Note: Descriptions are shown in the official language in which they were submitted.
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TOLL-LIKE RECEPTOR 9 EFFECTOR AGENTS AND USES THEREOF
Field of the Invention
This invention relates to cell surface toll-like receptor 9
(TLR9) effector agents such as TLR9 receptor binding agents and TLR9
ligand binding agents and their use in modulating an immune
response.
Background of the Iaveatioa
The immune system is armed with the means to discriminate
between self and non-self antigens. To this end, the immune system
has evolved a series of pattern-recognition receptors to identify
invading pathogens and initiate the host immune response. The toll-
like family of receptors function in this fashion to activate both
the innate and the adaptive arms of the immune response (Janeway and
Medzhitov, Ann. Rev. Immunol. 20: 197-216, (2002)). Mammalian toll-
like receptors (TLRs) were cloned based on sequence homology to the
Drosophila toll gene which plays a critical role in immunity to
infection with Aspergillus fumigatus (Lemaitre et al., Cell 86: 973-
983, (1996); Medzhitov et al., Nature 388: 394-397, (1997); Rock et
al., Proc. Natl. Acad Sci. (USA) 95: 588-593, (1998)). It has been
demonstrated that using a constitutively active form of human Toll
resulted in NF-K(3 activation and,upregulation of B7-1 as well as IL-
1, IL-8, and IL-6 cytokine message, suggesting a role for TLRs in
innate and adaptive immunity (Medzhitov et al., Nature 388: 394-397,
(1997)). At present, eleven TLR family members have been identified
in humans and nine in mice.
TLR9 has been identified as the receptor for the unmethylated
CpG dinucleotides found in bacterial but not human DNA (Hemmi et
al., Nature 408: 740-745, (2000); Krieg et al., Nature 374: 546-549,
(1995)). Expression profiling revealed TLR9 mRNA or protein in B
cells and plasmacytoid dendritic cells (Bauer et al., Proc. Natl.
Acad. Sci. (USA) 98: 9237-9242, (2001); Krug et al., Eur. J.
Immunol. 31: 3026-3037, (2001). Using synthetic CpG
oligonucleotides (ODN) for TLR9 stimulation/ligation, it was found
that CpG-0DN could act in an adjuvant fashion (Sun et al., J. Exp.
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Med. 187: 1145-1150 (1998); Lipford et al., Eur. J. Immunol. 27:
2340-2344, (1997); Chu et al., J. Exp. Med. 186: 1623-1631 (1997))
to stimulate cytokine production (Klinman et al., Proc. Natl. Acad.
Sci. (USA) 93: 2879-2883, (1996)) and mediate dendritic cell
maturation (Hartmann et al., Proc. Natl. Acad. Sci. (USA) 96: 9305-
9310, 1999; Bauer et al., J. Immunol. 166: 5000-5007, (2001).
Furthermore, it was found that cells from TLR9 deficient mice did
not proliferate or secrete cytokines in response to CpG stimulation,
and overall, the mice were resistant to lethal CpG-induced shock
(Hemmi et al . , supra) .
Conflicting data has been reported as to whether TLR9 can be
expressed at the cell surface, despite it sharing significant
homology with other members of the TLR family including putative
intracellular, extracellular, and transmembrane domain sequences (Du
et al., Eur. Cytokine Netw. 11:362-371, (2002); Hemmi et al.,
supra).
Prior to the discovery of TLR9, studies using fluorescently
labeled CpG-ODN to stimulate macrophage cell lines revealed that
ODNs were rapidly taken up into the endosomal compartment with
minimal localization at the plasma membrane (Hacker et al., EMBO J.
.17:6230-6240, (1998)). In primary cell assays, immobilized CpG-ODN
that could not be internalized were used to investigate the
potential of a cell-surface receptor capable of mediating CpG
triggered stimulation. While one group found that murine B cells
failed to become activated when cultured with these CpG-ODN (Krieg
et al., supra), another group found that immobilized CpG-ODN induced
human B cell proliferation and Ig secretion~comparable to free CpGs
(Lung et al., J. Clin. Invest. 9:1119-1129, (1996); Liang et al.,
J. Immunol. .265:1438-1445, (2000)).
Following the discovery of TLR9 and the identification of the
receptor-ligand relationship of TLR9 for CpG dinucleotides, (Hemmi
et al., supra; Du et al., supra) the cellular localization of TLR9
still remained unclear. Chuang et al. in J. Leukoc. Biol. 71:538-
544, (2002) using murine TLR9, generated data suggesting cell
surface expression on transfected human HEK293 cells. These authors
transiently transfected the cell line with TLR9-Flag, and found
expression of Flag at the cell surface using an anti-Flag antibody,
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thereby suggesting that TLR9 may also be at the cell surface. The
use of this artificial system resulting in TLR9 overexpression in
transformed cell lines does not evaluate the physiologic
localization of TLR9 in primary immune cells.
Similar results were found by Takeshita et al. (J. Immunol.
167:3555-3558, (2002)) using human TLR9 transiently transfected HEK
293 cells. These studies were criticized as the endogenous leader
sequence of TLR9 (comprised of the first 26 N-terminal amino acids)
had been replaced by the authors with a heterogeneous leader
sequence derived from the IgK gene (Ahmad-Nejad et al., Eur. J.
Immunol. 32:1958-1968, (2002)).
To further address the issue of TLR9 localization, Ahmad-Nejad
et al., supra, generated a murine anti-TLR9 mAb directed toward the
extracellular domain of human TLR9. Using this mAb to stain a
permeablized cell line, the authors reported intracellular TLR9
expression but not cell-surface expression following IFN-y
treatment.
TLR9 stimulation has been recognized as having an important
role in activating both innate and adaptive immune responses. These
responses play a role in autoimmune diseases, inflammatory diseases
and sepsis as well as adjuvant and anti-tumor effects. Accordingly,
a need exists for antagonistic or agonistic agents that can modulate
TLR9 biological activity.
Brief Description of i~he Drawings
Fig. 1 shows representative flow cytometry data for cell
surface TLR9+ cells in tonsillar cell populations.
Fig. 2 shows representative flow cytometry data for cell
surface TLR9+ cells in PBMC populations.
Fig. 3 shows a blockade of TLR9 staining using a TLR9 peptide.
Fig. 4 shows a control peptide does not prevent TLR9 staining.
Fig. 5 shows CpG-dependent binding of a TLR9 extracellular
domain to CpG-ODN.
Summary of the Invention
One aspect of the invention is a method of modifying antigen
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presenting cell function in a patient in need thereof comprising
administering to the patient a cell surface TLR9 binding agent that
specifically binds to human TLR9 in an amount effective to modify
antigen presenting cell function in the patient.
Another aspect of the invention is a method of identifying
TLR9 binding agents comprising the steps of contacting MHCII+CD19~ or
MHCII+CD19- primary human cells expressing TLR9 on their surface with
a putative binding agent; measuring the binding of the putative
binding agent to the cell surface and the effect on TLR9 biological
activity; and identifying TLR9 binding agents affecting TLR9
biological activity.
Another aspect of the invention is a method of modifying
antigen presenting cell function in a patient in need thereof
comprising administering to the patient a TLR9 ligand binding agent
in an amount effective to modify antigen presenting cell function in
the patient.
Another aspect of the invention is a TLR9 ligand binding agent
comprising residues 1 to 260 of the extracellular domain of human
TLR9, protein, a fragment thereof or its mature form.
Other aspects of the invention are a TLR9 ligand binding agent
comprising residues 1 to 260 of human TLR9 protein extracellular
domain, a fragment thereof or its mature form fused to a fusion
partner and its use in identifying TLR9 binding agents.
Detailed Description of the Invention
All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as though fully set forth.
