U.S. patent application number 12/190739 was filed with the patent office on 2009-02-19 for compositions comprising dc-sign blockers and methods of using dc-sign blockers for preventing or treating diseases of a mammal, including viral infections.
This patent application is currently assigned to Institut Pasteur. Invention is credited to Ali Amara, Fernando Arenzana-Seisdedos, Julie Dechanet-Merville, Thierry Delaunay, Franck Halary, Jean-Francois Moreau.
Application Number | 20090048209 12/190739 |
Document ID | / |
Family ID | 32719122 |
Filed Date | 2009-02-19 |
United States Patent
Application |
20090048209 |
Kind Code |
A1 |
Amara; Ali ; et al. |
February 19, 2009 |
COMPOSITIONS COMPRISING DC-SIGN BLOCKERS AND METHODS OF USING
DC-SIGN BLOCKERS FOR PREVENTING OR TREATING DISEASES OF A MAMMAL,
INCLUDING VIRAL INFECTIONS
Abstract
The present invention relates to methods and compositions for
preventing or treating diseases of a mammal, including viral
infections, wherein at least one symptom of the disease is mediated
at least in part by the binding of an effector molecule to a
DC-SIGN receptor present on cells of the mammal to be treated. The
invention also provides methods of identifying compositions,
wherein the compositions are useful for treating mammalian
diseases, including viral infections, for which at least one
symptom of the disease is mediated at least in part by the specific
binding of an effector molecule to a DC-SIGN receptor present on
the cells that express the DC-SIGN receptor, belonging to the
mammal to be treated. The invention further relates to compositions
and methods for targeting subject molecules to cells that express
the DC-SIGN receptor.
Inventors: |
Amara; Ali; (Paris, FR)
; Halary; Franck; (Bordeaux, FR) ;
Dechanet-Merville; Julie; (Merignac, FR) ; Moreau;
Jean-Francois; (Merignac, FR) ; Arenzana-Seisdedos;
Fernando; (Bievres, FR) ; Delaunay; Thierry;
(Le Hillan, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Institut Pasteur
|
Family ID: |
32719122 |
Appl. No.: |
12/190739 |
Filed: |
August 13, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10700507 |
Nov 5, 2003 |
7427469 |
|
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12190739 |
|
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|
|
60423581 |
Nov 5, 2002 |
|
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60425324 |
Nov 12, 2002 |
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
C07K 16/2851 20130101;
A61P 31/00 20180101; A61K 2039/505 20130101 |
Class at
Publication: |
514/54 |
International
Class: |
A61K 31/715 20060101
A61K031/715; A61P 31/00 20060101 A61P031/00 |
Claims
1-80. (canceled)
81. A method of blocking a DC-Specific ICAM-Grabbing Nonintegrin
(DC-SIGN) receptor in a mammal, wherein the method comprises
administering to the mammal an amount of a mannosylated molecule
that specifically binds to the DC-SIGN receptor.
82. The method of claim 81, wherein the mannosylated molecule is
mannan.
83. The method of claim 81, wherein the DC-SIGN receptor is chosen
from DC-SIGN and DC-S DC-Specific ICAM-Grabbing Nonintegrin Related
(DC-SIGNR).
84. A method of treating a viral infection of a mammal, wherein the
viral infection is mediated at least in part by the binding of a
viral effector molecule to at least one DC-SIGN receptor of the
mammal to be treated, wherein the method comprises administering to
the mammal an amount of a DC-SIGN blocker sufficient to
substantially inhibit the binding of the viral effector molecule to
the DC-SIGN receptor to thereby treat the viral infection, wherein
the DC-SIGN blocker is a mannosylated molecule that specifically
binds to the DC-SIGN receptor.
85. The method of claim 84, wherein the mannosylated molecule is
mannan.
86. The method of claim 84, wherein the DC-SIGN receptor is chosen
from DC-SIGN and DC-SIGNR.
87. The method of claim 84, wherein the viral infection is chosen
from Human Immunodeficiency Virus (HIV) infection, Simian
Immunodeficiency Virus (SIV) infection, and Ebola Virus
infection.
88. A method of inhibiting entry of a virus into a cell of a human
that expresses at least one DC-SIGN receptor of the human to be
treated, the method comprising administering to the human a
mannosylated molecule that specifically binds to the DC-SIGN
receptor, wherein the mannosylated molecule that specifically binds
to the DC-SIGN receptor is administered in an amount sufficient to
inhibit the binding of the virus effector molecule to the DC-SIGN
receptor, to thereby inhibit entry of the virus into the cell.
89. The method of claim 88, wherein the mannosylated molecule is
mannan.
90. The method of claim 88, wherein the DC-SIGN receptor is chosen
from DC-SIGN and DC-SIGNR.
91. The method of claim 88, wherein the viral infection is chosen
from HIV infection, SIV infection, and Ebola Virus infection.
Description
[0001] This is a divisional of application Ser. No. 10/700,507,
filed Nov. 5, 2003, which claims the right of priority under 35
U.S.C. .sctn. 119(e) based on Provisional Patent Application Nos.
60/423,581, filed Nov. 5, 2002, and 60/425,324, filed Nov. 12,
2002, all of which are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to methods and compositions for
preventing or treating diseases of a mammal, wherein at least one
symptom of the disease is mediated at least in part by the binding
of an effector molecule to a DC-SIGN receptor of the mammal to be
treated. The effector molecule may be a molecule on a foreign
organism. The foreign organism may be a virus.
[0004] The invention also relates to compositions, and to methods
of identifying compositions, wherein the compositions are useful
for treating mammalian diseases for which at least one symptom of
the disease is mediated at least in part by the binding of an
effector molecule to a DC-SIGN receptor of the mammal to be
treated.
[0005] The invention further relates to compositions and methods
for targeting subject molecules to cells expressing DC-SIGN
receptors, such as dendritic cells. These compositions and methods
are based on targeting complexes, in which one or more subject
molecules are covalently attached to one or more DC-SIGN blockers
and, by virtue of binding of one or more of the DC-SIGN blockers of
the targeting complex to DC-SIGN, the subject molecule is targeted
to cells expressing DC-SIGN receptors.
[0006] 2. Description of the Related Art
[0007] Human Cytomegalovirus (CMV) is a double strand DNA virus
belonging to the Herpesviridae family and a ubiquitous pathogen in
humans. CMV interaction with its host is characterized by a primary
infection followed by lifelong persistence in the host organism and
viral reactivation episodes. CMV infection is asymptomatic in most
immunocompetent individuals because of an efficient anti-viral
immune response. In contrast, CMV remains a major cause of
morbidity and mortality in newborn and immunocompromised patients,
namely in organ-transplanted recipients or AIDS patients. In many
cases, CMV disease is characterized by a wide viral spread toward
multiple organs (i.e., salivary glands, lung, kidney,
gastrointestinal tract, liver, retina, and the central nervous
system (CNS)).
[0008] In vitro, a number of cell types are susceptible to CMV
infection when considering virus entry and viral immediate early
gene expression. However, full replication of virus DNA and
subsequent production of infectious virions is limited to
permissive cells (i.e., fibroblasts, endothelial cells, the U373 MG
astrocytoma cell line, etc.; see for review Plachter et al., 1996).
In fibroblasts (the prototypic cell type for in vitro studies of
CMV infection) CMV entry occurs in sequential steps involving
several viral envelope (Env) glycoproteins. Initial attachment of
virus to host cells is mediated through interaction between Env
glycoproteins g8 (CMV g8) and/or CMV gM with cell surface heparan
sulfate proteoglycans (Compton et al., 1993; Kari and Gehrz, 1992).
Thereafter, binding of CMV gB with non-heparin cellular receptors
probably allows more stable attachment of the virus to the cell
surface (Boyle and Compton, 1998). Subsequent pH-independent fusion
events between viral envelope and cell membrane are necessary for
viral entry (Compton et al., 1992; Milne et al., 1998). Cell
proteins involved in CMV attachment and/or fusion have not been
identified precisely although two candidates have been proposed.
The first one is annexin II which interacts with CMV gB
(Pietropaolo and Compton, 1997). The second one is a 92.5 kDa
protein binding to CMV gH (Baldwin et al., 2000). Fusion events are
followed by penetration of the capsid which is transported to the
nucleus. In some permissive cells, such as retinal pigment
epithelial cells, CMV can also penetrate into cells by a mechanism
of endocytosis (Bodaghi et al., 1999).
[0009] Recently, dendritic cells (DC), which are refractory to
infection by laboratory-adapted CMV strains, were shown to be
permissive to CMV infection and replication when infected with
primary, clinical viral isolates (Riegler et al., 2000).
[0010] Dendritic cells are a diverse population of morphologically
similar cell types found in lymphoid or non-lymphoid tissues.
Dendritic cells function as antigen-presenting cells that
efficiently capture antigens in the peripheral tissues and process
them to form MHC-peptide complexes. After antigen uptake, these
immature dendritic cells acquire the unique capacity to migrate
from the periphery to the T cell areas of the secondary lymphoid
organs. Dendritic cells convert antigens from foreign cells and
infectious microorganisms into short peptides that are bound to
membrane proteins of the major histocompatibility complex (MHC).
These MHC-peptide complexes are formed intracellularly, but are
ultimately presented on the plasma membrane where they serve as
ligands for antigen-specific T cell receptors (TCR). In addition to
TCR ligand formation, dendritic cells carry out many other
functions, which allow them to control immunity at several points
(Steinman, 2000).
[0011] The mechanism of CMV entry into DC has not been investigated
yet. It was recently shown that DC express a lectin called DC-SIGN
(DC-Specific ICAM-Grabbing Nonintegrin). DC-SIGN, also called
CD209, is a ligand for IntraCellular Adhesion Molecule-2 (ICAM-2)
and ICAM-3 (Geijtenbeek et al., 2000a; Geijtenbeek et al., 2000c)
and is involved in the attachment of Human Immunodeficiency Virus-1
(HIV-1) (Geijtenbeek et al., 2000b) and Ebola (Alvarez et al.,
2002) to DC. DC-SIGN was originally cloned from a placental cDNA
library on the basis of its capacity to bind to the surface subunit
HIV-1 Env glycoprotein 120 (HIV-1 gp120) (Curtis et al., 1992).
DC-SIGN mediates HIV binding and internalization into DC conferring
to these cells the ability to transmit HIV to permissive CD4+ T
cells independently from HIV-1 replication (Geijtenbeek et al.,
2000b). These findings suggest that DC-SIGN efficiently captures
HIV-1 at mucosal sites of inoculation and facilitates its transport
to sites of infection by using the migratory ability of DC towards
lymphoid organs (Banchereau and Steinman, 1998). A homologue of
DC-SIGN, DC-SIGNR, was recently identified on the surface of
endothelial cells and shown to display the same HIV-1 binding and
trans-infection enhancement capacities shown by DC-SIGN (Bashirova
et al., 2001; Pohlmann et. al., 2001b). It has been suggested that
the DC-SIGN lectin may act as a receptor for other glycan ligands
present on other viral envelopes and on the cell walls of other
microbes, or even tumor cells (Steinman, 2000). The putative role
of DC-SIGN or DC-SIGNR in Herpesvirus attachment to DC or
endothelial cells has never been reported.
[0012] There exists a need in the art to develop methods and
compositions for modulating the specific binding of effector
molecules to the DC-SIGN receptor, for example on the dendritic
cells of mammals. Such methods and compositions are needed, for
example, to prevent and treat diseases such as viral infections;
for example CMV infections. In this regard, there is a need to
identify cell proteins involved in viral attachment and/or fusion.
Additionally, methods and compositions are needed that allow the
specific targeting of cells expressing DC-SIGN receptor, such as
dendritic cells or alveolar macrophages, to aid in therapy or
diagnosis.
SUMMARY OF THE INVENTION
[0013] The inventors analyzed the mechanisms of CMV attachment to
DC and the role of DC-SIGN in this process. They demonstrated that
CMV is able to bind DC and DC-SIGN-expressing THP-1 cells through
direct interaction of DC-SIGN with viral envelope CMV gB. Without
in any way limiting the invention, the inventors believe that this
binding is involved in: (1) the transmission of DC-SIGN-bound
infectious viral particles to different permissive cells and (2) an
enhanced infection and CMV replication in DC and DC-SIGN-expressing
THP-1 cells.
[0014] Accordingly, this invention identifies DC-SIGN as a receptor
involved in the binding of viruses other than HIV and Ebola virus
to dendritic cells. The invention further provides a number of
novel methods and compositions for treating diseases of mammals,
including viral infections.
