beta.R
antibodies.
[0297] In addition to goat polyclonal HVEM IgG and goat polyclonal
LT.beta.R IgG, two mouse monoclonal anti-HVEM antibodies, CW1 and
CW8, and two mouse monoclonal anti-LT.beta.R antibodies, BDA8 and
CDHlO were used. All bound to the appropriate receptor on the
surface of HT29 cells (FIG. 20A). The anti-HVEM antibodies CW1 and
CW8 were used on the basis of ELISA blocking studies in which CW8
inhibited binding of LIGHT to huHVEM, as did polyclonal goat
anti-HVEM, whereas CW1 had no effect on binding (FIG. 20B).
[0298] Because monoclonal anti-LT.beta.R antibody BDA8 has been
shown to inhibit LT.alpha.1.beta.2-mediated apoptosis of HT29
cells, whereas CDHlO has been shown to enhance this effect, it was
investigated whether these antibodies might affect LIGHT-mediated
apoptosis in the same way. In ELISA blocking studies, polyclonal
goat anti-LT.beta.R and BDA8 inhibited binding of LIGHT to
LT.beta.R, whereas CDHlO had no effect on binding (FIG. 20C).
[0299] Goat polyclonal anti-LT.beta.R induced slow apoptotic death
of HT29 cells in the presence of IFN.gamma. (FIG. 20D), whereas
goat polyclonal anti-HVEM did not affect cell viability. This
suggests that cross-linking of HVEM does not induce apoptosis in
this cell line.
[0300] Further, polyclonal anti-HVEM neither enhanced nor inhibited
polyclonal anti-LT.beta.R-dependent cell death (FIG. 20D).
Pre-incubation with polyclonal goat anti-LT.beta.R markedly
enhanced the sensitivity of HT29 cells to LIGHT-mediated killing
(FIG. 20E).
[0301] DHlO, which enhances killing by LT.alpha.1.beta.2, also
enhanced susceptibility of the cells to LIGHT, whereas
pre-incubation with BDA8, which inhibits killing by
LT.alpha.1.beta.2, resulted in reduced LIGHT-mediated cytotoxicity
(FIG. 20E). Pre-incubation of the cells with polyclonal goat
anti-HVEM or with the monoclonal anti-HVEM antibodies CW1 and CW8
had no effect on their susceptibility to killing by LIGHT.
[0302] Collectively, these data indicate that crosslinking of HVEM
did not induce apoptosis, that HVEM signaling did not synergize
with LT.beta.R signaling in induction of apoptosis, and that HVEM
signaling did not trigger protective events sufficient to interfere
with the LT.beta.R dependent apoptotic pathway.
Example 14
Binding Studies of Soluble, Homotrimeric Human LIGHT to LT.beta.R
and HVEM by ELISA and Surface Plasmon Resonance
[0303] Association and dissociation rates of the interaction of
LIGHT t66 (a soluble, homotrimeric p30) and LIGHT t66 mutants
(described above) with human HVEM: Fc and LT.beta.R: Fc were
determined by surface plasmon resonance. Receptor binding
characteristics were investigated by ELISA using human (hu) and
murine (mu) LT.beta.R:Fc and HVEM: Fc constructs as capture
molecules and M2 anti-FLAG antibody for detection (FIG. 17).
[0304] The capture molecule, human HVEM: Fc and LT.beta.R: Fc, (50
.mu.g/ml) was coupled to a CM5 sensor chip of a BIA-core 1000.TM.
(BIAcore Inc., Piscataway, N.J.) by amine coupling at pH 5.0. The
sensor surface was equilibrated with PBS (20 mM sodium
phosphate/150 mM NaCl, pH 7.4) and sensorgrams were collected at
25.degree. C. and a flow rate of 5 .mu.l/min. A 10 .mu.l injection
of LIGHT t66 or mutant was passed over the sensor surface; after
the association phase 800 seconds of dissociation data were
collected. The sensor surface was regenerated after each cycle with
a 10 .mu.l pulse of 10 mM glycine pH 2.0. Sets of 5 analyte
concentrations, 100 to 500 nM were collected, and analyzed by
nonlinear regression using the BIlAevaluation software (2.1).TM..
Association and dissociation data were fitted on the basis of the
simple AB.revreaction.A+B model.
[0305] LIGHT t66 bound hu HVEM:Fc and huLT.beta.R:Fc with
comparable affinity. LIGHT t66 bound muHVEM:Fc and muLT.beta.R:Fc
with tenfold lower affinity. The LIGHT t66 mutants L120Q and Q117T
bound huHVEM:Fc and huLT.beta.R:Fc with comparable affinity to
LIGHT t66. Interestingly, L120Q showed enhanced affinity for both
murine receptors. G119E showed reduced but significant affinity for
hu HVEM and undetectable binding to hu LT.beta.R, while Y173F had
reduced but significant binding to both hu LT.beta.R and hu HVEM.
G119E and Y 173F did not bind the murine receptors.
[0306] Analysis of binding of LIGHT t66 and its mutants to
huLT.beta.R:Fc and hu HVEM:Fc by surface plasmon resonance
confirmed and extended the data obtained by ELISA (FIG. 18).
Results are summarized in Table 1. Association and dissociation
phases for interaction of LIGHT t66 with both receptors fitted well
with the simple A+B.revreaction.AB model, with
ka=2.7.+-.0.5.times.10.sup.4 (M/s) for hu LT.beta.R:Fc and
1.2.+-.0.2.times.10.sup.4 (M/s) for huHVEM:Fc, and kd
s=1.2.+-.0.2.times.10.sup.-4 and 4.8.+-.5.1.times.10.sup.-5
s.sup.-1 hu LT.beta.R:Fc and huHVEM:Fc, respectively. The intrinsic
dissociation rate constants (kD), calculated from the ratio kd/ka,
were 4.5.+-.0.7 nM for huLT.beta.R:Fc and 3.9.+-.3.9 nM for
huHVEM:Fc (these data are the means of 5 measurements over a
concentration range 100 to 500 nM LIGHT t66).
[0307] G119E showed no detectable binding to huLT.beta.R:Fc and its
affinity for huHVEM:Fc was approximately 30-fold lower than that of
LIGHT t66 (KD=114 nM). The reduction in affinity of G119E for
huHVEM:Fc was due to an increase in dissociation rate
(kd=2.0.+-.0.4.times.10.sup.-3s.sup.-1). Y173F bound both
huLT.beta.R:Fc and hu HVEM:Fc with reduced affinity, again due to
increased dissociation rates. Affinity of Y173F for huLT8R (kD=31
nM) was 3 to 4 fold lower than that of LIGHT t66, while affinity of
Y173F for huHVEM:Fc was more than 40-fold lower (kD=180 nM).
