U.S. patent application number 15/072870 was filed with the patent office on 2016-10-06 for myeloid derived suppressor cell inhibiting agents.
The applicant listed for this patent is Colorado State University Research Foundation. Invention is credited to Steven W. Dow, Angela J. Henderson, Leah Mitchell.
Application Number | 20160287686 15/072870 |
Document ID | / |
Family ID | 44649851 |
Filed Date | 2016-10-06 |
United States Patent
Application |
20160287686 |
Kind Code |
A1 |
Dow; Steven W. ; et
al. |
October 6, 2016 |
MYELOID DERIVED SUPPRESSOR CELL INHIBITING AGENTS
Abstract
Myeloid derived suppressor cell (MDSC) inhibitory agents and
vaccine and/or adjuvant enhancers are provided. Improved vaccine
treatment regimens employing these agents are also provided. Cancer
vaccines and methods for inhibiting tumor growth and cancer
metastases are also presented. The myeloid derived suppressor cell
(MDSC) inhibiting agents are described as bisphosphonates (such as
liposomal clodronate) and CCR2 inhibitors and/or CCR2 antagonists.
Methods for enhancing antibody titer levels in response to an
antigen of interest are also provided.
Inventors: |
Dow; Steven W.; (Littleton,
CO) ; Henderson; Angela J.; (Fort Collins, CO)
; Mitchell; Leah; (Fort Collins, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Colorado State University Research Foundation |
Fort Collins |
CO |
US |
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|
Family ID: |
44649851 |
Appl. No.: |
15/072870 |
Filed: |
March 17, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13166017 |
Jun 22, 2011 |
9320735 |
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15072870 |
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13050614 |
Mar 17, 2011 |
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13166017 |
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61422984 |
Dec 14, 2010 |
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61315263 |
Mar 18, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/892 20180801;
A61K 39/395 20130101; A61K 31/165 20130101; A61K 31/683 20130101;
A61P 37/00 20180101; A61K 31/437 20130101; A61P 37/02 20180101;
A61K 31/537 20130101; A61P 35/00 20180101; A61K 31/454 20130101;
A61K 31/40 20130101; A61K 31/439 20130101; A61K 39/0011 20130101;
A61K 31/5386 20130101; A61K 31/4184 20130101; A61K 31/42 20130101;
A61K 31/17 20130101; A61K 31/438 20130101; A61K 31/4184 20130101;
A61K 2300/00 20130101; A61K 31/42 20130101; A61K 2300/00 20130101;
A61K 31/437 20130101; A61K 2300/00 20130101; A61K 31/438 20130101;
A61K 2300/00 20130101; A61K 31/5386 20130101; A61K 2300/00
20130101; A61K 39/395 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 31/537 20060101 A61K031/537; A61K 31/40 20060101
A61K031/40; A61K 31/439 20060101 A61K031/439; A61K 31/165 20060101
A61K031/165; A61K 31/17 20060101 A61K031/17; A61K 31/683 20060101
A61K031/683; A61K 31/454 20060101 A61K031/454 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 18, 2010 |
US |
PCT/US2011/29022 |
Claims
1. A myeloid derived suppressor cell inhibiting agent therapeutic
regimen preparation comprising: a first component myeloid derived
suppressor cell inhibiting agent; and a second component vaccine
antigen of interest, wherein said therapeutic regimen preparation
enhances antibody titer levels to the vaccine antigen of interest
in vivo greater than antibody titer levels in vivo with a vaccine
treatment regimen in the absence of the myeloid derived suppressor
cell inhibiting agent, said myeloid derived suppressor cell
inhibiting agent comprising a bisphosphonate or a CCR2
inhibitor.
2. The myeloid derived suppressor cell inhibiting agent of claim 1
wherein said agent inhibits inflammation induced migration of
myeloid derived suppressor cells to draining lymph nodes in
vivo.
3. The myeloid derived suppressor cell inhibiting agent of claim 1
wherein the myeloid derived suppressor cell inhibiting agent is a
CCR2 inhibitor.
4. The myeloid derived suppressor cell inhibiting agent of claim 1
wherein the bisphosphonate is clodronate, zoledronate,
palmidronate, etidronate, or other bisphosphonate drug and the CCR2
inhibitor is RS 1028595 or PF-04178903.
5. A method for enhancing immune response to a vaccine in an animal
comprising administering a myeloid derived suppressor cell
inhibiting agent to the animal as part of a vaccine treatment
regimen.
6. The method of claim 5 wherein the immune response is
demonstrated as an elevated antibody titer level in vivo, compared
to an antibody titer level in response to a vaccine treatment
regimen without a myeloid derived suppressor cell inhibiting
agent.
7. The method of claim 5 wherein the vaccine treatment regimen
provides for administration of the myeloid derived suppressor cell
inhibiting agent before, at the same time, after or before, during
and after the administration of the vaccine.
8. The method of claim 5 wherein the myeloid derived suppressor
cell inhibiting agent is a bisphosphonate or a CCR2 inhibitor.
9. The method of claim 5 wherein the myeloid derived suppressor
cell inhibiting agent is a small molecule agent capable of
inhibiting inflammatory response induced myeloid derived suppressor
cell activity.
10. The method of claim 5 comprising the steps of: (a) treating an
animal in need thereof with a therapeutic amount of a vaccine
containing a vaccine antigen of interest and a myeloid derived
suppressor cell inhibiting agent; and (b) enhancing immune response
in the animal.
11. The method of claim 10 wherein the myeloid derived suppressor
cell inhibiting agent is a bisphosphonate or a CCR2 inhibitor.
12. The method of claim 10 wherein the myeloid derived suppressor
cell inhibiting agent is administered before, at the same time and
after the vaccine is administered.
13. The method of claim 10 wherein vaccine contains alum,
incomplete Freunds adjuvant, Corrixa MPL adjuvant, liposomal
polyI:C adjuvant or cationic liposome-DNA complex adjuvant.
14. A method for inhibiting tumor growth in an animal comprising:
(a) administering a therapeutic amount of an anti-tumor preparation
containing adjuvant and an amount of myeloid derived suppressor
cell inhibiting agent sufficient to inhibit myeloid derived
suppressor cell activity; and (b) inhibiting tumor growth in said
animal.
15. The method of claim 14 wherein the myeloid derived suppressor
cell inhibiting agent is a bisphosphonate or a CCR2 inhibitor.
16. The method of claim 15 wherein the bisphosphonate is
clodronate, zoledronate, palmidronate, etidronate, or other
bisphosphonate drug and the CCR2 inhibitor is RS 1028595 or
PF-04178903.
17. The method of claim 16 wherein the bisphosphonate is liposomal
clodronate.
18. The method of claim 14 wherein the myeloid derived suppressor
cell inhibiting agent is administered before, at the same time, or
after the anti-tumor preparation, or before, at the same time and
after the anti-tumor preparation.
19. The method of claim 18 wherein the anti-tumor preparation
comprises alum, incomplete Freunds adjuvant, Corrixa MPL adjuvant,
liposomal polyI:C adjuvant or cationic liposome-DNA complex
adjuvant.
20. The method of claim 14 wherein the myeloid derived suppressor
cell inhibiting agent is RS 1028595.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 13/166,017, filed Jun. 22, 2011, now issued U.S. Pat. No.
9,320,735, granted Apr. 26, 2016; which is a continuation-in-part
application of U.S. patent application Ser. No. 13/050,614 filed
Mar. 17, 2011, now abandoned. U.S. application Ser. No. 13/166,017
is a continuing application of PCT/US11/29022, filed Mar. 18, 2011.
