U.S. patent application number 10/533924 was filed with the patent office on 2006-06-22 for dc-sign blockers and their use for preventing or treating viral infections.
Invention is credited to Ali Amara, Fernando Arenzana-Seisdedos, Philippe Despres, Jean-Louis Virelizier.
Application Number | 20060134100 10/533924 |
Document ID | / |
Family ID | 32314490 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134100 |
Kind Code |
A1 |
Amara; Ali ; et al. |
June 22, 2006 |
Dc-sign blockers and their use for preventing or treating 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)
; Arenzana-Seisdedos; Fernando; (Meudon, FR) ;
Despres; Philippe; (Garenne, FR) ; Virelizier;
Jean-Louis; (Paris, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Family ID: |
32314490 |
Appl. No.: |
10/533924 |
Filed: |
November 5, 2003 |
PCT Filed: |
November 5, 2003 |
PCT NO: |
PCT/IB03/05569 |
371 Date: |
November 21, 2005 |
Current U.S.
Class: |
424/133.1 ;
424/145.1; 424/159.1; 514/54 |
Current CPC
Class: |
Y02A 50/386 20180101;
G01N 33/6872 20130101; C07K 16/2851 20130101; G01N 33/566 20130101;
A61K 38/162 20130101; A61K 2039/505 20130101; C07K 16/2896
20130101; G01N 2333/70596 20130101; A61P 31/14 20180101; G01N
2500/00 20130101; G01N 2500/10 20130101; Y02A 50/53 20180101; A61P
31/18 20180101 |
Class at
Publication: |
424/133.1 ;
424/145.1; 424/159.1; 514/054 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/42 20060101 A61K039/42; A61K 31/715 20060101
A61K031/715 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 5, 2002 |
US |
60423582 |
Nov 12, 2002 |
US |
60425246 |
Claims
1. Use of an amount of a DC-SIGN modulator sufficient to
substantially modulate the binding of an effector molecule to a
DC-SIGN receptor for the preparation of a medicament for preventing
or treating a disease of a mammal, wherein at least one symptom of
the disease is mediated at least in part by the binding of the
effector molecule to the DC-SIGN receptor of the mammal to be
treated.
2. Use of an amount of a DC-SIGN blocker sufficient to
substantially inhibit the binding of an effector molecule to a
DC-SIGN receptor for the preparation of a medicament for preventing
or treating a disease of a mammal, wherein at least one symptom of
the disease is mediated at least in part by the binding of the
effector molecule to the DC-SIGN receptor of the mammal to be
treated.
3. The use of claim 2, wherein the DC-SIGN blocker is a blocking
derivative of the effector molecule.
4. The use of claim 2, wherein the DC-SIGN blocker is an
antibody.
5. The use of claim 4, wherein the antibody specifically binds
DC-SIGN.
6. The use of claim 4, wherein the antibody specifically binds the
effector molecule.
7. The use of claim 2, wherein the DC-SIGN blocker is a
mannosylated molecule that binds to a DC-SIGN receptor.
8. The use of claim 7, wherein the mannosylated molecule is
mannan.
9. Use of an amount of a DC-SIGN modulator sufficient to
substantially modulate the binding of a viral effector molecule to
a DC-SIGN receptor for the preparation of a medicament for
preventing or treating a viral infection of a mammal, wherein the
viral infection is mediated at least in part by the binding of the
viral effector molecule to the DC-SIGN receptor of the mammal to be
treated.
10. Use of an amount of a DC-SIGN blocker sufficient to
substantially inhibit the binding of a viral effector molecule to a
DC-SIGN receptor for the preparation of a medicament for preventing
or treating a viral infection of a mammal, wherein the viral
infection is mediated at least in part by the binding of the viral
effector molecule to the DC-SIGN receptor of the mammal to be
treated.
11. The use of claim 10, wherein the viral effector molecule is a
molecular constituent of the viral envelope.
12. The use of claim 11, wherein the molecular constituent of the
viral envelope is an envelope glycoprotein.
13. The use of claim 10, wherein the DC-SIGN blocker comprises a
binding moiety of the viral effector molecule.
14. The use of claim 12, wherein the DC-SIGN blocker comprises a
binding moiety of the envelope glycoprotein.
15. The use of claim 10, wherein the DC-SIGN blocker is an
antibody.
16. The use of claim 15, wherein the antibody is a monoclonal
antibody.
17. The use of claim 16, wherein the mammal is a human and the
monoclonal antibody is humanized.
18. The use of claim 15, wherein the antibody specifically binds
DC-SIGN.
19. The use of claim 15, wherein the antibody specifically binds
the viral effector molecule.
20. The use of claim 19, wherein the antibody specifically binds
the binding moiety of the viral effector molecule.
21. The use of claim 10, wherein the DC-SIGN blocker is a
mannosylated molecule that binds to a DC-SIGN receptor.
22. The use of claim 21, wherein the mannosylated molecule is
mannan.
23. The use of claim 10, wherein the viral infection is a
Flaviviridae virus infection and the viral effector molecule is a
Flaviviridae effector molecule.
24. The use of claim 23, wherein the mammal is a human.
25. The use of claim 23, wherein the Flaviviridae viral infection
is a Dengue virus infection and the Flaviviridae effector molecule
is a Dengue effector molecule.
26. The use of claim 25, wherein the Dengue virus effector molecule
is a molecular constituent of the Dengue virus envelope.
27. The use of claim 26, wherein the molecular constituent of the.
Dengue virus envelope is a Dengue virus envelope glycoprotein.
28. The use of claim 27, wherein the Dengue virus envelope
glycoprotein is Dengue virus E glycoprotein.
29. The use of claim 25, wherein the DC-SIGN blocker comprises a
binding moiety of the Dengue virus effector molecule.
30. The use of claim 28, wherein the DC-SIGN blocker comprises a
binding moiety of the Dengue virus E glycoprotein.
31. The use of claim 30, wherein the DC-SIGN blocker is a
recombinantly produced protein.
32. The use of claim 25, wherein the DC-SIGN blocker is an
antibody.
33. The use of claim 32, wherein the antibody is a monoclonal
antibody.
34. The use of claim 33, wherein the monoclonal antibody is
humanized.
35. The use of claim 32, wherein the antibody specifically binds
DC-SIGN.
36. The use of claim 32, wherein the antibody specifically binds
the Dengue virus effector molecule.
37. The use of claim 36, wherein the Dengue virus effector molecule
is Dengue virus E glycoprotein.
38. Use of an amount of a DC-SIGN modulator sufficient to
substantially modulate the binding of HIV or SIV to a DC-SIGN
receptor present on dendritic cells of a human or a simian for the
preparation of a medicament for preventing or treating an HIV or a
SIV infection of said human or said simian.
39. Use of an amount of a DC-SIGN blocker sufficient to
substantially inhibit the binding of HIV or SIV to a DC-SIGN
receptor present on dendritic cells of a human or a simian for the
preparation of a medicament for preventing or treating an HIV or a
SIV infection of said human or said simian.
40. The use of claim 39, wherein the DC-SIGN blocker comprises a
binding moiety of the Dengue virus E glycoprotein.
41. The use of claim 39, wherein an HIV infection of a human is
prevented or treated.
42. Use of an amount of a DC-SIGN modulator sufficient to
substantially modulate the binding of ICAM-3 present on T cells of
a mammal with DC-SIGN receptor present on dendritic cells of the
mammal for the preparation of a medicament for preventing or
treating inflammation in said mammal caused by specific binding of
ICAM-3 present on T cells of the mammal with DC-SIGN receptor
present on dendritic cells of the mammal.