[0308] Q117T bound both huHVEM:Fc and huLT.beta.R:Fc with similar
association rates to LIGHT t66, and slower dissociation rates, so
that affinity of this mutant for the receptors was increased
relative to LIGHT t66.
TABLE-US-00002 TABLE 1 Hu HVEM:Fc HuLT.beta.R:Fc ka (M.sup.-1
s.sup.-1) kd (s.sup.-1) kD (nM) ka (M.sup.-1s.sup.-1) kd (s.sup.-1)
kD (nM) LIGHT t66 1.2 .+-. 0.2 .times. 10.sup.4 4.8 .+-. 5.1
.times. 10.sup.-5 3.9 .+-. 3.9 2.7 .+-. 0.5 .times. 10.sup.4 1.2
.+-. 0.2 .times. 10.sup.-4 4.5 .+-. 0.7 Q117T 2.0 .+-. 0.7 .times.
10.sup.4 5.0 .+-. 4.5 .times. 10.sup.6.sup. 0.3 .+-. 0.3 .sup. 3.2
.+-. 0.7 .times. 10.sup.-4 4.5 .+-. 1.2 .times. 10.sup.-5 1.5 .+-.
0.7 G119E 1.8 .+-. 0.4 .times. 10.sup.4 2.0 .+-. 0.4 .times.
10.sup.-3 114 .+-. 14 No Binding Y173F 1.9 .+-. 0.2 .times.
10.sup.4 3.3 .+-. 0.1 .times. 10.sup.-3 173 .+-. 30 3.7 .+-. 0.8
.times. 10.sup.4 1.1 .+-. 0.4 .times. 10.sup.-3 31 .+-. 8
Example 15
Upregulation of ICAM by Soluble, Homotrimeric Human LIGHT is
Mediated by LT.beta.R
[0309] This example demonstrates that soluble, homotrimeric LIGHT
and two variations (point mutations at L120Q and Q117T) can
up-regulate ICAM expression.
[0310] NHDF cells were used at passage 5 or earlier. Cells
(180,000/4.2 cm.sup.2 dish) were incubated with cytokine in
complete DMEM (600 .mu.l/well) supplemented with insulin and
fibroblast growth factor. After 36 h cells were stained with a
monoclonal anti-ICAM (P2A4; 10 .mu.g/ml) followed by a goat
anti-mouse IgG-PE and analyzed by flow cytometry.
[0311] L120Q and Q117T (5 nM) induced up-regulation of ICAM
expression by NHDF cells to levels comparable to those achieved
using LIGHT t66 (4-fold induction) (FIG. 21). When used at 5 nM
Y173F caused a slight induction of ICAM expression, whereas at 20
nM induction was 2.5-fold. At 5 nM G119E caused no detectable
induction of ICAM expression and slight induction (<1.5-fold) at
20 nM.
Example 16
HVEM co-Localizes with TRAF2 and TRAF5, but not TRAF3 on the Cell
Membrane
[0312] This example demonstrates that HVEM, when co-expressed as a
recombinant protein in 293 cells with TRAF polypeptides,
co-localized and bound to TRAF2 and TRAF5 (but not TRAF3) on the
cell membrane.
[0313] In co-transfection experiments in 293 cells, it was
demonstrated (using confocal immunofluorescence microscopy) that
HVEM co-localized with TRAFs 2 and 5, but not with TRAF 3.
LT.beta.R co-localized with all three TRAFs examined; negative
control was co-transfection with the empty vector pBabe.
FLAG-tagged TRAFs were visualized with FITC. LT.beta.R and HVEM
were visualized using Texas Red. Co-localized proteins appeared
yellow. HVEM co-localized with TRAFs 2 and 5, but not TRAF3.
LT.beta.R co-localized with TRAFs 2,3, and 5.
[0314] It was also demonstrated that HVEM bound to TRAF2 and TRAF5
by experiments where immunoprecipitation of the recombinantly
expressed HVEM (by anti-HVEM antibody) co-precipitated TRAFs 2 and
5, but not TRAF3 (from lysates of the transfected 293 cells).
LT.beta.R precipitated only TRAF 3. See FIGS. 22A and 22B.
[0315] Confocal Immunofluorescence Microscopy Procedures
[0316] Twenty four hours post transfection 293T cells were seeded
in 8 well Lab-TekB.TM. chamber Sides (Lab-Tek catalogue number
177445) at 3.times.10.sup.4 cells/well and cultured for 18 to 36
hours at 37.degree. C. and 5% CO.sub.2. For staining, wells were
washed two times with PBS, fixed for 10 minutes at room temperature
in freshly prepared 2% paraformaldehyde in PBS pH 7.0, washed again
two times with PBS, and then permeabilized in methanol for 2
minutes at room temperature. Cells were washed in PBS, then blocked
for a minimum of 10 minutes at room temperature in PBS containing
3% BSA. Polyclonal goat anti-LT.beta.R was diluted to a final
concentration of 20 .mu.g/ml, polyclonal rat anti-HVEM was diluted
to a final concentration of 20 .mu.g/ml and mouse monoclonal
anti-FLAG, M2 (Sigma F3165), was diluted to a final concentration
of 5 .mu.g/ml. Antibodies were diluted in PBS, 3% BSA and 0.2%
Triton X 100 (PBS/BSA/Triton). Primary antibodies were added to the
wells for a final volume of 120 .mu.l/well and incubated in a
humidified chamber at room temperature for 1 hour. Wells were then
washed three times in PBS/BSA/Triton. FITC conjugated donkey anti
mouse antibodies in combination with Texas Red conjugated donkey
anti goat antibodies (both from Jackson Immuno-Research
Laboratories), or Texas Red conjugated donkey anti rabbit
antibodies (Jackson immuno Research Laboratories), were diluted to
a final concentration of 1:200 in PBS/BSA/Triton in a final volume
of 120 .mu.l/well. Slides were incubated in a humidified chamber at
room temperature in the dark for one hour and then washed three
times in PBS/BSA/Triton and the wells removed. Four microliters per
well of a mounting solution made of 80% glycerol in PBS was added
over the cells of each well and the slides were covered with a
24.times.55 microscope cover glass (Fisherbrand #12-544-18). Slides
were kept at 4.degree. C. in the dark for 1 to 7 days before
visualization.