[0015] A first object of the invention is to provide a method of
preventing or treating a disease of a mammal, where at least one
symptom of the disease is mediated at least in part by the binding
of an effector molecule to a DC-SIGN receptor of the mammal to be
treated, and where the method comprises administering to the mammal
an amount of a DC-SIGN modulator sufficient to substantially
modulate the binding of the effector molecule to the DC-SIGN
receptor to thereby prevent or treat the disease.
[0016] Another object of the invention is to provide a method of
preventing or treating a disease of a mammal, where at least one
symptom of the disease is mediated at least in part by the binding
of an effector molecule to a DC-SIGN receptor of the mammal to be
treated, and where the method comprises administering to the mammal
an amount of a DC-SIGN blocker sufficient to substantially inhibit
the binding of the effector molecule to the DC-SIGN receptor to
thereby prevent or treat the disease.
[0017] In some embodiments the DC-SIGN blocker is a blocking
derivative of the effector molecule. In other embodiments the
DC-SIGN blocker is an antibody.
[0018] Among embodiments of the invention where the DC-SIGN blocker
is an antibody are included embodiments where the antibody
specifically binds DC-SIGN and embodiments where the antibody
specifically binds the effector molecule.
[0019] In some embodiments the DC-SIGN blocker is a mannosylated
molecule that binds to a DC-SIGN receptor. The mannosylated
molecule may be mannan.
[0020] A further object of the invention is to provide a method of
preventing or treating a viral infection of a mammal, where the
viral infection is mediated at least in part by the binding of a
viral effector molecule to a DC-SIGN receptor of the mammal to be
treated, where the method comprises administering to the mammal an
amount of a DC-SIGN modulator sufficient to substantially modulate
the binding of the viral effector molecule to the DC-SIGN receptor
to thereby prevent or treat the viral infection.
[0021] Another object of the invention relates to preventing or
treating a viral infection of a mammal, where the viral infection
is mediated at least in part by the binding of a viral effector
molecule to a DC-SIGN receptor of the mammal to be treated, and
where the method comprises administering to the mammal an amount of
a DC-SIGN blocker sufficient to substantially inhibit the binding
of the viral effector molecule to the DC-SIGN receptor to thereby
prevent or treat the viral infection.
[0022] In some embodiments of the method, the DC-SIGN blocker
comprises a binding moiety of the viral effector molecule. In other
embodiments, the DC-SIGN blocker comprises a binding moiety of a
viral envelope glycoprotein. In other embodiments, the DC-SIGN
blocker is an antibody. The antibody may specifically bind DC-SIGN
or specifically bind the viral effector molecule. In additional
embodiments the DC-SIGN blocker is a mannosylated molecule that
binds to a DC-SIGN receptor. The mannosylated molecule may be
mannan.
[0023] Among embodiments of the invention in which the DC-SIGN
blocker is an antibody are included embodiments in which the
antibody is a monoclonal antibody; the mammal is a human and the
antibody is a monoclonal antibody that is humanized; the antibody
specifically binds DC-SIGN; the monoclonal antibody is mAb
1B10.2.6; the antibody specifically binds the viral effector
molecule; and the antibody specifically binds the binding moiety of
the viral effector molecule.
[0024] In further embodiments of the method, the viral effector
molecule is a molecular constituent of the viral envelope. In
certain embodiments, the molecular constituent of the viral
envelope is an envelope glycoprotein.
[0025] In additional embodiments of the method, the DC-SIGN blocker
comprises a binding moiety of the viral effector molecule. In some
embodiments of the invention in which the viral effector molecule
is a molecular constituent of the viral envelope, the DC-SIGN
blocker that is used comprises a binding moiety of the envelope
glycoprotein.
[0026] In a preferred aspect of the invention, the viral infection
is a CMV infection and the viral effector molecule is a CMV
effector molecule. In a further preferred aspect the mammal is a
human. In some embodiments the CMV effector molecule is a molecular
constituent of the CMV envelope. In further embodiments, the
molecular constituent of the CMV envelope is a CMV envelope
glycoprotein. In yet further embodiments, the CMV envelope
glycoprotein is CMV envelope glycoprotein B.
[0027] Included among embodiments of the invention in which the
viral infection is a CMV infection and the viral effector molecule
is a CMV effector molecule are embodiments where the DC-SIGN
blocker comprises a binding moiety of the CMV effector molecule;
the DC-SIGN blocker comprises a binding moiety of the CMV envelope
glycoprotein B; the DC-SIGN blocker is a recombinantly produced
protein; and the DC-SIGN blocker is an antibody. Among embodiments
where the DC-SIGN blocker is an antibody are embodiments where the
antibody is a monoclonal antibody; the mammal is a human and the
monoclonal antibody is humanized; the antibody specifically binds
DC-SIGN; the monoclonal antibody is mAb 1B10.2.6; and the antibody
specifically binds the CMV effector molecule. Among embodiments
where the antibody specifically binds the CMV effector molecule are
embodiments where the CMV effector molecule is CMV envelope
glycoprotein B.
[0028] In a further aspect, the invention provides a method of
preventing or treating an Ebola, HIV, or SIV infection of a human
or a simian, where the method comprises administering to the human
or simian an amount of a DC-SIGN modulator sufficient to
substantially modulate the binding of HIV or SIV to the DC-SIGN
receptor present on dendritic cells of the human or simian to
thereby prevent or treat the HIV or SIV infection.
[0029] In another aspect, the invention provides a method of
preventing or treating an Ebola, HIV, or SIV infection of a human
or a simian, where the method comprises administering to the human
or simian an amount of a DC-SIGN blocker sufficient to
substantially inhibit the binding of HIV or SIV to the DC-SIGN
receptor present on dendritic cells of the human or simian to
thereby prevent or treat the HIV or SIV infection. In a preferred
embodiment, the DC-SIGN blocker comprises a binding moiety of the
CMV envelope glycoprotein B. In another preferred embodiment an HIV
infection of a human is prevented or treated.
[0030] In a further aspect, the invention provides a method of
preventing or treating an Ebola, HIV, or SIV infection of a human
or a simian, where the method comprises administering to the human
or simian an amount of a DC-SIGN modulator sufficient to
substantially modulate the binding of HIV or SIV to the DC-SIGN
receptor present on dendritic cells of the human or simian to
thereby prevent or treat the HIV or SIV infection.
[0031] In another aspect, the invention provides a method of
preventing or treating an Ebola, HIV, or SIV infection of a human
or a simian, where the method comprises administering to the human
or simian an amount of a DC-SIGN blocker sufficient to
substantially inhibit the binding of HIV or SIV to the DC-SIGN
receptor present on dendritic cells of the human or simian to
thereby prevent or treat the HIV or SIV infection. In a preferred
embodiment the DC-SIGN blocker comprises a binding moiety of the
CMV envelope glycoprotein B. In another preferred embodiment, an
HIV infection of a human is prevented or treated.
[0032] In a further aspect, the invention provides a pharmaceutical
composition comprising:
[0033] a) a DC-SIGN modulator, and
[0034] b) at least one pharmaceutically acceptable excipient;
[0035] wherein the DC-SIGN blocker is present in the composition at
an achievable therapeutic concentration.
[0036] In a further aspect, the invention provides a pharmaceutical
composition comprising:
[0037] c) a DC-SIGN blocker, and
[0038] d) at least one pharmaceutically acceptable excipient:
[0039] wherein the DC-SIGN blocker is present in the composition at
an achievable therapeutic concentration.
[0040] In some embodiments of the pharmaceutical composition, the
DC-SIGN blocker is a derivative of a viral effector molecule. In
one embodiment, the DC-SIGN blocker comprises the binding moiety of
a CMV effector molecule. In another embodiment, the CMV effector
molecule is CMV envelope glycoprotein B.
[0041] In other embodiments of the pharmaceutical composition, the
DC-SIGN blocker is an antibody. Embodiments where the DC-SIGN
blocker is an antibody include embodiments where the antibody is a
monoclonal antibody; the monoclonal antibody is humanized; the
antibody specifically binds DC-SIGN; the monoclonal antibody is mAb
1B10.2.6; the antibody specifically binds the viral effector
molecule; or the antibody specifically binds the binding moiety of
the viral effector molecule.
[0042] In a further aspect, the invention provides a method of
identifying a DC-SIGN modulator, wherein the method comprises:
[0043] a) determining a baseline binding value by: [0044] i.
providing cultured cells comprising a DC-SIGN receptor; [0045] ii.
exposing the cultured cells to a marked viral effector molecule
binding moiety for a period of time sufficient to allow binding
equilibrium to be reached; and [0046] iii. determining the extent
of binding of the marked viral effector molecule binding moiety to
the cultured cells to thereby determine a baseline binding
value;
[0047] b) determining a test substance binding value by: [0048] i.
providing cultured cells comprising a DC-SIGN receptor; [0049] ii.
exposing the cultured cells to a marked viral effector molecule
binding moiety in the presence of a test substance for a period of
time sufficient to allow binding equilibrium to be reached; and
[0050] iii. determining the extent of binding of the marked viral
effector molecule binding moiety to the cultured cells to thereby
determine a test substance binding value; and
[0051] c) determining a test substance binding modulation value for
the test substance by dividing the test substance binding value by
the baseline binding value,
[0052] wherein a test substance binding inhibition value
representing an about 95% modulation of binding of the viral
effector molecule to dendritic cells by the test substance
indicates that the test substance is a substance that substantially
modulates the binding of a viral effector molecule to the DC-SIGN
receptor.
[0053] In a preferred aspect, the invention provides a method of
identifying a DC-SIGN blocker, wherein the method comprises:
[0054] a) determining a baseline binding value by: [0055] i.
providing cultured cells comprising a DC-SIGN receptor; [0056] ii.
exposing the cultured cells to a marked viral effector molecule
binding moiety for a period of time sufficient to allow binding
equilibrium to be reached; and [0057] iii. determining the extent
of binding of the marked viral effector molecule binding moiety to
the cultured cells to thereby determine a baseline binding
value;
[0058] b) determining a test substance binding value by: [0059] i.
providing cultured cells comprising a DC-SIGN receptor; [0060] ii.
exposing the cultured cells to a marked viral effector molecule
binding moiety in the presence of a test substance for a period of
time sufficient to allow binding equilibrium to be reached; and
[0061] iii. determining the extent of binding of the marked viral
effector molecule binding moiety to the cultured cells to thereby
determine a test substance binding value; and
[0062] c) determining a test substance binding inhibition value for
the test substance by dividing the test substance binding value by
the baseline binding value,
[0063] wherein a test substance binding inhibition value
representing an about 95% inhibition of binding of the viral
effector molecule to dendritic cells by the test substance
indicates that the test substance is a substance that substantially
inhibits the binding of a viral effector molecule to the DC-SIGN
receptor.
[0064] The method of identifying a DC-SIGN blocker includes
embodiments where the cultured cells are DC; the cultured cells are
THP-1 cells; the viral effector molecule is a CMV effector
molecule; and the CMV effector molecule is CMV envelope
glycoprotein B.
[0065] In a further aspect, the invention provides an isolated
DC-SIGN blocker identified by the above method of identifying a
DC-SIGN blocker.
[0066] In another aspect, the invention provides a method of
targeting a subject molecule to a cell expressing a DC-SIGN
receptor by exposing the cell to a targeting complex, where the
targeting complex comprises a subject molecule and a DC-SIGN
blocker, and where the exposure is under conditions which allow the
DC-SIGN blocker to bind to DC-SIGN on the cell expressing the
DC-SIGN receptor, thereby targeting the subject molecule to the
cell expressing a DC-SIGN receptor.
[0067] The method of targeting a subject molecule to a cell
expressing a DC-SIGN receptor includes embodiments where the
DC-SIGN blocker is an antibody; the DC-SIGN blocker is a monoclonal
antibody; the subject molecule is a protein; the subject molecule
is an antibody; the subject molecule is labeled; the exposure
occurs in vivo; and the exposure occurs in vitro.
[0068] In a further aspect, the invention provides an isolated
antibody, wherein the isolated antibody specifically binds DC-SIGN.
In one embodiment, the antibody is a DC-SIGN modulator. In a
preferred embodiment, the antibody is a DC-SIGN blocker. In a
further embodiment, the invention provides an isolated monoclonal
antibody, wherein the isolated monoclonal antibody specifically
binds DC-SIGN. In one embodiment, the monoclonal antibody is a
DC-SIGN modulator. In a preferred embodiment, the monoclonal
antibody is a DC-SIGN blocker. In the further preferred embodiment,
the isolated monoclonal antibody is mAb 1B10.2.6, produced by
hybridoma 1B10.2.6, deposited at the C.N.C.M. on Nov. 7, 2002,
under the accession number 1-2951.
