U.S. patent application number 10/512873 was filed with the patent office on 2005-07-14 for antibody and inhibitor and transformation method and transformation kit using the same.
Invention is credited to Matsumoto, Misako, Seya, Tsukasa.
Application Number | 20050153910 10/512873 |
Document ID | / |
Family ID | 29727832 |
Filed Date | 2005-07-14 |
United States Patent
Application |
20050153910 |
Kind Code |
A1 |
Matsumoto, Misako ; et
al. |
July 14, 2005 |
Antibody and inhibitor and transformation method and transformation
kit using the same
Abstract
The present invention provides a monoclonal antibody which
specifically binds to human Toll-like receptor 3. Further, the
present invention provides an inhibitor which (a) suppresses a
double-stranded RNA-mediated immune response in a cell which
expresses Toll-like receptor recognizing the double-stranded RNA
and produces type I interferon, and (b) includes an antibody, which
binds to the Toll-like receptor and inhibits production of the type
I interferon. Particularly, the antibody is a monoclonal antibody
against human Toll-like receptor 3. Further, the present invention
provides a transfection method comprising the step of infecting a
cell which expresses Toll-like receptor recognizing a
double-stranded RNA and produces type I interferon by expressing
Toll-like receptor recognizing a double-stranded RNA with a
recombined virus vector, in which a gene of interest has been
inserted, under the inhibitory condition for production of the type
I interferon by using an inhibitor including an antibody which
binds to the Toll-like receptor. Thus, it is possible to provide
(i) an inhibitor which inhibits the production of the type I
interferon induced by a virus double-stranded RNA so as to suppress
an immune response against the virus, and (ii) a transfection
method or a transfection kit whereby a transfection efficiency is
improved without enhancing an infectious capacity of a virus
vector.
Inventors: |
Matsumoto, Misako;
(Ikoma-shi, JP) ; Seya, Tsukasa; (Nara-shi,
JP) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Family ID: |
29727832 |
Appl. No.: |
10/512873 |
Filed: |
October 29, 2004 |
PCT Filed: |
February 17, 2003 |
PCT NO: |
PCT/JP03/01673 |
Current U.S.
Class: |
514/44R ;
424/143.1; 530/388.22 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/2896 20130101; A61P 31/12 20180101; C07K 16/28
20130101 |
Class at
Publication: |
514/044 ;
424/143.1; 530/388.22 |
International
Class: |
A61K 048/00; A61K
039/395; C07K 016/28 |
Claims
1. An antibody, specifically binding to human Toll-like receptor
3.
2. An inhibitor for suppressing a double-stranded RNA-mediated
immune response in a cell which expresses a Toll-like receptor
recognizing the double-stranded RNA and produces type I interferon,
the inhibitor comprising an antibody, which binds to the Toll-like
receptor and inhibits production of the type I interferon.
3. The inhibitor as set forth in claim 2, wherein the antibody is a
monoclonal antibody against human Toll-like receptor 3.
4. A transfection method, comprising the step of infecting a cell
which expresses a Toll-like receptor recognizing a double-stranded
RNA and produces type I interferon with a recombined virus vector,
in which a gene of interest has been inserted, under the inhibitory
condition for production of the type I interferon by using an
inhibitor including an antibody which binds to the Toll-like
receptor.
5. The method as set forth in claim 4, wherein the antibody is a
monoclonal antibody against human Toll-like receptor 3.
6. A transfection kit for transfecting a cell which expresses
Toll-like receptor recognizing a double-stranded RNA and produces
type I interferon, the transfection kit comprising: an inhibitor
including an antibody, which binds to the Toll-like receptor and
inhibits production of the type I interferon; and a recombined RNA
virus vector in which a gene of interest has been inserted.
7. The transfection kit as set forth in claim 6, wherein the
antibody is a monoclonal antibody against human Toll-like receptor
3.
8. A method for control production of type I interferon by using an
antibody which specifically binds to Toll-like receptor recognizing
a double-stranded RNA.
Description
TECHNICAL FIELD
[0001] The present invention relates to (i) an antibody
specifically bound to human Toll-like receptor 3, (ii) an inhibitor
for blocking a signaling induced by double-stranded RNA
(ribonucleic acid) so as to suppress a double-stranded RNA-mediated
immune response in a cell which produces type I interferon
(interferon-.alpha. and interferon-.beta.) by expressing the
Toll-like receptor recognizing the double-stranded RNA on the
surface, (iii) a transfection method or a transfection kit using
the antibody and the inhibitor so as to carry out transfection with
a recombined RNA virus vector in which a gene of interest is
inserted.
BACKGROUND ART
[0002] It is said that there have been viruses since so early stage
of birth of life and have been evolving while contributing to
evolution of organisms. There are many viruses having RNA as a gene
(RNA viruses), and 90% of plant viruses are RNA viruses.
[0003] Recently, a method for introducing genetically-engineered
genes into mammalian cells (a transfection method for animal cells)
has been being studied intensively. As the method for introducing
genes, a method utilizing a virus (such as a retrovirus) as a
vector (a virus vector) (called a virus method) in order to
transfect animal cells is widely used due to its relatively high
transfection efficiency. In such a method utilizing a virus vector,
a host cell is infected with the virus vector (a recombinant
virus), thereby introducing a gene of interest into the host cell,
wherein the virus vector is produced by partially recombining the
gene of the virus with the gene of interest to be introduced or a
promoter which can function in the host cell, or the like. Thus,
the host cell uptakes and expresses an exogenous gene (i.e. a
foreign gene).
[0004] In the virus method, it is necessary to use a virus vector
having a high infection efficiency in order to obtain a sufficient
transfection efficiency. The infection efficiency depends on many
factors in a virus and/or a host cell such as: an invasion
efficiency of the virus into the cell; a replication efficiency of
the virus in the cell (some cells cannot be used for some viruses:
this replication efficiency is referred to also as tropism); an
expression efficiency of the viral gene in the host cell (an
incorporation property into a genome, the number of viral gene
copies, and the like); and the like. In order to improve the
infection efficiency, various measures such as selection of cell
types, improvement of a vector, addition of a secondary factor such
as a T-antigen, and the like have been devised. However, the
improvement of the infection efficiency has not yet been achieved,
and is the largest factor which prevents application of the virus
vector as a multipurpose vector. When a highly infectious virus
vector is used, the gene recombinant is more likely to leak to the
outside of an experimental laboratory so as to affect the
environment.
[0005] Thus, there is great need for a technique for improving the
transfection efficiency by treating the host cell as necessary
without enhancing the infectious capacity of the virus vector
itself. Each of cells of plants, insects, invertebrates, and
vertebrates has an immune system for suppressing infection of an
RNA virus as a host defense mechanism of organisms (a bioregulation
mechanism). Thus, when it is possible to artificially depress the
immune function, it may be possible to further improve the
transfection efficiency in the virus method.