43. Use of an amount of a DC-SIGN blocker sufficient to
substantially inhibit the binding of ICAM-3 present on T cells of a
mammal with DC-SIGN receptor present on dendritic cells of the
mammal for the preparation of a medicament for preventing or
treating inflammation in said mammal caused by specific binding of
ICAM-3 present on T cells of the mammal with DC-SIGN receptor
present on dendritic cells of the mammal.
44. The use of claim 43, wherein the DC-SIGN blocker comprises a
binding moiety of the Dengue virus E glycoprotein.
45. The use of claim 43, wherein the mammal is a human.
46. A pharmaceutical composition comprising: a) A DC-SIGN blocker,
and b) at least one pharmaceutically acceptable excipient; wherein
the DC-SIGN blocker is present in the composition at an achievable
therapeutic concentration.
47. The pharmaceutical composition of claim 46, wherein the DC-SIGN
blocker is a derivative of a viral effector molecule.
48. The pharmaceutical composition of claim 46, wherein the DC-SIGN
blocker comprises the binding moiety of a Dengue virus effector
molecule.
49. The pharmaceutical composition of claim 48, wherein the Dengue
virus effector molecule is Dengue virus E glycoprotein.
50. The pharmaceutical composition of claim 46, wherein the DC-SIGN
blocker is an antibody.
51. The pharmaceutical composition of claim 50, wherein the
antibody is a monoclonal antibody.
52. The pharmaceutical composition of claim 51, wherein the
monoclonal antibody is humanized.
53. The pharmaceutical composition of claim 50, wherein the
antibody specifically binds DC-SIGN.
54. The pharmaceutical composition of claim 50, wherein the
antibody specifically binds the viral effector molecule.
55. The pharmaceutical composition of claim 54, wherein the
antibody specifically binds the binding moiety of the viral
effector molecule.
56. A method of identifying a DC-SIGN modulator, wherein the method
comprises: a) determining a baseline binding value by: i. providing
cultured cells comprising a DC-SIGN receptor; 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 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; b) determining a test
substance binding value by: i. providing cultured cells comprising
a DC-SIGN receptor; 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 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 c) determining a test substance binding modulation value for
the test substance by dividing the test substance binding value by
the baseline binding value, wherein a test substance binding
modulation 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.
57. A method of identifying a DC-SIGN blocker, wherein the method
comprises: a) determining a baseline binding value by: i. providing
cultured cells comprising a DC-SIGN receptor; 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 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; b) determining a test
substance binding value by: i. providing cultured cells comprising
a DC-SIGN receptor; 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 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 c) determining a test substance binding inhibition value for
the test substance by dividing the test substance binding value by
the baseline binding value, 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.
58. The method of claim 57 wherein the cultured cells are DC.
59. The method of claim 57, wherein the cultured cells are THP-1
cells.
60. The method of claim 57, wherein the viral effector molecule is
a Dengue virus effector molecule.
61. The method of claim 60, wherein the Dengue virus effector
molecule is Dengue virus E glycoprotein.
62. An isolated DC-SIGN blocker identified by the method of claim
57.
63. A method of targeting a subject molecule to a cell expressing a
DC-SIGN receptor by exposing the cell to a targeting complex,
wherein the targeting complex comprises a subject molecule and a
DC-SIGN blocker, wherein 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.
64. The method of claim 63, wherein the DC-SIGN blocker is an
antibody.
65. The method of claim 64, wherein the antibody is a monoclonal
antibody.
66. The method of claim 63, wherein the subject molecule is a
protein.
67. The method of claim 63, wherein the subject molecule is an
antibody.
68. The method of claim 63, wherein the subject molecule is
labeled.
69. The method of claim 63, wherein the exposure occurs in
vivo.
70. The method of claim 63, wherein the exposure occurs in
vitro.
71. A pharmaceutical composition comprising: a) A DC-SIGN
modulator, and b) at least one pharmaceutically acceptable
excipient; wherein the DC-SIGN modulator is present in the
composition at an achievable therapeutic concentration.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to methods, uses 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
or interaction of an effector molecule to a DC-SIGN receptor of the
mammal to be treated. The effector molecule may be a molecule of a
foreign organism. The foreign organism may be a virus.
[0003] 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.
[0004] 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.
[0005] 2. Description of the Related Art
[0006] Dengue is an acute febrile tropical disease and the virus
which causes it is an arbovirus which is transmitted by mosquitoes.
The vectors of the disease are mosquitoes of the Aedes genus, in
particular Aedes aegypti, which most commonly leave their larvae in
domestic and peridomestic areas. The responsible virus, isolated in
1951, has been classified into four different antigenic types
(DEN1, DEN2, DEN3 and DEN4). It belongs to the Flaviviridae family,
genus flavivirus.
[0007] More than two billion inhabitants live in endemic regions
and the number of individuals infected by the virus is thought to
be more than 100 million per year. Dengue is in particular
responsible for 500 000 hospitalizations and for several tens of
thousands of deaths annually, mostly children.
[0008] After an incubation of five to eight days, the clinical
signs generally begin suddenly and consist of the appearance of
undifferentiated fever (DF dengue fever) accompanied by severe
headaches, lumbago, muscle and joint pain and also shivering. From
the third to the fifth day of the febrile phase, a congestive
maculopapular rash may appear for three to four days (conventional
dengue).
[0009] In its severe form, the infection may result in the
appearance of a hemorrhagic- syndrome (DHF or dengue hemorrhagic
fever), characterized by increased vascular permeability and
deregulation of hemostasis. Although, in the majority of cases, the
disease generally evolves favorably within a week, it may turn out
to be fatal in the event of hypovolemic shock (DSS or dengue shock
syndrome). These complications may be due to the presence of
preexisting immunity, acquired in particular during a primary
infection with a heterologous dengue virus (different serotype).
Specifically, two different types of serological response are
identified in individuals infected with dengue: individuals who
have never suffered a flavivirus infection and have not been
vaccinated, against another flavivirus (yellow fever virus,
Japanese encephalitis virus for example) will exhibit a primary
response, characterized by a slow appearance of antibodies specific
for the virus responsible for the infection; individuals who have
already suffered a flavivirus infection (other dengue serotype for
example) or have been vaccinated against another flavivirus will
exhibit a secondary response, characterized by the rapid appearance
of antibodies.
[0010] The infectious agent is the dengue virus which belongs to
the Flaviviridae family, to which the yellow fever virus and the
Japanese encephalitis virus also belong (T. P. Monath et al.,
(1996) Flaviviruses in B. N. Fields, D. M. Knipe, P. M. Howly et
al. (eds.) "Fields Virology" Philadelphia: Lippincott Raven Press
Publishers). These viruses have a single-strand RNA with positive
polarity which comprises 11000 nucleotides and which encodes a
poly-protein of approximately 3400 amino acids. It is separated
into three structural proteins and seven nonstructural proteins
NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, during co-translational
and post-translational cleavage by viral and cellular proteases.
The NS1 nonstructural protein was identified for the first time in
1970 by P. K. Russel et al. (J. Immunol., (1970), 105, 838-845) and
characterized in 1985 by G. W. Smith et al. (J. Gen Virol., (1985),
66, 559-571). This glycoprotein, which is highly conserved in the
flavivirus genus (T. P. Monath already mentioned), in particular in
the four dengue virus serotypes, exists in an intracellular form
and in an extracellular form. The intracellular form is thought to
be involved in the early phases of replication of the virus (Hall
R. A. et al., J. Virol. (1999), 73, 10272-10280; Rice C. M. at al.,
J. Virol., (1997), 71, 291-298; Rice C. M. et al., J. Virol.,
(1996), 222, 159-168; Rice C. M. et al., J. Virol., (1997), 71,
9608-9617). Before being transported to the plasma membrane, the
NS1 protein undergoes dimerization. In mammalian cells, but not in
insect cells, a portion of the NS1 protein is released into the
extracellular medium, either primarily in the form of a soluble
protein, or secondarily in a microparticulate form. When it is in a
soluble form, the protein exists in the form of an oligomer, in
particular of a pentamer or of a hexamer (Crooks A. J. et al. J.
