U.S. patent application number 13/437739 was filed with the patent office on 2012-07-26 for immunological uses of immunomodulatory compounds for vaccine and anti-infectious disease therapy.
Invention is credited to Justin B. BARTLETT, Angus G. Dalgleish, Christine Galustian, Brendan Meyer, George W. Muller, Peter H. Schafer.
Application Number | 20120190110 13/437739 |
Document ID | / |
Family ID | 37806889 |
Filed Date | 2012-07-26 |
United States Patent
Application |
20120190110 |
Kind Code |
A1 |
BARTLETT; Justin B. ; et
al. |
July 26, 2012 |
IMMUNOLOGICAL USES OF IMMUNOMODULATORY COMPOUNDS FOR VACCINE AND
ANTI-INFECTIOUS DISEASE THERAPY
Abstract
Methods of enhancing immune response to an immunogen in a
subject are disclosed. Also disclosed are methods of reducing the
sensitivity to an allergen in a subject. The methods comprise the
administration of an immunomodulatory compound in specific dosing
regimens that result in enhanced immune response or reduced
sensitivity.
Inventors: |
BARTLETT; Justin B.;
(Warren, NJ) ; Muller; George W.; (Bridgewater,
NJ) ; Schafer; Peter H.; (Randolph, NJ) ;
Galustian; Christine; (Croydon Surrey, GB) ;
Dalgleish; Angus G.; (Cheam, GB) ; Meyer;
Brendan; (London, GB) |
Family ID: |
37806889 |
Appl. No.: |
13/437739 |
Filed: |
April 2, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11514447 |
Aug 31, 2006 |
|
|
|
13437739 |
|
|
|
|
60712823 |
Sep 1, 2005 |
|
|
|
Current U.S.
Class: |
435/375 |
Current CPC
Class: |
A61P 33/00 20180101;
A61P 35/02 20180101; A61K 39/39 20130101; A61K 2039/55511 20130101;
A61P 37/04 20180101; Y02A 50/388 20180101; A61P 1/16 20180101; Y02A
50/484 20180101; Y02A 50/30 20180101; A61P 31/12 20180101; A61P
31/10 20180101; Y02A 50/39 20180101; A61P 37/08 20180101; A61P
35/00 20180101; A61P 37/00 20180101; Y02A 50/401 20180101; A61P
31/04 20180101; Y02A 50/466 20180101; A61K 31/4035 20130101; A61P
31/20 20180101; Y02A 50/469 20180101 |
Class at
Publication: |
435/375 |
International
Class: |
C12N 5/0783 20100101
C12N005/0783 |
Claims
1. A method of reducing or inhibiting the immuno-suppressive
activity of a regulatory T cell comprising contacting the
regulatory T cell with an immunomodulatory compound for a time
sufficient for the reduction or inhibition of such suppressive
activity.
2-51. (canceled)
Description
[0001] This application claims priority to U.S. provisional
application No. 60/712,823, filed Sep. 1, 2005, the entirety of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the use of certain non-peptide
small molecules known as immunomodulatory compounds or IMiDs.RTM.
in various immunological applications, in particular as vaccine
adjuvants, particularly anticancer vaccine adjuvants. The invention
also relates to the uses of IMiDs.RTM. in combination with vaccines
to treat or prevent cancer or infectious diseases. This invention
also relates to other various uses of immunomodulatory compounds
such as reduction or desensitization of allergic reactions.
2. BACKGROUND
[0003] 2.1 Vaccines
[0004] Vaccines have traditionally consisted of live attenuated
pathogens, whole inactivated organisms or inactivated toxins. In
many cases, these approaches have been successful at inducing
immune protection based on antibody mediated responses. However,
certain pathogens, e.g., HIV, HCV, TB, and malaria, require the
induction of cell-mediated immunity (CMI). Non-live vaccines have
generally proven ineffective in producing CMI. In addition,
although live vaccines may induce CMI, some live attenuated
vaccines may cause disease in immunosuppressed subjects. As a
result of these problems, several new approaches to vaccine
development have emerged, such as recombinant protein subunits,
synthetic peptides, protein polysaccharide conjugates, and plasmid
DNA. While these new approaches may offer important safety
advantages, a general problem is that vaccines alone are often
poorly immunogenic. Therefore, there is a continuing need for the
development of potent and safe adjuvants that can be used in
vaccine formulations to enhance their immunogenicity. See, e.g.,
Edelman, Molecular Biotech. 21: 129-148 (2002); O'Hagan et al.,
Biomolecular Engineering, 18: 69-85 (2001); Singh et al., Pharm.
Res. 19(6): 715-28 (2000) for detailed review of the state of the
art in vaccine development.
[0005] Traditionally, the immunogenicity of a vaccine formulation
has been improved by injecting it in a formulation that includes an
adjuvant. Immunological adjuvants were initially described by Ramon
(1924, Ann. Inst. Pasteur, 38: 1) "as substances used in
combination with a specific antigen that produced a more robust
immune response than the antigen alone." A wide variety of
substances, both biological and synthetic, have been used as
adjuvants. However, despite extensive evaluation of a large number
of candidates over many years, the only adjuvants currently
approved by the U.S. Food and Drug administration are
aluminum-based minerals (generically called Alum). Alum has a
debatable safety record see, e.g. Malakoff, Science. 2000, 288:
1323), and comparative studies show that it is a weak adjuvant for
antibody induction to protein subunits and a poor adjuvant for CMI.
Moreover, Alum adjuvants can induce IgE antibody response and have
been associated with allergic reactions in some subjects (see,
e.g., Gupta et al. 1998, Drug Deliv. Rev. 32: 155-72; Relyveld et
al., 1998, Vaccine 16: 1016-23). Many experimental adjuvants have
advanced to clinical trials since the development of Alum, and some
have demonstrated high potency but have proven too toxic for
therapeutic use in humans. Thus, an on-going need exists for safe
and potent adjuvants.
[0006] Cancer vaccines have been a subject of much attention.
Recently, there appears to be an emergin consensus that cancer
vaccines are less likely to be successful in the context of high
tumor buden/load (see, e.g., Nature Medicine Commentary, 10(12):
1278 (2004) and Cancer Immunol. Immunother., 53(10): 844-54
(2004)). This is attributed to effective tumor-mediated immune
suppression due to the secretion of IL-10, TGF-b, and PGE-2, among
others.
[0007] On the other hand, recent evidence suggests that immediately
after tumor resection or ablation, there is leakage of tumor cells
in the peripheral blood. Therefore, the presence of tumor antigen
in the context of low tumor burden, without associated immune
suppression, may enable re-priming of the immune response. Thus, a
need exists for an agent that promotes the long-term anti-tumor
immunity, possibly through Th1 type cellular immune responses.
[0008] 2.2 Regulatory T Cells (T.sub.reg Cells)
[0009] T.sub.reg cells refer to a population of specialized T cells
that express CD4 and CD25. T.sub.reg cells are exceptional in that
their main function appears to be suppression of function of other
cells. In this regard, T.sub.reg cells are also referred to as
"suppressor cells." It has been reported that a further defining
characteristic of T.sub.reg cells is their expression of the
transcription factor Foxp3.
[0010] Due to the variety of their effect, T.sub.reg cells have
been a subject of a great deal of interest. It has been reported
that T.sub.reg cells may influence the outcome of infection,
autoimmunity, transplantation, cancer and allergy. It has been
suggested that the modes of suppression employed by T.sub.reg cells
range from the cytokines IL-10 and TGF-.beta. to cell-cell contact
via the inhibitory molecule CTLA-4. Recently, it has been reported
that dendritic cells (DC) may induce the activation and
proliferation of T.sub.reg cells, although DC are recognized as
powerful activators of immune response due, in part, to their
potency as antigen presentation cells (APC). See Yamazaki et J.
Exp. Med., 198: 235 (2003).
[0011] Generally, it is believed that T.sub.reg cells suppress the
immunity of the host, and thus preventing an immunogen (e.g., a
vaccine) from invoking effective immune response in the host. On
the other hand, the absence of T.sub.reg cells can lead to an
outburst of immune response, often resulting in inflammation or
autoimmunity. Therefore, to maximize the immunity acquired from an
immunogen, a balance needs to be achieved with regard to the level
or functionality of T.sub.reg cells.
[0012] 2.3 Gamma Delta (.gamma..delta.) T Cells
[0013] Human T cells bearing the .gamma..delta. T cell receptor
represent a unique lymphocyte population with characteristic tissue
distribution, being present in organized lymphoid tissue as well as
skin- and gut-associated lymphoid tissue. .gamma..delta. T cells
are activated in a non-MHC restricted manner by small
phosphorylated non-peptidic metabolites, including the prototypic
ligand isopentenyl pyrophosphate (IPP). Some .gamma..delta. T cell
ligands are microbial intermediates from the farnseylpyrophosphate
synthesis pathway, which is ubiquitous and essential for cell
survival. This unique antigen specificity has been suggested to be
best suited for activation of sentinel cells independently of
antigens derived from individual microbes (De Libero, Immunology
Today, 18: 22-26 (1997)). Recent data suggest that .gamma..delta. T
cells play a role in tumor surveillance, for example, of
spontaneous B cell lymphomas (Street et al, J Exp Med, 199: 879-884
(2004)), since these cells have been shown to recognize
intermediates of the melavonate pathway, an essential pathway
leading to cholesterol biosynthesis (Gober et al, J Exp Med, 197:
163-168 (2003)). These .gamma..delta. T cell tumor ligands can be
enhanced by treatment with amino-bisphosphonates (nitrogen
containing bisphosphonate drugs include pamidronate and zoledronate
and are used in myeloma treatment), suggesting that pretreatment
with these drugs could sensitize tumor cells to .gamma..delta. T
cell-mediated killing. .gamma..delta. T cells may also be able to
augment anti-tumor immunity by enhancing dendritic cell maturation
(Ismaili et al, Clin Immunol, 103: 296-302 (2002)).
[0014] In non-cancer settings, .gamma..delta. T cells play a role
in protection from viral infection, e.g., West Nile virus (Wang et
al, J Immunol, 171: 2524-2531 (2003)). Also, intraepithelial
.gamma..delta. T cells play a protective role in intestinal
inflammation (Chen et al, Proc. Natl. Acad. Sci. U.S.A., 99:
14338-14343 (2002); and Inagaki-Ohara et al, J Immunol, 173:
1390-1398 (2004)). Furthermore, .gamma..delta. TCR-bearing
dendritic epidermal cells play a role in wound repair (Jameson et
al. Science, 296: 747-749 (2002)).
[0015] 2.4 Immunomodulatory Compounds
[0016] A number of studies have been conducted with the aim of
providing compounds that can safely and effectively be used to
treat diseases associated with abnormal production of INF-.alpha..
See, e.g., Marriott, J. B., et al., Expert Opin. Biol. Ther.
1(4):1-8 (2001); G. W. Muller, et al., Journal of Medicinal
Chemistry, 39(17): 3238-3240 (1996); and G. W. Muller, et al.,
Bioorganic & Medicinal Chemistry Letters, 8: 2669-2674 (1998).
Some studies have focused on a group of compounds selected for
their capacity to potently inhibit TNF-.alpha. production by LPS
stimulated PBMC. L. G. Corral, et al., Ann. Rheum. Dis. 58 (suppl
I): 1107-1113 (1999). These compounds, which are referred to as
IMiDs.RTM. (Celgene Corporation) or Immunomodulatory Drugs, show
not only potent inhibition of TNF-.alpha. but also marked
inhibition of LPS induced monocyte IL1.beta. and IL12 production.
LPS induced IL6 is also inhibited by immunomodulatory compounds,
albeit partially. These compounds are potent stimulators of LPS
induced IL10. Id. Particular examples of IMiDs.RTM. include, but
are not limited to, the substituted 2-(2,6-dioxopiperidin-3-yl)
phthalimides and substituted
2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles described and claimed
in U.S. Pat. Nos. 6,281,230 and 6,316,471, both to G. W. Muller, et
al.
3. SUMMARY OF THE INVENTION
[0017] This invention relates to immunological and other uses of
IMiD.RTM.. In particular, this invention encompasses the use of
IMiDs.RTM. in combination of an immunogen (e.g., a vaccine) in
specific dosing regimen, providing an enhanced immune responses
from the immunogen as compared to the responses obtained when
IMiDs.RTM. are not used.
[0018] This invention also encompasses methods of reducing or
inhibiting proliferation or immuno-suppressive activity of
T.sub.reg cells comprising contacting the T.sub.reg cell with an
immunomodulatory compound of the invention.
[0019] This invention also encompasses methods of eliciting an
enhanced immune response from an immunogen. This invention also
encompasses methods of eliciting a reduced allergic response from
an allergen. The methods comprise administering an immunomodulatory
compound of the invention to a subject prior to the exposure of the
subject to an immunogen or an allergen. It should be noted that
IMiDs.RTM. can be additionally administered during and/or after the
subject's exposure to the immunogen or allergen.
[0020] Pharmaceutical compositions, dosing regimen, and combination
therapies using an immunomodulatory compound are also encompassed
by the invention.
4. BRIEF DESCRIPTION OF FIGURES
[0021] FIG. 1 is a non-limiting list of vaccines that may be used
in connection with methods of this invention.
[0022] FIG. 2A illustrates the effects of
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione on the
function of regulatory T cells.
[0023] FIG. 2B illustrates the effects of
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione on
the function of regulatory T cells.
[0024] FIG. 2C illustrates the effects of thalidomide on the
function of regulatory T cells.
[0025] FIG. 3 illustrates the effects of immunomodulatory compounds
of the invention and thalidomide on the expression of T.sub.reg
marker Foxp3 (FIG. 3A-DMSO control; FIG. 3B-1 .mu.M
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; FIG.
3C-0.01 .mu.M
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; FIG.
3D-1 .mu.M
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione;
FIG. 3E-0.01 .mu.M
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione;
FIG. 3F-1 .mu.M thalidomide; and FIG. 3G-0.01 .mu.M
thalidomide).
[0026] FIG. 4 illustrates the effects of
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione on
the number of regulatory T cells.
[0027] FIG. 5A illustrates the effects of
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-O-piperidine-2,6-dione on
the expression of .gamma..delta. T cells in PMBC activated with
IL-2 and IPP.
[0028] FIG. 5B illustrates the effects of
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione on the
expression of .gamma..delta. T cells in PMBC activated with IL-2
and IPP.
[0029] FIG. 5C illustrates the effects of
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione on
the expression of NKG2D in PMBC activated with IL-2 and IPP.
[0030] FIG. 5D illustrates the effects of
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione on the
expression of NKG2D in PMBC activated with IL-2 and IPP.
[0031] FIG. 6 illustrates the effects of
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione on the
apoptosis in .gamma..delta. T cells on day 4 (FIG. 6A), day 5 (FIG.
6B), day 6 (FIG. 6C), and day 7 (FIG. 6D) after the treatment.
[0032] FIGS. 7A and 7B illustrate the comparison of IFN-.gamma.
production in cells treated with .alpha.CD3 alone (FIG. 7A) and
those treated with .alpha.CD3 and
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (FIG.
7B) in freshly prepared .gamma..delta. T cells.
[0033] FIGS. 7C and 7D illustrate the comparison of TNF-.alpha.
production in cells treated with .alpha.CD3 alone (FIG. 7C) and
those treated with .alpha.CD3 and
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (FIG.
7D) in freshly prepared .gamma..delta. T cells.
[0034] FIGS. 7E and 7F illustrate the comparison of IFN-.gamma.
production in cells treated with IPP alone (FIG. 7E) and those
treated with IPP and
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (FIG.
7F) in freshly prepared .gamma..delta. T cells.
[0035] FIGS. 7G and 7H illustrate the comparison of INF-.alpha.
production in cells treated with IPP alone (FIG. 7G) and those
treated with IPP and
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (FIG.
7H) in freshly prepared .gamma..delta. T cells.
[0036] FIG. 8A illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=0.5:1
ratio) without preincubation with pamidronate.
[0037] FIG. 8B illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=0.5:1
ratio) without preincubation with pamidronate, but with treatment
with
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione.
[0038] FIG. 8C illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=1:1
ratio) without preincubation with pamidronate.
[0039] FIG. 8D illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=1:1
ratio) without preincubation with pamidronate, but with treatment
with
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione.
[0040] FIG. 8E illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=2:1
ratio) without preincubation with pamidronate.
[0041] FIG. 8F illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=2:1
ratio) without preincubation with pamidronate, but with treatment
with
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione.
[0042] FIG. 8G illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=0.5:1
ratio) with preincubation with pamidronate.
[0043] FIG. 8H illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=0.5:1
ratio) with preincubation with pamidronate and treatment with
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione.