The terms "agonist" and "agonistic" as used herein refer to or
describe a molecule that is capable of, directly or indirectly,
substantially inducing, promoting or enhancing TLR9 biological
activity or TLR9 receptor activation.
The terms "antagonist" or "antagonistic" as used herein refer
to or describe a molecule that is capable of, directly or
indirectly, substantially counteracting, reducing or inhibiting TLR
biolocial activity or TLR9 receptor activation.
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The term "antibodies" as used herein is meant in a broad sense
and includes immunoglobulin or antibody molecules including
polyclonal antibodies, monoclonal antibodies including murine,
human, humanized and chimeric monoclonal antibodies and antibody
fragments.
In general, antibodies are proteins or polypeptides that
exhibit binding specificity to a specific antigen. Intact
antibodies are heterotetrameric glycoproteins, composed of two
identical light chains and two identical heavy chains. Typically,
each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain disulfide
bridges. Each heavy chain has at one end a variable domain (VH)
followed by a number of constant domains. Each light chain has a
variable domain at one end (VL) and a constant domain at its other '
end; the constant domain of the light chain is aligned with the
first constant domain of the heavy chain and the light chain
variable domain is aligned with the variable domain of the heavy
chain. Antibody light chains of any vertebrate species can be
assigned to one of two clearly distinct types, namely kappa (x) and
lambda (A), based on the amino acid sequences of their constant
domains.
Immunoglobulins can be assigned to five major classes, namely
IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant
domain amino acid sequence. IgA and IgG are further sub-classified
as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.
The term "antibody fragments" means a portion of an intact
antibody, generally the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab')2 and Fv fragments, diabodies, single chain antibody molecules
and multispecific antibodies formed from at least two intact
antibodies.
"CDRs" are defined as the complementarity determining region
amino acid sequences of an antibody which are the hypervariable
regions of immunoglobulin heavy and light chains. See, e.g., Kabat
et al., Sequences of Proteins of Immunological Interest, 4th ed.,
U.S. Department of Health and Human Services, National Institutes of
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Health (1987). There are three heavy chain and three light chain
CDRs or CDR regions in the variable portion of an immunoglobulin.
Thus, "CDRs" as used herein refers to all three heavy chain CDRs, or
all three light chain CDRs or both all heavy and all light chain
CDRs, if appropriate.
CDRs provide the majority of contact residues for the binding
of the antibody to the antigen or epitope. CDRs of interest in this
invention are derived from donor antibody variable heavy and light
chain sequences, and include analogs of the naturally occurring
CDRs, which analogs also share or retain the same antigen binding
specificity andlor neutralizing ability as the donor antibody from
which they were derived.
The term "mimetibody" as used herein means a protein having
the generic formula (I):
(V1(n)-Pep(n)-Flex(n)-V2(n)-pHinge(n)-CH2(n)-CH3(n))(m)
(I)
where V1 is at least one portion of an N-terminus of an
immunoglobulin variable region, Pep is at least one bioactive
peptide that binds to cell surface TLR9, Flex is polypeptide that
provides structural flexablity by allowing the mimetibody to have
alternative orientations and binding properties, V2 is at least one
portion of a C-terminus of an immunoglobulin variable region, pHinge
is at least a portion of an immunoglobulin variable hinge region,
CH2 is at least a portion of an immunoglobulin CH2 constant region
and CH3 is at least a portion of an immunoglobulin CH3 constant
region, where n and m can be an integer between 1 and 10. A
mimetibody mimics properties and functions of different types of
immunoglobulin molecules such as IgGl, IgG2, IgG3, IgG4, IgA, IgM,
IgD and IgE. A mimetibody of the present invention affects TLR9
biological activity through binding to cell surface TLR9.
The term "monoclonal antibody" (mAb) as used herein means an
antibody (or antibody fragment) obtained from a population of
substantially homogeneous antibodies. Monoclonal antibodies are
highly specific, typically being directed against a single antigenic
determinant. The modifier "monoclonal" indicates the substantially
homogeneous character of the antibody and does not require
production of the antibody by any particular method. For example,
murine mAbs can be made by the hybridoma method of Kohler et al.,
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Nature 256: 495 (1975). Chimeric mAbs containing a light chain and
heavy chain variable region derived from a donor antibody (typically
murine) in association with light and heavy chain constant regions
derived from an acceptor antibody (typically another mamammlian
species such as human) can be prepared by the method disclosed in
U.S. Pat. No. 4,816,567. Humanized mAbs having CDRs derived from a
non-human donor immunoglobulin (typically murine) and the remaining
immunoglobulin-derived parts of the molecule being derived from one
or more human immunoglobulins, optionally having altered framework
support residues to preserve binding affinity, can be obtained by
the techniques disclosed in Queen et al., Proc. Nat1 Acad Sci (USA),
~6: 10029-10032, (1989) and Hodgson et al., Bio/Technology, 9: 421,
(1991) .
Fully human mAbs lacking any non-human sequences can be
prepared from human immunoglobulin transgenic mice by techniques
referenced in, e.g., Lonberg et al., Nature 368: 856-859, (1994);
Fishwild et al., Nature Biotechnology 14: 845-851, (1996)' and
Mendez et al., Nature Genetics .15: 146-156, (1997). Human mAbs can
also be prepared and optimized from phage display libraries by
techniques referenced in, e.g., Knappik et al., J. Mol. Biol. 296:
57-86, (2000) and Krebs et al., J. Immunol. Meth. 254: 67-84,
(2001) .
The term "TLR9 biological activity" or "TLR9 receptor
activation" as used herein refers to any activation of the innate or
adaptive arms of the immune response or any activities occurring as
a result of ligand binding to cell surface TLR9.
The present invention relates to agents that can bind
specifically to TLR9 on mammalian cell surfaces. The cell surface
TLR9 binding agents are useful as agonists or antagonists to modify
the function of TLR9 located on the cell surface. These binding
agents are useful as research reagents, diagnostic reagents and
potential therapeutic agents. In one embodiment of the invention,
the agents bind specifically to TLR9 on human cell surfaces.
In particular, the invention relates to the use of the
agonists or antagonists to modify the TLR9 biological activity of
distinct subsets of MHC ClassIl+CD19+ (MHCII+CD19+) human cells such
as MHCII+CD19+CD1231°W and MHC ClassII+CD19-human cells such as
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MHCII1°WCD19-CD123bright and MHCII1~"'CD19-CD1231~w~ These subsets
can be
antigen presenting cells such as B cells or dendritic cells. Cell
surface TLR9 agonists and antagonists include, but are not limited
to, any antibody, fragment or mimetibody; any soluble receptor,
fragment or mimetic; or any small organic molecule; or any
combination of the foregoing. TLR9-specific mAbs are included as
one type of such an agonist or antagonist.
Anti-TLR9 mAbs can be generated in normal mice using standard
hybridoma technology techniques (I~ohler et al., supra) well known to
those skilled in the art. Briefly, separate groups of mice are
immunized with human TLR9 (SEQ ID N0: 1) or a fragment such as the
extracellular domain (residues 1 through 819 of SEQ ID N0: 1)
emulsified in complete Freund's adjuvant (CFA). Each mouse receives
25 ug of the immunogen in CFA followed by an equal amount of the
immunogen in incomplete Freund's adjuvant two weeks later.
Alternatively, mice can receive two injections (two weeks apart) of
plasmid DNA encoding human TLR9 or a fragment thereof, such as the
extracellular domain (10 ug/mouse), followed by a booster injection
with human TLR9 protein or a fragment thereof, such as the
extracellular domain.
Three days prior to B cell fusion, protein or DNA-immunized
mice are given an intravenous injection of the immunogen in
phosphate-buffered saline (PBS) at 15 ug immunogen per mouse.
