U.S. patent application number 12/021118 was filed with the patent office on 2009-07-09 for methods of modulating immune function.
Invention is credited to Joshua Cabrera, Evan W. Newell, M. KAREN NEWELL ROGERS.
Application Number | 20090175838 12/021118 |
Document ID | / |
Family ID | 39674687 |
Filed Date | 2009-07-09 |
United States Patent
Application |
20090175838 |
Kind Code |
A1 |
NEWELL ROGERS; M. KAREN ; et
al. |
July 9, 2009 |
METHODS OF MODULATING IMMUNE FUNCTION
Abstract
The invention relates to methods for modulating the immune
function through targeting of CLIP molecules as well as gamma delta
T-cells. The result is wide range of new therapeutic regimens for
treating, inhibiting the development of, or otherwise dealing with,
a multitude of illnesses and conditions, including autoimmune
disease, transplant and cell graft rejection, cancer, bacterial
infection, HIV infection, and AIDS, as well as novel methods of
diagnosis and of introducing a treatment regimen into a
subject.
Inventors: |
NEWELL ROGERS; M. KAREN;
(Colorado Springs, CO) ; Newell; Evan W.; (Menlo
Park, CA) ; Cabrera; Joshua; (Colorado Springs,
CO) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE., SUITE 2400
AUSTIN
TX
78701
US
|
Family ID: |
39674687 |
Appl. No.: |
12/021118 |
Filed: |
January 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60886852 |
Jan 26, 2007 |
|
|
|
60906731 |
Mar 13, 2007 |
|
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|
Current U.S.
Class: |
424/93.71 ;
435/375; 514/23; 514/557 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
31/12 20180101; A61P 29/00 20180101; A61K 31/40 20130101; A61P
43/00 20180101; A61P 37/00 20180101; A61K 39/395 20130101; Y02A
50/401 20180101; Y02A 50/30 20180101; A61P 31/18 20180101; A61P
7/02 20180101; A61P 3/10 20180101; A61K 31/4706 20130101; A61K
45/06 20130101; A61P 21/04 20180101; A61K 31/03 20130101; A61P 9/00
20180101; A61P 25/00 20180101; A61K 31/7088 20130101; A61P 19/02
20180101; A61P 37/02 20180101; A61K 38/10 20130101; A61P 35/00
20180101; A61P 7/06 20180101; A61P 35/02 20180101; A61P 35/04
20180101; A61K 31/03 20130101; A61K 2300/00 20130101; A61K 31/40
20130101; A61K 2300/00 20130101; A61K 31/4706 20130101; A61K
2300/00 20130101; A61K 31/7088 20130101; A61K 2300/00 20130101;
A61K 38/10 20130101; A61K 2300/00 20130101; A61K 39/395 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/93.71 ;
435/375; 514/23; 514/557 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/02 20060101 C12N005/02; A61K 31/70 20060101
A61K031/70; A61K 31/19 20060101 A61K031/19 |
Claims
1. A method for treating a disorder associated with
.gamma..delta.T-cell expansion, activation and/or effector function
comprising contacting a CLIP molecule expressing cell with an
inhibitor of .gamma..delta.T-cell expansion, activation and/or
effector function in an effective amount to interfere with
.gamma..delta.T-cell expansion, activation and/or effector function
by the CLIP molecule expressing cell.
2. The method of claim 1, wherein the CLIP molecule is CLIP.
3. The method of claim 1, wherein the CLIP molecule is CD74.
4. The method of claim 1, wherein the disorder associated with
.gamma..delta.T-cell expansion and/or activation is autoimmune
disease.
5. The method of claim 1, wherein the disorder associated with
.gamma..delta.T-cell expansion and/or activation is HIV
infection.
6. The method of claim 1, wherein the CLIP molecule expressing cell
is a B-cell.
7. The method of claim 1, wherein the CLIP compound expressing cell
is a neuron, an oligodendrocyte, a microglial cell, or an
astrocyte.
8. The method of claim 1, wherein the CLIP compound expressing cell
is a hearT-cell, a pancreatic beta cell, an intestinal epithelial
cell, a lung cell, an epithelial cell lining the uterine wall, a
skin cell.
9. The method of claim 6, further comprising contacting the B-cell
with an anti-HLA class I or II antibody in an effective amount to
kill the B-cell.
10. The method of claim 1, wherein the inhibitor of
.gamma..delta.T-cell expansion, activation and/or effector function
is a CLIP expression inhibitor.
11. The method of claim 10, wherein the CLIP expression inhibitor
is an siRNA of a CLIP molecule or HLA-DO.
12. The method of claim 1, wherein the inhibitor of
.gamma..delta.T-cell expansion, activation and/or effector function
is a CLIP activity inhibitor.
13. The method of claim 12, wherein the CLIP activity inhibitor is
an agent that displaces CLIP.
14. The method of claim 12, wherein the agent that displaces CLIP
is chloroquine, a lysosomatropic agent, peptide/lipopeptide
antigen, small molecular compound pCP, chlorobenzene (CB), and
parachloroanisol (pCA), peptide HA306-318 (PKYVKQNTLKLAT) (SEQ ID
NO. 2), or peptide CO260-272 (IAGFKGEQGPKGE) (SEQ ID NO. 3).
15. The method of claim 12, wherein the agent that displaces CLIP
is FRIMAVLAS (SEQ ID NO. 1).
16. The method of claim 12, wherein the agent that displaces CLIP
is an HLA binding peptide.
17. The method of claim 12, wherein the agent that displaces CLIP
is a pharmacon that is a combination of a glycolytic inhibitor and
a halogenated alkyl ester.
18. The method of claim 12, wherein the CLIP activity inhibitor is
an anti-CLIP antibody or recombinant HLA-DM.
19. The method of claim 12, wherein the CLIP activity inhibitor is
an agent that inhibits CD74 processing, such as cystatin A, B or
C.
20. The method of claim 1, further comprising exposing the CLIP
molecule expressing cell to an MHC class I or II loading
peptide.
21. The method of claim 1, wherein the .gamma..delta.T-cell is a
v.gamma.9v.delta.2 T-cell.
22. A method for treating a subject having autoimmune disease
comprising administering to the subject a CLIP inhibitor in an
effective amount to reduce CLIP function in a CLIP molecule
expressing cell of the subject.
23.-31. (canceled)
32. A method for treating a subject infected with HIV comprising
administering to the subject a CLIP inhibitor in an effective
amount to reduce CLIP function in a CLIP molecule expressing cell
of the subject.
33.-41. (canceled)
42. A method for treating a subject having a cell or tissue graft,
comprising administering to the subject a CLIP inhibitor in an
effective amount to reduce CLIP function in the cell or tissue
graft, or hematopoeitic cells in the tissue graft, in order to
inhibit cell or tissue graft rejection in the subject.
43. (canceled)
44. A method for treating a subject having a disorder associated
with .gamma..delta.T-cell expansion, activation and/or effector
function comprising removing antigen non-specifically activated
B-cells and/or .gamma..delta.T-cells from the subject to treat the
disorder.
45. A method for inducing B-cell death, comprising: (a) inducing
CLIP molecule expression on an antigen non-specifically activated
B-cell; and (b) contacting the antigen non-specifically activated
B-cell with an anti-HLA class II antibody in an effective amount to
kill the antigen non-specifically activated B-cell.
46.-52. (canceled)
53. A method for displacing CLIP from the surface of a cell,
comprising administering a halogenated alkyl ester or as a
pharmacon a combination of a glycolytic inhibitor and a halogenated
alkyl ester to a subject to displace CLIP from the surface of the
cell.
54.-55. (canceled)
56. The method of claim 53, wherein the glycolytic inhibitor is
2-deoxy-D-glucose.
57. The method of claim 53, wherein the halogenated alkyl ester is
dichloroacetate or salts thereof.
58. A composition comprising a bifunctional compound of a
glycolytic inhibitor and a halogenated alkyl ester.
59.-62. (canceled)
63. A method for treating a subject having cancer comprising
contacting a B-cell or cancer cell with a CLIP inducing agent in an
effective amount to promote CLIP expression on the surface of the
B-cell or cancer cell.
64.-74. (canceled)
75. A method of killing a cancer cell in a subject comprising (a)
inducing cell surface expression of CLIP on a B-cell; (b)
contacting the B-cell of step (a) with a .gamma..delta.T-cell or NK
cell and (c) contacting the .gamma..delta.T-cell or NK cell with
said cancer cell.
76.-91. (canceled)
92. A method of activating a B-cell, a macrophage or dendritic cell
in an antigen non-specific fashion comprising inducing cell surface
expression of CLIP in said cell.
93.-103. (canceled)
104. A method of providing an activated .gamma..delta.T-cell to a
subject comprising: (a) obtaining a .gamma..delta.T-cell; (b)
contacting an antigen non-specifically activated B-cell with said
.gamma..delta.T-cell; and (c) transferring said
.gamma..delta.T-cell once activated to said subject.
105.-112. (canceled)
Description
[0001] The present application claims benefit of priority to U.S.
Provisional Application Ser. Nos. 60/886,852, filed Jan. 26, 2007,
and 60/906,731, filed Mar. 13, 2007, the entire contents of both of
which are hereby incorporated by reference.
BACKGROUND OF INVENTION
[0002] Major Histocompatiblity Complex (MHC)-encoded molecules are
key components of T-cell immunity. The significance of these
molecules as tissue compatibility molecules was first observed in
the late 1930's. Peter Gorer and George Snell observed that when
tumors were transplanted from a genetically non-identical member of
the same species, the tumors were always rejected, but when tumors
were transplanted between genetically identical members of the same
species, the tumor would "take" and would grow in the syngeneic
animal. The genetic complex responsible for the rejection was
subsequently found to be a series of genes that encode protein
products known as Major Histocompatibility molecules. These genes,
also known as immune response or IR genes, and their protein
products are responsible for all graft rejection. There are two
types of MHC molecules: MHC class I and MHC class II. All nucleated
cells express cell surface MHC class I. A subset of specialized
cells express class II MHC. Included in the specialized,
professional antigen-presenting cells (APCs) are B-cells,
macrophages, microglia, dendritic cells, and Langerhans cells among
others.
[0003] As stated above, B-cells express MHC class II. Once antigen
has been bound by the antigen receptor on the B-cell, the antigen
and its receptor are engulfed into an endosomal compartment. This
compartment fuses with another compartment known as the lysosome.
The B-cell is very efficient at breaking down antigens into smaller
parts and loading the parts into MHC class II in the lysosome. The
MHC is then trafficked to the cell surface where the B-cell can
effectively "show" the antigen to a CD4+ T-cell. The activated CD4
cell is also called a helper cell and there are two major
categories, Th1 and Th2.
[0004] The MHC molecules are tightly protected in the
endosomal/lysosomal compartments to insure that only antigens for
which one needs a response get presented to T-cells. MHC class II
molecules, prior to antigen loading, are associated with a molecule
called invariant chain, also known as CD74. The invariant chain is
associated with MHC class II (and recently shown to be associated
with certain MHC class I molecules) prior to antigen loading into
the antigen binding grooves of the MHC molecules. As antigen is
processed, the invariant chain gets cleaved by proteases within the
compartment. First an end piece is removed, and then another known
as CLIP (class II invariant chain associated peptide). CLIP fills
the groove that will ultimately hold the antigen until the antigen
is properly processed. For a detailed review of the invariant
chain, including CLIP, see Matza et al. (2003), incorporated herein
in its entirety. Despite the fact that this "chaperone" role for
invariant chain and CLIP has been identified, the full impact of
these molecules on immune signaling and activation has yet to be
determined.
SUMMARY OF INVENTION
[0005] The invention is based at least in part on the discovery
that inhibitors of .gamma..delta.T-cell expansion, activation
and/or effector function are useful in the treatment of disorders
such as HIV infection, autoimmune disease and tissue graft
rejection. The invention is also based on the discovery that the
same disorders can be treated by inhibiting CLIP presentation in
MHC on a cell surface.
[0006] The invention, in some aspects, is a method for treating a
disorder associated with .gamma..delta.T-cell expansion, activation
and/or effector function by contacting a CLIP molecule expressing
cell with an inhibitor of .gamma..delta.T-cell expansion,
activation and/or effector function in an effective amount to
interfere with .gamma..delta.T-cell expansion, activation and/or
effector function by the CLIP molecule expressing cell. In some
embodiments, the .gamma..delta.T-cell is a v.gamma.9v.delta.2
T-cell. Disorders associated with .gamma..delta.T-cell expansion
and/or activation include, for instance autoimmune disease, HIV
infection, and cell, tissue and graft rejection.
[0007] The CLIP molecule expressing cell is a B-cell in some
embodiments. In other embodiments, the CLIP compound expressing
cell is a neuron, an oligodendrocyte, a microglial cell, or an
astrocyte. In yet other embodiments, the CLIP compound expressing
cell is a heart-cell, a pancreatic .beta. cell, an intestinal
epithelial cell, a lung cell, an epithelial cell lining the uterine
wall, and a skin cell. When the cell is a B-cell, the method may
further involve contacting the B-cell with an anti-HLA class I or
II antibody in an effective amount to kill the B-cell.
[0008] In another aspect, the invention is a method for treating a
subject having autoimmune disease by administering to the subject a
CLIP inhibitor in an effective amount to reduce CLIP function in a
CLIP molecule expressing cell of the subject. In some embodiments,
the autoimmune disease is multiple sclerosis, systemic lupus
erythematosus, type 1 diabetes, viral endocarditis, viral
myocarditis, viral encephalitis, rheumatoid arthritis, Graves'
disease, autoimmune thyroiditis, autoimmune myositis, discoid lupus
erythematosus, Crohns disease, Sjogren's syndrome, Reiter's
syndrome, Rheumatoid arthritis, Lyme Disease, myasthenia gravis,
Kawasaki's disease, Celiac disease, Goodpasture's syndrome, or
aplastic anemia.
[0009] A method for treating a subject infected with HIV by
administering to the subject a CLIP inhibitor in an effective
amount to reduce CLIP function in a CLIP molecule expressing cell
of the subject is provided according to other aspects of the
invention. Optionally, the method further involves removing antigen
non-specifically activated B-cells and/or .gamma..delta.T-cells
from the subject.
[0010] In yet another aspect, the invention is a method for
treating a subject having a cell or tissue graft, by administering
to the subject a CLIP inhibitor in an effective amount to reduce
CLIP function in the cell or tissue graft, or hematopoeitic cells
in the tissue graft, in order to inhibiT-cell or tissue graft
rejection in the subject. In some embodiments, the graft tissue or
cell is heart, lung, kidney, skin, cornea, liver, neuronal tissue
or cell, stem cell, including hematopoetic or embryonic stem
cell.
[0011] In some embodiments, the CLIP molecule is CLIP. In other
embodiments, the CLIP molecule is CD74.
[0012] The inhibitor of .gamma..delta.T-cell expansion, activation
and/or effector function may be a CLIP expression inhibitor. CLIP
expression inhibitors include, for instance, an siRNA of a CLIP
molecule or HLA-DO as well as antisense molecules.
[0013] In other embodiments, the inhibitor of .gamma..delta.T-cell
expansion, activation and/or effector function is a CLIP activity
inhibitor. The CLIP activity inhibitor may be an agent that
displaces CLIP. Agents that displace CLIP include but are not
limited to chloroquine, a lysosomatropic agent, peptide/lipopeptide
antigen, small molecular compound pCP, chlorobenzene (CB), and
parachloroanisol (pCA), peptide HA306-318 (PKYVKQNTLKLAT) (SEQ ID
NO. 2), or peptide CO260-272 (IAGFKGEQGPKGE) (SEQ ID NO. 3). In
some embodiments, the agent that displaces CLIP is FRIMAVLAS (SEQ
ID NO. 1). In other embodiments, the agent that displaces CLIP is
an HLA binding peptide. In yet other embodiments, the agent that
displaces CLIP is a pharmacon that is a combination of a glycolytic
inhibitor and a halogenated alkyl ester. Alternatively, the CLIP
activity inhibitor is an anti-CLIP antibody or recombinant HLA-DM.
In some embodiments, the anti-CLIP antibody is specific for CLIP in
the context of MHC class II. In other embodiments, the anti-CLIP
antibody is specific for CLIP in the context of MHC class I. In
other embodiments, the CLIP activity inhibitor is an agent that
inhibits CD74 processing, such as cystatin A, B or C.
[0014] In some embodiments, the method further includes exposing
the CLIP molecule expressing cell to an MHC class I or II loading
peptide.
[0015] In other aspects, the invention is a method for treating a
subject having a disorder associated with .gamma..delta.T-cell
expansion, activation and/or effector function by removing antigen
non-specifically activated B-cells and/or .gamma..delta.T-cells
from the subject to treat the disorder.
[0016] In yet another aspect, the invention is a method for
inducing B-cell death, by inducing CLIP molecule expression on an
antigen non-specifically activated B-cell and contacting the
antigen non-specifically activated B-cell with an anti-HLA class II
antibody in an effective amount to kill the antigen
non-specifically activated B-cell.
[0017] In some embodiments, the B-cell is in vitro. In other
embodiments, the B-cell is in a subject. The subject may be
administered a CLIP inducing agent. CLIP inducing agents include
but are not limited to CLIP expression vectors and CLIP activators.
The subject may have an autoimmune disease or be infected with
HIV.
[0018] A method for displacing CLIP from the surface of a cell is
provided according to other aspects of the invention. The method
may be performed by administering a halogenated alkyl ester or as a
pharmacon a combination of a glycolytic inhibitor and a halogenated
alkyl ester to a subject to displace CLIP from the surface of the
cell. In some embodiments, the pharmacon is a single bifunctional
compound acting as a prodrug.
[0019] In other embodiments, the glycolytic inhibitor is a
2-deoxyglucose. Optionally, the 2-deoxyglucose is
2-deoxy-D-glucose. The halogenated alkyl ester may be, for
instance, dichloroacetate or salts thereof.
[0020] A composition of a bifunctional compound of a glycolytic
inhibitor and a halogenated alkyl ester is provided according to
other aspects of the invention. In some embodiments, the
bifunctional compound has the following structure:
##STR00001##
[0021] In other embodiments, the bifunctional compound has the
following structure:
##STR00002##
[0022] In yet other embodiments, the bifunctional compound has the
following structure:
##STR00003##
[0023] The bifunctional compound may have the following
structure:
##STR00004##
[0024] The invention is also based on the findings that diseases
such as cancer and infection by agents other than HIV can be
treated by promoting CLIP on the surface such that
.gamma..delta.T-cells can cause the killing of the cancerous or
infected cells. In other aspects of the invention, a method for
treating a subject having cancer by contacting a B-cell or cancer
cell with a CLIP inducing agent in an effective amount to promote
CLIP expression on the surface of the B-cell or cancer cell is
provided.
[0025] In some embodiments, the CLIP inducing agent is a CLIP
expression vector. In other embodiments the CLIP inducing agent is
a CLIP activator. The CLIP activator may be, for instance, nef or
an agent that increases nef expression, ectopic CLIP, a
palmitoylated protein or PAM, or an anti-CD40 or CD40L molecule in
combination with IL-4. In some embodiments the CLIP activator is a
HLA-DO molecule which promotes a higher HLA-DO: HLA-DM ratio. In
yet other embodiments, the CLIP activator is an anti-sense or siRNA
to HLA-DM.
[0026] In some embodiments, the cancer cell is a breast cancer
cell, a lung cancer cell, a head & neck cancer cell, a brain
cancer cell, an esophageal cancer cell, a liver cancer cell, a
prostate cancer cell, a stomach cancer cell, an ovarian cancer
cell, a uterine cancer cell, a cervical cancer cell, a testicular
cancer cell, a skin cancer cell, a colon cancer cell, a leukemia
cell, a lymphoma cell, a glioblastoma cell, a rhabdomyosarcoma
cell, a melanoma cell, or a Kaposi's sarcoma cell. In some
embodiments the cancer is primary, metastatic, recurrent or
multi-drug resistant.
[0027] The methods may involve treating the subject with a standard
anti-cancer therapy. Standard anti-cancer therapy includes for
instance chemotherapy, radiotherapy or hormonal therapy.
[0028] A method of killing a cancer cell in a subject by (a)
inducing cell surface expression of CLIP on a B-cell; (b)
contacting the B-cell of step (a) with a .gamma..delta.T-cell or NK
cell and (c) contacting the .gamma..delta.T-cell or NK cell with
said cancer cell is provided according to other aspects of the
invention. In some embodiments the step (a) is performed ex vivo
and in other embodiments step (b) is performed in vivo.
[0029] In some embodiments, the B-cell of step (a) is allogeneic to
the subject. In other embodiments the .gamma..delta.T-cell of step
(c) is allogeneic to the subject. In yet other embodiments, the NK
cell is allogeneic to the subject. In some embodiments the NK cell
is a .gamma..delta.+NK cell or NK T-cells.
[0030] A method for treating a subject having a non-HIV infection
by contacting a B-cell or a cell of the subject infected with a
non-HIV infectious agent with a CLIP inducing agent in an effective
amount to promote CLIP expression on the surface of the B-cell or
the cell infected with a non-HIV infectious agent is provided
according to other aspects of the invention.
[0031] In other aspects, the invention is a method of activating a
B-cell, a macrophage or dendritic cell in an antigen non-specific
fashion by inducing cell surface expression of CLIP in the cell. In
some embodiments, the cell is administered to a subject.
[0032] In some embodiments, the activated cell is allogeneic to
said subject. In other embodiments the activated cell is autologous
to said subject. In yet other embodiments the cell is a B-cell, a
macrophage or dendritic cell.
[0033] A method of inhibiting activation of a cell selected from a
.gamma..delta.T-cell, an NK cell or an NK T-cell in a subject by
depleting antigen non-specifically activated B-cells from said
subject is provided according to other aspects of the invention. In
one embodiment, the depletion comprises leukophoresis. In other
embodiments, the depletion comprises antibody ablation. The subject
may suffer from an autoimmune disease, HIV infection or be a
transplant recipient. In some embodiments, the .gamma..delta.T-cell
is a v.gamma.9v.delta.2 T-cell.
[0034] The invention also provides a method of providing an
activated .gamma..delta.T-cell to a subject by (a) obtaining a
.gamma..delta.T-cell; (b) contacting an antigen non-specifically
activated B-cell with said .gamma..delta.T-cell; and (c)
transferring said .gamma..delta.T-cell once activated to said
subject. In some embodiments, the .gamma..delta.T-cell is
allogeneic to said subject. In other embodiments, the
.gamma..delta.T-cell is autologous to said subject. In yet other
embodiments the .gamma..delta.T-cell is a v.gamma.9v.delta.2
T-cell.
[0035] In other aspects, the invention is a method of diagnosing
autoimmune disease or HIV infection comprising measuring levels of
at least one defensin in a subject exhibiting one or more
additional symptoms of autoimmune disease. In some embodiments, the
autoimmune disease is multiple sclerosis, systemic lupus
erythematosus, type 1 diabetes, viral endocarditis, viral
encephalitis, rheumatoid arthritis, Graves' disease, autoimmune
thyroiditis, autoimmune myositis, discoid lupus erythematosus. In
some embodiments, the defensin is LL37.
[0036] This invention is not limited in its application to the
details of construction and the arrangement of components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments and of being practiced or
of being carried out in various ways. Also, the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of "including,"
"comprising," or "having," "containing," "involving," and
variations thereof herein, is meant to encompass the items listed
thereafter and equivalents thereof as well as additional items.