[0044] FIG. 8I illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=1:1
ratio) with preincubation with pamidronate.
[0045] FIG. 8J illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=1:1
ratio) with preincubation with pamidronate and treatment with
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione.
[0046] FIG. 8K illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=2:1
ratio) with preincubation with pamidronate.
[0047] FIG. 8L illustrates the IFN-.gamma. production in response
to co-culture with RPMI cells with (tumor:.gamma..delta. T=2:1
ratio) with preincubation with pamidronate and treatment with
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione.
[0048] FIG. 9A illustrates the effects of immunomodulatory
compounds of the invention on the cytotoxicity of .gamma..delta. T
cells on MM cell lines where the compounds are preincubated with
tumor cells.
[0049] FIG. 9B illustrates the effects of immunomodulatory
compounds of the invention on the cytotoxicity of .gamma..delta. T
cells on MM cell lines where the compounds are not preincubated
with tumor cells, but are added during the chromium release assay
only.
5. DETAILED DESCRIPTION OF THE INVENTION
[0050] This invention is based, in part, on the inventors'
discovery that pre-treatment with immunomodulatory compounds of the
invention, before the introduction of an immunogen (e.g., a
vaccine) results in an enhanced immune response in a host, as
determined by the experiments described herein. Without being
limited by a particular theory, the invention encompasses
administration of an immunomodulatory compound to a host,
preferably prior to the introduction of an immunogen, to enhance
the function of dendritic cells as antigen presentation cells
and/or suppress the proliferation and/or function of T.sub.reg
cells, resulting in an enhanced immune response in the host. In
addition, without being limited by a particular theory,
immunomodulatory compounds of the invention augment the innate
anti-tumor activity of .gamma..delta. T cells. Furthermore, without
being limited by a particular theory, it is also believed that the
immunomodulatory compounds of the invention promote successful Th1
type cellular immune responses necessary for efficient long-term
anti-tumor activity, thereby delaying or prevent tumor
reoccurrence.
[0051] Accordingly, this invention encompasses methods of reducing
or inhibiting proliferation and/or immuno-suppressive activity of
regulatory T cells comprising contacting the regulatory T cells
with an immunomodulatory compound of the invention for a time
sufficient for the reduction or the inhibition of proliferation
and/or immuno-suppressive activity.
[0052] As used herein, and unless otherwise specified, the term
"reducing or inhibiting the proliferation," when used in connection
with regulatory T cells, means that the number of regulatory T
cells in a cell culture or a host treated with an immunomodulatory
compound of the invention is less than the number of regulatory T
cells in a cell culture or a host without the treatment with an
immunomodulatory compound of the invention, as determined by
methods known in the art, some of which are described herein. A
typical method involves the staining of a marker and analysis of
the stain using, for example, FACS analysis. Preferably, reduced
proliferation means the number of T cells in immunomodulatory
compound treated culture or host is about 10%, 20%, 30%, 50%, 70%,
or 90% or less than those in the culture or host without such
treatment.
[0053] As used herein, and unless otherwise specified, the teem
"reducing or inhibiting the immuno-suppressive activity," when used
in connection with regulatory T cells, means that the
immuno-suppressive activity of regulatory T cells, when treated or
contacted with an immunomodulatory compound of the invention, is
lower than those without such treatment or contact. The
immuno-suppressive activity can be determined using methods known
in the art including those described herein. Typically, the
immuno-suppressive activity of regulatory T cells can be assessed
by monitoring the proliferation of for example, anti-CD3 stimulated
CD25-cells in response to TCR signal. Preferably, reduced
immuno-suppressive activity means the activity of regulatory T
cells treated with an immunomodulatory compound of the invention is
about 10%, 20%, 30%, 50%, 70%, or 90% or less than the activity of
those without such treatment.
[0054] This invention also encompasses methods of eliciting an
enhanced immune response from an immunogen in a subject (e.g.,
human) comprising administering to the subject an immunomodulatory
compound of the invention prior to the administration of the
immunogen to the subject.
[0055] As used herein, and unless otherwise specified, the term
"immunogen" means any substance or organism that provokes an immune
response (produces immunity) when introduced to the body. In some
embodiments, an immunogen can be used in therapeutic settings in a
form of a vaccine.
[0056] As used herein, and unless otherwise specified, the term
"enhanced immune response" means that, when an immunogen is
administered in combination with an immunomodulatory compound
according to methods of this invention, there is an increased
antibody formation, measured using any standard methods known in
the art or described herein, in a subject that receives such an
administration as compared to a subject to which same amount of the
immunogen alone is administered. As used herein, the term
"administration in combination with," used in connection with two
or more therapeutic agents, means that such agents are administered
simultaneously, concurrently, or sequentially using the same or
different routes. Preferably, an enhanced immune response means
about 10%, 20%, 30%, 50%, 70%, or 100% or greater increase in
antibody formation.
[0057] In specific embodiments, an immunomodulatory compound is
administered to a subject about 30 days, 20 days, 15 days, 12 days,
10 days, 7 days, 5 days, 3 days, 1 day, 12 hours, or 5 hours prior
to the administration of the immunogen. In other embodiments, an
immunomodulatory compound is administered from about 30 days to
about 5 hours, from about 20 days to about 5 hours, from about 15
days to about 12 hours, from about 12 days to about 5 hours, from
about 10 days to about 12 hours, from about 7 days to about 12
hours, from about 5 days to about 12 hours, from about 5 days to
about 1 day, from about 3 days to about 12 hours, or from about 3
days to about 1 day prior to the administration of an
immunogen.
[0058] In other embodiments, methods of the invention further
comprises a second administration of an immunomodulatory compound
of the invention after the administration of an immunogen. Without
being limited by a particular theory, it is believed that
administering an immunomodulatory compound after the administration
of an immunogen can enhance the immune response obtained from the
immunogen by improving antigen presentation of host cells,
enhancing the activity of T cells (e.g., .alpha..beta. and
.gamma..delta. TCR positive), and generating cytotoxic effector
response and long term memory (e.g., Th1 type) immune response. In
these embodiments, there are at least two administrations of an
immunomodulatory compound of the invention--one pre-immunogen and
one post-immunogen.
[0059] In specific embodiments, an immunomodulatory compound of the
invention is administered to a subject about 30 days, 20 days, 15
days, 12 days, 10 days, 7 days, 5 days, 3 days, 1 day, 12 hours, or
5 hours after the administration of the immunogen. In other
embodiments, an immunomodulatory compound of the invention is
administered from about 5 hours to about 30 days, from about 5
hours to about 20 days, from about 12 hours to about 15 days, from
about 5 hours to about 12 days, from about 12 hours to about 10
days, from about 12 hours to about 7 days, from about 12 hours to
about 5 days, from about 1 day to about 5 days, from about 12 hours
to about 3 days, or from about 1 day to about 3 days after the
administration of an immunogen.
[0060] In another aspect, this invention encompasses methods of
eliciting a reduced allergic response in a subject comprising
administering to the subject an immunomodulatory compound of the
invention prior to the subject's exposure to an allergen. As used
herein, the term "subject's exposure to allergen" encompasses a
subject's exposure to an allergen which is foreseeable (e.g.,
intake of food or exposure to the naturally occurring allergens),
as well as allergy vaccination where an allergen is administered to
a subject according to a dosing scheme over a period of time.
Without being limited by a particular theory, it is believed that
immunomodulatory compounds not only preferentially induce Th1
immune response, but also inhibit and/or reverse Th2
differentiation, resulting in milder, non-acute immune response to
an allergen mediated by Th1 cells.
[0061] In specific embodiments, an immunomodulatory compound is
administered to a subject about 30 days, 20 days, 15 days, 12 days,
10 days, 7 days, 5 days, 3 days, 1 day, 12 hours, 5 hours, 2 hours,
or 30 minutes prior to the subject's exposure to an allergen. In
other embodiments, an immunomodulatory compound is administered
from about 30 days to about 30 minutes, from about 20 days to about
1 hour, from about 15 days to about 1 hour, from about 12 days to
about 30 minutes, from about 10 days to about 2 hours, from about 7
days to about 2 hours, from about 5 days to about 2 hours, from
about 5 days to about 1 hour, from about 1 day to about 30 minutes,
or from about 1 day to about 2 hours prior to the subject's
exposure to an allergen.
[0062] In other embodiments, methods of the invention further
comprises a second administration of an immunomodulatory compound
of the invention after the subject's exposure to an allergen.
Without being limited by a particular theory, it is believed that
administering an immunomodulatory compound after the subject's
exposure to an allergen can generate long term memory (e.g., Th1
type) immune response. In these embodiments, there are at least two
administrations of an immunomodulatory compound of the
invention--one pre-allergen and one post-allergen.
[0063] In specific embodiments, an immunomodulatory compound of the
invention is administered to a subject about 30 days, 20 days, 15
days, 12 days, 10 days, 7 days, 5 days, 3 days, 1 day, 12 hours, or
5 hours after the subject's exposure to an allergen. In other
embodiments, an immunomodulatory compound of the invention is
administered from about 5 hours to about 30 days, from about 5
hours to about 20 days, from about 12 hours to about 15 days, from
about 5 hours to about 12 days, from about 12 hours to about 10
days, from about 12 hours to about 7 days, from about 12 hours to
about 5 days, from about 1 day to about 5 days, from about 12 hours
to about 3 days, or from about 1 day to about 3 days after the
subject's exposure to an allergen.
[0064] 5.1 Immunogens And Vaccines
[0065] Various immunogens may be used in connection with methods of
this invention. The immunogens are usually administered to a
subject in a form of an immunogenic composition (e.g., a vaccine),
but may be administered in any form that is acceptable for use in
animals, in particular, humans.
[0066] 5.1.1 Immunogens
[0067] Immunogens that may be used in the immunogenic compositions
include antigens from an animal, a plant, a bacteria, a protozoan,
a parasite, a virus or a combination thereof. Immunogens may be any
substance that under appropriate conditions results in an immune
response in a subject, including, but not limited to, polypeptides,
peptides, proteins, glycoproteins, lipids, nucleic acids (e.g.,
RNAs and DNAs) and polysaccharides.
[0068] An immunogenic composition may comprise one or more
immunogens. The amount of the immunogen used in the compositions
may vary depending on the chemical nature and the potency of the
immunogen.
[0069] Immunogens may be any viral peptide, protein, polypeptide,
or a fragment thereof, derived from a virus.
[0070] Immunogens used in methods of the invention may be an
antigen of a pathogenic virus such as, but are not limited to:
adenovirdiae (e.g., mastadenovirus and aviadenovirus),
herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus
2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae
(e.g., levivirus, enterobacteria phase MS2, allolevirus),
poxyiridae (e.g., chordopoxyirinae, parapoxvirus, avipoxvirus,
capripoxvirus, leporipoxvirus, suipoxvirus, molluscipoxvirus, and
entomopoxyirinae), papovaviridae (e.g., polyomavirus and
papillomavirus), paramyxoviridae (e.g, paramyxovirus, parainfluenza
virus 1, mobillivirus (e.g. measles virus), rubulavirus (e.g.,
mumps virus), pneumonovirinae (e.g., pneumovirus, human respiratory
syncytial virus), and metapneumovirus (e.g, avian pneumovirus and
human metapneumovirus), picornaviridae (e.g., enterovirus,
rhinovirus, hepatovirus (e.g., human hepatitis A virus),
cardiovirus, and apthovirus, reoviridae (e.g., orthoreovirus,
orbivirus, rotavirus, cypovirus, fijivirus, phytoreovirus, and
oryzavirus), retroviridae (e.g., mammalian type B retroviruses,
mammalian type C retroviruses, avian type C retroviruses, type D
retrovirus group, BLV-HTLV retroviruses, lentivirus (e.g. human
immunodeficiency virus 1 and human immunodeficiency virus 2),
spumavirus), flaviviridae (e.g., hepatitis C virus), hepadnaviridae
(e.g., hepatitis B virus), togaviridae (e.g, alphavirus, e.g.,
sindbis virus) and rubivirus (e.g., rubella virus), rhabdoviridae
(e.g, vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus,
and necleorhabdovirus), arenaviridae (e.g, arenavirus, lymphocytic
choriomeningitis virus, Ippy virus, and lassa virus), and
coronaviridae (e.g., coronavirus and torovirus).
[0071] Immunogens used in methods of this invention may be an
infectious disease agent including, but not limited to, influenza
virus hemagglutinin (Genbank Accession No. JO2132; Air, 1981, Proc.
Natl. Acad. Sci. USA 78: 7639-7643; Newton et al., 1983, Virology
128: 495-501), human respiratory syncytial virus G glycoprotein
(Genbank Accession No. Z33429; Garcia et al., 1994, J. Virol.:
Collins et al., 1984, Proc. Natl. Acad. Sci. USA 81: 7683), core
protein, matrix protein or any other protein of Dengue virus
(Genbank Accession No. M19197; Hahn et al., 1988, Virology 162:
167-180), measles virus hemagglutinin (Genbank Accession No.
M81899; Rota et al., 1992, Virology 188: 135-142), herpes simplex
virus type 2 glycoprotein gB (Genbank Accession No. M14923; Bzik et
al., 1986, Virology 155:322-333), poliovirus I VPI (Emini et al.,
1983. Nature 304:699), envelope glycoproteins of HIV I (Putney et
al. 1986, Science 234: 1392-1395), hepatitis B surface antigen
(Itoh et al. 1986, Nature 308: 19; Neurath et al., 1986, Vaccine 4:
34), diptheria toxin (Audibert et al., 1981, Nature 289: 543),
streptococcus 24M epitope (Beachey, 1985, Adv. Exp. Med. Biol.
185:193), gonococcal pilin (Rothbard and Schoolnik, 1985, Adv. Exp.
Med. Biol. 185:247), pseudorabies virus g50 (gpD), pseudorabies
virus II (gpB), pseudorabies virus gill (gpC), pseudorabies virus
glycoprotein H, pseudorabies virus glycoprotein E, transmissible
gastroenteritis glycoprotein 195, transmissible gastroenteritis
matrix protein, swine rotavirus glycoprotein 38, swine parvovirus
capsid protein, Serpulina hydrodysenteriae protective antigen,
bovine viral diarrhea glycoprotein 55, Newcastle disease virus
hemagglutinin-neuraminidase, swine flu hemagglutinin, swine flu
neuraminidase, foot and mouth disease virus, hog cholera virus,
swine influenza virus, African swine fever virus. Mycoplasma
hyopneumoniae, infectious bovine rhinotracheitis virus (e.g.,
infectious bovine rhinotracheitis virus glycoprotein E or
glycoprotein G), or infectious laryngotracheitis virus (e.g.,
infectious laryngotracheitis virus glycoprotein G or glycoprotein
I), a glycoprotein of La Crosse virus (Gonzales-Scarano et al.,
1982, Virology 120: 42), neonatal calf diarrhea virus (Matsuno and
Inouye, 1983, Infection and Immunity 39: 155), Venezuelan equine
encephalomyelitis virus (Mathews and Roehrig, 1982, J. Immunol.
129: 2763), punta toro virus (Dalrymple et al., 1981, in
Replication of Negative Strand Viruses, Bishop and Compans (eds.),
Elsevier, N.Y., p. 167), murine leukemia virus (Steeves et al.,
1974. J. Virol. 14:187), mouse mammary tumor virus (Massey and
Schochetman, 1981, Virology 115: 20), hepatitis B virus core
protein and/or hepatitis B virus surface antigen or a fragment or
derivative thereof (see. e.g., U.K. Patent Publication No. GB
2034323A published Jun. 4, 1980; Ganem and Varmus, 1987. Ann. Rev.
Biochem. 56:651-693; Tiollais et al. 1985. Nature 317:489-495),
antigen of equine influenza virus or equine herpesvirus (e.g.,
equine influenza virus type A/Alaska 91 neuraminidase, equine
influenza virus type A/Miami 63 neuraminidase, equine influenza
virus type A/Kentucky 81 neuraminidase equine herpesvirus type 1
glycoprotein B, and equine herpesvirus type 1 glycoprotein D,
antigen of bovine respiratory syncytial virus or bovine
parainfluenza virus (e.g., bovine respiratory syncytial virus
attachment protein (BRSV G), bovine respiratory syncytial virus
fusion protein (BRSV F), bovine respiratory syncytial virus
nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3
fusion protein, and the bovine parainfluenza virus type 3
hemagglutinin neuraminidase), bovine viral diarrhea virus
glycoprotein 48 or glycoprotein 53.