Spleens from immunized mice are harvested and B cell fusion carried
out using the methods of Kohler et al., (supra). Fused cells are
selected using medium containing hypoxanthine-aminopterin-thymidine
(HAT) and wells are screened for the presence of anti-TLR9
antibodies by enzyme-linked immunosorbent assay (ELISA). Positive
wells are expanded and cloned by limiting dilution.
Another aspect of the invention is a method of identifying
TLR9 binding agents comprising the steps of contacting MHCII+CD19+ or
MHCII+CD19- primary human cells expressing TLR9 on their surface with
a putative binding agent; measuring the binding of the putative
binding agent to the cell surface and the effect on TLR9 biological
activity; and identifying TLR9 binding agents affecting TLR9
biological activity. The TLR9 binding agents that can be identified
by this method of the invention include small organic molecules,
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oligonucleotides, nucleic acids, peptides, antibodies and other
proteins.
The present invention also relates to TLR9 ligand binding
agents and their use. TLR9 ligand binding agents function as
antagonists by binding TLR9 ligands, thereby preventing the ligands
from binding to TLR9 located on the cell surface. These binding
agents are useful as research reagents, diagnostic reagents and
potential therapeutic agents. In one embodiment of the invention,
the agents bind specifically to human TLR9 ligands.
TLR9 ligand binding agents of the invention include residues 1
to 260 of the extracellular domain of human TLR9 protein, a fragment
thereof or its mature form lacking a leader sequence. Also included
are fusion proteins where residues 1 to 260 of the extracellular
domain of human TLR9 protein, a fragment thereof or the mature form
are fused to a fusion partner such as the Fc portion of an
immunoglobulin molecule or a mimetibody. An exemplary TLR9 ligand
binding agent of the invention is a fusion construct including
residues 1 to 260 of the extracellular domain of human TLR9 protein
fused to an IgG1 Fc region having the amino acid sequence shown in
SEQ ID NO: 2.
As mentioned above, one embodiment of the TLR9 ligand binding
agents of the invention is the mature form of the 260 residue
extracellular domain of human TLR9 protein as well as a fusion
construct containing the mature form. The mature secreted form of
this extracellular domain fragment will lack the signal sequence.
The signal sequence cleavage site for this extracellular domain
fragment is predicted to be at residue 25 of SEQ ID NO: 2. However,
it will be recognized by those skilled in the art that the actual
signal sequence cleavage site can vary from the predicted cleavage
site. Thus, another exemplary TLR9 ligand binding agent of the
invention is a fusion construct including the mature form of the 260.
residue extracellular domain of human TLR9 protein fused to an IgGI
Fc region. One example is the fusion protein having the amino acid
sequence shown in SEQ ID N0: 11.
The exemplary ligand binding agents of the invention can be
expressed using standard recombinant protein expression platforms,
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e.g., mammalian cell expression systems, and utilize either stable
cell lines or transient transfection production procedures.
The TLR9 ligand binding agents of the invention can also be
used in a method of identifying TLR9 binding agents by contacting a
TLR ligand binding agent with a putative TLR9 binding agent;
measuring the binding of the putative TLR9 binding agent to the TLR9
ligand binding agent and the effect on TLR9 biological activity; and
identifying TLR9 binding agents affecting TLR9 biological activity.
The TLR9 binding agents that can be identified by this method of
the invention include small organic molecules, oligonucleotides,
nucleic acids, peptides, antibodies and other proteins.
TLR9, like other TLRs, may consist of TLR heterodimers or as
yet unidentified adapter molecules. Therefore, primary cell
populations expressing TLR9 in its natural form, unlike TLR9
transfected cell lines, represent an ideal tool for the selection of
agonistic or antagonistic TLR9-specific mAbs. Without the use of
primary cell populations expressing TLR9 for mAb screening, it is
possible that mAbs directed toward significant epitopes of TLR9
would be missed. For screening purposes, primary cells would be
incubated with hybridoma supernatants or purified hybridoma-
generated mAbs with or without bacterial DNA. Cytokine production,
or lack thereof would be used to identify both agonistic and
antagonistic TLR9-specific mAbs.
A cell surface TLR9 agonist is useful for treating a number of
mammalian disease states including, but not limited to, pathologic
conditions related to bacterial, viral, parasitic, or fungal
infections particularly Herpes simplex virus (HSV), Human papilloma
virus (HPV) and Chlamydia; treatment and/or augmentation of other
therapies used to treat cancer; and in treatment of pathologies
associated with allergic responses such as asthma.
While not wishing to be bound to any particular theory, it is
thought that TLR9 agonists will be useful as an adjuvant in all
types of infections (bacterial, viral, parasitic, and fungal).
Further, given the effectiveness of the TLR9 agonist CpG to treat
genital infections such as herpes simplex virus (Pyles et al., J.
Virol. 76: 11387-11396, (2002)), a TLR9 agonist is likely to be
effective in treating a variety of genital infections including HSV,
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HPV and Chlamydia. TLR9 agonists could also be used topically to
prevent or treat symptoms associated with genital tract infections.
Also, again not wishing to be bound to any particular theory,
TLR9 agonists will be useful to treat cancer because of their potent
effects on innate immunity. TLR9 agonists will be useful either as
monotherapy or in combination with cancer cytotoxics or anti-cancer
mAbs since bacterial DNA has been shown to have potent anti-tumor
effects (Tokunaga et al., J. Natl. Cancer Inst. 72: 955-962, (1984))
and a synthetic single-stranded DNA was also found to have anti-
tumor properties (Tokunaga et al., Jpn. J. Cancer Res. 79:682-686,
(1988)).
Further, and again not wishing to be bound by any particular
theory, TLR9 agonists will be useful in treating diseases that have
a Th2-mediated immunopathology, e.g., asthma, allergy, pulmonary
fibrosis and ulcerative colitis. CpG-ODNs and immunostimulatory
sequences (ISS) have been shown to prevent the development of
allergic airway responses in animal models (Kline et al., J.
Immunol. 160: 2555-2559, (1998)) by inducing a potent Th1 response.
Therefore, TLR9 agonists are also expected to be useful in this
regard. For specific desensitization to allergens, the allergen
could be conjugated to the TLR9 mAb as described for ragweed-
conjugated ISS (Santeliz et al., J. Allergy Clin. Immunol. 209: 455-
462, (2002)). In contrast to synthetic ODN or ISS, TLR9-specific
mAbs would have a longer plasma half-life and would selectively
target the cell surface TLR9 molecules.
A cell surface TLR9 antagonist is useful for treating a number
of mammalian disease states including, but not limited to,
autoimmune disorders such as systemic lupus erythematosus, Sjogren's
syndrome, Scleroderma and CREST syndrome, multiple sclerosis, Th1-
cell mediated inflammatory disease, sarcoidosis, cystic fibrosis and
rheumatoid arthritis, inflammatory conditions such as chronic
obstructive pulmonary disease (COPD), inflammatory bowel disease and
sepsis.
While not wishing to be bound to any particular theory, it is
thought that TLR9 antagonists will be useful for treating the
autoimmune diseases mentioned above due to the likely role of TLR9
stimulation in the aberrant activation of autoreactive B cells
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(Leadbetter et al., Nature 416: 603-607, (2002)) coupled with the
fact that DNA methylation is known to be decreased in cells from
autoimmune humans and mice (Richardson et al., Arthritis Rheum. 33:
1665-1673, (1990)). In addition, the association between infectious
disease illnesses and flare-ups of multiple sclerosis can be
correlated with release of bacterial DNA that binds TLR9 (Ichikawa
et al., J. Immunol. 169: 2781-2787, (2002)), suggesting a potential
therapeutic benefit of TLR9 antagonists in multiple sclerosis.