BRIEF DESCRIPTION OF DRAWINGS
[0037] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component may be
labeled in every drawing. In the drawings:
[0038] FIG. 1 depicts % B-cell Death in resistant C57B16 versus
sensitive Coxsackievirus infected mice from 1 to 5 days post
infection.
[0039] FIGS. 2A-B are dot plots representing flow cytometric
analysis of 5 day cultures in which CD40 Ligand activated B-cells
were co-cultured with autologous PMBCs for 5 days.
[0040] FIGS. 3A-C depict CLIP displacement from the surface of
model B-cells lines (Daudi and Raji) in response to thymic nuclear
protein (TNP) mixture. FIG. 3A is a 3 hour reaction. FIG. 3B is a
24 hour reaction. FIG. 3C is a 48 hour reaction.
[0041] FIG. 4 depicts that 2-Deoxyglucose and dichloroacetate
affects B-cell surface CLIP.
[0042] FIG. 5 depicts CLIP displacement from the surface of model
B-cells lines (Daudi and Raji) in response to a synthetic peptide
FRIMAVLAS (SEQ ID NO. 1).
DETAILED DESCRIPTION
[0043] The present invention provides new insights into the role of
invariant chain (CD74) and CLIP in disease and presents novel
approaches to modulating the immune function through targeting of
invariant chain/CD74 and CLIP. The result is wide range of new
therapeutic regimens for treating or inhibiting the development or
progression of a multitude of illnesses and conditions, including
autoimmune disease, transplant and cell graft rejection, cancer,
bacterial infection, HIV infection, as well as novel methods of
diagnosis and of introducing a treatment regimen into a
subject.
[0044] It has been discovered herein that B-cells, in addition to
producing antibodies, can also be activated in a somewhat antigen
non-specific, bystander fashion. For example, during a viral or
bacterial infection, non-antigen specific B-cells in the area of
the antigen-specific B-cell that were in close proximity to an
inflammatory or inciting lesion could manage to become activated in
a bystander fashion. In those cases, CLIP would remain in the
groove and get transported to the cell surface of the B-cell. Its
presence on the cell surface is dangerous because if CLIP gets
plucked out of the groove by a self antigen, the B-cell would be in
a position to present self antigens to self-reactive T-cells, a
process that could lead to autoreactivity and autoimmune disease.
For some B-cells this may result in death to the B-cell by a nearby
killer cell, perhaps a natural killer (NK) cell. However, if this
doesn't remove the potentially autoreactive B-cell and it
encounters a CD4+ T-cell that can recognize that antigen (most
likely one that was not in the thymus) the CLIP might be removed,
in which case the B-cell might receive additional help from a
T-cell specific for the antigen that now begins to occupy the
groove (antigen binding location in the MHC molecule).
Alternatively, a nearby cell whose job it is to detect damaged self
cells, may become activated by the self antigen-presenting B-cell.
Such a damage detecting cell is, for example, a gamma delta-cell,
also referred to as a .gamma..delta.T-cell (.gamma..delta. refers
to the chains of its receptor), which can then seek out other sites
of inflammation (for example in the brain in MS, in the heart for
autoimmune myocarditis, in the pancreas in the case of Type I
Diabetes). Alternatively, the .gamma..delta.T-cell might attempt to
kill the CD4 T-cell that may respond to self antigens. In either
event, activation of a .gamma..delta.T-cell could be bad.
[0045] An example of the necessity for selective B-cell death when
the antigen receptor has not been bound by a real bona fide antigen
is in Coxsackievirus. Most people that contract Coxsackievirus get
a flu-like disease and then they recover, but in a genetic manner,
some people (especially young men) contract Coxsackievirus and then
go on to develop autoimmune myocarditis. In some genetically inbred
strains of mice, the mice are resistant to myocarditis
post-infection; in other strains of mice, the mice succumb. One
difference was that the mice that were susceptible had a particular
isoform of MHC class II. Mice on the resistant background having
the other isoform of class II inserted, both artificially and
genetically, showed susceptibility simply on the basis of the
isoform, and it was shown that susceptibility depended on the
presence of .gamma..delta.T-cells (Huber et al., 1999).
[0046] Moreover, it was observed that in the mice that did not
develop autoimmune disease, during the course of infection, all of
their B-cells died. Even with such B-cell death, the animals
survive as new B-cells are produced continually. However, the
animals susceptible to autoimmune disease had no B-cell death.
Further support for this notion is the .gamma..delta.knock-out mice
(they genetically have no .gamma..delta.T-cells) do not get EAE,
the mouse version of multiple sclerosis, nor do they get Type 1
diabetes. NK cell knock-out animals get worse disease in both
cases. In addition, the invariant chain knock-out animals are
resistant to the animal models of autoimmune diseases as well.
Although not bound by mechanism, it is believed according to the
invention that removal of .gamma..delta.T-cells is a therapeutic
treatment for MS, and that NK cells kill the antigen non-specific
B-cells in normal people and animals, thereby preventing disease.
There appears to be a reciprocity of function between these two
regulatory cell types.
[0047] Many therapies to block autoimmune and transplant disease
involve eliminating or inhibiting B-cells. No one knows the
mechanism by which these B-cell depleting therapies make people
better. The inventor has observed that .gamma..delta.T-cell
activation is often associated with proteins that have been lipid
modified. It turns out the invariant chain is fatty acid acylated
(e.g., palmitoylated). As described in the examples below, antigen
non-specifically activated human B-cells were treated with
anti-CLIP antibodies and subjected to flow cytometry. It was
surprisingly found that these antigen-non-specifically activated
B-cells express cell surface CLIP. Thus, the inventor recognized
that B-cell surface expression of CLIP is likely how
.gamma..delta.T-cells get activated. For example, if there is
inflammation at a given site, the long-lived .gamma..delta.T-cell
kills the type of CD4 helper T-cell that could improve disease (the
Th2 CD4+ T-cells; these likely also express CLIP on their surfaces,
making them a target for .gamma..delta.T-cells), at the site of
injury. They attack the inflamed tissue as well as kill the Th2
cells, leaving behind B-cells that can now present self antigens
(that load the CLIP binding site) to Th1 cells. The Th1 cells go on
to activate additional CD8 killer cells and to attack the tissues
as well. Once the .gamma..delta.T-cell is activated, it searches
for damaged tissue. Importantly, CLIP can preferentially associate
with certain isoforms of MHC class II (1-E in mouse, HLA-DR in
humans) and to certain MHC class I's (for example, but not limited
to, CD1). Interestingly, many autoimmune diseases map to the same
HLA-DR alleles and not to the other isoforms.
[0048] The invention, thus, involves treatments for autoimmune
disease, transplantation, and infectious disease. In a particular
example, during HIV infection, the AIDS virus encodes and induces
expression of the HIV pathogenic factor, the nef gene product. This
factor is known to increase viral replication in HIV infection. In
addition the nef gene product down regulates the surface expression
of MHC molecules while upregulating the cell surface expression of
CD74. The nef gene product appears to selectively increase the cell
surface expression of CLIP and CD74. If the ectopic/cell surface
expression of Ii chain or its products is centrally important in
activating .gamma..delta.T-cells, the nef gene could promote
activation of .gamma..delta.T-cells that kill traditional
HIV-affected CD4 T-cells and the activation of NK cells that may
promote viral replication or syncytial formation. Hence, it is
distinctly possible that the killer of the CD4 T-cells in AIDS are
.gamma..delta.T-cells. AIDS is characterized by an early bout of
polyclonal B-cell activation and symptoms of autoimmune disease,
followed by the slow loss of T-cells in waves. These particular
.gamma..delta.T-cells secrete an anti-biotic like protein called
defensins, specifically one produced by the .gamma..delta.T-cells
in MS, known as LL-37. It is a protein that can be detected in the
serum of people with activated .gamma..delta.T-cells--this could be
important as a diagnostic tool for a variety of diseases, including
Multiple Sclerosis, other autoimmune diseases, as well as HIV
disease.
[0049] Nef is a pathogenicity factor for HIV-1 infection.
Expression of the gene is known to increase viral replication. CD4
T-cells are important targets of HIV infection. Expression of the
nef gene has been shown to down-regulate the level of CD4 and MHC
class I on T-cells, while increasing the level of expression of MHC
class II CD74. In some experiments, researchers have shown a
decrease in cell surface expression of mature major
histocompatibility complex class II (MHC-II) molecules, while
demonstrating an up-regulation of surface expression of the
invariant chain (Ii) associated with immature MHC-II
(Stumptner-Cuvelette et al., 2001). Furthermore, the investigators
identified acidic residues, located at the base of the flexible
C-proximal loop of Nef, that are critical for increased Ii
expression. The authors of these studies conclude that Nef
functions may contribute directly to the impaired
CD4(+)-T-helper-cell responses found in HIV-1-infected patients
with progressive disease. The ability of Nef to interfere with
MHC-II antigen presentation might play a role in AIDS pathogenesis.
Importantly, interpreted through the perspective provided by the
present invention, the ability of nef to increase CD74 expression
has important implications for the activation of
.gamma..delta.T-cells, NK cells, and NK T-cells. In other words,
nef may promote .gamma..delta.T-cell-mediated cell death of
infected CD4 cells as a result of anomalous CD74/Ii/CLIP expression
on the cell surface of CD4 T-cells.
[0050] Toll-like receptor activation, resulting from pathogenic
infection, can induce a powerful immune response. On susceptible
genetic backgrounds, when TLRs recognize predominantly microbial
products, the activation of these receptors by pathogens can induce
the activation of cells that are implicated in autoimmunity. The
involvement of microbial products, self antigens, and other TLR
ligands in the onset of specific autoimmune diseases remains
unclear. In the present invention, the inventors suggest that the
binding of TLR ligands on the appropriate genetic background will
result in the ectopic expression of CLIP molecules, including but
not limited to CLIP and intact CD74. It is the ectopic expression
of these molecules that results in activation of
.gamma..delta.T-cells, NK cells, or NKT-cells that mediate
autoimmune sequalae. Reciprocally, the TLR ligands can be used to
promote recognition and killing of tumor cells by the same effector
cells (.gamma..delta.T-cells, NK cells, and/or NKT-cells).
Cell Types
[0051] A. B-Cells
[0052] B lymphocytes are the precursors of antibody-producing
cells. These cells express a cell surface form of antibody that is
their receptor for antigen (also known as membrane immunoglobulin).
Once antigen is bound by that receptor, the B-cell is stimulated,
the antigen that is bound by the receptor gets engulfed along with
the receptor, where the complex is internalized in the B-cell's
endosomal system. Once inside the cell, the antigen-containing
endosome fuses with a lysosome where antigen is broken down and
loaded onto special molecules for transport to the cell surface. At
the B-cell surface, the newly processed antigen is associated with
a molecular complex of MHC molecules that can be recognized by T
lymphocytes. Recognition of antigen and MHC as a complex is a
requirement for T-cell activation and a normal immune response.
[0053] B-cell maturation. B lymphocyte precursors, like all
lymphoid precursors, are born in the bone marrow where they are
derived from an even earlier precursor known as the hematopoietic
"stem" cell. Once the precursor B-cell has been given the "go
ahead" to develop and mature, there is transcription and
translation of the heavy chain for the "first-to-be-produced"
antibody, immunoglobulin M or IgM, for short. In fact, the
generation of messenger RNA for IgM designates the point at which
that precursor cell is destined to be a B lymphocyte. The newly
formed heavy chain eventually pairs with a precursor chain for a
true light chain and then switches to become associated with one of
two types of mature light chains, known as kappa or lambda, light
chains. In both mice and humans, ninety-five percent of light
chains are kappa chains. Once the heavy chain covalently bonds by
way of disulfide bonds to the light chains, the molecule gets
transported to the B-cell surface and the B-cell is now said to be
a pre-B-cell--still resident in the bone marrow, but not fully
mature. At this stage, the B-cell is particularly vulnerable to
deletion. When pre-B-cells in the bone marrow encounter antigens
(most likely "self" antigens) for which they are specific, the
pre-B-cell dies. This process of "clonal abortion" likely occurs to
protect the pre-B-cell from becoming self-reactive and from
producing self-reactive antibodies. Presumably in the bone marrow,
the majority of antigens are self. The suicidal death of the B-cell
prevents the maturation of self-reactive B-cells and likely
reflects that developmental pressure not to allow self-reactive
B-cells to enter the rest of the body.
[0054] The second isotype of heavy chain produced within the B-cell
while still in the bone marrow is the heavy chain for IgD
molecules. This particular isotype, like the membrane version of
IgM, gets transported to the cell surface once its transcription
and translation have occurred and once it has been connected with
light chains. Providing the B-cell has not been removed by
deletion, the developmental expression of cell surface IgM and cell
surface IgD signal the newly matured B-cell to exit the bone marrow
and to go to the peripheral lymphoid tissues, including the
circulation, the spleen, and the germinal centers of lymph nodes.
Once the peripheral immune system has been populated with mature
B-cells, the B-cells have a quite limited life span, unless they
encounter the antigen for which they are specific. Once that
happens and the B-cell receives the necessary growth factors or
other factors necessary for its development into an immunoglobulin
secreting plasma cell, the B-cell is protected from cell death. The
large numbers of those that don't encounter antigen die by
"neglect." Those that remain, for example, the daughter cells of
the activated and expanding antigen-specific clones of B-cells, are
thought to be memory B-cells. The memory cells are also thought to
express the cell surface molecule CD27 and are thought to live for
a long time, unlike the naive, non-primed B-cell. Recognizing
antigen in the periphery is the first life-saving step for the
B-cell in this case.
[0055] Once the peripheral B-cell encounters antigen, unless the
antigen is a particular kind of bacterial (polymeric) antigen, the
B-cell will need additional "help" in the form of cytokines and
even perhaps direct contact with CD4.sup.+ T-cells. The mechanism
by which the CD4.sup.+ T-cell "helps" B-cells to finish their
peripheral maturation process is not completely understood. Most
studies suggest that actual "cognate" interactions occur between
the B-cell and the T-cell, while others suggest the T-cell's
production of cytokines is sufficient. Regardless, for the B-cell
to secrete all forms and isotypes of antibody with the exception of
IgM and membrane IgD, the B-cell needs T-cell "help" to produce all
forms of IgG, IgA, and IgE. The only T-independent antigens are
those that are the highly polymeric type. When they are sufficient
to signal B-cell activation and differentiation into an
antibody-secreting cell, the B-cell will only make IgM.
Reiterating, for production of all other isotypes of antibody, the
B-cell will need T-cell help because most B-cell responses to
antigen are said to be T-dependent.
[0056] As the price of a given geographic quantity of land is often
said to be dependent on "location, location, location," the same
may be true for the isotype of antibody that the CD4+ T-cell will
eventually help the B-cell to produce. For example, when B-cells
secrete antibody that will reside in secretions, the isotype of the
antibody will be IgA. Ultimately the B-cell that matures near areas
of secretions, for example, near salivary glands, the
genito-urinary areas, or in the lactating breast, will produce
mature forms of IgA. These molecules exists as dimers of a typical
antibody (2H, 2L chains) such that a mature IgA molecule will have
two typical antibody units bounded by a small protein known as a
secretory component and another small molecule called the J chain.
Most scientists that study secretory IgA suggest that the two
molecule structures protect the newly synthesized antibodies from
the harsh and degradative environment of the secretory tracts. This
type of antibody is very important to the newborn that acquires a
front-line of defense from Mother's milk. The baby orally ingests
Mom's secreted IgA and vicariously receives the protection from
specific antigens that Mom's B-cells have already encountered.
[0057] B-cells as Antigen Presenting Cells. A unique feature of
B-cells involves the ability of the membrane immunoglobulin (that
B-cell antigen receptor) not only to recognize and bind to antigens
very specifically and deliver an activation to the cell, but also
to engulf the antigen into unique endosomal compartments which fuse
with lysosomal compartments where the "receptor-engaged" antigen is
broken down into fragments, loaded into MHC class II molecules,
transported to the cell membrane and there antigens associated with
MHC are available to be recognized by T lymphocytes. The ability of
professional antigen presenting cells to present antigens to the T
lymphocytes depends on processing the antigen in the
endosomal/lysosomal compartment. The B lymphocyte is an
extraordinary APC in that the engulfment of antigen by this
particular APC, and unlike other phagocytes, is antigen
specific.
[0058] B. "Professional" Antigen Presenting Cells
[0059] The professional antigen-presenting cells (APCs) are a
subset of specialized cells express class II MHC, and include
B-cells, macrophages, microglia, dendritic cells, and Langerhans
cells among others. Traditionally, the professional
antigen-presenting cells of the myeloid lineage, dendritic cells
and macrophages, have been viewed primarily as accessory cells,
functioning simply to assist T-cell activation. Recently, however,
it has become clear that myeloid-lineage APCs exert a profound
influence on T-cells, regulating both the nature of the response
(humoral versus cellular immunity) and, in some cases, even whether
a response occurs at all (activation versus anergy).
[0060] C. T-Cells
[0061] T lymphocytes, like B lymphocytes, arise from hematopoeitic
stem cells in the bone marrow. However, unlike B-cells, the
pre-T-cells travel to another peripheral lymphoid tissue, the
thymus, where T lymphocyte maturation processes occur.
Interestingly, the thymus, as a T-cell development organ, reaches
its maximum size and capacity in very early childhood around the
age of 2 to 3 years and, at puberty, the thymus begins to
involute-shrinking to a small rudiment of what it had been earlier.
No one has unraveled exactly how the pre-T-cell is recruited to
homes to the thymus, but research has shown that once the cells
arrive they may stay as long as two weeks before the mature,
appropriate cells leave the thymus to circulate throughout the
periphery.
[0062] The thymus is the place where the pre-T-cell develops the
ability to recognize an enormous repertoire of antigens presented
by either MHC class I or MHC class II. The pre-T-cells enter the
thymus without receptors for antigen and MHC, without CD4, and
without CD8. In the thymus, T-cells acquire T-cell receptors for
antigen, and either CD4 or CD8. During the process, those T-cells
that will recognize antigen and MHC class I become CD8.sup.+
T-cells and those that recognize MHC class II and antigen become
CD4.sup.+ T-cells. Both CD4 and CD8 positive cells have cell
surface T-cell receptors for antigen. If a T-cell, either a CD4+ or
a CD8+ T-cell, recognizes "self" antigen and self MHC class I or
self MHC class II in the thymus, that T-cell is deleted. For most
of the CD4.sup.+ and CD8.sup.+ T-cells have T-cell receptors that
consist of an alpha chain and a beta chain. There are other, more
recently described T-cells that express receptors that are called
gamma/delta T-cell receptors. Interestingly, like the B-cell
receptor for antigen, each of the T-cell receptor chains have
variable and constant regions. T-cells, like B-cells, have antigen
receptors that can bind millions of different antigens (but in the
case of T-cells only so long as the processed antigens are
associated with MHC molecules). The diversity of T-cell receptors
is provided by the large number of possible variable regions the
T-cell receptor can have. Like the variable regions in B-cell
receptors, the development of the T-cell receptor variable regions
result from recombination of segments of DNA. However, unlike the
B-cell receptor for antigen, T-cell receptor recombination occurs
in the thymus, not in the bone marrow.
[0063] The developmental maturation of T-cells in the thymus
results in a high percentage of thymocyte cell death. Waves of
cortisone kill many of the pre-T-cells that don't meet the
necessary requirements for recognition and survival. In addition to
cortisone-dependent thymocyte cell death, recognition of antigen in
the thymus deletes some potentially self-reactive T-cells from the
repertoire. The process of antigen-specific T-cell death in the
thymus is commonly referred to as "negative" selection. NOTE that
CD4+ T-cells in us would only get deleted if they recognize self
MHC class I or MHC class II plus self antigen (like CLIP). (Those
that could recognize CLIP and someone else's MHC class I or class
II will not have been deleted--see below the discussion of
invariant chain (Ii) and CLIP). Those CD4+ or CD8+ T-cells that
recognize SELF antigens associated with either class I or class II
molecules will be deleted in the thymus. Those cells that meet all
of the survival criterion, e.g. appropriate recognition of antigen
and either MHC class I for the developing CD8.sup.+ T or MHC class
II for the developing CD4.sup.+ T-cell.
[0064] D. NK Cells
[0065] Natural killer cells (NK cells) are a population of
lymphocytes which represent a very early line of defense against
viruses and tumor cells. NK cells can be characterized by the
presence of CD56 and CD16 markers and by the absence of the CD3
marker. NK cells are involved in non specific anti-tumoral immunity
of antigens, to prevent the establishment of primitive or
metastatic tumors in the immunocompetent or immunosuppressed host.
NK cells appear to play a key role against tumor cells or negative
class I MHC cell variants. Because of their non-specific cytotoxic
properties for antigen and their efficacy, NK cells constitute a
particularly important population of effector cells in the
development of immunoadoptive approaches for the treatment of
cancer or infectious diseases. NK cells have also been used for
experimental treatment of different types of tumors.
Treatments and Diagnosis
[0066] Thus, in some aspects the invention relates to a method for
treating a disorder associated with .gamma..delta.T-cell expansion,
activation and/or effector function by contacting a CLIP compound
expressing cell with an inhibitor of .gamma..delta.T-cell
expansion, activation and/or effector function in an effective
amount to interfere with .gamma..delta.T-cell expansion, activation
and/or effector function by the CLIP compound expressing cell.
Alternatively the invention relates to treating such disorders in a
subject by administering to the subject a CLIP inhibitor in an
effective amount to reduce CLIP function in a CLIP compound
expressing cell of the subject.
[0067] A disorder associated with .gamma..delta.T-cell expansion,
activation and/or effector function is one in which the expansion,
activation or function of .gamma..delta.T-cells places a pathogenic
role in the disease. For instance the expansion and activation of
the .gamma..delta.T-cells by a cell expressing CLIP on the surface
in the context of an MHC molecule causes such cells to accumulate
in higher than normal amounts, such that they can act on other
T-cells or directly attack host tissue. An example of a disorder
associated with .gamma..delta.T-cell expansion, activation and/or
effector function is autoimmune disease. It is believed that,
according to an aspect of the invention, cells having CLIP on the
surface in the context of MHC may cause the expansion and/or
activation of these cells. Once the .gamma..delta.T-cells are
activated they may recognize CLIP in the context of MHC on host
tissue such as neurons, pancreatic B-cells and heart tissue. The
result of that recognition may be the killing of the T-cell. The
.gamma..delta.T-cells may also cause further production of antigen
non-specific B-cells which are capable of picking up host antigen
and further producing a host specific immune response. Other
disorders associated with .gamma..delta.T-cell expansion,
activation and/or effector function include HIV infection and
rejection of transplanted cells, tissues or grafts. The activated
.gamma..delta.T-cell can mediate the killing of the transplanted
cells and tissue as well as host T-cells, that are critical in
advancing the HIV infection.
[0068] A CLIP molecule expressing cell is a cell that has MHC class
I or II on the surface and includes a CLIP molecule within that
MHC. Such cells include B-cell, a neuron, an oligodendrocyte, a
microglial cell, or an astrocyte, a heart-cell, a pancreatic .beta.
cell, an intestinal epithelial cell, a lung cell, an epithelial
cell lining the uterine wall, and a skin cell.