[0072] Immunogens used in methods of this invention may also be a
cancer antigen or a tumor antigen. Any cancer or tumor antigen
known to one skilled in the art may be used in accordance with the
immunogenic compositions of the invention including, but not
limited to, KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990,
J. Immunol. 142: 3662-3667; Bumal, 1988. Hybridoma 7(4): 407-415),
ovarian carcinoma antigen (CA125) (Yu et al. 1991, Cancer Res.
51(2): 468-475), prostatic acid phosphate (Tailor et al., 1990,
Nucl. Acids Res. 18(16): 4928), prostate specific antigen (Henttu
and Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2): 903-910;
Israeli et al., 1993, Cancer Res. 53: 227-230), melanoma-associated
antigen p97 (Estin et al., 1989, J. Natl. Cancer Instit. 81(6):
445-446), melanoma antigen gp75 (Vijayasardahl et al., 1990, J.
Exp. Med. 171(4): 1375-1380), high molecular weight melanoma
antigen (HMW-MAA) (Natali et al., 1987, Cancer 59: 55-63; Mittelman
et al., 1990, J. Clin. Invest. 86: 2136-2144), prostate specific
membrane antigen, carcinoembryonic antigen (CEA) (Foon et al.,
1994, Proc. Am. Soc. Clin. Oncol. 13: 294), polymorphic epithelial
mucin antigen, human milk fat globule antigen, colorectal
tumor-associated antigens such as: CEA, TAG-72 (Yokata et al.,
1992, Cancer Res. 52: 3402-3408), C017-1A (Ragnhammar et al., 1993,
Int. J. Cancer 53: 751-758); GICA 19-9 (Herlyn et al., 1982, J.
Clin. Immunol. 2: 135), CTA-1 and LEA, Burkitt's lymphoma
antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83: 1329-1336),
human B-lymphoma antigen-CD20 (Reff et al., 1994. Blood 83:
435-445). CD33 (Sgouros et al., 1993, J. Nucl. Med. 34: 422-430),
melanoma specific antigens such as ganglioside GD2 (Saleh et al.
1993. J. Immunol., 151, 3390-3398), ganglioside GD3 (shitara et
al., 1993, Cancer Immunol. Immunother. 36: 373-380), ganglioside
GM2 (Livingston et al., 1994, J. Clin. Oncol. 12: 1036-1044),
ganglioside GM3 (Hoon et al., 1993. Cancer Res. 53: 5244-5250),
tumor-specific transplantation type of cell-surface antigen (TSTA)
such as virally-induced tumor antigens including T-antigen DNA
tumor viruses and Envelope antigens of RNA tumor viruses, oncofetal
antigen-alpha-fetoprotein such as CEA of colon, bladder tumor
oncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:
2210-2188), differentiation antigen such as human lung carcinoma
antigen L6, L20 (Hellstrom et al., 1986, Cancer Res. 46:
3917-3923), antigens of fibrosarcoma, human leukemia T cell
antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. of
Immunospecifically. 141: 1398-1403), neoglycoprotein,
sphingolipids, breast cancer antigen such as EGFR (Epidermal growth
factor receptor), HER2 antigen (p185.sup.HER2), polymorphic
epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem.
Sci. 17: 359), malignant human lymphocyte antigen-APO-1 (Bernhard
et al., 1989, Science 245: 301-304), differentiation antigen
(Feizi, 1985, Nature 314: 53-57) such as I antigen found in fetal
erythrocytes, primary endoderm, I antigen found in adult
erythrocytes, preimplantation embryos, I (Ma) found in gastric
adenocarcinomas, M18, M39 found in breast epithelium, SSEA-1 found
in myeloid cells, VEP8, VEP9, Myl, VIM-D5, D.sub.156-22 found in
colorectal cancer. TRA-1-85 (blood group H), C14 found in colonic
adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in
gastric cancer, Y hapten, Ley found in embryonal carcinoma cells,
TL5 (blood group A), EGF receptor found in A431 cells, E.sub.1
series (blood group B) found in pancreatic cancer, FC10.2 found in
embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514
(blood group Le.sup.a) found in Adenocarcinoma. NS-10 found in
adenocarcinomas, CO-43 (blood group Le.sup.b), G49 found in EGF
receptor of A431 cells, MH2 (blood group ALe.sup.b/Le.sup.y) found
in colonic adenocarcinoma, 19.9 found in colon cancer, gastric
cancer mucins, T.sub.5A.sub.7 found in myeloid cells, R.sub.24
found in melanoma, 4.2, G.sub.D3, D1.1 OFA-1, G.sub.M2, OFA-2,
G.sub.D2, and M1:22:25:8 found in embryonal carcinoma cells, and
SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos. In one
embodiment, the antigen is a T cell receptor derived peptide from a
Cutaneous T cell Lymphoma (see, Edelson, 1998. The Cancer Journal
4: 62).
[0073] In a preferred embodiment, the immunogenic composition used
in methods of this invention is a cancer vaccine. Examples of
cancer vaccines include, but are not limited to: antigen modified
denritic cell (DC) vaccines such as, but not limited to, Provenge,
Neuvenge, Immunovex, Telomerase vaccine, Uvidem, Collidem,
DCVax-prostate, and DCVax-brain: peptide vaccines such as, but not
limited to, Theratope, L-BLP25, Oncophage (HSPPC-96), GTOPO-99,
IGN-101, FavId, Panvac-VF, Prostvac-VF, Avicine, EP-2101, MyVax,
Biovaxid, Mitumomab (IMC-BEC2), IMG-GP75, HER-2 DNA/Protein
AutoVac, Zyc 300, and HER-2 protein AutoVac: whole tumor cell
vaccines such as, but not limited to, Canvaxin, Ony-P, Melacine,
GVAX, GVAX and MDX-010, and Oncovax; and viral vector vaccines such
as, but not limited to, ALVAC-CEA/B&1. Allovectin-7, ALVAC,
Lovaxin C, AdhTAP(OS-1), TroVax, and MVA-MUC1-IL2 (TG4010).
Characteristics of these vaccines are summarized in Tables 1-4.
[0074] Immunogens may comprise a virus, against which an immune
response is desired. In certain cases, the immunogenic composition
used in methods of this invention comprise recombinant or chimeric
viruses. In other cases, the immunogenic composition comprises a
virus which is attenuated. Production of recombinant, chimeric and
attenuated viruses may be performed using standard methods known to
one skilled in the art. This invention also encompasses a live
recombinant viral vaccine or an inactivated recombinant viral
vaccine to be formulated in accordance with the invention. A live
vaccine may be preferred because multiplication in the host leads
to a prolonged stimulus of similar kind and magnitude to that
occurring in natural infections, and therefore, confers
substantial, long-lasting immunity. Production of such live
recombinant virus vaccine formulations may be accomplished using
conventional methods involving propagation of the virus in cell
culture or in the allantois of the chick embryo followed by
purification.
[0075] Recombinant virus may be non-pathogenic to the subject to
which it is administered. In this regard, the use of genetically
engineered viruses for vaccine purposes may require the presence of
attenuation characteristics in these strains. The introduction of
appropriate mutations (e.g., deletions) into the templates used for
transfection may provide the novel viruses with attenuation
characteristics. For example, specific missense mutations which are
associated with temperature sensitivity or cold adaptation can be
made into deletion mutations. These mutations should be more stable
than the point mutations associated with cold or temperature
sensitive mutants and reversion frequencies should be extremely
low.
[0076] Alternatively, chimeric viruses with "suicide"
characteristics may be constructed for use in the immunogenic
compositions. Such viruses would go through only one or a few
rounds of replication within the host. When used as a vaccine, the
recombinant virus would go through limited replication cycle(s) and
induce a sufficient level of immune response but it would not go
further in the human host and cause disease.
[0077] Alternatively, inactivated (killed) virus may be formulated
in accordance with the invention. Inactivated vaccine formulations
may be prepared using conventional techniques to "kill" the
chimeric viruses. Inactivated vaccines are "dead" in the sense that
their infectivity has been destroyed. Ideally, the infectivity of
the virus is destroyed without affecting its immunogenicity. In
order to prepare inactivated vaccines, the chimeric virus may be
grown in cell culture or in the allantois of the chick embryo,
purified by zonal ultracentrifugation, inactivated by formaldehyde
or .beta.-propiolactone, and pooled.
[0078] Completely foreign epitopes, including antigens derived from
other viral or non-viral pathogens can also be engineered into the
virus for use in immunogenic compositions. For example, antigens of
non-related viruses such as HIV (gp160, gp120, gp41) parasite
antigens (e.g. malaria), bacterial or fungal antigens or tumor
antigens can be engineered into the attenuated strain. Typically
such methods include inoculating embryonated eggs, harvesting the
allantoic fluid, concentrating, purifying and separating the whole
virus, using for example zonal centrifugation,
ultracentrifttgation, ultrafiltration, and chromatography in a
variety of combinations.
[0079] Virtually any heterologous gene sequence may be constructed
into the chimeric viruses for use in immunogenic compositions.
Preferably, heterologous gene sequences are moieties and peptides
that act as biological response modifiers. Preferably, epitopes
that induce a protective immune response to any of a variety of
pathogens, or antigens that bind neutralizing antibodies may be
expressed by or as part of the chimeric viruses. For example,
heterologous gene sequences that can be constructed into the
chimeric viruses include, but are not limited to, influenza and
parainfluenza hemagglutinin neuraminidase and fusion glycoproteins
such as the FIN and F genes of human PIV3. In addition,
heterologous gene sequences that can be engineered into the
chimeric viruses include those that encode proteins with
immuno-modulating activities. Examples of immuno-modulating
proteins include, but are not limited to, cytokines, interferon
type 1, gamma interferon, colony stimulating factors, interleukin
-1, -2, -4, -5, -6, -12, and antagonists of these agents.
[0080] Other heterologous sequences may be derived from tumor
antigens, and the resulting chimeric viruses be used to generate an
immune response against the tumor cells leading to tumor regression
in vivo. In accordance with the present invention, recombinant
viruses may be engineered to express tumor-associated antigens
(TAAs), including but not limited to, human tumor antigens
recognized by T cells (Robbins and Kawakami, 1996. Curr. Opin.
Immunol. 8:628-636, incorporated herein by reference in its
entirety); melanocyte lineage proteins, including gp100,
MART-1/MelanA, TRP-1 (gp75) and tyrosinase; tumor-specific widely
shared antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-1,
N-acetylglucosaminyltransferase-V and p15; tumor-specific mutated
antigens, such as .beta.-catenin. MUM-1 and CDK4; non-melanoma
antigens for breast, ovarian, cervical and pancreatic carcinoma.
HER-2/neu, human papillomavirus-E6, -E7, MUC-1.
[0081] 5.1.2 Vaccines and Target Diseases
[0082] A wide variety of vaccines may be used in connection with
methods of this invention. A non-limiting list of vaccines that can
be used in connection with this invention is provided in FIG. 1.
Target diseases for methods of the invention includes cancer, other
infectious or inflammatory diseases.
[0083] Methods of the invention can be used in the treatment of
cancers, including, but not limited to, neoplasms, tumors,
metastases, or any disease or disorder characterized by
uncontrolled cell growth. Specific examples of cancer include, but
are not limited to: cancers of the skin, such as melanoma; lymph
node; breast; cervix; uterus; gastrointestinal tract; lung; ovary;
prostate; colon; rectum; mouth; brain; head and neck; throat;
testes; kidney; pancreas; bone; spleen; liver; bladder; larynx;
nasal passages; and AIDS-related cancers. Methods of the invention
are particularly useful for treating cancers of the blood and bone
marrow, such as multiple myeloma and acute and chronic leukemias,
for example, lymphoblastic, myelogenous, lymphocytic, myelocytic
leukemias, and myelodysplastic syndromes including but not limited
to 5q minus syndrome, or myelodysplastic syndromes associated with
other cytopenic abnormalities. The methods of the invention can be
used for treating, preventing or managing either primary or
metastatic tumors.
[0084] Other specific cancers include, but are not limited to,
advanced malignancy, amyloidosis, neuroblastoma, meningioma,
hemangiopericytoma, multiple brain metastase, glioblastoma
multiforms, glioblastoma, brain stem glioma, poor prognosis
malignant brain tumor, malignant glioma, recurrent malignant
glioma, anaplastic astrocytoma, anaplastic oligodendroglioma,
neuroendocrine tumor, rectal adenocarcinoma, Dukes C & D
colorectal cancer, unresectable colorectal carcinoma, metastatic
hepatocellular carcinoma, Kaposi's sarcoma, karotype acute
myeloblastic leukemia. Hodgkin's lymphoma, non-Hodgkin's lymphoma,
cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuse large
B-Cell lymphoma, low grade follicular lymphoma, metastatic melanoma
(localized melanoma, including, but not limited to, ocular
melanoma), malignant mesothelioma, malignant pleural effusion
mesothelioma syndrome, peritoneal carcinoma, papillary serous
carcinoma, gynecologic sarcoma, soft tissue sarcoma, scelroderma,
cutaneous vasculitis, Langerhans cell histiocytosis,
leiomyosarcoma, fibrodysplasia ossificans progressive, hormone
refractory prostate cancer, resected high-risk soft tissue sarcoma,
unrescectable hepatocellular carcinoma, Waldenstrom's
macroglobulinemia, smoldering myeloma, indolent myeloma, fallopian
tube cancer, androgen independent prostate cancer, androgen
dependent stage 1V non-metastatic prostate cancer,
hormone-insensitive prostate cancer, chemotherapy-insensitive
prostate cancer, papillary thyroid carcinoma, follicular thyroid
carcinoma, medullary thyroid carcinoma, and leiomyoma. In a
specific embodiment, the cancer is metastatic. In another
embodiment, the cancer is refractory or resistance to chemotherapy
or radiation.
[0085] Infectious diseases are caused by infectious agents such as,
but not limited to, viruses, bacteria, fungi protozoa, helminths,
and parasites.
[0086] Examples of viruses that have been found in humans include,
but are not limited to, Retroviridae (e.g., human immunodeficiency
viruses, such as HIV-1 (also referred to as HTLV-III, LAV or
HTLV-III/LAV, or HIV-III: and other isolates, such as HIV-LP);
Picornaviridae (e.g., polio viruses, hepatitis A virus;
enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);
Calciviridae (e.g., strains that cause gastroenteritis);
Togaviridae (e.g., equine encephalitis viruses, rubella viruses);
Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow
fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae
(e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae
(e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza
viruses, mumps virus, measles virus, respiratory syncytial virus);
Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g.,
Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses);
Arena viridae (e.g., hemorrhagic fever viruses); Reoviridae (e.g.,
reoviruses, orbiviurses and rotaviruses); Birnaviridae;
Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);
Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae
(most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1
and 2, varicella zoster virus, cytomegalovirus (CMV), herpes
virus); Poxyiridae (variola viruses, vaccinia viruses, pox
viruses); and Iridoviridae (e.g., African swine fever virus); and
unclassified viruses (e.g., the etiological agents of Spongiform
encephalopathies, the agent of delta hepatitis (thought to be a
defective satellite of hepatitis B virus), the agents of non-A,
non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted, e.g., Hepatitis C); Norwalk and related
viruses, and astroviruses.
[0087] Retroviruses that results in infectious diseases in animals
and humans include both simple retroviruses and complex
retroviruses. The simple retroviruses include the subgroups of
B-type retroviruses, C-type retroviruses and D-type retroviruses.
An example of a B-type retrovirus is mouse mammary tumor virus
(MMTV). The C-type retroviruses include subgroups C-type group A
(including Rous sarcoma virus (RSV), avian leukemia virus (ALV),
and avian myeloblastosis virus (AMV)) and C-type group B (including
murine leukemia virus (MLV), feline leukemia virus (FeLV), murine
sarcoma virus (MSV), gibbon ape leukemia virus (GALV), spleen
necrosis virus (SNV), reticuloendotheliosis virus (RV) and simian
sarcoma virus (SSV)). The D-type retroviruses include Mason-Pfizer
monkey virus (MPMV) and simian retrovirus type 1 (SRV-1). The
complex retroviruses include the subgroups of lentiviruses, T-cell
leukemia viruses and the foamy viruses. Lentiviruses include HIV-1,
but also include HIV-2, SIV, Visna virus, feline immunodeficiency
virus (Hy), and equine infectious anemia virus (EIAV). The T-cell
leukemia viruses include HTLV-1, HTLV-II. simian T-cell leukemia
virus (STLV), and bovine leukemia virus (BLV). The foamy viruses
include human foamy virus (HFV), simian foamy virus (SFV) and
bovine foamy virus (BFV).