[0317] Cells were observed using a BioRad MRC-1024.TM. confocal
microscope with a Krypton/Argon ion Laser and a 60.times. Nikon.TM.
objective. Images were acquired using the LaserSharp.TM. operation
system and were analyzed and manipulated in Adobe Photoshop
5.0.TM..
[0318] HT29 cells (10.sup.6/ml in DMEM/3% BSA) were stained with
mouse monoclonal anti-HVEM or anti-LT.beta.R antibody for 30 min at
4.degree. C. followed by goat anti-mouse IgG coupled to
phycoerythrin (PE) for 30 min at 4.degree. C. and analyzed by flow
cytometry using a FACSCAN (Becton Dickinson, Mountain View,
Calif.).
Example 17
Inhibition of HCMV by Lymphotoxins and LIGHT
[0319] This example describes data demonstrating that human CMV is
inhibited by lymphotoxins LT.alpha. and LT.alpha.1.beta.2 and
LIGHT.
[0320] NHDF were seeded into 96 well plates at 1.times.10.sup.4
cells/well or 1.5.times.10.sup.5 cells in 12 well plates and
infected the following day with HCMV (MOI=0.01). Virus was allowed
to adsorb for 2 hours at 37.degree. C., cells were then washed
twice and cultured in medium with the indicated reagents. For
analysis of virus production and protein expression, 12 well
cultures were harvested and centrifuged to collect the cell pellet
for protein analysis by western blotting as described (Benedict et
al., (1999) J. Immunol. 126:6967), and the supernatant was assayed
for infectious virus by plaque assay. To control for equal protein
loading, the blots were reprobed with an antibody to human
.beta.-actin (MAB810, Chemicon, Temecula, Calif.).
[0321] The replication cycle of HCMV in normal human diploid
fibroblasts (NHDF) reaches completion after .about.72 hours. At a
low multiplicity of infection (MOI.ltoreq.0.1), HCMV infection
appears in focal areas of cytopathicity typified by cell rounding
and detachment, that spreads throughout the culture by day 6-7
(FIG. 23a). The addition of LT.alpha., LT.alpha.1.beta.2 or LIGHT
to the cultures completely inhibited the cytopathic effect of HCMV
(FIG. 23b,e,g).
[0322] The anti-HCMV activity of LT.alpha. was neutralized when
excess soluble decoy receptor, TNFR1-Fc was added (FIG. 23c).
Further, point mutations in LT.alpha. (LT.alpha.Y108F)
(Williams-Abbott et al. (1997) J. Biol. Chem. 272:19451) or LIGHT
(G119E) (Rooney et al., (2000) J. Biol. Chem. 275:14307), mutations
that disrupt binding to their specific receptors TNFR1 and
LT.beta.R, abolished the inhibitory action of these cytokines (FIG.
23d,f). Cytokine mediated inhibition was equivalent whether added
at the time of virus infection or several hours after virus
adsorption, indicating that the cytokines did not disrupt cell
attachment or entry of HCMV.
[0323] To assess the effect of LT.alpha., LT.alpha.1.beta.2 and
LIGHT on HCMV protein expression, both the major immediate early
protein (IE1/pp72) and the late tegument protein pp28 were analyzed
by western blot (FIG. 24a). LT.alpha.1.beta.2 and LIGHT showed
similar relative anti-viral activity (IC50 for inhibition of pp28
expression of .about.1 and 0.4 nM respectively), while LT.alpha.
was .gtoreq.40 fold more effective (IC50.about.0.01 nM, FIG. 24b),
which is consistent with the higher receptor-binding affinity of
secreted LT.alpha. compared with the normally membrane anchored
LT.alpha.1.beta.2 and LIGHT (Rooney et al., (2000) J. Biol. Chem.
275:14307).
[0324] Activation of the LT.beta.R via addition of an agonistic
polyclonal IgG elicited the inhibitory effect on HCMV protein
expression (FIG. 24a,b), indicating that this receptor mediated the
anti-viral effect seen with LT.alpha.1.beta.2 and LIGHT. Similarly,
the agonistic anti-TNFR1 mAb, H398, inhibited HCMV replication.
Consistent with reduction in cytopathicity and viral protein
expression mediated by these cytokines, production of infectious
virus was also significantly reduced (FIG. 24c).
[0325] Obvious cell death was not detected in NHDF treated with
lymphotoxins whether infected with HCMV or not. Dermal fibroblasts
express receptors for FasL and TRAIL, and in the presence of
cycloheximide undergo apoptosis following treatment with pM levels
of ligand. Surprisingly, FasL and TRAIL were unable to reduce virus
production (FIG. 24c), or inhibit virus protein expression and
cytopathicity indicating the antiviral effect is specific to LIGHT
and lymphotoxins in this model. Together, these results suggested
that the mechanism of blocking virus spread was probably not by
inducing death of infected cells.
Example 18
Non-Apoptotic and Reversible Effect of Lymphotoxin and Light on
HCMV Replication
[0326] This example describes data demonstrating that anti-viral
effect of lymphotoxins and LIGHT is reversible, does not involve
death of virus-infected cells and occurs late in the viral
replication cycle.
[0327] Although cell death or cytopathic effects were not observed
in HCMV-infected fibroblasts treated with lymphotoxins or LIGHT,
some expression of immediate early protein 1 (IE1) was always
detectable 7 days post-infection (FIGS. 24a and 25a), even at high
concentrations of cytokine that completely block infectious virus
production (FIG. 25b). This suggested that receptor signaling may
block viral spread by inhibiting gene expression downstream of the
immediate early genes. If this is the case, cytokine treated cells
may harbor HCMV genome in a restricted expression state.
[0328] To test this hypothesis, NHDF infected with HCMV and treated
with LT.alpha. for 7 days were washed and then supplemented with
medium without cytokine for an additional 7 days. Immediate early
protein 1 (IE1/pp72) and viral glycoproteins gB and gH (early and
early-late expressed genes, respectively) (Chambers et al., (1999)
J. Virol. 73:5757) could be detected by western blot during the
initial 7 days of infection in the presence of LT.alpha., when no
cytopathicity or virus production was seen. However, expression of
the true late protein pp28 (Kerry et al., (1997) J. Virol. 71:981)
was undetectable (FIG. 25a). This level of protein most likely
represents expression in cells initially infected at low MOI. A
vigorous reemergence of viral protein expression (FIG. 25a) with
high levels of pp28, concurrent with the release of infectious
virions (FIG. 25b), occurred after this initial 7 day period when
the culture was replaced with fresh medium lacking cytokine. Thus,
the block to HCMV appears to be late in the replication cycle based
on Western blot analysis of these representative viral proteins and
the short lag-time before cytopathicity (.about.24 hours) and
appearance of virus in the culture supernatant (.about.48 hours)
after removal of cytokine. LT.alpha.1.beta.2, anti-LT.beta.R
antibodies and LIGHT similarly inhibited HCMV. Together, these
results demonstrate that the anti-viral effect of lymphotoxins and
LIGHT is reversible and does not involve death of virus-infected
cells.