Also, and again not wishing to be bound to any particular
theory, it it thought that TLR9 antagonists will be useful for
treatment of inflammatory diseases due to the fact that blockade of
the interaction between bacterial DNA and TLR9 can alleviate the
Th1-driven inflammatory response during bacterial infections. For
example, the appearance of certain bacterial strains in the sputum
of patients with COPD is associated with disease exacerbation (Sethi
et al., New Engl. J. Med. 347: 465-471, (2002)), suggesting that a
TLR9 antagonist may have therapeutic benefit in inflammatory
diseases such as COPD, emphysema and sarcoidosis. The potential
role of bacterial species as initiators of the inflammatory process
in inflammatory bowel disease support the use of TLR9-specific
antagonists to block prolonged cell activation.
Further, and again not wishing to be bound to any particular
theory, it it thought that TLR9 antagonists will be useful for
treatment of sepsis due to the fact that release of free bacterial
DNA is likely to contribute to the cytokine storm during bacterial
sepsis. Therefore, a TLR9 antagonist may be more efficient in
treating bacterial sepsis than targeting individual cytokines.
Because TLR9 can be expressed at the cell surface,
identification of a peptide agonist may be suitable for a mimetibody
approach. This approach may result in increased potency compared to
agonist mAbs, CpG-ODN, or ISS, and therefore will likely require
less dosing and may be less expensive than other therapies (e. g.,
CpG-based therapies) which target TLR9.
The discovery of surface localization of TLR9 further allows
for the generation of and uses for agents binding TLR9 as targeting
moieties to specifically identify, activate, or destroy cells
displaying this marker on their surface. The invention therefore
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further describes the use of subsets of MFiCII+CD19+ and MHCII+CD19'~
primary human cells to identify compounds and compositions capable
of specifically binding TLR9 and, more particularly, those agents
capable of modifying TLR9 biological activity.
The mode of administration for therapeutic use of the binding
agents of the invention may be any suitable route which delivers the
agent to the host. The proteins, antibodies, antibody fragments and
mimetibodies and pharmaceutical compositions of these agents are
particularly useful for parenteral administration, i.e.,
subcutaneously, intramuscularly, intradermally, intravenously or
intranasally.
Binding agents of the invention may be prepared as
pharmaceutical compositions containing an effective amount of the
binding agent as an active ingredient in a pharmaceutically
acceptable carrier. An aqueous suspension or solution containing
the binding agent, preferably buffered at physiological pH, in a
form ready for injection is preferred. The compositions for
parenteral administration will commonly comprise a solution of the
binding agent of the invention or a cocktail thereof dissolved in an
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be employed, e.g., 0.4o saline,
0.3o glycine and the like. These solutions are sterile and
generally free of particulate matter. These solutions may be
sterilized by conventional, well known sterilization techniques
(e. g., filtration). The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions such as pH adjusting and buffering agents,
etc. The concentration of the binding agent of the invention in
such pharmaceutical formulation can vary widely, i.e., from less
than about 0.5~, usually at or at least about 1~ to as much as 15 or
20~ by weight and will be selected primarily based on fluid volumes,
viscosities, etc., according to the particular mode of
administration selected.
Thus, a pharmaceutical composition of the invention for
intramuscular injection could be prepared to contain 1 mL sterile
buffered water, and between about 1 ng to about 100 mg, e.g, about
50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg,
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of a binding agent of the invention. Similarly, a pharmaceutical
composition of the invention for intravenous infusion could be made
up to contain about 250 ml of sterile Ringer's solution, and about 1
mg to about 30 mg and preferably 5 mg to about 25 mg of a binding
agent of the invention. Actual methods for preparing parenterally
administrable compositions are well known or will be apparent to
those skilled in the art and are described in more detail in, for
example, "Remington's Pharmaceutical Science", 15th ed., Mack
Publishing Company, Easton, Pa.
The binding agents of the invention, when in a pharmaceutical
preparation, can be present in unit dose forms. The appropriate
therapeutically effective dose can be determined readily by those of
skill in the art. A determined dose may, if necessary, be repeated
at appropriate time intervals selected as appropriate by a physician
during the treatment period.
The protein, TLR9 mAb or mimetibody binding agents of the
invention can be lyophilized for storage and reconstituted in a
suitable carrier prior to use. This technique has been shown to be
effective with conventional immunoglobulins and protein preparations
and art-known lyophilization and reconstitution techniques can be
employed.
The present invention will now be described with reference to
the following specific, non-limiting Examples.
Example 1
TLR9 Surface Expression is Toasillar Cells
Human tonsil samples, harvested from pediatric donors, were
obtained from the National Disease Research Interchange
(Philadelphia, PA). Tissue samples were dissected into small pieces
and incubated with 1mg/ml Collagenase D (Boehringer Mannheim,
Mannheim, Germany) for one hour at 37°-C. Subsequently, samples
were
dissociated by passage through a cell strainer and then washed two
times to remove the collagenase. One million cells were stained per
condition for flow cytometry.
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Cells were stained using a commercially available unlabelled
mouse anti-human TLR9 mAb (Imgenix, San Diego, CA) followed by a
goat anti-mouse IgG F(ab')2-Cy5 (Jackson ImmunoResearch, West Grove,
PA) detecting reagent for single color fluorescence. However, use
of this secondary detecting reagent prevented multi-parameter
staining with other mouse anti-human lineage marker mAbs.
Therefore, for multi-parameter staining, the mouse anti-human TLR9
mAb was directly conjugated with allophycocyanin (APC) (Molecular
Probes Eugene, OR) and used in combination with other cell surface
marker mouse anti-human mAbs: MHCII-PerCP, CD123-PE, and CD19-FITC
all purchased from BD-Pharmingen (San Diego, CA). In every case,
the percentage of TLR9 positive cells determined by directly
conjugated mAb staining mirrored that found with the indirect two-
step staining process (n=4). Mouse anti-human IgG (BD-Pharmingen)
was used with the secondary detecting reagent or labeled with APC
for use as an isotype control. Cells were read on a BD FACSCaliburTM
System and samples were analyzed using CellquestTM Pro software (BD
Biosciences, San Jose, CA).
In Fig. 1, a dot plot depicting forward scatter (FSC-H) and
side scatter (SSC-H) of the tonsil samples is shown in (A). A
histogram displaying TLR9 staining (bold open line where staining is
marked by R2) relative to control levels of staining with an isotype
control mAb (gray shaded area) is shown in (B). Comparative dot
plot flow staining for the total cell population (C) vs. the TLR9+
cell population (D) (R2 gate) for MHC ClassII and CD19 levels to
elucidate the cell-surface phenotype of the TLR9+ cells.
Using flow cytometry on unpermeablized cells, TLR9 staining
was found on the cell surface of a subset of live cell gated tonsil
cells. Six different experiments were performed and a summary of
the data is shown in Table 1. The percentage of TLR9+ cells varied
from 2.2 to 9.50 of live gated tonsil cell preparations. Clearly,
variability exists among the different samples in the proportion of
cells that are positive for TLR9. This likely represents individual
donor variation, as samples were harvested from patients with
varying degrees of tonsillitis andlor tonsil hypertrophy. To
determine which cell populations in the tonsil samples exhibited
surface expression of TLR9, multi-parameter staining with the pan
antigen presenting cell marker MHC Class II (MHCII) and the pan B
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cell marker CD19 was performed. Table 1 shows summary data from
flow cytometric staining of MHCII and CD19 expression on TLR9+ live
gated cells. In these experiments it was found that regardless of
the overall percentage of TLR9+ cells, greater than 950 of the TLR9+
cells were B cells as indicated by their CD19 expression. The
remainder of the TLR9+ population expressed a phenotype of
MHCII1°wCDl9'. Overall, these data identify B cells as the primary
cell population displaying TLR9 surface expression in the tonsil.