[0088] Examples of RNA viruses that are antigenic or immunogenic in
vertebrate animals include, but are not limited to, the following:
members of the family Reoviridae, including the genus Orthoreovirus
(multiple serotypes of both mammalian and avian retroviruses), the
genus Orbivirus (Bluetongue virus, Eugenangee virus, Kemerovo
virus, African horse sickness virus, and Colorado Tick Fever
virus), the genus Rotavirus (human rotavirus, Nebraska calf
diarrhea virus, murine rotavirus, simian rotavirus, bovine or ovine
rotavirus, avian rotavirus); the family Picornaviridae, including
the genus Enterovirus (poliovirus, Coxsackie virus A and B, enteric
cytopathic human orphan (ECHO) viruses, hepatitis A virus, Simian
enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirus
muris, Bovine enteroviruses, Porcine enteroviruses), the genus
Cardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the
genus Rhinovirus (Human rhinoviruses including at least 113
subtypes; other rhinoviruses), the genus Apthovirus (Foot and Mouth
disease (FMDV); the family Calciviridae, including Vesicular
exanthema of swine virus, San Miguel sea lion virus, Feline
picornavirus and Norwalk virus; the family Togaviridae, including
the genus Alphavirus (Eastern equine encephalitis virus, Semliki
forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong
virus, Ross river virus, Venezuelan equine encephalitis virus,
Western equine encephalitis virus), the genus Flavirius (Mosquito
borne yellow fever virus. Dengue virus, Japanese encephalitis
virus, St. Louis encephalitis virus, Murray Valley encephalitis
virus, West Nile virus, Kunjin virus, Central European tick borne
virus. Far Eastern tick borne virus, Kyasanur forest virus, Louping
III virus, Powassan virus, Omsk hemorrhagic fever virus), the genus
Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease
virus. Hog cholera virus, Border disease virus); the family
Bunyaviridae, including the genus Bunyvirus (Bunyamwera and related
viruses. California encephalitis group viruses), the genus
Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever
virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever
virus, Nairobi sheep disease virus), and the genus Uukuvirus
(Uukuniemi and related viruses); the family Orthomyxoviridae,
including the genus Influenza virus (Influenza virus type A (many
human subtypes), Swine influenza virus, and Avian and Equine
Influenza viruses, influenza type B (many human subtypes), and
influenza type C (possible separate genus)); the family
paramyxoviridae, including the genus Paramyxovirus (Parainfluenza
virus type 1, Sendai virus, Hemadsorption virus, Parainfluenza
viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the
genus Morbillivirus (Measles virus, subacute sclerosing
panencephalitis virus, distemper virus, Rinderpest virus), the
genus Pneumovirus (respiratory syncytial virus (RSV), Bovine
respiratory syncytial virus and Pneumonia virus of mice); forest
virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross
river virus, Venezuelan equine encephalitis virus, Western equine
encephalitis virus), the genus Flavirius (Mosquito borne yellow
fever virus. Dengue virus, Japanese encephalitis virus, St. Louis
encephalitis virus, Murray Valley encephalitis virus, West Nile
virus, Kunjin virus, Central European tick borne virus, Far Eastern
tick borne virus, Kyasanur forest virus, Louping III virus,
Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus
(Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog
cholera virus, Border disease virus); the family Bunyaviridae,
including the genus Bunyvirus (Bunyamwera and related viruses,
California encephalitis group viruses), the genus Phlebovirus
(Sandfly fever Sicilian virus, Rift Valley fever virus), the genus
Nairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep
disease virus), and the genus Uukuvirus (Uukuniemi and related
viruses); the family Orthomyxoviridae, including the genus
Influenza virus (Influenza virus type A, many human subtypes);
Swine influenza virus, and Avian and Equine Influenza viruses;
influenza type B (many human subtypes), and influenza type C
(possible separate genus); the family paramyxoviridae, including
the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus,
Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle
Disease Virus, Mumps virus), the genus Morbillivirus (Measles
virus, subacute sclerosing panencephalitis virus, distemper virus,
Rinderpest virus), the genus Pneumovirus (respiratory syncytial
virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus
of mice); the family Rhabdoviridae, including the genus
Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus),
the crenus Lyssavirus (Rabies virus), fish Rhabdoviruses, and two
probable Rhabdoviruses (Marburg virus and Ebola virus); the family
Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),
Tacaribe virus complex, and Lassa virus; the family Coronoaviridae,
including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus,
Human enteric corona virus, and Feline infectious peritonitis
(Feline coronavirus).
[0089] Illustrative DNA viruses that are antigenic or immunogenic
in vertebrate animals include, but are not limited to: the family
Poxyiridae, including the genus Orthopoxvirus (Variola major,
Variola minor, Monkey pox Vaccinia, Cowpox, Buffalopo Rabbitpox,
Ectromelia), the genus Leporipoxvirus (Myxoma, Fibroma), the genus
Avipoxvirus (Fowlpox, other avian poxvirus), the genus
Capripoxvirus (sheeppox, goatpox), the genus Suipoxvirus
(Swinepox), the genus Parapoxvirus (contagious postular dermatitis
virus, pseudocowpox, bovine papular stomatitis virus); the family
Iridoviridae (African swine fever virus, Frog viruses 2 and 3,
Lymphocystis virus of fish); the family Herpesviridae, including
the alpha-Herpesviruses (Herpes Simplex Types 1 and 2,
Varicella-Zoster, Equine abortion virus, Equine herpes virus 2 and
3, pseudorabies virus, infectious bovine keratoconjunctivitis
virus, infectious bovine rhinotracheitis virus, feline
rhinotracheitis virus, infectious laryngotracheitis virus), the
Beta-herpesviruses (Human cytomegalovirus and cytomegaloviruses of
swine, monkeys and rodents), the gamma-herpesviruses (Epstein-Barr
virus (EBV), Marek's disease virus, Herpes saimiri, Herpesvirus
ateles, Herpesvirus sylvilagus, guinea pig herpes virus, Lucke
tumor virus); the family Adenoviridae, including the genus
Mastadenovirus (Human subgroups A, B, C, D, E and ungrouped; simian
adenoviruses (at least 23 serotypes), infectious canine hepatitis,
and adenoviruses of cattle, pigs, sheep, frogs and many other
species), the genus Aviadenovirus (Avian adenoviruses), and
non-cultivatable adenoviruses; the family Papoviridae, including
the genus Papillomavirus (Human papilloma viruses, bovine papilloma
viruses, Shope rabbit papilloma virus, and various pathogenic
papilloma viruses of other species), the genus Polyomavirus
(polyomavirus, Simian vacuolating agent (SV-40), Rabbit vacuolating
agent (RKV), K virus, BK virus, JC virus, and other primate polyoma
viruses such as Lymphotrophic papilloma virus); the family
Parvoviridae including the genus Adeno-associated viruses, the
genus Parvovirus (Feline panleukopenia virus, bovine parvovirus,
canine parvovirus, Aleutian mink disease virus, etc). Finally, DNA
viruses may include viruses which do not fit into the above
families such as Kuru and Creutzfeldt-Jacob disease viruses and
chronic infectious neuropathic agents.
[0090] Bacterial infections or diseases that can be treated by
methods of the present invention are caused by bacteria including,
but not limited to, bacteria that have an intracellular stage in
its life cycle, such as mycobacteria (e.g., Mycobacteria
tuberculosis, M. bovis, M. avium, M. leprae, or M. africanum),
rickettsia, mycoplasma, chlamydia, and legionella. Other examples
of bacterial infections contemplated include, but are not limited
to, infections caused by Gram positive bacillus (e.g., Listeria,
Bacillus such as Bacillus anthracis, Erysipelothrix species), Gram
negative bacillus (e.g., Bartonella, Brucella, Campylobacter,
Enterobacter, Escherichia, Francisella, Hemophilus, Klebsiella,
Morganella, Proteus, Providencia, Pseudomonas, Salmonella,
Serratia, Shigella, Vibrio, and Yersinia species), spirochete
bacteria (e.g., Borrelia species including Borrelia burgdorferi
that causes Lyme disease), anaerobic bacteria (e.g., Actinomyces
and Clostridium species), Gram positive and negative coccal
bacteria, Enterococcus species, Streptococcus species, Pneumococcus
species, Staphylococcus species, Neisseria species. Specific
examples of infectious bacteria include, but are not limited to:
Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,
Mycobacteria tuberculosis, M. avium, M. intracellulare, kansaii, M.
gordonae, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus viridans, Streptococcus, faecalis, Streptococcus
bovis, Streptococcus pneumoniae, Haemophilus influenzae, Bacillus
antracis, corynebacterium diphtheriae, Erysipelothrix
rhusiopathiae, Clostridium perfringens, Clostridium tetani,
Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella
multocida, Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia,
and Actinomyces israelli.
[0091] Fungal diseases that can be treated by methods of the
present invention include, but are not limited to, aspergilliosis,
crytococcosis, sporotrichosis, coccidioidomycosis,
paracoccidioidomycosis, histoplasmosis, blastomycosis, zygomycosis,
and candidiasis.
[0092] Parasitic diseases that can be treated by methods of the
present invention include, but are not limited to, amebiasis,
malaria, leishmania, coccidia, giardiasis, cryptosporidiosis,
toxoplasmosis, and trypanosomiasis. Also encompassed are infections
by various worms such as, but not limited to, ascariasis,
ancylostomiasis, trichuriasis, strongyloidiasis, toxoccariasis,
trichinosis, onchocerciasis, filaria, and dirofilariasis. Also
encompassed are infections by various flukes such as, but not
limited to, schistosomiasis, paragonimiasis, and clonorchiasis.
Parasites that cause these diseases can be classified based on
whether they are intracellular or extracellular. An "intracellular
parasite," as used herein, is a parasite whose entire life cycle is
intracellular. Examples of human intracellular parasites include
Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Toxoplasma
gondii. Babesia spp., and Trichinella spiralis. An "extracellular
parasite," as used herein, is a parasite whose entire life cycle is
extracellular. Extracellular parasites capable of infecting humans
include Entamoeba histolytica, Giardia lamblia. Enterocytozoon
bieneusi, Naegleria and Acanthamoeba as well as most helminths. Yet
another class of parasites is defined as being mainly extracellular
but with an obligate intracellular existence at a critical stage in
their life cycles. Such parasites are referred to herein as
"obligate intracellular parasites." These parasites may exist most
of their lives or only a small portion of their lives in an
extracellular environment, but they all have at least one obligate
intracellular stage in their life cycles. This latter category of
parasites includes Trypanosoma rhodesiense and Trypanosoma
gambiense, Isospora spp., Cryptosporidium spp, Eimeria spp.,
Neospora spp., Sarcocystis spp., and Schistosoma spp.
[0093] 5.2 Allergens
[0094] This invention encompasses methods of reducing or inhibiting
allergic reaction to an allergen in a subject comprising
administering to the subject an immunomodulatory compound of the
invention prior to the subject's exposure to an allergen.
Optionally, in addition to the administration before the exposure
to an allegen, an immunomodulatory compound may be administered
during and/or after the subject's exposure to an allergen. It is
contemplated that any types of exposure to allergens including, but
not limited to, the subject's exposure to naturally occurring
allergens, exposure by the intake of food, and exposure through
allergy vaccine administration, are encompassed by methods of this
invention.
[0095] Examples of allergens (e.g., naturally occurring or those
contained in allergy vaccines) include, but are not limited to,
allergens from:
[0096] mites such as, but not limited to, Dermatophagoides farinae,
Dermatophagoides pteronyssinus, Acarus siro, Blomia tropicalis,
Chortoglyphus arcuatas, Euroglyphus maynei, Lepidoglyphus
destructor, Tyrophagus putrescentiae, and Glyphagus demesticus;
[0097] venoms such as, but not limited to, Bombus spp., Vespa
crabro, Apis mellifera, Dolichovespula spp., Polistes spp., Vespula
spp., Dolichovespula maculata, and Dolichovespula arenaria;
[0098] insects such as, but not limited to, Camponotus
pennsylvanicus, Solenopsis invicta, Solenopsis richteri,
Periplaneta americana, Blattella germanica, Blatta orientals,
Tebanus spp., Musca domestics, Ephemeroptera spp., Culicidae sp.,
and Heterocera spp.;
[0099] epithelia, dander, hair and features such as, but not
limited to, Serinus canaria, Felts catus (domesticus), Bos taurus,
Gallus gallus (domesticus), Canis jarniliaris, Anas platyrhynchos,
Meriones unguiculatus, Capra hircus, Anser domesticus, Cavia
porcellus (cobava), Mesocrietus auratus, Sus scrota, Equus
caballus, Mus musculus, Psitiacidae, Columba fasciata, Oryctolagus
cuniculus, Rattus norvegicus, and Ovis aries;
[0100] fungi such as, but not limited to, Cephalosporhun
acremonium, Alternaria tenuis, Aspergillus glaucus, Aspergillus
flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus
niger, Aspergillus terreus, Aspergillus versicolor, Aureobasidium
pullulans (Pullularia pullulans), Drechslera sorokiniana,
Helminthosporium sativum, Botrytis cinerea, Candida albicans,
Chaetomium globosum, Cladosporium herbarum, Cladosporium
sphaerospermum (Homodendrum hordei), Drechslera spicifera
(Curvularia spicifera), Epicoccum nigrum (Epicoccum purpurascens),
Epidermophyton floccosum, Fusarium inonilifbrme, Fusarium solani,
Geotrichum candidum, Gliocladium viride, Helminthosporium solani,
Microsporum canis, Mucor circinelloides f. circinelloides, Mucor
circinelloides f. lusitanicus, Mucor plumbeus, Mycogone perniciosa,
Neurospora intermedia, Nigrospora oryzae, Paecilomyces varioiii,
Penicillum brevi-compactum, Penicillum camembertii, Penicillum
chrysogenum, Penicillum digitatum, Penicillum expansum, Penicillum
notatum, Penicillum roquefortii, Phoma betae, Phoma herbarum,
Rhizopus oryzae, Rhizopus stolonifer, Rhodotorula mucilaginosa,
Saccharomyces cerevisiae, Scopulariopsis brevicaults, Serpula
lacrymans, Setosphaeria rostrata, Stemphyliutn botryosum,
Stemphylium solani, Trichoderma harzianum, Trichophyton
mentagrophytes, Trichophyton rubrum, and Trichothecium roseum;
[0101] smuts such as, but not limited to Ustilago nuda, Ustilago
cynodontis, Ustilago maydis, Sporisorium cruentum, Ustilago avenae,
and Ustilago
[0102] grasses such as, but not limited to, Paspalum notatum,
Cynodon dactylon, Poa compressa, Bromus inermis, Phalaris
arundinacea, Zea mays, Elyirigia repens (Agropyron repens), Sorghum
haelpense, Poa pratensis, Fesluca pratensis (elailor), Avena
saliva, Dactylis glomerata, Agrostis gigantea (alba), Secale
cereale, Leymus (Elymus) condensatus, Lolium perenne ssp.