BRIEF DESCRIPTION OF THE DRAWINGS
[0069] The invention will be more fully described with reference to
the drawings in which:
[0070] FIG. 1 shows that DC-SIGN-expressing cells bind CMV on their
surface. (A) Detection of DC-SIGN. Anti-DC-SIGN 1B10.2.6 mAb (bold
line). Irrelevant isotypic control mAb (dotted line). Mean
fluorescence intensity (MFI) values are indicated. (B) Binding of
CMV AD169 strain to cells expressing or not DC-SIGN was revealed by
an anti-envelope glycoprotein B (CMV gB) mAb. Incubation in low pH
buffer (200 PFU/cell) prior to staining abrogates detection of CMV
gB.
[0071] FIG. 2 depicts DC-SIGN-dependent trans-infection of CMV
permissive cells. (A) MD-DC, (B) DC-SIGN.sup.+ vs parental THP-1
cells or (C) parental vs DC-SIGN.sup.+ Hela cells were pre-treated
with EGTA (5 mM), mannan (20, 1, 0.05 .mu.g/ml), anti-DC-SIGN
1B10.2.6 mAb (20, 1, 0.05 .mu.g/ml) or an isotypic control (20, 1,
0.05 .mu.g/ml) prior to incubation with ADGFP strain (1 PFU/cell).
After removal of unbound virus and competitors, ADGFP-challenged
cells were co-cultured for 3 days with MRC-5 cells. Infection of
MRC-5 cells was assessed by counting the number of GFP-expressing
cells by flow cytometry.
[0072] FIG. 3 shows that DC-SIGN enhances CMV trans-infection of
MRC-5 cells and retains long term infectious virus. (A) MD-DC, (B)
parental or DC-SIGN.sup.+ THP-1 cells, were incubated with ADGFP in
the absence or in the presence either of anti-DC-SIGN 1B1 0.2.6 mAb
or an isotypic, control mAb. Thereafter, cells were cocultured with
reporter MRC-5 cells. (C) DC-SIGN.sup.+ and parental THP-1 cells
were incubated (4 hours) with ADGFP (MOI=1) and washed thereafter.
At days 0, 2, 4, 6, and 8 after pulse, ADGFP-challenged cells were
co-cultured with reporter MRC-5 cells for 3 days. In (A), (B), and
(C), MRC-5 cells were incubated with the corresponding amount of
cell-free virus to monitor kinetic and extent of infection. Values
represent the percentage of MRC-5 cells expressing GFP.
[0073] FIG. 4 shows that DC-SIGN mediates CMV transmission to
different permissive cells but does not allow transmission of HSV-1
and VZV. (A) and (B), DC-SIGN.sup.+ THP-1 cells were exposed either
to anti-DC-SIGN 1B10.2.6 mAb or an isotypic control mAb prior to
infection with ADGFP. ADGFP-pulsed DC-SIGN.sup.+ THP-1 cells were
co-cultured for 24 hours either with MRC-5 cells (A) or U373 MG
cells (B). GFP- (grey bars) or CMV/EA/EA-expressing cells (open
bars) were counted to evaluate CMV infection. (C) DC-SIGN.sup.+
THP-1 cells incubated with anti-DC-SIGN 1B10.2.6 mAb (open bars),
an isotypic control mAb (dashed bars) or left untreated (grey bars)
were exposed for 1 hour to TB40/E, AD169 or Towne CMV isolates,
HSV-1, or VZV. After removal of unbound virus and mAb, cells were
cocultured with the MRC-5 reporter cell line for 5 days. (D) MRC-5
cells were exposed for 5 days to the identical amount of cell-free
viruses as used in (C) to infect DC-SIGN.sup.+ THP-1 cells.
IEA/EA-expressing cells were detected by immunocytochemistry using
specific antibodies for each herpesvirus.
[0074] FIG. 5 shows that the DC-SIGN cytoplasmic domain is required
for CMV transmission. (A) Cell surface expression of wild type (wt)
or mutated DC-SIGN (.DELTA.35 and .DELTA.20) receptors analyzed by
immunostaining (1B10.2.6 mAb) and flow cytometry. (8) CMV-binding
capacity of THP-1 expressing wt or truncated DC-SIGN revealed by
anti-CMV gB mAb. (C) Parental, or DC-SIGN.sup.+ cells were
incubated, either at 4.degree. C. or at 37.degree. C. for 2 hours,
with ADGFP CMV (MOI=0.1) and co-cultured with MRC-5 cells for 3
days. Infection was assessed by estimating the number of
GFP-expressing cells.
[0075] FIG. 6 shows that DC-SIGN expression renders susceptible
cells permissive to CMV infection. A) Cells were pre-treated for 30
minutes with medium (grey bars), anti-DC-SIGN 1B10.2.6 mAb (open
bars) or an isotypic control mAb (dashed bars) and thereafter
incubated with ADGFP strain (1 PFU/cell). HEK 293T cells were
transiently transfected either with a DC-SIGN cDNA plasmid or a
control plasmid (pcDNA3.1). (B) HEK 293T cells were transiently
transduced with DC-SIGN or DC-SIGNR cDNAs and incubated with ADGFP.
In (A) and (B), GFP-expressing cells were quantified by flow
cytometry at day 3 after infection. (C) THP-1, DC-SIGN.sup.+ THP-1
cells or MD-DC were pre-treated as described in (A) (same symbols)
and infected with TB40/E CMV (MOI=1) for 3 days. CMV infection was
assessed by immunostaining with specific CMV IEA/EA mAb. The total
number of CMV IEA/EA-positive cells on the slide was determined by
extrapolating the number of positive cells contained in the optical
field of the microscope (.times.10 objective). (D) MD-DC, MRC-5,
parental and DC-SIGN.sup.+ THP-1 cells were pre-treated and
infected as described in (C). Non-internalized viral particles were
removed by short incubation in a low pH buffer. At day 14 after
infection, virions released in culture supernatants were titrated
on MRC-5 cells by plaque assays. Figures on top of histograms
indicate infection fold amplification which result from dividing
the absolute number of CMV particles collected in supernatants by
the absolute number of CMV particles used to infect cells (20,000
PFU).
[0076] FIG. 7 depicts data that identify CMV gB as a viral ligand
for DC-SIGN and characterization of the DC-SIGN/CMV gB interaction
by SPR. (A) Binding of CMV gB to DC-SIGN. DC-SIGN.sup.+ THP-1 cells
or MD-DC were pre-treated with medium (black closed circles),
anti-DC-SIGN 1B10.2.6 mAb (open circles) or an isotypic, control
mAb (grey closed circles), and thereafter incubated with
biotinylated CMV gB. Cell-bound CMV gB was revealed by PE-labelled
streptavidin. Incubation of parental THP-1 cells with biotinylated
CMV gB is also shown (black closed triangles). (B) Competition
assay of biotinylated HIV-1 gp120 binding to DC-SIGN. Parental
(upper panel) or DC-SIGN.sup.+ THP-1 cells (all other panels) were
incubated with 2 Pg/mL of biotinylated HIV-1 gp120. DC-SIGN.sup.+
THP-1 cells were left untreated or pre-incubated with potential
competitors (unlabelled HIV-1 gp120, mannan, anti-DC-SIGN 1B10.2.6
mAb, control isotypic mAb, or envelope glycoproteins from CMV gB,
HSV-1 gB and gD, or VZV gE and gB) before incubation with
biotinylated HIV-1 gp120. MFI of biotinylated HIV-1 gp120 staining
is indicated in the upper-right corner of the histograms. In each
panel, control staining (dotted line), biotinylated HIV-1 gp120
labelling in the absence of competitor (gray-filled profile) or
after pre-incubation with competitors (black-filled histogram), are
shown. (C) Binding of CMV gB to DC-SIGNR. HEK 293T cells were
transiently transfected either with a control plasmid or plasmids
encoding DC-SIGN or DC-SIGNR cDNAs. Transfected cells were
incubated with increasing concentrations of biotinylated-CMV gB
(dashed bars), biotinylated-HIV-1 gp120 (black bars) or
biotinylated-BSA (open bars). Binding of biotinylated proteins was
revealed by PE-conjugated streptavidin and analyzed by flow
cytometry. Values are represented as MFI. (D) SPR analysis of
DC-SIGN/CMV gB interaction. The recombinant soluble CRD of DC-SIGN
at (from bottom to top) 0.13, 0.21, 0.36, 0.6 or 1 .mu.M was
injected over surfaces coated with HIV-1 gp120 (left panel), CMV gB
(middle panel) or HSV-1 gB (right panel) to analyze the association
phase, after which running, buffer alone was injected to analyze
the dissociation phase. Binding responses (Response Unit, RU) are
reported as a function of time. Dissociation constants (Kd) are
indicated for left and middle panels.
DETAILED DESCRIPTION OF THE INVENTION
[0077] This invention relates to a method of preventing or treating
a disease of a mammal, where at least one symptom of the disease is
mediated at least in part by the binding of an effector molecule to
a DC-SIGN receptor of the mammal to be treated. The method
comprises administering to the mammal an amount of a DC-SIGN
blocker sufficient to substantially inhibit the binding of the
effector molecule to the DC-SIGN receptor to thereby prevent or
treat the disease.
[0078] "Mammal" for purposes of the invention refers to any animal
classified within the class mammalia. Nonlimiting examples of
mammals include: humans and simians; pet animals, such as dogs,
cats, ferrets, and guinea pigs; farm animals, such as pigs, cows,
horses, sheep, goats, and llamas; and zoo animals, such as bears,
zebras, elephants, and water buffalo. The mammal is preferably
human.
[0079] As used herein a "disease" is any pathological condition of
a mammal, which results, for example, from infection, genetic
defect, or exposure to a substance in the environment. The methods
and compositions of the invention are useful for preventing or
treating diseases that are characterized in that at least one
symptom of the disease is mediated at least in part by the binding
of an effector molecule to the DC-SIGN receptor present on cells
such as dendritic cells or alveolar macrophages of the mammal.
Specific examples of such diseases include viral infection. A
specific example of viral infections that can be treated by the
method is CMV infection of a human.
[0080] In the case of humans, "DC-Specific ICAM-Grabbing
Nonintegrin receptor" or "DC-SIGN receptor" refers generically to
DC-SIGN (described in Curtis et al., 1992) and/or DC-SIGNR
(described in Pohlmann et al., 2001.), and/or a homologue of
DC-SIGN or DC-SIGNR. One of skill in the art will recognize that
there may be some situations in which use of one or the other of
these forms of DC-SIGN receptor is preferable or even necessary.
One of skill in the art will recognize that human DC-SIGN protein
can be obtained from many sources. For example, human DC-SIGN can
be purified from human dendritic cells which are obtained from an
in vivo source, such as human blood, or purified from an in vitro
source, such as human dendritic cells produced in tissue culture
from human dendritic cell precursor cells. It is also possible to
express human DC-SIGN using a recombinant system, using either a
cultured dendritic cell as a host or a suitable heterologous cell
type, such as COS-7 or HeLa cells, or bacteria such as E. coli.
[0081] In the case of nonhuman mammals, "DC-SIGN receptor" refers
to homologues of a human DC-SIGN receptor. One of skill in the art
will recognize that such proteins may be identified in any of a
number of different ways. These include expression cloning,
polymerase chain reaction using degenerate oligonucleotide primers,
and low stringency screening of a bacterial or bacteriophage
library.
[0082] Dendritic cells are a diverse population of morphologically
similar cell types found in lymphoid or non-lymphoid tissues.
Dendritic cells function as antigen-presenting cells that
efficiently capture antigens in the peripheral tissues and process
them to form MHC-peptide complexes. Dendritic cells are also
involved in the early activation of non-MHC-restricted
.gamma..delta. and CDI-restricted T cells specific for various
mycobacterial glycolipids, including CAM (Kaufmann, 2001 and Moody,
et al., 2000). After antigen uptake, these immature dendritic cells
acquire the unique capacity to migrate from the periphery to the T
cell areas of the secondary lymphoid organs. Dendritic cells
convert antigens from foreign cells and infectious microorganisms
into short peptides that are bound to membrane proteins of the
major histocompatibility complex (MHC). These MHC-peptide complexes
are formed intracellularly but are ultimately presented on the
plasma membrane where they serve as ligands for antigen-specific T
cell receptors (TCR). In addition to TCR ligand formation,
dendritic cells carry out many other functions, which allow them to
control immunity at several points (Steinman, 2000).
[0083] Alveolar macrophages and dendritic cells are examples of
cells expressing a DC-SIGN receptor. Endothelial cells are an
example of cells expressing DC-SIGNR.