Chrom. (1990), 502, 59-68 and J. Gen. Virol. (1994), 75, 3453-3460
and Glamand et al., J. Virol. (1999), 73, 6106-6110).
[0011] No specific treatment exists and the care given to the
patient is uniquely symptomatic. In the case of conventional
dengue, the treatment is based on the administration of analgesics
and antipyretics. In the case of DHF, the treatment consists of an
infusion to compensate for the plasma leakage, combined with
correction of hydroelectric problems and reinitiation of
diuresis.
[0012] There is no commercially available vaccine against the
dengue virus. On the other hand, protection assays with attenuated
strains of the 4 dengue virus serotypes have been carried out by N.
Bhamarapravati et: al. (Dengue and Dengue haemorrhagic fever
(1997), 367-377), with unsatisfactory results. Prevention is
therefore based solely on combating the vector. This combat
combines larval destruction and "adulticide" spraying.
[0013] The pathogenesis of severe forms of dengue (DEN) virus
infection is not completely understood. Substantial T cell
activation is observed in severe DEN disease. A number of cytokines
and chemokines are found to be elevated in dengue hemorrhagic fever
and/or dengue shock syndrome. Macrophages have long been recognized
as an important component of DEN pathogenesis. As emphasized by
Palucka, immature human dendritic cells (DC), in contrast to other
leukocytes, were preferentially permissive for DEN infection (Wu et
al., Nat.Med. 6: 816,2000; for review: Palucka, Nat. Med 6: 748,
2000). Unlike monocytes/macrophages, DEN virus infection was not
enhanced by specific antibody (Marovich et al., JID Symp.Proc.
6:219, 2001). Immature DCs can undergo maturation in response to
DEN virus infection. Upregulation of surface markers B7-1, B7-2,
HLA-DR CD11b and DC83 and cytokine production were observed
following infection of DCs. There is growing evidence that DEN
infection can induce functional maturity in DC. Such infection
causes upregulation of surface markers B7-1, B7-2, HLA-DR CD11b and
DC83 and stimulates cytokine production (Ho et al., Immunology 166:
1499, 2001). Immature DCs exposed to DEN virus produce TNF-.alpha.,
which may perturb endothelial cell function.
[0014] Dendritic cells (DC) are specialized Antigen Presenting
Cells (APC) involved in the initiation of T cell-dependent immune
responses as consequence of their high expression of MHC and
costimulatory molecules. Myeloid DC are distributed throughout the
body in an immature state, exhibiting a high capacity for antigen
uptake and processing. Once activated by inflammatory stimuli or
infectious agents, DC undergo a maturation process, migrate to
lymphoid organs, and acquire their capacity to activate naive T
lymphocytes.
[0015] An important question is which of DC-specific molecules DEN
virus uses as a receptor for entry. The human DC-specific adhesion
receptor DC-SIGN (ICAM-grabbing non integrin or CD-209), a type 11
integral protein, is of particular interest because its expression
is largely restricted to immature DCs. DC-SIGN has been shown to be
the ligand of ICAM-3, which enables transient DC-T cell
interactions, thus facilitating primary immune response
(Geijtenbeek et al., Nature 1: 353, 2000). DC-SIGN appeared to be a
critical mediator of the migratory and T cell-interacting
capabilities exhibited by maturing immature myeloid
monocyte-derived DCs. DC-SIGN expression is ILK dependent and is
negatively regulated by IFN-.gamma., IFN-.gamma., TGF-.beta., and
anti-inflammatory agents (Relloso et al. J.Immunol. 168: 2634,
2002). DC-SIGN polymorphisms might also explain why some patients
mount protective immunity whereas other do not.
[0016] DC-SIGN, a C-type lectin, has a single carbohydrate
recognition domain that interacts with proteins with either mannose
or galactose side chains in a calcium-dependent manner (Drickammer,
Curr.Opin.Immunol. 13: 585, 1999). DC-SIGN is now believed to bind
to high-mannose oligosaccharides that are present on the viral
glycoproteins and thereby may capture enveloped viruses (Feinberg
et al., Science 294: 2163, 2001). For example, DC-SIGN binds to the
HIV envelope glycoprotein gp120 (Geijtenbeek et al., Cell, 100:
587, 2000) and thereby mediates rapid internalization of intact HIV
into a nonlysosomal compartment (Kwon et al., Immunity, 16: 135,
2002).
[0017] 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 Dengue virus 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
[0018] The inventors sought to determine whether the DC-specific
adhesion receptor DC-SIGN has the ability to promote DEN virus
infection of human DC cells. The data reported herein showed that
DC-SIGN specific antibodies have a blocking action at the level of
DEN-1 virus infection. These results establish a novel function of
DC-SIGN, as a Dengue virus binding protein, likely through
interaction with the E glycoprotein. The process of
DC-SIGN-mediated Dengue virus infectivity to DCs provides a new
mechanism for targeting the design of anti-viral compounds.
[0019] Accordingly, this invention identifies DC-SIGN as a receptor
involved in the binding of viruses other than HIV to dendritic
cells. The invention further provides a number of novel methods,
uses and compositions for treating diseases of mammals, including
viral infections.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] In some embodiments the DC-SIGN blocker is a mannosylated
molecule that binds to a DC-SIGN receptor. The mannosylated
molecule may be mannan.
[0025] 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.
[0026] Another 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 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] In a preferred aspect of the invention the viral infection
is a Flaviviridae infection and the viral effector molecule is a
Flaviviridae effector molecule. In a more preferred embodiment, the
viral infection is a Dengue virus infection and the viral effector
molecule is a Dengue virus effector molecule. In a further
preferred aspect the mammal is a human. In some embodiments the
Dengue virus effector molecule is a molecular constituent of the
Dengue virus envelope. In further embodiments the molecular
constituent of the Dengue virus envelope is a Dengue virus envelope
glycoprotein. In yet further embodiments the Dengue virus envelope
glycoprotein is Dengue virus E glycoprotein.
[0032] Included among embodiments of the invention in which the
viral infection is a Dengue virus infection and the viral effector
molecule is a Dengue virus effector molecule are embodiments where
the DC-SIGN blocker comprises a binding moiety of the Dengue virus
effector molecule; the DC-SIGN blocker comprises a binding moiety
of the Dengue virus E glycoprotein; 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
Dengue virus effector molecule. Among embodiments where the
antibody specifically binds the Dengue virus effector molecule are
embodiments where the Dengue virus effector molecule is Dengue
virus E glycoprotein.
[0033] In a further aspect the invention provides a method of
preventing or treating an 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.
[0034] In another aspect the invention provides a method of
preventing or treating an 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 Dengue virus E
glycoprotein. In another preferred embodiment an HIV infection of a
human is prevented or treated.
[0035] In a further aspect the invention provides a method of
preventing or treating inflammation in a mammal caused by specific
binding of ICAM-3 present on T cells of the mammal with DC-SIGN
receptor present on dendritic cells of the mammal, wherein the
method comprises administering to the mammal an amount of a DC-SIGN
modulator sufficient to substantially modulate the binding 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.