[0069] The CLIP molecule, as used herein, refers to intact CD74
(also referred to as invariant chain), as well as the naturally
occurring proteolytic fragments thereof. SEQ ID NO:5 (Met Arg Met
Ala Thr Pro Leu Leu Met) is one of the naturally occurring
proteolytic fragments thereof. The function of the CLIP molecule in
this invention is mainly as an MHC class II chaperone. MHC class II
molecules are heterodimeric complexes that present foreign
antigenic peptides on the cell surface of antigen-presenting cells
(APCs) to CD4.sup.+ T-cells. MHC class II synthesis and assembly
begins in the endoplasmic reticulum (ER) with the non-covalent
association of the MHC .alpha. and .beta. chains with trimers of
CD74. CD74 is a non-polymorphic type II integral membrane protein;
murine CD74 has a short (30 amino acid) N-terminal cytoplasmic
tail, followed by a single 24 amino acid transmembrane region and
an .about.150 amino acid long lumenal domain. Three MHC class II
.alpha..beta. dimers bind sequentially to a trimer of the CD74 to
form a nonameric complex (.alpha..beta.Ii).sub.3, which then exits
the ER. After being transported to the trans-Golgi, the
.alpha..beta.Ii complex is diverted from the secretory pathway to
the endocytic system and ultimately to acidic endosome or
lysosome-like structures called MHC class II compartments (MIIC or
CIIV).
[0070] The N-terminal cytoplasmic tail of CD74 contains two
extensively characterized dileucine-based endosomal targeting
motifs. These motifs mediate internalization from the plasma
membrane and from the trans-Golgi network. In the endocytic
compartments, the CD74 chain is gradually proteolytically
processed, leaving only a small fragment, the class II-associated
CD74 chain peptide (CLIP), bound to the released .alpha..beta.
dimers. The final step for MHC class II expression requires
interaction of .alpha..beta.-CLIP complexes with another class
II-related .alpha..beta. dimer, called HLA-DM in the human system.
This drives out the residual CLIP, rendering the .alpha..beta.
dimers ultimately competent to bind antigenic peptides, which are
mainly derived from internalized antigens and are also delivered to
the endocytic pathway. The peptide-loaded class II molecules then
leave this compartment by an unknown route to be expressed on the
cell surface and surveyed by CD4.sup.+ T-cells.
[0071] The methods of this aspect of the invention my able
accomplished using an inhibitor of .gamma..delta.T-cell expansion,
activation and/or effector function. Inhibitors of
.gamma..delta.T-cell expansion, activation and/or effector function
are any molecules that reduce the presence of a CLIP molecule on
the MHC, either directly or indirectly. An example of a
.gamma..delta.T-cell expansion, activation and/or effector function
is a CLIP expression inhibitor. CLIP expression inhibitors are
compounds that inhibit the expression of a CLIP molecule RNA. For
instance CLIP expression inhibitors include antisense and siRNA.
For instance, antisense or siRNA directed to a CLIP molecule or
HLA-DO are useful as CLIP expression inhibitors. Antisense and
siRNA as well as other expression inhibitors are described in more
detail below.
[0072] Another type of .gamma..delta.T-cell expansion, activation
and/or effector function is a CLIP activity inhibitor. CLIP
activity inhibitors include agents that displace CLIP, anti-CLIP
molecule antibodies, recombinant HLA-DM and agents that inhibit
CD74 processing. Many molecules are useful for displacing CLIP
molecules. For instance compounds such as chloroquine, a
lysosomatropic agent, or peptide/lipopeptide antigen are known to
have such function. Other CLIP displacers include the small
molecular compound pCP, chlorobenzene (CB), parachloroanisol (pCA),
the peptides HA306-318 (PKYVKQNTLKLAT) (SEQ ID NO. 2), CO260-272
(IAGFKGEQGPKGE) (SEQ ID NO. 3), HLA binding peptides, and FRIMAVLAS
(SEQ ID NO. 1).
[0073] Another agent that displaces CLIP is a halogenated alkyl
ester. The halogenated alkyl ester is particularly useful in
combination with a glycolytic inhibitor. The combination of agents
may be administered separately or together. In some instances the
combination of agents is in the form of a prodrug bifunctional
molecule. Such materials are described in more detail below.
[0074] Anti-CLIP antibodies, which include antibodies that bind to
CLIP molecules are also useful as agents that displace CLIP. Such
antibodies are described in more detail below.
[0075] Other inhibitors of CLIP activity are agents that inhibit
CD74 processing. Agents that inhibit CD74 processing are known in
the art and include cystatin, A, B, or C.
[0076] In the methods of this aspect of the invention the CLIP
expressing cell may also be exposed to an MHC class I or II loading
peptide or an anti-MHC antibody. The purpose of exposing the cell
to an MHC class I or II loading peptide or an anti-MHC class II
antibody is to prevent the cell, once CLIP has been removed, from
picking up a self antigen, which could be presented in the context
of MHC. An MHC class I or II loading peptide is one that fits
within the MHC groove, and in some embodiments will not provoke an
interaction with other immune cells. For instance peptides such as
FRIMAVLAS (SEQ ID NO. 1) function quite well as MHC class I or II
loading peptides. One advantage of FRIMAVLAS (SEQ ID NO. 1) is that
it functions as both a CLIP molecule displacer and an MHC class I
or II loading peptide and thus only needs to be administered
once.
[0077] An anti-MHC class II antibody may be administered in order
to engage a B-cell and kill it. Once CLIP has been removed, the
antibody will be able to interact with the MHC and cause the B-cell
death. This prevents the B-cell with an empty MHC from picking up
and presenting self antigen or from getting another CLIP molecule
in the surface that could lead to further .gamma..delta.T-cell
expansion and activation. MHC is Major Histocompatibility Complex.
MHC-encoded molecule class I (HLA-A, B, or C, HLA-E, F, or G, CD1a,
b, c, or d), or Class II (HLA-DR, DP, or DQ; HLA-DM, HLA-DO) are
generally useful in the invention.
[0078] The methods may also involve the removal of antigen
non-specifically activated B-cells and/or .gamma..delta.T-cells
from the subject to treat the disorder. The methods can be
accomplished as described above alone or in combination with known
methods for depleting such cells.
[0079] A subject shall mean a human or vertebrate mammal including
but not limited to a dog, cat, horse, goat and primate, e.g.,
monkey. Thus, the invention can also be used to treat diseases or
conditions in non human subjects. Preferably the subject is a
human.
[0080] As used herein, the term treat, treated, or treating when
used with respect to a disorder refers to a prophylactic treatment
which increases the resistance of a subject to development of the
disease or, in other words, decreases the likelihood that the
subject will develop the disease as well as a treatment after the
subject has developed the disease in order to fight the disease,
prevent the disease from becoming worse, or slow the progression of
the disease compared to in the absence of the therapy.
[0081] A. AIDS or HIV-1 Infection
[0082] According to an embodiment of the invention, the methods
described herein are useful in inhibiting the development of and/or
treating AIDS or HIV-1 infections. In a specific embodiment,
treatment is by inhibiting or reducing the expression or activity
of CLIP molecules in, or blocking CLIP presentation by, CD4 T-cells
of a subject infected with HIV. In particular, CLIP molecules can
be blocked by treatment with an agent that removes CLIP molecules
from MHC, by treatment with an agent that prevents processing of
CLIP molecules, or by contacting a CD4 T-cell bearing CLIP with an
anti-CLIP antibody or binding peptide.
[0083] Examples of agents that remove CD74 or CLIP are chloroquine,
a lysosomatropic agent, or peptide/lipopeptide antigen. Examples of
agents that reduce expression of CD74 and/or a proteolytic product
thereof are antisense or siRNA to CLIP molecule/CD74 or CLIP or
HLA-DO.
[0084] In another embodiment, treatment is by killing or inhibiting
the function of antigen non-specifically activated B-cells and/or
.gamma..delta.T-cells, in some cases v.gamma.9v.delta.2 T-cells, in
a subject infected with HIV. Inhibiting function can include (a)
removing antigen non-specifically activated B-cells and/or
.gamma..delta.T-cells (or NK or NKT-cells) from the subject, (b)
removing TLR ligand-activated B-cells, (c) removing other antigen
presenting cells that results in cell surface expression of
invariant chain/CD74 or CLIP so as to remove any of those that can
express Ii/CD74 or CLIP on the cell surface, (d) selectively
removing the cells that have any form of invariant chain on the
surface, (e) inhibiting antigen non-specific activation of B-cells
in the subject, or (f) by antibody depletion of the B-cells and/or
the .gamma..delta.T-cells. An anti-CLIP antibody can be obtained
from BD Pharmingen or another commercial antibody source. Examples
of antibodies are described below. An anti-.gamma..delta.T-cell
antibody also an be obtained from BD Pharmingen.
[0085] In accordance with another embodiment, the methods of this
invention can be applied in conjunction with, or supplementary to,
the customary treatments of AIDS or HIV-1 infection. Historically,
the recognized treatment for HIV-1 infection is nucleoside analogs,
inhibitors of HIV-1 reverse transcriptase (RT). Intervention with
these antiretroviral agents has led to a decline in the number of
reported AIDS cases and has been shown to decrease morbidity and
mortality associated with advanced AIDS. Prolonged treatment with
these reverse transcriptase inhibitors eventually leads to the
emergence of viral strains resistant to their antiviral effects.
Recently, inhibitors of HIV-1 protease have emerged as a new class
of HIV-1 chemotherapy. HIV-1 protease is an essential enzyme for
viral infectivity and replication. Protease inhibitors have
exhibited greater potency against HIV-1 in vitro than nucleoside
analogs targeting HIV-1 RT. Inhibition of HIV-1 protease disrupts
the creation of mature, infectious virus particles from chronically
infected cells. This enzyme has become a viable target for
therapeutic intervention and a candidate for combination
therapy.
[0086] Knowledge of the structure of the HIV-1 protease also has
led to the development of novel inhibitors, such as saquinavir,
ritonavir, indinavir and nelfinavir. NNRTIs (non-nucleoside reverse
transcriptase inhibitors) have recently gained an increasingly
important role in the therapy of HIV infection. Several NNRTIs have
proceeded onto clinical development (i.e., tivirapine, loviride,
MKC-422, HBY-097, DMP 266). Nevirapine and delaviridine have
already been authorized for clinical use. Every step in the life
cycle of HIV-1 replication is a potential target for drug
development.
[0087] Many of the antiretroviral drugs currently used in
chemotherapy either are derived directly from natural products, or
are synthetics based on a natural product model. The rationale
behind the inclusion of deoxynucleoside as a natural based
antiviral drugs originated in a series of publications dating back
as early as 1950, wherein the discovery and isolation of thymine
pentofuranoside from the air-dried sponges (Cryptotethia crypta) of
the Bahamas was reported. A significant number of nucleosides were
made with regular bases but modified sugars, or both acyclic and
cyclic derivatives, including AZT and acyclovir. The natural
spongy-derived product led to the first generation, and subsequent
second--third generations of nucleosides (AZT, DDI, DDC, D4T, 3TC)
antivirals specific inhibitors of HIV-1 RT.
[0088] A number of non-nucleoside agents (NNRTIS) have been
discovered from natural products that inhibit RT allosterically.
NNRTIs have considerable structural diversity but share certain
common characteristics in their inhibitory profiles. Among NNRTIs
isolated from natural products include: calanoid A from calophylum
langirum; Triterpines from Maporonea African a. There are
publications on natural HIV integrase inhibitors from the marine
ascidian alkaloids, the lamellarin.
[0089] The role of HIV protease in the production of functionally
infectious particle has been described as a critical process for
retrovirus as well as HIV replication. The natural product,
Pepstatin A, is a metabolite of streptomycin testaceus and
Streptomyces argentolus var. toyonakensis was shown to inhibit
HIV-1 Protease enzyme. The key strategy used in the development of
the current HIV-1 protease inhibitors was to substitute the
scissile P1-P1 amide bond by a nonhydrozable isoster with
tetrahedral geometry; the designs were guided by assays and based
on substrate specificity. It eventually led to the optimization of
peptidomimetic lead structure and the development of novel class of
protease inhibitors including indinavir, Saqunovir, nelfinavir and
retinovir.
[0090] B. Transplant/Graft Rejection The success of surgical
transplantation of organs and tissue is largely dependent on the
ability of the clinician to modulate the immune response of the
transplant recipient. Specifically the immunological response
directed against the transplanted foreign tissue must be controlled
if the tissue is to survive and function. Currently, skin, kidney,
liver, pancreas, lung and heart are the major organs or tissues
with which allogeneic transplantations are performed. It has long
been known that the normally functioning immune system of the
transplant recipient recognizes the transplanted organ as
"non-self" tissue and thereafter mounts an immune response to the
presence of the transplanted organ. Left unchecked, the immune
response will generate a plurality of cells and proteins that will
ultimately result in the loss of biological functioning or the
death of the transplanted organ.
[0091] This tissue/organ rejection can be categorized into three
types: hyperacute, acute and chronic. Hyperacute rejection is
essentially caused by circulating antibodies in the blood that are
directed against the tissue of the transplanted organ (transplant).
Hyperacute rejection can occur in a very short time--often in
minutes--and leads to necrosis of the transplant. Acute graft
rejection reaction is also immunologically mediated and somewhat
delayed compared to hyperacute rejection. The chronic form of graft
rejection that can occur years after the transplant is the result
of a disease state commonly referred to as Graft Arterial Disease
(GAD). GAD is largely a vascular disease characterized by
neointimal proliferation of smooth muscle cells and mononuclear
infiltrates in large and small vessels. This neointimal growth can
lead to vessel fibrosis and occlusion, lessening blood flow to the
graft tissue and resulting in organ failure. Current
immunosuppressant therapies do not adequately prevent chronic
rejection. Most of the gains in survival in the last decade are due
to improvements in immunosuppressive drugs that prevent acute
rejection. However, chronic rejection losses remain the same and
drugs that can prevent it are a critical unmet medical need.
[0092] According to an embodiment of the invention, the methods
described herein are useful in inhibiting cell graft or tissue
graft rejection. Thus, the methods are useful for such grafted
tissue as heart, lung, kidney, skin, cornea, liver, neuronal tissue
or cell, or with stem cells, including hematopoetic or embryonic
stem cells, for example. In accordance herewith, treatment can be
by inhibiting or reducing the cell surface expression of CLIP
molecules in cells of grafted tissue or cells, or by blocking CLIP
molecule presentation by cells of a grafted tissue or cell.
[0093] Inhibiting or reducing cell surface expression of CLIP
molecules includes treatment with an agent that removes or blocks
CLIP molecules from MHC or that prevents processing of CD74 to
CLIP. Examples of agents that remove CLIP molecules from MHC are
chloroquine, a lysosomatropic agent, or peptide/lipopeptide
antigen. Examples of agents that reduce expression of CLIP
molecules are antisense or siRNA. To block CLIP molecule
presentation, one can contact the grafted tissue with an anti-CLIP
molecule antibody. Each of these methods is described above in more
detail.
[0094] In accordance with another embodiment, the methods of this
invention can be applied in conjunction with, or supplementary to,
the customary treatments of transplant/graft rejection. Tissue
graft and organ transplant recipients are customarily treated with
one or more cytotoxic agents in an effort to suppress the
transplant recipient's immune response against the transplanted
organ or tissue. Current immunosuppressant drugs include:
cyclosporin, tacrolimus (FK506), sirolimus (rapamycin),
methotrexate, mycophenolic acid (mycophenolate mofetil),
everolimus, azathiprine, steroids and NOX-100. All of these drugs
have side effects (detailed below) that complicate their long-term
use. For example, cyclosporin (cyclosporin A), a cyclic polypeptide
consisting of 11 amino acid residues and produced by the fungus
species Tolypocladium inflatum Gams, is currently the drug of
choice for administration to the recipients of allogeneic kidney,
liver, pancreas and heart (i.e., wherein donor and recipient are of
the same species of mammals) transplants. However, administration
of cyclosporin is not without drawbacks as the drug can cause
kidney and liver toxicity as well as hypertension. Moreover, use of
cyclosporin can lead to malignancies (such as lymphoma) as well as
opportunistic infection due to the "global" nature of the
immunosuppression it induces in patients receiving long term
treatment with the drug, i.e., the hosts normal protective immune
response to pathogenic microorganisms is downregulated thereby
increasing the risk of infections caused by these agents. FK506
(tacrolimus) has also been employed as an immunosuppressive agent
as a stand-alone treatment or in combination. Although its
immunosuppressive activity is 10-100 times greater than
cyclosporin, it still has toxicity issues. Known side effects
include kidney damage, seizures, tremors, high blood pressure,
diabetes, high blood potassium, headache, insomnia, confusion,
seizures, neuropathy, and gout. It has also been associated with
miscarriages. Methotrexate is commonly added to the treatment of
the cytotoxic agent. Methotrexate is given in small doses several
times after the transplant. Although the combination of cyclosporin
and methotrexate has been found to be effective in decreasing the
severity of transplant rejection, there are side effects, such as
mouth sores and liver damage. Severe transplant rejection can be
treated with steroids. However, the side effects of steroids can be
extreme, such as weight gain, fluid retention, elevated blood
sugar, mood swings, and/or confused thinking.
[0095] Rapamycin, a lipophilic macrolide used as an anti-rejection
medication can be taken in conjunction with other anti-rejection
medicines (i.e., cyclosporin) to reduce the amount of toxicity of
the primary cytotoxic agent, but it too has specific side effects,
such as causing high cholesterol, high triglycerides, high blood
pressure, rash and acne. Moreover, it has been associated with
anemia, joint pain, diarrhea, low potassium and a decrease in blood
platelets.
[0096] When used in combination with the therapies of the invention
the dosages of known therapies may be reduced in some instances, to
avoid side effects.
[0097] C. Autoimmune Disease
[0098] "Autoimmune Disease" refers to those diseases which are
commonly associated with the nonanaphylactic hypersensitivity
reactions (Type II, Type III and/or Type IV hypersensitivity
reactions) that generally result as a consequence of the subject's
own humoral and/or cell-mediated immune response to one or more
immunogenic substances of endogenous and/or exogenous origin. Such
autoimmune diseases are distinguished from diseases associated with
the anaphylactic (Type I or IgE-mediated) hypersensitivity
reactions.
[0099] According to an embodiment of the invention, the methods
described herein are useful in inhibiting the development of an
autoimmune disease comprising inhibiting, in a subject, the cell
surface expression of CLIP molecules by an antigen presenting cell
or blocking CLIP molecule presentation to a .gamma..delta.T-cell
such as a v.gamma.9v.delta.2 T-cell. Thus, the methods are useful
for such autoimmune diseases as multiple sclerosis, systemic lupus
erythematosus, type 1 diabetes, viral endocarditis, viral
encephalitis, rheumatoid arthritis, Graves' disease, autoimmune
thyroiditis, autoimmune myositis, and discoid lupus
erythematosus.
[0100] In accordance herewith, treatment can be by inhibiting or
reducing the cell surface expression of CLIP molecules in cells, or
by blocking CLIP molecules presentation. Inhibiting or reducing
cell surface expression of CLIP molecules includes treatment with
an agent that removes or blocks CLIP molecules from MHC or that
prevents processing of CD74 to CLIP. Examples of agents that remove
CLIP molecules from MHC are chloroquine, a lysosomatropic agent, or
peptide/lipopeptide antigen. Examples of agents that reduce
expression of CLIP molecules are antisense or siRNA. To block CLIP
presentation, one can contact the grafted tissue with an anti-CLIP
molecule antibody. Each of these methods is described above in more
detail.
[0101] D. Diagnosing Autoimmune Disease, HIV and Tissue Graft
Rejection
[0102] According to an embodiment of the invention, the methods
described herein are useful in diagnosing autoimmune disease, HIV
infection and tissue graft rejection such as multiple sclerosis,
systemic lupus erythematosus, type 1 diabetes, viral endocarditis,
viral encephalitis, rheumatoid arthritis, Graves' disease,
autoimmune thyroiditis, autoimmune myositis, and discoid lupus
erythematosus. The method involves measuring levels of at least one
defensin in a subject exhibiting one or more additional symptoms of
these diseases. In a specific embodiment, the defensin is LL37.
[0103] Defensins are family of potent antibiotics made within the
body by neutrophils and macrophages, and play important roles
against invading microbes. They act against bacteria, fungi and
viruses by binding to their membranes and increasing membrane
permeability. On a chemical level, the defensins are small peptides
unusually rich in the amino acid cysteine (Cys). The human
defensins are classified into the .alpha.-defensins and
.beta.-defensins on the basis of their sequence homology and their
Cys residues. In accordance with this invention, defensins can
serve as a marker for these diseases. For example
.gamma..delta.T-cell mediated autoimmunity may be diagnosed by
determining the blood levels of defensins that are produced by
.gamma..delta.T-cells that are destructive. Levels of defensin at
or above detectable levels means there are activated
.gamma..delta.T-cells and they are likely a pathogenic component of
any form of autoimmune disease.
[0104] E. Cancer
[0105] According to an embodiment of the invention, the methods
described herein are useful in treating cancers, tumors, and other
conditions involving rapidly dividing cell populations that are
typically uncontrolled. A "rapidly dividing cell," as used herein,
is a cell which is undergoing mitotic growth. Such cells are well
known in the art and include, but are not limited to, tumor cells,
cancer cells, lymphocytes (T-cells or B-cells), bacteria, and
pancreatic beta (.beta.) cells. In these aspects of the invention
it is desirable to activate .gamma..delta.T-cells that can kill the
rapidly dividing cells. The methods may be accomplished using a
CLIP inducing agent.
[0106] A CLIP inducing agent as used herein refers to a compound
that results in increased CLIP molecule presentation on the cell
surface in the context of MHC. CLIP inducing agents include, for
instance, CLIP expression vectors and CLIP activators. A CLIP
expression vector is a vector that when administered to the cells
causes production of a CLIP molecule protein. The CLIP molecule
protein may be CD74, for instance. In the case that CD74 is
produced it is desired that the CD74 be produced in the cell such
that it can be processed intracellularly to produce a CLIP
associated with MHC. Alternatively it may be processed in other
cells that are capable of secreting it such that CD74 protein is
capable of interacting with MHC on the surface. The expression
vector may also produce a CLIP peptide either intracellularly or
extracellularly.
[0107] CLIP activators include for instance soluble nef or an agent
that increases nef expression such as a nef expression vector. CLIP
activators also include ectopic CLIP, palmitoylated protein or PAM,
and an anti-CD40 or CD40L molecule in combination with IL-4. An
HLA-DO molecule which promotes a higher HLA-DO:HLA-DM ratio is also
a CLIP activator. A high HLA-DO:HLA-DM ratio causes the cell to
produce more CLIP on the cell surface. Another molecule that is
capable of achieving a high HLA-DO:HLA-DM ratio is an anti-sense or
siRNA to HLA-DM. These compounds are described in more detail
herein.
[0108] In a specific embodiment of the invention, cancer cells in a
subject are killed by (1) treating the patient directly with
soluble CLIP protein (can be made by proteolytic cleavage of
recombinant invariant chain, simply making recombinant CLIP, or
synthetic CLIP to activate a Th 1 response; (2) (a) inducing cell
surface expression of CLIP on a B-cell, preferably ex vivo; (b)
contacting the B-cell of step (a) with a .gamma..delta.T-cell or NK
cell, preferably in vivo, and (c) contacting the
.gamma..delta.T-cell or NK cell with the cancer cell. In particular
embodiments, the .gamma..delta.T-cell is a v.gamma.9v.delta.2
T-cell. In still other particular embodiments, the B-cell of step
(a) and/or the .gamma..delta.T-cell of step (c) and/or the NK cell
is allogeneic to the subject.