[0006] Recently, it is a problem to resolve the mechanism of the
immune system how an innate immunity (basic immunity) system of
plant, insects, invertebrates, and vertebrates detects and prevents
virus invasion. A bioregulation mechanism (such as production of
antibodies, and onset against virus-infected cells by lymph cells
(called cytotoxic T lymph cells (CTLs)) has been developed by an
acquired immune system appeared in the vertebrates. However, in
order that the bioregulation mechanism functions sufficiently, it
is necessary to help of the innate immunity such as an
antigen-presenting cell. With completion of Genome Projects in
various organisms, molecules involved in the innate immunity
systems critical to the host defense (infection control) mechanism
against bacteria and viruses are being identified. It has not been
clarified for a long time which molecule regulates the host defense
mechanism against the viral infection according to the innate
immunity system and how to regulate the host defense mechanism by
the molecule in human. However, only recently, it is gradually
clarified to analyze the host defense mechanism at molecular
level.
[0007] An initial immune response against a virus or a bacteria has
been conventionally considered to be non-specific. However, a
receptor group called microbial receptors was identified, so that
it was clarified that: an immunocompetent cell of the innate
immunity system such as macrophage and a dendritic cell detects
foreign substances entered via a receptor, induces release of
cytokine and activates lymph cells by expression of sub-stimulating
molecules.
[0008] A Toll-like receptor which recognizes various microbial
components and transmits a danger signal into a host is one of the
foregoing microbial receptors, and such Toll-like receptors exist
in plants, insects, mammals, and the like regardless of kinds. The
Toll-like receptor is a homologue of a membrane protein (Drosophila
Toll) involved in both development and immunity of Drosophila.
Eleven members of the Toll-like receptors are found in human, and
twelve members of the Toll-like receptors are found in mice. These
Toll-like receptors constitute a group of a receptor family called
a Toll-like receptor family. The Toll-like receptor has been
noticed as a microbial receptor recently, and it has been clarified
that the Toll-like receptor is involved in recognition of various
microbial components.
[0009] Further, recently, it has been clarified that: a Toll-like
receptor 3 (TLR3) which is one (kind) of the Toll-like receptors
recognizes double-stranded RNA so as to activate a nucleic factor
.kappa. B (hereinafter, referred to as "NF-.kappa.B") (L.
Alexopoulou, A. C. Holt, R. Medzhitov, R. A. Flavell, Nature 413
(2001) 732-738). That is, it was found that the Toll-like receptor
3 is a receptor involved in a double-stranded RNA-mediated
signaling.
[0010] While, in the immune response of animal cells, it is known
that type I interferon (interferon-.alpha. or interferon-.beta.)
which is one (kind) of cytokines plays an important role in
defending against viral infection. Thus, it is considered that it
is possible to drop an immune function against various kinds of
viruses, when it is possible to prevent production of the type I
interferon. Further, it is known that: when fibroblasts are
stimulated with poly-(inosinic acid :cytidylic acid) (hereinafter,
referred to as "poly(I):poly(C)" which is a synthesis analog of a
viral double-stranded RNA (double-stranded RNA produced by a
virus), transcription of the type I interferon is induced.
[0011] However, it has not been clarified how the animal cells
recognize the viral double-stranded RNA and which signaling pathway
produces the type I interferon. It was not known that signaling
pathways involving in the production of the type I interferon exist
in a downstream of the human Toll-like receptor 3.
DISCLOSURE OF INVENTION
[0012] The object of the present invention is to provide (i) an
antibody against a Toll-like receptor having a function for
inhibiting production of type I interferon which is induced by a
viral double-stranded RNA, (ii) an inhibitor capable of suppressing
immune response against a specific virus by inhibiting the
production of the type I interferon induced by the viral
double-stranded RNA, and (iii) a transfection method or a
transfection kit by which it is possible to improve a transfection
efficiency without enhancing an infection efficiency of a virus
vector.
[0013] The inventors of the present invention created a monoclonal
antibody (mAb) against the TLR3, and found that: signaling pathways
involved in the production of the type I interferon exist in a
downstream of the TLR3, and it is possible to block the signaling
pathways by the monoclonal antibody (mAb) against the TLR3. That
is, the inventors found that the monoclonal antibody against the
TLR3 has a function for inhibiting the production of the type I
interferon, thereby completing the present invention.
[0014] That is, the antibody according to the present invention is
an antibody specifically bound to a human Toll-like receptor 3.
Further, in order to achieve the foregoing object, the inhibitor
according to the present invention is an inhibitor for suppressing
a double-stranded RNA-mediated immune response in a cell which
expresses a Toll-like receptor recognizing the double-stranded RNA
and produces type I interferon, and the inhibitor comprises an
antibody, preferably, a monoclonal antibody against the human TLR3,
which binds to the Toll-like receptor and inhibits production of
the type I interferon.
[0015] When the antibody or the inhibitor is used, the antibody
binds to the Toll-like receptor recognizing the double-stranded RNA
so that it is possible to inhibit the binding between the
double-stranded RNA and the Toll-like receptor, thereby preventing
the type I interferon from being produced in a downstream of
signaling pathways involved in the immune response against the
double-stranded RNA. Thus, the antibody suppresses the immune
response in TLR3-expressing cells induced by the double-stranded
RNA.
[0016] Thus, this antibody enables us to amplify RNA viral
infection by suppressing the immune response. The suppression of
the immune response is not observed in uninfected cells even in
case of single-stranded RNA. However, the single-stranded RNA virus
has a double-stranded RNA phase during a process of gene
replication, so that it is possible to amplify (promote)
single-stranded RNA viral infection. Thus, it is feasible to
improve a transfection efficiency with a RNA virus vector such as a
Sendai-virus vector, a retrovirus vector, and the like without
enhancing an infectious efficiency of the virus vector. Note that,
examples of the RNA virus include a negative-stranded RNA virus
such as Sendai-virus, a positive-stranded RNA virus, and a
double-stranded RNA virus. Each of these viruses replicates a large
number of double-stranded RNAs in cells, so that infection of these
viruses would be amplified by the antibody or the inhibitor.
[0017] Further, the antibody or the inhibitor can bind to the
Toll-like receptor recognizing double-stranded RNA thereby
suppressing the immune response in upstream of the signaling
pathway, which leads to selective suppression of the immune
response against the RNA virus. As a result, it is possible to
maintain an immune function against antigens other than the RNA
virus, e.g., a DNA (deoxyribo nucleic acid) virus, bacteria, and
the like.
[0018] Further, in order to achieve the foregoing object, the
transfection method according to the present invention comprises
the step of infecting a cell which expresses a Toll-like receptor
recognizing a double-stranded RNA and produces type I interferon
with a recombined virus vector, in which a gene of interest has
been inserted, under the inhibitory condition for production of the
type I interferon by using the inhibitor of the present invention
(which binds to the Toll-like receptor recognizing the
double-stranded RNA and inhibits the production of the type I
interferon).
[0019] Further, the transfection kit according to the present
invention relates to a kit for transfecting a cell which expresses
a Toll-like receptor recognizing a double-stranded RNA and produces
type I interferon, and wherein the transfection kit comprises: an
inhibitor including an antibody, which binds to the Toll-like
receptor and inhibits production of the type I interferon; and a
recombined RNA virus vector in which a gene of interest has been
inserted.
[0020] According to them, as described above, it is possible to
amplify (promote) infection of single-stranded RNA virus and
double-stranded RNA virus with the inhibitor. Thus, it is possible
to improve a transfection efficiency using RNA virus vector such as
retrovirus vector without enhancing an infection efficiency of the
virus vector.