[0036] In another aspect the invention provides a method of
preventing or treating inflammation in a mammal caused by specific
binding of ICAM-3 present on T cells of the mammal with DC-SIGN
receptor present on dendritic cells of the mammal, wherein the
method comprises administering to the mammal an amount of a DC-SIGN
blocker sufficient to substantially inhibit the binding 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. In a preferred embodiment the DC-SIGN blocker
comprises a binding moiety of the Dengue virus E glycoprotein. In
another preferred embodiment the mammal is a human.
[0037] A further object of the invention is the use of:
[0038] an amount of a DC-SIGN modulator sufficient to substantially
modulate the binding of an effector molecule to a DC-SIGN receptor
for the preparation of a medicament for preventing or treating a
disease of a mammal, wherein at least one symptom of the disease is
mediated at least in part by the binding of the effector molecule
to the DC-SIGN receptor of the mammal to be treated.
[0039] an amount of a DC-SIGN blocker sufficient to substantially
inhibit the binding of an effector molecule to a DC-SIGN receptor
for the preparation of a medicament for preventing or treating a
disease of a mammal, wherein at least one symptom of the disease is
mediated at least in part by the binding of the effector molecule
to the DC-SIGN receptor of the mammal to be treated.
[0040] an amount of a DC-SIGN modulator sufficient to substantially
modulate the binding of a viral effector molecule to a DC-SIGN
receptor for the preparation of a medicament for preventing or
treating a viral infection of a mammal, wherein the viral infection
is mediated at least in part by the binding of the viral effector
molecule to the DC-SIGN receptor of the mammal to be treated.
[0041] an amount of a DC-SIGN blocker sufficient to substantially
inhibit the binding of a viral effector molecule to a DC-SIGN
receptor for the preparation of a medicament for preventing or
treating a viral infection of a mammal, wherein the viral infection
is mediated at least in part by the binding of the viral effector
molecule to the DC-SIGN receptor of the mammal to be treated.
[0042] an amount of a DC-SIGN modulator sufficient to substantially
modulate the binding of HIV or SIV to a DC-SIGN receptor present on
dendritic cells of a human or a simian for the preparation of a
medicament for preventing or treating an HIV or SIV infection of
said human or said simian.
[0043] an amount of a DC-SIGN blocker sufficient to substantially
inhibit the binding of HIV or SIV to a DC-SIGN receptor present on
dendritic cells of a human or a simian for the preparation of a
medicament for preventing or treating an HIV or SIV infection of a
human or a simian.
[0044] an amount of a DC-SIGN modulator sufficient to substantially
modulate the binding of ICAM-3 present on T cells of a mammal with
DC-SIGN receptor present on dendritic cells of the mammal to
prepare a drug for preventing or treating inflammation in said
mammal caused by specific binding of ICAM-3 present on T cells of
the mammal with DC-SIGN receptor present on dendritic cells of the
mammal.
[0045] an amount of a DC-SIGN blocker sufficient to substantially
inhibit the binding of ICAM-3 present on T cells of the mammal with
DC-SIGN receptor present on dendritic cells of the mammal for the
preparation of a medicament for preventing or treating inflammation
in a mammal caused by specific binding of ICAM-3 present on T cells
of the mammal with DC-SIGN receptor present on dendritic cells of
the mammal. In a preferred embodiment the DC-SIGN blocker comprises
a binding moiety of the Dengue virus E glycoprotein. In another
preferred embodiment the mammal is a human.
[0046] The preferred embodiments listed for the hereabove methods
apply to the uses.
[0047] In a further aspect the invention provides a pharmaceutical
composition comprising:
[0048] a) A DC-SIGN modulator, and
[0049] b) at least one pharmaceutically acceptable excipient;
[0050] wherein the DC-SIGN modulator is present in the composition
at an achievable therapeutic concentration.
[0051] In another aspect the invention provides a pharmaceutical
composition comprising:
[0052] c) A DC-SIGN blocker, and
[0053] d) at least one pharmaceutically acceptable excipient;
[0054] wherein the DC-SIGN blocker is present in the composition at
an achievable therapeutic concentration.
[0055] In some embodiments of the pharmaceutical composition the
DC-SIGN blocker is a derivative of a viral effector molecule. In
one embodiment DC-SIGN blocker comprises the binding moiety of a
Dengue virus effector molecule. In another embodiment the Dengue
virus effector molecule is Dengue virus E glycoprotein.
[0056] 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.
[0057] In a further aspect the invention provides a method of
identifying a DC-SIGN modulator, wherein the method comprises:
[0058] a) determining a baseline 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 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 baseline binding value;
[0062] b) determining a test substance binding value by:
[0063] i. providing cultured cells comprising a DC-SIGN
receptor;
[0064] 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
[0065] 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
[0066] c) determining a test substance binding modulation value for
the test substance by dividing the test substance binding value by
the baseline binding value,
[0067] 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.
[0068] In a preferred aspect the invention provides a method of
identifying a DC-SIGN blocker, wherein the method comprises:
[0069] a) determining a baseline binding value by:
[0070] i. providing cultured cells comprising a DC-SIGN
receptor;
[0071] 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
[0072] 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;
[0073] b) determining a test substance binding value by:
[0074] i. providing cultured cells comprising a DC-SIGN
receptor;
[0075] 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
[0076] 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
[0077] c) determining a test substance binding inhibition value for
the test substance by dividing the test substance binding value by
the baseline binding value,
[0078] 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.
[0079] 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 Dengue virus effector
molecule; and the Dengue virus effector molecule is Dengue virus E
glycoprotein.
[0080] In a further aspect the invention provides an isolated
DC-SIGN blocker identified by the above method of identifying a
DC-SIGN blocker.
[0081] 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.
[0082] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] The invention will be more fully described with reference to
the drawings in which:
[0084] FIG. 1 depicts DEN-1 virus infection of human DCs ex-vivo.
DCs infected with DEN-1 virus strain FGA/NA dld (5 Ap6l FFU/cell)
were fixed with 3% PFA in PBS 40 h post-infection and permeabilized
with 0.1% Triton X-100 in PBS. Intracellular DEN proteins were
visualized with anti-DEN-1 virus HMAF by indirect
immunofluorescence and nuclei were stained with Hoechst 33258. DEN
virus-infected DCs (viral antigens) and chromatin condensation
(apoptotic nuclei) were observed by fluorescence. Apoptotic DCs are
indicated (arrows).
[0085] FIG. 2 depicts apoptotic DNA fragmentation in DCs infected
with DEN-1 virus. Infected DCs were assayed simultaneously for the
presence of DEN antigens by indirect indirect immunofluorescence
(Viral antigens) as described in the legend of FIG. 1 and for
apoptosis by the TUNEL assay (TUNEL). TUNEL-positive cells were
observed by fluorescence. TUNEL-positive cells are indicated
(arrows). Low (A) or high (B) magnification.
[0086] FIG. 3 shows that Anti-DC-SIGN Mab 1B10.2.6 blocks DEN-1
virus infection of human DCs. Prior to infection, DCs were
incubated with anti-DC-SIGN Mab 110 (20 .mu.g/ml) or anti-DEN E Mab
9D12 (dilution 1:50) (Desprbs et al. Virology, 196: 209-219, 1993)
for 20 min. Antibody-treated DCs were infected with DEN-1 virus
strain FGA/NA did in the presence of Mab for 2 hrs. Viral antigens
were detected by indirect immunofluorescence as described in the
legend of FIG. 1. The percentages of infected DCs 42 h
post-infection are indicated.