[0109] In accordance with an embodiment of the invention, the
cancer cells are killed by inducing cell surface expression of CLIP
in the cancer cell. One method of inducing cell surface expression
of CLIP in the cancer cell is to treat the cancer cells with nef or
an agent that increases nef expression. Thus one can use gene
targeting to express the nef gene in the cancer cell by exposing
the cancer cell to the soluble products of recombinant nef
expressed in a mammalian vector. Another method is to treat the
cancer cells with the protein product of the nef gene, the nef
protein, Another method is to treat the tumor directly with CLIP,
and gene target nef to the tumor so that only the tumor expresses
invariant chain/CD74, or proteolytic products such as CLIP.
[0110] Another method is to treat the cancer cells with agent that
will induce ectopic CLIP. For this procedure one can determine if
the cancer in question expresses Toll-like receptors (TLRs) and can
then treat the cancer patient with ligands for the TLR expressed on
the cancer cell. The invention predicts that TLR engagement will
result in the ectopic expression of CLIP on the surface of the
cancer cell. Examples of inflammatory mediators that can be used to
treat tumors include palmitoylated proteins, such as PAM--a
synthetic TLR2 ligand, Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4) and
other palmitoylated proteins from bacterial products. These ligands
will promote an increase in cell surface invariant chain/CD74 CLIP
and thereby make the tumor cell a target for .gamma..delta.T-cell
killing.
[0111] Still another method is to treating the cancer cells with an
inflammatory mediator such as palmitoylated proteins, such as
PAM--a synthetic TLR2 ligand, Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4),
or other palmitoylated proteins from bacterial products.
[0112] In further embodiments, the method further comprising
treating the subject with a standard anti-cancer therapy, for
example chemotherapy, radiotherapy or hormonal therapy.
[0113] In any of the foregoing treatments, the agent can be
introduced into the patient by any conventional means.
[0114] In particular embodiments, the cancer cell is a breast
cancer cell, a lung cancer cell, a head & neck cancer cell, a
brain cancer cell, an esophageal cancer cell, a liver cancer cell,
a prostate cancer cell, a stomach cancer cell, an ovarian cancer
cell, a uterine cancer cell, a cervical cancer cell, a testicular
cancer cell, a skin cancer cell, a colon cancer cell, a leukemia
cell, or a lymphoma cell. In other embodiments, the cancer cell is
a glioblastoma cell, a rhabdomyosarcoma cell, a melanoma cell, or a
Kaposi's sarcoma cell. In still other embodiments, the cancer is
primary, metastatic, recurrent or multi-drug resistant.
[0115] Thus, the methods of the invention, in some embodiments, are
useful for inducing cell death in many types of mammalian cells,
and particularly in tumor cells. The term "cell death" is herein to
refer to either of the processes of apoptosis or cell lysis. In
both apoptosis and cell lysis, the cell dies, but the processes
occur through different mechanisms and/or different metabolic
states of the cell. Apoptosis is a process of cell death in which
the cell undergoes shrinkage and fragmentation, followed by
phagocytosis of the cell fragments. Apoptosis is well known in the
art and can be assessed by any art-recognized method. For example,
apoptosis can easily be determined using flow cytometry, which is
able to distinguish between live and dead cells.
[0116] In one set of embodiments, the invention includes a method
of treating a subject susceptible to or exhibiting symptoms of
cancer. In some cases, the cancer is drug-resistant or multi-drug
resistant. As used herein, a "drug-resistant cancer" is a cancer
that is resistant to conventional commonly-known cancer therapies.
Examples of conventional cancer therapies include treatment of the
cancer with agents such as methotrexate, trimetrexate, adriamycin,
taxotere, doxorubicin, 5-fluorouracil, vincristine, vinblastine,
pamidronate disodium, anastrozole, exemestane, cyclophosphamide,
epirubicin, toremifene, letrozole, trastuzumab, megestrol,
tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate,
etc. A "multi-drug resistant cancer" is a cancer that resists more
than one type or class of cancer agents, i.e., the cancer is able
to resist a first drug having a first mechanism of action, and a
second drug having a second mechanism of action.
[0117] In one embodiment, the methods of the invention can be used
in conjunction with one or more other forms of cancer treatment,
for example, in conjunction with an anti-cancer agent,
chemotherapy, radiotherapy, etc. (e.g., simultaneously, or as part
of an overall treatment procedure). As another non-limiting
example, a cell may be manipulated to increase the amount of UCP or
Fas in the plasma membrane, and also exposed to another form of
cancer treatment. The term "cancer treatment" as used herein, may
include, but is not limited to, chemotherapy, radiotherapy,
adjuvant therapy, vaccination, or any combination of these methods.
Parameters of cancer treatment that may vary include, but are not
limited to, dosages, timing of administration or duration or
therapy; and the cancer treatment can vary in dosage, timing, or
duration. Another treatment for cancer is surgery, which can be
utilized either alone or in combination with any of the previously
treatment methods. One of ordinary skill in the medical arts can
determine an appropriate treatment for a subject.
[0118] A "tumor cell," as used herein, is a cell which is
undergoing unwanted mitotic proliferation. A tumor cell, when used
in the in vitro embodiments of the invention, can be isolated from
a tumor within a subject, or may be part of an established cell
line. A tumor cell in a subject may be part of any type of cancer.
Cancers include, but are not limited to, biliary tract cancer;
bladder cancer; brain cancer including glioblastomas and
medulloblastomas; breast cancer; cervical cancer; choriocarcinoma;
colon cancer; endometrial cancer; esophageal cancer; gastric
cancer; hematological neoplasms including acute lymphocytic and
myelogenous leukemia; multiple myeloma; AIDS-associated leukemias
and adult T-cell leukemia lymphoma; intraepithelial neoplasms
including Bowen's disease and Paget's disease; liver cancer; lung
cancer; lymphomas including Hodgkin's disease and lymphocytic
lymphomas; neuroblastomas; oral cancer including squamous cell
carcinoma; ovarian cancer including those arising from epithelial
cells, stromal cells, germ cells and mesenchymal cells; pancreatic
cancer; prostate cancer; rectal cancer; sarcomas including
leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and
osteosarcoma; skin cancer including melanoma, Kaposi's sarcoma,
basocellular cancer, and squamous cell cancer; testicular cancer
including germinal tumors such as seminoma, non-seminoma,
teratomas, choriocarcinomas; stromal tumors and germ cell tumors;
thyroid cancer including thyroid adenocarcinoma and medullar
carcinoma; and renal cancer including adenocarcinoma and Wilms'
tumor. Commonly encountered cancers include breast, prostate, lung,
ovarian, colorectal, and brain cancer. In general, an effective
amount of a composition for treating a cancer will be that amount
necessary to inhibit mammalian cancer cell proliferation in situ.
Those of ordinary skill in the art are well-schooled in the art of
evaluating effective amounts of anti-cancer agents.
[0119] In some cases, the cancer treatment may include treatment
with an anti-cancer agent or drug, for example, a
conventionally-known anti-cancer agent or drug. Examples of
suitable anti-cancer agents and drugs include, but are not limited
to, methotrexate, trimetrexate, adriamycin, taxotere,
5-fluorouracil, vincristine, vinblastine, pamidronate disodium,
anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene,
letrozole, trastuzumab, megestrol, tamoxifen, paclitaxel,
docetaxel, capecitabine, and goserelin acetate. Additional examples
of suitable anti-cancer agents and drugs include, but are not
limited to, 20-epi-1,25 dihydroxyvitamin D3, 4-ipomeanol,
5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin,
aclarubicin, acodazole hydrochloride, acronine, acylfulvene,
adecypenol, adozelesin, aldesleukin, all-tk antagonists,
altretamine, ambamustine, ambomycin, ametantrone acetate, amidox,
amifostine, aminoglutethimide, aminolevulinic acid, amrubicin,
amsacrine, anagrelide, andrographolide, angiogenesis inhibitors,
antagonist D, antagonist G, antarelix, anthramycin,
anti-dorsalizing morphogenetic protein-1, antiestrogen,
antineoplaston, antisense oligonucleotides, aphidicolin glycinate,
apoptosis gene modulators, apoptosis regulators, apurinic acid,
ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin,
asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,
axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine,
azetepa, azotomycin, baccatin III derivatives, balanol, batimastat,
benzochlorins, benzodepa, benzoylstaurosporine, beta lactam
derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF
inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride,
bisaziridinylspermine, bisnafide, bisnafide dimesylate, bistratene
A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists,
breflate, brequinar sodium, bropirimine, budotitane, busulfan,
buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C,
calusterone, camptothecin derivatives, canarypox IL-2, caracemide,
carbetimer, carboplatin, carboxamide-amino-triazole,
carboxyamidotriazole, carest M3, carmustine, cam 700, cartilage
derived inhibitor, carubicin hydrochloride, carzelesin, casein
kinase inhibitors, castanospermine, cecropin B, cedefingol,
cetrorelix, chlorambucil, chlorins, chloroquinoxaline sulfonamide,
cicaprost, cirolemycin, cisplatin, cis-porphyrin, cladribine,
clomifene analogs, clotrimazole, collismycin A, collismycin B,
combretastatin A4, combretastatin analog, conagenin, crambescidin
816, crisnatol, crisnatol mesylate, cryptophycin 8, cryptophycin A
derivatives, curacin A, cyclopentanthraquinones, cycloplatam,
cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin
hydrochloride, decitabine, dehydrodidemnin B, deslorelin,
dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil,
dezaguanine, dezaguanine mesylate, diaziquone, dichloroacetate,
didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine,
dioxamycin, diphenyl spiromustine, docosanol, dolasetron,
doxifluridine, doxorubicin hydrochloride, droloxifene, droloxifene
citrate, dromostanolone propionate, dronabinol, duazomycin,
duocarmycin SA, ebselen, ecomustine, edatrexate, edelfosine,
edrecolomab, eflomithine, eflomithine hydrochloride, elemene,
elsamitrucin, emitefur, enloplatin, enpromate, epipropidine,
epirubicin hydrochloride, epristeride, erbulozole, erythrocyte gene
therapy vector system, esorubicin hydrochloride, estramustine,
estramustine analog, estramustine phosphate sodium, estrogen
agonists, estrogen antagonists, etanidazole, etoposide, etoposide
phosphate, etoprine, fadrozole, fadrozole hydrochloride,
fazarabine, fenretinide, filgrastim, finasteride, flavopiridol,
flezelastine, floxuridine, fluasterone, fludarabine, fludarabine
phosphate, fluorodaunorunicin hydrochloride, flurocitabine,
forfenimex, formestane, fosquidone, fostriecin, fostriecin sodium,
fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine,
ganirelix, gelatinase inhibitors, gemcitabine, gemcitabine
hydrochloride, glutathione inhibitors, hepsulfam, heregulin,
hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic
acid, idarubicin, idarubicin hydrochloride, idoxifene, idramantone,
ifosfamide, ilmofosine, ilomastat, imidazoacridones, imiquimod,
immunostimulant peptides, insulin-like growth factor-1 receptor
inhibitor, interferon agonists, interferon alpha-2A, interferon
alpha-2B, interferon alpha-N1, interferon alpha-N3, interferon
beta-IA, interferon gamma-IB, interferons, interleukins,
iobenguane, iododoxorubicin, iproplatin, irinotecan, irinotecan
hydrochloride, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-N triacetate, lanreotide, lanreotide acetate,
leinamycin, lenograstim, lentinan sulfate, leptolstatin, leukemia
inhibiting factor, leukocyte alpha interferon, leuprolide acetate,
leuprolide/estrogen/progesterone, leuprorelin, levamisole,
liarozole, liarozole hydrochloride, linear polyamine analog,
lipophilic disaccharide peptide, lipophilic platinum compounds,
lissoclinamide 7, lobaplatin, lombricine, lometrexol, lometrexol
sodium, lomustine, lonidamine, losoxantrone, losoxantrone
hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium
texaphyrin, lysofylline, lytic peptides, maytansine, mannostatin A,
marimastat, masoprocol, maspin, matrilysin inhibitors, matrix
metalloproteinase inhibitors, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, merbarone, mercaptopurine, meterelin, methioninase,
methotrexate sodium, metoclopramide, metoprine, meturedepa,
microalgal protein kinase C inhibitors, MIF inhibitor,
mifepristone, miltefosine, mirimostim, mismatched double stranded
RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone,
mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide,
mitosper, mitotane, mitotoxin fibroblast growth factor-saporin,
mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid a/mycobacterium cell wall SK, mopidamol, multiple drug
resistance gene inhibitor, multiple tumor suppressor 1-based
therapy, mustard anticancer agent, mycaperoxide B, mycobacterial
cell wall extract, mycophenolic acid, myriaporone,
n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine,
napavin, naphterpin, nartograstim, nedaplatin, nemorubicin,
neridronic acid, neutral endopeptidase, nilutamide, nisamycin,
nitric oxide modulators, nitroxide antioxidant, nitrullyn,
nocodazole, nogalamycin, n-substituted benzamides,
O6-benzylguanine, ocreotide, okicenone, oligonucleotides,
onapristone, ondensetron, oracin, oral cytokine inducer,
ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran,
paclitaxel analogs, paclitaxel derivatives, palauamine,
palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,
parabactin, pazelliptine, pegaspargase, peldesine, peliomycin,
pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole,
peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin,
piritrexim, piroxantrone hydrochloride, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, plicamycin, plomestane,
porfimer sodium, porfiromycin, prednimustine, procarbazine
hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic
carcinoma antiandrogen, proteasome inhibitors, protein A-based
immune modulator, protein kinase C inhibitor, protein tyrosine
phosphatase inhibitors, purine nucleoside phosphorylase inhibitors,
puromycin, puromycin hydrochloride, purpurins, pyrazofurin,
pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene
conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl
protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor,
retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin,
riboprine, ribozymes, RII retinamide, RNAi, rogletimide,
rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl,
safingol, safingol hydrochloride, saintopin, sarcnu, sarcophytol A,
sargramostim, SDI 1 mimetics, semustine, senescence derived
inhibitor 1, sense oligonucleotides, signal transduction
inhibitors, signal transduction modulators, simtrazene, single
chain antigen binding protein, sizofuran, sobuzoxane, sodium
borocaptate, sodium phenylacetate, solverol, somatomedin binding
protein, sonermin, sparfosate sodium, sparfosic acid, sparsomycin,
spicamycin D, spirogermanium hydrochloride, spiromustine,
spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell
inhibitor, stem-cell division inhibitors, stipiamide,
streptonigrin, streptozocin, stromelysin inhibitors, sulfinosine,
sulofenur, superactive vasoactive intestinal peptide antagonist,
suradista, suramin, swainsonine, synthetic glycosaminoglycans,
talisomycin, tallimustine, tamoxifen methiodide, tauromustine,
tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase
inhibitors, teloxantrone hydrochloride, temoporfin, temozolomide,
teniposide, teroxirone, testolactone, tetrachlorodecaoxide,
tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline,
thioguanine, thiotepa, thrombopoietin, thrombopoietin mimetic,
thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid
stimulating hormone, tiazofurin, tin ethyl etiopurpurin,
tirapazamine, titanocene dichloride, topotecan hydrochloride,
topsentin, toremifene citrate, totipotent stem cell factor,
translation inhibitors, trestolone acetate, tretinoin,
triacetyluridine, triciribine, triciribine phosphate, trimetrexate,
trimetrexate glucuronate, triptorelin, tropisetron, tubulozole
hydrochloride, turosteride, tyrosine kinase inhibitors,
tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, velaresol, veramine,
verdins, verteporfin, vinblastine sulfate, vincristine sulfate,
vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate
sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate,
vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin,
vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin
stimalamer, and zorubicin hydrochloride, as well as salts,
homologs, analogs, derivatives, enantiomers and/or functionally
equivalent compositions thereof.
[0120] In another embodiment, cells may be removed from a tumor or
other rapidly dividing cell mass (e.g., a tumor from a subject, a
tumor growing in vitro, etc.) and exposed in some fashion to the
methods described herein, including treatment with TLR ligands or
inflammatory agents to increase cell surface expression of
invariant chain/CD74, and/or CLIP. After suitable exposure, the
exposed cells may be introduced into a subject. Exposure of the
cells may alter the immunological profile of the tumor cells in
some fashion, for example, such that a subject's immune system is
able to recognize the tumor cells. The subject's immune system,
after interacting with the exposed cells, may then be able to
recognize tumors present within the subject, thus causing the
cancer (or other rapidly dividing cell mass) to decrease. If the
subject has a tumor, the cells may be injected into the tumor,
proximate the tumor, and/or systemically or locally delivered in a
region of the body away from the tumor. In some cases, a tumor may
be removed from a subject, then the exposed cells may be inserted,
e.g., into the cavity created upon removal of the tumor, or to
another site within the body. Optionally, other cancer treatment
methods, such as radiation or exposure to conventional anti-cancer
agents, may also be used in conjunction with these methods. In some
cases, the subject may not have a cancer or tumor, but the cells
may be injected to stimulate the immune system to produce
antibodies against future cancers and/or other uncontrolled
cellular growths, i.e., "immunizing" the subject from cancer and/or
other uncontrolled cellular growths. In some the cancer cells are
antigenic and can be targeted by the immune system. Thus, the
combined administration of the methods of the invention and cancer
medicaments, particularly those which are classified as cancer
immunotherapies, can be very useful for stimulating a specific
immune response against a cancer antigen.
[0121] A "cancer antigen" as used herein is a compound, such as a
peptide, associated with a tumor or cancer cell surface, and which
is capable of provoking an immune response when expressed on the
surface of an antigen-presenting cell in the context of an MHC
molecule. Cancer antigens, such as those present in cancer vaccines
or those used to prepare cancer immunotherapies, can be prepared
from crude cancer cell extracts, e.g., as described in Cohen et al.
(1994), or by partially purifying the antigens, using recombinant
technology, or de novo synthesis of known antigens. Cancer antigens
can be used in the form of immunogenic portions of a particular
antigen, or in some instances, a whole cell or a tumor mass can be
used as the antigen. Such antigens can be isolated or prepared
recombinantly or by any other means known in the art.
[0122] The methods of the invention can be used in combination with
immunotherapeutics, according to another embodiment. The goal of
immunotherapy is to augment a subject's immune response to an
established tumor. One method of immunotherapy includes the use of
adjuvants. Adjuvant substances derived from microorganisms, such as
Bacillus Calmette-Guerin, can heighten the immune response and
enhance resistance to tumors in animals. Immunotherapeutic agents
are often medicaments which derive from antibodies or antibody
fragments that specifically bind to or otherwise recognize a cancer
antigen. Binding of such agents can promote an immune response,
such as an antigen-specific immune response. Antibody-based
immunotherapy may function by binding to the cell surface of a
cancer cell, which can stimulate the endogenous immune system to
attack the cancer cell.
[0123] As used herein, a "cancer antigen" is broadly defined as an
antigen expressed by a cancer cell. The antigen can be expressed at
the cell surface of the cancer cell. In many cases, the antigen is
one which is not expressed by normal cells, or at least not
expressed at the same level or concentration as in cancer cells. As
examples, some cancer antigens are normally silent (i.e., not
expressed) in normal cells, some are expressed only at certain
stages of differentiation, and others are only temporally expressed
(such as embryonic and fetal antigens). Other cancer antigens are
encoded by mutanT-cellular genes, such as oncogenes (e.g.,
activated ras oncogene), suppressor genes (e.g., mutant p53),
fusion proteins resulting from internal deletions or chromosomal
translocations, or the like. Still other cancer antigens can be
encoded by viral genes, such as those carried on RNA and DNA tumor
viruses. The differential expression of cancer antigens in normal
and cancer cells can be exploited in order to target cancer cells
in some cases. As used herein, the terms "cancer antigen" and
"tumor antigen" are used interchangeably.
[0124] The theory of immune surveillance is that a prime function
of the immune system is to detect and eliminate neoplastic cells
before a tumor forms. A basic principle of this theory is that
cancer cells are antigenically different from normal cells and thus
can elicit immune reactions similar to those that cause rejection
of immunologically incompatible allografts. Studies have confirmed
that tumor cells differ, qualitatively or quantitatively, in their
expression of antigens. For example, "tumor-specific antigens" are
antigens that are specifically associated with tumor cells but not
normal cells. Examples of tumor specific antigens are viral
antigens in tumors induced by DNA or RNA viruses.
"Tumor-associated" antigens are present in both tumor cells and
normal cells but are present in a different quantity or a different
form in tumor cells. Examples of such antigens are oncofetal
antigens (e.g., carcinoembryonic antigen), differentiation antigens
(e.g., T and Tn antigens), and oncogene products (e.g.,
HER/neu).
[0125] Different types of cells that can kill tumor targets in
vitro and in vivo have been identified: natural killer cells (NK
cells), cytolytic T lymphocytes (CTLs), lymphokine-activated killer
cells (LAKs), and activated macrophages. NK cells can kill tumor
cells without having been previously sensitized to specific
antigens, and the activity does not require the presence of class I
antigens encoded by the major histocompatibility complex (MHC) on
targeT-cells. NK cells are thought to participate in the control of
nascent tumors and in the control of metastatic growth. In contrast
to NK cells, CTLs can kill tumor cells only after they have been
sensitized to tumor antigens and when the target antigen is
expressed on the tumor cells that also express MHC class I. CTLs
are thought to be effector cells in the rejection of transplanted
tumors and of tumors caused by DNA viruses. LAK cells are a subset
of null lymphocytes distinct from the NK and CTL populations.
Activated macrophages can kill tumor cells in a manner that is not
antigen-dependent, nor MHC-restricted, once activated. Activated
macrophages are thought to decrease the growth rate of the tumors
they infiltrate. In vitro assays have identified other immune
mechanisms such as antibody-dependent, cell-mediated cytotoxic
reactions, and lysis by antibody plus complement. However, these
immune effector mechanisms are thought to be less important in vivo
than the function of NK, CTLs, LAK, and macrophages in vivo (for a
review, see Piessens, 1996).
[0126] In some cases, the immunotherapeutic agent may function as a
delivery system for the specific targeting of toxic substances to
cancer cells. For example, the agent may be conjugated to toxins
such as ricin (e.g., from castor beans), calicheamicin,
maytansinoids, radioactive isotopes such as iodine-131 and
yttrium-90, chemotherapeutic agents, and/or to biological response
modifiers. In this way, the toxic substances can be concentrated in
the region of the cancer and non-specific toxicity to normal cells
can be minimized.
[0127] In certain instances, the immunotherapeutic agent may be
directed towards the binding of vasculature, such as those which
bind to endothelial cells. This is because solid tumors are
generally dependent upon newly formed blood vessels to survive, and
thus most tumors are capable of recruiting and stimulating the
growth of new blood vessels. As a result, one strategy of many
cancer medicaments is to attack the blood vessels feeding a tumor
and/or the connective tissues (or stroma) supporting such blood
vessels.