[0084] One of skill in the art will appreciate that dendritic cells
may be obtained from an in vivo source, such as the blood of a
mammal, or grown in vitro, by culturing dendritic cell precursor
cells under appropriate conditions. Dendritic cell precursor cells
include monocytes prepared according to Example 3.
[0085] An "effector molecule" is any molecule that specifically
binds to the DC-SIGN receptor present on cells of a mammal, such as
the dendritic cells or the alveolar macrophages of a mammal, and
thereby mediates a symptom that is associated with a disease of
that mammal. Examples of effector molecules are ligands present on
viruses that bind to receptors on cells of a mammal and thereby
facilitate the entry of the virus into a cell of the mammal. In
cases where the effector molecules are ligands present on viruses,
the effector molecules can be referred to as "viral effector
molecules." Examples of this type of ligand include gp120 of HIV
and envelope glycoprotein B of CMV, which bind with the DC-SIGN
receptor present on cells such as dendritic cells or alveolar
macrophages of a human to facilitate, in the case of CMV, the
transmission of DC-SIGN-bound infectious viral particles to
different permissive cells and an enhanced infection and CMV
replication in DC and DC-SIGN-expressing THP-1 cells. CMV envelope
glycoprotein B is thus a "CMV effector molecule." Other types of
effector molecules are ligands that are endogenous to the mammal.
This type of ligand includes both ligands that are bound to the
surface of other cells of the mammal and soluble ligands, which may
be localized to the extracellular space of a particular tissue or
circulating systemically.
[0086] A "symptom" is any pathological manifestation of the disease
to be treated. A symptom is caused at least in part by the binding
of an effector molecule to the DC-SIGN receptor present on the
dendritic cells of the mammal to be treated if a modulation (a
reduction or an increase) in the binding of the effector molecule
to the DC-SIGN receptor causes a determinable reduction in the
occurrence or severity of the symptom, or both. In a preferred
embodiment of the invention the symptom is no longer present or is
prevented from occurring following the reduction in the binding of
the effector molecule to the DC-SIGN receptor.
[0087] An effector molecule is said to "specifically bind" to the
DC-SIGN receptor present on cells such as the dendritic cells or
the alveolar macrophages of the mammal to be treated if such
binding is not competitively inhibited by the presence of unrelated
molecules (e.g., fetal calf serum), but is inhibited by antibodies
to DC-SIGN (e.g., 1B10.2.6) and/or additional effector
molecules.
[0088] An example of an effector molecule that specifically binds
to the DC-SIGN receptor present on cells such as the dendritic
cells or the alveolar macrophages of a mammal to be treated is CMV
envelope glycoprotein B.
[0089] One of skill in the art will appreciate that these assays
may be used to identify other effector molecules that specifically
bind to the DC-SIGN receptor present on cells such as dendritic
cells of a mammal to be treated. It will also be clear to one of
skill in the art that other equivalent assays may be substituted
for those specifically disclosed in the Examples.
[0090] Once an effector molecule is known to specifically bind to
the DC-SIGN receptor, the binding of the effector molecule to
DC-SIGN can be referred to simply as "binding." It will be
understood by one of skill in the art that such binding is
specific. In this regard, the "modulation" of binding may be
discussed. Modulation can include "inhibition" or
"enhancement."
[0091] "Modulation" means the act of regulating. It includes the
act of inducing variations of a property of a molecule. In the
context of the present invention, "modulation" means the act of
regulating and varying the binding of effector molecules to their
receptors. This modulation may serve to either inhibit or enhance
binding, or to impose other regulatory controls.
[0092] In the context of the present invention "inhibition" of
binding means a reduction in the total amount of effector molecule
that binds to DC-SIGN over a fixed period of time. Inhibition of
binding of the effector molecule is achieved by providing a DC-SIGN
blocker. A "DC-SIGN blocker" is any molecule that substantially
inhibits the binding of a given effector molecule at a
concentration at which the effector molecule specifically binds to
DC-SIGN. In a preferred embodiment, the DC-SIGN blocker used is a
monoclonal antibody that specifically binds DC-SIGN. In another
preferred embodiment, the DC-SIGN blocker used comprises a binding
moiety of the CMV envelope glycoprotein B.
[0093] In the context of the present invention, "enhancement" of
binding means an increase in the total amount of effector molecule
that binds to DC-SIGN over a fixed period of time. Enhancement of
binding of the effector molecule is achieved by providing a DC-SIGN
enhancer. A "DC-SIGN enhancer" is any molecule that substantially
enhances the binding of a given effector molecule at a
concentration at which the effector molecule specifically binds to
DC-SIGN.
[0094] A "binding moiety" is that portion of a molecule that
substantially retains the ability to bind to a second molecule when
other portions of the molecule are removed or modified or when the
binding moiety is placed into a heterologous context. For example,
in the case of an effector molecule as defined herein, a binding
moiety of the effector molecule can be defined. A binding moiety of
an effector molecule is that portion of the effector molecule that
substantially retains the ability to bind to DC-SIGN when other
portions of the molecule are removed or modified or when the
binding moiety is placed into a heterologous context. In this
context, "substantially retains" can be defined by one of skill
based on the specific properties of the binding moiety that are
sought.
[0095] "Substantially inhibit" means greater than 80% inhibition,
greater than 90% inhibition, greater than 95% inhibition, or
greater than 99% inhibition. In a preferred embodiment of the
present invention, about 90% binding inhibition is obtained.
[0096] "Inhibition" is measured by comparing the extent of effector
molecule binding to DC-SIGN in the presence of a DC-SIGN blocker
with the extent of effector molecule binding to DC-SIGN in the
absence of a DC-SIGN blocker. The ratio of extent of binding in the
presence of the DC-SIGN blocker compared to the extent of binding
in the absence of the DC-SING blocker is then determined. The
percent inhibition is then the proportional reduction in the amount
of binding. For example, a ratio of 0.1 represents a 90% reduction
in binding.
[0097] The term "treat," "treating," or "treatment" refers to the
administration of therapy to an individual who already manifests at
least one symptom of a disease. Such an individual includes an
individual who is diagnosed as having a known disease.
[0098] The term "prevent," "preventing," or "prevent" refers to the
administration of therapy on a prophylactic or preventative basis
to an individual who may ultimately acquire the disease but who has
not yet done so (i.e., those needing preventative measures). Such
individuals may be identified on the basis of risk factors that are
known to correlate with the subsequent occurrence of the
disease.
[0099] The term "therapeutic benefit" refers to an improvement of
at least one symptom of a disease, a slowing of the progression of
a disease, as manifested by a slowing in the increase in severity
of at least one symptom of a disease, or a cessation in the
progression of at least one symptom of a disease. The therapeutic
benefit is determined by comparing a symptom of a disease before
and after a DC-SIGN blocker is administered.
[0100] The term "antibody" refers to any antibody that can be made
by any technique known in the art. Suitable antibodies are obtained
by immunizing a host animal with peptides comprising all or a
portion of the target protein. Suitable host animals include a
mouse, a rat, sheep, a goat, a hamster, a rabbit, etc. The origin
of the protein immunogen may be a mammal such as a mouse, human,
rat, monkey, etc. or a microorganism, including a bacteria or a
virus. The host animal will generally be a different species than
the immunogen, e.g. human protein used to immunize mice, etc.
[0101] The immunogen may comprise the complete protein, or
fragments and derivatives thereof. Preferred immunogens comprise
all or a part of one of the subject proteins, where these residues
contain the post-translation modifications, such as glycosylation,
found on the native target protein. Immunogens comprising the
extracellular domain are produced in a variety of ways known in the
art, e.g. expression of cloned genes using conventional recombinant
methods, isolation from tumor cell culture supernatants, etc.
[0102] For preparation of polyclonal antibodies, the first step is
immunization of the host animal with the target protein, where the
target protein will preferably be in substantially pure form,
comprising less than about 1% contaminant. The immunogen may
comprise the complete target protein, fragments, or derivatives
thereof. To increase the immune response of the host animal, the
target protein may be combined with an adjuvant, where suitable
adjuvants include alum, dextran, sulfate, large polymeric anions,
oil & water emulsions, e.g., Freund's adjuvant, Freund's
complete adjuvant, and the like. The target protein may also be
conjugated to synthetic carrier proteins or synthetic antigens. A
variety of hosts may be immunized to produce the polyclonal
antibodies. Such hosts include rabbits, guinea pigs, rodents such
as mice or rats, sheep, goats, and the like. The target protein is
administered to the host, usually intradermally, with an initial
dosage followed by one or more, usually at least two, additional
booster dosages. Following immunization, the blood from the host
will be collected, followed by separation of the serum from the
blood cells. The immunoglobulin (Ig) present in the resultant
antiserum may be further fractionated using known methods, such as
ammonium salt fractionation, DEAE chromatography, and the like.
[0103] Monoclonal antibodies are produced by conventional
techniques. Generally, the spleen and/or lymph nodes of an
immunized host animal provide a source of plasma cells. The plasma
cells are immortalized by fusion with myeloma cells to produce
hybridoma cells. Culture supernatant from individual hybridomas is
screened using standard techniques to identify those producing
antibodies with the desired specificity. Suitable animals for
production of monoclonal antibodies to the human protein include
mouse, rat, hamster, etc. To raise antibodies against a mouse
protein, the animal will generally be a hamster, guinea pig,
rabbit, etc. The antibody may be purified from the hybridoma cell
supernatants or ascites fluid by conventional techniques, e.g.,
affinity chromatography using protein according to the subject
invention bound to an insoluble support, protein A sepharose,
etc.
[0104] The antibody may be produced as a single chain, instead of
the normal multimeric structure. Single chain antibodies are
described in Jost et al. (1994) J.B.C. 269:26267-73, and others.
DNA sequences encoding the variable region of the heavy chain and
the variable region of the light chain are ligated to a spacer
encoding at least about 4 amino acids of small neutral amino acids,
including glycine and/or serine. The protein encoded by this fusion
allows assembly of a functional variable region that retains the
specificity and affinity of the original antibody.
[0105] Also provided are "artificial" antibodies, e.g., antibodies
and antibody fragments produced and selected in vitro. In some
embodiments, such antibodies are displayed on the surface of a
bacteriophage or other viral particle. In many embodiments, such
artificial antibodies are present as fusion proteins with a viral
or bacteriophage structural protein, including, but not limited to,
M13 gene III protein. Methods of producing such artificial
antibodies are well known in the art. See, e.g., U.S. Pat. Nos.
5,516,637; 5,223,409; 5,658,727; 5,667,988; 5,498,538; 5,403,484;
5,571,698; and 5,625,033.
[0106] For in vivo use, particularly for injection into humans, it
is desirable to decrease the antigenicity of the antibody. An
immune response of a recipient against the blocking agent will
potentially decrease the period of time that the therapy is
effective. Methods of humanizing antibodies are known in the art.
The humanized antibody may be the product of an animal having
transgenic human immunoglobulin constant region genes (see for
example International Patent Applications WO 90/10077 and WO
90/04036). Alternatively, the antibody of interest may be
engineered by recombinant DNA techniques to substitute the CH1,
CH2, CH3, hinge domains, and/or the framework domain with the
corresponding human sequence (see WO 92/02190).
[0107] The use of Ig cDNA for construction of chimeric
immunoglobulin genes is known in the art (Liu et al. (1987)
P.N.A.S. 84:3439 and (1987) J. Immunol. 139:3521). mRNA is isolated
from a hybridoma or other cell producing the antibody and used to
produce cDNA. The cDNA of interest may be amplified by the
polymerase chain reaction using specific primers (U.S. Pat. Nos.
4,683,195 and 4,683,202). Alternatively, a library is made and
screened to isolate the sequence of interest. The DNA sequence
encoding the variable region of the antibody is then fused to human
constant region sequences. The sequences of human constant regions
genes may be found in Kabat et al. (1991) Sequences of Proteins of
Immunological Interest, N.I.H. publication no. 91-3242. Human C
region genes are readily available from known clones. The choice of
isotype will be guided by the desired effector functions, such as
complement fixation, or activity in antibodydependent cellular
cytotoxicity. Preferred isotypes are IgG1, IgG3, and IgG4. Either
of the human light chain constant regions, kappa or lambda, may be
used. The chimeric, humanized antibody is then expressed by
conventional methods.
[0108] In yet other embodiments, the antibodies may be fully human
antibodies. For example, xenogeneic antibodies which are identical
to human antibodies may be employed. By xenogenic human antibodies
is meant antibodies that are the same has human antibodies, i.e.,
they are fully human antibodies, with exception that they are
produced using a non-human host, which has been genetically
engineered to express human antibodies. See e.g. WO 98/50433; WO
98/24893 and WO 99/53049, the disclosures of which are herein
incorporated by reference.