[0087] FIG. 4 depicts Flavivirus infections of THP-1 and
THP-1l/DC-SIGN cells. Cells infected with DEN-1 virus strain FGA/NA
did (5 AP61FFU/cell), YF virus strain 17D-204 (50 VEROFFU/cell), or
WN virus strain IS-98-ST1 (5 AP61FFU/cell) were assayed 40 h
post-infection for the presence of viral antigens by indirect
immunofluorescence as described in the legend of FIG. 1. Viral
antigens were visualized with either anti-DEN-1 virus HMAF (AB
.alpha.-DEN-1), anti-YF virus HMAF (AB .alpha.-YF), or anti-WN
virus HMAF (Ab .alpha.-WN). (A) THP-1 cells mock-infected (top), or
infected with flavivirus (bottom) and infected THP-1/DC cells
(bottom) 40 post-infection (m.o.i., multiplicity of infection). (B)
The percentages of infected cells are indicated. Values represent
the mean of triplicate assays.+-.SD.
[0088] FIG. 5 shows that Mannan, EDTA and antibody specific DC-SIGN
block DEN-1 virus infection of THP-1/DC/SIGN cells. Prior to
infection, THP-1/DC/SIGN cells were incubated with either Mab 9D12
(dilution 1:50), Mab BD12.5 (20 .mu.g/ml), Mab 1B10.2.6 (20
.mu.g/ml), EDTA (5 mM), mannan (20 .mu.g/ml), or mock-treated
(control). Treated cells were infected with DEN-1 virus strain
FGA/NA dld (5 AP61FFU/cell) in the presence of reagents for 2 hrs.
Viral antigens were detected by indirect immunofluorescence as
described in the legend of FIG. 1. The percentages of infected
cells 48 h post-infection are indicated. Values represent the mean
of triplicate assays.+-.SD.
[0089] FIG. 6 depicts DEN-1 virus infection of THP-1 cell clone
expressing mutant form of DC-SIGN. THP-1 .DELTA.35 cell clone
(THP-1/DC-SIGN Mutant 35) infected with DEN-1 virus strain FGH/NA
dld (5 AP61 FFU/cell) was assayed 40 h post-infection for the
presence of viral antigens by indirect immunofluorescence as
described in the legend of FIG. 1.
[0090] FIG. 7 depicts the comparative DEN-1, DEN-2, DEN-3and DENA
virus infection of THP-1 cell clone expressing DC-SIGN (THP/DC-SIGN
cells) vs THP-1 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0091] 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.
[0092] "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.
[0093] 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 examples of viral infections that can be treated by the
method is Dengue virus infection of a human.
[0094] In the case of humans "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
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.
[0095] In the case of non human 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.
[0096] 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 histo-compatibility 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).
[0097] Alveolar macrophages and dendritic cells are examples of
cells expressing a DC-SIGN receptor. Endothelial cells are an
example of cells expressing DC-SIGNR.
[0098] 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 2.
[0099] 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 gp129 of HIV
and glycoprotein E of Dengue virus, 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 Dengue virus,
virus entry into DC-SIGN expressing cells. Dengue virus E
glycoprotein is thus a "Dengue virus 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.
[0100] 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.
[0101] 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
molecule.
[0102] 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
Dengue virus E glycoprotein. The binding of E glycoprotein present
on the surface of Dengue virus to DC-SIGN is not inhibited by 0.2%
bovine serum albumin as shown in FIGS. 3 and 5. However, binding is
inhibited by either antibody specific to DC-SIGN as shown in FIGS.
3 and 5, or soluble mannan added to the media as shown in FIG. 5,
or EDTA added to the media as shown in FIG. 5.
[0103] 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.
[0104] 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".
[0105] "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.
[0106] 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 Dengue virus E glycoprotein.
[0107] 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.
[0108] 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.
[0109] "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.
[0110] "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.
[0111] 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.
[0112] The term "prevent," "preventing" and "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.
[0113] 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.
[0114] 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 mouse,
rat sheep, goat, hamster, rabbit, etc. The origin of the protein
immunogen may be mouse, human, rat, monkey, or microorganism such
as a bacteria or virus etc. The host animal will generally be a
different species than the immunogen, e.g. human protein used to
immunize mice, etc.
[0115] 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.
[0116] 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, e.g.
mice, 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 Ig present in the resultant antiserum may be
further fractionated using known methods, such as ammonium salt
fractionation, DEAE chromatography, and the like.
[0117] 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.
[0118] 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.
[0119] 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
anti-bodies 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.
[0120] 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).
[0121] 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 antibody-dependent 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.
[0122] 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 anti-bodies 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.
[0123] 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 H chain, followed by a translational stop codon
to yield the truncated molecule.
[0124] Consensus sequences of H and L 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 V
region segments to human 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.
[0125] 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. SV40 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
[0126] An example of a disease that can be prevented or treated
utilizing the present invention is Dengue virus infection. The
results presented in the examples demonstrate for the first time a
role for DC-SIGN in Dengue virus binding to human dendritic
cells.
[0127] The results described herein, including the results
described in the examples, show that highly purified DEN-1 virus
bearing mosquito N-linked oligosaccharides is able to replicate in
human DC and produce progeny virus. Apoptotic cell death was also
observed among DEN-1 virus-infected DCs. The C-lectin molecule,
DC-SIGN, is expressed at the surface of DCs. The experiments
described herein sought to determine whether the DC-specific
adhesion receptor DC-SIGN has the ability to promote DEN virus
infection of human DC cells. The results showed that DC-SIGN
specific antibody has a blocking action at the level of DEN-1 virus
infection. A novel function of DC-SIGN has been therefore
identified as DEN virus binding protein possibly through
interaction with the E glycoprotein. The process of
DC-SIGN-mediated DEN virus infectivity to DCs provides a new
mechanism for targeting the design of anti-viral compounds.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] In some embodiments the DC-SIGN blocker is a mannosylated
molecule that binds to a DC-SIGN receptor. The mannosylated
molecule may be mannan.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] In a preferred embodiment, the viral infection is a
Flaviviridae infection and the viral effector molecule is a
Flaviviridae effector molecule. In a more preferred embodiment, the
viral infection is a Dengue virus infection and the viral effector
molecule is a Dengue virus effector molecule. In a further
preferred aspect the mammal is a human. In some embodiments the
Dengue virus effector molecule is a molecular constituent of the
Dengue virus envelope. In further embodiments the molecular
constituent of the Dengue virus envelope is a Dengue virus envelope
glycoprotein. In yet further embodiments the Dengue virus envelope
glycoprotein is Dengue virus E glycoprotein.
[0138] Included among embodiments of the invention in which the
viral infection is a Dengue virus infection and the viral effector
molecule is a Dengue virus effector molecule are embodiments where
the DC-SIGN blocker comprises a binding moiety of the Dengue virus
effector molecule; the DC-SIGN blocker comprises a binding moiety
of the Dengue virus E glycoprotein; 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
Dengue virus effector molecule. Among embodiments where the
antibody specifically binds the Dengue virus effector molecule are
embodiments where the Dengue virus effector molecule is Dengue
virus E glycoprotein.
[0139] In a 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.
[0140] It is interesting that both Dengue virus 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 HIV
infection. Specifically, it is an object of the invention to
provide a method of preventing or treating an 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 HIV or SIV with DC-SIGN receptor
present on dendritic cells of the human or simian to thereby
prevent or treat the HIV or SIV infection.
[0141] 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.
[0142] 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.
[0143] The invention also provides the use of an amount of a
DC-SIGN modulator sufficient to substantially modulate the binding
of an effector molecule to a DC-SIGN receptor for the preparation
of a medicament for preventing or treating a disease of a mammal,
wherein at least one symptom of the disease is mediated at least in
part by the binding of the effector molecule to the DC-SIGN
receptor of the mammal to be treated.