U.S. patent application Ser. No. 13/050,614 claims priority to U.S.
Provisional Patent Application No. 61/315,263, filed Mar. 18, 2010
and U.S. Provisional Patent Application No. 61/422,984, filed Dec.
14, 2010. Reference is also made here to co-pending application,
U.S. application Ser. No. 12/393,612, filed Feb. 26, 2009 (claiming
priority to U.S. Provisional Application 61/031,410, filed Feb. 26,
2008), entitled, "Liposomal Delivery of Bisphosphonates". The
entire disclosure and contents of the above applications are hereby
incorporated by reference
FIELD OF INVENTION
[0002] The present invention relates to materials that inhibit
and/or eliminate vaccine-induced immunosuppressive macrophages.
More particularly, the present invention relates to adjuvant
additives that enhance vaccine response through inhibition and/or
elimination of vaccine-induced immunosuppressive macrophages. The
invention also relates to the field of vaccines and adjuvant
additives, as additives for conventional vaccines that improve
immune response to a vaccine are provided. The present invention
also relates to methods for enhancing immune response to a
vaccine.
BACKGROUND OF THE INVENTION
[0003] Immunologic adjuvants are added to vaccines to stimulate the
immune system's response to the target antigen, but do not in
themselves confer immunity. Adjuvants can act in various ways in
presenting an antigen to the immune system. Adjuvants can act as a
depot for the antigen presenting the antigen over a long period of
time, thus maximizing the immune response before the body clears
the antigen. Examples of depot type adjuvants are oil emulsions.
Adjuvants can also act as an irritant which causes the body to
recruit and amplify immune response. A tetanus, diphtheria, and
pertussis vaccine, for example, contains minute quantities of
toxins produced by each of the target bacteria, but also contains
some aluminum hydroxide. Such aluminum salts are common adjuvants
in vaccines sold in the United States and have been used in
vaccines for over 70 years. The body's immune system develops an
antitoxin to the bacteria's toxins, not to the aluminum, but would
not respond enough without the help of the aluminum adjuvant.
[0004] Although immunological adjuvants have traditionally been
viewed as substances that aid the immune response to antigen,
adjuvants have also evolved as substances that can aid in
stabilizing formulations of antigens, especially for vaccines
administered for animal health
[0005] Vaccine preparations have been observed to demonstrate less
than robust immune response in vivo, creating a need for the
development of enhanced vaccine preparations. However, the exact
mechanisms working to inhibit and/or reduce less than robust
response to vaccines in vivo remain under study.
[0006] All vaccines induce inflammation and any inflammation that
is sustained for more than a few hours will result in recruitment
of myeloid cells (monocytes and neutrophils) to the site of
vaccination and to the vaccine draining lymph nodes. Certain
subpopulations of these cells are also referred to as vaccine
elicited myeloid cells (MDSC). These cells are also referred to as
myeloid derived suppressor cells (MDSC). In certain contexts, prior
reports indicate that these inflammatory cells (especially
monocytes) recruited to vaccine-draining lymph nodes may actually
augment immune response, though information presented in the
present disclosure suggests otherwise.
[0007] Clodronate is a bisphosphonate drug that kills osteoclasts
and other macrophages via induction of apoptosis. When clodronate
is incorporated within liposomes (LC), uptake by phagocytic cells
such as macrophages is greatly enhanced, resulting in selective
targeting of macrophages for killing..sup.24, 25, 36 Some studies
report that repeated LC administration is capable of depleting both
tumor associated macrophages and myeloid suppressor cells..sup.13,
32, 44
[0008] A need remains in the medical arts for improved vaccine
preparations with enhanced ability to provoke robust immune
response.
SUMMARY OF THE INVENTION
[0009] The present invention, in a general and overall sense,
provides a family of adjuvants and vaccine/adjuvant additives,
myeloid derived suppressor cell inhibiting agents, found to enhance
and/or augment the immunoactivity of a vaccine or cancer/tumor
inhibiting treatment. The adjuvants and vaccine/adjuvant additives
may be used in combination with virtually any conventional adjuvant
and/or vaccine, or as an adjuvant alone, to provide an improved
therapeutic preparation as a vaccine, as well as together with any
variety of cancer treatment therapies (chemotherapy, radiation,
cancer vaccine). Methods and compositions are provided that
increase the effectiveness of a vaccine and cancer/tumor treatment
in a manner that is independent of the type of vaccine adjuvant
included in the preparation.
[0010] Adjuvants and vaccine Adjuvant Additives/MDSC Inhibiting
Agents and Improved Vaccine Preparations:
[0011] In one aspect, the adjuvants and vaccine adjuvant additives
described here comprise myeloid derived suppressor cell (MDSC)
inhibiting and/or blocking agents. By way of example, suitable
drugs to block the suppressive effects of MDSC include tyrosine
kinase inhibitors (eg, sunitinib), MDSC differentiating agents (eg,
all-trans retinoic acid), reactive nitrogen inhibitors (eg,
aminoguanidine or similar drugs); arginase enzyme inhibitors,
indoleamine deoxygenase enzyme inhibitors, reactive oxygen species
inhibitors, TGF-b inhibitors, IL-10 inhibitors, VEGF inhibitors,
and PGE2 synthesis inhibitors.
[0012] In some embodiments, the myeloid derived suppressor cell
inhibiting agents may be further described as comprising a
bisphosphonate drug, such as clodronate, zoledronate, pamidronate,
etidronate, or any other type of drug that is capable of depleting
or inhibiting macrophages, and that when provided with an adjuvant
containing vaccine, provides for an enhanced immune response in an
animal greater than the observed immune response in the animal
given the adjuvant vaccine preparation without the myeloid derived
suppressor cell inhibiting agent. In some embodiments, the
bisphosphonate drug is a liposomal conjugated agent, such as
liposomal clodronate.
[0013] Additional examples of suitable vaccine/adjuvant additives
(e.g., MDSC depleting agents) of the invention include
liposome-encapsulated bisphosphonate drugs, antibodies targeted to
MDSC, liposomes encapsulating other apoptosis inducing agents, or
liposomes encapsulating siRNA or other RNA targeting molecules that
induce MDSC apoptosis. By way of further example, the vaccine
additive of the invention may comprise virtually any agent
demonstrated to deplete and/or inhibit the migration, accumulation
or activity of myeloid derived suppressor cells (MDSC), thus
providing for an inhibition of the immunosuppressive activity of
the MDSCs. Additional vaccine additives (MDSC depleting agents)
include drugs that block monocyte release from bone marrow (CCL2 or
CCR2 inhibitors, competitors or agonists, M-CSF inhibitors, GM-CSF
inhibitors). In other embodiments, the adjuvant additives consist
of drugs that inhibit the recruitment and/or migration of MDSC to
sites of vaccine inflammation. These drugs would consist most
specifically of small molecule inhibitors of the receptor for CCL2
(MCP-1), which is known as CCR2. These CCR2 receptor inhibitors
block the egress of monocytes from the bone marrow into the
bloodstream, and also inhibit the accumulation of monocytes at
sites of vaccine-induced inflammation, such as vaccine-draining
lymph nodes or the skin site of vaccination. Specific inhibitors in
this family include RS102895 (Sigma-Aldrich) and other similar
molecules.
[0014] Other similar drugs would include other small molecule
chemokine/cytokine inhibitors, such as inhibitors of M-CSF, GM-CSF,
IL-3, or IL-8, or receptors for these cytokines and chemokines.
Other candidates for inhibition would include the S100 family of
proteins, including especially S100A8/A9.