multillorum, Lolium perenne, Amhoxanthum odoratum, Phleum pratense,
Holcus lanatus, Triticum aestivum, and Elymus (Agropyron)
smithii;
[0103] weeds such as, but not limited to, Atriplex polycarpa,
Baccharis halimilblia, Baccharis sarothroides, Hymenoclea salsola,
Amaranthus hybridus, Xanthium strumarium (commune), Rumex crispus,
Eupathium capillifolium, Solidago spp., Amaranthus tuberculatus
(Acnida tamariscina), Allenrolfea occidentalis, Chenopodium botrys,
Kochia scoparia, Chenopodium album, Iva xanthriblia, Iva
angustijblia, Chenopodium ambrosioides, Artemisia vulgaris,
Artemisia ludoviciana, Urtica dioica, Amaranthus spinosus, Plantago
lanceolakt, Iva axillaris, Atriplex lentilbrmis, Ambrosia dumosa,
Ambrosia acanthicarpa, Ambrosia trifida, Ambrosia artemisiijblia,
Ambrosia conlertiflora, Ambrosia bidentata, Ambrosia psilostachya,
Salsola kali (pestifer), Artemisia californica, Artemisia frigida,
Artemisia tridentata, Atriplex wrightii, Atriplex confertifblia,
and Artemisia annua;
[0104] trees such as, but not limited to, Acasia spp., Alnus
glutinosa, Alnus rubra, Alnus incana ssp. rugosa, Alnus
rhombilblia, Fraxinus velutina, Fraxinus pennsylvanica, Fraxinus
latifblia, Fraxinus americana, Populus tremuloides, Myrica
cerifera, Fagus grandifolia (americana), Casuarina equisettj blia,
Betula lenta, Betula pendula, Betula nigra, Betula occudentalis
(fantinalis), Betula populifblia, Acer negundo, Cryptomeria
japonica, Juniperus ashei (sabinoides), Juniperus virginiana,
Tamarix gallica, Populus balsamifera ssp. trichocarpa, Populus
deltoides, Populus fremontii, Populus wislizeni, Populus monilifera
(sargentii), Cupressus arizonoca, Taxodium distichum, Cupressus
sempervirens, Ulmus americana, Ulmus crassyblia, Ulmus pumila,
Eucalyptus globulus, Celtis occidentalis, Corylus americana,
Corylus avellana, Carya ovata, Carya laciniosa, Carya alba,
Junperus monosperma, Juniperus princhotii, Juniperus scopulorum,
Juniperus occidentalis, Robinia pseudoacacia, Mangifera indica,
Acer macrophyllum, Acer rubrum, Acer saccharum, Melaleuca
quinquenervia (leucadendron), Prosopis glandulosa (juliflora),
Broussonetia papyrifera, Morus rubra, Morus alba, Quercus gambelii,
Quercus velutina, Quercus macrocarpa, Quercus kelloggii, Quercus
agrifolia, Quercus lobata, Quercus ilex, Quercus stellata, Quercus
rubra, Quercus dumosa, Quercus virginiana, Quercus nigra, Quercus
garryana, Quercus alba, Olea europaea, Elaegnus angustifblia,
Citrus sinensis, Arecastrum romanzolfianum (Cocos plumosa), Carya
illnoensis, Schinus molle, Schinus terebinthifolius, Pinus taeda,
Pinus strobus, Pinus palustris, Pinus ponderosa, Pinus elliottii,
Pinus virginiana, Pinus monticola, Pinus echinata, Populus nigra,
Populus alba, Ligustrum vulgare, Liquidambar styraciflua, Platanus
occidentalis, Platanus orientalis, Platanus racemosa, Platanus
acerifolia, Juglans nigra, Juglans californica, Juglans regia,
Salix lasiolepsis, Salix nigra, and Salix discolor;
[0105] flowers such as, but not limited to, Chrysanthemum
leucantheinum, Taraxacum qfficinale, and Helianthus annuus;
[0106] farm plants such as, but not limited to, Medicago sativa,
Ricinus communis, Trifolium pratense, Brassica spp., and Beta
vulgaris;
[0107] plant food such as, but not limited to, Prunus dulcis, Mains
pumila, Prunus arnieniaca, Musa paradisiaca (sapientum), Hordeutn
vulgare, Phaseolus lunatus, Phaseolus vulgaris, Phaseolus sp.,
Phaseolus sp., Phaseolus vulgaris, Rubus allegheniensis, Vaccinium
sp., Brassica oleracea var. botrytis, Fagopyrum esculentum,
Brassica oleracea vur. capitata, Theobroma cacao, Cucumis meld,
Daucus carota, Brassica oleracea var. botrytis Apium graveolens
var. dulce, Prunus sp., Cinnamomum vertim, Coffea arabic, Zea mays,
Vaccinium macrocarpon, Cucumis sativus, Allium sativum, Zingiber
qfficinale, Vitis.sp., Citrus paradisi, Humulus lupulus, Citrus
limon, Lactuca sativa, Agaricus campestris, Brassica sp., Myristica
fragrans, Avena saliva, Olea europaea, Allium cepa var. cepa,
Citrus sinensis, Vigna unguiculata, Pisum sativum, Prunus persica,
Pyrus communis, Piper nigrum, Capsicum annuutn var. annuum, Ananas
comosus. Ipomoea batatas, Solanum tuberosum, Rubus idaeus var.
idaeus, Oryza saliva, Secale cereale, Sesamum orientale (indicurn),
Glycine max, Spinacia oleracea, Cucurbita pepo var. melopepo,
Fragaria chiloensis, Lycopersicon esculentum (lycopersicum),
Brassica rapa var. rapa, Vanilla planifblia, Citrullus lanatus var.
lanatus, and Triticun aestivum;
[0108] fish and shellfish such as, but not limited to, Micropterus
sp., Ictalurus punctatus, Itiercenaria mercenaria, Gadus morhua,
Callinectes sapidus, Platichthys sp., Hippoglossus sp., Homarus
americanus, Scomber scombrus, Crassostrea virginica, Sebastes
marinus, Salmo salar, Clupeiformes, Pecten magellanicus, Penaeus
sp., Salvelinus sp., and Thunnus sp.;
[0109] animal foods such as, but not limited to, Bos taurus, Ovis
aries, and Sus scrota;
[0110] poultry products such as, but not limited to, chicken
(Gallus gallus) products and turkey (Meleagris gallopavo)
products;
[0111] dairy products such as, but not limited to, bovine casein
and bovine milk;
[0112] nuts such as, but not limited to, Bertholletia excelsa,
Anacardium occidentale, Cocos inicifera, Corylus americana, Arachis
hypogaea, Carya illinoensis, Juglans nigra, and Juglans regia;
[0113] miscellaneous allergens such as, but not limited to, those
from Gossypium hirsutum, Linum usitcnissimum, Acaia senegal,
Sterculia urens, Astragalus gummifer, Ceiba pentancira, Iris
germanica var. florentina, Chrysanthemum cinerariifblium, Bomhvx
mori, and Nicotiana tabacum;
[0114] dust such as, but not limited to, barley grain dust, corn
grain dust, house dust, mattress dust, oat grain dust, wheat grain
dust, and upholstery dust.
[0115] 5.3 Immunomodulatory Compounds
[0116] As used herein and unless otherwise indicated, the terms
"immunomodulatory compounds of the invention" and "IMiDs.RTM."
(Celgene Corporation) encompass certain small organic molecules
that inhibit LPS induced monocyte TNF-.alpha., IL-1.beta., 1L-12,
IL-6, MIP-1.alpha., MCP-1, GM-CSF, G-CSF, and COX-2 production.
Specific immunomodulatory compounds are discussed below.
[0117] TNF-.alpha. is an inflammatory cytokine produced by
macrophages and monocytes during acute inflammation. TNF-.alpha. is
responsible for a diverse range of signaling events within cells.
Without being limited by a particular theory, one of the biological
effects exerted by the immunomodulatory compounds of the invention
is the reduction of myeloid cell TNF-.alpha. production.
Immunomodulatory compounds of the invention may enhance the
degradation of TNF-.alpha. mRNA.
[0118] Further, without being limited by theory, immunomodulatory
compounds used in the invention may also be potent co-stimulators
of T cells and increase cell proliferation dramatically in a dose
dependent manner. Immunomodulatory compounds of the invention may
also have a greater co-stimulatory effect on the CD8+ T cell subset
than on the CD4+ T cell subset. In addition, the compounds
preferably have anti-inflammatory properties against myeloid cell
responses, yet efficiently co-stimulate T cells to produce greater
amounts of IL-2, IFN-.gamma., and to enhance T cell proliferation
and CD8+ T cell cytotoxic activity. Further, without being limited
by a particular theory, immunomodulatory compounds used in the
invention may be capable of acting both indirectly through cytokine
activation and directly on Natural Killer ("NK") cells and Natural
Killer T ("NKT") cells, and increase the NK cells' ability to
produce beneficial cytokines such as, but not limited to,
IFN-.gamma., and to enhance NK and NKT cell cytotoxic activity.
[0119] Specific examples of immunomodulatory compounds include
cyano and carboxy derivatives of substituted styrenes such as those
disclosed in U.S. Pat. No. 5,929,117;
1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and
1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such
as those described in U.S. Pat. Nos. 5,874,448 and 5,955,476; the
tetra substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolines
described in U.S. Pat. No. 5,798,368; 1-oxo and
1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines (e.g., 4-methyl
derivatives of thalidomide), substituted
2-(2,6-dioxopiperidin-3-yl) phthalimides and substituted
2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles including, but not
limited to, those disclosed in U.S. Pat. Nos. 5,635,517, 6,281,230,
6,316.471, 6,403,613, 6,476,052 and 6,555,554; 1-oxo and
1,3-dioxoisoindolines substituted in the 4- or 5-position of the
indo line ring (e.g.,
4-(4-amino-1,3-dioxoisoindoline-2-yl)-4-carbamoylbutanoic acid)
described in U.S. Pat. No. 6,380,239; isoindoline-1-one and
isoindoline-1,3-dione substituted in the 2-position with
2,6-dioxo-3-hydroxypiperidin-5-yl (e.g.,
2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindolin-1-
-one) described in U.S. Pat. No. 6,458,810; a class of
non-polypeptide cyclic amides disclosed in U.S. Pat. Nos. 5,698,579
and 5,877,200; and isoindole-imide compounds such as those
described in U.S. patent publication no. 2003/0045552 published on
Mar. 6, 2003, U.S. patent publication no. 2003/0096841 published on
May 22, 2003, and International Application No. PCT/USO 1/50401
(International Publication No. WO 02/059106). The entireties of
each of the patents and patent applications identified herein are
incorporated herein by reference. Immunomodulatory compounds do not
include thalidomide.
[0120] Various immunomodulatory compounds of the invention contain
one or more chiral centers, and can exist as racemic mixtures of
enantiomers or mixtures of diastereomers. This invention
encompasses the use of stereomerically pure forms of such
compounds, as well as the use of mixtures of those fauns. For
example, mixtures comprising equal or unequal amounts of the
enantiomers of a particular immunomodulatory compounds of the
invention may be used in methods and compositions of the invention.
These isomers may be asymmetrically synthesized or resolved using
standard techniques such as chiral columns or chiral resolving
agents. See, e.g., Jacques, J., et al., Enantiomers, Racemates and
Resolutions (Wiley-Interscience, New York, 1981); Wilen, S. H. et
al., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of
Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H., Tables
of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel,
Ed., Univ. of Notre Dame Press, Notre Dame, Ind., 1972).
[0121] Preferred immunomodulatory compounds of the invention
include, but are not limited to, 1-oxo- and 1,3
dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines substituted with
amino in the benzo ring as described in U.S. Pat. No. 5,635,517
which is incorporated herein by reference. These compounds have the
structure I:
##STR00001##
in which one of X and Y is C.dbd.O, the other of X and Y is C.dbd.O
or CH.sub.2, and R.sup.2 is hydrogen or lower alkyl, in particular
methyl. Specific immunomodulatory compounds include, but are not
limited to:
##STR00002##
1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;
##STR00003##
1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; and
##STR00004##
1,3-dioxo-2-(3-methyl-2,6-dioxopiperidin-3-yl)-4-aminoisoindole,
and optically pure isomers thereof. The compounds can be obtained
via standard, synthetic methods (see e.g., U.S. Pat. No. 5,635,517,
incorporated herein by reference). The compounds are also available
from Celgene Corporation, Warren, N.J.
[0122] As used herein, and unless otherwise indicated, the term
"optically pure" means a composition that comprises one optical
isomer of a compound and is substantially free of other isomers of
that compound. For example, an optically pure composition of a
compound having one chiral center will be substantially free of the
opposite enantiomer of the compound. An optically pure composition
of a compound having two chiral centers will be substantially free
of other diastereomers of the compound. A typical optically pure
compound comprises greater than about 80% by weight of one
enantiomer of the compound and less than about 20% by weight of
other enantiomers of the compound, more preferably greater than
about 90% by weight of one enantiomer of the compound and less than
about 10% by weight of the other enantiomers of the compound, even
more preferably greater than about 95% by weight of one enantiomer
of the compound and less than about 5% by weight of the other
enantiomers of the compound, more preferably greater than about 97%
by weight of one enantiomer of the compound and less than about 3%
by weight of the other enantiomers of the compound, and most
preferably greater than about 99% by weight of one enantiomer of
the compound and less than about 1% by weight of the other
enantiomers of the compound.
[0123] Other specific immunomodulatory compounds of the invention
belong to a class of substituted 2-(2,6-dioxopiperidin-3-yl)
phthalimides and substituted
2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles, such as those
described in U.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and
6,476,052, and International Patent Application No. PCT/US97/13375
(International Publication No. WO 98/03502), each of which is
incorporated herein by reference. Representative compounds are of
formula:
##STR00005##
in which:
[0124] one of X and Y is C.dbd.O and the other of X and Y is
C.dbd.O or CH.sub.2;
[0125] (i) each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4,
independently of the others, is halo, alkyl of 1 to 4 carbon atoms,
or alkoxy of 1 to 4 carbon atoms or (ii) one of R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 is --NHR.sup.5 and the remaining of R',
R.sup.2, R.sup.3, and R.sup.4 are hydrogen;
[0126] R.sup.5 is hydrogen or alkyl of 1 to 8 carbon atoms;
[0127] R.sup.6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl,
or halo;
[0128] provided that R.sup.6 is other than hydrogen if X and Y are
C.dbd.O and (i) each of R', R.sup.2, R.sup.3, and R.sup.4 is fluoro
or (ii) one of R', R.sup.2, R.sup.3, or R.sup.4 is amino. Compounds
representative of this class are of the formulas:
##STR00006##
wherein R.sup.1 is hydrogen or methyl. In a separate embodiment,
the invention encompasses the use of enantiomerically pure forms
(e.g. optically pure (R) or (S) enantiomers) of these
compounds.
[0129] Still other specific immunomodulatory compounds of the
invention belong to a class of isoindole-imides disclosed in U.S.
Patent Application Publication Nos. 2003/0096841 and US
2003/0045552, and International Application No. PCT/US01/50401
(International Publication No. WO 02/059106), each of which are
incorporated herein by reference. Representative compounds are of
formula II:
##STR00007##
and pharmaceutically acceptable salts, hydrates, solvates,
clathrates, enantiomers, diastereomers, racemates, and mixtures of
stereoisomers thereof, wherein:
[0130] one of X and Y is C.dbd.O and the other is CH.sub.2 or
C.dbd.O;
[0131] R.sup.1 is H, (C.sub.1-C.sub.8)alkyl,
(C.sub.3-C.sub.7)cycloalkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, benzyl, aryl,
(C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl,
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl, C(O)R.sup.3,
C(S)R.sup.3, C(O)OR.sup.4, (C.sub.1-C.sub.8)alkyl-N(R.sup.6).sub.2,
(C.sub.1-C.sub.8)alkyl-OR.sup.5,
(C.sub.1-C.sub.8)alkyl-C(O)OR.sup.5, C(O)NHR.sup.3, C(S)NHR.sup.3,
C(O)NR.sup.3R.sup.3', C(S)NR.sup.3R.sup.3' or
(C.sub.1-C.sub.8)alkyl-O(CO)R.sup.5;
[0132] R.sup.2 is H. F, benzyl, (C.sub.1-C.sub.8)alkyl,
(C.sub.2-C.sub.8)alkenyl, or (C.sub.2-C.sub.8)alkynyl;
[0133] R.sup.3 and R.sup.3 are independently
(C.sub.1-C.sub.8)alkyl, (C.sub.3-C.sub.7)cycloalkyl,
(C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, benzyl, aryl,
(C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl,
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl,
(C.sub.0-C.sub.8)alkyl-N(R.sup.6).sub.2,
(C.sub.1-C.sub.8)alkyl-OR.sup.5,
(C.sub.1-C.sub.8)alkyl-C(O)OR.sup.5,
(C.sub.2-C.sub.8)alkyl-O(CO)R.sup.5, or C(O)OR';
[0134] R.sup.4 is (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, (C.sub.1-C.sub.4)alkyl-OR.sup.5, benzyl,
aryl, (C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl, or
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl;
[0135] R.sup.5 is (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, benzyl, aryl, or
(C.sub.2-C.sub.5)heteroaryl:
[0136] each occurrence of R.sup.6 is independently H,
(C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, benzyl, aryl,
(C.sub.2-C.sub.8)heteroaryl, or
(C.sub.0-C.sub.8)alkyl-C(O)O--R.sup.5 or the R.sup.6 groups can
join to form a heterocycloalkyl group;
[0137] n is 0 or 1; and
[0138] * represents a chiral-carbon center.
In specific compounds of formula II, when n is 0 then R.sup.1 is
(C.sub.3-C.sub.7)cycloalkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, benzyl, aryl,
(C.sub.0-C.sub.4alkyl-(C.sub.1-C.sub.6)heterocycloalkyl,
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.8)heteroaryl, C(O)R.sup.3,
C(O)OR.sup.4, (C.sub.1-C.sub.8)alkyl-N(R.sup.6).sub.2,
(C.sub.1-C.sub.8)alkyl-OR.sup.5,
(C.sub.1-C.sub.8)alkyl-C(O)OR.sup.5, C(S)NHR.sup.3, or
(C.sub.1-C.sub.8)alkyl-O(CO)R.sup.5;
[0139] R.sup.2 is H or (C.sub.1-C.sub.8)alkyl: and
[0140] R.sup.3 is (C.sub.1-C.sub.8)alkyl,
(C.sub.3-C.sub.7)cycloalkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, benzyl, aryl,
(C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl,
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl,
(C.sub.5-C.sub.8)alkyl-N(R.sup.6).sub.2;
(C.sub.0-C.sub.8)alkyl-NH--C(O)O--R.sup.5:
(C.sub.1-C.sub.8)alkyl-OR.sup.5,
(C.sub.1-C.sub.8)alkyl-C(O)OR.sup.5,
(C.sub.1-C.sub.8)alkyl-O(CO)R.sup.5, or C(O)OR.sup.5; and the other
variables have the same definitions.