[0144] The invention also provides use of an amount of a DC-SIGN
blocker sufficient to substantially inhibit the binding of an
effector molecule to a DC-SIGN receptor for the preparation of a
medicament for preventing or treating a disease of a mammal,
wherein at least one symptom of the disease is mediated at least in
part by the binding of the effector molecule to the DC-SIGN
receptor of the mammal to be treated. In some embodiments the
DC-SIGN blocker is a blocking derivative of the effector molecule.
In further embodiments the DC-SIGN blocker is an antibody. In yet
further embodiments the antibody specifically binds DC-SIGN. In
other embodiments the antibody specifically binds the effector
molecule.
[0145] In other embodiments the DC-SIGN blocker is a mannosylated
molecule that binds to a DC-SIGN receptor; the mannosylated
molecule being preferably mannan.
[0146] The invention also provides the use of an amount of a
DC-SIGN modulator sufficient to substantially modulate the binding
of a viral effector molecule to a DC-SIGN receptor for the
preparation of a medicament for preventing or treating a viral
infection of a mammal, wherein the viral infection is mediated at
least in part by the binding of the viral effector molecule to the
DC-SIGN receptor of the mammal to be treated.
[0147] The invention also provides the use of an amount of a
DC-SIGN blocker sufficient to substantially inhibit the binding of
a viral effector molecule to a DC-SIGN receptor for the preparation
of a medicament for preventing or treating a viral infection of a
mammal, wherein the viral infection is mediated at least in part by
the binding of the viral effector molecule to the DC-SIGN receptor
of the mammal to be treated. In some embodiments of said use the
viral effector molecule is a molecular constituent of the viral
envelope. In other embodiments the molecular constituent of the
viral envelope is an envelope glycoprotein.
[0148] In further embodiments 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 the envelope
glycoprotein. In other embodiments the DC-SIGN blocker is an
antibody. The antibody is a monoclonal antibody. In other
embodiments the mammal is a human and the monoclonal antibody is
humanized. The antibody may specifically bind DC-SIGN or
specifically binds the viral effector molecule. In additional
embodiments the antibody specifically binds the binding moiety of
the viral effector molecule. In additional the DC-SIGN blocker is a
mannosylated molecule that binds to a DC-SIGN receptor. The
mannosylated molecule may be mannan.
[0149] In a preferred embodiment the viral infection is a
Flaviviridae virus infection and the viral effector molecule is a
Flaviviridae effector molecule. In a more preferred embodiment, the
Flaviviridae viral infection is a Dengue virus infection and the
Flaviviridae effector molecule is a Dengue effector molecule. In a
further preferred aspect the mammal is human. In some embodiments
the Dengue virus effector molecule is a molecular constituent of
the Dengue virus envelope. In further embodiments the molecular
constituent of the Dengue virus envelope is a Dengue virus envelope
glycoprotein. In yet further embodiments the Dengue virus envelope
glycoprotein is Dengue virus E glycoprotein.
[0150] Included among embodiments of said use in which the viral
infection is a Dengue virus infection and the viral effector
molecule is a Dengue virus effector molecule are embodiments where
the DC-SIGN blocker comprises a binding moiety of the Dengue virus
effector molecule; the DC-SIGN blocker comprises a binding moiety
of the Dengue virus E glycoprotein; 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
Dengue virus effector molecule. Among embodiments where the
antibody specifically binds the Dengue virus effector molecule are
embodiments where the Dengue virus effector molecule is Dengue
virus E glycoprotein.
[0151] The invention also provides the use of an amount of a
DC-SIGN modulator sufficient to substantially modulate the binding
of HIV or SIV to a DC-SIGN receptor present on dendritic cells of a
human or a simian for the preparation of a medicament for
preventing or treating an HIV or a SIV infection of said human or
said simian.
[0152] The invention also provides the use of an amount of a
DC-SIGN blocker sufficient to substantially inhibit the binding or
interaction of HIV or SIV to a DC-SIGN receptor present on
dendritic cells of a human or a simian for the preparation of a
medicament for preventing or treating an HIV or a SIV infection of
said human or said simian. In some embodiments the DC-SIGN blocker
comprises a binding moiety of the Dengue virus E glycoprotein. In
further embodiments an HIV infection of a human is prevented or
treated.
[0153] The invention also provides the use of an amount of a
DC-SIGN modulator sufficient to substantially modulate the binding
of ICAM-3 present on T cells of a mammal with DC-SIGN receptor
present on dendritic cells of the mammal for the preparation of a
medicament for preventing or treating inflammation in said mammal
caused by specific binding of ICAM-3 present on T cells of the
mammal with DC-SIGN receptor present on dendritic cells of the
mammal.
[0154] The invention also provides the use of an amount of a
DC-SIGN blocker sufficient to substantially inhibit the binding or
interaction of ICAM-3 present on T cells of a mammal with DC-SIGN
receptor present on dendritic cells of the mammal for the
preparation of a medicament for preventing or treating inflammation
in said mammal caused by specific binding of ICAM-3 present on T
cells of the mammal with DC-SIGN receptor present on dendritic
cells of the mammal. In some embodiments, the DC-SIGN blocker
comprises a binding moiety of the Dengue virus E glycoprotein. In
other embodiments the mammal is a human.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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 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 micro-organisms 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] Therapeutically useful agents, such as growth factors (e.g.,
BMPs, TGF-.beta., FGF, 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] Techniques for making the PC-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.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] In a preferred embodiment the subject molecule is an
antibody.
[0174] 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.
[0175] The targeting complex of the invention can be exposed to a
cell expressing DC-SIGN, such as 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.
[0176] 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
Flaviviruses
[0177] The production and purification of DEN type-1 (DEN-1) virus
strain FGA/NA d1d (GenBank accession number AF226686) (Duarte dos
Santos et al. Virology, 274: 292, 2000) and West Nile (WN) virus
strain IS-98-ST1 (GenBank accession number AF481864) (Mashimo et
al., PNAS, 99: 11311, 2002) from mosquito Aedes pseudoscutellaris
AP61 cell monolayers and virus titration on AP61 cells by focus
immunodetection assay (FIA) were performed as previously described
(Despres et al., Virology, 196: 209, 1993). Yellow fever (YF) virus
vaccine strain 17D-204 (STAMARIL, Pasteur Vaccins, Lot E113)
(GenBank accession number: X07755) was propagated twice in African
green monkey kidney VERO cell monolayers, purified in sucrose
gradients and titrated on VERO cells. Infectivity titers were
expressed as focus forming units (FFU).
[0178] It is noteworthy that FGA/NA d1d E glycoprotein has two
N-linked glycosylation sites at positions Asn.sub.67 and
Asn.sub.153. Both N-glycosylation sites of the DEN-1 glycoprotein E
appear to be utilized during the N-glycosylation process (Courageot
et al., J.Virol., 74: 564-572). IS-98-ST1 E glycoprotein has a
single N-linked glycosylation site that appeared to be utilized
(Despres, personal communication). Whereas 17D-204 E protein is not
N-glycosylated. The flavivirus M protein is not glycosylated.
[0179] In these experiments, we tested DEN-1 and WN
virion-associated E glycoproteins which bear mature N-linked
oligosaccharides from mosquito AP61 cells.