[0015] The MDSC depleting/inhibiting agents of the invention may be
administered orally, i.v., s.c., i.m., or i.p. at the time of
vaccination, before the time of vaccination, after the time of
vaccination, or before, at the same time and after vaccination.
[0016] Methods of Vaccination with MDSC inhibiting Agents/Vaccine
Additive Enhancing Agents:
[0017] In another aspect, improved methods of vaccinating an animal
are provided. Surprisingly, the present inventors found that the
inhibition of a particular population of myeloid derived suppressor
cells (the suppressive population of myeloid derived suppressor
cells) from moving to the site of vaccination and/or lymph nodes
resulted in a demonstrable increase in immune response (resulting
in an observable increase in antibody titer in vivo production) in
the treated animal. A vast improvement in vivo for inducing a
significant and robust immune response was observed. Despite prior
teaching that inflammatory monocytes recruited to vaccine-draining
lymph nodes may actually augment immune response, the present data
and inventive preparations and methods demonstrates the opposite is
in fact the case. Inhibiting the recruitment of inflammatory
monocytes is suggested by the present data to augment immune
response.
[0018] In some embodiments, the method comprises administering the
vaccine adjuvant additive (e.g., the myeloid derived suppressor
cell (MDSC) depleting agent) before, at the same time or slightly
after (1 day, 2, days, 3 days, immediately after, several hours
after) the time that a vaccine is administered. While not wanting
to be limited to any particular mechanism of action, it is proposed
that the administration of the vaccine additive at the same time or
after the vaccine acts to deplete and/or inhibit the influx of
myeloid cells (monocytes and neutrophils) to the site of
inflammation (typically the site of vaccination) and to vaccine
draining lymph nodes in vivo. The possible routes of drug
administration include oral, i.v., s.c., i.m., i.p. or topical at
the site of vaccination. Other preferable routes of drug
administration would be to mix the adjuvant additive with the
vaccine itself, and provide the preparation administered together
to an animal.
[0019] In some embodiments, the combined vaccination and
administration of the myeloid derived suppressor cell inhibiting
agent (myeloid cell depletion approach) is repeated each time the
vaccine is administered.
[0020] The presently described vaccine additives are provided
together with a vaccine that includes an adjuvant. Alternatively,
the additive may be provided whenever a conventional supplied to an
animal, either after or at the same time adjuvant is. By way of
example, conventional adjuvants include alum, other compounds of
aluminum, Bacillus of Calmette and Guerin (BCG), TiterMax.RTM.
adjuvant, Ribi.RTM., Freund's Complete Adjuvant (FCA) and a new
adjuvant disclosed by the United States Department of Agriculture's
(USDA) National Wildlife Research Center on their web site at
aphis.usda.gov/ws/nwrc/pzp.htm based on Johne's antigen. Alum is
generally considered to be any salt of aluminum, in particular, the
salts of inorganic acids. Hydroxide and phosphate salts are
particularly useful as adjuvants. A suitable alum adjuvant is sold
under the trade name, Imject.RTM. Alum (Pierce Chemical Company)
that consists of an aqueous solution of aluminum hydroxide (45
mg/ml) and magnesium hydroxide (40 mg/ml) plus inactive
stabilizers. Alum is a particularly advantageous adjuvant since it
already has regulatory approval and it is widely accepted in the
art.
[0021] The amount of vaccine and/or adjuvant additive to be used
depends on the amount and type of the particular antigen used, and
on the type of additional adjuvant that may be included with the
vaccine, as well as any other treatment being provided along with
the vaccine. One Attorney skilled in the art can readily determine
the amount of vaccine additive needed in a particular application
by assessing antibody titer levels and performing a standard dose
response curve.
[0022] The vaccine regimen of the present invention may include any
variety of vaccine antigen, such as a recombinant protein/peptide,
a live vector vaccine, killed organism, or cell vaccine. In this
regard, the present myeloid derived suppressor cell agents may be
employed as part of a regimen for cancer as well as infectious
disease immunization.
[0023] Vaccines for Use as part of the Vaccine Treatment
Regimen
[0024] It is anticipated that the vaccine regimen of the present
invention may include any variety of different antigens, such as
recombinant protein/peptides, live vector antigens, killed
organism, or cell vaccine. By way of example, the vaccines with
which the present additives may be provided include I.) any
infectious agent (bacterial, viral, fungal, protozoal); 2.)
vaccines for allergy, 3.) vaccines for autoimmune disorders; 4.)
vaccines for toxins; 5.) vaccines for addictive substances (eg.,
nicotine, alcohol, caffeine, etc.).
[0025] The vaccine additives/adjuvant agents may be administered
i.p., i.v., mucosally, orally, s.c., or i.m. by injection, together
with a conventional vaccine to boost immune response. Examples of
conventional adjuvants that may be included with the vaccine as
part of the herein described vaccination regimen include alum, or
whole killed organism or cell vaccine plus adjuvant, or replicating
or non-replicating viral vectored or bacterial vectored
vaccines.
[0026] The vaccine treatment regimen of the invention provides for
the administration of the adjuvant/vaccine additive agent (the
myeloid derived suppressor cell inhibiting agent) provided just
before, at the same time, just after a conventional vaccine, or
before, at the same time and after the conventional vaccine is
provided to an animal. The myeloid derived suppressor cell
inhibiting agent may be admixed with the vaccine, given adjacent to
the vaccine, or given systemically to an animal in order to boost
vaccine immune response.
[0027] In other embodiments, the vaccine regimen (boosting system)
may be administered with cancer vaccines, infectious disease
vaccines, with toxoid vaccines, or vaccines against autoimmune
antigens. In another aspect, a method for inhibiting tumor growth
is provided, such as for inhibiting histiocytic sarcoma and other
cancers. An enhanced anti-cancer vaccine treatment preparation that
incorporates the myeloid derived suppressor cell agents of the
invention is this also provided.
BRIEF DESCRIPTION OF DRAWINGS
[0028] Other objects and advantages of the present invention will
become apparent to those skilled in the art upon reading the
following detailed description of preferred embodiments, in
conjunction with the accompanying drawings, wherein like reference
numerals have been used to designate like elements, and
wherein:
[0029] FIG. 1. Effects of liposomal clodronate (LC) administration
on antibody responses following vaccination.
[0030] FIG. 2. Effects of liposomal clodronate administration on
IFN-g responses by spleen cells from vaccinated mice.
[0031] FIG. 3. Co-administration of LC with a vaccine results in
elimination of Gr-1+ myeloid suppressor cells (MSC) in draining
lymph nodes.
[0032] FIG. 4. Efficient systemic depletion of phagocytic cells
following LC injection.
[0033] FIG. 5. LC Treatment elicits potent antitumor activity in
mouse tumor models (square=Control; circle=Lip-PBS;
trianagle=Lip-chloronate (p<0.02).
[0034] FIG. 6. Tumor regression in dogs with soft tissue sarcoma
following LC treatment.
[0035] FIG. 7. Regression of lung and pleural metastases in
histiocytic sarcoma after LC+ chemo.
[0036] FIG. 8. LC depletes tumor-associated macrophages and myeloid
cells.
[0037] FIG. 9. LC also depletes MDSC in multiple tissue sites.
[0038] FIG. 10. Antitumor activity elicited by LC treatment is T
cell dependent.
[0039] FIG. 11. CD8 T cells are required for LC-induced antitumor
activity.
[0040] FIG. 12. Rapid kinetics of MDSC expansion following
vaccination.
[0041] FIG. 13. Depletion of myeloid cells and vaccine
responses.