[0021] Note that, in the present specification, the term "antibody
or antibodies against . . . " means "antibody or antibodies
specifically bound to . . . ".
[0022] For a fuller understanding of the nature and advantages of
the present invention, reference should be made to the ensuing
detailed description taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1(a), FIG. 1(b), and FIG. 1(c) are graphs each of which
shows a result of flow cytometric analysis in which mouse cells is
analyzed by using a monoclonal antibody against the TLR3. FIG. 1(a)
shows a case using normal mouse cells as a control. FIG. 1(b) shows
a case using mouse cells expressing TLR2. FIG. 1(c) shows a case
using mouse cells expressing the TLR3.
[0024] FIG. 2 shows a result obtained by immunoblotting the TLR3
that has been immunoprecipitated with an anti-Flag monoclonal
antibody, an anti-TLR2 monoclonal antibody, or an anti-TLR3
monoclonal antibody.
[0025] FIG. 3(a), FIG. 3(b), and FIG. 3(c) are graphs each of which
shows results of flow cytometric analysis in which two kinds of
human fibroblasts are analyzed by using monoclonal antibodies
against various kinds of TLRs. FIG. 3(a) shows a case using a
monoclonal antibody against human TLR2. FIG. 3. (b) shows a case
using a monoclonal antibody against human TLR3. FIG. 3(c) shows a
case using a monoclonal antibody against human TLR4.
[0026] FIG. 4 is a graph showing a result obtained by measuring
concentrations of interferon-.beta. when human lung fibroblasts
pretreated with the anti-TLR2 monoclonal antibody or the anti-TLR3
monoclonal antibody is stimulated with poly(I):poly(c).
[0027] FIG. 5 is a graph showing a result of analysis on whether or
not NF-.kappa.B is activated by stimulation of poly(I):poly(C) via
various TLRs.
[0028] FIG. 6 is a graph showing a result of analysis on whether or
not interferon-.beta. promoter is activated by stimulation of
poly(I):poly(C) via various TLRs via poly(I):poly(c).
[0029] FIG. 7 is a graph showing a result of analysis on whether or
not NF-.kappa.B is activated by poly(I):poly(c), a single-stranded
RNA, and a double-stranded DNA, via the TLR3.
[0030] FIG. 8 is a graph showing a result of analysis on whether or
not interferon-.beta. promoter is activated by poly(I):poly(c), a
single-stranded RNA, and a double-stranded DNA, via the TLR3.
BEST MODE FOR CARRYING OUT THE INVENTION
[0031] First, the inventors of the present invention confirmed
that: human fibroblasts selectively express TLR3 on their cell
surface, and type I interferon, particularly interferon-.beta., is
produced upon viral infection or treatment with poly(I):poly(c)
which is double-stranded RNA. Next, the inventors generated a
monoclonal antibody against human TLR3 in order to identify the
function of TLR3 and the ligands for TLR3. Then, production of
interferon-.beta. by poly(I):poly(c) was suppressed by the
monoclonal antibody against human TLR3. Thus, it was found that
human TLR3 is a receptor for the double-stranded RNA produced by an
RNA virus.
[0032] By the foregoing study, the inventors obtained such novel
and original finding that "double-stranded RNA-mediated
interferon-.beta. production can be interrupted by binding the
monoclonal antibody against the human TLR3, and the monoclonal
antibody partially inhibits the double-stranded RNA-mediated
cellular responses".
[0033] The present invention was completed on the basis of the
foregoing finding. The inhibitor according to the present invention
is an inhibitor for suppressing a double-stranded RNA-mediated
immune response (particularly, immune response to viral infection)
in a cell which expresses a Toll-like receptor (particularly, human
Toll-like receptor 3) recognizing the double-stranded RNA and
produces type I interferon (particularly, interferon-.beta.), and
wherein the inhibitor comprises an antibody (particularly, a
monoclonal antibody against human Toll-like receptor 3), which
binds to the Toll-like receptor and inhibits production of the type
I interferon.
[0034] First, the Toll-like receptor is described as follows.
[0035] Mammalian Toll-like receptors (hereinafter, referred to as
TLRs as required) recognize a variety of microbial nucleic
acid-derivatives, metabolites, and products to induce activation of
NF-.kappa.B and other signaling pathways. Ten members of the TLR
family have been so far identified in humans, and are called human
TLR1 through human TLR10.
[0036] Each TLR protein comprises an extracellular domain
containing leucine-rich repeats (LRRs) domains, a C-terminal
flanking region (LRRCT), and an intercellular domain containing a
cytoplasmic signaling domain, that is, a so-called
Toll/interleukin-1 receptor homology domain (Toll/IL-1R domain: TIR
domain) (see L. A. O' Neil and C. A. Dinarello, Immunol. Today 21
(2000) 206-209). A typical LRR has a repeat structure consisting of
24 amino acids containing conserved asparagine residual groups and
leucine residual groups, and is included in various proteins of
bacteria, yeasts, plants, and animals, so that LRR domain is
considered to act upon protein-protein interaction.
[0037] The ligands namely pathogen-associated molecular pattern
(PAMP) and their elicited immune responses, though all are not
clearly identified yet, differ among the TLRs.
[0038] As the TLR recognizing the double-stranded RNA, human TLR3
and mouse TLR3 were identified. It has been confirmed that the TLR3
recognizes double-stranded RNA by the study using a TLR3-knock-out
mouse of the aforementioned document (L. Alexopoulou, A. C. Holt,
R. Medzhitov, R. A. Flavell, Nature 413 (2001) 732-738) and the
study (described later) performed by the inventors of the present
invention.
[0039] The human TLR3 is an I-type membrane protein consisting of
904 amino acids. The extracellular LRR domain of the TLR3 comprises
23 LRRs whose motifs are conserved in more preferable manner than
those of other TLRs. The intercellular TIR domain of the TLR3 is
slightly different from that of other TLR in that amino acids in a
conserved region essential for the receptor signaling are
different. The TLR3 gene exists in a long arm q35 of chromosome IV.
Further, in terms of a genome structure, although other TLRs are
encoded by one or two exons, an open reading frame (ORF) of the
TLR3 is encoded by four exons. Further, only the TIR domain of the
TLR3 is split into two exons.
[0040] As cells according to the present invention, any cells can
be used as long as the cells express TLR recognizing a viral
double-stranded RNA and produce the type I interferon. It is
preferable to use cells which express TLR recognizing the viral
double-stranded RNA on their surface and produce the type I
interferon when recognizing the double-stranded RNA.
[0041] According to the study performed by the inventors of the
present invention, the human TLR3 is expressed in various dendritic
cell (DC) subsets. Further, it has been reported that the human
TLR3 is expressed in human intestinal epithelial cells (M. Muzio,
D. Bosisio, N. Polentarutti, G. D'amico, A. Stoppacciro, R.
Mancinelli C. van't Veer, G. Penton-Rol, L. P. Ruco, P. Allavena,
and A. Mantovani: J. Immunol. 164 (2000) 5998-6004, and E. Cario
and D. K. Podolsky: Infect. Immun. 68 (2000) 7010-7017). These
facts suggest that the function of the human TLR3 is closely
connected with responses to microbial nuclear products in the
innate immune system. Thus, the present invention is effective with
respect to cells which express the human TLR3 and produce the type
I interferon, particularly, cells which express the human TLR3 on
their surface and produce interferon-.beta. when recognizing an RNA
virus. Examples of such cells include: human fibroblasts such as
human lung fibroblasts, human foreskin fibroblasts, and the like;
human dendritic cells; human intestinal epithelial cells; and the
like. Particularly, fibroblasts are known to produce
interferon-.beta. upon viral infection or treatment with
double-stranded RNA through different signaling pathways, so that
its effect is expected to be great. Further, examples of the cells
which express the mouse TLR3 so as to produce the interferon-.beta.
include mouse fibroblasts and the like.