Although previous data had suggested that B cells could express TLR9
mRNA (Bauer et al., Proc. Natl. Acad. Sci. (USA) 9~: 9237-9242,
(2001); Krug et al., Eur. J. Immunol. 31: 3026-3037, (2001)), the
data presented here provide direct visual evidence for TLR9 cell
surface protein expression on human primary tonsil cell populations.
Table d: Relative frequency of TLR9 positive cells in tonsil
samples. The proportion of live cell gated TLR9+ tonsil cells is
shown (column one) relative to the isotype control (column two).
Within those TLR9+ populations, the proportion of cells that are
MHCII+CD19+ (column three) or MHCII1°'"CD19' (column four) are
shown.
1 2 3 4.
Total Isotype TLR9+ TLR9+
TLR9'' coxitrol NgiCII'"CD19+NgiCIh"CD19-
2 . 2 0 0 ~ ND ND
9.50 1.8o ND ND
3.8o Oo 95.90 4.10
9.2o Oo 96.20 3.80
2.20 0.50 96.4 3.60
2.70 1.20 96.10 3.90
Example 2
TLR9 Surface Expression on Peripheral Blood Mononuclear Cells
Human peripheral blood mononuclear cells (PBMC) were isolated
from whole blood samples using Ficoll gradient centrifugation. One
million cells were stained per condition for flow cytometry.
In Fig. 2, a dot plot depicting FSC-H and SSC-H of the PBMC
samples is shown in (A). A histogram displaying TLR9 staining (bold
open line where staining is marked by R2) relative to control levels
of staining with an isotype control mAb (gray shaded area) is shown
in (B). C-J show comparative dot plots of flow staining for the
total cell population vs. the TLR9+ cell population (R2 gate) for
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MHCII and CD19 (C, D), CD123 (E, F), CDllc (G, H), CD14 (I, J) to
elucidate the cell-surface phenotype of the TLR9+ cells.
In six experiments, TLR9 staining was evident on a subset of
PBMC. The proportion of TLR9~' cells ranged from 1 to 13.30 of total
live gated cells, relative to the isotype control (Table 2). To
determine which cell populations) were expressing TLR9, multi-
parameter staining with MHCII, CD19, and CD123 was performed.
CD123, also known as IL-3 receptor alpha, is expressed on a variety
of cell types, and at very high levels on plasmacytoid dendritic
cells (Dzionek et al., J. Immunol. 165: 6037-6046, (2000)). Because
plasmacytoid dendritic cells are also CD19- and MHCII1°w (O~Doherty
et al., Immunology 82: 487-493, (1994); Grouard et al., J. Exp. Med.
185: 1101-1111, (1997)), four color flow cytometric staining can be
used to determine whether TLR9 is expressed on the cell surface of
cells expressing markers of plasmacytoid dendritic cells. The
results of four color flow cytometric staining for TLR9 population
subtyping are shown in Table 2. In these experiments, it was found
that sizeable populations of both MHCII+CD19+CD1231°w and
MHCII1°"CD19-
CD123bright Cells displayed surface expression of TLR9. However,
unlike tonsil cells, the majority of TLR9+ cells in PBMC lack CD19
expression (87o and 890). Furthermore, a portion of the TLR9+ cell
population is CD123brig''t and MHCII1°WCD19-, a cell surface phenotype
suggestive of plasmacytoid dendritic cells (Dzionek et al., supra).
The majority of the TLR9+ population expresses both CDllc (column
5) and CD14 (column 6). These data provide direct evidence for TLR9
cell surface protein expression on human PBMC populations.
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Table 2: Relative frequency of TLR9 positive cells in PBMC samples.
The proportion of live cell gated TLR9+ PBMC is shown (column one)
relative to the isotype control (column two). Within the TLR9+
population, the proportion of cells that are MHCII+CD19+CD123'-~w
(column three) or MHCII1°WCD19-CD123bright (Column four) are shown. The
relative proportions of MHCII+CD19-CD1231~w expressing either CDllc
(column 5) or CD14 (column 6) are shown for experiments 5 and 6.
The average percentage and standard deviation values are shown in
the last row for the populations of interest.
1 2 3 4 5 6
Total TLR9+ TLR9+ TLR9+ TLR9i
IsotypeNRICII+CD19+
TLR9'" control NgiCIh'CD19-NgiCII'"CD19-NgiCII+CD
CD1231' CD123b=isht CD1231'"CDllc'"CD1231'~C
Exp. 5.9a l.Oo 12.50 31.9a
1
Exp. l.Oo 0.20 l3.Oo 6.10
2
Exp. 7.10 0.60 11.20 3.5s
3
Exp. 8.40 0.70 14.10 2.40
4
Exp. 13.30 0.80 2.50 2.60 89.30 71.7
5
Exp. 4.40 l.Oo 26.2 9.3a 63.3 54.3
6
Average 6.74.2 0.60.4 13.37.6 9.311.4 76.318.4 63.01~
Example 3
LPS Mediated Up-Regulation of PBMC TLR9 Surface Expression
Whole PBMC were cultured overnight in either media alone or in
media containing l0uglml of bacterial lipopolysaccharide (LPS).
Following culture, TLR9 levels were analyzed via flow cytometry.
Prior to LPS stimulation, 4.4~ of the PBMC population expressed
cell-surface TLR9 (Table 2). After 18 hours in culture, 6.9~ of the
control PBMC cultured in media alone and 10.90 of the PBMC cultured
in LPS had detectable levels of cell-surface TLR9 (Table 3).
Importantly, PBMC stimulated for 18 hours with LPS expressed
approximately 4-fold higher cell-surface levels of TLR9 (159.4 Mean
Fluorescence Intensity (MFI)) relative to those PBMC in media alone
(40.7 MFI) (Table 3). These data demonstrate that activation of
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PBMC with LPS results in the upregulation of TLR9 expression. These
data suggest a cross-regulatory mechanism of expression for TLR4 and
TLR9, both of which have ligands that are derived from bacterial
components.
Table 3: Mean fluorescence intensity of TLR9 staining on cultured
PBMC. PBMC populations from Exp. 6 (Table II) were cultured
overnight in either media alone or media with 10~.g/ml of LPS. The
relative frequency of TLR9+ cells and the mean fluorescence
intensity of the staining are shown. Overnight culture with LPS
upregulates the level of TLR9 expression greater than 3.5-fold on
PBMC.
1 2 3 4
MAb Isotype TLR9 Isotype TLR9
Culture control staining control LPS staining
condition Media Media LPS
Exp. 6 0.80 6.90 0.80 10.90
PBMC post
culture
Mean 31.6 40.7 29.0 159.4
fluorescence
intensity
of
staining
Example 4
hmnunofluorescence~Detection of PBMC Cell Surface TLR9
To observe visually whether the mouse anti-human TLR9 mAb was
recognizing TLR9 at the cell-surface or rather inside the cell,
cytospins of PBMC from the LPS stimulated cultures were made.
Cytospins of PBMC stained with anti-CD19-FITC (a B cell marker) and
either anti-TLR9-APC or an isotype control-APC were viewed by
fluorescence microscopy. Images of individual slide fields of these
cytospins were viewed and captured at 40X magnification under a
wavelength of light capable of detecting FITC (green fluorescence)
and under light capable of detecting APC (far red fluorescence).
CD19-FITC staining was observed on LPS stimulated PBMC cells,
while no staining was detectable on those same cells with the mouse
isotype control mAb labeled with APC (data not shown). Importantly,
staining was observed with the mouse anti-human TLR9-APC (data not
shown) on cytospins of LPS stimulated PBMC. CD19-FITC was also
observed on the corresponding microscopy field (data not shown).
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However, TLR9 staining was observed on larger cells not found to be
staining with CD19. These data are consistent with the data
obtained by flow cytometry where the TLR9+ cells found in PBMC
samples were observed to be larger cells, few of which were CD19+.