Example 19
NF.kappa.B but not TRAF3 or FADD is Necessary for Lymphotoxin and
LIGHT Anti-HCMV Activity
[0329] This example describes data demonstrating that anti-HCMV
activity of TNFR1 and LT.beta.R requires activation of NFkB.
[0330] LT.beta.R and TNFR1 are capable of activating both apoptotic
and non-apoptotic signaling pathways that can be distinguished by
introducing dominant negative (dn) mutants of key signaling
molecules. Mutant signaling molecules were introduced into NHDF
with retroviral vectors containing dominant acting forms of
I.kappa.B.alpha., TRAF3, FADD, or a control empty vector (LXSN).
The I.kappa.B.alpha. mutant (I.kappa.B.alpha.M) contains two point
mutations at serine 32 and 36 to alanine that deletes critical
phosphorylation sites targeted by cytokine-activated serine kinases
(Van Antwerp et al., (1996) Science 274:787). When introduced into
NHDF, I.kappa.B.alpha.M protein cannot be phosphorylated and
degraded by the ubiquitination pathway, thus retaining NF.kappa.B
in its latent cytoplasmic state, which in turn prevents nuclear
translocation and transcriptional activation of NF.kappa.B target
genes, such as ICAM1 (FIG. 26a). The TRAF3 dn mutant can inhibit
LT.beta.R induced death in HT29 carcinoma cells, but not NF.kappa.B
activation (Force et al. (2000) J. Biol. Chem. 275:11121; Rooney et
al. (2000) J. Biol. Chem. 275:14307). The FADD dn mutant has the
death effector domain required for caspase 8 recruitment to TNFR1
and Fas deleted.
[0331] Stable expression of I.kappa.B.alpha.M (S32,36A), TRAF3.D11
and FADD.dn were generated by transduction with replication
incompetent retroviral vectors. Retroviral vectors were produced by
a three plasmid CaPO.sub.4 transfection method into 293T cells as
described (Force et al. (1997) J. Biol. Chem. 272:30835). The
retroviral vector expression plasmids LXSN, I.kappa.B.alpha.M (a
dominant negative mutant of IicBa cloned into LXSN (Van Antwerp et
al., (1996) Science 274:787), and TRAF3.D11 dominant negative
acting mutants inserted into pBABE retroviral vector have been
described (Force et al., (1997) Proc. Natl. Acad. Sci. 94:2460).
The pBABE-FADD.dn retroviral vector was generated from a plasmid
containing full length FADD (gift of V. Dixit) by PCR amplification
of a truncated FADD coding sequence lacking an intact death
effector domain (nucleotide 240-627) with the addition of a 5' SnaB
I site and a 3' EcoR I site. After amplification, the PCR product
was digested, ligated into pBABE-FLAG (pBABE-puro containing an
N-terminal FLAG epitope tag inserted at the BamH I/SnaB I site) and
verified by DNA sequencing (ABI Prism 310 Perkin-Elmer, Foster
City, Calif.). Retroviral vector transduction frequency was greater
than 99% as gauged by resistance of cells to drug selection.
[0332] I.kappa.B.alpha.M expressing fibroblasts, when compared to
control vector transduced cells, were refractory to the anti-viral
effects of lymphotoxins and LIGHT as detected by viral protein
expression (FIG. 26b) or virus production (FIG. 26c).
I.kappa.B.alpha.M did not significantly alter HCMV replication in
the absence of cytokine. By contrast, the TRAF3 dn mutant did not
block the antiviral effect of either LT.alpha.1.beta.2 or LIGHT.
Surprisingly, the FADD dn mutant actually enhanced the effect of
LT.alpha., but also partially diminished virus replication in the
absence of cytokine. These results demonstrate that anti-HCMV
signaling mediated by both TNFR1 and LT.beta.R requires activation
of NF.kappa.B and confirm that inhibition of HCMV replication does
not involve apoptosis of infected cells.
Example 20
Interferon-.beta. Mediates Lymphotoxin and LIGHT Dependent
Antiviral Activity
[0333] This example demonstrates that IFN.beta. mediates the
antiviral activity of lymphotoxins and LIGHT. This example also
demonstrates that the level of IFN.beta. induction needed to
mediate anti-viral activity (e.g., inhibit CMV replication) is
greater than that produced by virus alone.
[0334] Supernatant from LT.alpha. treated HCMV infected
fibroblasts, but not from cells treated with LT.alpha. or virus
alone was capable of transferring antiviral activity to newly
infected cells (FIG. 27a). However, neutralization of LT.alpha. in
these supernatants by TNFR1-Fc failed to block the antiviral
activity as measured by IE1 protein expression (FIG. 27a). This
result suggested that a secondary mediator was responsible for the
antiviral activity induced by lymphotoxins.
[0335] Neutralizing antibodies to IFN.beta. added to the medium
along with TNFR1-Fc ablated the transfer of antiviral activity,
implicating type I interferon (FIG. 27a). Inclusion of
anti-IFN.beta. neutralizing antibodies upon initial treatment of
HCMV infected cells with LT.alpha. also reversed the block to viral
gene expression (FIG. 27b) and virion production (FIG. 27c).
Anti-IFN.alpha. showed minimal (<5% compared to anti-IFN.beta.)
but consistent neutralizing activity; anti-IFN.alpha. was without
effect (FIG. 27b). Additionally, anti-IFN.beta. was able to block
the antiviral activity of LIGHT or LT.beta.R agonistic antibodies
indicating that a similar signaling mechanism is initiated by both
LT.beta.R and TNFR1.
[0336] For analysis of IFN.alpha./.beta. mRNA induction by RT-PCR,
NHDF (.about.80% confluent) were harvested 4 hours after infection
and total RNA was isolated (Rneasy mini kit, Qiagen). RNA was
treated with DNAse I, and 2 mg was used for reverse transcription.