[0042] FIG. 14 Co-administration of LC depletes vaccine-induced
myeloid cell accumulation in vaccine-draining lymph nodes.
[0043] FIG. 15. Co-administration of LC with a conventional vaccine
markedly alters vaccine responses.
[0044] FIGS. 16A-16B. Co-administration of LC enhances CD4 T cell
responses to tumor vaccine.
[0045] FIG. 17. LC co-administration significantly improves tumor
vaccine efficacy: whole cell tumor vaccines.
[0046] FIG. 18. LC co-administration significantly improves tumor
vaccine efficacy: tumor cell membrane vaccine. A20 HA tumor growth;
10.sup.6 A20 HA on flank; 10 .mu.g MPF/vaccine/week.
[0047] FIG. 19. Impact of MDSC depletion with liposomal clodronate
on antibody responses to vaccination with various adjuvants. Key to
adjuvants: IFA=incomplete Freund's adjuvant; Alum=Alhydrogel;
MPL=Corrixa MPL adjuvant; DPIC=liposomal polyLC adjuvant;
CLDC=cationic liposome-DNA complex adjuvant
[0048] FIG. 20. Impact of MDSC depletion with liposomal clodronate
on cytokine responses to following vaccination with various
adjuvants.
[0049] FIG. 21. Vaccination triggers recruitment of myeloid cells
(monocytes and neutrophils) into draining lymph nodes.
[0050] FIG. 22. Administration of liposomal clodronate (LC)
efficiently depletes inflammatory myeloid cells from the vaccine
draining lymph nodes.
[0051] FIG. 23. LC administration generates significant increases
in antibody responses to vaccination when administered at the time
of vaccination of within 1-2 days after vaccination.
[0052] FIG. 24. Administration of LC at the time of vaccination
significantly increases T cell responses to vaccination.
[0053] FIG. 25. Vaccination induces the recruitment of inflammatory
myeloid cells into the draining LN, which results in suppression of
T cell proliferative responses compared to mice that were
vaccinated and treated concurrently with LC to deplete inflammatory
myeloid cells.
[0054] FIG. 26. Vaccination induces production of the chemokine
MCP-1 (CLL2).
[0055] FIG. 27. Vaccine responses are increased in CCR2-/- mice
that are impaired in their ability to mobilize monocytes in
response to inflammation.
[0056] FIG. 28. Elimination of inflammatory myeloid cells using LC
significantly improves the activity of cancer vaccines
[0057] FIG. 29. Combined vaccination and inflammatory myeloid cells
depletion increases IFN-.gamma. responses by CD4 T cells from
vaccinated mice. A20 HA vaccine study. IFN-.gamma. release in
response to SFERFEIFPKE peptide (Class II restricted HA
peptide)
[0058] FIG. 30. Combined vaccination and inflammatory myeloid cells
depletion increases IFN-.gamma. responses by CD8 T cells from
vaccinated mice.
[0059] FIG. 31. Inflammatory myeloid cells depletion augments
generation of tumor-specific antibodies following tumor
vaccination. Serum 1:100 ab IgG against fixed A20HA cells.
[0060] FIG. 32. Combined vaccination and LC administration also
significantly increases T cell responses to vaccination against MCA
sarcoma in mice. IFN-.gamma. released in response to whole MCA
cells effected: target 100:1 splenocytes: live MCA cells 24
hours.
[0061] FIG. 33. Effects of co-administration of RS102895 (small
molecule inhibitor, Tocris Bioscience), on antibody responses
following vaccination via two different modes of administration
i.p. (intraperitoneally) or SQ (subcutaneous). Mice were vaccinated
SQ with standard vaccine adjuvant (CLDC) and 5 ug ovalbumin, and
boosted 10 days later. The first group of mice (n=4 per group)
received the vaccine only (cross-hatch bar). A second group of mice
was vaccinated and also treated 1 day before, on the same day, and
1 day after with 5 mg/kg of the CCR2 antagonist RS102895
(horizontal line bar), administered i.p. A third group of mice was
vaccinated and also treated 1 day before, on the same day, and 1
day after with 5 mg/kg of the CCR2 antagonist RS102895,
administered s.c. at the site of vaccination (vertical line bar).
Antibody titers to ovalbumin were determined 2 weeks after the
boost and plotted as endpoint dilution titers for all animals in
all groups. Mice that were vaccinated and treated with RS102895 by
either route developed significantly higher antibody titers than
mice that received the vaccine alone without the CCR2 antagonist.
These data demonstrate the effectiveness of a CCR2 antagonist in
increasing immune response as measured by antibody titer level in
mice. This enhanced immune response and increase in antibody titer
level may be achieved with the administration of the CCR2
antagonist by either an intraperitoneal (IP) (injection into the
peritoneum of an animal) or subcutaneous (SQ) administration (under
the skin). In addition, administration of the adjuvant additive is
demonstrated to be equally as robust administered by either route.
Thus, the present adjuvant additives, especially the CCR2
antagonists, may be administered according to techniques routinely
used in administering vaccines.
[0062] FIG. 34. Effects of co-administration of RS102895 (small
molecule inhibitor, Tocris BioSciences), on spleen lymphocyte
production of IFN-.gamma. from vaccinated animals (T cell
response). Mice were vaccinated s.c. with standard vaccine adjuvant
(CLDC) and 5 ug ovalbumin and boosted 10 days later. A first group
of mice (n=4 per group) received the vaccine only (cross-hatch). A
second group of mice was vaccinated and also treated 1 day before,
on the same day, and 1 day after with 5 mg/kg of the CCR2
antagonist RS102895, administered i.p. (horizontal line bar). A
third group of mice was vaccinated and also treated 1 day before,
on the same day, and 1 day after with 5 mg/kg of the CCR2
antagonist RS102895, administered s.q. at the site of vaccination
(vertical line bar). The mice were sacrificed 2 weeks after the
booster vaccination and spleen cells were incubated in vitro with
ovalbumin (50 ug/ml) for 72 hours. Release of IFN-.gamma. into the
supernatants was determined by ELISA. Lymphocytes from mice that
were vaccinated and treated with RS102895 by either route produced
significantly higher amounts of IFN-.gamma. than lymphocytes from
mice that were vaccinated without the RS102895 additive. The data
shows IFN-.gamma. production from spleen lymphocytes from
vaccinated animals without a CCR2 antagonist was about 450 pg/ml
IFN-.gamma., while with the CCR2 antagonist administered i.p.,
IFN-.gamma. production was about 2,300 pg/ml. Spleen cells from
animals provided the CCR2 antagonist SQ demonstration an
IFN-.gamma. production of about 1,400 pg/ml. This data demonstrates
that co-administration of RS102895 by either route triggered
significantly greater T cell responses to vaccination compared to
levels achieved without the RS102895. These data are consistent
with the idea that recruitment of inflammatory monocytes during
vaccination significantly suppresses vaccine responses.
DETAILED DESCRIPTION OF THE INVENTION
[0063] The present invention embraces a unique class of agents
described herein as inhibitory myeloid derived suppressor cell
(MDSC) agents that are useful in enhancing immune response to
adjuvinated vaccine preparations. As part of a vaccine regimen, the
MDSI agents of the invention may be included with any variety of
vaccines, such as recombinant, live vectored, killed organism, or
cell vaccine. It is also envisioned that the present MDSI agents
may be provided with cancer and infectious disease treatment
methods to enhance the effectiveness of these treatment methods
(anti-tumor, anti-viral, etc., effectiveness).