Importantly, the TLR9 staining observed on the cytospins of the LPS
stimulated PBMC cultures appeared to be at the cell-surface, a
finding consistent with the flow cytometric analysis of TLR9
expression.
Example 5
Specificity of the Fluoresceatly Cor~,jugated TLR9 mAb
A commercially available mouse anti-human TLR9 mAb (Imgenix,
San Diego, CA). This antibody was made by immunizing a mouse with a
15-mer peptide of TLR9 having the amino acid sequence
CPRHFPQLHPDTfSHLS (SEQ ID NO: 3) conjugated to keyhole limpet
hemocyanin (KLH) using standard hybridoma technology. The peptide
represented residues 268-284 of human TLR9 located in the putative
extracellular domain. To test the specificity of the flow
cytometric staining observed with the mouse anti-human TLR9 mAb, the
immunizing peptide was synthesized and compared with a control
peptide (residues 31-45 of human prostate specific antigen (PSA))
having the amino acid sequence CEF~HSQPWQVLVASR (SEQ ID NO: 4) for
the ability to block the TLR9 staining observed by flow cytometry.
The TLR9 peptide or control peptide was preincubated with the mouse
anti-human TLR9 mAb or the isotype control mAb for 15 minutes prior
to its addition to the PBMC preparation. Labeled PBMCs were then
analyzed by flow cytometry. Flow cytometry methods,
instrumentation, software, mAbs and PBMC preparations were as
described in the preceding Examples.
No effect on mouse isotype control staining was observed with
either the TLR9 peptide or the control peptide (data not shown).
Importantly, preincubation of the mouse anti-human TLR9 mAb with the
TLR9 peptide reduced the level of TLR9 staining to close to
background levels (those levels observed with the isotype control
mAb) (Fig. 3). Histograms shown are gated on live cells and show
fluorescence for the mouse anti-human TLR9 mAb (gray histogram), the
mouse anti-human TLR9 mAb - preincubated with the TLR9 peptide (bold
black line), and the mouse isotype-APC mAb (thin stippled line).
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Preincubation of mouse anti-human TLR9 mAb with a TLR9 peptide
reduces the fluorescence staining observed for TLR9 to near
background levels observed with the isotype control.
In contrast, preincubation of the mouse anti-human TLR9 mAb
with the control peptide had no effect on TLR9 staining (Fig. 4).
Because the TLR9 peptide and not an irrelevant peptide can block the
fluorescence observed with the mouse anti-human TLR9 mAb, these data
confirm the specificity of the TLR9 staining. Histograms shown are
gated on live cells and show fluorescence for the mouse anti-human
TLR9 mAb (gray histogram), the mouse anti-human TLR9 mAb
preincubated with a control peptide (bold black line), and the mouse
isotype-APC mAb (thin stippled line). Preincubation of mouse anti-
human TLR9 mAb with a control peptide has no effect on the
fluorescence staining observed with the TLR9 mAb. Together these
data presented in Figs. 3 and 4 confirm the specificity of the PBMC
labeling observed with the TLR9 mAb and that the TLR9+ PBMC
populations observed in the preceding Examples are not artifactual.
Example 6
Increased TLR9 Transcript Levels in a Mouse Model of Chronic Lung
Inflammation
Gene transcript levels, as assessed by real time PCR, are
generally regarded by those of ordinary skill in the art as a proxy
for gene (protein) expression levels. Real time-PCR was used to
quantify TLR9 gene transcript levels in the lung tissues of SP-
C/TNF-a transgenic and wild-type mice. SP-C/TNF-a transgenic mice
overexpress TNF-a in alveolar type II cells. This TNF-a
overexpression is controlled in these mice by the Human surfactant
protein C promoter (Fujita et al., Am. J. Physiol. - Lung C 280:L39-
49, (2001)). Histopathological studies have revealed chronic lung
inflammation in SP-C/TNF-a transgenic mice. Additionally,
physiological assessments have demonstrated that SP-C/TNF-a
transgenic mice exhibit increased lung volumes and a decrease in
elastic recoil characteristic of emphysema (Fujita et al., supra).
The SP-C/TNF-a transgenic mice are an accepted mouse model for
chronic lung inflammation.
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Total RNA for real time-PCR analysis was extracted from mouse
lung tissue samples using TrizolTM (Invitrogen Corp., Carlsbad, CA)
according to the manufacturer's instructions. cDNAs were prepared
using the OmniscriptTM kit (Qiagen Inc., Valencia, CA) according to
manufacturer's instructions. TaqManTM real time-PCR was then
performed in 50 ml volumes on 96-well plates using the Universal
Master Mix (Applied Biosystems) and ABI PRISIMrM 7000HT
instrumentation. The TaqManTM real-time PCR technology and ABI
instrumentation detect accumulation of PCR products continuously
during the PCR process and allow accurate transcript quantitation in
the early exponential phase of PCR. Primer ExpressTM software was
used to design the probe sequence 5'-CGTCGCTGCGACCATGCC-3' (SEQ ID
N0: 5), the forward primer sequence 5'-ACTTGATGTGGGTGGGAATTG-3' (SEQ
ID NO: 6) and the reverse primer sequence 5'-
GCCACATTCTATACAGGGATTGG-3' (SEQ ID N0: 7). cDNA levels were
normalized against transcipt levels for the thioredoxin reductase
housekeeping gene. The thermal cycling protocol started with a 50°C
annealing step for two minutes, followed by ten minutes at 95°C to
denature the DNA and activate the AmpliTaq GoldTM polymerase. This
was followed by 40 cycles of 95°C for 15 seconds and 60°C for
one
minute during which the AmpliTaq GoldTM polymerase cleaves the probe
and the fluorescence data is collected. The data collection and
transcript quanitation in the early exponential phase is performed
by the ABI PRISIMrM 7000HT instrumentation and associated software.
The results are presented in Table 4 and show that TLR9 mRNA
transcript levels are increased in the lung tissue of SP-C/TNF-a
transgenic mice as compared to the lung tissue of age-matched, wild-
type'control mice. Peak levels of TLR9 mRNA expression were
observed in 9 week-old transgenic mice, an age that correlates with
a marked inflammatory response in the lungs (Fujita et al., supra).
These data demonstrate that TLR9 transcript levels, and presumably
expression, are increased in the lungs of SP-C/TNF-a transgenic
mice. Lastly, the data indicates a role for TLR9 in TNF-a driven
lung inflammation.
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Table 4: Real time PCR quantitative analysis of TLR9 expression in
transgenic SP-C/TNF-a mice.
Age (weeks) 4 6 9 14
TLR9 fold 1.2 3.22 5.23 2.88
increase*
*Relative fold increase in mRNA expression compared to a reference
wild-type mouse given a value of 1. cDNA sample TLR9 levels were
normalized against transcript levels of the thioredoxin reductase
housekeeping gene.
Example 7
Generation of Anti-TLR9 mAbs
Separate groups of mice will be immunized with plasmid DNA
encoding the extracellular domain of TLR9 (residues 1 through 818 of
SEQ ID NO: 1). Each mouse will receive three 15 ~.g doses of
plasmid DNA diluted in PBS (150 mM NaCl; pH 7.4), each dose to be
injected intradermally in the ears two weeks apart. After the
plasmid DNA injections, mice will be boosted twice at biweekly
intervals (15 ~.g per mouse injected intradermally) with a Fc fusion
or mimetibody construct containing the 260 residue extracellular
domain fragment of TLR9 or its mature form such as the fusion
protein having the sequence shown in SEQ ID N0: 11. Spleens from
immunized mice will be harvested and B cell fusions carried out
using standard hybridoma methods of Kohler et al., supra). Three
days prior to B cell fusion, mice will be given an intravenous
injection of 15 ~.g of the protein used for boosting. Fused cells
will be selected using HAT medium and will be screened for the
presence of anti-TLR9 antibodies by ELISA. Fused cells testing
positive will be expanded and cloned by limiting dilution. Anti-
TLR9 antibody nucleic acid and protein sequences will be determined
by standard techniques.