For PCR analysis, volumes of RT reactions and cycles of PCR were
determined empirically to ensure analysis was within the linear
range. Primer sequences used for PCR were: Pactin,
5'-tgacggggtcacccacactgtgcccatcta-3' and
5'-ctagaagcatttgcggtggacgatggag-3'; IFN.beta.,
5'-gtcagtgtcagaagctcctgtggc-3' and 5'-ctatggtccaggcacagtgactg-3',
IFN.alpha., 5'-gaatctctcctttacctg-3' and 5'-ctgacaacctcccaggcac-3',
IE1, 5' gcatagaatcaaggagcacatgc-3' and
5'-gtgatcaatgtgcgtgagcacc-3'. IFN.alpha. primers were designed to
hybridize to conserved sequences present in all subtypes. For real
time PCR, (GeneAmp 5700 sequence detection system, PE Biosystems,
Foster city, CA) the primers and Taqman.RTM. probe (PE Biosystems)
for detection of IFN.beta. were:
5'-gacatccctgaggagattaagca-3',5'-ggagcatctcatagatggtcaatg-3', probe
sequence 5'VIC-cgtcctccttctggaactgctgcag-TAMRA3'.
[0337] For determination of fold differences in message levels, the
cycle number (Ct) which the relative fluorescence (Rn) crossed the
manually set threshold value was determined using the analysis
software provided with the 5700 SDS. Fold differences were
calculated as follows:
[log.sub.2IFN.beta.C.sub.t(HCMV+LT.alpha.)-C.sub.t(HCMV)]/[log.sub.2.bet-
a.actin C.sub.t(HCMV+LT.alpha.)-C.sub.t(HCMV).
[0338] A high level induction of IFN.beta. mRNA was observed only
in NHDF that were both infected with HCMV and treated with
LT.alpha. (FIG. 28a), LIGHT or LT.alpha.1.beta.2 (FIG. 28b). The
level of IFN.beta. message increased proportional to the amount of
infectious virus suggesting the production of IFN.beta. occurred in
virus-infected cells (FIG. 28a). The induction of IFN.beta. was
rapid, peaking at 4-6 hr after infection, whereas trace levels of
IFN.alpha. mRNA were detectable at 8-10 hours after infection (FIG.
28c). Treatment of NHDF with polyI:C, a potent IFN inducer,
stimulated IFN.alpha. and .beta. mRNA in the absence of
lymphotoxins or virus. IFN.beta. was not detected in uninfected
cells treated with lymphotoxins, but was minimally induced by HCMV
alone. However, this level of IFN.beta. induction with virus alone
was not sufficient to restrict CMV replication (FIG. 27).
[0339] Quantitative real time RT-PCR revealed an induction of
IFN.beta. mRNA 48-103 fold (mean=77.+-.26; n=4) above the level
seen with virus alone (FIG. 28d). NHDF-I.kappa.B.alpha.M had
significantly reduced levels of IFN.beta. mRNA after exposure to
HCMV and LT.alpha. (8.6 fold induction vs 103 fold in LSXN control
cells) (FIGS. 28d and 28e), consistent with the refractory response
to the antiviral effects of LT-related ligands. Importantly, no
difference was seen in the ability of recombinant IFN.beta. to
inhibit HCMV replication in NHDF-I.kappa.B.alpha.M cells indicating
that NF.kappa.B activation is critical for the induction of
IFN.beta., and not for subsequent IFN.beta. mediated antiviral
effects.
Example 21
Production and Characterization of an Anti-Mouse LT.beta.R Antibody
Having Agonist Activity
[0340] This example describes synthesis of an anti-mouse LT.beta.R
monoclonal antibody. This example also describes data demonstrating
that the anti-mouse LT.beta.R monoclonal antibody has LT.beta.R
agonist activity.
[0341] Monoclonal antibodies to mouse LT.beta.R were prepared by
immunization of Sprague-Dawley female rats with mouse LT.beta.R-Fc
fusion protein in Freund's adjuvant. Purified LT.beta.R-Fc was
produced by the standard baculovirus expression system and purified
by Protein G affinity chromatography (Force et al., (1995) J.
Immunol. 155:5280). Hybridomas were produced by fusion of rat
splenocytes with mouse P3X myeloma cell line by standard
techniques. Specific antibody secreting cells were selected by a
standard solid phase ELISA assay that employed plate bound mouse
LT.beta.R-Fc and detection of rat IgG with goat anti-Rat IgG
conjugated to peroxidase (Immunochemical techniques. Part I:
Hybridoma technology and monoclonal antibodies. Methods Enzymol.
(1986)121:1-947). Specificity of monoclonal antibodies was assessed
by staining a human cell line (HEK293) transfected with mouse
LT.beta.R cDNA using standard methods of DNA transfection. The rat
anti mouse LT.beta.R antibodies 3C8 (IgG1), 3H4 (IgG1), 4H8 (IgG2a)
were purified from ascities fluid by ammonium sulfate precipitation
and protein G affinity chromatography.
[0342] In order to examine activity of the LT.beta.R antibodies,
induction of Vascular cell adhesion molecule-1 (VCAM 1) with
antibody to the mouse LT.beta.R was studied. Mouse fibroblasts were
stimulated with either rat anti-LT.beta.R (3 .mu.g/ml 3C8), human
TNT (10 ng/ml) or left unstimulated for 16 hrs in tissue culture
medium. The cells were harvested and stained with anti-mouse VCAM1
and FITC labeled second antibody. The fluorescence staining
intensity was measured by flow cytometry.
[0343] The results show that both TNF and anti-LT.beta.R monoclonal
antibody stimulate increased expression of VCAM1 on mouse
fibroblasts (FIG. 30). LT.beta.R antibody is therefore an agonist
of LT.beta.R and, as such, is useful in practicing the methods of
the invention.
[0344] Additional cell-based and in vivo animal assays for
identifying such antibodies are described herein (see above and
Example 22), and also are known in the art (see e.g., Rennert et
al., (1998) Immunity 1:71; Force et al., (2000) J. Biol. Chem.
275:11121).
Example 22
LT.alpha..beta. is Critical for Host Defense Against Murine CMV
[0345] This example describes data demonstrating that each of
LT.alpha..beta. and LIGHT participate in mediating host defense to
MCMV. This example also describes data demonstrating that an
anti-mouse LT.beta.R monoclonal antibody having agonist activity
protects against CMV infection in mice.
[0346] The antiviral activity that lymphotoxins exhibit against
human CMV suggested these ligands may be conserved across species.
The capacity of LT.alpha.-deficient mice (LT.alpha./-) to respond
to infection with mouse CMV (MCMV) was studied. MCMV replicates in
most visceral organs and to high levels in spleen, liver, lung and
salivary glands. Depending on virus dose, and the strain and age of
the mice, MCMV can cause death from acute shock at 2-3 days post
infection. However, the most common course is death at 5-7 days as
a result of multiorgan disease, and in particular hepatic
failure.