[0064] By way of further example, the vaccine with which the
present MDSI agents may be included to enhance effectiveness
include vaccines for: 1.) any infectious agent (bacterial, viral,
fungal, protozoal); 2.) vaccines for allergy, 3.) vaccines for
autoimmune disorders; 4.) vaccines for toxins; 5.) vaccines for
addictive substances (eg., nicotine, alcohol, caffeine, etc.).
[0065] The composition or compositions described herein, may be
administered either systemically or locally, by any method standard
in the art, for example, subcutaneously, intravenously,
parenterally, intraperitoneally, intradermally, intramuscularly,
topically, enterally, rectally, nasally, buccally, vaginally or by
inhalation spray, by drug pump or contained within transdermal
patch or an implant. Dosage formulations of the composition
described herein may comprise conventional non-toxic,
physiologically or pharmaceutically acceptable carriers or vehicles
suitable for the method of administration and are well known to an
individual having ordinary skill in this art.
[0066] The compositions described herein may be administered
independently one or more times to achieve, maintain or improve
upon a therapeutic effect. It is well within the skill of an
artisan in the pharmacological or medical arts to determine dosage
or whether a suitable dosage of the composition(s) described herein
should optionally comprise a single administered dose or multiple
administered doses. An appropriate dosage depends on the subject's
health, the elicitation of the immune responses and/or treatment of
the cancer or pathogen associated disease, the route of
administration and the formulation used, among other factors.
[0067] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion. The present
examples, along with the methods, procedures, treatments,
molecules, and specific compounds described herein are presently
representative of preferred embodiments. One skilled in the art
will appreciate readily that the present invention is well adapted
to carry out the objects and obtain the ends and advantages
mentioned, as well as those objects, ends and advantages inherent
herein.
DEFINITIONS
[0068] The term "comprising" means "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0069] As used herein, the term, "a" or "an" may mean one or more.
As used herein in the claim(s), when used in conjunction with the
word "comprising", the words "a" or "an" may mean one or more than
one. As used herein "another" or "other" may mean at least a second
or more of the same or different claim element or components
thereof.
[0070] As used herein, the term "adjuvant" has its conventional
meaning, i.e., the ability to enhance the immune response to a
particular antigen. Such ability is manifested by a significant
increase in immune-mediated protection. An enhancement of humoral
immunity is typically manifested by a significant increase (usually
>10%) in the titer of antibody raised to the antigen. Similarly,
enhancement of cellular immunity is typically manifested by a
significant increase (usually >10%) in the number of responding
CD8+ or CD4+ T cells. The term "about" in relation to a numerical
value x means, for example, x.+-.10%.
[0071] As used here, the term "myeloid derived suppressor cell
inhibiting agent" may be described as an agent that is capable of
inhibiting inflammation induced activity (migration, accumulation,
other activity) of a population of myeloid cells recognized as
myeloid derived suppressor cells.
[0072] The term "antibody" is used in the broadest sense and
includes monoclonal antibodies (including full length or intact
monoclonal antibodies), polyclonal antibodies, multivalent
antibodies, multispecific antibodies (e.g., bispecific antibodies),
and antibody fragments (see below) so long as they exhibit the
desired biological activity.
[0073] The term "concurrently" is used herein to refer to
administration of two or more therapeutic agents, where at least
part of the administration overlaps in time. Accordingly,
concurrent administration includes a dosing regimen when the
administration of one or more agent(s) continues after
discontinuing the administration of one or more other agent(s).
[0074] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Included in this definition are benign
and malignant cancers as well as dormant tumors or
micrometastatses. Examples of cancer include but are not limited
to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular examples of such cancers include squamous cell cancer,
lung cancer (including small-cell lung cancer, non-small cell lung
cancer, adenocarcinoma of the lung, and squamous carcinoma of the
lung), cancer of the peritoneum, hepatocellular cancer, gastric or
stomach cancer (including gastrointestinal cancer), pancreatic
cancer, glioblastoma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
colorectal cancer, endometrial or uterine carcinoma, salivary gland
carcinoma, kidney or renal cancer, liver cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma and various types
of head and neck cancer, as well as B-cell lymphoma (including low
grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic
NHL; high grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), and Meigs' syndrome.
[0075] By "metastasis" is meant the spread of cancer from its
primary site to other places in the body. Cancer cells can break
away from a primary tumor, penetrate into lymphatic and blood
vessels, circulate through the bloodstream, and grow in a distant
focus (metastasize) in normal tissues elsewhere in the body.
Metastasis can be local or distant. Metastasis is believed to be a
sequential process, contingent on tumor cells breaking off from the
primary tumor, traveling through the bloodstream, and stopping at a
distant site. At the new site, the cells establish a blood supply
and can grow to form a life-threatening mass. Both stimulatory and
inhibitory molecular pathways within the tumor cell regulate this
behavior, and interactions between the tumor cell and host cells in
the distant site are also significant.
[0076] By "micrometastasis" is meant a small number of cells that
have spread from the primary tumor to other parts of the body.
Micrometastasis may or may not be detected in a screening or
diagnostic test.
[0077] "Cancer recurrence" herein refers to a return of cancer
following treatment, and includes return of cancer in the primary
organ, as well as distant recurrence, where the cancer returns
outside of the primary organ.
[0078] A subject at "high risk of cancer recurrence" is one who has
a greater chance of experiencing recurrence of cancer. For example,
relatively young subjects (e.g., less than about 50 years old),
those with positive lymph nodes, particularly 4 or more involved
lymph nodes (including 4-9 involved lymph nodes, and 10 or more
involved lymph nodes), and those with tumors greater than 2 cm in
diameter, e.g., in breast cancer patients. A subject's risk level
can be determined by a skilled physician. Generally, such high risk
subjects will have lymph node involvement (for example with 4 or
more involved lymph nodes); however, subjects without lymph node
involvement are also high risk, for example if their tumor is
greater or equal to 2 cm.
[0079] "Decrease in risk of cancer recurrence" is meant reducing
the likelihood of experiencing recurrence of cancer relative to an
untreated patient (i.e., relative to a patient not treated with a
regimen that includes the MDSC inhibiting agent), or relative to a
control treatment protocol, such as treatment only with the
chemotherapeutic agent, such as those used in the standard of care
for colorectal cancer, e.g., leucovorin, 5-fluorouracil,
oxaliplatin, irinotecan or a combination thereof. Cancer recurrence
is monitored for at least about two months, four months, six
months, nine months, or at least about 1 year, or at least about 2
years, or at least about 3 years, or at least about 4 years, or at
least about 5 years, or at least about 10 years, etc., following
the initiation of treatment or following the initial diagnosis.
[0080] "Initiation of treatment" refers to the start of a treatment
regimen following surgical removal of a tumor. In one embodiment,
such may refer to administration of one or more chemotherapeutic
agents following surgery. Alternatively, this can refer to an
initial administration of a treatment that includes the MDSC
inhibiting agent and one or more chemotherapeutic agent.
[0081] By "curing" cancer is herein is meant the absence of cancer
recurrence at about 2, 3, 4 or about 5 years after beginning
adjuvant therapy, depending on the type of cancer.
[0082] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0083] The term "anti-cancer therapy" refers to a therapy useful in
treating cancer. Examples of anti-cancer therapeutic agents
include, but are limited to, e.g., surgery, chemotherapeutic
agents, growth inhibitory agents, cytotoxic agents, agents used in
radiation therapy, anti-angiogenesis agents, apoptotic agents,
anti-tubulin agents, and other agents to treat cancer, such as
anti-HER-2 antibodies, anti-CD20 antibodies, an epidermal growth
factor receptor (EGFR) antagonist (e.g., a tyrosine kinase
inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (Tarceva.RTM.),
platelet derived growth factor inhibitors (e.g., Gleevec.RTM.