Example 8
CpG-Dependent Binding of EC260-Fc to CpG 0ligodinucletotide
A human TLR9 extracellular domain Fc fusion construct was made
as follows. A cDNA fragment encoding amino acids 1 to 260 of human
TLR9 was amplified by polymerase chain reaction and cloned into a
FcHA6His-Fly FLY cell expression vector resulting in a fusion
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protein containing the first 260 amino acids of TLR9 fused in-frame
with an Fc fragment of human IgG1 followed by a hemagglutinin tag
and a hexa-histidine tag at the C-terminus. The complete coding
sequence of the fusion protein was then excised and cloned into a
pcDNA3.1/(+) vector (Invitrogen, Carlsbad, CA) at the EcoR1 and Xho1
sites.
HEK293 cells were transfected with the pcDNA3.1/(+)-EC260-
FcHAhexaHis vector and selected in 400 ug/ml 6418. The EC260-Fc
protein was detectable from cells and as a soluble dimer protein in
culture supernatant. The amino acid sequence of the fusion protein
construct is shown in SEQ ID NO: 2. The secreted form of the fusion
protein will lack the signal sequence and is predicted to have the
amino acid sequence shown in SEQ ID NO: 11.
Culture supernatant from stable HEK293 cells expressing and
secreting EC260-Fc was harvested and cleared by centrifugation.
Protein A-Sepharose (PAS) beads (Amersham Biosciences, Piscataway,
NJ) were added to the cleared supernatant and incubated at 4°-C
for
>2 hours to allow for binding of the EC260-Fc construct to the
beads. After incubation, the beads were pelleted by centrifugation
and washed twice with saline.
The synthetic immunostimulatory CpG oligodinucleotide (ODN)
ODN2006 (SEQ ID N0: 8) was end-labeled with 33P using T4 kinase
(Promega, Madison, WI) and 33P-y-ATP (Amersham Biosciences,
Piscataway, NJ). The end-labeled ODNs were then separated from free
33P-y-ATP by G25 (Amersham Biosciences, Piscataway, NJ) column
chromatography.
The CpG-dependent binding of ODNs by PAS-bound EC260-Fc
protein was examined by incubating PAS-EC260-Fc beads with 33P-
ODN2006 in binding buffer (lOmM Tris.HCl, pH6; 50mM NaCl, 1mM MgCl,
0.5mM EDTA, 1mM DTT, 0.1o NP-40, 0.030 BSA, 5o Glycerol) in the
presence of excess non-specific DNA from salmon testes (50 ug/ml) at
room temperature for 2 hours followed by washing three times with
saline. Bead-bound radioactivity was determined in a TopCount
scintillation counter (PerkinElmer, Boston, MA). To determine if
binding is CpG dependent, separate binding reactions were run
including a 50-fold excess of unlabeled ODN2006 or an inactive oligo
where the CpG dinucleotides of ODN2006 were changed to GpC
dinucleotides (ODN2006GC, SEQ ID N0: 9) or the CpG to GpC changes
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were made in addition to changes in the flanking sequences (SEQ ID
N0: 10.
The results shown in Fig. 5 indicate that a significant amount
of 33P-ODN2006 was detected on the PAS-EC260-Fc beads. The results
suggest that the binding is CpG-dependent since ODN2006 competed
well for binding while the inactive oligos ODN2006-GC and
ODN2006GCmf did not.
Example 9
Effect of EC260-Fc on CpG-Induced Cytokine Production
An eactracellular domain fragment of hTLR9 (residues 1 to 260)
generated using a transient transfection protocol was tested for its
ability to compete with the TLR9 ligand CpG ODN for cytokine
production in human PBMCs. Human PBMC secrete a variety of
cytokines and chemokines including IFN-y, IFN-a, TNF-a, IL-10, IL-
12, IL-8, MCP-1, MIP1-a and RANTES in response to CpG stimulation.
Human PBMCs were isolated using standard Ficoll gradient and
stimulated with either CpG alone or with CpG pre-incubated for 1
hour at 37°-C with the mature form of the 1 to 260-Fc fusion protein
domain of human TLR9 (SEQ ID NO: 11). Culture supernatants were
harvested at 24 hours after stimulation and cytokine levels were
analyzed using Luminex.
The percent inhibition in cytokine production observed when
the CpG-ODN were pre-cultured with the TLR9-Fc reagent prior to the
addition to PBMC cultures, relative to CpG-ODN stimulation alone is
shown in the Table 5 below. The results indicate that the human
TLR9 domain consisting of amino acids 26-260 is capable of
interfering with CpG-induced IFN-y, IL-10, IL-6, MCP-1, MIP1-a,
RANTES and TNF-a production, indicating that the TLR9-Fc fusion
protein could serve as a sink to minimize the ability of bacterial
CpG to stimulate the secretion of inflammatory cytokines during
bacterial infections.
Table 5: Percent inhibition of cytokine production compared to CpG
stimulation alone.
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The present invention now being fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
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cEN5022seplist.txt
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530 535 540
Pro Arg Leu Glu Ala Leu Asp Leu Ser Tyr Asn Ser Gln Pro Phe Gly
545 550 555 560
Met Gln Gly Val Gly His Asn Phe Ser Phe Val Ala His Leu Arg Thr
565 570 575
Leu Arg His Leu Ser Leu Ala His Asn Asn Ile His Ser Gln Val Ser
580 585 590
Gln Gln Leu Cys Ser Thr Ser Leu Arg Ala Leu Asp Phe Ser Gly Asn
595 600 605
Ala Leu Gly His Met Trp Ala Glu Gly Asp Leu Tyr Leu His Phe Phe
610 615 620
Gln Gly Leu Ser Gly Leu Ile Trp Leu Asp Leu Ser Gln Asn Arg Leu
625 630 635 640
His Thr Leu Leu Pro Gln Thr Leu Arg Asn Leu Pro Lys Ser Leu Gln
645 650 655
Val Leu Arg Leu Arg Asp Asn Tyr Leu Ala Phe Phe Lys Trp Trp Ser
660 665 670
Leu His Phe Leu Pro Lys Leu Glu Val Leu Asp Leu Ala Gly Asn Gln
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675 680 685
Leu Lys Ala Leu Thr Asn Gly Ser Leu Pro Ala Gly Thr Arg Leu Arg
690 695 700
Arg Leu Asp Val Ser Cys Asn Ser Ile Ser Phe Val Ala Pro Gly Phe
705 710 715 720
Phe Ser Lys Ala Lys Glu Leu Arg Glu Leu Asn Leu Ser Ala Asn Ala
725 730 735
Leu Lys Thr Val Asp His Ser Trp Phe Gly Pro Leu Ala Ser Ala Leu
740 745 750
Gln Ile Leu Asp Val Ser Ala Asn Pro Leu His Cys Ala Cys Gly Ala
755 760 765
Ala Phe Met Asp Phe Leu Leu Glu Val Gln Ala Ala Val Pro Gly Leu
770 775 780
Pro Ser Arg Val Lys Cys Gly Ser Pro Gly Gln Leu Gln Gly Leu Ser
785 790 795 800
Ile Phe Ala Gln Asp Leu Arg Leu Cys Leu Asp Glu Ala Leu Ser Trp
805 810 815
Asp Cys Phe Ala Leu Ser Leu Leu Ala Val Ala Leu Gly Leu Gly Val
820 825 830
Pro Met Leu His His Leu Cys Gly Trp Asp Leu Trp Tyr Cys Phe His