Example 2
Human Mononuclear Cells
[0180] Human DCs from purified mononuclear cells, human monocytic
cell line THP-1 (ATCC TIB 202) and DC-SIGN expressing cell clone,
THP/DC-SIGN, were obtained from Ali Amara (Immunologie Virale)
(Kwon et al., Immunity, 16: 135, 2002). Immature DC, THP-1 and
THP/DC-SIGN cells were cultured in RPMI 1640 culture medium
supplemented with 10% heat-inactivated fetal calf serum (FCS)
(Eurobio, lot 160402), 2 mM L-glutamine and antibiotics
Peni/Strepto.
[0181] DC were adhered to poly-L-lysine (Sigma)-coated glass
Lab-tek chambers (Nalge Nunc International) (5.times.10.sup.4 cells
per cm.sup.2). THP-1 and THP/DC-SIGN cells were adhered to
poly-L-lysine-treated glass Lab-tek chambers or polylysine-treated
12-well flasks (5.times.10.sup.4 cells per cm.sup.2).
Example 3
Virus Infections
[0182] Cells were washed once with RPMI 1640, incubated with highly
purified virus in RPMI 1640 supplemented with 0.2% bovine serum
albumine (BSA, pH 7.5) (Sigma) for 2 hrs at 37.degree. C. and
placed into fresh media supplemented with 2% FCS, 2 mM L-glutamine
and antibiotics Peni/Strepto at 37.degree. C. for 40 hrs.
[0183] The percentage of cells expressing viral antigen was
determined using either DEN-1 virus-specific hyperimmune mouse
ascites fluid (HMAF) 9801 (strain Hawai), WN virus-specific HMAF
0801 (strain IS-98-ST1), or YF virus-specific HMAF 9803 (strain
FNV) with dilution of 1:50, by indirect immunofluorescence as
previously described (Despres et al., J.Virol., 70: 4090,
1996).
Example 4
Inhibition of DC-SIGN-Mediated Virus Binding
[0184] Adherent cells were incubated either with EDTA (5 mM),
mannan (20 .mu.g/ml), anti-DC-SIGN Mab 1B10.2.6 (20 .mu.g/mi),
anti-LMCV Mab 12.5 (isotype control, 20 .mu.g/ml), or DEN
E-specific Mab 9D12 (dilution 1:50) in RPMI 1640 supplemented with
0.2% BSA for 20 min at room temperature before binding the virus
for 2 hrs. Two hours after infection, cells were washed with RPMI
1640 and incubated with RMPI 1640 2% FCS for 40 hrs. Anti-DC-SIGN
Mab 1B10.2.6 and anti-LMCV Mab 12.5 were obtained from Ali
Amara.
Example 5
Immunofluorescence Assay
[0185] Briefly, cells were fixed with 3% paraformaldehyde (PFA) in
PBS for 20 min at room temperature, incubated with 50 mM NH.sub.4Cl
in PBS for 20 min and permeabilized with 0.1% Triton X-100 in PBS
for 5 min. Intracellular viral antigens were stained with
anti-flavivirus HMAF. The secondary antibody used was a
FITC-conjugated goat anti-mouse IgG (Sigma). Cells were observed
with a fluorescence microscope.
Example 6
In Situ Detection of Apoptotic Cells
[0186] To assess the nuclear changes associated with apoptotic cell
death, PFA-fixed cells on glass slides were treated with 0.1
.mu.g/ml Hoechst 33258 (Sigma) in 0.1% citrate buffer (pH 6.0) for
10 min at room temperature. Cells were considered to be apoptotic
if their nuclei exhibited margination and condensation of
chromatin. Cells were observed with a fluorescence microscope.
[0187] Apoptosis-induced DNA breaks were detected by the
deoxyterminal transferase-mediated dUTP nick-end labeling (TUNEL)
method (Despres et al., J.Virol., 70: 4090, 1996). A TUNEL assay
was performed with streptavidin-CY.TM. 3 conjugate (Jackson
Immunoresearch). Cells were observed with a fluorescence
microscope.
Example 7
DEN Virus Infection of Human DC Ex-Vivo
[0188] We have examined whether DEN-1 virus strain FGA/NA d1d
replicates in DCs. Inoculation with 5AP61FFU/cell was needed to
infect 50% of human DCs to DEN virus within the 40 hrs as examined
by IF assay using anti-DEN-1 HMAF (FIG. 1). Infective particles
accumulated in FGA/NA d1d-infected DCs to 9 (.+-.3).times.10.sup.4
APG1FFU/ml (for 50,000 DCs) at 48 h postinfection. Unlike DEN-1
virus, lower than 1% of the DCs were infected with WN virus strain
IS-98-ST1 (m.o.i. of 5AP61FFU/cell) or YF vaccine strain (m.o.i. of
50VEROFFU/cell) at 40 h postinfection (data not shown).
[0189] Infection of DCs with DEN-1 virus strain FGA/NA d1d results
in apoptosis after 40 h of infection as judged by Hoescht 33258
staining (FIG. 1) and TUNEL method (FIGS. 2A & B).
Example 8
Anti-DC-SIGN Mab 1B10.2.6 Blocks DEN-1 Virus Infection of DCs
[0190] We analyzed the effect of anti-DC-SIGN-specific Mab 1B10.2.6
on DEN-1 virus strain FGA/NA d1d infectivity. FIG. 3 indicates that
20 .mu.g/ml of Mab 1B10.2.6 blocked FGA/NA d1d infection of DC
cells as examined by IF assay. As a positive control, anti-E Mab
9D12 reduced the infectivity of DEN-1 virus by 70%. The production
of infectious particles in FGA/NA did-infected DCs treated with Mab
1B10.2.6 was very low (<5AP61FFU/ml).
Example 9
DEN Virus Infection of Human Monocytes THP-1 and THP/DC-SIGN
1. Specificity of the Interaction Between DC-SIGN and DEN Virus
[0191] We further examined the specificity of the interaction
between DC-SIGN and DEN virus. To investigate this issue, human
monocytic THP-1 cells were first infected with DEN-1 virus strain
FGA/NA did. At an m.o.i of 5AP61FFU/cell, less than 1% of THP-1
were positive for DEN antigens as examined by IF assay (FIGS. 4A
& B). Similarly, YF virus vaccine strain 17D-204 (m.o.i. of
50VEROFFU/cell) failed to replicate in THP-1 cells (FIGS. 4A &
B). Whereas an m.o.i of 5 AP61FFU/cell was needed to infect about
of 70% of THP-1 cells with WN virus strain IS-98-ST1 (FIGS. 4A
& B).
2. Role of DC-SIGN in DEN-1 Virus Infectivity at THP-1
[0192] To determine whether DC-SIGN confers DEN-1 virus infectivity
at THP-1, we tested the DC-SIGN-expressing human monocytic THP cell
line, THP/DC-SIGN (Kwon et al., Immunity, 16: 135, 2002). At an
m.o.i. of 5 AP61FFU/cell, more than 50% of THP/DC-SIGN cells were
positive for FGA/NA d1d antigens after 48 h of infection (FIG. 4).
Mortality of THP/DC-SIGN cells in which DEN-1 virus was replicating
occurred after a 96-h period of infection. Unlike DEN-1 virus, YF
virus vaccine strain 17D-204 did not replicate in THP/DC-SIGN cells
at an m.o.i. as high as 50VEROFFU/cell. It is also of interest that
DC-SIGN could mediate enhancement of WN virus infection by
monocytic cells (FIG. 4).