[0109] Antibody fragments, such as Fv, F(ab').sub.2, and Fab may be
prepared by cleavage of the intact protein, e.g., by protease or
chemical cleavage. Alternatively, a truncated gene is designed. For
example, a chimeric gene encoding a portion of the F(ab').sub.2
fragment would include DNA sequences encoding the CH1 domain and
hinge region of the heavy (H) chain, followed by a translational
stop codon to yield the truncated molecule.
[0110] Consensus sequences of H and light (L) chain J regions may
be used to design oligonucleotides for use as primers to introduce
useful restriction sites into the J region for subsequent linkage
of variable (V) region segments to human constant (C) region
segments. C region cDNA can be modified by site directed
mutagenesis to place a restriction site at the analogous position
in the human sequence.
[0111] Expression vectors include plasmids, retroviruses, YACs,
EBV-derived episomes, and the like. A convenient vector is one that
encodes a functionally complete human CH or CL immunoglobulin
sequence, with appropriate restriction sites engineered so that any
VH or VL sequence can be easily inserted and expressed. In such
vectors, splicing usually occurs between the splice donor site in
the inserted J region and the splice acceptor site preceding the
human C region, and also at the splice regions that occur within
the human CH exons. Polyadenylation and transcription termination
occur at native chromosomal sites downstream of the coding regions.
The resulting chimeric antibody may be joined to any strong
promoter, including retroviral LTRs, e.g. SV-40 early promoter,
(Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcoma virus
LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murine
leukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native
Ig promoters, etc.
[0112] An example of a disease that can be prevented or treated
utilizing the present invention is CMV infection. The results
presented in the examples demonstrate for the first time a role for
DC-SIGN in CMV binding to human dendritic cells.
[0113] The experiment described herein, including the results
described in the examples, provides new insights into the
mechanisms of interaction of CMV with DC and the transmission of
CMV infection to other cell targets. The data show that DC-SIGN
accounts for most of the binding of CMV to DC and mediates the
attachment of CMV virions when expressed in DC-SIGN negative cells.
Interaction of CMV with DC-SIGN occurs through specific binding
with at least one CMV envelope glycoprotein, CMV gB. DC-SIGN is a
type II membrane protein in which the extracellular domain
encompasses the CRD and a stalk that mediates tetramerization
(Mitchell et al., 2001). Like DC-SIGN, CMV gB is also present in
multimeric complexes in the CMV envelope (Scheffczik et al., 2001).
CMV gB-DC-SIGN interactions analyzed by SPR were conducted using
recombinant soluble forms of DC-SIGN CRD, which are monomers. The
affinity of CMV gB for DC-SIGN measured by SPR was 0.3 pM and was
comparable to that estimated for HIV-1 gp120 (Mitchell et al.,
2001). This relatively low affinity is likely due to the inability
of CRD to multimerize. The estimated affinity (Kd) of HIV-1 gp120
for the natural DC-SIGN molecule is 1.4 nM (versus 5 nM for CD4)
(Curtis et al., 1992). These findings suggest that, like HIV-1
gp120, CMV gB would display high affinity for oligomerized
DC-SIGN.
[0114] DC-SIGN-bound CMV retains infectious capacity since, upon
binding onto DC-SIGN.sup.+ THP-1 cells or MD-DC, CMV is transmitted
to permissive cells where the virus replicates actively. DC are
receptive to CMV infection by primary, non-adapted CMV isolates and
refractory to infection by adapted, CMV laboratory strains. The
capacity of DC to transmit CMV to permissive cell targets can be
dissociated from the ability of CMV to infect and replicate in DC.
This result was confirmed using DC-SIGN.sup.+ HeLa cells which were
capable of transmitting CMV to permissive target cells while CMV IE
antigen expression was never detected in these refractory cells.
This DC-SIGN function is reminiscent of the aptitude to transmit
infection to CD4+ T lymphocytes shown either by HIV-1-pulsed-DC or
-DC-SIGN-transduced cells.
[0115] Binding to, and transfer of HIV-1 from, DC-SIGN.sup.+ cells
appear to be separable steps (Pohlmann et al., 2001a). Recently, it
has been shown that efficient transmission of HIV to CD4+ T
lymphocytes from DC-SIGN-expressing THP-1 cells requires
internalization signals encoded in the cytoplasmic domain of the
lectin (Kwon et al., 2002). The requirement of DC-SIGN cytoplasmic
signals for efficient trans-infection (named trans-enhancement)
becomes particularly evident when low amounts of virus are used as
an inoculum (Geijtenbeek et al., 2000b). Similarly to HIV,
sub-optimal inoculums of CMV become highly infectious when
transferred from DC-SIGN.sup.+ THP-1 cells. Moreover, DC-SIGN
.DELTA.35 and .DELTA.20 failed to support CMV transmission to
highly susceptible cells and incubation of CMV with wild type- or
truncated-DC-SIGN-expressing cells at 4.degree. C. prevented CMV
transmission to permissive cells. These findings suggest that
endocytosis of the receptor is required for efficient transmission
of CMV to permissive cells. However, the experiments reported here
do not rule out the involvement of putative transduction of
intracellular signals in this phenomenon since deletion of DC-SIGN
cytoplasmic domains or inhibition of cell signal activation at
4.degree. C. may preclude DC-SIGN-dependent cell activation.
Overall, these findings suggest that in the natural CMV infection,
DC-SIGN promotes take up of CMV and permits enhancement of CMV
transmission by interstitial DC to other cells. The hypothesis of
in vivo CMV transport by DC raised the question of the stability of
DC-SIGN-bound CMV particles. As previously described for HIV, the
experiments described herein provide evidence that DC-SIGN.sup.+
THP-1 cells can transmit CMV to other cell targets after five days
in culture whereas cell-free virus loses infectivity upon
incubation at 37.degree. C. for 24 to 48 hours. The ability of DC
to transmit infection long time after exposure supports the
hypothesis that DC transport small amounts of CMV from entry sites
to target organs where they could transmit infectious CMV particles
by cell-to-cell contact.
[0116] A striking feature of DC-SIGN-CMV interactions is the
capacity of the lectin to facilitate the infection of
low-susceptible cells to CMV infection. Thus, THP-1 cells that do
not normally support CMV replication become productively infected
as they express DC-SIGN. CMV attachment to host cells is supposed
to occur namely through low affinity interactions with heparan
sulfate proteoglycans (Compton et al., 1993; Kari and Gehrz, 1992).
However, beyond this primary site of binding, the existence of an
alternative cellular co-factor, required for a strong attachment of
CMV on cell membranes as well as for its entry into cells, is
postulated (Boyle and Compton, 1998). Annexin II, which binds to
CMV gB (Pietropaolo and Compton, 1997) and a 92.5 kDa protein which
binds to CMV gH (Baldwin et al., 2000), have been proposed to play
this role. It is unlikely that DC-SIGN is the exclusive CMV
receptor that ultimately determines entry of the enveloped virions
and replication in CMV-infection susceptible cells. Indeed, CMV
entry and infection occurs in a number of cell types (i.e., MRC-5
fibroblasts and U373 MG astrocytoma cells) where DC-SIGN is not
expressed. The putative CMV receptor in these cells might be
different from a lectin, although the existence of yet unidentified
DC-SIGN-like molecules accounting for binding and entry of CMV
cannot be formally ruled out.
[0117] The capacity of DC-SIGN to promote in cis CMV replication in
otherwise low-susceptible cells may result from any of three not
mutually exclusive hypotheses. DC-SIGN has the capacity to capture
and internalize HIV-1 in DC (Kwon et al., 2002). By analogy,
DC-SIGN might promote internalization and trafficking of CMV to an
intracellular compartment from where it could initiate the
infectious cycle. Alternatively, attachment of CMV to DC-SIGN, or
DC-SIGNR, might facilitate the interaction with the authentic
cellular receptor, which ultimately would account for CMV entry.
Such a function would be reminiscent of the facilitating effect
shown by DC-SIGN on HIV infection of T lymphocytes displaying low
levels of CCR5 (Lee et al., 2001). Finally, differentiation of
THP-1 cells with TPA was shown to induce permissiveness to CMV
replication (Weinshenker et al., 1988). Similarly, signal
transduction through DC-SIGN could lead to cellular differentiation
and subsequent CMV replication.
[0118] Regarding CMV infection, the in cis capacity of DC-SIGN to
facilitate viral entry is likely of biological relevance since the
blockade by specific anti-DC-SIGN antibodies drastically reduces
infectiveness of DC by primary, CMV isolates. The capacity of DC to
support CMV infection may be related to the amount of DC-SIGN
expressed at their surface. Thus, immature DC which express high
levels of DC-SIGN can be infected by CMV (Raftery et al., 2001;
Riegler et al., 2000) while matured DC that display low DC-SIGN
expression show reduced susceptibility to CMV. Expression of
DC-SIGN on immature DC of intestinal and genital mucosae
(Geijtenbeek et al., 2000b; Jameson et al., 2002) may confer to
this co-factor a crucial role for the infection of these primary
target cells at the anatomical sites where initial CMV transmission
or propagation most probably take place. A recent study described a
monocyte-derived macrophage circulating subset, expressing DC
markers in vivo (Soderberg-Naucler et al., 1997). This subset was
shown to harbor latent CMV which reactivates upon allogeneic
stimulation. It appears necessary to investigate the expression of
DC-SIGN by these cells which could represent a biological link
between this newly identified dendritic-like subset and the results
described herein. As recently reported, CMV-infected DC display
decreased antigen presentation and differentiation capacities
(Andrews et al., 2001; Raftery et al., 2001). Hence, by promoting
DC-mediated trans-infection of target cells as well as
cis-infection of DC, DC-SIGN could be involved, apart from virus
propagation, in CMV-mediated altered immune response.
[0119] The data reported herein show that DC-SIGNR is also able to
bind CMV gB and to promote cis-infection of apparently low
susceptible cells. This DC-SIGN homologue is mainly expressed on EC
(Bashirova et al., 2001; Pohlmann et al., 2001b), which are known
to be preferential targets of CMV in vivo and replicate primary,
non-adapted CMV strains in vitro (Kahl et al., 2000). The
expression of DC-SIGNR on placental EC and macrophages (Soilleux et
al., 2001) could be involved in the materno-fetal transmission of
CMV during congenital infections. Similarly, DC-SIGNR expressed in
liver EC may be implicated in CMV-induced hepatitis, one of the
most frequent clinical forms of this infection.
[0120] Murine CMV shares many essential characteristics with its
human counterpart and has been a widely studied model for CMV
infection. It has been shown that infection of DC by murine CMV
prevents delivery of the signals required for T cell activation.
The impairment of DC functions by murine CMV is supposed to be
detrimental for the host immune responses (Andrews et al., 2001).
The cloning of several homologues of DC-SIGN in mice (Park et al.,
2001), should provide this model with an invaluable tool for
studying the implication of DC-SIGN-like molecules in the dynamic
of CMV dissemination, the role of the different subsets of DC in
the course of CMV propagation, and eventually the causes of
CMV-induced immunosuppression.
[0121] In accordance with these results, the invention provides a
method of preventing or treating a disease of a mammal, where at
least one symptom of the disease is mediated at least in part by
the binding of an effector molecule to a DC-SIGN receptor of the
mammal to be treated, and where the method comprises administering
to the mammal an amount of a DC-SIGN blocker sufficient to
substantially inhibit the binding of the effector molecule to the
DC-SIGN receptor to thereby prevent or treat the disease.
[0122] In some embodiments the DC-SIGN blocker is a blocking
derivative of the effector molecule. In other embodiments the
DC-SIGN blocker is an antibody.
[0123] Among embodiments of the invention where the DC-SIGN blocker
is an antibody are included embodiments where the antibody
specifically binds DC-SIGN and embodiments where the antibody
specifically binds the effector molecule.
[0124] In some embodiments the DC-SIGN blocker is a mannosylated
molecule that binds to a DC-SIGN receptor. The mannosylated
molecule may be mannan.
[0125] The invention also provides a method of preventing or
treating a viral infection of a mammal, where the viral infection
is mediated at least in part by the binding of a viral effector
molecule to a DC-SIGN receptor of the mammal to be treated, where
the method comprises administering to the mammal an amount of a
DC-SIGN blocker sufficient to substantially inhibit the binding of
the viral effector molecule to the DC-SIGN receptor to thereby
prevent or treat the viral infection.