(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib),
interferons, cytokines, antagonists (e.g., neutralizing antibodies)
that bind to one or more of the following targets ErbB2, ErbB3,
ErbB4, PDGFR-beta, BlyS, APRIL, TRAIL/Apo2, and other bioactive and
organic chemical agents, etc. Combinations of two or more of these
agents are also included in the invention.
[0084] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include is a chemical compound useful in the treatment of cancer.
Examples of chemotherapeutic agents include alkylating agents such
as thiotepa and CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1.sup.3 dynemicin,
including dynemicin A; bisphosphonates, such as clodronate; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, carminomycin, carzinophilin, chromomycinis,
dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; eflornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.RTM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11) (including the
treatment regimen of irinotecan with 5-FU and leucovorin);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment
regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g.,
erlotinib (Tarceva.RTM.)) and VEGF-A that reduce cell proliferation
and pharmaceutically acceptable salts, acids or derivatives of any
of the above.
[0085] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); epidermal
growth factor; hepatic growth factor; fibroblast growth factor;
prolactin; placental lactogen; tumor necrosis factor-alpha and
-beta; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-alpha; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;
insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-alpha, -beta
and -gamma colony stimulating factors (CSFs) such as macrophage-CSF
(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF
(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor
necrosis factor such as TNF-alpha or TNF-beta; and other
polypeptide factors including LIF and kit ligand (KL). As used
herein, the term cytokine includes proteins from natural sources or
from recombinant cell culture and biologically active equivalents
of the native sequence cytokines.
[0086] By "reduce or inhibit" is meant the ability to cause an
overall decrease preferably of 20% or greater, more preferably of
50% or greater, and most preferably of 75%, 85%, 90%, 95%, or
greater. Reduce or inhibit can refer to the symptoms of the
disorder being treated, the presence or size of metastases or
micrometastases, the size of the primary tumor or the presence or
the size of the dormant tumor.
[0087] The term "intravenous infusion" refers to introduction of a
drug into the vein of an animal or human patient over a period of
time greater than approximately 5 minutes, preferably between
approximately 30 to 90 minutes, although, according to the
invention, intravenous infusion is alternatively administered for
10 hours or less.
[0088] The term "intravenous bolus" or "intravenous push" refers to
drug administration into a vein of an animal or human such that the
body receives the drug in approximately 15 minutes or less,
preferably 5 minutes or less.
[0089] The term "subcutaneous administration" refers to
introduction of a drug under the skin of an animal or human
patient, preferable within a pocket between the skin and underlying
tissue, by relatively slow, sustained delivery from a drug
receptacle. The pocket may be created by pinching or drawing the
skin up and away from underlying tissue.
[0090] The term "subcutaneous infusion" refers to introduction of a
drug under the skin of an animal or human patient, preferably
within a pocket between the skin and underlying tissue, by
relatively slow, sustained delivery from a drug receptacle for a
period of time including, but not limited to, 30 minutes or less,
or 90 minutes or less. Optionally, the infusion may be made by
subcutaneous implantation of a drug delivery pump implanted under
the skin of the animal or human patient, wherein the pump delivers
a predetermined amount of drug for a predetermined period of time,
such as 30 minutes, 90 minutes, or a time period spanning the
length of the treatment regimen.
[0091] The term "subcutaneous bolus" refers to drug administration
beneath the skin of an animal or human patient, where bolus drug
delivery is preferably less than approximately 15 minutes, more
preferably less than 5 minutes, and most preferably less than 60
seconds. Administration is preferably within a pocket between the
skin and underlying tissue, where the pocket is created, for
example, by pinching or drawing the skin up and away from
underlying tissue.
[0092] The term "therapeutically effective amount" refers to an
amount of a compound, preparation or regimen effective to treat a
disease or disorder in a mammal. In the case of cancer, the
therapeutically effective amount of the drug may reduce the number
of cancer cells; reduce the tumor size; inhibit (i.e., slow to some
extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the disorder. For the treatment of tumor dormancy
or micrometastases, the therapeutically effective amount of the
drug may reduce the number or proliferation of micrometastases;
reduce or prevent the growth of a dormant tumor; or reduce or
prevent the recurrence of a tumor after treatment or removal (e.g.,
using an anti-cancer therapy such as surgery, radiation therapy, or
chemotherapy). To the extent the drug may prevent growth and/or
kill existing cancer cells, it may be cytostatic and/or cytotoxic.
For cancer therapy, efficacy in vivo can, for example, be measured
by assessing the duration of survival, disease free survival (DFS),
time to disease progression (TTP), duration of progression free
survival (PFS), the response rates (RR), duration of response, time
in remission, and/or quality of life. The effective amount may
improve disease free survival (DFS), improve overall survival (OS),
decrease likelihood of recurrence, extend time to recurrence,
extend time to distant recurrence (i.e., recurrence outside of the
primary site), cure cancer, improve symptoms of cancer (e.g., as
gauged using a cancer specific survey), reduce appearance of second
primary cancer, etc.
[0093] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented, including those in which the
occurrence or recurrence of cancer is to be prevented.
[0094] The following examples are presented to illustrate certain
embodiments of the invention.
Example 1
Effects of Liposomal Clodronate (LC) Administration on Antibody
Responses and IFN Following Vaccination
[0095] The present example is provided to demonstrate the enhanced
antibody response and IFN-.gamma. of an animal to a vaccine
containing a conventional adjuvant in conjunction with the MDSC
inhibitory additive described herein.
[0096] Mice (n=3 per group) were immunized s.c. with 10 ug
ovalbumin in a commercial adjuvant (CLDC). At the indicated time
points, the mice were also administered liposomal clodronate (LC)
an MDSC inhibitory additive, in order to assess the effects of LC
administration on conventional vaccine responses. Antibody
responses in serum were assessed after 2 rounds of treatment had
been administered and anti-ova titers were determined by ELISA.
[0097] As demonstrated in FIG. 1, antibody titers in animal given
vaccine alone resulted in an ova endpoint titler level of about
10.sup.(5). The administration of the MDSC inhibitor, LC, 2 days
after administration of the Ova vaccine did not appreciably change
this antibody titer. However, LC administered on the same day as
the vaccine resulted in a measurable increase of antibody titler of
about 10.sup.(6). Even more significantly, LC administration 1 day
before administration of the vaccine resulted in a measurable
antibody titler level of over 10.sup.(7). The antibody titer in
animals administered the LC vaccine additive 2 days prior to the
vaccine was about 10.sup.(6), with the antibody title in animals
administered the LC additive 3 days prior to the vaccine also being
about 10.sup.(6).
[0098] Mice were vaccinated with Ova, with or without the
administration of LC, as described in as described above. After two
treatments, the mice were euthanized and the spleen cells were
collected and restimulated in vitro with Ova and 18 hours later,
culture supernatants were analyzed for release of IFN-.gamma..
Pre-treatment of mice by one day with LC resulted in a significant
increase in IFN-.gamma. production in lymphocytes of vaccinated
mice response to Ova restimulation.
[0099] The present results demonstrate the enhanced immune response
in increased antibody titer levels and IFN-.gamma., provided upon
administration for the MDSC inhibiting agents, such as LC, with a
vaccine preparation containing an adjuvant.