3. Effects of Mannan, ETDA and DC-SIGN Specific Mab 1B10.2.6 on
DC-SIGN-Mediated DEN Virus Binding
[0193] We tested the effects of mannan, ETDA and DC-SIGN specific
Mab 1B10.2.6 on DC-SIGN-mediated DEN virus binding. In these
experiments, Mab BDi2.5 served as a negative control whereas anti-E
Mab 9D12 was used as a positive control. THP/DC-SIGN cells were
infected with DEN-1 virus strain FGA/NA did at an m.o.i. of
5AP61FFU/cell. When THP/DC-SIGN cells were preincubated with EDTA
or DC-SIGN specific Mab 1B10.2.6, infectivity of DEN-1 virus was
essentially abolished (FIG. 5). Dose of 20 .mu.g/ml mannan was
needed to reduce FGANNA d1d infectivity by 75%. It is therefore
reasonable to conclude that DC-SIGN is capable of promoting DEN
virus infection of mononuclear cells
[0194] The cytoplasmic tail of DC-SIGN contains two defined
putative internalization motifs, a dileucine-based motif and a
tyrosine-based motif. We next tested THP-1 cell clone .DELTA.35
expressing mutant form of DC-SIGN in which the cytoplasmic domain
of the molecule was truncated (Kwon et al., Immunity, 16: 135,
2002). The truncation removed 35 amino acids that include both the
dileucine motif and the tyrosine-based motif. We found that DEN-1
virus infectivity is mostly preserved in THP-1 cell clone .DELTA.35
(FIG. 6). Thus, the cytosolic tail does not contribute to the
enhancement of DEN-1 virus infection by DC-SIGN.
4. Comparative Interaction Between DC and Different DEN Virus
Types
[0195] THP/DC-SIGN cells were infected with DEN virus [DEN-1 virus
strain FGA/NA did (French Guiana); DEN-2 virus strain Jam
(Jamaica); DEN-3 virus strain PaH 881 (Thailand); DEN-4 virus
strain 63632 (Birmanie)] at the multiplicities of infection (MOI)
from 0.1 to 10AP61FFU/cell as specified in example 3. The
percentage (%) of DEN antigen-positive THP cells expressing DC-SIGN
was determined by indirect immunofluorescence using specific
anti-DEN HMAF at 40 h post-infection (FIG. 7). No DEN antigen was
detected in THP cells infected with each DEN virus strain.
[0196] The following Table sums up the results. TABLE-US-00001 DEN
virus infection of DC-SIGN-expressing THP/DC-SIGN cells Virus MOI
0.1 MOI 1 MOI 10 DEN-1 25% 40% 50% DEN-2 2.5% 6% 14% DEN-3 27.5%
49% 70% DEN-4 1.5% 2.5% 5%
Deposits
[0197] The Hela cell line denoted "Hela DC-SIGN Flap" was deposited
at the C.N.C.M., 28 rue du Docteur Roux, 75724 PARIS Cedex 15,
France, on Oct. 30, 2002, under the accession number 1-2949.
[0198] The DC-SIGN clone denoted "DC-SIGN human clone2" was
deposited at the C.N.C.M., 28 rue du Docteur Roux, 75724 PARIS
Cedex 15, France, on Oct. 30, 2002, under the accession number
1-2950.
[0199] The hybridoma denoted "1B10.2.6" was deposited at the
C.N.C.M., 28 rue du Docteur Roux, 75724 PARIS Cedex 15, France, on
Nov. 7, 2002, under the accession number 1-2951.
References
[0200] Bhamarapravati, N. et: al. (Dengue and Dengue haemorrhagic
fever (1997), 367-377).
[0201] Courageot et al., J.Virol., 74: 564-572.
[0202] Crooks A. J. et al. J. Chrom. (1990), 502, 59-68.
[0203] Crooks A. J. et al. J. Gen. Virol. (1994), 75,
3453-3460.
[0204] Curtus et al., PNAS USA (1992), 89, 8356-8360.
[0205] Despres et al., J.Virol., 70: 4090, 1996.
[0206] Despres et al., Virology, 196: 209, 1993.
[0207] Drickammer, Curr.Opin.Immunol. 13: 585, 1999.
[0208] Duarte dos Santos et al., Virology, 274: 292, 2000.
[0209] Feinberg, H., Mitchell, D. A., Drickamer, K. & Weis, W.
I. (2001). Structural basis for selective recognition of
oligosaccharides by DC-SIGN and DC-SIGNR. Science 294, 2163-6.
[0210] Geijtenbeek, T. B. et al. (2000). DC-SIGN, a dendritic
cell-specific HIV-1-binding protein that enhances trans-infection
of T cells. Cell 100, 587-97.
[0211] Geijtenbeek et al., Nature 1: 353, 2000.
[0212] Glamand et al., J. Virol. (1999), 73, 6106-6110.
[0213] Gorman et al. (1982) P.N.A.S. 79:6777.
[0214] Grosschedl et al. (1985) Cell 41:885.
[0215] Hall R. A. et al., J. Virol. (1999), 73, 10272-10280.
[0216] Ho et al., Immunology 166: 1499, 2001.
[0217] Jost et al. (1994) J.B.C. 269:26267-73.
[0218] Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, N.I.H. publication no. 91-3242.
[0219] Kaufmann, S. H. E. (2001). How can immunology contribute to
the control of tuberculosis, Nat. Rev. Immunol. 1, 20-30.
[0220] Kwon et al., Immunity, 16: 135, 2002.
[0221] Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J. Immunol.
139:3521).
[0222] Marovich et al., JID Symp.Proc. 6:219, 2001.
[0223] Mashimo et al., PNAS, 99: 11311, 2002.
[0224] Monath, T. P. et al., (1996) Flaviviruses in B. N. Fields,
D. M. Knipe, P. M. Howly et al. (eds.) "Fields Virology"
Philadelphia: Lippincott Raven Press Publishers.
[0225] Moody, D. B. et al. (2000). CD1c-mediated T-cell recognition
of isoprenoid glycolipids in Mycobacterium tuberculosis infection.
Nature 404, 884-8.
[0226] Okayama et al. (1983) Mol. Cell. Bio. 3:280).
[0227] Palucka, Nat. Med 6: 748, 2000.
[0228] Pohlmann, S., et al., PNAS USA, (2001), 98, 2670-2675.
[0229] Relloso et al., J.Immunol. 168, 2634, 2002.
[0230] Rice C. M. at al., J. Virol., (1997), 71, 291-298.
[0231] Rice C. M. et al., J. Virol., (1997), 71, 9608-9617):
[0232] Rice C. M. et al., J. Virol., (1996), 222,159-168.
[0233] Russel, P. K. et al. (J. Immunol., (1970), 105,
838-845).
[0234] Smith, G. W. et al. (J. Gen Virol., (1985), 66,
559-571).
[0235] Steinman, R. M. (2000). DC-SIGN: A Guide to Some Mysteries
of Dendritic Cells. Cell 100, 491-94.
[0236] Wu et al., Nat.Med. 6: 816, 2000.
[0237] WO 90/10077
[0238] WO 90/04036.
[0239] WO 92/02190.
[0240] WO 98/50433.
[0241] WO 98,24893.
[0242] WO 99/53049.
[0243] U.S. Pat. No. 4,683,195.
[0244] U.S. Pat. No. 4,683,202.
[0245] U.S. Pat. No. 5,516,637.
[0246] U.S. Pat. No. 5,223,409.
[0247] U.S. Pat. No. 5,658,727.
[0248] U.S. Pat. No. 5,667,988.
[0249] U.S. Pat. No. 5,498,538
[0250] U.S. Pat. No. 5,403,484
[0251] U.S. Pat. No. 5,571,698.
[0252] U.S. Pat. No. 5,625,033.
[0253] The entire contents of all references, patents and published
patent applications cited throughout this application are herein
incorporated by reference in their entirety.
* * * * *