[0042] In the human fibroblasts, the interferon-.beta. is produced
merely by adding poly(I):poly(C) to the cells. However, in mouse
embryonic fibroblasts, DEAE-dextran is essential to producing
interferon-.beta. in addition to the stimulation with
poly(I):poly(C) in general. This suggests a possibility that the
human fibroblasts and the mouse fibroblasts are different from each
other in terms of localization of the expressed receptor protein
and a possibility that they are different from each other in terms
of mechanisms of interferon-.beta. production by poly(I):poly(C).
Thus, it can be considered that the inhibitor according to the
present invention can inhibit the interferon-.beta. production more
effectively in the cells which express the human TLR3 on their cell
surface than in the cells which express the mouse TLR3.
[0043] Next, the antibody bound to TLR is described as follows.
[0044] As the antibody according to the present invention, any
antibody can be used as long as the antibody can be bound to TLR,
and a polyclonal antibody against TLR etc. may be used. It is
preferable to use a monoclonal antibody against TLR, particularly,
a monoclonal antibody against the human TLR3 because of the
following reasons: properties of the monoclonal antibody are
homogenous; it is easy to supply the monoclonal antibody; the
monoclonal antibody can be varied into a human antibody in the
future; the monoclonal antibody can be semi-permanently stored as
the state of hybridoma; and the like. By using such a monoclonal
antibody against TLR3, it is possible to effectively suppress the
type I interferon production elicited by the double-stranded
RNA.
[0045] The monoclonal antibody is generated by the following
method. First, TLR protein, fragments, or other derivatives, or
analogs thereof, or cells expressing them are used as an immunogen
so as to immunize mouse splenetic lymph cells, and the immunized
mouse splenetic lymph cells are fused with mouse myeloma cells so
as to produce hybridoma. Next, the monoclonal antibody can be
produced by the hybridoma. Various methods for immunization known
in the art can be used for the present invention: for example, a
hybridoma method (Kohler,G. and Milstein,C., Nature 256,495-497
(1975)), a trioma method, human B-cells hybridoma method (Kozbor,
Immunology Today 4, 72 (1983)), and EBV-hybridoma method
(Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc.,77-96
(1985)).
[0046] Note that, the inhibitor according to the present invention
may include not only the antibody but also other component which
does not interrupt a function of the antibody.
[0047] Next, transfection using the inhibitor according to the
present invention is described as follows.
[0048] The transfection method according to the present invention
relates to an infection method comprising subjecting a recombined
RNA virus vector, in which a gene of interest has been inserted, to
cells (particularly, human fibroblasts) which express TLR
(particularly, human TLR3) recognizing the double-stranded RNA and
produce the type I interferon (particularly, interferon-.beta.),
under the inhibitory condition for production of the type I
interferon by using the inhibitor according to the present
invention.
[0049] Further, the transfection kit according to the present
invention is a kit for transfecting cells which express TLR
recognizing the double-stranded RNA and produce the type I
interferon, and comprises the inhibitor according to the present
invention and a recombined RNA virus vector in which a gene of
interest has been inserted.
[0050] The target cells are not particularly limited as long as the
cells express TLR recognizing the double-stranded RNA and produce
the type I interferon, and a foreign gene can be introduced into
the cells by the RNA virus vector. However, in case where the
inhibitor used comprises the monoclonal antibody against the human
TLR3, it is preferable to use the cells which express the human
TLR3, and it is more preferable to use cells which express the
human TLR3 on their cell surface, for example, human fibroblasts,
human dendritic cells, human intestinal epithelial cells, and the
like. Further, it is considered that a useful transfectant can be
obtained when human stem cells are used as target cells.
[0051] As the recombined RNA virus vector, any virus vector can be
used as long as the virus vector is prepared by inserting a foreign
gene (a gene of interest) into the virus gene of the virus vector
having RNA as a gene. A method for inserting the foreign gene into
the virus gene is not particularly limited, and various known
methods can be used.
[0052] The RNA virus vector may be a virus vector having a
single-stranded RNA as a gene (single-stranded RNA virus vector),
or a virus vector having a double-stranded RNA as a gene
(double-stranded RNA virus vector). Examples of the virus vector
include Sendai virus vector, retrovirus vector, and the like. Among
the foregoing virus vectors, a virus vector, such as Sendai virus
vector, which produces a large amount of double-stranded RNAs, can
be particularly effective in the present invention. Further, the
retrovirus vector is preferable gene transfer means particularly in
a gene therapy desired to express a gene for an extended period of
time since the retrovirus vector is highly infectious, and enables
the foreign gene to be introduced into cells with a high
efficiency, and enables the foreign gene to be integrated into a
chromosome DNA stably.
[0053] It is preferable to design the virus vector in various
manners so as not to exert a bad influence to organisms in which
the gene is introduced. For example, it is preferable that a virus
vector used to introduce a gene replicates itself in cells and the
replication function of the vector is defective so as to prevent
infection (gene transfer) from being limitlessly repeated.
Generally, it is possible to produce the replication-defective
virus vector, in accordance with a method for preparing a virus
vector packaged with viral particles by using virus producing cells
(packaging cells).
[0054] In the transfection method according to the present
invention, the method for inhibiting the type I interferon
production in cells by using the inhibitor of the present invention
is not particularly limited. For example, the inhibitor is added to
cells cultured in a culture medium.
[0055] Further, in terms of inhibition of the type I interferon
production, it is preferable to use the inhibitor of the present
invention before infecting the cells with the recombined RNA virus
vector in which the gene of interest has been inserted. The
inhibitor and the recombined RNA vector may be used simultaneously.
In case of using the inhibitor and the recombined RNA vector
simultaneously, these are added to the cells cultured in the
culture medium by mixture or separately.
[0056] Next, the present invention is further detailed on the basis
of Examples, but the present invention is not limited to them.
[0057] [Cell Culture]
[0058] In the following Examples, normal human lung fibroblasts
MRC-5 which had been obtained from Riken Cell Bank in Institute of
Physical and Chemical Research (Tsukuba-shi Kouyadai 3-1-1,
Ibaraki-ken Japan) was used as the human fibroblasts. Further, the
normal human lung fibroblasts MRC-5 was maintained in MEM (Minimum
Essential Medium: improved Eagle medium) supplemented with 10%
heat-inactivated FCS (Fetal Calf Serum: product of JRH
Bio-sciences) and antibiotics.
[0059] Further, human foreskin FS-4 fibroblasts (see J. Vilcek, M.
Kohase, D. Henriksen-DeStefano, J.Cell.Physiol. 130 (1987) 37-43)
and human embryonic kidney (HEK) 293 cells were maintained in DEM
supplemented with 10% FCS and antibiotics.