Example 2
Co-Administration of LC with a Vaccine Results in Elimination of
Gr-1+ Myeloid Derived Suppressor Cells (MDSC) in Draining Lymph
Nodes
[0100] The present example demonstrates that the use of the vaccine
additive (such as LC) together with a vaccine will block and/or
eliminate the presence of myeloid derived suppressor cells (MDSC)
in draining lymp nodes in vivo. Removing the vaccine-inhibiting
activity of the MDSCs to provide a more robust immune response to
the vaccine. More specifically, the present example demonstrates
the effects of liposomal clodronate (LC) on the immunosuppressive
MDSC population of cells, which are elicited by the vaccine and
typically accumulate in draining lymph nodes. In addition, the
elimination of interference with the recruitment of monocytic
and/or neutrophilic MDSC to tumor and lymp node tissues in response
to tumor derived chemokines is demonstrated when the vaccine
additive (MDSC inhibiting agent) is administered along with a
vaccine.
[0101] Mice (n=3 per group) were immunized with vaccine alone (Vax
alone), LC alone, or with LC plus the vaccine (Vax+LC) and the
draining lymph nodes were collected 48 h later and the cells in the
lymph nodes were analyzed by flow cytometry to determine the number
of Gr-1+MSC.
[0102] As demonstrated in the data presented at FIG. 3, when mice
were vaccinated alone, there was a large increase in the number of
MSSC (7,000+/-50 Cells). However, when the mice were co-vaccinated
with a conventional vaccine together with LC (Vax+LC), the numbers
of MDSC that accumulated in the draining lymph node were
significantly suppressed (500+/-10 Cells), by at least 14-fold,
compared to administration of vaccine without LC.
Example 3
Depletion of Immunosuppressive Myeloid Derived Suppressor Cells
Generates Tumor Immunity and Elicits Antitumor Activity In Vivo
[0103] The present example demonstrates the utility of the present
invention for providing an enhancement of vaccine activity in tumor
bearing animals. In addition, the present example demonstrates the
utility of the present vaccine adjuvant additives (MCSC inhibiting
agents) for reducing tumor growth in vivo. In addition, the present
example demonstrates the utility of the invention for providing an
enhanced vaccine preparation for tumor bearing animals through
depletion of suppressive myeloid derived cells.
[0104] Two populations of myeloid cells with opposing functions are
generated in response to inflammation. These two different subsets
of MDSCs suppress immunity via different mechanisms. The balance
between these two populations regulates innate and adaptive
immunity in an animal. The present inventors have found that
removing the suppressive population of myeloid derived suppressor
cells that are stimulated during inflammation has a marked impact
on new and adaptive immune responses in vivo.
[0105] It has been reported that immature myeloid cells generated
by inflammation suppresses immune responses.
[0106] FIG. 4 demonstrates that injection of LC, and MDSC
suppressing agent, results in the efficient and systemic depletion
of phagocyte cells. As shown in FIG. 4A, before treatment
demonstrated a population of macrophages of 8.05% of total spleen
cells, while administration of LC after 24 hours resulted in a
marked reduction in splenic macrophages to only 0.37% of the total
cells.
[0107] FIG. 5 demonstrates the effect of depleting phagocytic
myeloid cells in mice with established syngeneic tumors.
Administration of liposomal clodronate (LC) (-.tangle-solidup.-)
significantly reduced tumor area size at 15 days post treatment
(tumor area less than 10 mm.sup.2 at about 18 days), while tumor
area continued to increase in mice treated with liposomes with
phosphate buffered saline (tumor area 30 mm.sup.2 at about 18 days)
(Lip-PBS) (- -), similar to the continued tumor area growth seen in
non-treated Control mice with tumors (-.box-solid.-).
[0108] FIG. 6 demonstrates a marked regression of tumor size in
dogs with soft tissue sarcoma following LC treatment. Soft tissue
sarcoma is locally invasive, with surgery being the primary
treatment. These sarcomas are also typically chemoresistant.
[0109] Dogs having this spontaneous form of soft tissue sarcoma
given the LC treatment demonstrated a significant decrease in tumor
size at about 40 days post treatment (tumor size pre-treatment of
about 9.5 cm, tumor size 40 days post LC treatment about 8 cm) (See
FIG. 6). These dogs were not vaccinated.
[0110] FIG. 7 demonstrates the marked regression of lung and
pleural metastasis in histiocytic sarcoma after LC and chemotherapy
treatment (lomustine). Histiocytic sarcomas are very aggressive
locally and rapidly metastatic, and chemotherapy resistant. FIG. 7
(top panel, "Before LC Treatment"), shows significant metastasis of
the sarcoma (see heavily occluded image of lung tissue from sarcoma
cancer cell proliferation and metastasis), while treatment with LC
resulted in almost complete ablation of the occluded lung area
after 14 days post treatment (See FIG. 14, Bottom Panel, "Day 14
After Treatment"). These dogs were not vaccinated.
[0111] FIG. 8 demonstrates that administration of the MDSC
depleting agent (LC) mediates effects locally in a tumor bearing
animal by significantly depleting tumor associated macrophages
(Control 11+/-4% CD 11b/F4/80+ cells, LC 2.5+/-1% CD 11b/F4/80+
cells).
[0112] FIG. 9 demonstrates that administration of the MDSC
depleting agent (LC) is also capable of depleting the MDSC
population for cells systemically, as it evidenced by suppression
in multiple sites. The MSC/ul blood in Control (non-treated
animals) was about 4.1.times.10.sup.5, while MSC in blood from
LC-treated animal was about 1.9.times.10.sup.5.
[0113] FIG. 10 demonstrates that the antitumor activity elicited by
LC treatment is T cell dependent (RAG-/-LC=- -; RAG-/-Lip
(Control)=-.box-solid.-; WT LC=-.tangle-solidup.-).
[0114] (RAG-/- is recombinase activating gene knockout mouse; WT is
wild Type) FIG. 11 demonstrates CD8 cells are required for LC
induced antitumor activity. The tumor area in animals treated with
WT LC (- -) remained relatively unchanged up to 20 days after
treatment (about 10 mm.sup.2), while tumor area significantly
increased in animals from the CD 8-/- lip Control group (--) (about
110 mm.sup.2 tumor area after 20 days), CD 8-/-LC (-.box-solid.-)
(about 75 mm.sup.2 tumor area after 20 days), and WT lip Control
group (circle) (about 60 mm.sup.2 tumor area after 20 days).
[0115] FIG. 12 illustrates the rapid kinetics of MDSC expansion in
vivo (Control Gr-1 hi vs Gr-1 mid, Day 2, day 6, Day 8). This data
speaks to the mechanism by which it is believed that the vaccine
recruits MDSC to the lymph nodes, namely via the bloodstream. So,
while not intending to be limited to any specific mechanism of
action or to eliminate consideration of other physiological or
other factors, the MDSC depleting agents that work are acting to
deplete the MDSC in the bloodstream before they get to the draining
lymph nodes.
[0116] FIG. 13 demonstrates that the concurrent depletion of
myeloid cells (by administration of an MDSC depleting agent like
LC) at the time of vaccination significantly enhances vaccine
responses. The data shows that LC treatment 2 days prior to
administration of the Ova vaccine resulted in a significant
enhancement of antibody titer, compared to mice that received the
vaccine only. However, the greatest enhancement in antibody
response occurred when LC was administered on the same day as the
vaccine, or one or two days afterwards.