[0126] In some embodiments of the method, the DC-SIGN blocker
comprises a binding moiety of the viral effector molecule. In other
embodiments, the DC-SIGN blocker comprises a binding moiety of a
viral envelope glycoprotein. In other embodiments, the DC-SIGN
blocker is an antibody. The antibody may specifically bind DC-SIGN
or specifically bind the viral effector molecule. In additional
embodiments the DC-SIGN blocker is a mannosylated molecule that
binds to a DC-SIGN receptor. The mannosylated molecule may be
mannan.
[0127] Among embodiments of the invention in which the DC-SIGN
blocker is an antibody are included embodiments in which the
antibody is a monoclonal antibody; the mammal is a human and the
antibody is a monoclonal antibody that is humanized; the antibody
specifically binds DC-SIGN; the monoclonal antibody is mAb
1B10.2.6; the antibody specifically binds the viral effector
molecule; and the antibody specifically binds the binding moiety of
the viral effector molecule.
[0128] In further embodiments of the method, the viral effector
molecule is a molecular constituent of the viral envelope. In
certain embodiments, the molecular constituent of the viral
envelope is an envelope glycoprotein.
[0129] In additional embodiments of the method, the DC-SIGN blocker
comprises a binding moiety of the viral effector molecule. In some
embodiments of the invention in which the viral effector molecule
is a molecular constituent of the viral envelope, the DC-SIGN
blocker that is used comprises a binding moiety of the envelope
glycoprotein.
[0130] In a preferred embodiment, the viral infection is a CMV
infection and the viral effector molecule is a CMV effector
molecule. In a further preferred aspect, the mammal is a human. In
some embodiments, the CMV effector molecule is a molecular
constituent of the CMV envelope. In further embodiments, the
molecular constituent of the CMV envelope is a CMV envelope
glycoprotein. In yet further embodiments, the CMV envelope
glycoprotein is CMV envelope glycoprotein B.
[0131] Included among embodiments of the invention in which the
viral infection is a CMV infection and the viral effector molecule
is a CMV effector molecule are embodiments where the DC-SIGN
blocker comprises a binding moiety of the CMV effector molecule;
the DC-SIGN blocker comprises a binding moiety of the CMV envelope
glycoprotein B; the DC-SIGN blocker is a recombinantly produced
protein; and the DC-SIGN blocker is an antibody. Among embodiments
where the DC-SIGN blocker is an antibody are embodiments where the
antibody is a monoclonal antibody; the mammal is a human and the
monoclonal antibody is humanized; the antibody specifically binds
DC-SIGN; the monoclonal antibody is mAb 1B10.2.6; and the antibody
specifically binds the CMV effector molecule. Among embodiments
where the antibody specifically binds the CMV effector molecule are
embodiments where the CMV effector molecule is CMV envelope
glycoprotein B.
[0132] In one preferred embodiment of the invention the effector
molecule and the DC-SIGN blocker are the same. In a second
preferred embodiment the effector molecule and the DC-SIGN blocker
are different.
[0133] It is interesting that CMV, Ebola, and HIV (as well as SIV)
can bind to DC-SIGN. HIV binding to dendritic cells is mediated by
the binding of the gp120 glycoprotein of HIV with DC-SIGN. Thus,
gp120 is a viral effector molecule. The invention thus provides a
method for the prevention and treatment of an Ebola invention and
an HIV infection. Specifically, it is an object of the invention to
provide a method of preventing or treating an Ebola, HIV or SIV
infection of a human or a simian. The method comprises
administering to the human or simian an amount of a DC-SIGN blocker
that is sufficient to inhibit the interaction of Ebola, HIV, or SIV
with DC-SIGN receptor present on dendritic cells of the human or
simian to thereby prevent or treat the Ebola, HIV, or SIV
infection.
[0134] DC-SIGN is also believed to have a critical role in
mediating the known loose adhesion that takes place between
dendritic cells and T cells in the apparent absence of foreign
antigen. This adhesion is thought to be necessary to provide an
opportunity for the TCR to scan the dendritic cell surface and
identify the very small amounts of TCR ligand which are present,
and in turn to become activated by this ligand. For this reason,
the interaction between DC-SIGN on dendritic cells, and ICAM-3 on T
cells, is likely to be critically important for the process of T
cell activation and stimulation. This model suggests that the
DC-SIGN-ICAM-3 interaction may have a role in mediating and/or
potentiating other stimulatory effects of dendritic cells on T
cells.
[0135] For this reason DC-SIGN blockers may be potent
anti-inflammatory agents, by blocking the interaction of the ICAM-3
effector molecule with DC-SIGN. Accordingly, the invention also
provides a method of preventing or treating inflammation in a
mammal caused by interaction of ICAM-3 present on T cells of the
mammal with DC-SIGN receptor present on dendritic cells of the
mammal. The method comprises administering to the mammal an amount
of a DC-SIGN blocker that is sufficient to inhibit the interaction
of ICAM-3 present on T cells of the mammal with DC-SIGN receptor
present on dendritic cells of the mammal to thereby prevent or
treat inflammation.
[0136] The invention also provides pharmaceutical compositions
comprising a DC-SIGN blocker. Such compositions may be suitable for
pharmaceutical use and administration to patients. The compositions
typically contain a purified DC-SIGN blocker at a therapeutically
achievable concentration and a pharmaceutically acceptable
excipient. As used herein, the phrase "pharmaceutically acceptable
excipient" includes any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, that are compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. The
compositions can also contain other active compounds providing
supplemental, additional, or enhanced therapeutic functions. The
pharmaceutical compositions can also be included in a container,
pack, or dispenser together with instructions for
administration.
[0137] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. Methods
to accomplish the administration are known to those of ordinary
skill in the art. The administration may, for example, be
intravenous, intramuscular, subcutaneous, or via inhalation.
[0138] Solutions or suspensions used for subcutaneous application
typically include one or more of the following components: a
sterile diluent, such as water for injection, saline solution,
fixed oils, polyethylene glycols, glycerin, propylene glycol or
other synthetic solvents; antibacterial agents, such as benzyl
alcohol or methyl parabens; antioxidants, such as ascorbic acid or
sodium bisulfite; chelating agents, such as ethylenediaminetetra
acetic acid; buffers, such as acetates, citrates or phosphates; and
agents for the adjustment of tonicity, such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide. Such preparations can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0139] Pharmaceutical compositions suitable for injection include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersion. For intravenous administration, suitable carriers
include physiological saline, bacteriostatic water, Cremophor.RTM.
ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
In all cases, the composition must be sterile and should be fluid
to the extent that easy syringability exists. It must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion, and by the use of surfactants.
Prevention of the action of microorganisms can be achieved by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In
many cases, one may include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0140] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0141] For administration by inhalation, the DC-SIGN blocker
containing compositions are delivered in the form of an aerosol
spray from pressured container or dispenser, which contains a
suitable propellant, e.g., a gas such as carbon dioxide, or a
nebulizer.
[0142] In one embodiment, a purified DC-SIGN blocker is prepared
with carriers that will protect it against rapid elimination from
the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions containing LAM can also
be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811.
[0143] Therapeutically useful agents, such as growth factors (e.g.,
BMPs, TGF-.beta., FGF, and IGF), cytokines (e.g., interleukins and
CDFs), antibiotics, and any other therapeutic agent beneficial for
the condition being treated can optionally be included in or
administered simultaneously or sequentially with the DC-SIGN
blocker.
[0144] It is especially advantageous to formulate compositions in
dosage unit form for ease of administration and uniformity of
dosage. "Dosage unit form" as used herein refers to physically
discrete units suited as unitary dosages for the subject to be
treated; each unit containing a predetermined quantity of active
compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of
the active compound and the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an active compound for the treatment of individuals.
[0145] Toxicity and therapeutic efficacy of compositions comprising
a DC-SIGN blocker can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD50 (the dose lethal to 50% of the population) and
the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD50/ED50. DC-SIGN blockers which exhibit large therapeutic indices
are preferred.
[0146] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage can vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any DC-SIGN blocker used in the present invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC50 (i.e., the concentration of the test DC-SIGN blocker which
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Levels in plasma can be measured, for example, by
high performance liquid chromatography. The effects of any
particular dosage can be monitored by a suitable bioassay.
[0147] A targeting complex of the present invention comprises at
least one DC-SIGN blocker molecule covalently attached to at least
one subject molecule. In some embodiments, a single DC-SIGN blocker
molecule is covalently linked to a single subject molecule. In
other embodiments, more than one DC-SIGN blocker molecule can be
covalently linked to a single subject molecule. The multiple
DC-SIGN blocker molecules can each be independently covalently
linked to the subject molecule; alternatively, one or more of the
more than one DC-SIGN blocker molecules can be covalently linked
only to one or more other DC-SIGN blocker molecules, at least one
of which is itself covalently linked to the subject molecule.
[0148] In other embodiments, multiple subject molecules are
covalently linked to a single DC-SIGN blocker molecule. The
multiple subject molecules can each be independently covalently
linked to the DC-SIGN blocker molecule; alternatively, one or more
of the more than one subject molecules can be covalently linked
only to one or more other subject molecules, at least one of which
is itself covalently linked to the DC-SIGN blocker molecule.
[0149] Additional embodiments of the invention utilize compositions
of more than one of the various types of DC-SIGN blockers described
immediately above. There is no limit to the diversity of such
compositions which can be used. One of skill in the art will
appreciate that the composition to be used for a particular
application will be dictated by many factors and that a suitable
composition can thus be appropriately chosen for each application
of the invention.
[0150] Techniques for making the DC-SIGN blockers of the invention
are well known and widely practiced by those of skill in the
biochemistry art, and thus need not be detailed here. However, one
of skill in the art will recognize that any suitable technique
which results in the formation of a covalent bond between a subject
molecule and a DC-SIGN blocker molecule can be used.
[0151] Subject molecules can be any molecule of interest.
Nonlimiting examples include: small organic molecules, proteins,
nucleic acids, carbohydrates, and lipids. One of ordinary skill in
the art will appreciate that any known derivatives and composites
of one or more of these classes of molecules can also be used.
[0152] In the case in which the subject molecule is a protein,
nucleic acid, carbohydrate, or lipid, the subject molecule can be
obtained from a natural source, i.e., purified from an organism,
which comprises the molecule. Alternatively, the subject molecule
can be obtained from a recombinant source, i.e., from a recombinant
organism, which has been engineered to produce a subject molecule
of choice. In some cases, the recombinant organism that is used to
produce the recombinant subject molecule is one that comprises the
subject molecule, as the organism occurs in nature, in
nonrecombinant form. In other cases, the subject molecule is one
that does not naturally occur in the recombinant organism.
[0153] The subject molecules of the invention also include
derivatives of small organic molecules, proteins, nucleic acids,
carbohydrates, and lipids. As used here, a derivative is a form of
small organic molecule, protein, nucleic acid, carbohydrate, or
lipid that is modified from its natural state by adding,
subtracting, or altering one or more chemically reactive sites
present on the small organic molecule, protein, nucleic acid,
carbohydrate, or lipid. Techniques for making derivatives of small
organic molecules, proteins, nucleic acids, carbohydrates, and
lipids are well known and widely practiced by those of skill in the
biochemistry art, and thus need not be detailed here.
[0154] In a preferred embodiment, the subject molecule is an
antibody.
[0155] The subject molecule can also be a molecule that is
antigenic. A molecule is antigenic when it is capable of
specifically interacting with an antigen recognition molecule of
the immune system, such as an immunoglobulin (antibody) or T cell
antigen receptor. An antigenic polypeptide contains at least about
5, and preferably at least about 10, amino acids. An antigenic
portion of a molecule can be that portion that is immunodominant
for antibody or T cell receptor recognition, or it can be a portion
used to generate an antibody to the molecule by conjugating the
antigenic portion to a carrier molecule for immunization. A
molecule that is antigenic need not be itself immunogenic, i.e.,
capable of eliciting an immune response without a carrier.
[0156] The targeting complex of the invention can be exposed to a
dendritic cell either in vivo or in vitro. In vivo exposure is
achieved by administering the targeting complex in a pharmaceutical
composition as described herein or in any suitable equivalent
formulation known in the art. In that case, the targeting complex
will bind to DC-SIGN on the surface of dendritic cells in vivo. In
vitro exposure occurs when dendritic cells grown in vitro are
exposed to the targeting complex.
[0157] The following examples aid in describing certain aspects of
the invention. One of ordinary skill in the art will recognize the
numerous modifications and variations that may be performed without
altering the spirit or scope of the present invention. Such
modifications and variations are believed to be encompassed within
the scope of the invention. The examples do not in any way limit
the invention.