[0347] LT.alpha.-/- mice backcrossed to C57BL/6 mice for 8
generations (Banks et al., (1995) J. Immunol. 155:1685) and
wildtype C57BL/6 mice were purchased from the Jackson Laboratories
(Bar Harbor, Me.). The transgenic line expressing a soluble mouse
LT.beta.R/human IgG1-Fc fusion protein (LT.beta.R-Fc Tg) on a
Balb/c background was provided by R. Ettinger and H. O. McDevitt
(Ettinger et al., (1996) Proc. Natl. Acad. Sci. 93:13102). These
animals constitutively express the murine LTbR-Fc under the control
of the human CMV promoter. The construct is specifically mutated in
the CH2 domain to block binding to Fc receptors and complement
activation. Serum levels of this soluble chimeric receptor were
determined by ELISA (Ettinger et al., (1996) Proc. Natl. Acad. Sci.
93:13102); studies used animals with serum levels >1.2 .mu.g/ml.
Non-transgenic littermates served as controls. All mice were bred
and housed under specific pathogen-free conditions and in
accordance with institutional guidelines. In all experiments mice
were age (6-12 weeks) and sex-matched.
[0348] Murine cytomegalovirus (MCMV; Smith strain) viral stocks
were prepared from salivary gland extracts as described (Reddehase
et al., (1985) J. Virol. 55:264). To determine both lethal and
sublethal virus doses for acute MCMV infection in the different
mouse strains, virus doses ranging from 5.times.10.sup.3 to
1.times.10.sup.6 PFU were injected by the intraperitoneal route
into groups of 4 to 6 mice per virus dose and the mice monitored
daily for morbidity and mortality over a period of 14 days.
[0349] LT.alpha.-/- mice (C57/BL6 background) were profoundly
susceptible to lethal infection with MCMV requiring .about.100 fold
less virus than age matched C57/BL6 mice (FIG. 29 upper panel). At
viral doses >4.times.10.sup.4 PFU (LD50 for LT.alpha.-/-
mice=.about.3.times.10.sup.4 PFU) the LT.alpha.-/- mice succumbed
at 5 to 7 days indicating hepatic failure as the likely cause of
death. Similarly, when high doses of MCMV (>3.times.10.sup.6
PFU; LD50 for C57/BL6=.about.2-3.times.10.sup.6 PFU) were used to
infect C57/BL6 controls, these mice also died between 5-7 days.
[0350] LT.alpha.-/- mice have defects in the development of
peripheral lymphoid organs and also lack most of their NK and NK-T
cells (Lizuka et al., (1999) Proc. Natl. Acad. Sci. USA 96:6336);
Elewaut et al., (2000) J. Immunol. 165:671), which could account
for their susceptibility to MCMV. However, mice expressing the
LT.beta.R-Fc decoy as a transgene, which have a normal complement
of lymph nodes and NK and NK-T cells, were also susceptible to MCMV
compared to transgene negative littermates (FIG. 29, lower panel).
This result indicates that the developmental abnormalities in the
LT.alpha.-/- mice are not likely to be responsible for the
susceptibility of these mice to MCMV, and implicates
LT.alpha..beta. and LIGHT as potentially critical effector
molecules in host defense to MCMV.
[0351] Normal mice treated with mouse LT.beta.R-Fc fusion protein,
a soluble injectable neutralizing agent specific for
LT.alpha.1.beta.2 or LIGHT, or mouse HVEM-Fc, which binds LT.alpha.
or LIGHT, results in profound susceptibility of normal C57B1/6 mice
to MCMV at a dose of virus that is 10 times below the amount that
causes a lethal infection in this strain (Table 2). Treatment of
mice with LT.beta.R-Fc 3 days before infection with MCMV does not
cause this susceptibility. By contrast, LT.alpha. deficient mice
treated with a rat monoclonal antibody to mouse LT.beta.R (a pool
of 3C8 with 3H4 and 4H8) are protected compared to control
LT.alpha.-/- mice as measured by the substantial delay in time
(prolonged from death at 5 days to 9 days) to the lethality caused
by MCMV (Table 3). The administration of anti-LT.beta.R antibodies
allows LT.alpha. deficient mice to resist lethal infection with
MCMV demonstrating that antibodies to LT.beta.R can be protective
in vivo in MCMV infection.
TABLE-US-00003 TABLE 2 Resistance to MCMV is dependent on
LT.alpha.1.beta.2 and LIGHT MCMV only LT.beta.R-Fc LT.beta.R-Fc-72
hrs* HVEM-Fc 4/4 0/4 4/4 0/4 Normal C57/B16 mice were injected IP
with 100 .mu.g of purified mouse LT.beta.R-Fc or mouse HVEM-Fc and
4 hrs later infected IP with 2 .times. 10.sup.5 PFU MCMV (Smith
strain). Morbidity and mortality were assessed daily for 13 days.
Data represent number of surviving mice per total mice in each
group at day 13. Death occurred at 5-6 days post infection.
*LT.beta.R-Fc was administered 3 days prior to virus infection.
TABLE-US-00004 TABLE 3 LT.beta.R signaling protects against lethal
MCMV infection Time Anti-LT.beta.R/no virus MCMV Anti-LT.beta.R +
MCMV Day 5 4/4 0/4 0/4 Day 14 4/4 0/4 0/4* LT.alpha.-/- mice
(groups of 4) were administered 100 .mu.g of rat anti-mouse
LT.beta.R monoclonal antibody IP, followed by IP infection with
MCMV 8 .times. 10.sup.4 PFU (Smith strain) 4 hrs later. Animal
morbidity and mortality was assessed daily. Data represent number
of surviving mice per total mice in each group. *Mice survived
until day 9.
[0352] Together, these results reveal the role of lymphotoxins in
establishing host defense to CMV. LT.beta.R and TNFR1 agonists that
mimic lymphotixins, including antibodies that activate LT.beta.R,
are therefore useful for treatment of HCMV primary infection or
reactivation of infection, such as in immune compromised patients
with cancer or HIV that suffer from CMV hepatitis, nephritis,
retinitis or disseminated disease. Treatment of patients with
anti-LT.beta.R antibody can inhibit HCMV reactivation caused by
bone marrow transplantation or other tissue grafts. Finally,
anti-LT.beta.R antibody can be useful in treating disorders
associated with CMV such as, arteriosclerosis or multiple
sclerosis.