[0141] In other specific compounds of formula II, R.sup.2 is H or
(C.sub.1-C.sub.4)alkyl.
[0142] In other specific compounds of formula II, R.sup.1 is
(C.sub.1-C.sub.8)alkyl or benzyl.
[0143] In other specific compounds of formula II, R.sup.1 is H,
(C.sub.1-C.sub.8)alkyl, benzyl, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2OCH.sub.3, or
##STR00008##
[0144] In another embodiment of the compounds of formula II,
R.sup.1 is
##STR00009##
[0145] wherein Q is O or S, and each occurrence of R.sup.7 is
independently H,(C.sub.1-C.sub.8)alkyl,
(C.sub.3-C.sub.7)cycloalkyl, (C.sub.2-C.sub.8)alkenyl,
(C.sub.2-C.sub.8)alkynyl, benzyl, aryl, halogen,
(C.sub.0-C.sub.4)alkyl-(C.sub.1-C.sub.6)heterocycloalkyl,
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl,
(C.sub.0-C.sub.8)alkyl-N(R.sup.6).sub.2,
(C.sub.1-C.sub.8)alkyl-OR.sup.5,
(C.sub.1-C.sub.8)alkyl-C(O)OR.sup.5,
(C.sub.1-C.sub.8)alkyl-O(CO)R.sup.5, or C(O)OR.sup.5, or adjacent
occurrences of R.sup.7 can be taken together to form a bicyclic
alkyl or aryl ring.
[0146] In other specific compounds of formula II, R.sup.1 is
C(O)R.sup.3.
[0147] In other specific compounds of formula II, R.sup.3 is
(C.sub.0-C.sub.4)alkyl-(C.sub.2-C.sub.5)heteroaryl,
(C.sub.1-C.sub.8)alkyl, aryl, or
(C.sub.0-C.sub.4)alkyl-OR.sup.5.
[0148] In other specific compounds of formula II, heteroaryl is
pyridyl, furyl, or thienyl.
[0149] In other specific compounds of formula II, R.sup.1 is
C(O)OR.sup.4.
[0150] In other specific compounds of formula II, the H of
C(O)NHC(O) can be replaced with (C.sub.1-C.sub.4)alkyl, aryl, or
benzyl.
[0151] Further examples of the compounds in this class include, but
are not limited to:
[2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethy-
l]-amide;
(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol--
4-ylmethyl)-carbamic acid tert-butyl ester;
4-(aminomethyl)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione;
N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmet-
hyl)-acetamide;
N-{(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl)methyl}cyclopropyl-
-carboxamide;
2-chloro-N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}a-
cetamide;
N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-3-pyridy-
lcarboxamide;
3-{1-oxo-4-(benzylamino)isoindolin-2-yl}piperidine-2,6-dione;
2-(2,6-dioxo(3-piperidyl))-4-(benzylamino)isoindoline-1,3-dione:
N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}propanamid-
e;
N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-3-pyrid-
ylcarboxamide;
N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}heptanamid-
e;
N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-2-furyl-
carboxamide;
{N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)carbamoyl}methyl
acetate;
N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)pentanami-
de;
N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-2-thienylcarbo-
xamide;
N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(bu-
tylamino)carboxamide;
N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(octylamin-
o)carboxamide; and
N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(benzylami-
no)carboxamide.
[0152] Still other specific immunomodulatory compounds of the
invention belong to a class of isoindole-imides disclosed in U.S.
Patent Application Publication Nos. 2002/0045643, International
Publication No. WO 98/54170, and U.S. Pat. No. 6,395,754, each of
which is incorporated herein by reference. Representative compounds
are of formula III:
##STR00010##
and pharmaceutically acceptable salts, hydrates, solvates,
clathrates, enantiomers, diastereomers, racemates, and mixtures of
stereoisomers thereof, wherein:
[0153] one of X and Y is C.dbd.O and the other is CH.sub.2 or
C.dbd.O;
[0154] R is H or CH.sub.2OCOR'';
[0155] (i) each of R.sup.1, R.sup.2, R.sup.3, or R.sup.4,
independently of the others, is halo, alkyl of 1 to 4 carbon atoms,
or alkoxy of 1 to 4 carbon atoms or (ii) one of R', R.sup.2,
R.sup.3, or R.sup.4 is nitro or --NHR.sup.5 and the remaining of
R.sup.1, R.sup.2, R.sup.3, or R.sup.4 are hydrogen;
[0156] R.sup.5 is hydrogen or alkyl of 1 to 8 carbons
[0157] R.sup.6 hydrogen, alkyl of 1 to 8 carbon atoms, benzo,
chloro, or fluoro;
[0158] R' is R.sup.7--CHR.sup.10--N(R.sup.8R.sup.9);
[0159] R.sup.7 is m-phenylene or p-phenylene or
--(C.sub.nH.sub.2n)-- in which n has a value of 0 to 4;
[0160] each of R.sup.8 and R.sup.9 taken independently of the other
is hydrogen or alkyl of 1 to 8 carbon atoms, or R.sup.8 and R.sup.9
taken together are tetramethylene, pentamethylene, hexamethylene,
or --CH.sub.2CH.sub.2X.sub.1CH.sub.2CH.sub.2-- in which X.sub.1 is
--O--, --S--, or --NH--;
[0161] R.sup.10 is hydrogen, alkyl of to 8 carbon atoms, or phenyl;
and
[0162] * represents a chiral-carbon center.
[0163] Other representative compounds are of formula:
##STR00011##
wherein:
[0164] one of X and Y is C.dbd.O and the other of X and Y is
CO.dbd.O or CHI;
[0165] (i) each of R.sup.1, R.sup.2, R.sup.3, or R.sup.4,
independently of the others, is halo, alkyl of 1 to 4 carbon atoms,
or alkoxy of 1 to 4 carbon atoms or (ii) one of R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 is --NHR.sup.3 and the remaining of R.sup.1,
R.sup.2, R.sup.3, and R.sup.4 are hydrogen;
[0166] R.sup.5 is hydrogen or alkyl of 1 to 8 carbon atoms;
[0167] R.sup.6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzo,
chloro, or fluoro;
[0168] R.sup.7 is m-phenylene or p-phenylene or
--(C.sub.nH.sub.2n)--in which n has a value of 0 to 4;
[0169] each of R.sup.8 and R.sup.9 taken independently of the other
is hydrogen or alkyl of 1 to 8 carbon atoms, or R.sup.8 and R.sup.9
taken together are tetramethylene, pentamethylene, hexamethylene,
or --CH.sub.2CH.sub.2X.sup.1CH.sub.2CH.sub.2-- in which X.sup.1 is
--O--, --S--, or --NH--; and
[0170] R.sup.10 is hydrogen, alkyl of to 8 carbon atoms, or
phenyl.
[0171] Other representative compounds are of formula:
##STR00012##
in which
[0172] one of X and Y is C.dbd.O and the other of X and Y is
C.dbd.O or CH.sub.2;
[0173] each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4,
independently of the others, is halo, alkyl of 1 to 4 carbon atoms,
or alkoxy of 1 to 4 carbon atoms or (ii) one of R', R.sup.2,
R.sup.3, and R.sup.4 is nitro or protected amino and the remaining
of R', R.sup.2, R.sup.3, and R.sup.4 are hydrogen; and
[0174] R.sup.6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzo,
chloro, or fluoro.
[0175] Other representative compounds are of formula:
##STR00013##
in which:
[0176] one of X and Y is C.dbd.O and the other of X and Y is
C.dbd.O or CH.sub.2;
[0177] (i) each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4,
independently of the others, is halo, alkyl of 1 to 4 carbon atoms,
or alkoxy of 1 to 4 carbon atoms or (ii) one of R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 is --NHR.sup.5 and the remaining of R.sup.1,
R.sup.2, R.sup.3, and R.sup.4 are hydrogen;
[0178] R.sup.5 is hydrogen, alkyl of 1 to 8 carbon atoms, or
CO--R.sup.7--CH(R.sup.10)NR.sup.8R.sup.9 in which each of R.sup.7,
R.sup.8, R.sup.9, and R.sup.10 is as herein defined; and
[0179] R.sup.6 is alkyl of 1 to 8 carbon atoms, benzo, chloro, or
fluoro. Specific examples of the compounds are of formula:
##STR00014##
in which:
[0180] one of X and Y is C.dbd.O and the other of X and Y is
C.dbd.O or CH.sub.2;
[0181] R.sup.6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl,
chloro, or fluoro;
[0182] R.sup.7 is m-phenylene, p-phenylene or --(C.sub.nH.sub.2n)--
in which n has a value of 0 to 4; each of R.sup.8 and R.sup.9 taken
independently of the other is hydrogen or alkyl of 1 to 8 carbon
atoms, or R.sup.8 and R.sup.9 taken together are tetramethylene,
pentamethylene, hexamethylene, or
--CH.sub.2CH.sub.2X.sup.1CH.sub.2CH.sub.2-- in which X.sup.1 is
--O--, --S-- or --NH--; and
[0183] R.sup.10 is hydrogen, alkyl of 1 to 8 carbon atoms, or
phenyl.
[0184] Other specific immunomodulatory compounds of the invention
include, but are not limited to,
1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3y1) isoindolines and
1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such
as those described in U.S. Pat. Nos. 5,874,448 and 5,955,476, each
of which is incorporated herein by reference. Representative
compounds are of formula:
##STR00015##
wherein:
[0185] Y is oxygen or H.sup.2 and
[0186] each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4,
independently of the others, is hydrogen, halo, alkyl of 1 to 4
carbon atoms, alkoxy of 1 to 4 carbon atoms, or amino.
[0187] Other specific immunomodulatory compounds of the invention
include, but are not limited to, the tetra substituted
2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolines described in U.S.
Pat. No. 5,798,368, which is incorporated herein by reference.
Representative compounds are of formula:
##STR00016##
wherein each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4,
independently of the others, is halo, alkyl of 1 to 4 carbon atoms,
or alkoxy of 1 to 4 carbon atoms.
[0188] Other specific immunomodulatory compounds of the invention
include, but are not limited to, 1-oxo and
1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines disclosed in
U.S. Pat. No. 6,403,613, which is incorporated herein by reference.
Representative compounds are of formula:
##STR00017##
in which
[0189] Y is oxygen or H.sub.2,
[0190] a first of R.sup.1 and R.sup.2 is halo, alkyl, alkoxy,
alkylamino, dialkylamino, cyano, or carbamoyl, the second of
R.sup.1 and R.sup.2, independently of the first, is hydrogen, halo,
alkyl, alkoxy, alkylamino, dialkylamino, cyano, or carbamoyl,
and
[0191] R.sup.3 is hydrogen, alkyl, or benzyl.
[0192] Specific examples of the compounds are of formula:
##STR00018##
wherein
[0193] a first of R.sup.1 and R.sup.2 is halo, alkyl of from 1 to 4
carbon atoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in
which each alkyl is of from 1 to 4 carbon atoms, cyano, or
carbamoyl;
[0194] the second of R.sup.1 and R.sup.2, independently of the
first, is hydrogen, halo, alkyl of from 1 to 4 carbon atoms, alkoxy
of from 1 to 4 carbon atoms, alkylamino in which alkyl is of from 1
to 4 carbon atoms, dialkylamino in which each alkyl is of from 1 to
4 carbon atoms, cyano, or carbamoyl; and
[0195] R.sup.3 is hydrogen, alkyl of from 1 to 4 carbon atoms, or
benzyl. Specific examples include, but are not limited to,
1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.
[0196] Other representative compounds are of formula:
##STR00019##
wherein:
[0197] a first of R.sup.1 and R.sup.2 is halo, alkyl of from 1 to 4
carbon atoms, alkoxy of from 1 to 4 carbon atoms, dialkylamino in
which each alkyl is of from 1 to 4 carbon atoms, cyano, or
carbamoyl;
[0198] the second of R.sup.1 and R.sup.2, independently of the
first, is hydrogen, halo, alkyl of from 1 to 4 carbon atoms, alkoxy
of from 1 to 4 carbon atoms, alkylamino in which alkyl is of from 1
to 4 carbon atoms, dialkylamino in which each alkyl is of from 1 to
4 carbon atoms, cyano, or carbamoyl; and
[0199] R.sup.3 is hydrogen, alkyl of from 1 to 4 carbon atoms, or
benzyl.
[0200] Other specific immunomodulatory compounds of the invention
include, but are not limited to, 1-oxo and 1,3-dioxoisoindolines
substituted in the 4- or 5-position of the indoline ring described
in U.S. Pat. No. 6,380,239 and co-pending U.S. application Ser. No.
10/900,270, filed Jul. 28, 2004, which are incorporated herein by
reference. Representative compounds are of formula:
##STR00020##
in which the carbon atom designated C* constitutes a center of
chirality (when n is not zero and R.sup.1 is not the same as
R.sup.2); one of X.sup.1 and X.sup.2 is amino, nitro, alkyl of one
to six carbons, or NH--Z, and the other of X.sup.1 or X.sup.2 is
hydrogen; each of R.sup.1 and R.sup.2 independent of the other, is
hydroxy or NH--Z; R.sup.3 is hydrogen, alkyl of one to six carbons,
halo, or haloalkyl; Z is hydrogen, aryl, alkyl of one to six
carbons, formyl, or acyl of one to six carbons; and n has a value
of 0, 1, or 2; provided that if X' is amino, and n is 1 or 2, then
R.sup.1 and R.sup.2 are not both hydroxy; and the salts
thereof.
[0201] Further representative compounds are of formula:
##STR00021##
in which the carbon atom designated C* constitutes a center of
chirality when n is not zero and R.sup.1 is not R.sup.2; one of
X.sup.1 and X.sup.2 is amino, nitro, alkyl of one to six carbons,
or NH--Z, and the other of X.sup.1 or X.sup.2 is hydrogen; each of
R.sup.1 and R.sup.2 independent of the other, is hydroxy or NH--Z;
R.sup.3 is alkyl of one to six carbons, halo, or hydrogen; Z is
hydrogen, aryl or an alkyl or acyl of one to six carbons; and n has
a value of 0, 1, or 2.
[0202] Specific examples include, but are not limited to,
2-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric
acid and
4-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric
acid, which have the following structures, respectively, and
pharmaceutically acceptable salts, solvates, prodrugs, and
stereoisomers thereof:
##STR00022##
[0203] Other representative compounds are of formula:
##STR00023##
in which the carbon atom designated C* constitutes a center of
chirality when n is not zero and R.sup.1 is not R.sup.2; one of
X.sup.1 and X.sup.2 is amino, nitro, alkyl of one to six carbons,
or NH--Z, and the other of X.sup.1 or X.sup.2 is hydrogen; each of
R.sup.1 and R.sup.2 independent of the other, is hydroxy or NH--Z;
R.sup.3 is alkyl of one to six carbons, halo, or hydrogen; Z is
hydrogen, aryl, or an alkyl or acyl of one to six carbons; and n
has a value of 0.1, or 2; and the salts thereof.
[0204] Specific examples include, but are not limited to,
4-carbamoyl-4-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoind-
ol-2-yl}-butyric acid,
4-carbamoyl-2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoind-
ol-2-yl}-butyric acid,
2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-4-p-
henylcarbamoyl-butyric acid, and
2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-pen-
tanedioic acid, which have the following structures, respectively,
and pharmaceutically acceptablesalts, solvate, prodrugs, and
stereoisomers thereof:
##STR00024##
[0205] Other specific examples of the compounds are of formula:
##STR00025##
wherein:
[0206] one of X.sup.1 and X.sup.2 is nitro, or NH--Z, and the other
of X.sup.1 or X.sup.2 is hydrogen;
[0207] each of R.sup.1 and R.sup.2, independent of the other, is
hydroxy or NH--Z;
[0208] R.sup.3 is alkyl of one to six carbons, halo, or
hydrogen;
[0209] Z is hydrogen, phenyl, an acyl of one to six carbons, or an
alkyl of one to six carbons; and
[0210] n has a value of 0, 1, or 2; and
[0211] if --COR.sup.2 and --(CH.sub.2).sub.nCOR.sup.1 are
different, the carbon atom designated C* constitutes a center of
chirality.