EXAMPLES
Example 1
Herpesviruses
[0158] AD169, Towne (CMV laboratory strains) and TB40/E (CMV
clinical isolate) were provided by Dr. S. Michelson (Institut
Pasteur, Paris, France) and Dr. C. Sinzger (Tubingen, Germany),
respectively. ADGFP is a genetically modified AD169 strain encoding
an Enhanced Green Fluorescent Protein driven by the CMV
immediate-early gene promoter (Borst et al., 2001). VZV and HSV-1
clinical isolates were obtained from Dr. Isabelle Garrigue
(Laboratory of Virology, CHU Pellegrin, Bordeaux, France).
Example 2
Reagents, Antibodies and Viral Glycoproteins
[0159] Mannan and EGTA were purchased from Sigma-Aldrich
Corporation (Saint Louis, Mo.). Soluble viral envelope
glycoproteins were produced and purified from mammalian or insect
cells. HIV-1 gp120 (MN isolate) was obtained from the NIBSC
repository (Medical Research Council, United Kingdom). VZV gB and
VZV gE (Jacquet et al., 1995) were gifts from Dr. A. Jacquet,
(Department of Applied Genetics, Gosselies, Belgium). HSV-1 gB and
HSV-1 gD (Sisk et al., 1994) were provided by Dr. G. H. Cohen
(University of Pennsylvania, Philadelphia, PN). Expression and
purification of CMV gB (gift of Dr Claude Meric, Aventis Pasteur,
Marcy L'Etoile, France) were previously described (Norais et al.,
1996; Pass et al., 1999). Anti-CMV gB (clone 1-M-12, IgG1) and
anti-DC-SIGNR (clone 120604, IgG2a) mAbs were purchased from
Biodesign International (Saco, Me.) and R&D Systems
(Minneapolis, Minn.), respectively. Anti-LIF 7D2 (Taupin et al.,
1993) and anti-SDF-1 K15C monoclonal antibodies (mAbs) (Amara et
al., 1999) were used as isotypic controls.
Example 3
Cells
[0160] MRC-5 (Bio Merieux S. A., Marcy I'Etoile, France) and U373
MG (ECCC, Salisbury, United Kingdom) are CMV-, HSV-1- and
VZV-permissive cell lines, from fibroblastic and astrocytic origin,
respectively. Parental and DC-SIGN.sup.+ THP-1 cells (wild type and
.DELTA.35 and .DELTA.20 mutants lacking the first 35 and 20 amino
acids of the cytoplasmic domain, respectively) (Kwon et al., 2002)
were a gift from Dr. D. R. Littmann (Skirball Institute of
Biomolecular Medicine, New York, N.Y., USA). DC-SIGN.sup.+ HeLa
cells were generated by infecting HeLa cells with an HIV-derived
vector (TRIP-.DELTA.U3 vector, a gift from Dr. P. Charneau,
Institut Pasteur, Paris) encoding a human DC-SIGN cDNA. MD-DC were
generated from peripheral blood monocytes treated with 20 ng/mL
IL-4 (Schering-Plough, Kenilworth, N.J.) and 100 ng/mL GM-CSF
(Leucomax.RTM., Novartis-Pharma, Rueil Malmaison, France) (Romani
et al., 1994). At day 5, virtually the totality of cells displayed
the phenotype CD1a.sup.+, HLA-DR.sup.+, CD80.sup.low, CD86.sup.low,
CD83.sup.-, and CD14.sup.-, characteristic of immature MD-DC.
Example 4
DC-SIGN cDNA and Anti-DC-SIGN Antibodies
[0161] DC-SIGN cDNA was isolated from human immature MD-DC by
RT-PCR. For expression in mammalian cells, human DC-SIGN was
subcloned at the EcoR1/Xba 1 sites of the pcDNA3 myc-His (version
A) plasmid (Invitrogen, Carsbad, CA). The DC-SIGNR cDNA was a gift
from Dr R. W. Doms, (University of Pennsylvania, Philadelphia, PN).
Anti-DC-SIGN clone 1B10.2.6 (IgG2a) was obtained by immunizing
BALB/c mice with HEK 293T cells transfected with DC-SIGN cDNA,
screened by indirect immunofluorescent staining and FACS analysis
on DC-SIGN.sup.+ HeLa cells and used as purified
immunoglobulins.
Example 5
Infection Assays
[0162] For trans-infection experiments, cells were incubated with
viral suspensions (CMV, VZV, or HSV-1, MOI=1) for 2 hours, at
37.degree. C. Thereafter, unbound viral particles were removed by
extensive washes and cells were co-cultured with sub-confluent
MRC-5 or U373 MG cell monolayers. After 24 to 72 hours, infected
MRC-5 or U373-MG cells were fixed, permeabilized and stained with
specific mAbs directed against IEA- or EA-CMV (mAbs E13 and 2A2,
respectively), VZV (mAb 2013) or HSV (mAb CHA-437) (Argen Biosoft,
Varilhes, France). When indicated, MD-DC or THP-1 (parental or
DC-SIGN.sup.+) cells were incubated with EGTA (5 mM), mannan, or
anti-DC-SIGN (1B10.2.6 mAb) for 30 minutes at 4.degree. C. prior
challenge with infectious preparations. Infection by ADGFP strain
was assessed by counting GFP-expressing cells at day 3. For long
term infectivity experiments DC-SIGN.sup.+ or parental THP-1 cells
were incubated with ADGFP (MOI=1) for 4 hours at 37.degree. C.
After extensive washes, infected cells were incubated at 37.degree.
C. and an aliquot of these cells was added to a sub-confluent MRC-5
cell culture every 2 days during the assay.
[0163] To assess the effect in cis of DC-SIGN during infection,
cells were incubated with low titers of CMV (MOI=0.1) for 2 hours
at 37.degree. C. Non-internalized viral particles were removed by
washes in low pH citrate buffer (pH=3). The number of infected
cells was determined by immunocytochemistry 72 hours after
infection. Supernatants from infected cells kept in culture for 14
days were harvested to quantify de novo generated virions by plaque
assay titration.
Example 6
HIV-1 gp120 Binding Competition and CMV g8 Direct Binding
Assays
[0164] DC-SIGN.sup.+ THP-1 cells were washed two times, resuspended
in ice-cold binding buffer (1 mM CaCl.sub.2, 2 mM MgCl.sub.2, and
0.1% Bovine Serum Albumin in PBS) at 10.sup.6 cells/mL and
pre-treated or not for 15 minutes with competitors (20 .mu.g/ml).
Thereafter, recombinant biotinylated CXCR4-tropic (MN isolate)
HIV-1 gp120 (2 .mu.g/ml; Immunodiagnostics Inc., Woburn, Mass.) was
added for 30 minutes at 4.degree. C. After extensive washing,
cell-bound biotinylated HIV-1 gp120 was revealed by flow cytometry
using FITC-conjugated Streptavidin (Immunotech SA, Marseille,
France). For CMV gB binding experiments, recombinant soluble CMV gB
and Bovine Serum Albumine (BSA; Amersham Pharmacia Biotech,
Uppsala, Sweden) were biotinylated with sulfo-NHS biotin, according
to manufacturer's instructions (Pierce, Rockford, Ill.).
Example 7
Analysis of DC-SIGN Interactions with Viral Envelope Glycoproteins
by SPR
[0165] The cDNA coding for the DC-SIGN CRD (amino acids 254-404)
was obtained by PCR and cloned into pET15b (Novagen). The protein
was expressed in Escherichia coli C41 (DE3) as inclusion bodies.
Refolding of the protein has been done by dilution and dialysis as
described (Mitchell et al., 2001). Purification of refolded DC-SIGN
CRD has been achieved in two steps: first on a Ni-NTA (QIAGEN)
column equilibrated in 25 mM Tris Cl pH 7.8, 150 mM NaCl, 4 mM
CaCl.sub.2 (Loading Buffer) and eluted with a linear gradient of
imidazole and second on a Mannose-agarose column equilibrated in
Loading Buffer, and eluted in buffer where CaCl.sub.2 was replaced
by EDTA (10 mM). Pooled fractions are then concentrated and
dialyzed against Loading Buffer.
[0166] Four flow cells of a Biacore B1 sensor chip were activated
as described (Amara et ai., 1999). The first flow cell was then
blocked with 50 .mu.l of 1M ethanolamine pH 8.5 and served as a
control surface. The three other ones were treated with soluble
gp120, gB CMV, or gB HSV (concentration range 1-10 .mu.g/ml in 10
mM acetate buffer pH 5). Typically, this procedure permitted the
coupling of approximately 250-350 resonance units (RU) of proteins.
For binding assays, DC-SIGN CRD was diluted in Loading Buffer and
was allowed to react with the sensor chip (at 30 .mu.l/min). In a
typical analysis, DC-SIGN CRD (0.13 to 1 .mu.M, see figure legend)
was injected over the four flow cells for 8 minutes, after which
the complexes were rinsed with buffer to analyze the dissociation
phase. The surface was then regenerated with a 6 minute pulse of
running buffer containing 50 mM EDTA instead of CaCl.sub.2. Sets of
sensorgrams were analyzed using the BlAevaluation 3 software.
Example 8
Expression of DC-SIGN at the Cell Membrane Enables Binding of
CMV
[0167] The capacity of CMV to bind DC-SIGN was investigated.
Parental and DC-SIGN.sup.+ THP-1 cells, or immature
monocyte-derived DC (MD-DC) were incubated on ice with increasing
concentrations of CMV and the presence of cell-bound virions was
quantified by flow cytometry using a mAb directed against the CMV
gB. While parental THP-1 cells failed to bind detectable amounts of
CMV, both DC-SIGN-expressing THP-1 and MD-DC absorbed CMV virions
in a dose dependent manner (FIG. 1B). Prevention of CMV gB
antibody-labeling by acidic washes proved the existence of
cell-bound virions (FIG. 1B). Abrogation of virion attachment
observed following pre-incubation of cells with mannan, a complex
sugar that binds to the Carbohydrate Recognition Domain (CRD) of
lectins, suggests that the CMV-DC-SIGN interaction is accounted for
by the glycosylated residues of CMV envelope glycoproteins.
Example 9
Transmission of CMV Infection to Permissive Cells is Mediated by
DC-SIGN
[0168] MD-DC, THP-1, or HeLa cells expressing DC-SIGN were
incubated with a mutant CMV strain encoding a GFP (ADGFP) (Borst et
al., 2001). HeLa cells were selected for their refractoriness to
CMV infection (Einhorn et al., 1982; Tsutsui et al., 1987), which
persists despite transduction with DC-SIGN (our unpublished
observations). MD-DC (FIG. 2A), DC-SIGN.sup.+ THP-1 (FIG. 2B) and
DC-SIGN.sup.+ HeLa cells (FIG. 2C), in contrast to parental THP-1
or HeLa cells, conveyed CMV infection as proved by the expression
of GFP in MRC-5 cells. Trans-infection of MRC-5 cells was prevented
by pre-incubating MD-DC, DC-SIGN.sup.+ THP-1 or DC-SIGN.sup.+ HeLa
cells either with EGTA or mannan before being pulsed with CMV.
Moreover, the anti-DC-SIGN mAb 1B10.2.6, which blocks HIV
transmission (data not shown), also inhibited efficiently the
transmission of CMV from DC-SIGN.sup.+ cells to MRC-5 cells. We
conclude that trans-infection of CMV to susceptible cells is
accounted for by DC-SIGN and does not require productive infection
by DC-SIGN-expressing cells.
[0169] The capacity of DC-SIGN to enhance infectiveness of CMV was
assessed. To this end, MRC-5 cells were either incubated with low
titers of cell-free CMV or co-cultured with MD-DC (FIG. 3A) or
DC-SIGN.sup.+ THP-1 cells (FIG. 3B) previously pulsed with
identical amount of CMV. Co-culture of MRC-5 with CMV-pulsed
DC-SIGN.sup.+ cells led to a substantial enhancement of MRC-5
infections as compared to MRC-5 exposed to cell-free virus. The
enhancement of CMV infectivity conferred by DC-SIGN.sup.+ cells
pulsed with CMV was abrogated by specific anti-DC-SIGN mAb 1B10.2.6
(FIGS. 3A and 38). To determine if DC-SIGN-bound CMV retains
infectivity over a more prolonged period of time than free virus,
DC-SIGN.sup.+ THP-1 cells were pulsed with CMV, washed and cultured
at 37.degree. C. for different periods before co-culture with MRC-5
cells. In parallel, cell-free virus was incubated for the same
period of time at 37.degree. C. before being added to MRC-5 cells.