[0060] Further, in the following Examples, interleukin 3 (IL-3)
dependent murine cell line Ba/F3 was cultured in RPMI (Roswell Park
Memorial Institute) culture medium containing 10% FCS, 5 ng/ml
murine IL-3, 100 .mu.M 2-mercaptoethanol (2-ME), and
antibiotics.
[0061] [Reagent]
[0062] Poly(I):poly(C), polycytidylic acid (poly C), polyuridylic
acid (poly U), and poly(dI):poly(dC) were purchased from Amersham
Pharmacia Biotech. Polymyxin B, LPS from Escherichia coli serotype
0111 :B4, and mouse IgG1 were from Sigma. The mycoplasma
lipopeptide MALP-2 was prepared in accordance with a method recited
by M. Nishiguchi, M. Matsumoto, T. Takao, M. Hoshino, Y.
Shimonishi, S. Tsuji, N. A. Begum, O. Takuchi, S. Akira, K.
Toyoshima, T. Seya: J. Immunol. 166 (2001) 2610-2616. Note that,
the mycoplasma lipopeptide MALP-2 is lipopeptide consists of
N-terminal fourteen amino acids of M161Ag.
[0063] These reagents, except for LPS, were treated with polymyxin
B (10 .mu.g/ml) for 1 h at 37.degree. C. before stimulation of the
cells.
[0064] [Monoclonal Antibody against Human Toll-Like Receptor 4]
[0065] The monoclonal antibody against human TLR4 (HTA125) was a
gift from Dr. Kensuke Miyake (Institute of Medical Science,
University of Tokyo) (as to the production method thereof, see R.
Shimazu, S. Akashi, H. Ogata, Y. Nagai, K. Fukudome, K. Miyake, M.
Kimoto: J. Exp. Med. 189 (1999) 1777-1782).
[0066] [Complementary DNA Expression Vectors Encoding Human
TLRs]
[0067] Complementary DNA expression vectors (pEFBOS expression
vectors) encoding human TLR1, TLR2, and TLR3 were generated in
accordance with the following method. First, a human monocyte was
cultured in the presence of recombined human GM-CSF
(granulocyte-macrophage colony stimulating factor) so as to obtain
a complementary DNA (cDNA) library. Subsequently, the human TLR1,
TLR2, and TLR3 were generated from the obtained cDNA library in
accordance with PCR (polymerase chain reaction) method, and thus
generated human TLR1, TLR2, and TLR3 were cloned in plasmid pEFBOS,
so as to obtain three types of cDNA expression vectors, namely,
pEFBOS expression vector encoding human TLR1 (human TLR1 expression
vector), pEFBOS expression vector coding human TLR2 (human TLR2
expression vector), and pEFBOS expression vector coding human TLR3
(human TLR3 expression vector). Note that, plasmid pEFBOS was a
gift from Dr. Shigekazu Nagata (Osaka University). The human TLR4
expression vector was a gift from Dr. Kensuke Miyake (Institute of
Medical Science, University of Tokyo) (see R. Shimazu, S. Akashi,
H. Ogata, Y. Nagai, K. Fukudome, K. Miyake, M. Kimoto: J. Exp. Med.
189 (1999) 1777-1782). These plasmids were prepared with a Plasmid
Maxi kit (Qiagen).
[0068] [Stable Transfectants]
[0069] Murine cell Ba/F3 cells were transfected with pFEBOS
expression vectors encoding human TLR2 or TLR3 together with
pSV2neo plasmid (registered in RIKEN GenBank of Institute of
Physical and Chemical Research) by electroporation so as to obtain
a transfectant in which human TLR2 has been introduced or a
transfectant in which human TLR3 has been introduced. The
transfectants were selected with G418 for 10 days so as to obtain
murine cells Ba/F3 stably expressing human TLR2 (stable
transfectant) and murine cells Ba/F3 stably expressing human TLR3
(stable transfectant). Expression of each TLR was confirmed by
intercellular staining for the flag epitope, which had been
attached to the COOH-terminus of each TLR.
EXAMPLE 1
[0070] In order to identify ligands for TLR3 by checking expression
of TLR3 protein level and localization of TLR3 in terms of protein,
the monoclonal antibody against human TLR3 was generated as the
inhibitor according to the present invention.
[0071] That is, first, BALB/c mice were immunized with Ba/F3 cells
stably expressing Flag-tagged (fluorescence-tagged) human TLR3, and
then, spleen cells of the mice were fused with NS-1 myeloma cells
so as to obtain an antibody producing hybridoma. From the antibody
producing hybridoma, a monoclonal antibody against TLR3 was chosen
by cell-surface staining of the same TLR3 transfectants used for
immunization, so as to establish a monoclonal antibody against
TLR3. The monoclonal antibody was named as TLR3.7. In the
monoclonal antibody TLR3.7, immunoglobulin subclass was IgG1 and an
L-chain was type .kappa..
[0072] Further, as a control, a monoclonal antibody against TLR2
was generated in the same manner as in the monoclonal antibody
TLR3.7. The monoclonal antibody was named as TLR2.45. In the
monoclonal antibody TLR2.45, immunoglobulin subclass was IgG1 and
an L-chain was type .kappa..
[0073] Next, it was confirmed that the monoclonal antibody TLR3.7
recognized human TLR3 by using two assessment criteria in the
supernatants of hybridomas.
[0074] As first assessment, the monoclonal antibody TLR3.7 was
screened in accordance with flow cytometry.
[0075] The flow cytometry was performed as follows. The murine
cells Ba/F3 stably expressing Flag-tagged (fluorescence-tagged)
human TLR2 and TLR3 were incubated with the monoclonal antibody (1
.mu.g) against TLR together with human IgG (10 .mu.g) for 30
minutes at 4.degree. C. in FACS (fluorescence activation cell
sorter) buffer. Further, the FACS buffer is DPBS (Dulbecco's
Phosphoric acid Buffer Solution) containing 0.5% BAS (Bovine Serum
Albumin) and 0.1% sodium azide. After the cells were washed twice
with the FACS buffer, FITC (fluorescence isothiocyanate)-labeled
secondary antibody (American Qualex) was added and further
incubated for 30 minutes at 4.degree. C. The cells were then
analyzed on a flow cytometer (FACS Calibur: product of Becton
Dickinson).
[0076] Results of the flow cytometry are shown in FIG. 1(a), FIG.
1(b), and FIG. 1(c). Shaded histograms of FIG. 1(b) and FIG. 1(c)
respectively show results obtained by staining the murine Ba/F3
cells, stably expressing Flag-tagged (fluorescence-tagged) human
TLR2 and TLR3, with anti-TLR3 monoclonal antibody (TLR3.7) and
FITC-labeled secondary antibody (American Qualex). Further, an open
histogram of FIG. 1(c) represents cells labeled with an
isotype-matched control antibody. Further, FIG. 1(a) shows a result
obtained by staining the murine Ba/F3 cells (indicated by "BAF3" in
FIG. 1(a), used as a control, with anti-TLR3 monoclonal antibody
and the FITC-labeled secondary antibody.