[0212] Other representative compounds are of formula:
##STR00026##
wherein:
[0213] one of X.sup.1 and X.sup.2 is alkyl of one to six
carbons;
[0214] each of R.sup.1 and R.sup.2, independent of the other, is
hydroxy or NH--Z;
[0215] R.sup.3 is alkyl of one to six carbons, halo, or
hydrogen;
[0216] Z is hydrogen, phenyl, an acyl of one to six carbons, or an
alkyl of one to six carbons; and
[0217] n has a value of 0, 1, or 2; and
[0218] if --COR.sup.2 and --(CH.sub.2).sub.nCOR.sup.1 are
different, the carbon atom designated C* constitutes a center of
chirality.
Still other specific immunomodulatory compounds of the invention
include, but are not limited to isoindoline-1-one and
isoindoline-1,3-dione substituted in the 2-position with
2,6-dioxo-3-hydroxypiperidin-5-yl described in U.S. Pat. No.
6,458,810, which is incorporated herein by reference.
Representative compounds are of formula:
##STR00027##
wherein:
[0219] the carbon atoms designated * constitute centers of
chirality;
[0220] X is --C(O)-- or --CH.sub.2--;
[0221] R.sup.1 is alkyl of 1 to 8 carbon atoms or --NHR.sup.3;
[0222] R.sup.2 is hydrogen, alkyl of 1 to 8 carbon atoms, or
halogen; and
[0223] R.sup.3 is hydrogen,
[0224] alkyl of 1 to 8 carbon atoms, unsubstituted or substituted
with alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1
to 4 carbon atoms.
[0225] cycloalkyl of 3 to 18 carbon atoms.
[0226] phenyl, unsubstituted or substituted with alkyl of 1 to 8
carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or
alkylamino of 1 to 4 carbon atoms,
[0227] benzyl, unsubstituted or substituted with alkyl of 1 to 8
carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or
alkylamino of 1 to 4 carbon atoms, or --COR.sup.4 in which
[0228] R.sup.4 is hydrogen,
[0229] alkyl of 1 to 8 carbon atoms, unsubstituted or substituted
with alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1
to 4 carbon atoms,
[0230] cycloalkyl of 3 to 18 carbon atoms,
[0231] phenyl, unsubstituted or substituted with alkyl of 1 to 8
carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or
alkylamino of 1 to 4 carbon atoms, or
[0232] benzyl, unsubstituted or substituted with alkyl of 1 to 8
carbon atoms, alkoxy of 1 to 8 carbon atoms, halo, amino, or
alkylamino of 1 to 4 carbon atoms.
[0233] All of the compounds described can either be commercially
purchased or prepared according to the methods described in the
patents or patent publications disclosed herein. Further, optically
pure compounds can be asymmetrically synthesized or resolved using
known resolving agents or chiral columns as well as other standard
synthetic organic chemistry techniques.
[0234] Compounds used in the invention may be small organic
molecules having a molecular weight less than about 1,000 g/mol,
and are not proteins, peptides, oligonucleotides, oligosaccharides
or other macromolecules.
[0235] It should be noted that if there is a discrepancy between a
depicted structure and a name given that structure, the depicted
structure is to be accorded more weight. In addition, if the
stereochemistry of a structure or a portion of a structure is not
indicated with, for example, bold or dashed lines, the structure or
portion of the structure is to be interpreted as encompassing all
stereoisomers of it.
[0236] 5.4 Methods of Treatment and Prevention
[0237] This invention encompasses methods of treating and/or
preventing (e.g., prophylactic treatment such as vaccination) of
various disorders using the dosing regimen involving an
immunomodulatory compounds of the invention as described
herein.
[0238] In one embodiment, this invention encompasses treatment or
prevention of cancer. Examples of cancer that can be treated or
prevented using methods of the invention include those described in
Section 5.1.2, above. In some embodiments, cancers to be treated or
prevented using methods of the invention are metastatic. In other
embodiment, specific cancers that can be treated or prevented using
methods of the invention are sarcoma, carcinoma, melanoma, lymphoma
and leukemia.
[0239] In another embodiment, this invention encompasses methods of
vaccinating against cancer by reducing the inhibition of anti-tumor
immune response in a subject (e.g., a human) comprising
administering to the subject an immunomodulatory compound of the
invention prior to the administration of a cancer vaccine. This
invention also encompasses methods of enhancing immune response to
a cancer vaccine in a subject comprising administering to the
subject an immunomodulatory compound of the invention prior to the
administration of a cancer vaccine. Examples of cancer vaccines
that can be used in connection with methods of the invention
include those listed in Tables 1-4. In specific embodiment, cancers
against which vaccination is performed are sarcoma, carcinoma,
melanoma, lymphoma and leukemia. In another specific embodiment,
the cancer vaccine is an antigen modified dendritic cell vaccine, a
peptide vaccine, a whole tumor cell vaccine, or a viral vector
vaccine.
[0240] In another embodiment, this invention also encompasses
treatment or prevention of an infectious disease. Examples of
infectious diseases that can be treated or prevented using methods
of the invention are described in Section 5.1.2, above. In some
embodiments, infectious diseases that can be treated or prevented
using methods of the invention include those caused by viruses,
bacteria, fungi, and parasites.
[0241] In another embodiment, this invention encompasses methods of
vaccinating against an infectious disease by reducing the
inhibition of immune response in a subject (e.g., a human)
comprising administering to the subject an immunomodulatory
compound of the invention prior to the administration of a vaccine
against an infectious disease. This invention also encompasses
methods of enhancing immune response to a vaccine against an
infectious disease in a subject comprising administering to the
subject an immunomodulatory compound of the invention prior to the
administration of the vaccine. Examples of infectious diseases
against which a subject can be vaccinated according to methods of
the invention are described in Section 5.1.1, above. In a specific
embodiment, infectious diseases are those caused by viruses,
bacteria, fungi, and parasites. In a specific embodiment, the
vaccine against an infectious disease is hepatitis B vaccine.
[0242] 5.5 Methods of Administration
[0243] Methods encompassed by this invention comprise administering
one or more immunomodulatory compounds, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or prodrug thereof, to a
subject (e.g., a human) prior to the exposure to or administration
of an immunogen or an allergen.
[0244] Any route of administration may be used. For example, an
immunomodulatory compound can be orally, parenterally,
transdermally, rectally, sublingually, mucosally, or nasally
administered. In addition, an immunomodulatory compounds can be
administered in a form of pharmaceutical composition and/or unit
dosage form. Suitable dosage forms include, but are not limited to,
capsules, tablets (including rapid dissolving and delayed release
tablets), powder, syrups, oral suspensions and solutions for
parenteral administration. Pharmaceutical compositions may contain
one of more pharmaceutically acceptable excipients. See, e.g., Rowe
et al., Handbook of Pharmaceutical Excipients, 4.sup.th Ed. (2003),
entirety of which is incorporated herein by reference. In addition,
an immunomodulatory compound of the invention may be included in a
kit, which may comprise an immunogen or an allergen, one or more
other active ingredients, and devices and directions for
administration. Other ingredients (e.g., immunogen, allergen, and
other active ingredients) may be included in the same formulation
with the immunomodulatory compound of the invention, or in separate
formulations.
[0245] The specific amount of the agent will depend on the specific
agent used, the type of disease or disorder being treated or
managed, and the amount(s) of an immunomodulatory compound of the
invention and any optional additional agents concurrently
administered to the patient. Typical dosage forms of the invention
comprise an immunomodulatory compound of the invention or a
pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug
thereof in an amount of from about 0.001 to about 150 mg. In
particular, dosage forms comprise an immunomodulatory compound of
the invention or a pharmaceutically acceptable salt, solvate,
stereoisomer, or prodrug thereof in an amount of about 0.001, 0.01,
0.1, 1, 2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50, 100, 150 or 200
mg. In a particular embodiment, a dosage form comprises
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione in an
amount of about 0.001, 0.01, 0.1, 1, 2, 5, 10, 25 or 50 mg.
[0246] In some embodiments, this invention encompasses
administration of racemic mixture, optically pure (R)-isomer, or
optically pure (S)-isomer of
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione. In one
specific embodiment, the racemic
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione is
administered at an amount of 1, 2, 5, 10, or 25 mg per day. As
(S)-isomer of
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione is
reported to have a higher potency than the racemic mixture, a lower
dose can be given when (S)-isomer is used. For examples,
(S)-4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione can
be administered at an amount of 0.01, 0.1, 1. 2.5, 5, or 10 mg per
day. (R)-isomer of
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione can be
administered at an amount comparable to the racemic mixture.
[0247] In a specific embodiment, a dosage form comprises
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione in
an amount of about 0.001, 0.01, 0.1, 1, 5, 10, 25 or 50 mg. Typical
dosage forms comprise the second active ingredient in an amount of
1 .mu.g to about 1000 mg, from about 0.01 to about 500 mg, from
about 0.1 to about 350 mg, or from about 1 to about 200 mg. This
invention also encompasses the use of racemic mixture, (S)-isomer,
and (R)-isomer of
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione.
Typically, racemic
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione
can be administered at an amount of 1, 5, 10, 15, 25, or 50 mg per
day. Optical isomers also can be administered at an amount
comparable to racemic mixture. Doses can be adjusted depending on
the type of disease or disorder being treated, prevented or
managed, and the amount(s) of an immunomodulatory compound of the
invention and any optional additional agents concurrently
administered to the patient, which are all within the skill of the
art.
6. EXAMPLES
6.1 Effects of IMiDs on Regulatory T Cells
[0248] An assay in which the ability of isolated T.sub.reg a to
suppress anti-CD3 mAb activated CD4+CD25-cells was performed.
Results showed that pre-incubation of T.sub.reg with
4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione
(Actimid.TM.) and
3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione
(lenalidomide), but not thalidomide, inhibits the suppressive
function of these cells. The inhibition of T regulatory cell
function and production by these compounds was shown to be not due
to any cytotoxic or apoptotic effects of the IMiDs on the cells,
but the inhibition of function was associated with a decrease in
FOXP3 expression in CTLA4+CD25.sup.highCD4+ cells.
[0249] 6.1.1 Effects on T.sub.reg Function
[0250] Regulatory T cells were isolated by the Dynal T regulatory
cell isolation kit, and treated for 24 hours with varying
concentrations of an immunomodulatory compound (Actimid.TM. or
lenalidomide) or DMSO. The cells were washed and incubated at a 1:2
ratio with CD25.sup.-CD4.sup.+ cells, which were also isolated by
the Dynal T regulatory cell isolation kit. Results were expressed
as the mean % change in proliferation compared to the cpms obtained
from DMSO treated CD25.sup.+ cells incubated with CD25.sup.- cells.
As shown in FIG. 2, pre-treatment of CD25.sup.+CD4.sup.+ cells with
the IMiDs tested significantly increased the proliferation of
CD25.sup.- cells in the presence of CD25.sup.+CD4.sup.+ cells as
compared to the DMSO treated CD4.sup.+CD25.sup.+ cells. Thalidomide
showed little effect under these assay conditions. The results
suggest that the IMiDs tested reduce or inhibit the suppressive
activity of regulatory T cells.
[0251] 6.1.2 Effects on Foxp3 Expression
[0252] CD4.sup.+CD25.sup.+ cells were incubated for 24 hours with
varying concentrations of DMSO, Actimid.TM., lenalidomide, or
thalidomide and then washed twice with RPMI medium. Cells were
stained with CD152-PE, CD4-PERCP, and CD25-APC. Intracellular Foxp3
staining and CD 152 staining were carried out after permeabilizing
the CD4.sup.-CD25.sup.+ cells. Results were expressed as percentage
of expression of Foxp3 in the CD4.sup.+CD25.sup.+ population or the
CD4.sup.+CD25.sup.- population. As shown in FIG. 3, cells
pre-treated with the IMiDs showed inhibition of Foxp3 expression,
while DMSO and thalidomide showed little effects. The results show
that the inhibition of Treg cells by the IMiDs tested may be
associated with the compounds' ability to inhibit Foxp3
expression.
[0253] 6.1.3 Effects on Level of T.sub.reg Cells
[0254] PBMCs were treated with 150 U/ml of IL-2. Some of the
cultures were also treated with Actimid.TM. or lenalidomide. Cells
were stained with CD25-FITC/CD152-PE/CD4-PerCP/NKG2D-APC and
analyzed using a FACSCalibur. As shown in FIG. 4, levels of CD4,
CD25high, CD152high expressing cells are reduced in groups
pre-treated with an IMiD as compared to the untreated group. The
results suggest that the IMiDs of the invention also decrease the
levels of regulatory T cells or inhibits the proliferation of such
cells.
6.2 Effects on Acquired Antibody Resistance
[0255] Rituximab-resistant cell lines (RRCL) were generated by
chronic exposure of Raji cells to escalating doses of rituximab
alone (2R) or along with human complement (4RH). Functional assays
including antibody-dependant cellular cytotoxicity (ADCC) and
complement-mediated cytotoxicity (CMC) were performed to
demonstrate resistance to rituximab. To study the effects of
lenalidomide-priming of PBMC's against RRCL, peripheral blood
mononuclear cells from healthy donors were cultured with either
DMSO or lenalidomide (at final concentrations of 10 or 20
.mu.g/ml), with or without IL-2 (201 U/ml), over a 5-day period at
37.degree. C., 5% CO.sub.2. Parentral Raji, and RRCL (2R and 4RH)
were labeled with .sup.51Cr and exposed to either rituximab or
trastuzumab (Isotype control at 20 .mu.g/ml) in the presence of an
IMiD or control stimulated-PBMCs (Effector:Target ratio of 40:1).
.sup.51Cr release was measured and the percentage of lysis was
calculated. Statistical differences were analyzed by chi-square
test.
[0256] In vitro exposure of PBMC to IMiD+/-IL-2 improved
rituximab-associated ADCC in RRCL. Exposure of PBMC to IMiD+/-IL-2
for 5 days led to a statistically significant increase in
rituximab-mediated ADCC in 2R cells [IMiD mean % lysis
26.9+/-1.18%] [IMiD+IL-2 mean % lysis 38.4+/-4.14%] when compared
to control-stimulated PBMC's [mean % lysis 17.6+/-5.6%]. Similar
effects were observed in 4RH cells. The mean % of lysis by ADCC for
combination IMiD/IL-2 exposed PBMC's on 4RH cells was found to be
highest at 38.4+/-4.1%, as compared to IMiD (mean % lysis
26.5+/-1.83%) or vehicle exposed PBMC's (mean % lysis 17.6+/-5.69%)
(P=0.01). These results suggest that modulation (e.g.,
PBMC-priming) of the immune system by the IMiD of the invention
(+/-IL-2) improves rituximab anti-tumor activity and may partially
overcome rituximab resistance in RRCL via augmentation of ADCC.
6.3 Effects on Growth Arrest and Apoptosis
[0257] Direct effects of IMiDs on NHL tumor cells were tested by
treating Raji cells with IMiDs alone, or in combination with anti
CD20 antibodies B1 or rituxan. IMiD 1 alone caused up to 40%
inhibition of proliferation at 10 .mu.M in Raji cells, which
corresponded to G1 arrest. In combination with B1. Actimid.TM.
showed a small additive effect at 10 .mu.M, while lenalidomide
effects were minimal up to 10 .mu.M. In combination with rituxan,
Actimid.TM. showed a slight additive effect at 10 .mu.M, and
lenalidomide showed the same at 50 .mu.M.
[0258] A co-culture assay of PBMC and NHL tumor cells were
developed as an in vitro model of tumor-host immune system
interaction, to further explore the anti-tumor potential of IMiDs
in NHL cells. This assay is non-radioactive and flow cytometry
based. Using Raji and PBMC, it was shown that pre-treatment of PMBC
with an IMiD can enhance the PBMC activity in inducing Raji cell
apoptosis in a dose dependent manner. In addition, it was shown
that pre-treatment of Raji cells with an IMiD can further enhance
the apoptosis induced by PBMC pre-treated with an IMiD. These
results suggest that the IMiDs of the invention directly induce NHL
tumor cell growth arrest and effectively enhance tumor cell
apoptosis induced by PBMC.
6.4 Effects on HSC Expansion
[0259] The ability of IMiDs to enhance the expansion of
hematopoietic stem cells (HSC) ex vivo in combination with growth
factors were tested. It was shown that the IMiDs of the invention
dramatically enhance the expansion of CD34+cells in a serum-free
system, achieving up to 100-fold expansion after 14 days in
culture. In addition, the IMiDs of the invention enabled a
preferential expansion of CD34+CD38- cells, a more immature
phenotype.
[0260] IMiDs showed similar activities on HSC from all sources
tested: bone marrow, cord blood and peripheral blood (steady-state
or G-CSF-mobilized). It was also shown that IMiDs can efficiently
expand CD34+ cells isolated from frozen cord blood units.