Our findings show that CMV remains infectious for 4-5 days when
bound to DC-SIGN whereas cell-free virus retains its infectivity
only for 2 days (FIG. 3C).
[0170] In parallel, the detection by immunostaining of early
markers of CMV replication (intranuclear immediate early and early
antigens, IEA and EA, respectively) has been done (FIG. 4A). The
findings obtained by this alternative assay confirmed the role of
DC-SIGN in the transmission of CMV to permissive cells and
validated the trans-infection assay. Transmission of CMV from
DC-SIGN.sup.+ cells is not restricted to a particular permissive
cell type since DC-SIGN.sup.+ THP-1 cells also transmitted
infectious virions to the U373 MG astrocytoma cell line (FIG. 4B).
We next aimed at determining if other members of the herpesviridae
family have the same capacity as CMV to interact with DC-SIGN. To
this purpose, DC-SIGN.sup.+ THP-1 cells were exposed to a clinical
isolate of CMV, HSV-1, or VZV and thereafter co-cultured with MRC-5
cells, which are fully susceptible to the three viruses (FIG. 4D).
Expression of CMV-, but not HSV-1- or VZV-EA or --IEA in MRC-5
cells is compatible with a high degree of specificity for the
interaction of DC-SIGN with CMV envelope glycoproteins (FIG.
4C).
Example 10
DC-SIGN Cytoplasmic Tail is Critical for Enhanced Transmission of
CMV
[0171] The role proposed for DC-SIGN internalisation for
trans-enhancement of HIV infection was assessed for CMV
transmission from DC-SIGN.sup.+ cells to susceptible cells. To this
end, THP-1 cells expressing mutant forms of DC-SIGN (Kwon et al.,
2002) encoding either combined deletion of dileucine- and
tyrosine-based motifs (DC-SIGN .DELTA.35), or the dileucine-based
motif only (DC-SIGN .DELTA.20), which are putative internalization
motifs required for DC-SIGN endocytosis, were exposed to low MOI
CMV infection. Both DC-SIGN mutants were expressed in THP-1 cells
with similar efficiency as the wild type counterpart (FIG. 5A).
Moreover, they displayed roughly comparable capacities to bind CMV
particles (FIG. 5B). Parental and DC-SIGN-expressing (either wt or
mutated) THP-1 cells were then assessed for their ability to
transmit CMV to permissive MRC-5 cells. We found that following
incubation with ADGFP CMV at 37.degree. C., DC-SIGN .DELTA.35- or
DC-SIGN .DELTA.20-expressing THP-1 cells showed a marked decreased
capacity to transmit CMV as compared to DC-SIGN.sup.+ THP-1 cells
(FIG. 5C). Incubation on ice of DC-SIGN wt-expressing THP-1 cells
with CMV prevented virus transmission to MRC-5 cells (FIG. 5C).
These results suggest that, similarly to HIV infection,
trans-enhancement of CMV infection by DC-SIGN-expressing cells
requires the cytoplasmic domain of DC-SIGN.
Example 11
DC-SIGN Expression Renders Low-Susceptible Cells Sensitive to CMV
Infection and Mediates the Infection of MD-DC by Primary CMV
Isolates
[0172] We next investigated whether DC-SIGN is involved in cis in
the entry of CMV into host cells. Two complementary approaches were
developed for this purpose. First, using either HEK 293T or THP-1
cells transduced with DC-SIGN, we evaluated their capacity to
support CMV infection. It has been previously reported that
undifferentiated THP-1 cells are unable to support CMV IE gene
expression despite virus entry (Lashmit et al., 1998; Weinshenker
et al., 1988). We confirmed this finding and show that the HEK 293T
cell line similarly appears to be poorly susceptible to CMV
infection (ADGFP virus). In sharp contrast with these findings,
both HEK 293T and THP-1 cells expressing DC-SIGN were highly
susceptible to CMV infection. Indeed, more than 40% of
DC-SIGN.sup.+ THP-1 cells were positive for GFP after 2 hours of
contact with CMV ADGFP followed by a 2 day incubation while no GFP
expression was found in parental cells (FIG. 6A). Similarly to
DC-SIGN, the homologous DC-SIGNR lectin was capable of rendering
HEK 293T cells susceptible to CMV infection (FIG. 6B). Conclusive
evidence about the role played by DC-SIGN in the infectiveness of
transduced cells came from the drastic reduction of the CMV IE gene
expression levels in both DC-SIGN.sup.+ THP-1 and HEK 293T cells in
the presence of anti-DC-SIGN mAb (FIG. 6A).
[0173] MD-DC, which show natural expression of DC-SIGN, were used
to confirm and extend the findings observed in the first set of
experiments. By opposition to THP-1 cells, MD-DC are known to be
permissive to infection by primary CMV isolates. Detection of IEA
and EA in a substantial number of MD-DC when incubated with TB40/E
proved the susceptibility of these cells to non-adapted, clinical
CMV strains. Amazingly, pre-incubation of MD-DC with the
anti-DC-SIGN 1B10.2.6 mAb prevented their infection by CMV with
roughly the same efficiency as it did in DC-SIGN.sup.+ THP-1 cells
(FIG. 6C).
[0174] Full replication of CMV in DC-SIGN-expressing cells was then
assessed by quantifying the progeny of infectious virions. MRC-5,
MD-DC, DC-SIGN.sup.+ or parental THP-1 cells were incubated with
low titers of a primary CMV strain, washed in acidic buffer to
remove non internalized virus, and thereafter cultured for 14 days.
The generation of infectious CMV virions from these cells was
quantified by plaque assay titration on MRC-5 cells. Accumulation
of CMV virions was detected in culture supernatants from MD-DC and
DC-SIGN.sup.+ THP-1 cells (FIG. 6D). The amount of infectious
virions released by MD-DC cells or DC-SIGN-expressing THP-1 cells
were 10 and 16 times, respectively, more elevated than the number
of input virus used at day 0 and were comparable to amounts
released by MRC-5 cells (FIG. 6D). Pre-incubation of MD-DC cells or
DC-SIGN.sup.+ THP-1 cells with the specific anti-DC-SIGN 1B10.2.6
mAb precluded detectable generation of CMV infectious virions thus
demonstrating the involvement of DC-SIGN in the cis-infection of
DC-SIGN-expressing cells (FIG. 6D).
[0175] Hence, these results imply that in cis cell surface
expression of DC-SIGN not only potentiates the expression of CMV IE
gene products but also confers to CMV low susceptible cells the
capacity to support a full replicative cycle in the host cell.
These findings suggest a crucial biological role of DC-SIGN in the
propagation of the CMV natural infection by DC.
[0176] We identified CMV glycoprotein B as a viral ligand of
DC-SIGN. Since DC-SIGN was shown to bind HIV particles through a
specific interaction between the Carbohydrate Recognition Domain
(CRD) of DC-SIGN and sugar moities of HIV-1 gp120 (Mitchell et al.,
2001), we searched for an equivalent of HIV-1 gp120 on CMV
particles. The human CMV virion is known to harbor several
different envelope glycoproteins. Among them, CMV gB, gH, and gM
were shown to be directly involved in two early events of the CMV
infection: CMV attachment and fusion between viral and cellular
membranes (Compton et al., 1993; Kari and Gehrz, 1992; Milne et
al., 1998). The reasons for focusing our research on CMV gB are
manifold. First, CMV gB is the most abundant and the most
extensively N- and O-glycosylated envelope glycoprotein of CMV
(Gibson, 1983). Second, it has been demonstrated that sequence
variations in CMV gB from different strains of human CMV are lower
than in other CMV envelope glycoproteins (Chou and Dennison, 1991).
Third, CMV gB has been proposed to play central roles in virion
penetration into cells, transmission from cell to cell, and fusion
of infected cells (Navarro et al., 1993).
[0177] Recombinant, biotinylated CMV gB was directly bound and
detected on DC-SIGN-expressing THP-1 cells or MO-DC, but not on
parental THP-1 (FIG. 7A) and similar findings were observed with
unlabelled CMV gB (data not shown). The attachment of CMV gB to
cells was specifically abrogated by pre-incubation with the
blocking anti-DC-SIGN 1B10.2.6 mAb. Further authentification of CMV
gB as a CMV DC-SIGN ligand came from a competition assay with other
viral envelope glycoproteins. In this assay we pre-incubated
DC-SIGN.sup.+ THP-1 cells with purified HIV-1 gp120, CMV gB, HSV-1
gB, VZV gB, HSV-1 gD or VZV gE. Following exposure to each single
envelope glycoprotein, cells were incubated with biotinylated HIV-1
gp120, which binding to DC-SIGN.sup.+ THP-1 cells was evidenced by
immunostaining and FACS analysis. Among the herpesvirus proteins
assessed, only CMV gB decreased the binding of biotinylated HIV-1
gp120 on DC-SIGN. This competitive effect of CMV gB was almost as
efficient as that shown by unlabelled HIV-1 gp120, mannan, or
anti-DC-SIGN mAb 1B10.2.6 (FIG. 7B). Pre-treatment of DC-SIGN.sup.+
THP-1 cells and MD-DC with recombinant CMV gB before incubation
with CMV virions also efficiently blocked transmission of CMV to
susceptible MRC-5 cells (data not shown). To investigate whether
DC-SIGNR could also bind to CMV gB, we incubated HEK 293T cells
transiently transfected with cDNA encoding DC-SIGN or DC-SIGNR in
the presence of biotinylated-HIV-1 gp120, -CMV gB, or -BSA (FIG.
7C). No binding was observed when incubating transfected cells with
the control BSA. In contrast, both HIV-1 gp120 and CMV gB
efficiently bound to HEK 293T cells expressing either DC-SIGN or
DC-SIGNR. Both interactions were calcium-dependent since they were
blocked by EGTA (data not shown). Surprisingly, at low
concentrations CMV gB displayed a higher apparent affinity than
HIV-1 gp120 for DC-SIGNR, whereas both viral glycoproteins bound to
DC-SIGN-expressing cells with comparable efficiency. Together,
these results demonstrated that CMV gB is a CMV ligand for DC-SIGN
and DC-SIGNR. It deserves to be investigated whether this capacity
is restricted to CMV gB or is shared by other CMV envelope
glycoproteins.
Example 12
Characterization of DC-SIGN-Glycoproteins Interactions
[0178] The surface plasmon resonance (SPR) technology was used to
further analyze the characteristics of DC-SIGN binding to HIV-1
gp120 and CMV gB in vitro. Typical sensorgrams were obtained by
injection of a concentration range of recombinant soluble CRD
domain of DC-SIGN (0.13 to 1 .mu.M) over surfaces functionalized
with HIV-1 gp120 (FIG. 7D, left panel), CMV gB (FIG. 7D, middle
panel) or HSV-1 gB (FIG. 7D, right panel). Visual inspection of the
binding curves immediately showed that DC-SIGN binds to HIV-1 gp120
and CMV gB, while only displaying negligible binding to HSV-1 gB.
Binding of DC-SIGN CRD to both HIV-1 gp120 and CMV gB was strongly
inhibited by the anti-DC-SIGN 1B10.2.6 mAb and EDTA (data not
shown). The binding curves were then individually fitted to a
Langmuir model (A+B=AB). This analysis returned an average on rate
k.sub.on=3.33.times.10.sup.3 M.sup.-1 S.sup.-1, and off rate
k.sub.off=1.01.times.10.sup.-3 S.sup.-1, thus giving a equilibrium
dissociation constant of 0.30 .mu.M for HIV-1 gp120, and
k.sub.on=4.4.times.10.sup.-3M.sup.-1 S.sup.-1,
k.sub.off=1.26.times.10.sup.-3 S.sup.-1, leading to an equilibrium
dissociation constant of 0.29 .mu.M for CMV gB. Since the affinity
that characterize the DC-SIGN CRD binding to HIV-1 gp120 and to CMV
gB are similar, the higher binding level observed with the HIV-1
gp120 activated surface compared to the CMV gB surface (FIG. 7D,
left and middle panels) may simply reflect a difference in
immobilization or in glycan density between both proteins.
DEPOSITS
[0179] The Hela cell line denoted "Hela DC-SIGN Flap" was deposited
at the C.N.C.M. on Oct. 30, 2002, under the accession number
I-2949.
[0180] The DC-SIGN clone denoted "DC-SIGN human clone2" was
deposited at the C.N.C.M. on Oct. 30, 2002, under the accession
number I-2950.
[0181] The hybridoma denoted "1B10.2.6" was deposited at the
C.N.C.M. on Nov. 7, 2002, under the accession number I-2951.
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[0243] The entire contents of all references, patents and published
patent applications cited throughout this application are herein
incorporated by reference in their entirety.
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