[0077] As shown in FIG. 1(c), the peak of fluorescence of the
murine Ba/F3 cells stably expressing the Flag-tagged human TLR3
(indicated by "BAF3/TLR3-Flag" in FIG. 1(c)) was shifted by the
monoclonal antibody TLR3.7. Thus, the monoclonal antibody TLR3.7
was found to react with the murine Ba/F3 cells stably expressing
the Flag-tagged human TLR3.
[0078] On the other hand, as shown in FIG. 1(b), the peak of
fluorescence of the murine Ba/F3 cells stably expressing the
Flag-tagged human TLR2 (indicated by "BAF3/TLR2-Flag" in FIG. 1(b))
was not shifted by the monoclonal antibody TLR3.7. Thus, the
monoclonal antibody TLR3.7 was found not to react with the murine
Ba/F3 cells stably expressing the human Flag-tagged TLR2.
[0079] Thus, in the transfection experiment, it was found that the
monoclonal antibody TLR3.7 shows specificity to TLR3 and does not
react with the murine Ba/F3 cells stably expressing TLR2.
[0080] As second assessment, a monoclonal antibody was chosen by
immunoprecipitation using cell-lysates of the murine Ba/F3 cells
expressing the Flag-tagged human TLR3. That is, immunoprecipitation
with anti-flag antibody or anti-human TLR3 antibody was performed
from the cell-lysates of transfectants as follows so as to judge
expression of the Flag-tagged human TLR3 with anti-flag antibody.
First, the murine Ba/F3 cells stably expressing the Flag-tagged
human TLR2 was lysed using lysis buffer (Promega). Subsequently,
TLR3 was immunoprecipitated with an anti-flag monoclonal antibody
(M2: indicated by "anti-Flag M2" in FIG. 2) or an anti-TLR3
monoclonal antibody (TLR3.7), and subjected to SDS-PAGE (sodium
dodecyl sulfate-polyacrylamide gel electrophoresis) under reducing
conditions, followed by immuno-blotting (Western-blotting) with
anti-flag monoclonal antibody. Further, anti-TLR2 monoclonal
antibody (TLR2.45) was used as a negative-control antibody for
immunoprecipitation. An arrowhead of FIG. 2 indicates TLR3 with
molecular mass of 116 kDa. A result of the immunoblotting is shown
in FIG. 2. As a result, the specificity of the monoclonal antibody
TLR3.7 against TLR3 was confirmed also in analysis of
immunoprecipitation.
[0081] Note that, after a number of trials, the inventors of the
present invention established a monoclonal antibody against human
TLR3 that recognized the 116 kDa TLR3 protein. The difficulty of
screening a monoclonal antibody against human TLR3 was found due to
a low expression level of human TLR3 on the murine Ba/F3 cells as
shown in FIG. 1(c).
[0082] Next, the inventors of the present invention searched for
TLR3-positive human cells/cell lines (expressing TLR3 on their
surface) by flow cytometry using the monoclonal antibody against
various TLRs.
[0083] The flow cytometry was performed as follows. Normal human
lung fibroblasts MRG-5 and normal human foreskin fibroblasts FS-4
were incubated with the monoclonal antibody (1 .mu.g) against TLR
together with human IgG (10 .mu.g) for 30 minutes at 4.degree. C.
in FACS (fluorescence activation cell sorter) buffer. Further, the
FACS buffer was DPBS (Dulbecco's Phosphoric acid Buffer Solution)
containing 0.5% BSA (Bovine Serum Albumin) and 0.1% sodium
azide.
[0084] After the cells were washed twice with the FACS buffer, FITC
(fluorescence isothiocyanate)-labeled secondary antibody (American
Qualex) was added and further incubated for 30 minutes at 4.degree.
C. The cells were then analyzed on a flow cytometer (FACS Calibur:
product of Becton Dickinson). Results thereof are shown in FIG.
3(a), FIG. 3(b), and FIG. 3(c). FIG. 3(a) shows a result of
analysis on expression of TLR2 in the cells MRC-5 and FS-4 in
accordance with flow cytometry using the monoclonal antibody
TLR2.45 against human TLR2. FIG. 3(b) shows a result of analysis on
expression of TLR3 in the cells MRC-5 and FS-4 in accordance with
flow cytometry using the monoclonal antibody TLR3.75 against human
TLR3. FIG. 3(c) shows a result of analysis on expression of TLR4 in
the cells MRC-5 and FS-4 in accordance with flow cytometry using
the monoclonal antibody HTA125 against human TLR4.
[0085] The result of the flow cytometry of FIG. 3(b) shows that
TLR3 exists on cell surface of the human lung fibroblasts MRC-5 and
the human foreskin fibroblasts FS-4. Thus, it was found that TLR3
is expressed on a cell surface of fibroblasts and inside the
fibroblasts (this has not been found until the present study is
carried out).
[0086] While, as shown in FIG. 3(a) and FIG. 3(c), neither TLR2 nor
TLR4 was detected on the cell surface of the human lung fibroblasts
MRC-5 and the human foreskin fibroblasts FS-4. These cell lines
however expressed the mRNA (messenger RNA) of TLR1, 2, 3, 5, and 6
by RT-PCR (reverse transcription PCR), although their proteins were
barely detected by flow cytometry.
[0087] Human fibroblasts expressing TLR3 naturally produce
interferon-.beta. upon viral infection or stimulation with
poly(I):poly(C), a synthetic double-stranded RNA analog. Therefore,
as an experiment for inhibiting the interferon-.beta. production on
the basis of the double-stranded RNA recognition, the inventors of
the present invention examined whether or not the interferon-.beta.
production by stimulation of poly(I):poly(C) in the human
fibroblasts is inhibited by anti-TLR3 monoclonal antibody.
[0088] That is, first, the human lung fibroblasts MRC-5 cells in
24-well plates (7.5.times.104 cells/wells) were pre-treated with 20
.mu.g/ml of anti-TLR2 monoclonal antibody (TLR2.45: referred to as
"Anti-TLR2 mAb" in FIG. 4), or anti-TLR3 monoclonal antibody
(TLR3.7: referred to as "Anti-TLR3 mAb" in FIG. 4) for 1 hour at
37.degree. C., then stimulated with polymyxin B-treated
poly(I):poly(C) (5 or 10 .mu.g/ml) for 24 hours. The concentrations
of interferon-.beta. in the supernatants of the culture media were
measured by ELISA (enzyme-linked immuno-sorbent assay) (TFB Inc.).
A result of the measurement is shown in FIG. 4.
[0089] As apparent from FIG. 4, the pre-treatment of the human lung
fibroblasts MRC-5 cells with anti-TLR3 monoclonal antibody
inhibited of interferon-.beta. production by poly(I):poly(C), while
the human lung fibroblasts treated with anti-TLR2 monoclonal
antibody did not. This indicates that TLR3, expressed on the cell
surface, participates in the recognition of double-stranded RNA and
triggers signaling toward the downstream leading to
interferon-.beta. production. The monoclonal antibody resulted in
loss of function of TLR3, which is consistent with a previous
report (L. Alexopoulou, A. C. Holt, R. Medzhitov, R. A. Flavell:
Nature 413 (2001) 732-738) with different approaches. Further, the
result offered the possibility of direct blocking of
poly(I):poly(C)-mediated interferon-.beta. induction by anti-TLR3
monoclonal antibody.