835 840 845
Leu Cys Leu Ala Trp Leu Pro Trp Arg Gly Arg Gln Ser Gly Arg Asp
850 855 860
Glu Asp Ala Leu Pro Tyr Asp Ala Phe Val Val Phe Asp Lys Thr Gln
865 870 875 880
Ser Ala Val Ala Asp Trp Val Tyr Asn Glu Leu Arg Gly Gln Leu Glu
885 890 895
Glu Cys Arg Gly Arg Trp Ala Leu Arg Leu Cys Leu Glu Glu Arg Asp
900 905 910
Trp Leu Pro Gly Lys Thr Leu Phe Glu Asn Leu Trp Ala Ser Val Tyr
915 920 925
Page 4
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Gly Ser Arg Lys Thr Leu Phe Val Leu Ala His Thr Asp Arg Val Ser
930 935 940
Gly Leu Leu Arg Ala Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu
945 950 955 960
Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Ser Pro Asp Gly Arg
965 970 975
Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val
980 985 990
Leu Leu Trp Pro His Gln Pro Ser Gly Gln Arg Ser Phe Trp Ala Gln
995 1000 1005
Leu Gly Met Ala Leu Thr Arg Asp Asn His His Phe Tyr Asn Arg
1010 1015 1020
Asn Phe Cys Gln Gly Pro Thr Ala Glu
1025 1030
<210> 2
<211> 514
<212> PRT
<213> Artificial
<220>
<223> TLR9 1-260 Fc Fusion
<400> 2
Met Gly Phe Cys Arg Ser Ala Leu His Pro Leu Ser Leu Leu Val Gln
1 5 10 15
Ala Ile Met Leu Ala Met Thr Leu Ala Leu Gly Thr Leu Pro Ala Phe
20 25 30
Leu Pro Cys Glu Leu Gln Pro His Gly Leu Val Asn Cys Asn Trp Leu
35 40 45
Phe Leu Lys Ser Val Pro His Phe Ser Met Ala Ala Pro Arg Gly Asn
50 55 60
Val Thr Ser Leu Ser Leu Ser Ser Asn Arg Ile His His Leu His Asp
65 70 75 80
Ser Asp Phe Ala His Leu Pro Ser Leu Arg His Leu Asn Leu Lys Trp
85 90 95
Asn Cys Pro Pro Val Gly Leu Ser Pro Met His Phe Pro Cys His Met
Page 5
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100 105 110
Thr Ile Glu Pro Ser Thr Phe Leu Ala Val Pro Thr Leu Glu Glu Leu
115 120 125
Asn Leu Ser Tyr Asn Asn Ile Met Thr Val Pro Ala Leu Pro Lys Ser
130 135 140
Leu Ile Ser Leu Ser Leu Ser His Thr Asn Ile Leu Met Leu Asp Ser
145 150 155 160
Ala Ser Leu Ala Gly Leu His Ala Leu Arg Phe Leu Phe Met Asp Gly
165 170 175
Asn Cys Tyr Tyr Lys Asn Pro Cys Arg Gln Ala Leu Glu Val Ala Pro
180 185 190
Gly Ala Leu Leu Gly Leu Gly Asn Leu Thr His Leu Ser Leu Lys Tyr
195 200 205
Asn Asn Leu Thr Val Val Pro Arg Asn Leu Pro Ser Ser Leu Glu Tyr
210 215 220
Leu Leu Leu Ser Tyr Asn Arg Ile Val Lys Leu Ala Pro Glu Asp Leu
225 230 235 240
Ala Asn Leu Thr Ala Leu Arg Val Leu Asp Val Gly Gly Asn Cys Arg
245 250 255
Arg Cys Asp His Gly Ser Pro Lys Ser Cys Asp Lys Thr His Thr Cys
260 265 270
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
275 280 285
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
290 295 300
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
305 310 315 320
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
325 330 335
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
340 345 350
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Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
355 360 365
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
370 375 380
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
385 390 395 400
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
405 410 415
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
420 425 430
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
435 440 445
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
450 455 460
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
465 470 475 480
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Asp Arg
485 490 495
Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Arg Ser His His His His
500 505 510
His His
<210> 3
<211> 17
<212> PRT
<213> Homo Sapiens
<400> 3
Cys Pro Arg His Phe Pro Gln Leu His Pro Asp Thr Phe Ser His Leu
1 5 10 15
ser
<210> 4
<211> 15
<212> PRT
<213> Homo Sapiens
Page 7
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<400> 4
Cys Glu Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg
1 5 10 15
<210> 5
<211> 18
<212> DNA
<213> Mus musculus
<400> 5
cgtcgctgcg accatgcc 1g
<210> 6
<211> 21
<212> DNA
<213> Mus musculus
<400> 6
acttgatgtg ggtgggaatt g 21
<210> 7
<211> 23
<212> DNA
<213> Mus musculus
<400> 7
gccacattct atacagggat tgg 23
<210> 8
<211> 24
<212> DNA
<213> Artificial
<220>
<223> oligodinucleotide
<400> 8
tcgtcgtttt gtcgttttgt cgtt 24
<210> 9
<211> 24
<212> DNA
<213> Artificial
<220>
<223> oligodinucleotide
<400> 9
tgctgctttt gtgcttttgt gctt 24
<210> 10
<211> 24
<212> DNA
<213> Artificial
Page 8
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<220>
<223> oligodinucleotide
<400> 10
tcttgcgttt ttgcgttttt gcgt 24
<210> 11
<211> 489
<212> PRT
<213> Artificial
<220>
<223> Predicted Mature Form of TLR9 EC260-Fc Fusion
<400> 11 ,
Leu Gly Thr Leu Pro Ala Phe Leu Pro Cys Glu Leu Gln Pro His Gly
1 5 10 15
Leu Val Asn Cys Asn Trp Leu Phe Leu Lys Ser Val Pro His Phe Ser
20 25 30
Met Ala Ala Pro Arg Gly Asn Val Thr Ser Leu Ser Leu Ser Ser Asn
35 40 45
Arg Ile His His Leu His Asp Ser Asp Phe Ala His Leu Pro Ser Leu
50 55 60
Arg His Leu Asn Leu Lys Trp Asn Cys Pro Pro Val Gly Leu Ser Pro
65 70 75 80
Met His Phe Pro Cys His Met Thr Ile Glu Pro Ser Thr Phe Leu Ala
85 90 95
Val Pro Thr Leu Glu Glu Leu Asn Leu Ser Tyr Asn Asn Ile Met Thr
100 105 110
Val Pro Ala Leu Pro Lys Ser Leu Ile Ser Leu Ser Leu Ser His Thr
115 120 125
Asn Ile Leu Met Leu Asp Ser Ala Ser Leu Ala Gly Leu His Ala Leu
130 135 140
Arg Phe Leu Phe Met Asp Gly Asn Cys Tyr Tyr Lys Asn Pro Cys Arg
145 150 155 160
Gln Ala Leu Glu Val Ala Pro Gly Ala Leu Leu Gly Leu Gly Asn Leu
165 170 175
Thr His Leu Ser Leu Lys Tyr Asn Asn Leu Thr val Val Pro Arg Asn
180 185 190
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Leu Pro Ser Ser Leu Glu Tyr Leu Leu Leu Ser Tyr Asn Arg Ile Val
195 200 205
Lys Leu Ala Pro Glu Asp Leu Ala Asn Leu Thr Ala Leu Arg Val Leu
210 215 220
Asp Val Gly Gly Asn Cys Arg Arg Cys Asp His Gly Ser Pro Lys Ser
225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
245 250 255
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
305 310 315 320
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
355 360 ~ 365
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
385 390 395 400
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
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435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460
Ser Pro Gly Lys Ile Asp Arg Ser Tyr Pro Tyr Asp Val Pro Asp Tyr
465 470 475 480
Ala Arg Ser His His His His His His
485
Page 11