[0128] In another set of embodiments, the combined administration
of the methods of the invention and an apoptotic chemotherapeutic
agent may be used. An "apoptotic chemotherapeutic agent," as used
herein, includes molecules which function by a variety of
mechanisms to induce apoptosis in rapidly dividing cells. Apoptotic
chemotherapeutic agents are a class of chemotherapeutic agents
which are well known to those of ordinary skill in the art.
Chemotherapeutic agents include those agents disclosed in Goodman
and Gilman's Chapter 52, and the introduction thereto, (1990),
incorporated herein by reference. Suitable chemotherapeutic agents
may have various mechanisms of action. Classes of suitable
chemotherapeutic agents include, but are not limited to: (a)
alkylating agents, such as nitrogen mustard (e.g., mechlorethamine,
cylophosphamide, ifosfamide, melphalan, chlorambucil),
ethylenimines and methylmelamines (e.g., hexamethylmelamine,
thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,
carmustine, which is also known as BCNU, lomustine which is also
known as CCNU, semustine, which is also known as methyl-CCNU,
chlorozotocin, streptozocin), and triazines (e.g., dicarbazine,
which is also known as DTIC); (b) antimetabolites, such as folic
acid analogs (e.g., methotrexate), pyrimidine analogs (e.g.,
5-fluorouracil floxuridine, cytarabine, and azauridine and its
prodrug form azaribine), and purine analogs and related materials
(e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); (c) natural
products, such as the vinca alkaloids (e.g., vinblastine,
vincristine), epipodophyllotoxins (e.g., etoposide, teniposide),
antibiotics (e.g., dactinomycin, which is also known as
actinomycin-D, daunorubicin, doxorubicin, bleomycin, plicamycin,
mitomycin, epirubicin, which is 4-epidoxorubicin, idarubicin which
is 4-dimethoxydaunorubicin, and mitoxanthrone), enzymes (e.g.,
L-asparaginase), and biological response modifiers (e.g.,
interferon .alpha.); (d) miscellaneous agents, such as the platinum
coordination complexes (e.g., cisplatin, carboplatin),
dichloroacetate and its derivatives, substituted ureas (e.g.,
hydroxyurea), methylhydrazine derivatives (e.g., procarbazine),
adrenocortical suppressants (e.g., mitotane, aminoglutethimide)
taxol; (e) hormones and antagonists, such as adrenocorticosteroids
(e.g., prednisone or the like), progestins (e.g.,
hydroxyprogesterone caproate, medroxyprogesterone acetate,
megestrol acetate), estrogens (e.g., diethylstilbestrol, ethinyl
estradiol, or the like), antiestrogens (e.g., tamoxifen), androgens
(e.g., testosterone propionate, Fluoxymesterone, or the like),
antiandrogens (e.g., flutamide), and gonadotropin-releasing hormone
analogs (e.g., leuprolide), and (f) DNA damaging compounds, such as
adriamycin. The combined administration of the methods of the
invention and an apoptotic chemotherapeutic agent effective to
inhibit growth of the tumor cell is that amount effective to induce
apoptosis of the tumor cell in some cases.
[0129] In yet another set of embodiments, the methods of the
invention may be used in conjunction with a vaccine, such as a
cancer vaccine. Cancer vaccines are medicaments which are intended
to stimulate an endogenous immune response against cancer cells.
Currently-produced vaccines predominantly activate the humoral
immune system (i.e., the antibody dependent immune response). Other
vaccines currently in development are focused on activating the
cell-mediated immune system including cytotoxic T lymphocytes which
are capable of killing tumor cells. Cancer vaccines generally
enhance the presentation of cancer antigens to both antigen
presenting cells (e.g., macrophages and dendritic cells) and/or to
other immune cells such as T-cells, B-cells, and NK cells.
[0130] Although cancer vaccines may take one of several forms,
their purpose is to deliver cancer antigens and/or cancer
associated antigens to antigen presenting cells (APC) in order to
facilitate the endogenous processing of such antigens by APC and
the ultimate presentation of antigen presentation on the cell
surface in the context of MHC class I molecules. One form of cancer
vaccine is a whole cell vaccine which is a preparation of cancer
cells which have been removed from a subject, treated ex vivo and
then reintroduced as whole cells in the subject. Lysates of tumor
cells can also be used as cancer vaccines to elicit an immune
response in certain cases. Another form of cancer vaccine is a
peptide vaccine which uses cancer-specific or cancer-associated
small proteins to activate T-cells. Cancer-associated proteins are
proteins which are not exclusively expressed by cancer cells (i.e.,
other normal cells may still express these antigens). However, the
expression of cancer-associated antigens is generally consistently
upregulated with cancers of a particular type. Yet another form of
cancer vaccine is a dendritic cell vaccine which includes whole
dendritic cells that have been exposed to a cancer antigen or a
cancer-associated antigen in vitro. Lysates or membrane fractions
of dendritic cells may also be used as cancer vaccines in some
instances. Dendritic cell vaccines are able to activate
antigen-presenting cells directly. Other non-limiting examples of
cancer vaccines include ganglioside vaccines, heat-shock protein
vaccines, viral and bacterial vaccines, and nucleic acid
vaccines.
[0131] Other cancer vaccines can take the form of dendritic cells
which have been exposed to cancer antigens in vitro, have processed
the antigens and are able to express the cancer antigens at their
cell surface in the context of MHC molecules for effective antigen
presentation to other immune system cells.
[0132] In some embodiments, cancer vaccines may be used along with
adjuvants. Adjuvants are substances which activate the subject's
immune system, and can be used as an adjunct therapy in any of the
systems or methods of the invention. Adjuvants include, for
example, alum, QS-Stimulon (Aquila), MF-59 (Chiron), Detox (Ribi),
Optivax (Vaxcels) and LeIF (Corixa).
[0133] F. Infection
[0134] According to an embodiment of the invention, the methods
described herein are useful in treating a intracellular bacterial
infection in a subject by inducing cell surface expression of CLIP
in an antigen presenting cell or by treating directly with soluble,
synthetic CLIP or activating .gamma..delta.T-cells. Specific
methods of inducing cell surface expression of CLIP in the cell
have been described above under the description related to methods
of treating cancer. Examples are treating the cell with nef or an
agent that increases nef expression, with ectopic CLIP, or with an
inflammatory mediator such as Pam(3)Cys-Ser-(Lys)(4)
(Pam(3)CSK(4).
[0135] The present invention would have applications therefore in
the prevention and treatment of diseases against which a T-cell
response would be effective. The following pathogenic virus
classes, which are mentioned by way of example, are specifically
contemplated as targets for T-cell selecting peptide
administration: influenza A, B and C, parainfluenza,
paramyxoviruses, Newcastle disease virus, respiratory syncytial
virus, measles, mumps, parvoviruses, Epstein-Barr virus,
rhinoviruses, coxsackieviruses, echoviruses, reoviruses,
rhabdoviruses, lymphocytic choriomeningitis, coronavirus,
polioviruses, herpes simplex, human immunodeficiency viruses,
cytomegaloviruses, papillomaviruses, virus B, varicella-zoster,
poxviruses, rubella, rabies, picornaviruses, rotavirus and Kaposi
associated herpes virus.
[0136] In addition to the viral diseases mentioned above, the
present invention is also useful in the prevention, inhibition, or
treatment of bacterial infections, including, but not limited to,
the 83 or more distinct serotypes of pneumococci, streptococci such
as S. pyogenes, S. agalactiae, S. equi, S. canis, S. bovis, S.
equinus, S. anginosus, S. sanguis, S. salivarius, S. mitis, S.
mutans, other viridans streptococci, peptostreptococci, other
related species of streptococci, enterococci such as Enterococcus
faecalis, Enterococcus faecium, staphylococci, such as
Staphylococcus epidermidis, Staphylococcus aureus, Hemophilus
influenzae, pseudomonas species such as Pseudomonas aeruginosa,
Pseudomonas pseudomallei, Pseudomonas mallei, brucellas such as
Brucella melitensis, Brucella suis, Brucella abortus, Bordetella
pertussis, Borellia species, such as Borellia burgedorferi
Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella
catarrhalis, Corynebacterium diphtheriae, Corynebacterium ulcerans,
Corynebacterium pseudotuberculosis, Corynebacterium
pseudodiphtheriticum, Corynebacterium urealyticum, Corynebacterium
hemolyticum, Corynebacterium equi, etc. Listeria monocytogenes,
Nocordia asteroides, Bacteroides species, Actinomycetes species,
Treponema pallidum, Leptospirosa species, Haemophilus species,
Helicobacter species, including Helicobacter pylori, Treponema
species and related organisms. The invention may also be useful
against gram negative bacteria such as Klebsiella pneumoniae,
Escherichia coli, Proteus, Serratia species, Acinetobacter,
Yersinia pestis, Francisella tularensis, Enterobacter species,
Bacteriodes and Legionella species, Shigella species, Mycobacterium
species (e.g., Mycobacterium tuberculosis, Mycobacterium bovis or
other mycobacteria infections), Mycobacterium avium complex (MAC),
Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium
kansaii, Yersinia infections (e.g., Yersinia pestis, Yersinia
enterocolitica or Yersinia pseudotuberculosis) and the like. In
addition, the invention in contemplated to be of use in controlling
protozoan, helminth or other macroscopic infections by organisms
such as Cryptosporidium, Entamoeba, Plamodiium, Giardia,
Leishmania, Trypanasoma, Trichomonas, Naegleria, Isospora belli,
Toxoplasma gondii, Trichomonas vaginalis, Wunchereria, Ascaris,
Schistosoma species, Cyclospora species, for example, and for
Chlamydia trachomatis and other Chlamydia infections such as
Chlamydia psittaci, or Chlamydia pneumoniae, for example. Of course
it is understood that the invention may be used on any pathogen
against which an effective antibody can be made.
[0137] Fungal and other mycotic pathogens (some of which are
described in Human Mycoses (1979; Opportunistic Mycoses of Man and
Other Animals (1989); and Scrip's Antifungal Report (1992), are
also contemplated as a target of administration of a T-cell
selecting peptide. Fungi disease contemplated in the context of the
invention include, but are not limited to, Aspergillosis, Black
piedra, Candidiasis, Chromomycosis, Cryptococcosis, Onychomycosis,
or Otitis extema (otomycosis), Phaeohyphomycosis, Phycomycosis,
Pityriasis versicolor, ringworm, Tinea barbae, Tinea capitis, Tinea
corporis, Tinea cruris, Tinea favosa, Tinea imbricata, Tinea
manuum, Tinea nigra (palmaris), Tinea pedis, Tinea unguium,
Torulopsosis, Trichomycosis axillaris, White piedra, and their
synonyms, to severe systemic or opportunistic infections, such as,
but not limited to, Actinomycosis, Aspergillosis, Candidiasis,
Chromomycosis, Coccidioidomycosis, Cryptococcosis,
Entomophthoramycosis, Geotrichosis, Histoplasmosis, Mucormycosis,
Mycetoma, Nocardiosis, North American Blastomycosis,
Paracoccidioidomycosis, Phaeohyphomycosis, Phycomycosis,
pneumocystic pneumonia, Pythiosis, Sporotrichosis, and
Torulopsosis, and their synonyms, some of which may be fatal. Known
fungal and mycotic pathogens include, but are not limited to,
Absidia spp., Actinomadura madurae, Actinomyces spp., Allescheria
boydii, Alternaria spp., Anthopsis deltoidea, Apophysomyces
elegans, Arnium leoporinum, Aspergillus spp., Aureobasidium
pullulans, Basidiobolus ranarum, Bipolaris spp., Blastomyces
dermatitidis, Candida spp., Cephalosporium spp., Chaetoconidium
spp., Chaetomium spp., Cladosporium spp., Coccidioides immitis,
Conidiobolus spp., Corynebacterium tenuis, Cryptococcus spp.,
Cunninghamella bertholletiae, Curvularia spp., Dactylaria spp.,
Epidermophyton spp., Epidermophyton floccosum, Exserophilum spp.,
Exophiala spp., Fonsecaea spp., Fusarium spp., Geotrichum spp.,
Helminthosporium spp., Histoplasma spp., Lecythophora spp.,
Madurella spp., Malassezia furfur, Microsporum spp., Mucor spp.,
Mycocentrospora acerina, Nocardia spp., Paracoccidioides
brasiliensis, Penicillium spp., Phaeosclera dematioides,
Phaeoannellomyces spp., Phialemonium obovatum, Phialophora spp.,
Phoma spp., Piedraia hortai, Pneumocystis carinii, Pythium
insidiosum, Rhinocladiella aquaspersa, Rhizomucor pusillus,
Rhizopus spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis,
Sporothrix schenckii, Syncephalastrum racemosum, Taeniolella
boppii, Torulopsosis spp., Trichophyton spp., Trichosporon spp.,
Ulocladium chartarum, Wangiella dermatitidis, Xylohypha spp.,
Zygomyetes spp. and their synonyms. Other fungi that have
pathogenic potential include, but are not limited to, Thermomucor
indicae-seudaticae, Radiomyces spp., and other species of known
pathogenic genera.
[0138] In some aspects, the invention provides methods and kits
that include anti-CLIP molecule and anti-HLA binding molecules such
as peptides, antibodies, antibody fragments and small molecules.
CLIP and HLA binding molecules bind to CLIP molecules and HLA
respectively on the surface of cells. The binding molecules are
referred to herein as isolated molecules that selectively bind to
CLIP molecules and HLA. A molecule that selectively binds to CLIP
and HLA as used herein refers to a molecule, e.g., small molecule,
peptide, antibody, fragment, that interacts with CLIP and HLA. In
some embodiments the molecules are peptides.
[0139] The peptides minimally comprise regions that bind to CLIP
and HLA. CLIP and HLA-binding regions, in some embodiments derive
from the CLIP and HLA-binding regions of known or commercially
available antibodies, or alternatively, they are functionally
equivalent variants of such regions. The term "antibody" herein is
used in the broadest sense and specifically covers intact
monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies) formed from at least two
intact antibodies, antibody fragments, so long as they exhibit the
desired biological activity, and antibody like molecules such as
scFv. A native antibody usually refers to heterotetrameric
glycoproteins composed of two identical light (L) chains and two
identical heavy (H) chains. Each heavy and light chain has
regularly spaced intrachain disulfide bridges. Each heavy chain has
at one end a variable domain (VH) followed by a number of constant
domains. Each light chain has a variable domain at one end (VL) and
a constant domain at its other end; the constant domain of the
light chain is aligned with the first constant domain of the heavy
chain, and the light-chain variable domain is aligned with the
variable domain of the heavy chain. Particular amino acid residues
are believed to form an interface between the light- and
heavy-chain variable domains.
[0140] Numerous CLIP and HLA antibodies are available commercially
for research purposes. Certain portions of the variable domains
differ extensively in sequence among antibodies and are used in the
binding and specificity of each particular antibody for its
particular antigen. However, the variability is not evenly
distributed throughout the variable domains of antibodies. It is
concentrated in three or four segments called
"complementarity-determining regions" (CDRs) or "hypervariable
regions" in both in the light-chain and the heavy-chain variable
domains. The more highly conserved portions of variable domains are
called the framework (FR). The variable domains of native heavy and
light chains each comprise four or five FR regions, largely
adopting a .beta.-sheet configuration, connected by the CDRs, which
form loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., 1991). The constant domains are not
necessarily involved directly in binding an antibody to an antigen,
but exhibit various effector functions, such as participation of
the antibody in antibody-dependenT-cellular toxicity.
[0141] A hypervariable region or CDR as used herein defines a
subregion within the variable region of extreme sequence
variability of the antibody, which form the antigen-binding site
and are the main determinants of antigen specificity. According to
one definition, they can be residues (Kabat nomenclature) 24-34
(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region
and residues (Kabat nomenclature 31-35 (H1), 50-65 (H2), 95-102
(H3) in the heavy chain variable region. Kabat et al., 1991).
[0142] An "intact" antibody is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (C.sub.L) and heavy chain constant domains, C.sub.H1,
C.sub.H2 and C.sub.H3. The constant domains may be native sequence
constant domains (e.g., human native sequence constant domains) or
amino acid sequence variant thereof. Preferably, the intact
antibody has one or more effector functions. Various techniques
have been developed for the production of antibody fragments.
Traditionally, these fragments were derived via proteolytic
digestion of intact antibodies (see, e.g., Morimoto et al., 1992;
and Brennan et al., 1985). However, these fragments can now be
produced directly by recombinant hosT-cells. For example, the
antibody fragments can be isolated from antibody phage libraries.
Alternatively, Fab'-SH fragments can be directly recovered from E.
coli and chemically coupled to form F(ab').sub.2 fragments (Carter
et al., 1992). According to another approach, F(ab').sub.2
fragments can be isolated directly from recombinant hosT-cell
culture.
[0143] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments. Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0144] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen-binding site on the surface of the VH-VL
dimer. Collectively, the six CDRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0145] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0146] The term "Fc region" is used to define the C-terminal region
of an immunoglobulin heavy chain which may be generated by papain
digestion of an intact antibody. The Fc region may be a native
sequence Fc region or a variant Fc region. Although the boundaries
of the Fc region of an immunoglobulin heavy chain might vary, the
human IgG heavy chain Fc region is usually defined to stretch from
an amino acid residue at about position Cys226, or from about
position Pro230, to the carboxyl-terminus of the Fc region. The Fc
region of an immunoglobulin generally comprises two constant
domains, a CH2 domain and a CH3 domain, and optionally comprises a
CH4 domain. By "Fc region chain" herein is meant one of the two
polypeptide chains of an Fc region.
[0147] The "hinge region," and variations thereof, as used herein,
includes the meaning known in the art, which is illustrated in, for
example, Janeway et al., 1999).
[0148] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
The heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called .alpha., .delta., .epsilon.,
.gamma., and .mu., respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0149] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0150] The peptides useful herein are isolated peptides. As used
herein, the term "isolated peptides" means that the peptides are
substantially pure and are essentially free of other substances
with which they may be found in nature or in vivo systems to an
extent practical and appropriate for their intended use. In
particular, the peptides are sufficiently pure and are sufficiently
free from other biological constituents of their hosts cells so as
to be useful in, for example, producing pharmaceutical preparations
or sequencing. Because an isolated peptide of the invention may be
admixed with a pharmaceutically acceptable carrier in a
pharmaceutical preparation, the peptide may comprise only a small
percentage by weight of the preparation. The peptide is nonetheless
substantially pure in that it has been substantially separated from
the substances with which it may be associated in living
systems.
[0151] The CLIP and HLA binding molecules bind to CLIP and HLA,
preferably in a selective manner. As used herein, the terms
"selective binding" and "specific binding" are used interchangeably
to refer to the ability of the peptide to bind with greater
affinity to CLIP and HLA and fragments thereof than to non-CLIP and
HLA derived compounds. That is, peptides that bind selectively to
CLIP and HLA will not bind to non-CLIP and HLA derived compounds to
the same extent and with the same affinity as they bind to CLIP and
HLA and fragments thereof, with the exception of cross reactive
antigens or molecules made to be mimics of CLIP and HLA such as
peptide mimetics of carbohydrates or variable regions of
anti-idiotype antibodies that bind to the CLIP and HLA-binding
peptides in the same manner as CLIP and HLA. In some embodiments,
the CLIP and HLA binding molecules bind solely to CLIP and HLA and
fragments thereof.
[0152] "Isolated antibodies" as used herein refer to antibodies
that are substantially physically separated from other cellular
material (e.g., separated from cells which produce the antibodies)
or from other material that hinders their use either in the
diagnostic or therapeutic methods of the invention. Preferably, the
isolated antibodies are present in a homogenous population of
antibodies (e.g., a population of monoclonal antibodies).
Compositions of isolated antibodies can however be combined with
other components such as but not limited to pharmaceutically
acceptable carriers, adjuvants, and the like.
[0153] In one embodiment, the CLIP and HLA peptides useful in the
invention are isolated intact soluble monoclonal antibodies
specific for CLIP and HLA. As used herein, the term "monoclonal
antibody" refers to a homogenous population of immunoglobulins that
specifically bind to an identical epitope (i.e., antigenic
determinant).
[0154] In other embodiments, the peptide is an antibody fragment.
As is well-known in the art, only a small portion of an antibody
molecule, the paratope, is involved in the binding of the antibody
to its epitope (see, in general, Clark (1986); Roitt (1991); and
Pier et al. (2004)). The pFc' and Fc regions of the antibody, for
example, are effectors of the complement cascade and can mediate
binding to Fc receptors on phagocytic cells, but are not involved
in antigen binding. An antibody from which the pFc' region has been
enzymatically cleaved, or which has been produced without the pFc'
region, designated an F(ab').sub.2 fragment, retains both of the
antigen binding sites of an intact antibody. An isolated
F(ab').sub.2 fragment is referred to as a bivalent monoclonal
fragment because of its two antigen binding sites. Similarly, an
antibody from which the Fc region has been enzymatically cleaved,
or which has been produced without the Fc region, designated an Fab
fragment, retains one of the antigen binding sites of an intact
antibody molecule. Proceeding further, Fab fragments consist of a
covalently bound antibody light chain and a portion of the antibody
heavy chain denoted Fd (heavy chain variable region). The Fd
fragments are the major determinant of antibody specificity (a
single Fd fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation.
[0155] The terms Fab, Fc, pFc', F(ab').sub.2 and Fv are employed
with either standard immunological meanings (Klein, 1982; Clark,
1986; Roitt, 1991; and Pier et al., 2004).
[0156] The anti-CLIP and HLA antibodies of the invention may
further comprise humanized antibodies or human antibodies.
Humanized forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of
antibodies) which contain minimal sequence derived from non-human
immunoglobulin. Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a complementary
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found
neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones et
al., 1986; Riechmann et al., 1988; Presta, 1992).
[0157] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source that is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al., 1986;
Riechmann et al., 1988); Verhoeyen et al., 1988), by substituting
rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567), wherein
substantially less than an intact human variable domain has been
substituted by the corresponding sequence from a non-human species.
In practice, humanized antibodies are typically human antibodies in
which some CDR residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0158] Various forms of the humanized antibody or affinity matured
antibody are contemplated. For example, the humanized antibody or
affinity matured antibody may be an antibody fragment, such as a
Fab, which is optionally conjugated with one or more cytotoxic
agent(s) in order to generate an immunoconjugate. Alternatively,
the humanized antibody or affinity matured antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0159] As an alternative to humanization, human antibodies can be
generated. A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any techniques for making human
antibodies. This definition of a human antibody specifically
excludes a humanized antibody comprising non-human antigen-binding
residues. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (JH) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.
(1993); Jakobovits et al. (1993); Bruggermann et al. (1993); and
U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.
[0160] Human monoclonal antibodies also may be made by any of the
methods known in the art, such as those disclosed in U.S. Pat. Nos.
5,567,610, 5,565,354, and 5,571,893, Kozber (1984); Brodeur et al.
(1987), and Boerner et al. (1991).
[0161] The invention also encompasses the use of single chain
variable region fragments (scFv). Single chain variable region
fragments are made by linking light and/or heavy chain variable
regions by using a short linking peptide. Any peptide having
sufficient flexibility and length can be used as a linker in a
scFv. Usually the linker is selected to have little to no
immunogenicity. An example of a linking peptide is multiple GGGGS
residues, which bridge the carboxy terminus of one variable region
and the amino terminus of another variable region. Other linker
sequences may also be used.