TABLE-US-00005 SEQUENCE LISTING <110> Ware, Carl F.
<120> LIGAND FOR HERPES SIMPLEX VIRUS ENTRY MEDIATOR AND
METHODS OF USE <130> <140> <141> <160> 6
<170> PatentIn Ver. 2.0 <210> 1 <211> 29
<212> DNA <213> Artificial Sequence <220> 223>
Description of Artificial Sequence: Forward primer sequence
<400> 1 cggagatctg agttcatcct gctagctgg 29 <210> 2
<211> 31 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence:
Backward primer sequence <400> 2 ataggatccc ttggtctggt
gctgacattc c 31 <210> 3 <211> 29 <212> DNA
<213> Artificial Sequence <220> <223> Description
of ArtificialSequence:Forward primer sequence <400> 3
gacgtcagat cttcccacct ttcctccta 29 <210> 4 <211> 29
<212> DNA <213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Backward primer
sequence <400> 4 gaacagagat ctcattgctc ctggctctg 29
<210> 5 <211> 1169 <212> DNA <213> Homo
sapiens <220> <221> CDS <222> (49)..(771)
<400> 5 gaggttgaag gacccaggcg tgtcagccct gctccagaga ccttgggc
atg gag ga 57 Met Glu Glu 1 agt gtc gta cgg ccc tca gtg ttt gtg gtg
gat gga cag acc gac atc Ser Val Val Arg Pro Ser Val Phe Val Val Asp
Gly Gln Thr Asp Ile 105 5 10 15 cca ttc acg agg ctg gga cga agc cac
cgg aga cag tcg tgc agt gtg 153 Pro Phe Thr Arg Leu Gly Arg Ser His
Arg Arg Gln Ser Cys Ser Val 20 25 30 35 gcc cgg gtg ggt ctg ggt ctc
ttg ctg ttg ctg atg ggg get ggg ctg 201 Ala Arg Val Gly Leu Gly Leu
Leu Leu Leu Leu Met Gly Ala Gly Leu 40 45 50 gcc gtc caa ggc tgg
ttc ctc ctg cag ctg cac tgg cgt cta gga gag 249 Ala Val Gln Gly Trp
Phe Leu Leu Gln Leu His Trp Arg Leu Gly Glu 55 60 65 atg gtc acc
cgc ctg cct gac gga cct gca ggc tcc tgg gag cag ctg 297 Met Val Thr
Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp Glu Gln Leu 70 75 80 ata
caa gag cga agg tct cac gag gtc aac cca gca gcg cat ctc aca 345 Ile
Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala His Leu Thr 85 90
95 ggg gcc aac tcc agc ttg acc ggc agc ggg ggg ccg ctg tta tgg gag
393 Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu
100 105 110 115 act cag ctg ggc ctg gcc ttc ctg agg ggc ctc agc tac
cac gat ggg 441 Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr
His Asp Gly 120 125 130 gcc ctt gtg gtc acc aaa gct ggc tac tac tac
atc tac tcc aag gtg 489 Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr
Ile Tyr Her Lys Val 135 140 145 cag ctg ggc ggt gtg ggc tgc ccg ctg
ggc ctg gcc agc acc atc acc 537 Gln Leu Gly Gly Val Gly Cys Pro Leu
Gly Leu Ala Ser Thr Ile Thr 150 155 160 cac ggc ctc tac aag cgc aca
ccc cgc tac ccc gag gag ctg gag ctg 585 His Gly Leu Tyr Lys Arg Thr
Pro Arg Tyr Pro Glu Glu Leu Glu Leu 165 170 175 ttg gtc agc cag cag
tca ccc tgc gga cgg gcc acc agc agc tcc cgg 633 Leu Val Ser Gln Gln
Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg 180 185 190 195 gtc tgg
tgg gac agc agc ttc ctg ggt ggt gtg gta cac ctg gag gct 681 Val Trp
Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala 200 205 210
ggg gag gag gtg gtc gtc cgt gtg ctg gat gaa cgc ctg gtt cga ctg 729
Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu 215
220 225 cgt gat ggt acc cgg tct tac ttc ggg gct ttc atg gtg tga 771
Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val 230 235 240
aggaaggagc gtggtgcatt ggacatgggt ctgacacgtg gagaactcag agggtgcctc
831 aggggaaaga aaactcacga agcagaggct gggcgtggtg gctctcgcct
gtaatcccag 891 cactttggga ggccaaggca ggcggatcac ctgaggtcag
gagttcgaga ccagcctggc 951 taacatggca aaaccccatc tctactaaaa
atacaaaaat tagccggacg tggtggtgcc 1011 tgcctgtaat ccagctactc
aggaggctga ggcaggataa ttttgcttaa acccgggagg 1071 cggaggttgc
agtgagccga gatcacacca ctgcactcca acctgggaaa cgcagtgaga 1131
ctgtgcctca aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1169 <210> 6
<211> 240 <212> PRT <213> Homo sapiens
<400> 6 Met Glu Glu Ser Val Val Arg Pro Ser Val Phe Val Val
Asp Gly Gln 1 5 10 15 Thr Asp Ile Pro Phe Thr Arg Leu Gly Arg Ser
His Arg Arg Gln Ser 20 25 30 Cys Ser Val Ala Arg Val Gly Leu Gly
Leu Leu Leu Leu Leu Met Gly 35 40 45 Ala Gly Leu Ala Val Gln Gly
Trp Phe Leu Leu Gin Leu His Trp Arg 50 55 60 Leu Gly Glu Met Val
Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp 65 70 75 80 Glu Gln Leu
Ile Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala 85 90 95 His
Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu 100 105
110 Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr
115 120 125 His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr
Ile Tyr 130 135 140 Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu
Gly Leu Ala Ser 145 150 155 160 Thr Ile Thr His Gly Leu Tyr Lys Arg
Thr Pro Arg Tyr Pro Glu Glu 165 170 175 Leu Glu Leu Leu Val Ser Gln
Gln Ser Pro Cys Gly Arg Ala Thr Ser 180 185 190 Ser Ser Arg Val Trp
Trp Asp Ser Ser Phe Leu Gly Gly Val Val His 195 200 205 Leu Glu Ala
Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu 210 215 220 Val
Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val 225 230
235 240
[0353] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
16129DNAArtificial SequenceDescription of Artificial Sequence
Primer 1cggagatctg agttcatcct gctagctgg 29231DNAArtificial
SequenceDescription of Artificial Sequence Primer 2ataggatccc
ttggtctggt gctgacattc c 31329DNAArtificial SequenceDescription of