[0090] The foregoing result indicates that the specific recognition
of the double-stranded RNA by extracellular TLR3 on the basis of
direct or indirect bond between TLR3 and the double-stranded RNA is
essential for induction of type I interferon-.beta.. Further, the
result also indicates that: the monoclonal antibody bound to the
TLR3 inhibits the specific recognition of the double-stranded RNA
by the extracellular TLR2, so that the type I interferon production
is inhibited.
[0091] Thus, it is found that the monoclonal antibody against TLR3
plays a role as an inhibitor for suppressing virus-dependent
cellular response occurring via another signaling pathway involving
double-stranded RNA-TLR3 recognition which can occur in host
cells.
[0092] [Verification on Gain-of-Function by Poly(I):poly(C)]
[0093] Fibroblasts produce interferon-.beta. upon viral infection
or stimulation by poly(I):poly(C) which is synthesized analog of
double-stranded RNA. Therefore, in order to examine the possible
role of the TLRs in the recognition of the double-stranded RNA,
first, it was confirmed that human fibroblasts induced production
of interferon-.beta. upon poly(I):poly(C) stimulation.
Specifically, the human lung fibroblasts MRC-5 (cell numbers:
7.5.times.104) was stimulated with poly(I):poly(C) of various
concentrations, ranging from 0 to 20 .mu.g/ml, for 4 or 24 hours.
Further, the human foreskin fibroblasts FS-4 (cell numbers:
7.5.times.104) was stimulated with poly(I):poly(C) of various
concentrations, ranging from 0 to 20 .mu.g/ml, for 4 hours. Table 1
shows a result of measurement of amounts of interferon-.beta.
produced.
1 TABLE 1 Interferon-.beta. (IU/ml) Poly(I):poly(C) MRC-5 FS-4
(.mu.g/ml) (4 h) (24 h) (4 h) 0 0 0 0 5 9.2 15 16.5 10 13.5 24.3
27.0 20 16.0 42.7 43.0
[0094] As apparent from Table 1, stimulation of the human lung
fibroblasts MRC-5 and the human foreskin fibroblasts FS-4 by
poly(I):poly(C) induced secretion of interferon-.beta..
[0095] In epithelial cells, poly(I):poly(C) often mimics viral
double-stranded RNA to induce activation of NF-.kappa.B following
secretion of interferon-.beta. and cytokines critical to the host
defense against viral infection.
[0096] Then, gain-of-function studies were next performed using
human cell lines expressing various TLRs so as to examine how TLR3
relates to immune response mechanism in which NF-.kappa.B and
interferon-.beta. promoter were activated by the recognition of the
double-stranded RNA. That is, it was verified whether an immune
function was gained or not by poly(I):poly(C) by using human
embryonic kidney (HEK) 293 cells expressing various human TLRs
transfected with vectors and using a reporter gene assay with the
NF-.kappa.B and interferon-.beta..
[0097] The reporter gene assay was carried out as follows. First,
HEK293 cells (1.times.106 cells/wells) were transiently transfected
in 6-well plates using Lipofectamine 2000 reagent (cationic lipids
for gene transfer: product of Gibco, BRL) with human TLR1
expression vector, human TLR2 expression vector, human TLR3
expression vector, human TLR4 expression vector (0.5 or 1 .mu.g),
or empty vector, together with a reporter gene.
[0098] As the reporter gene, a luciferase-linked NF-.kappa.B
reporter gene (Stratagene, 0.5 .mu.g) or p-125 luc reporter plasmid
(0.5 .mu.g) was used. The p-125 luc reporter plasmid was provided
by Dr. Tadatsugu Taniguchi (Graduate School of Medicine and Faculty
of Medicine, University of Tokyo) (see T. Taniguchi, K. Ogasawara,
A. Takaoka, N. Tanaka, Annu. Rev Immunol. 19 (2001) 623-655). The
p-125 luc reporter contains the human interferon-.beta. promoter
region (-125 through +19) inserted into the Picagene luciferase
reporter plasmid (Toyo Ink). Thus, the p-125 luc reporter plasmid
can be used as the interferon-.beta. reporter gene.
[0099] The total amount of transfected DNA was kept constant by
adding empty vector. Further, the plasmid pCMV.beta. (Clontech,
0.0025 .mu.g) was used as an internal control.
[0100] Twenty-four hours after transfection, cells were harvested,
seeded into 24-well plates (2.times.104/ml), and stimulated with
medium alone, lipopolysaccharide (LPS, concentration: 100 ng/ml)
from polymixin B, polymixin B-treated mycroplasma lipopeptide,
MALP-2 (100 nM), or polymixin B-treated poly(I):poly(C) (50
.mu.g/ml) for 6 hours.
[0101] The cells were lysed using lysis buffer (Promega) and both
luciferase and .beta.-galactosidase activities were measured
according to the manufacturer's instructions.
[0102] Table 5 shows a result of measurement in case of using the
NF-.kappa.B reporter gene as a reporter gene, and Table 6 shows a
result of measurement in case of using the interferon-.beta.
reporter gene as a reporter gene. Data of Table 5 and Table 6 show
average values of relative stimulations.
[0103] HEK293 cells transfected with human TLR3 responded to
poly(I):poly(C) so as to activate NF-.kappa.B as shown in FIG. 5.
While, as shown in FIG. 5, HEK293 cells transfected with other
human TLRs (human TLR1, TLR2, and TLR4) did not. However, as shown
in FIG. 5, human TLR2-expressing cells responded to mycroplasma
lipopeptide, MALP-2, a control TLR2 ligand.
[0104] Further, as shown in FIG. 6, human TLR3-expressing cells
markedly responded to poly(I):poly(C), and activated
interferon-.beta. promoter. Hence, poly(I):poly(C) induced both
NF-.kappa.B promoter activation and interferon-.beta. promoter
activation through TLR3. In contrast, as shown in FIG. 6, cells
which express human TLR2 or human TLR4 did not respond to LPS,
MALP-2, or poly(I):poly(C), and did not activate interferon-.beta.
promoter.
[0105] Specificity of poly(I):poly(C) to TLR3 was next examined.
First, HEK293 cells were transfected with a reporter gene in the
same manner as the foregoing transfection by using human TLR3
expression vector (0.5 or .mu.g) or empty vector. Twenty-four hours
after transfection, cells were harvested, seeded into 24-well
plates (2.times.105/ml), and stimulated with medium alone,
poly(I):poly(C) (concentration: 50 ng/ml), poly(U), poly(C),
poly(dI):poly(dC) (50 .mu.g/ml) for 6 hours.
[0106] The cells were lysed using lysis buffer (Promega) and both
luciferase and .beta.-galactosidase activities were measured
according to the manufacturer's instructions, and evaluated the
degree of the activation of luciferase and .beta.-galactosidase
promoters.
[0107] FIG. 7 shows a result of measurement in case of using the
NF-.kappa.B reporter gene as a reporter gene, and FIG. 8 shows a
result of measurement in case of using the interferon-.beta.
reporter gene as a reporter gene. Data of FIG. 7 and FIG. 8 are
expressed as fold stimulation based on mean plus standard deviation
for a representative stimulation experiment from a minimum of three
independent experiments.