[0162] All or any portion of the heavy or light chain can be used
in any combination. Typically, the entire variable regions are
included in the scFv. For instance, the light chain variable region
can be linked to the heavy chain variable region. Alternatively, a
portion of the light chain variable region can be linked to the
heavy chain variable region, or portion thereof. Also contemplated
are scFvs in which the heavy chain variable region is from the
antibody of interest, and the light chain variable region is from
another immunoglobulin.
[0163] The scFvs can be assembled in any order, for example,
V.sub.H-linker-V.sub.L or V.sub.L-linker-V.sub.H. There may be a
difference in the level of expression of these two configurations
in particular expression systems, in which case one of these forms
may be preferred. Tandem scFvs can also be made, such as
(X)-linker-(X)-linker-(X), in which X are polypeptides form the
antibodies of interest, or combinations of these polypeptides with
other polypeptides. In another embodiment, single chain antibody
polypeptides have no linker polypeptide, or just a short,
inflexible linker. Possible configurations are V.sub.L-V.sub.H and
V.sub.H-V.sub.L. The linkage is too short to permit interaction
between V.sub.L and V.sub.H within the chain, and the chains form
homodimers with a V.sub.L/V.sub.H antigen binding site at each end.
Such molecules are referred to in the art as "diabodies."
[0164] Single-chain variable regions may be produced either
recombinantly or synthetically. For synthetic production of scFv,
an automated synthesizer can be used. For recombinant production of
scFv, a suitable plasmid containing polynucleotide that encodes the
scFv can be introduced into a suitable host-cell, either
eukaryotic, such as yeast, plant, insect or mammalian cells, or
prokaryotic, such as E. coli, and the expressed protein may be
isolated using standard protein purification techniques.
[0165] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al. (1993).
[0166] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological
activity.
[0167] Peptides, including antibodies, can be tested for their
ability to bind to CLIP and HLA using standard binding assays known
in the art. As an example of a suitable assay, CLIP and HLA can be
immobilized on a surface (such as in a well of a multi-well plate)
and then contacted with a labeled peptide. The amount of peptide
that binds to the CLIP and HLA (and thus becomes itself immobilized
onto the surface) may then be quantitated to determine whether a
particular peptide binds to CLIP and HLA. Alternatively, the amount
of peptide not bound to the surface may also be measured. In a
variation of this assay, the peptide can be tested for its ability
to bind directly to a CLIP and HLA-expressing cell.
[0168] The invention also encompasses small molecules that bind to
CLIP and HLA. Such binding molecules may be identified by
conventional screening methods, such as phage display procedures
(e.g., methods described in Hart et al., 1994). Hart et al. report
a filamentous phage display library for identifying novel peptide
ligands. In general, phage display libraries using, e.g., M13 or fd
phage, are prepared using conventional procedures such as those
described in the foregoing reference. The libraries generally
display inserts containing from 4 to 80 amino acid residues. The
inserts optionally represent a completely degenerate or biased
array of peptides. Ligands having the appropriate binding
properties are obtained by selecting those phage which express on
their surface a ligand that binds to the target molecule. These
phage are then subjected to several cycles of reselection to
identify the peptide ligand expressing phage that have the most
useful binding characteristics. Typically, phage that exhibit the
best binding characteristics (e.g., highest affinity) are further
characterized by nucleic acid analysis to identify the particular
amino acid sequences of the peptide expressed on the phage surface
in the optimum length of the express peptide to achieve optimum
binding. Phage-display peptide or antibody library is also
described in Brissette et al. (2006).
[0169] Alternatively, binding molecules can be identified from
combinatorial libraries. Many types of combinatorial libraries have
been described. For instance, U.S. Pat. Nos. 5,712,171 (methods for
constructing arrays of synthetic molecular constructs by forming a
plurality of molecular constructs having the scaffold backbone of
the chemical molecule and modifying at least one location on the
molecule in a logically-ordered array); 5,962,412 (methods for
making polymers having specific physiochemical properties); and
5,962,736 (specific arrayed compounds).
[0170] Other binding molecules may be identified by those of skill
in the art following the guidance described herein. Library
technology can be used to identify small molecules, including small
peptides, which bind to CLIP and HLA and interrupt its function.
One advantage of using libraries for antagonist identification is
the facile manipulation of millions of different putative
candidates of small size in small reaction volumes (i.e., in
synthesis and screening reactions). Another advantage of libraries
is the ability to synthesize antagonists which might not otherwise
be attainable using naturally occurring sources, particularly in
the case of non-peptide moieties.
[0171] Small molecule combinatorial libraries may also be
generated. A combinatorial library of small organic compounds is a
collection of closely related analogs that differ from each other
in one or more points of diversity and are synthesized by organic
techniques using multi-step processes. Combinatorial libraries
include a vast number of small organic compounds. One type of
combinatorial library is prepared by means of parallel synthesis
methods to produce a compound array. A "compound array" as used
herein is a collection of compounds identifiable by their spatial
addresses in Cartesian coordinates and arranged such that each
compound has a common molecular core and one or more variable
structural diversity elements. The compounds in such a compound
array are produced in parallel in separate reaction vessels, with
each compound identified and tracked by its spatial address.
Examples of parallel synthesis mixtures and parallel synthesis
methods are provided in PCT published patent application WO
95/18972, U.S. Pat. No. 5,712,171 and corresponding PCT published
patent application WO 96/22529, which are hereby incorporated by
reference.
[0172] The CLIP and HLA binding molecules described herein can be
used alone or in conjugates with other molecules such as detection
or cytotoxic agents in the detection and treatment methods of the
invention, as described in more detail herein.
[0173] Typically, one of the components usually comprises, or is
coupled or conjugated to a detectable label. A detectable label is
a moiety, the presence of which can be ascertained directly or
indirectly. Generally, detection of the label involves an emission
of energy by the label. The label can be detected directly by its
ability to emit and/or absorb photons or other atomic particles of
a particular wavelength (e.g., radioactivity, luminescence, optical
or electron density, etc.). A label can be detected indirectly by
its ability to bind, recruit and, in some cases, cleave another
moiety which itself may emit or absorb light of a particular
wavelength (e.g., epitope tag such as the FLAG epitope, enzyme tag
such as horseradish peroxidase, etc.). An example of indirect
detection is the use of a first enzyme label which cleaves a
substrate into visible products. The label may be of a chemical,
peptide or nucleic acid molecule nature although it is not so
limited. Other detectable labels include radioactive isotopes such
as .sup.32P or .sup.3H, luminescent markers such as fluorochromes,
optical or electron density markers, etc., or epitope tags such as
the FLAG epitope or the HA epitope, biotin, avidin, and enzyme tags
such as horseradish peroxidase, .beta.-galactosidase, etc. The
label may be bound to a peptide during or following its synthesis.
There are many different labels and methods of labeling known to
those of ordinary skill in the art. Examples of the types of labels
that can be used in the present invention include enzymes,
radioisotopes, fluorescent compounds, colloidal metals,
chemiluminescent compounds, and bioluminescent compounds. Those of
ordinary skill in the art will know of other suitable labels for
the peptides described herein, or will be able to ascertain such,
using routine experimentation. Furthermore, the coupling or
conjugation of these labels to the peptides of the invention can be
performed using standard techniques common to those of ordinary
skill in the art.
[0174] Another labeling technique which may result in greater
sensitivity consists of coupling the molecules described herein to
low molecular weight haptens. These haptens can then be
specifically altered by means of a second reaction. For example, it
is common to use haptens such as biotin, which reacts with avidin,
or dinitrophenol, pyridoxal, or fluorescein, which can react with
specific anti-hapten antibodies.
[0175] Conjugation of the peptides including antibodies or
fragments thereof to a detectable label facilitates, among other
things, the use of such agents in diagnostic assays. Another
category of detectable labels includes diagnostic and imaging
labels (generally referred to as in vivo detectable labels) such as
for example magnetic resonance imaging (MRI): Gd(DOTA); for nuclear
medicine: .sup.201Tl, gamma-emitting radionuclide 99 mTc; for
positron-emission tomography (PET): positron-emitting isotopes,
.sup.18F-fluorodeoxyglucose (.sup.18FDG), .sup.18F-fluoride,
copper-64, gadodiamide, and radioisotopes of Pb(II) such as
.sup.203Pb; .sup.111In.
[0176] The conjugations or modifications described herein employ
routine chemistry, which chemistry does not form a part of the
invention and which chemistry is well known to those skilled in the
art of chemistry. The use of protecting groups and known linkers
such as mono- and hetero-bifunctional linkers are well documented
in the literature and will not be repeated here.
[0177] As used herein, "conjugated" means two entities stably bound
to one another by any physiochemical means. It is important that
the nature of the attachment is such that it does not impair
substantially the effectiveness of either entity. Keeping these
parameters in mind, any covalent or non-covalent linkage known to
those of ordinary skill in the art may be employed. In some
embodiments, covalent linkage is preferred. Noncovalent conjugation
includes hydrophobic interactions, ionic interactions, high
affinity interactions such as biotin-avidin and biotin-streptavidin
complexation and other affinity interactions. Such means and
methods of attachment are well known to those of ordinary skill in
the art.
[0178] A variety of methods may be used to detect the label,
depending on the nature of the label and other assay components.
For example, the label may be detected while bound to the solid
substrate or subsequent to separation from the solid substrate.
Labels may be directly detected through optical or electron
density, radioactive emissions, nonradiative energy transfers, etc.
or indirectly detected with antibody conjugates,
streptavidin-biotin conjugates, etc. Methods for detecting the
labels are well known in the art.
[0179] The conjugates also include an antibody conjugated to a
cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an
enzymatically active toxin of bacterial, fungal, plant or animal
origin, or fragments thereof, or a small molecule toxin), or a
radioactive isotope (i.e., a radioconjugate). Other antitumor
agents that can be conjugated to the antibodies of the invention
include BCNU, streptozoicin, vincristine and 5-fluorouracil, the
family of agents known collectively LL-E33288 complex described in
U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S.
Pat. No. 5,877,296). Enzymatically active toxins and fragments
thereof which can be used in the conjugates include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes.
[0180] For selective destruction of the cell, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc.sup.99m or I.sup.123, or a
spin label for nuclear magnetic resonance (NMR) imaging (also known
as magnetic resonance imaging, mri), such as iodine-123,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0181] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et
al., 1978). "Monoclonal Antibodies in Immunoscintigraphy" (Chatal,
1989) describes other methods in detail.
[0182] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., (1987). .sup.14C-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a cleavable linker facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., 1992; U.S. Pat. No. 5,208,020) may be
used.
[0183] Additionally the methods of the invention may involve the
administration of a glycolytic inhibitor and or a halogenated alkyl
ester. The glycolytic inhibitor and or a halogenated alkyl ester
function as CLIP activity inhibitors that displace CLIP from the
MHC on the cell surface. Preferred glycolytic inhibitors are
2-deoxyglucose compounds, defined herein as 2-deoxy-D-glucose, and
homologs, analogs, and/or derivatives of 2-deoxy-D-glucose. While
the levo form is not prevalent, and 2-deoxy-D-glucose is preferred,
the term "2-deoxyglucose" is intended to cover inter alia either
2-deoxy-D-glucose and 2-deoxy-L-glucose, or a mixture thereof.
[0184] Examples of 2-deoxyglucose compounds useful in the invention
are: 2-deoxy-D-glucose, 2-deoxy-L-glucose; 2-bromo-D-glucose,
2-fluoro-D-glucose, 2-iodo-D-glucose, 6-fluoro-D-glucose,
6-thio-D-glucose, 7-glucosyl fluoride, 3-fluoro-D-glucose,
4-fluoro-D-glucose, 1-O-propyl ester of 2-deoxy-D-glucose,
1-O-tridecyl ester of 2-deoxy-D-glucose, 1-O-pentadecyl ester of
2-deoxy-D-glucose, 3-O-propyl ester of 2-deoxy-D-glucose,
3-O-tridecyl ester of 2-deoxy-D-glucose, 3-O-pentadecyl ester of
2-deoxy-D-glucose, 4-O-propyl ester of 2-deoxy-D-glucose,
4-O-tridecyl ester of 2-deoxy-D-glucose, 4-O-pentadecyl ester of
2-deoxy-D-glucose, 6-O-propyl ester of 2-deoxy-D-glucose,
6-O-tridecyl ester of 2-deoxy-D-glucose, 6-O-pentadecyl ester of
2-deoxy-D-glucose, and 5-thio-D-glucose, and mixtures thereof.
[0185] Glycolytic inhibitors particularly useful herein can have
the formula:
##STR00005##
wherein X represents an O or S atom; R.sub.1 represents a hydrogen
atom or a halogen atom; R.sub.2 represents a hydroxyl group, a
halogen atom, a thiol group, or CO--R.sub.6; and R.sub.3, R.sub.4,
and R.sub.5 each represent a hydroxyl group, a halogen atom, or
CO--R.sub.6 wherein R.sub.6 represents an alkyl group of from 1 to
20 carbon atoms, and wherein at least two of R.sub.3, R.sub.4, and
R.sub.5 are hydroxyl groups. The halogen atom is preferably F, and
R.sub.6 is preferably a C.sub.3-C.sub.15 alkyl group. A preferred
glycolytic inhibitor is 2-deoxy-D-glucose. Such glycolytic
inhibitors are described in detail in application U.S. Ser. No.
10/866,541, filed Jun. 11, 2004, by M. K. Newell et al., the
disclosure of which is incorporated herein by reference.
[0186] In some embodiments of the invention, one can remove CLIP by
administering as a pharmacon a combination of a glycolytic
inhibitor and a halogenated alkyl ester. The combination is
preferably combined as a single bifunctional compound acting as a
prodrug, which is hydrolyzed by one or more physiologically
available eterases. Because of the overall availability of the
various esterases in physiological conditions, one can form an
ester by combining the glycolytic inhibitor and the halogenated
alkyl ester. The prodrug will be hydrolyzed by a physiologically
available esterase into its two functional form.
[0187] In other particular embodiments, the halogenated alkyl ester
has the formula: R.sup.7.sub.mCH.sub.1-mX.sub.2R.sup.8.sub.nCOOY
where R.sup.7 is methyl, ethyl, propyl or butyl, m and n are each
is 0 or 1, R.sup.8 is CH or CHCH, X is a halogen, for example
independently selected from chlorine, bromine, iodine and fluorine.
When used as a separate compound, Y is an alkali metal or alkaline
earth metal ion such as sodium, potassium, calcium, and magnesium,
ammonium, and substituted ammonium where the substituent is a mono-
or di-lower alkyl radical of 1-4 carbon atoms and ethylene
diammonium. When used combined with the glycolytic inhibitor as a
prodrug, Y is esterified with the glycolytic inhibitor as described
in the Methods and Materials section below.
[0188] Preferred prodrugs are those prepared by esterification of
dichloroacetic acid, exemplified by the following structures:
##STR00006##
[0189] In certain embodiments, the method for treating a subject
involves administering to the subject an effective amount of a
nucleic acid molecule to treat the disorder. In certain of these
embodiments, the method for treatment involves administering to the
subject an effective amount of a small interfering nucleic acid
molecule such as antisense, RNAi, or siRNA oligonucleotide to
reduce the level of CLIP molecule, HLA-DO, or HLA-DM expression.
The nucleotide sequences of CLIP molecules, HLA-DO, and HLA-DM are
all well known in the art and can be used by one of skill in the
art using art recognized techniques in combination with the
guidance set forth below to produce the appropriate siRNA
molecules. Such methods are described in more detail below.
[0190] The invention features the use of small nucleic acid
molecules, referred to as small interfering nucleic acid (siNA)
that include, for example: microRNA (miRNA), small interfering RNA
(siRNA), double-stranded RNA (dsRNA), and short hairpin RNA (shRNA)
molecules. An siNA of the invention can be unmodified or
chemically-modified. An siNA of the instant invention can be
chemically synthesized, expressed from a vector or enzymatically
synthesized as discussed herein. The instant invention also
features various chemically-modified synthetic small interfering
nucleic acid (siNA) molecules capable of modulating gene expression
or activity in cells by RNA interference (RNAi). The use of
chemically-modified siNA improves various properties of native siNA
molecules through, for example, increased resistance to nuclease
degradation in vivo and/or through improved cellular uptake.
Furthermore, siNA having multiple chemical modifications may retain
its RNAi activity. The siNA molecules of the instant invention
provide useful reagents and methods for a variety of therapeutic
applications.
[0191] Chemically synthesizing nucleic acid molecules with
modifications (base, sugar and/or phosphate) that prevent their
degradation by serum ribonucleases can increase their potency (see
e.g., Eckstein et al., International Publication No. WO 92/07065;
Perrault et al, 1990; Pieken et al., 1991; Usman and Cedergren,
1992; Usman et al., International Publication No. WO 93/15187; and
Rossi et al., International Publication No. WO 91/03162; Sproat,
U.S. Pat. No. 5,334,711; and Burgin et al., supra; all of these
describe various chemical modifications that can be made to the
base, phosphate and/or sugar moieties of the nucleic acid molecules
herein). Modifications which enhance their efficacy in cells, and
removal of bases from nucleic acid molecules to shorten
oligonucleotide synthesis times and reduce chemical requirements
are desired. (All these publications are hereby incorporated by
reference herein).
[0192] There are several examples in the art describing sugar, base
and phosphate modifications that can be introduced into nucleic
acid molecules with significant enhancement in their nuclease
stability and efficacy. For example, oligonucleotides are modified
to enhance stability and/or enhance biological activity by
modification with nuclease resistant groups, for example, 2'amino,
2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H, nucleotide base
modifications (for a review see Usman and Cedergren, 1992; Usman et
al., 1994; Burgin et al., 1996). Sugar modification of nucleic acid
molecules have been extensively described in the art (see Eckstein
et al., International Publication PCT No. WO 92/07065; Perrault et
al, 1990; Pieken et al., 1991; Usman and Cedergren, 1992; Usman et
al. International Publication PCT No. WO 93/15187; Sproat, U.S.
Pat. No. 5,334,711; Beigelman et al., 1995; Beigelman et al.,
International PCT publication No. WO 97/26270; Beigelman et al.,
U.S. Pat. No. 5,716,824; Usman et al., 1994, molecule comprises one
or more chemical modifications.
[0193] In one embodiment, one of the strands of the double-stranded
siNA molecule comprises a nucleotide sequence that is complementary
to a nucleotide sequence of a target RNA or a portion thereof, and
the second strand of the double-stranded siNA molecule comprises a
nucleotide sequence identical to the nucleotide sequence or a
portion thereof of the targeted RNA. In another embodiment, one of
the strands of the double-stranded siNA molecule comprises a
nucleotide sequence that is substantially complementary to a
nucleotide sequence of a target RNA or a portion thereof, and the
second strand of the double-stranded siNA molecule comprises a
nucleotide sequence substantially similar to the nucleotide
sequence or a portion thereof of the target RNA. In another
embodiment, each strand of the siNA molecule comprises about 19 to
about 23 nucleotides, and each strand comprises at least about 19
nucleotides that are complementary to the nucleotides of the other
strand.
[0194] In some embodiments an siNA is an shRNA, shRNA-mir, or
microRNA molecule encoded by and expressed from a genomically
integrated transgene or a plasmid-based expression vector. Thus, in
some embodiments a molecule capable of inhibiting mRNA expression,
or microRNA activity, is a transgene or plasmid-based expression
vector that encodes a small-interfering nucleic acid. Such
transgenes and expression vectors can employ either polymerase II
or polymerase III promoters to drive expression of these shRNAs and
result in functional siRNAs in cells. The former polymerase permits
the use of classic protein expression strategies, including
inducible and tissue-specific expression systems. In some
embodiments, transgenes and expression vectors are controlled by
tissue specific promoters. In other embodiments transgenes and
expression vectors are controlled by inducible promoters, such as
tetracycline inducible expression systems.
[0195] In some embodiments, a small interfering nucleic acid of the
invention is expressed in mammalian cells using a mammalian
expression vector. The recombinant mammalian expression vector may
be capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid). Tissue
specific regulatory elements are known in the art. Non-limiting
examples of suitable tissue-specific promoters include the myosin
heavy chain promoter, albumin promoter, lymphoid-specific
promoters, neuron specific promoters, pancreas specific promoters,
and mammary gland specific promoters. Developmentally-regulated
promoters are also encompassed, for example the murine hox
promoters and the a-fetoprotein promoter.
[0196] As used herein, a "vector" may be any of a number of nucleic
acid molecules into which a desired sequence may be inserted by
restriction and ligation for transport between different genetic
environments or for expression in a hosT-cell. Vectors are
typically composed of DNA although RNA vectors are also available.
Vectors include, but are not limited to, plasmids, phagemids and
virus genomes. An expression vector is one into which a desired DNA
sequence may be inserted by restriction and ligation such that it
is operably joined to regulatory sequences and may be expressed as
an RNA transcript. In some embodiments, a virus vector for
delivering a nucleic acid molecule is selected from the group
consisting of adenoviruses, adeno-associated viruses, poxviruses
including vaccinia viruses and attenuated poxviruses, Semliki
Forest virus, Venezuelan equine encephalitis virus, retroviruses,
Sindbis virus, and Ty virus-like particle. Examples of viruses and
virus-like particles which have been used to deliver exogenous
nucleic acids include: replication-defective adenoviruses (e.g.,
Xiang et al., 1996; Eloit et al., 1997; Chengalvala et al., 1997),
a modified retrovirus (Townsend et al., 1997), a nonreplicating
retrovirus (Irwin et al., 1994), a replication defective Semliki
Forest virus (Zhao et al., 1995), canarypox virus and highly
attenuated vaccinia virus derivative (Paoletti, 1996),
non-replicative vaccinia virus (Moss, 1996), replicative vaccinia
virus (Moss, 1994), Venzuelan equine encephalitis virus (Davis et
al., 1996), Sindbis virus (Pugachev et al., 1995), and Ty
virus-like particle (Allsopp et al., 1996). In preferred
embodiments, the virus vector is an adenovirus.
[0197] Another preferred virus for certain applications is the
adeno-associated virus, a double-stranded DNA virus. The
adeno-associated virus is capable of infecting a wide range of cell
types and species and can be engineered to be
replication-deficient. It further has advantages, such as heat and
lipid solvent stability, high transduction frequencies in cells of
diverse lineages, including hematopoietic cells, and lack of
superinfection inhibition thus allowing multiple series of
transductions. The adeno-associated virus can integrate into human
cellular DNA in a site-specific manner, thereby minimizing the
possibility of insertional mutagenesis and variability of inserted
gene expression. In addition, wild-type adeno-associated virus
infections have been followed in tissue culture for greater than
100 passages in the absence of selective pressure, implying that
the adeno-associated virus genomic integration is a relatively
stable event. The adeno-associated virus can also function in an
extrachromosomal fashion.
[0198] In general, other preferred viral vectors are based on
non-cytopathic eukaryotic viruses in which non-essential genes have
been replaced with the gene of interest. Non-cytopathic viruses
include retroviruses, the life cycle of which involves reverse
transcription of genomic viral RNA into DNA with subsequent
proviral integration into hosT-cellular DNA. Adenoviruses and
retroviruses have been approved for human gene therapy trials. In
general, the retroviruses are replication-deficient (i.e., capable
of directing synthesis of the desired proteins, but incapable of
manufacturing an infectious particle). Such genetically altered
retroviral expression vectors have general utility for the
high-efficiency transduction of genes in vivo. Standard protocols
for producing replication-deficient retroviruses (including the
steps of incorporation of exogenous genetic material into a
plasmid, transfection of a packaging cell lined with plasmid,
production of recombinant retroviruses by the packaging cell line,
collection of viral particles from tissue culture media, and
infection of the targeT-cells with viral particles) are provided in
Kriegler (1990) and Murry (1991).