[0261] Global gene expression (Affymetrix) analysis of
IMiDs-expanded CD34+ cells revealed that the IMiDs of the invention
modulate several genes involved in cell differentiation, cell
adhesion and cell self-renewal. The IMiDs of the invention also
upregulated many genes involved in immune responses and antigen
presentation.
6.5 Effects on T Cell Differentiation
[0262] Effects of IMiDs on T cell differentiation were investigated
using various methods. It was demonstrated that, in combination
with anti-CD3 stimulation, the IMiD of the invention directly
increases expression of Th1 transcription factor T-bet via enhanced
T-bet RNA transcription at 4 hours after stimulation. A concomitant
decrease in expression of Th2 transcription factor GATA-3 was also
observed. The regulation of two key transcription factors by the
IMiD favors Th1 differentiation of human naive CD4.sup.+ T cells.
Enhancement of T-bet by the IMiD results in increased tyrosine
phosphorylation of T-bet, increased expression of IL-12R.beta.2,
and increased IFN-.gamma. production, compared to treatment with
anti-CD3 alone.
[0263] A similar effect of the IMiD on T-bet and GATA-3 was also
observed in differentiated human Th2 cells in vitro under Th2
polarizing condition. The intracellular cytokine staining of IL-4
and IFN-.gamma. on re-stimulated Th2 cells showed that the IMiD
reduced the number of IL-4 producing cells and increased the number
of IFN-.gamma. producing cells in the presence of plate bound
anti-CD3 antibody. The effect of the IMiD on polarized Th2 cells
includes reversal of Th2 cell differentiation and enforcement of
IFN-.gamma. expression in IL-4 positive cells, which is greatly
enhanced by addition of exogenous IL-12. These results suggest that
the IMiDs of the invention not only preferentially induce Th1
immune response by enhancing T-bet, but also inhibit Th2 lineage
commitment by reducing GATA-3 expression.
6.6 Effects on T Cell Activation
[0264] The Gab proteins, including Gab1, Gab2 and Gab3 comprise a
growing family of phospho-tyrosine regulated scaffolding molecules
involved in RTK signal transduction. Phosphorylation of Gab1 in B
cells is associated with PI3-kinase activity and cell
proliferation. While Gab1 is expressed in B cells, only Gab2 is
expressed in T cells. Although Gab2 is tyrosine phosphorylated upon
TCR activation by ZAP-70, it functions as a negative regulator of
TCR signaling via a Shp-2 dependent mechanism. Overexpression of
Gab2 in T cells results in the inhibition of IL-2 production
(Yamasaki et al., J. Biol. Chem., 2001). The effect of lenalidomide
on Gab2 phosphorylation and activation in anti-CD3/CD28 stimulated
Jurkat T cells was examined. Lenalidomide inhibited Gab2
phosphorylation dose-dependently (with approximately 50% inhibition
at about 1 .mu.M) in a manner that correlated with T cell
costimulation and enhancement of IL-2 production. The results show
that the mechanism of action of lenalidomide is therefore
consistent with inhibition of phosphorylation of Gab2 in
anti-CD3/CD28-stimulated T cells.
6.7 Effects on .gamma..delta. T Cells
[0265] 6.7.1 Materials and Methods
[0266] Phenotyping of PBMC preparations stimulated with IL-2 and
IPP.+-.IMiDs: PBMC preparations were obtained and treated weekly
with IL-2 and IPP (150 units/ml and 10 uM respectively). Expression
of .delta..gamma. TCR and NKG2D were measured by FACS over a period
of three weeks.
[0267] Generation of .gamma..delta. T cells: PBMC preparations were
treated with IL-2 (150 units/ml) and IPP (25 uM) weekly. Cultures
were split and replenished weekly with fresh IL-2 and IPP and %
.gamma..delta. TCR+ve cells determined by FACS. After 3-4 weeks
.gamma..delta. T cells were purified by negative magnetic
separation using CD4+and CD8+ Dynalbeads and maintained in
IL-2.
[0268] Measurement of cytokine production in purified
.gamma..delta. T cells and fresh .gamma..delta. cells in PBMC
preparations: Purified .gamma..delta. T cells were stimulated with
IPP.+-.IMiDs (10 .mu.g/mL) or with the MM cell line RPMI-8226
(.+-.IMiDs (10 .mu.g/mL)) in 24 well plates and were incubated 8-72
hours. Cell-free supernatants were collected and stored at
-70.degree. C. until assayed by ELISA. IFN-.gamma., TNF-.alpha. and
IL-2 were measured by ELISA (BD pharmingen). For fresh
.delta..gamma. preps, PBMCs were stimulated with plate bound
anti-CD3 (1.25 .mu.g/ml) for 48 hours and the expression of
TNF-.alpha., IFN-.gamma., IL-2 and IL-4 were measured by
intracellular FACS on cells stained for .gamma..delta. TCR.
[0269] Measurement of apoptosis in .delta..gamma. cells: Gamma
delta T cells were treated with a single dose of 25 nM IPP and
weekly with 150 U/ml of IL-2 for 4 weeks and 3 days. Cells were
then either left untreated or treated with Actimid.TM., IPP or
Actimid.TM. and IPP. Apoptosis was assessed by staining of cells
with annexin V PE and 7-AAD at various time points and analysis
using a FACSCalibur.
[0270] Cytotoxicity assays: Gamma delta T cells were treated with a
single dose of 25 .mu.M IPP and weekly with 150 U/ml of IL-2 for 3
weeks and 1 day. RPMI-8226 target cells were incubated overnight
with 50 nM pamidronate then treated with 3 MBq 51Cr. Target, and
effector cells were incubated at different ratios and chromium
release was assayed after 4 hours. To determine the effects of
Actimid.TM., the compound was either included in the 22 day
preincubation before the assay and in the chromium release step, or
was included during the chromium release assay.
[0271] 6.7.2 Effects on the Expression of .gamma..delta. T Cells
and NKG2D
[0272] PBMCs were treated with a single dose of 25 uM IPP and then
weekly with 150 U/ml of IL-2. In addition, some cultures were
treated with 10 .mu.M Actimid.TM. or lenalidomide. IL-2 treated
cells were stained with CD25 FITC/CD4 PE/CD3 PerCP/NKG2D APC, and
IL-2 plus IPP treated cells were stained with .delta..gamma. TCR
FITC/alpha beta TCR PE/CD3 PerCP/NKG2D APC and analysed using a
FACSCalibur.
[0273] As shown in FIG. 5, cells treated with an immunomodulatory
compound of the invention exhibited higher .gamma..delta. T cells
and NKG2D expression. The results show that immunomodulatory
compounds of the invention enhance the expression of .gamma..delta.
T cells and NKG2D in PMBCs activated with IL-2 and IPP.
[0274] 6.7.3 Effects of Apoptosis of .gamma..delta. T Cells
[0275] Gamma delta T cells were treated with a single dose of 25
.mu.M IPP and weekly with 150 U/ml of IL-2 for 31 days. Cells were
then either left untreated or treated with Actimid.TM., IPP, or
Actimid.TM. and IPP in combination. Apoptosis was assessed by
staining of cells with annexin V PE and 7-AAD at the stated time
points and analysis using a FACSCalibur. Annexin V PE
negative/7-AAD negative cells are designated live, annexin V PE
positive/7-AAD negative early apoptotic, annexin V PE
positive/7-AAD positive late apoptotic and annexin V PE
negative/7-AAD positive dead.
[0276] As shown in FIG. 6, Actimid.TM. offered protection against
apotosis in .gamma..delta. T cells with or without IPP. The results
suggest that immunomodulatory compounds of the invention protect
against apotosis of .gamma..delta. T cells.
[0277] 6.7.4 Effects on Cytokine Production by .gamma..delta. T
Cells
[0278] The effects of Actimid.TM. on IFN-.gamma., TNF-.alpha., and
IL-4 were examined in freshly prepared .gamma..delta. T cells and
.gamma..delta. T cell lines stimulated with IPP. As shown in FIG.
7A, Actimid.TM. enhanced the production of both IFN-.gamma. and
TNF-.alpha. in TCR .gamma..delta. cells from within a freshly
prepared PMBC population. In addition, as shown in FIG. 7B,
Actimid.TM. enhanced the production of IFN-.gamma., but not IL-4,
in .gamma..delta. T cells stimulated with IPP. The results show
that immunomodulatory compounds of the invention stimulate the
production of IFN-.gamma. and TNF-.alpha., but not IL-4.
[0279] 6.7.5 Effects on IFN-.gamma. Production in Response to
Varying Tumor to .gamma..delta. T Cells Ratio
[0280] Tumor cells pre-incubated with (FIG. 8B) or without (FIG.
8A) pamidronate were incubated with .delta..gamma. T cells at
different tumor (RPMI-8226 MM) to .gamma..delta. T cells ratios as
indicated in FIG. 8. Some of the cells were further treated by
Actimid.TM.. Intracellular IFN-gamma production was measured by
flow cytometry.
[0281] As shown in FIGS. 8A and 8B, Actimid.TM. augmented
IFN-.gamma. production by .gamma..delta. T cells. IFN-.gamma.
production increased with increasing tumor to .gamma..delta. T
cells ratio. The results show that immunomodulatory compounds of
the invention enhance the production of IFN-.gamma. by
.gamma..delta. T cells, and the effects increase in response to
increasing tumor to .gamma..delta. T cells ratio.
[0282] 6.7.6 Effects on Cytotoxicity of .gamma..delta. T Cells
[0283] Gamma delta cells were treated with a single dose of 25 uM
IPP and weekly with 150 U/ml of IL-2 for 22 days. RPMI-8226 target
cells were incubated overnight with 50 .mu.M pamidronate, then
treated with 3 MBq 51 Cr. Target and effector cells were incubated
at various ratios with fresh Actimid.TM. and chromium release
assayed after 4 hours. Actimid.TM. was also added to some wells for
the 22 day pretreatment with IL-2 and IPP (FIG. 9A) or just for the
4 hr chromium release assay (FIG. 9B).
[0284] As shown in FIG. 9, the addition of Actimid.TM. during
either the pretreatment or the chromium release assay enhanced the
cytotoxicity of .gamma..delta. T cells toward RPMI-8226 MM cell
lines, although a better effect was observed with the addition of
Actimid.TM. during the pretreatment of period. The results suggest
that immunomodulatory compounds of the invention enhance the
cytotoxicity of .gamma..delta. T cells toward tumor cells, and the
effects may be improved by pretreating the tumor cells with the
compounds of the invention.
6.8 Effects on Invariant NKT Cells
[0285] The establishment of highly purified primary invariant NKT
(iNKT) cell lines from health donors and multiple myeloma (MM)
patients has been tested, and the effects of IMiD 2 on iNKT cells
were further explored. iNKT cells derived from peripheral blood or
bone marrow mononuclear cells were enriched with anti-TCRV.alpha.
24 mAb or anti-6B11 mAb and further expanded by several rounds of
stimulation with .alpha.-GalCer-loaded dendritic cells. Phenotype
analysis confirmed 95% purity in expanded iNKT cell lines. No
significant phenotypic difference was observed in iNKT cells
between healthy donors and MM patients.
[0286] Majority of iNKT cells expressed CD161 and CD28, whereas
CD56 expression was at very low level. Following anti-CD3 or
a-GalCer-loaded dendritic cells stimulation, iNKT cells showed
strong proliferative activity as measured by .sup.3H-TdR
incorporation assay and production of IFN-.gamma. measured by
ELISA.
[0287] Next, the effects of IMiD 2, which is known to enhance T
cell costimulation and NK cell activity, on iNKT cells were
evaluated. From the tests, it was observed that IMiD 2 enhances
anti-CD3 mediated proliferation of expanded iNKT cells by 1.4 fold,
and the enhanced expression and fluorescent intensity of CD25 (MFI
68.6 versus 28.5) on iNKT cells treated with IMiD 2 compare to
untreated iNKT cells. Additionally, compared to the control group
stimulated with .alpha.-GalCer-loaded dendritic cells alone, IMiD 2
plus .alpha.-GalCer-loaded DC also enhanced the production of IL-2.
These results provide the preclinical feasibility and rationale to
clinically evaluate the efficacy of adoptive transfer of iNKT cells
in MM. Additionally, the results demonstrate the ability of the
IMiDs of the invention to augment the immunoreactivity of iNKT
cells, suggestive of their use in enhancing iNKT cell mediated
immunotherapy in myeloma.
[0288] 6.9 Use with Hepatitis B Vaccine
[0289] A two-center, randomized, double-blind, placebo-controlled
trial is designed. A single dose of Hepatitis B vaccine is
administered to subjects. An IMiD or placebo is administered to 64
patients for 7 days prior to and 7 days after the vaccine.
Collection of blood samples for immune analysis is performed prior
to the initiation of the IMiD administration, at the time of
vaccination, and 7, 14, and 28 days after vaccination. Safety
assessments is performed at day 14, the last day of study drug.
[0290] Subjects may opt for 2.sup.nd and 3.sup.rd doses of vaccine
in order to complete the usual course of hepatitis B vaccination.
Opting for additional vaccinations is not a requirement of this
study. Patients opting to receive the second (day 28) and 3.sup.rd
(6 month) vaccination may have their blood samples collected prior
to the 2'' and 3.sup.rd and 1 month after 3.sup.rd vaccination. The
28 day blood draw serves as the blood draw prior to the 2'' dose of
vaccine. The blood draws one month after the 2'' and 3.sup.rd dose
are not required for subjects wishing to receive the 2'' and
3.sup.rd dose of vaccine.
[0291] The effect of the IMiD on the response to hepatitis B
vaccine in subjects with plasma cell dyscrasias, as measured by
change in antibody titer against hepatitis B surface antigen
(HbSAg), can be determined following the above procedures. In
addition, serum and blood cells can be collected to: a) assess the
development of T cell responses against HbSAg following
vaccination; b) identify phenotypic changes in peripheral blood
cells following the IMiD administration especially with regard to
CD3, CD4, CD8 T cells, and NK and NKT cells: and c) determine
changes in gene expression profile of immune cells before and after
the treatment of the IMiD using micro array protocols.
[0292] 6.10 T.sub.reg Cell Phenotyping and Functional Analyses from
Patients Undergoing Lenalidomide Treatment
[0293] Patients with any malignancy which are selected for
lenalidomide treatment in are asked to participate in this study.
The cycle of dosing for the patients selected for lenalidomide
treatment is 3 weeks of dosing with 25 mg lenalidomide daily,
followed by 1 week without dosing, followed by three more weeks of
dosing, in repeated cycles. Forty ml samples of blood are collected
into heparin tubes and 5 mls into serum tubes at time points from 1
hour to 24 hrs before the first administration of lenalidomide (25
mg/dose) and at 21 days and 49 days after dosing.
[0294] The blood in the heparin tubes is layered onto histopaque
and spun for 25 minutes at 600 g to separate the buffy coat layer.
The buffy coat containing the peripheral blood mononuclear cells
and malignant hematological cells is isolated. The cells isolated
are subjected to the following procedures:
[0295] 6.10.1 Phenotype Analysis Using a FACScalibur Machine
[0296] Dominant phenotypes of the PBMCs freshly isolated from each
patient are analyzed, and the percentage of cells in the patients
that are of a regulatory T cell phenotype (CD4.sup.+CD25.sup.+
positive cells, staining positive also for FOXP3 and CTLA-4) is
measured.
[0297] 6.10.2 Isolation of CD4.sup.+CD25.sup.+ cells from the
patients PBMCs
[0298] CD4.sup.+CD25.sup.+ cells and CD4.sup.+CD25.sup.- cells are
isolated from the patients' PBMCs using standard magnetic bead kits
(Invitrogen). The ability in-vitro of the CD4.sup.+CD25.sup.+ cells
to inhibit the proliferation of CD4.sup.+CD25.sup.- cells, upon
stimulation with anti-CD3, is assessed.
[0299] 6.10.3 Analysis of Serum
[0300] Serums are analysed for TGF-beta, IL-10. IL-4. IL-6,
IFN-.gamma. and INF-.alpha. concentrations, using methods described
herein as well as those well-known in the art.
[0301] All of the references cited herein are incorporated by
reference in their entirety. While the invention has been described
with respect to the particular embodiments, it will be apparent to
those skilled in the art that various changes and modifications may
be made without departing from the spirit and scope of the
invention as recited by the appended claims.
[0302] The embodiments of the invention described above are
intended to be merely exemplary, and those skilled in the art will
recognize, or will be able to ascertain using no more than routine
experimentation, numerous equivalents of specific compounds,
materials, and procedures. All such equivalents are considered to
be within the scope of the invention and are encompassed by the
appended claims.
* * * * *