[0108] As shown in FIG. 7 and FIG. 8, TLR3-mediated NF-.kappa.B or
interferon-.beta. promoter activity was induced by poly(I):poly(C),
and TLR3-mediated NF-.kappa.B or interferon-.beta. promoter
activity was not induced by neither single-stranded RNA (poly(U) or
poly(C)) or double-stranded DNA (poly (dI);poly(dC)).
[0109] As described above, TLR3 recognized the double-stranded RNA
so as to mediate the NF-.kappa.B or interferon-.beta. promoter
activity by poly(I):poly(C) stimulation, but neither
single-stranded RNA nor double-stranded DNA induced the
TLR3-mediated signaling.
[0110] Thus, it is concluded that TLR3 recognizes very specific
structural features in double-stranded RNA, for example, the
presence or absence of a hydroxyl group bound to the 2' carbon in
.beta.-D-ribose, so that TLR3 selectively recognizes
double-stranded RNA, which is unique to viruses, and transmits the
signaling from viruses to inside cells.
[0111] In this way, the inventors of the present invention found
that: TLR3 recognizes double-stranded RNA so as to activate
NF-.kappa.B and interferon-.beta. promoter, which promotes
production of interferon-.beta.. Thus, interferon-.beta. production
could be promoted and viral infection could be suppress by
promoting the signaling mediated by TLR3. Further, by searching
medicaments for promoting the signaling, it would be possible to
produce a new inhibitor for viral-infection. Since many of
refractory diseases are mediated by viruses, it would be possible
to cure such refractory diseases by suppressing viral
infection.
[0112] Still another object, feature, superior point of the present
invention are described as follows. Still another object of the
present invention is to apply the foregoing technique to anticancer
immune therapy (innate immune therapy of cancer) and viral
infectious diseases targeting TLR3 (Toll-like receptor 3).
[0113] As described above, the inventors of the present invention
generated a monoclonal antibody against human Toll-like receptor 3
and found that the antibody specifically inhibits production of
double-stranded RNA-mediated interferon-.beta. (IFN-.beta.). Thus,
it would be possible to provide a new method for controlling
production of virus-dependent IFN-.beta.. It is expected that
symptoms of various infectious diseases, cancer (hepatic cancer,
cervical cancer, and the like), kidney cancer, and the like induced
by viral infection would be improved by a threshold value of
IFN-.beta.. In case of cancer, it is known that reduction and/or
regression of the cancer could be occurred according to the
changing IFN-.beta. sensitivity. Therefore it would be possible to
control proliferation of cancerous cells with the antibody of the
present invention.
[0114] Conventionally, bioregulation mechanisms against bacterial
infection have been discussed in terms of "acquired immunity
system" mediated by specific T cells and B cells. However, "innate
immunity" in host defense (infection control) has come to the front
since Toll-like receptor was found several years ago.
Double-stranded RNA specifically produced by viral infection
activates an immune system (particularly, dendritic cells) via
TLR3, so that it has been technically suggested that it is possible
to use double-stranded RNA to control immunity upon infection. Main
cellular responses against double-stranded RNA are IFN-.beta.
production and dendritic cells maturation. However, clinical
applications of their effects have not been considered yet.
[0115] It would be possible to develop immune therapy for cancer
and viral infectious disease by controlling the signaling involving
double-stranded RNA-TLR3. Several immune therapies for cancer and
viral infectious disease are known: (1) "anticancer immune therapy"
developed by using lymph cells (immune therapy with mediation of an
acquired immunity system), (2) peptide therapy, and the like. In
the technique (1) "anticancer immune therapy", LAK, TIL, adoptive
immunity, and the like are used.
[0116] However, the technique (1) "anticancer immune therapy" does
not necessarily bring about high curing effects. Further, the
technique (2) "peptide therapy" does not give high selectivity.
Further, the technique (2) "peptide therapy" is effective for
melanoma, but is less effective for solid tumor. Further, general
treatment effective for viral infection has not been developed
yet.
[0117] However, when an immune therapy controlling a signaling
system of double-stranded RNA-TLR3 is developed by using the
present invention, it would be possible to obtain higher
selectivity than that of the peptide therapy, and it would be
possible to reduce adverse effects. Further, when the immune
therapy controlling the signaling involving double-stranded
RNA-TLR3 is developed, it would be possible to obtain effects on
solid tumors which are partially caused by viruses, such as
post-hepatitis C hepatic cancer, cervical cancer, lymphoma, renal
cell carcinona of kidney, and the like.
[0118] Further, it was found that ligands for TLR3 (i.e.
double-stranded RNA) are produced in virus infection, so that it
would be possible to establish a new anti-virus therapy controlling
the signaling involving double-stranded RNA-TLR3 by further
studying the foregoing mechanism.
[0119] When the immune therapy controlling the signaling involving
double-stranded RNA-TLR3 is developed, it would be possible to
apply the therapy to alleviation of various symptoms induced by
double-stranded RNA production due to viral proliferation.
Particularly, it would be possible to suppress onset or degeneracy
of symptoms that is caused by certain kinds of cancer (kidney
cancer, post-hepatitic hepatic cancer) and viral infectious disease
(hepatitis B or C virus, measles virus, rotavirus, influenza virus,
herpes virus, and the like).
[0120] Further, as described above, it is possible to
quantitatively analyze expression of TLR3 in cells on the basis of
flow cytometry using the antibody of the present invention. Thus,
according to the flow cytometry using the antibody of the present
invention, it is possible to screen cells expressing TLR3 and to
detect cells aberrantly expressing TLR3.
[0121] Note that, in analyzing the expression of TLR3 using the
antibody of the present invention, it is preferable to adopt the
aforementioned method, that is, the method in which: a secondary
antibody against anti-TLR3 antibody is labeled with fluorescence so
as to measure its fluorescence intensity on the basis of flow
cytometry, thereby measuring an antigen antibody reaction between
TLR3 and the antibody in cells. However, it is possible to adopt
other method. Examples of other method include: a method in which
anti-TLR3 antibody is directly labeled with fluorescent so as to
measure its fluorescent intensity by flow cytometry; an ELISA
method using an enzyme label (enzyme-linked immune adsorption
assay); a method in which anti-TLR3 antibody is labeled with
radioactive isotopes so as to measure its radioactive intensity;
and the like.
[0122] The invention being thus described, it will be obvious that
the same way may be varied in many ways. Such variations are not to
be regarded as a departure from the spirit and scope of the
invention, and all such modifications as would be obvious to one
skilled in the art are intended to be included within the scope of
the following claims.
[0123] Industrial Applicability
[0124] According to the antibody of the present invention, it is
possible to suppress an immune response induced by double-stranded
RNA. Thus, the antibody facilitates double-stranded RNA viral
infection, and facilitates single-stranded RNA viral infection
which has a double-stranded RNA phase during a process of gene
replication. Hence, it is possible to improve an transfection
efficiency using RNA virus vector such as retrovirus vector without
enhancing an infectious capacity of the virus vector, and it is
possible to prevent occurrence of excessive immune response.
Further, according to the foregoing arrangement, it is possible to
suppress immune response selectively, so that it is possible to
maintain an immune function of an antigen other than RNA virus, for
example, DNA (deoxyribo nucleic acid) viruses or bacteria and the
like.
[0125] Further, the antibody of the present invention can be used
as an auxiliary agent which improves a transfection efficiency in a
transfection method or a transfection kit using RNA virus
vector.
* * * * *