Artificial Sequence Primer 3gacgtcagat cttcccacct ttcctccta
29429DNAArtificial SequenceDescription of Artificial Sequence
Primer 4gaacagagat ctcattgctc ctggctctg 2951171DNAHomo
sapiensCDS(50)..(773) 5gaggttgaag gacccaggcg tgtcagccct gctccagaga
ccttgggcat atg gag tga 59 Met Glu Glu 1agt gtc gta cgg ccc tca gtg
ttt gtg gtg gat gga cag acc gac atc 107Ser Val Val Arg Pro Ser Val
Phe Val Val Asp Gly Gln Thr Asp Ile 5 10 15cca ttc acg agg ctg gga
cga agc cac cgg aga cag tcg tgc agt gtg 155Pro Phe Thr Arg Leu Gly
Arg Ser His Arg Arg Gln Ser Cys Ser Val20 25 30 35gcc cgg gtg ggt
ctg ggt ctc ttg ctg ttg ctg atg ggg gct ggg ctg 203Ala Arg Val Gly
Leu Gly Leu Leu Leu Leu Leu Met Gly Ala Gly Leu 40 45 50gcc gtc caa
ggc tgg ttc ctc ctg cag ctg cac tgg cgt cta gga gag 251Ala Val Gln
Gly Trp Phe Leu Leu Gln Leu His Trp Arg Leu Gly Glu 55 60 65atg gtc
acc cgc ctg cct gac gga cct gca ggc tcc tgg gag cag ctg 299Met Val
Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp Glu Gln Leu 70 75 80ata
caa gag cga agg tct cac gag gtc aac cca gca gcg cat ctc aca 347Ile
Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala His Leu Thr 85 90
95ggg gcc aac tcc agc ttg acc ggc agc ggg ggg ccg ctg tta tgg gag
395Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu Leu Trp
Glu100 105 110 115act cag ctg ggc ctg gcc ttc ctg agg ggc ctc agc
tac cac gat ggg 443Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser
Tyr His Asp Gly 120 125 130gcc ctt gtg gtc acc aaa gct ggc tac tac
tac atc tac tcc aag gtg 491Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr
Tyr Ile Tyr Ser Lys Val 135 140 145cag ctg ggc ggt gtg ggc tgc ccg
ctg ggc ctg gcc agc acc atc acc 539Gln Leu Gly Gly Val Gly Cys Pro
Leu Gly Leu Ala Ser Thr Ile Thr 150 155 160cac ggc ctc tac aag cgc
aca ccc cgc tac ccc gag gag ctg gag ctg 587His Gly Leu Tyr Lys Arg
Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu 165 170 175ttg gtc agc cag
cag tca ccc tgc gga cgg gcc acc agc agc tcc cgg 635Leu Val Ser Gln
Gln Ser Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg180 185 190 195gtc
tgg tgg gac agc agc ttc ctg ggt ggt gtg gta cac ctg gag gct 683Val
Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His Leu Glu Ala 200 205
210ggg gag gag gtg gtc gtc cgt gtg ctg gat gaa cgc ctg gtt cga ctg
731Gly Glu Glu Val Val Val Arg Val Leu Asp Glu Arg Leu Val Arg Leu
215 220 225cgt gat ggt acc cgg tct tac ttc ggg gct ttc atg gtg tga
773Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val 230 235
240aggaaggagc gtggtgcatt ggacatgggt ctgacacgtg gagaactcag
agggtgcctc 833aggggaaaga aaactcacga agcagaggct gggcgtggtg
gctctcgcct gtaatcccag 893cactttggga ggccaaggca ggcggatcac
ctgaggtcag gagttcgaga ccagcctggc 953taacatggca aaaccccatc
tctactaaaa atacaaaaat tagccggacg tggtggtgcc 1013tgcctgtaat
ccagctactc aggaggctga ggcaggataa ttttgcttaa acccgggagg
1073cggaggttgc agtgagccga gatcacacca ctgcactcca acctgggaaa
cgcagtgaga 1133ctgtgcctca aaaaaaaaaa aaaaaaaaaa aaaaaaaa
11716240PRTHomo sapiens 6Met Glu Glu Ser Val Val Arg Pro Ser Val
Phe Val Val Asp Gly Gln1 5 10 15Thr Asp Ile Pro Phe Thr Arg Leu Gly
Arg Ser His Arg Arg Gln Ser 20 25 30Cys Ser Val Ala Arg Val Gly Leu
Gly Leu Leu Leu Leu Leu Met Gly 35 40 45Ala Gly Leu Ala Trp Gln Gly
Trp Phe Leu Leu Gln Leu His Trp Arg 50 55 60Leu Gly Glu Met Val Thr
Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp65 70 75 80Glu Gln Leu Ile
Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala 85 90 95His Leu Thr
Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu 100 105 110Leu
Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr 115 120
125His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr
130 135 140Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu
Ala Ser145 150 155 160Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro
Arg Tyr Pro Glu Glu 165 170 175Leu Glu Leu Leu Val Ser Gln Gln Ser
Pro Cys Gly Arg Ala Thr Ser 180 185 190Ser Ser Arg Val Trp Trp Asp
Ser Ser Phe Leu Gly Gly Val Val His 195 200 205Leu Glu Ala Gly Glu
Glu Val Val Val Arg Val Leu Asp Glu Arg Leu 210 215 220Val Arg Leu
Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val225 230 235
240728DNAArtificial SequenceDescription of Artificial Sequence
Forward primer 7tatggattca tggaacctct cccaggat 28830DNAArtificial
SequenceDescription of Artificial Sequence Backward primer
8tatggattcg gaggagcagg tggtgtctgt 30921DNAArtificial
SequenceDescription of Artificial Sequence Forward primer sequence
9acgctgggcc tggcctnctg a 211020DNAArtificial SequenceDescription of
Artificial Sequence Backward primer 10actctcccat aacagcggcc
201123DNAArtificial SequenceDescription of Artificial Sequence
Forward primer 11gagctggccn tgctgagggg cct 231220DNAArtificial
SequenceDescription of Artificial Sequence Backward primer
12cagctgagtc tcccataaca 201322DNAArtificial SequenceDescription of
Artificial Sequence Forward primer 13caggccntcc tgaggggcct ca
221421DNAArtificial SequenceDescription of Artificial Sequence
Backward primer 14gcccagctga gtctcccata a 211520DNAArtificial
SequenceDescription of Artificial Sequence Forward primer
15ttccccgagg agctggagct 201620DNAArtificial SequenceDescription of
Artificial Sequence Backward primer 16gcggggtgtg cgcttgtaga 20
* * * * *