[0199] Various techniques may be employed for introducing nucleic
acid molecules of the invention into cells, depending on whether
the nucleic acid molecules are introduced in vitro or in vivo in a
host. Such techniques include transfection of nucleic acid
molecule-calcium phosphate precipitates, transfection of nucleic
acid molecules associated with DEAE, transfection or infection with
the foregoing viruses including the nucleic acid molecule of
interest, liposome-mediated transfection, and the like. For certain
uses, it is preferred to target the nucleic acid molecule (e.g., an
small interfering nucleic acid molecule) to particular cells. In
such instances, a vehicle used for delivering a nucleic acid
molecule of the invention into a cell (e.g., a retrovirus, or other
virus; a liposome) can have a targeting molecule attached thereto.
For example, a molecule such as an antibody specific for a surface
membrane protein on the targeT-cell or a ligand for a receptor on
the targeT-cell can be bound to or incorporated within the nucleic
acid molecule delivery vehicle. Especially preferred are monoclonal
antibodies. Where liposomes are employed to deliver the nucleic
acid molecules of the invention, proteins that bind to a surface
membrane protein associated with endocytosis may be incorporated
into the liposome formulation for targeting and/or to facilitate
uptake. Such proteins include capsid proteins or fragments thereof
tropic for a particular cell type, antibodies for proteins which
undergo internalization in cycling, proteins that target
intracellular localization and enhance intracellular half life, and
the like. Polymeric delivery systems also have been used
successfully to deliver nucleic acid molecules into cells, as is
known by those skilled in the art. Such systems even permit oral
delivery of nucleic acid molecules.
[0200] In addition to delivery through the use of vectors, nucleic
acids of the invention may be delivered to cells without vectors,
e.g., as "naked" nucleic acid delivery using methods known to those
of skill in the art.
[0201] Other inhibitor molecules that can be used include
ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs), triple
helix forming oligonucleotides, antibodies, and aptamers and
modified form(s) thereof directed to sequences in gene(s), RNA
transcripts, or proteins. Antisense and ribozyme suppression
strategies have led to the reversal of a tumor phenotype by
reducing expression of a gene product or by cleaving a mutant
transcript at the site of the mutation (Carter and Lemoine, 1993;
Lange et al., 1993; Valera et al., 1994; Dosaka-Akita et al., 1994;
Feng et al., 1995; Quattrone et al., 1995; Lewin et al., 1998). For
example, neoplastic reversion was obtained using a ribozyme
targeted to an H-Ras mutation in bladder carcinoma cells (Feng et
al., 1995). Ribozymes have also been proposed as a means of both
inhibiting gene expression of a mutant gene and of correcting the
mutant by targeted trans-splicing (Sullenger and Cech, 1994; Jones
et al., 1996). Ribozyme activity may be augmented by the use of,
for example, non-specific nucleic acid binding proteins or
facilitator oligonucleotides (Herschlag et al., 1994; Jankowsky and
Schwenzer, 1996). Multitarget ribozymes (connected or shotgun) have
been suggested as a means of improving efficiency of ribozymes for
gene suppression (Ohkawa et al., 1993).
[0202] Triple helix approaches have also been investigated for
sequence-specific gene suppression. Triple helix forming
oligonucleotides have been found in some cases to bind in a
sequence-specific manner (Postel et al., 1991; Duval-Valentin et
al., 1992; Hardenbol and Van Dyke, 1996; Porumb et al., 1996).
Similarly, peptide nucleic acids have been shown to inhibit gene
expression (Hanvey et al., 1991; Knudsen and Nielson, 1996; Taylor
et al., 1997). Minor-groove binding polyamides can bind in a
sequence-specific manner to DNA targets and hence may represent
useful small molecules for future suppression at the DNA level
(Trauger et al., 1996). In addition, suppression has been obtained
by interference at the protein level using dominant negative mutant
peptides and antibodies (Herskowitz, 1987; Rimsky et al., 1989;
Wright et al., 1989). In some cases suppression strategies have led
to a reduction in RNA levels without a concomitant reduction in
proteins, whereas in others, reductions in RNA have been mirrored
by reductions in protein.
[0203] The diverse array of suppression strategies that can be
employed includes the use of DNA and/or RNA aptamers that can be
selected to target, for example CLIP or HLA-DO. Suppression and
replacement using aptamers for suppression in conjunction with a
modified replacement gene and encoded protein that is refractory or
partially refractory to aptamer-based suppression could be used in
the invention.
[0204] The active agents of the invention are administered to the
subject in an effective amount for treating disorders such as
autoimmune disease, cancer, HIV infection, other infections, and
graft rejection. An "effective amount", for instance, is an amount
necessary or sufficient to realize a desired biologic effect. An
"effective amount for treating cancer," for instance, is an
effective amount of a compound of the invention could be that
amount necessary to (i) kill a cancer cell; (ii) inhibit the
further growth of the cancer, i.e., arresting or slowing its
development; and/or (iii) sensitize a cancer cell to an anti-cancer
agent or therapeutic. According to some aspects of the invention,
an effective amount is that amount of a compound of the invention
alone or in combination with another medicament, which when
combined or co-administered or administered alone, results in a
therapeutic response to the disease, either in the prevention or
the treatment of the disease. The biological effect may be the
amelioration and or absolute elimination of symptoms resulting from
the disease. In another embodiment, the biological effect is the
complete abrogation of the disease, as evidenced for example, by
the absence of a symptom of the disease. or a tumor or a biopsy or
blood smear which is free of cancer cells.
[0205] The effective amount of a compound of the invention in the
treatment of a disease described herein may vary depending upon the
specific compound used, the mode of delivery of the compound, and
whether it is used alone or in combination. The effective amount
for any particular application can also vary depending on such
factors as the disease being treated, the particular compound being
administered, the size of the subject, or the severity of the
disease or condition. One of ordinary skill in the art can
empirically determine the effective amount of a particular molecule
of the invention without necessitating undue experimentation.
Combined with the teachings provided herein, by choosing among the
various active compounds and weighing factors such as potency,
relative bioavailability, patient body weight, severity of adverse
side-effects and preferred mode of administration, an effective
prophylactic or therapeutic treatment regimen can be planned which
does not cause substantial toxicity and yet is entirely effective
to treat the particular subject.
[0206] Pharmaceutical compositions of the present invention
comprise an effective amount of one or agent, dissolved or
dispersed in a pharmaceutically acceptable carrier. The phrases
"pharmaceutical or pharmacologically acceptable" refers to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
such as, for example, a human, as appropriate. Moreover, for animal
(e.g., human) administration, it will be understood that
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biological
Standards.
[0207] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art (see, for example, Remington's Pharmaceutical Sciences
(1990), incorporated herein by reference). Except insofar as any
conventional carrier is incompatible with the active ingredient,
its use in the therapeutic or pharmaceutical compositions is
contemplated.
[0208] The agent may comprise different types of carriers depending
on whether it is to be administered in solid, liquid or aerosol
form, and whether it need to be sterile for such routes of
administration as injection. The present invention can be
administered intravenously, intradermally, intraarterially,
intralesionally, intratumorally, intracranially, intraarticularly,
intraprostaticaly, intrapleurally, intratracheally, intranasally,
intravitreally, intravaginally, intrarectally, topically,
intratumorally, intramuscularly, intraperitoneally, subcutaneously,
subconjunctival, intravesicularlly, mucosally, intrapericardially,
intraumbilically, intraocularally, orally, topically, locally,
inhalation (e.g., aerosol inhalation), injection, infusion,
continuous infusion, localized perfusion bathing targeT-cells
directly, via a catheter, via a lavage, in cremes, in lipid
compositions (e.g., liposomes), or by other method or any
combination of the forgoing as would be known to one of ordinary
skill in the art (see, for example, Remington's Pharmaceutical
Sciences (1990), incorporated herein by reference). In a particular
embodiment, intraperitoneal injection is contemplated.
[0209] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, the an active compound may comprise between
about 2% to about 75% of the weight of the unit, or between about
25% to about 60%, for example, and any range derivable therein. In
other non-limiting examples, a dose may also comprise from about 1
microgram/kg/body weight, about 5 microgram/kg/body weight, about
10 microgram/kg/body weight, about 50 microgram/kg/body weight,
about 100 microgram/kg/body weight, about 200 microgram/kg/body
weight, about 350 microgram/kg/body weight, about 500
microgram/kg/body weight, about 1 milligram/kg/body weight, about 5
milligram/kg/body weight, about 10 milligram/kg/body weight, about
50 milligram/kg/body weight, about 100 milligram/kg/body weight,
about 200 milligram/kg/body weight, about 350 milligram/kg/body
weight, about 500 milligram/kg/body weight, to about 1000
mg/kg/body weight or more per administration, and any range
derivable therein. In non-limiting examples of a derivable range
from the numbers listed herein, a range of about 5 mg/kg/body
weight to about 100 mg/kg/body weight, about 5 microgram/kg/body
weight to about 500 milligram/kg/body weight, etc., can be
administered, based on the numbers described above.
[0210] In any case, the composition may comprise various
antioxidants to retard oxidation of one or more component.
Additionally, the prevention of the action of microorganisms can be
brought about by preservatives such as various antibacterial and
antifungal agents, including but not limited to parabens (e.g.,
methylparabens, propylparabens), chlorobutanol, phenol, sorbic
acid, thimerosal or combinations thereof.
[0211] The agent may be formulated into a composition in a free
base, neutral or salt form. Pharmaceutically acceptable salts,
include the acid addition salts, e.g., those formed with the free
amino groups of a proteinaceous composition, or which are formed
with inorganic acids such as for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic, tartaric
or mandelic acid. Salts formed with the free carboxyl groups also
can be derived from inorganic bases such as for example, sodium,
potassium, ammonium, calcium or ferric hydroxides; or such organic
bases as isopropylamine, trimethylamine, histidine or procaine.
[0212] In embodiments where the composition is in a liquid form, a
carrier can be a solvent or dispersion medium comprising but not
limited to, water, ethanol, polyol (e.g., glycerol, propylene
glycol, liquid polyethylene glycol, etc.), lipids (e.g.,
triglycerides, vegetable oils, liposomes) and combinations 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 by dispersion in carriers such as, for example liquid
polyol or lipids; by the use of surfactants such as, for example
hydroxypropylcellulose; or combinations thereof such methods. In
many cases, it will be preferable to include isotonic agents, such
as, for example, sugars, sodium chloride or combinations
thereof.
[0213] Subject doses of the compounds described herein typically
range from about 0.1 .mu.g to 10,000 mg, more typically from about
1 .mu.g/day to 8000 mg, and most typically from about 10 .mu.g to
100 .mu.g. Stated in terms of subject body weight, typical dosages
range from about 0.1 .mu.g to 20 mg/kg/day, more typically from
about 1 to 10 mg/kg/day, and most typically from about 1 to 5
mg/kg/day. The absolute amount will depend upon a variety of
factors including the concurrent treatment, the number of doses and
the individual patient parameters including age, physical
condition, size and weight. These are factors well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation. It is preferred generally that a maximum
dose be used, that is, the highest safe dose according to sound
medical judgment.
[0214] Multiple doses of the molecules of the invention are also
contemplated. In some instances, when the molecules of the
invention are administered with a cancer medicament a
sub-therapeutic dosage of either the molecules or the cancer
medicament, or a sub-therapeutic dosage of both, is used in the
treatment of a subject having, or at risk of developing, cancer.
When the two classes of drugs are used together, the cancer
medicament may be administered in a sub-therapeutic dose to produce
a desirable therapeutic result. A "sub-therapeutic dose" as used
herein refers to a dosage which is less than that dosage which
would produce a therapeutic result in the subject if administered
in the absence of the other agent. Thus, the sub-therapeutic dose
of a cancer medicament is one which would not produce the desired
therapeutic result in the subject in the absence of the
administration of the molecules of the invention. Therapeutic doses
of cancer medicaments are well known in the field of medicine for
the treatment of cancer. These dosages have been extensively
described in references such as Remington's Pharmaceutical
Sciences, 18.sup.th Ed., 1990; as well as many other medical
references relied upon by the medical profession as guidance for
the treatment of cancer. Therapeutic dosages of antibodies have
also been described in the art.
[0215] A variety of administration routes are available. The
particular mode selected will depend, of course, upon the
particular active agents selected, the particular condition being
treated and the dosage required for therapeutic efficacy. The
methods of this invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of protection
without causing clinically unacceptable adverse effects. Preferred
modes of administration are parenteral routes. The term
"parenteral" includes subcutaneous, intravenous, intramuscular,
intraperitoneal, and intrasternal injection, or infusion
techniques. Other routes include but are not limited to oral,
nasal, dermal, sublingual, and local.
[0216] The formulations of the invention are administered in
pharmaceutically acceptable solutions, which may routinely contain
pharmaceutically acceptable concentrations of salt, buffering
agents, preservatives, compatible carriers, adjuvants, and
optionally other therapeutic ingredients.
[0217] The compounds of the invention can be administered by any
ordinary route for administering medications. Depending upon the
type of cancer to be treated, compounds of the invention may be
inhaled, ingested or administered by systemic routes. Systemic
routes include oral and parenteral. Inhaled medications are
preferred in some embodiments because of the direct delivery to the
lung, particularly in lung cancer patients. Several types of
metered dose inhalers are regularly used for administration by
inhalation. These types of devices include metered dose inhalers
(MDI), breath-actuated MDI, dry powder inhaler (DPI),
spacer/holding chambers in combination with MDI, and nebulizers.
Preferred routes of administration include but are not limited to
oral, parenteral, intramuscular, intranasal, intratracheal,
intrathecal, intravenous, inhalation, ocular, vaginal, and rectal.
For use in therapy, an effective amount of the compounds of the
invention can be administered to a subject by any mode that
delivers the nucleic acid to the affected organ or tissue.
"Administering" the pharmaceutical composition of the present
invention may be accomplished by any means known to the skilled
artisan.
[0218] According to the methods of the invention, the compound may
be administered in a pharmaceutical composition. In general, a
pharmaceutical composition comprises the compound of the invention
and a pharmaceutically-acceptable carrier.
Pharmaceutically-acceptable carriers for peptides, monoclonal
antibodies, and antibody fragments are well-known to those of
ordinary skill in the art. As used herein, a
pharmaceutically-acceptable carrier means a non-toxic material that
does not interfere with the effectiveness of the biological
activity of the active ingredients, e.g., the ability of the
peptide to bind to CLIP and HLA.
[0219] Pharmaceutically acceptable carriers include diluents,
fillers, salts, buffers, stabilizers, solubilizers and other
materials which are well-known in the art. Exemplary
pharmaceutically acceptable carriers for peptides in particular are
described in U.S. Pat. No. 5,211,657. Such preparations may
routinely contain salt, buffering agents, preservatives, compatible
carriers, and optionally other therapeutic agents. When used in
medicine, the salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically-acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically
and pharmaceutically-acceptable salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,
salicylic, citric, formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts.
[0220] The compounds of the invention may be formulated into
preparations in solid, semi-solid, liquid or gaseous forms such as
tablets, capsules, powders, granules, ointments, solutions,
depositories, inhalants and injections, and usual ways for oral,
parenteral or surgical administration. The invention also embraces
pharmaceutical compositions which are formulated for local
administration, such as by implants.
[0221] Compositions suitable for oral administration may be
presented as discrete units, such as capsules, tablets, lozenges,
each containing a predetermined amount of the active agent. Other
compositions include suspensions in aqueous liquids or non-aqueous
liquids such as a syrup, elixir or an emulsion.
[0222] When the compounds described herein (including peptide and
non-peptide varieties) are used therapeutically, in certain
embodiments a desirable route of administration may be by pulmonary
aerosol. Techniques for preparing aerosol delivery systems
containing compounds are well known to those of skill in the art.
Generally, such systems should utilize components which will not
significantly impair the biological properties of the peptides
(see, for example, Sciarra and Cutie, "Aerosols," in Remington's
Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712;
incorporated by reference). Those of skill in the art can readily
determine the various parameters and conditions for producing
aerosols without resort to undue experimentation.
[0223] The compounds of the invention may be administered directly
to a tissue. Preferably, the tissue is one in which the CLIP
expressing cells are found. Direct tissue administration may be
achieved by direct injection. The compounds may be administered
once, or alternatively they may be administered in a plurality of
administrations. If administered multiple times, the compounds may
be administered via different routes. For example, the first (or
the first few) administrations may be made directly into the
affected tissue while later administrations may be systemic.
[0224] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a subject to be treated.
Pharmaceutical preparations for oral use can be obtained as solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Optionally the oral formulations may also be formulated
in saline or buffers for neutralizing internal acid conditions or
may be administered without any carriers.
[0225] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0226] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. Microspheres formulated for oral
administration may also be used. Such microspheres have been well
defined in the art. All formulations for oral administration should
be in dosages suitable for such administration.
[0227] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0228] For administration by inhalation, the compounds for use
according to the present invention may be conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch. Techniques for
preparing aerosol delivery systems are well known to those of skill
in the art. Generally, such systems should utilize components which
will not significantly impair the biological properties of the
active agent (see, for example, Sciarra and Cutie, "Aerosols," in
Remington's Pharmaceutical Sciences, 18th edition, 1990, pp
1694-1712; incorporated by reference). Those of skill in the art
can readily determine the various parameters and conditions for
producing aerosols without resort to undue experimentation.
[0229] The compounds, when it is desirable to deliver them
systemically, may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0230] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like. Lower doses will result from other forms of
administration, such as intravenous administration. In the event
that a response in a subject is insufficient at the initial doses
applied, higher doses (or effectively higher doses by a different,
more localized delivery route) may be employed to the extent that
patient tolerance permits. Multiple doses per day are contemplated
to achieve appropriate systemic levels of compounds.
[0231] In yet other embodiments, the preferred vehicle is a
biocompatible microparticle or implant that is suitable for
implantation into the mammalian recipient. Exemplary bioerodible
implants that are useful in accordance with this method are
described in PCT International Application No. PCT/US/03307
(Publication No. WO 95/24929, entitled "Polymeric Gene Delivery
System"). PCT/US/0307 describes a biocompatible, preferably
biodegradable polymeric matrix for containing a biological
macromolecule. The polymeric matrix may be used to achieve
sustained release of the agent in a subject. In accordance with one
aspect of the instant invention, the agent described herein may be
encapsulated or dispersed within the biocompatible, preferably
biodegradable polymeric matrix disclosed in PCT/US/03307. The
polymeric matrix preferably is in the form of a microparticle such
as a microsphere (wherein the agent is dispersed throughout a solid
polymeric matrix) or a microcapsule (wherein the agent is stored in
the core of a polymeric shell). Other forms of the polymeric matrix
for containing the agent include films, coatings, gels, implants,
and stents. The size and composition of the polymeric matrix device
is selected to result in favorable release kinetics in the tissue
into which the matrix device is implanted. The size of the
polymeric matrix device further is selected according to the method
of delivery which is to be used, typically injection into a tissue
or administration of a suspension by aerosol into the nasal and/or
pulmonary areas. The polymeric matrix composition can be selected
to have both favorable degradation rates and also to be formed of a
material which is bioadhesive, to further increase the
effectiveness of transfer when the device is administered to a
vascular, pulmonary, or other surface. The matrix composition also
can be selected not to degrade, but rather, to release by diffusion
over an extended period of time.
[0232] Both non-biodegradable and biodegradable polymeric matrices
can be used to deliver the agents of the invention to the subject.
Biodegradable matrices are preferred. Such polymers may be natural
or synthetic polymers. Synthetic polymers are preferred. The
polymer is selected based on the period of time over which release
is desired, generally in the order of a few hours to a year or
longer. Typically, release over a period ranging from between a few
hours and three to twelve months is most desirable. The polymer
optionally is in the form of a hydrogel that can absorb up to about
90% of its weight in water and further, optionally is cross-linked
with multivalent ions or other polymers.
[0233] In general, the agents of the invention may be delivered
using the bioerodible implant by way of diffusion, or more
preferably, by degradation of the polymeric matrix. Exemplary
synthetic polymers which can be used to form the biodegradable
delivery system include: polyamides, polycarbonates, polyalkylenes,
polyalkylene glycols, polyalkylene oxides, polyalkylene
terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl
esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides,
polysiloxanes, polyurethanes and co-polymers thereof, alkyl
cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, polymers of acrylic and methacrylic
esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,
hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,
cellulose acetate, cellulose propionate, cellulose acetate
butyrate, cellulose acetate phthalate, carboxylethyl cellulose,
cellulose triacetate, cellulose sulphate sodium salt, poly(methyl
methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),
poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl
acetate, poly vinyl chloride, polystyrene and
polyvinylpyrrolidone.
[0234] Examples of non-biodegradable polymers include ethylene
vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and
mixtures thereof.
[0235] Examples of biodegradable polymers include synthetic
polymers such as polymers of lactic acid and glycolic acid,
polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid),
poly(valeric acid), and poly(lactide-cocaprolactone), and natural
polymers such as alginate and other polysaccharides including
dextran and cellulose, collagen, chemical derivatives thereof
(substitutions, additions of chemical groups, for example, alkyl,
alkylene, hydroxylations, oxidations, and other modifications
routinely made by those skilled in the art), albumin and other
hydrophilic proteins, zein and other prolamines and hydrophobic
proteins, copolymers and mixtures thereof. In general, these
materials degrade either by enzymatic hydrolysis or exposure to
water in vivo, by surface or bulk erosion.
[0236] Bioadhesive polymers of particular interest include
bioerodible hydrogels described by Sawhney et al. (1993), the
teachings of which are incorporated herein, polyhyaluronic acids,
casein, gelatin, glutin, polyanhydrides, polyacrylic acid,
alginate, chitosan, poly(methyl methacrylates), poly(ethyl
methacrylates), poly(butylmethacrylate), poly(isobutyl
methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), and poly(octadecyl acrylate).
[0237] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the compound, increasing
convenience to the subject and the physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include polymer base systems such as
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono-di- and tri-glycerides; hydrogel
release systems; silastic systems; peptide based systems; wax
coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which the platelet reducing agent is contained in a form within a
matrix such as those described in U.S. Pat. Nos. 4,452,775,
4,675,189, and 5,736,152 and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.
In addition, pump-based hardware delivery systems can be used, some
of which are adapted for implantation.
[0238] Use of a long-term sustained release implant may be
particularly suitable for treatment of chronic diseases or
recurrent cancer. Long-term release, as used herein, means that the
implant is constructed and arranged to delivery therapeutic levels
of the active ingredient for at least 30 days, and preferably 60
days. Long-term sustained release implants are well-known to those
of ordinary skill in the art and include some of the release
systems described above.
[0239] Therapeutic formulations of the peptides or antibodies may
be prepared for storage by mixing a peptide or antibody having the
desired degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences, 1980), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
EXAMPLES
[0240] The following examples are provided to illustrate specific
instances of the practice of the present invention and are not
intended to limit the scope of the invention. As will be apparent
to one of ordinary skill in the art, the present invention will
find application in a variety of compositions and methods.
Example 1
B-Cell Apoptosis after Coxsackievirus Infection
[0241] During the course of Coxsackievirus infection, animals that
recover from the virus without subsequent autoimmune sequalae have
high percentages of splenic B-cell apoptosis during the infection
in vivo (FIG. 1). Those animals susceptible to
Coxsackievirus-mediated autoimmune disease have non-specifically
activated B-cells that do not undergo apoptosis, at least not
during acute infection, nor during the time period prior to
autoimmune symptoms indicating that a common feature in the
development of autoimmune disease is failure of non-specifically
activated B-cells to die.
Example 2
Activated B-Cells in HIV Disease Mediate NK Cell Activation
[0242] The inventors experimentally induced polyclonal activation
of peripheral blood human B-cells in an antigen-independent fashion
using a combination of CD40 engagement (CD40Ligand bearing
fibroblasts) and culture in recombinant IL-4. They isolated the
activated B-cells and return them to co-culture with autologous
peripheral blood mononuclear cells (PBMCs). After five days of
co-culture, they observed a striking increase in the percentage of
activated NK cells in the PBMC culture (NK cells accounting for up
to 25-50%, FIG. 2A, of the surviving PBMCs), and a dramatic
apoptotic loss of the activated B-cells (FIG. 2B). These data
indicate that antigen-independent activated B-cells in HIV disease
initially activate NK cells.
Example 3
Antigen-Independent B-Cell Activation Affecting NK Cell
Activity
[0243] Elements of HIV infection that provide an
antigen-independent activation signal to B-cells that results in NK
cell activation and polyclonal B-cell activation are examined.
[0244] Antigen-independent activation of B-cells: Human B-cells:
PBMCs are prepared from 5 normal and 5 HIV-infected adult donors
using standard Ficoll-Hypaque density-gradient techniques.
Irradiated (75 Gy) human CD40L-transfected murine fibroblasts
(LTK-CD40L), are plated in six-well plates (BD Bioscience, Franklin
Lakes, N.J.) at a concentration of 0.1.times.10.sup.6 cells/well,
in RPMI complete medium and cultured overnight at 37.degree. C., 5%
CO2. After washing twice with PBS, 2.times.10.sup.6 cells/mL PBMC
are co-cultured with LTK-CD40L cells in the presence of recombinant
human interleukin-4 (rhIL-4; 4 ng/mL; Peprotech, Rocky Hill, N.J.)
or with purified HIV derived gp 120 protein in complete Dulbecco's
medium (Invitrogen), supplemented with 10% human AB serum (Gemini
Bio-Product, Woodland, Calif.) Cultured cells are transferred to
new plates with freshly prepared, irradiated LTK-CD40L cells every
3 to 5 days. Before use, dead cells are removed from the
CD40-B-cells by Ficoll density centrifugation, followed by washing
twice with PBS. The viability of this fraction is expected to be
>99%, and >95% of the cells, using this protocol, have been
shown to be B-cells that are more than 95% pure CD19+ and CD20+
after 2 weeks of culture. This protocol yields a viability of
>99%, and >95% of the cells have been shown to be B-cells
that are more than 95% pure CD19+ and CD20+ after 2 weeks of
culture.
[0245] The activated B-cells are co-cultured with autologous PBMC
at a ratio of 1:10 and cultured for five days. Harvested cells are
stained with fluorochrome-conjugated antibodies (BD Pharmingen) to
CD56, CD3, CD19, CD4, and CD8. Cells are analyzed flow
cytometrically to determine the percentage of NK cells (percent
CD56+, CD3-) resulting from co-culture comparing non-infected to
infected samples. NK cells are counter-stained for NK killing
ligand KIR3DS1, NKG2D, FaL, or PD1. Similarly the percent surviving
large and small C19+ cells are quantitated flow cytometrically.
[0246] B-cell activation in HIV: To determine if activated NK or
CD3 T-cells promote polyclonal B-cell activation, the inventors
perform reciprocal co-culture experiments in which the inventors
purposely activate NKs or CD3+ T-cells and co-culture 1:10 in PBMC
from the autologous donors. PBMCs are prepared from HIV infected or
uninfected adult donors using standard Ficoll-Hypaque
density-gradient techniques. To activate NKs and CD3+ T-cells,
PBMCs are cultured in RPMI with 10% FCS, 1 mM penicillin, 1 mM
Glutamax, and 1% W/V glucose at 2.0-4.0.times.10.sup.6/mL for 3
days with 1:40,000 OKT3, 100 U/mL IL-2, or no stimulation
(resting). After 3 days stimulation, non-adherent PBMCs are gently
harvested and immune cell subsets are purified by MACS technology
according to manufacturers protocol (Miltenyi Biotec, Auburn
Calif.). In brief, NK cells are first selected using the
CD56+multisort kit, followed by bead release, and depletion with
anti-CD3 beads. T-cells are obtained by depleting non-adherent
PBMCs with CD56 beads with or without anti-CD4 or anti-CD8 beads
for isolation of each individual subset. Purity of cell fractions
are confirmed for each experiment by flow cytometry using CD56,
CD3, CD4, CD8 and CD14 antibodies. Following culture for 5 days,
the inventors use flow cytometry to determine relative changes in
CD19+, CD4, CD8, NK, CD3, and CD69 as a marker for activation.
[0247] The inventors examine the NK cells from the co-culture
experiments for KIR3DS1 and other killer cell ligands including
NKG2D ligand, PD1, and FasL that are indicative of killer cell
functions.
[0248] Antigen-independent activation of mouse B-cells: Mouse
spleens are removed from C57B16 mice, red cells are removed using
buffered ammonium chloride, T-cells are depleted with an
anti-T-cell antibody cocktail (HO13, GK1.5 and 30H12) and
complement. T depleted splenocytes are washed and fractionated
using Percoll density gradient centrifugation. The inventors
isolate the B-cells at the 1.079/1.085 g/ml density interface
(resting B-cells) and wash to remove residual Percoll. The cells
are cultured in the presence of LPS or
tri-palmitoyl-S-glyceryl-cystinyl N-terminus (Pam(3)Cys), agonists
of TLR2, on B-cells. The activated B-cells are co-cultured with
total spleen cells at a ratio of 1:10 B-cell:total spleen cells.
After five days in culture, the remaining cells are analyzed for
expansion of cell subsets including those expressing mouse CD56,
CD3, B220, CD4 and CD8. These cell surface molecules are analyzed
flow cytometrically. CD56+CD3- cells are counterstained for NKG2D
and other death-inducing receptors.
Example 4
NK Cell Killing of Activated CD4+ T-Cells
[0249] The ability of NK cells to lyse activated CD4 T-cells as
targets as a result of NK cell activation and changes in the CD4
T-cell target is examined.
[0250] Activation of Human NK and CD3+ T-cells: PBMCs are prepared
from HIV infected or uninfected adult donors using standard
Ficoll-Hypaque density-gradient techniques. NKs and CD3+ T-cells
are activated and isolated as disclosed herein. T-cells and NK
cells are routinely between 80-95% pure with less than 1% monocyte
contamination. T-cell activation in OKT3-stimulated PBMCs is
confirmed by assays using .sup.3H-thymidine incorporation. NK cell
activation is confirmed by increase in size and granularity by flow
cytometry, by staining for CD56+ and CD3- fow cytometrically, and
by lytic activity as measured by chromium release of
well-established NK targets. The inventors load well-established NK
cell targets or the non-specifically activated B-cells as disclosed
herein with 51Chromium. The inventors use chromium release as a
measurement of targeT-cell death.
[0251] Activation of mouse NK and CD3+ T-cells: The inventors
isolate splenocytes as disclosed herein. The red blood
cell-depleted spleen cells are cultured in recombinant mouse IL-2
or with 145.2C11 (anti-mouse CD3, Pharmingen) for 3 days. After
stimulation, the cells are harvested and purified using Cell-ect
Isolation kits for either NK, CD4, or CD8+ T-cells. The cells are
then co-cultured with 51-Chromium-labelled, well-established NK
cell targets or with 51-Chromium-labelled non-specifically
activated B-cells as disclosed herein.
Example 5
Chronically Activated HIV Infected (or HIV-Specific CD4 T-Cells) as
the Intercellular Targets of Activated Killer Cells
[0252] Chronically activated CD4+ T-cells become particularly
susceptible to killer cells as a consequence of the chronic immune
stimulation resulting from HIV infection.
[0253] The inventors isolate NK cells from uninfected or
HIV-infected individuals using the CD56+multisort kit as disclosed
herein. The inventors activate the cells in IL-2 as disclosed
herein. The inventors perform co-culture experiments with these
cells added back to PBMC at a 1:10 ratio from autologous donors.
Prior to co-culture, the inventors examine the NK cells from HIV
infected and uninfected donors for deat-inducing receptor: ligand
pairs killer, including KIR3DS1, FasL, and NKG2D ligands that are
indicative of killer cell functions. In parallel, the inventors
stain pre- and post-coculture PBMCs from the autologous donors of
HIV infected or uninfected donors.
Example 6
TNP MIXTURE Displaces CLIP from Model B-Cell Lines
[0254] Kinetics of CLIP displacement from the surface of model
B-cells lines (Daudi and Raji) in response to thymic nuclear
protein mixture was determined.
[0255] Results were expressed in histogram analyses (FIGS. 3A-C).
The Y axis represents cell number of the 5000 live cells versus the
X axis which is a reflection of relative Fitc fluorescence. The
distance between the histogram from the isotype control staining
versus the histogram reflecting the specific stain is a measure of
level of cell surface CLIP on a population of live Raji or Daudi
cells as indicated.
[0256] At three hours, on both cell lines, the inventors see
evidence by diminished ratio of Isotype to CLIP staining, that the
TNP mixtures at 200 .mu.g/ml cause a reduction in detectable cell
surface CLIP.
[0257] At 24 hours, the effect was less, and may have caused an
increase in detectable CLIP. Noticeably at 24 hours, the TNP
mixture caused death of the B-cell lines at the 200 .mu.g/ml
concentrations and by 48 hours all of the cells treated with 200
micrograms were dead and the 50 .mu.g concentrations also resulted
in significant toxicity.
[0258] At 3 hours, treatment with 200 micrograms TNP/ml, there was
2.5 times the number of dead cells as determined by Trypan blue
exclusion. Cell death in the flow cytometric experiments was,
determined by forward versus side scatter changes (decreased
forward scatter, increased side scatter).
[0259] Cell Culture Conditions: The Raji and Daudi cell lines were
purchased from American Type Culture Collection, were thawed, and
grown in RPMI 1640 medium supplemented with standard supplements,
including 10% fetal calf serum, gentamycin, penicillin,
streptomycin, sodium pyruvate, HEPES buffer, 1-glutamine, and
2-ME.
[0260] Protocol: Cells were plated into a 12-well plate with 3 mls
total volume containing approximately 0.5.times.10.sup.6/well for
Daudi cells and 1.0.times.10.sup.6/well for Raji cells. Treatment
groups included no treatment as control; 50 .mu.g/ml TNP mixture;
200 .mu.g/ml TNP mixture; 50 .mu.g of control bovine albumin; or
200 .mu.g/ml bovine albumin as protein controls.
[0261] The cells were incubated at 37.degree. C. in an atmosphere
containing 5% CO.sub.2 and approximately 92% humidity. The cells
were incubated for 3, 24, and 48 hours. At each time point, the
cells from that experimental time were harvested and stained for
flow cytometric analysis of cell surface expression of CLIP (MHC
Class II invariant peptide, human) by using the commercially
available (Becton/Dickinson/PHarmingen) anti-human CLIP FITC
(Catalogue # 555981).
[0262] Harvested cells were stained using standard staining
procedure that called for a 1:100 dilution of Fitc-anti-human CLIP
or isotype control. Following staining on ice for 25 minutes, cells
were washed with PBS/FCS and resuspended in 100 microliters and
added to staining tubes containing 400 microliters of PBS. Samples
were acquired and analyzed on a Coulter Excel Flow Cytometer.
Example 7
MKN1 (bioCLIP) Alters Cell Surface CLIP and CD74 Levels
[0263] The ability of MKN1 (bioCLIP) to alter cell surface CLIP and
CD74 levels was determined using Raji or Daudi cells.
[0264] Data were analyzed by histogram with Y axis represents cell
number of the 5000 live cells versus the X axis which is a
reflection of relative FITC fluorescence with either antibodies to
CLIP or CD74. The distance between the histogram from the isotype
control staining versus the histogram reflecting the specific stain
and is a measure of level of cell surface CLIP or CD74 when
staining a population of live Raji or Daudi cells.
[0265] The results show that treatment with MKN1 (bioCLIP) alters
cell surface CLIP and CD74 levels.
[0266] Cell Culture Conditions: The Raji and Daudi cell lines were
purchased from American Type Culture Collection, were thawed, and
grown in RPMI 1640 medium supplemented with standard supplements,
including 10% fetal calf serum, gentamycin, penicillin,
streptomycin, sodium pyruvate, HEPES buffer, 1-glutamine, and
2-ME.
[0267] Protocol: Cells were plated into a 12-well plate with 3 mls
total volume containing approximately 0.5.times.10.sup.6/mL for
Daudi cells and 0.5.times.10.sup.6/mL for Raji cells. Treatment
groups included no treatment as control; MKN 3 and MKN 5 at 50
.mu.M final concentration based on the reported molarity of the
synthesized compounds.
TABLE-US-00001 Peptide 1: MKN.1 (19 mer) Biotin at N-Terminal =
Biotinylated CLIP (SEQ ID NO. 4) SGG GSK MRM ATP LLM QAL Y 5-10 mg
Obtained @ >95% purity (ELIM Pharmaceuticals)
[0268] The cells were incubated at 37.degree. C. in an atmosphere
containing 5% CO.sub.2 and approximately 92% humidity. The cells
were incubated for 24 and 48 hours. At each time point, the cells
from that experimental time were harvested and stained for flow
cytometric analysis of cell surface expression of CLIP (MHC Class
II invariant peptide, human) by using the commercially available
(Becton/Dickinson/Pharmingen) anti-human CLIP FITC (Catalogue #
555981) versus Streptavidin, and for CD74 using the commercially
available (Becton/Dickinson/Pharmingen) anti-human CC74 FITC
antibody.
[0269] Harvested cells were stained using standard staining
procedure that called for a 1:100 dilution of Fitc-anti-human CLIP
or CD74 antibody (FITC, Pharmingen, Cat # 554647) or isotype
control. Following staining on ice for 25 min, cells were washed
with PBS/FCS and resuspended in 100 microliters and added to
staining tubes containing 400 .mu.l of PBS. Samples were acquired
and analyzed on a Coulter Excel Flow Cytometer.
Example 8
2-Deoxyglucose and Dichloroacetate Cause Removal of B-Cell Surface
CLIP
[0270] The ability of 2-deoxyglucose and dichloroacetate affect
B-cell surface CLIP was determined.
[0271] Results are expressed in histogram analyses (FIG. 4). The Y
axis represents cell number of the 5000 live cells versus the X
axis which is a reflection of relative Fitc fluorescence with
either antibodies to CLIP. The distance between the histogram from
the isotype control staining versus the histogram reflecting the
specific stain and is a measure of level of cell surface CLIP when
staining a population of live Raji or Daudi cells as indicated.
[0272] These results show that treatment equimolar amounts of
2-deoxyglucose and dichloroacetate decrease (remove) cell surface
CLIP from both B-cell lines optimally at 48 hours.
[0273] Cell Culture Conditions: The Raji and Daudi cell lines were
purchased from American Type Culture Collection, were thawed, and
grown in RPMI 1640 medium supplemented with standard supplements,
including 10% fetal calf serum, gentamycin, penicillin,
streptomycin, sodium pyruvate, HEPES buffer, 1-glutamine, and
2-ME.
[0274] Protocol: Cells were plated into a 12-well plate with 3 mls
total volume containing approximately 0.5.times.10.sup.6/ml for
Daudi cells and 0.5.times.10.sup.6/ml for Raji cells. Treatment
groups included no treatment as control; MKN 3 and MKN 5 at 50
.mu.M final concentration based on the reported molarity of the
synthesized compounds.
[0275] The cells were incubated at 37.degree. C. in an atmosphere
containing 5% CO.sub.2 and approximately 92% humidity. The cells
were incubated for 4, 24 and 48 hours with or without
2-deoxyglucose and dichloroacetate at 1 mg/ml of each compound. At
each time point, the cells from that experimental time were
harvested and stained for flow cytometric analysis of cell surface
expression of CLIP (MHC Class II invariant peptide, human) by using
the commercially available (Becton/Dickinson/PHarmingen) anti-human
CLIP FITC (Catalogue #555981).
[0276] Harvested cells were stained using standard staining
procedure that called for a 1:100 dilution of FITC-anti-human CLIP
(FITC, Pharmingen, Cat # 555981) or isotype control. Following
staining on ice for 25 min, cells were washed with PBS/FCS and
resuspended in 100 .mu.l and added to staining tubes containing 400
.mu.l of PBS. Samples were acquired and analyzed on a Coulter Excel
Flow Cytometer.
Example 9
Competing Peptides Induce Cell Surface Expression of CD1d
[0277] The ability of synthetic peptides to compete with binding of
CLIP peptides and result in the cell surface expression of CD1d was
determined.
[0278] Results: The results shown in FIG. 5 are expressed in
histogram analyses. The Y axis represents cell number of the 5000
live cells versus the X axis which is a reflection of relative Fitc
fluorescence versus Streptavidin-PE (eBioscience, Cat. #12-4317)
that will bind with high affinity to cell-bound biotinylated
peptides. The distance between the histogram from the isotype
control staining versus the histogram reflecting the specific stain
and is a measure of level of cell surface CD1d.
[0279] At four hours, on both cell lines, significant evidence that
the biotinylated synthetic peptide bound with high affinity to the
human B-cell lines, Raji and Daudi, at 4 hours was observed. Less
binding is observed at 24 hours. The cells were counter-stained the
cells with FITC-Anti-CD1d and found that treatment and binding of
Biotinylated FRIMAVLAS (SEQ ID NO. 1) resulted in cell surface
expression of CD1d on both cell lines, marginally at 4 hours and
slightly more at 24 hours.
[0280] Cell Culture Conditions: The Raji and Daudi cell lines were
purchased from American Type Culture Collection, were thawed, and
grown in RPMI 1640 medium supplemented with standard supplements,
including 10% fetal calf serum, gentamycin, penicillin,
streptomycin, sodium pyruvate, HEPES buffer, 1-glutamine, and
2-ME.
[0281] Protocol: Cells were plated into a 12 well plate with 3 mls
total volume containing approximately 1.5.times.10.sup.6/well for
Daudi cells and 3.0.times.10.sup.6/well for Raji cells. Treatment
groups included no treatment as control and biotinylated FRIMVLAS
(SEQ ID NO. 1) (also referred to as MKN 5) at 50 .mu.M final
concentration based on the reported molarity of the synthesized
compounds.
[0282] The cells were incubated at 37.degree. C. in an atmosphere
containing 5% CO.sub.2 and approximately 92% humidity. The cells
were incubated for 4 and 24 hours. At each time point, the cells
from that experimental time were harvested and stained for flow
cytometric analysis of cell surface expression of CD1d by staining
with PE anti-human CD1d (eBioscience, clone 51.5, cat. #
12-00016-71).
[0283] Harvested cells were stained using standard staining
procedure that called for a 1:100 dilution of PE anti-CD1d.
Following staining on ice for 25 min, cells were washed with
PBS/FCS and resuspended in 100 .mu.l and added to staining tubes
containing 400 microliters of PBS. Samples were acquired and
analyzed on a Coulter Excel Flow Cytometer.
Example 10
Preparation of a Prodrug Ester
[0284] The history of prodrug administration includes the use of
esterases available throughout the digestive track in humans. The
esterase acts as a hydrolase in ester hydrolysis, cleaving the
carboxylate ester from the alcohol. The purpose of this approach
has generally included the improvement of absorption, metabolism,
and overall bioavailability. The approach has been used in various
drugs including, but not limited to, enalapril, Valacyclovir,
heroin, and chloramphenicol.
[0285] [3,4,6-trihydroxytetrahydro-2H-pyran-2-yl]methyl
dichloroacetate can be prepared by mixing 2-deoxy-D-glucose,
dichloroacetate, and sulfuric acid and refluxing the solution.
After cooling the solution, one can then add water and diethyl
ether for mixing and allow the aqueous and organic layers to
separate in order to remove the aqueous layer. The product can then
be extracted using sodium bicarbonate until a neutral pH is
acquired and then dried over anhydrous sodium sulfate. The diethyl
ether will be evaporated over a warm sand bath and the product will
be allowed to cool to room temperature.
[0286] [3,4,6-trihydroxytetrahydro-2H-pyran-2-yl]methyl
dichloroacetate can be prepared by mixing 2-deoxy-D-glucose,
anhydrous dichloroacetate, and acetic anhydride and then stirring
and refluxing the solution. After the reflux, the contents can be
mixed with ice and cold water, then suction filtered and
recrystallized in ethanol.
[0287] The Mechanism for Acid-Catalyzed Esterification: The
mechanism for the Acid-Catalyzed Esterification of Dichloroacetic
Acid is as follows, with R=Halogen, R'=the remainder of
2-deoxy-D-glucose, or an analog or homolog thereof.
Overall Reaction
##STR00007##
[0288] Step 1: The Carboxylic Acid is Protonated on its Carbonyl
Oxygen
##STR00008##
[0289] Step 2: Protonation of the Carboxylic Acid Increases the
Positive Character of its Carbonyl Group. a Molecule of the Alcohol
Acts as a Nucleophile and Attacks the Carbonyl Carbon
##STR00009##
[0290] Step 3: the Oxonium Ion Formed in Step 2 Loses a Proton to
Give the Tetrahedral Intermediate in its Neutral Form
##STR00010##
[0291] Step 4: the Tetrahedral Intermediate is Hydroxylated
##STR00011##
[0292] Step 5: the Intermediate Loses a Molecule of Water to Give
the Protonated Form of the Ester
##STR00012##
[0293] Step 6: Deprotonation of the Species Formed in Step 5 Gives
the Neutral Form of the Ester Product
##STR00013##
[0295] Having thus described several aspects of at least one
embodiment of this invention, it is to be appreciated various
alterations, modifications, and improvements will readily occur to
those skilled in the art. Such alterations, modifications, and
improvements are intended to be part of this disclosure, and are
intended to be within the spirit and scope of the invention.
Accordingly, the foregoing description and drawings are by way of
example only.
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Sequence CWU 1
1
519PRTArtificial SequenceSynthetic peptide 1Phe Arg Ile Met Ala Val
Leu Ala Ser1 5213PRTArtificial SequenceSynthetic peptide 2Pro Lys
Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr1 5 10313PRTArtificial
SequenceSynthetic peptide 3Ile Ala Gly Phe Lys Gly Glu Gln Gly Pro
Lys Gly Glu1 5 10419PRTArtificial SequenceSynthetic peptide 4Ser
Gly Gly Gly Ser Lys Met Arg Met Ala Thr Pro Leu Leu Met Gln1 5 10
15Ala Leu Tyr59PRTArtificial SequenceSynthetic peptide 5Met Arg Met
Ala Thr Pro Leu Leu Met1 5
* * * * *