Example 4
Coadministration of Liposomal Bisphoshonates Depletes
Vaccine-Induced MSC Accumulation/Novel Vaccine Adjuvants/Vaccine
Additives
[0117] The present example is provided to demonstrate the utility
of the invention as a vaccine additive, and demonstrates the
utility of suppressing myeloid suppressor cells as a method for
enhancing the vaccine activity and effectiveness in a healthy
individual.
[0118] In this study, accumulation of MDSC cells in the lymph nodes
of control animals and in animals treated with an MDSC depleting
agent, LC, was measured. As demonstrated in FIG. 14, the number of
cells (11b+Gr-1+) in the control animals was about 1000 (no
vaccine, no LC). The number of cells in animals treated with the
vaccine alone was about 7,500+/-500. The animals treated with the
MDSC depleting agent (LC) alone, demonstrated a cell number of only
about 2,500, with a similar number of cells being observed in
animals receiving vaccine plus the MDSC depleting agent (LC). This
data demonstrates that administration of an MDSC depleting agent
will prevent MDSC accumulation in lymph nodes, thus eliminating the
"block" typically resulting during a robust immune vaccine
response.
[0119] FIG. 15 demonstrates that total IgG titers in animals
treated with a vaccine is significantly enhanced in the presence of
an MDSC inhibiting agent, such as LC. The enhancement of IgG titer
levels in animals treated with MDSC inhibiting agent (LC) on the
same day or 1 day after the vaccine treatment was increased 10-fold
over control or vaccine only receiving animals. The IgG titer
levels were increased about 5-fold in animals treated with an MDSC
inhibiting agent (LC) either 2 days or 3 days after vaccine
treatment, compared to controls and vaccine only treated
animals.
[0120] FIG. 16 demonstrates the enhanced CD4 T cell response to
tumor cell lysates prepared from lysed lymphoma A20 cells. The
combination of vaccine and MDSC inhibiting agent (LC) resulted in
an IFN-g (pg/ml) level of about 425+/-124, while vaccine alone
resulted in only a level of about 225 pg/ml+/- about 50. (FIG.
16A). FIG. 16 B provides results achieved in animals when their
spleen cells were restimulated with whole tumor cells, which tends
to induce immune responses by CD8+ T cells. Similar enhancement of
IFNg levels in MDSC depleting agent treated animal s receiving the
vaccine were observed, compared to animals treated with vaccine
alone.
[0121] Tumor size in animals treated with the whole tumor vaccines
were also examined after treatment with vaccine or MDSC inhibiting
agent (LC) plus vaccine treatment. The results of this study are
presented in FIG. 17. The greatest suppression of tumor size and
growth was again observed in animals treated with the MDSC
inhibiting agent (LC) in combination with the whole cell tumor
vaccines.
[0122] Tumor size in animals treated with a tumor cell membrane
vaccine was also examined. These results are presented in FIG. 18.
Similarly, the greatest suppression of tumor size and growth was
again observed in animals treated with the MDSC inhibiting agent
(LC) in combination with the tumor cell membrane vaccines.
Example 5
The MDSC Depleting Agents in Combination with Numerous Different
Adjuvants Provides an Enhancement of Vaccine Adjuvancy
[0123] The present example demonstrates the utility of the present
invention for enhancing the immune response of an animal to a
vaccine containing a diverse group of adjuvants, including by way
of example, IFA (incomplete Freund's adjuvant), Alum, Alhydroge;
MPL (Corrixa MPL adjuvant), DPIC (liposomal polyI:C adjuvant); and
CLDC (cationic liposome-DNA complex adjuvant).
[0124] Mice (n=4 per group) were vaccinated with 5 ug Ova protein
admixed with adjuvant s.c. Half of the groups of animals were
vaccinated and treated at the same time by i.v. administration of
liposomal clodronate (LC), which is an effective myeloid derived
suppressor cell (MDSC) depleting agent. (The optimal timing of LC
administration relative to vaccine delivery was previously
determined). The mice were then boosted with vaccine +/-LC 10 days
later, then antibody responses to Ova were assessed by endpoint
dilution ELISA 7 days after the boost. The data indicated that
co-administration of LC with vaccination generated a strong
increase in antibody titers for all 5 different adjuvants. The
magnitude of the LC effect was greatest with the Alum adjuvant.
These results are presented in FIG. 19. The ability of LC
co-administration to enhance vaccine humoral responses with
conventional adjuvants was relatively independent of adjuvant
composition. It should also be noted that vaccination with Ova+LC
elicited only modest humoral immune responses. Key to adjuvants:
IFA=incomplete Freund's adjuvant; Alum=Alhydrogel; MPL=Corrixa MPL
adjuvant; DPIC=liposomal polyLC adjuvant; CLDC=cationic
liposome-DNA complex adjuvant.
[0125] Mice (n=4 per group) were vaccinated with 5 ug Ova protein
admixed with adjuvant s.c. Half of the groups of animals were
vaccinated and treated at the same time by i.v. administration of
liposomal clodronate (LC), which is an effective myeloid derived
suppressor cell (MDSC) depleting agent. The optimal timing of LC
administration relative to vaccine delivery was determined in
previous studies. The mice were then boosted with vaccine +/-LC 10
days later, and the mice were euthanized 7 days later for
assessment of T cell responses to Ova restimulation in vitro.
Spleen cells were incubated with 50 ug/ml Ova in triplicate wells
for 72 h, then supernatants were collected and IFN-.gamma.
concentrations determined by ELISA. The results are presented at
FIG. 20. The data indicated that co-administration of LC with
conventional vaccines generated stronger T cell recall IFN-.gamma.
responses than immunization with vaccine alone. This effect was
observed for all 5 different adjuvants, and was particularly
evident in the case of vaccines that elicited relatively small
IFN-g responses on their own (eg, IFA, Alum, MPL). Thus, the
ability of LC co-administration to enhance T cell responses is
demonstrated to be independent of adjuvant composition.
Example 6
Vaccination Triggers Recruitment of MDSC Cells that can be Blocked
with Adjuvant/Vaccine Additive (Myeloid Derived Suppressor Cell
Inhibiting Agent)
[0126] The present example is provided to demonstrate the utility
of the present invention for blocking the vaccine inhibitory action
of infiltrating myeloid derived suppressor cells (MDSC) (also known
as vaccine elicited myeloid cells, or MDSC), with a detectable
augmentation of vaccine immunity in vivo. In particular, the
present example presents data demonstrating that the interference
with the MDSC recruitment will augment vaccine immunity by
interfering with the recruitment of MDSC to the lymph nodes after
vaccination. In addition, the data presented here presents direct
in vivo data demonstrating that the presence of the MDSC in the
vaccine draining lymph nodes interferes with T cell responses. T
cell activation in the lymph nodes is essential to developing a
good vaccine response. The present example also provides evidence
of the utility of the present invention for augmenting the
effectiveness of a cancer vaccine by including the vaccine/adjuvant
additives (the MDSC inhibitory agents) in vaccination.
[0127] Mice were vaccinated in the footpad and 24 h later, the
vaccine draining LN was collected and cellular responses assessed
using flow cytometry. A strong infiltrate of Cd11b+/Gr-1+ myeloid
cells (MDSC) in the lymph nodes (LNs) of vaccinated mice was
observed. FIG. 21 demonstrates the increase in myeloid cell
infiltration in vaccinated animals (Right panel) compared to
non-vaccinated animals (Left panel).
[0128] Next, a study was done to demonstrate that treatment with
the adjuvant/vaccine additive (MDSC inhibiting agent) blocked the
infiltration of cells to lymph nodes (LN). Mice (n=4 per group)
were vaccinated and at the same time treated with LC alone, or LC+
vaccine. In the draining LNs of mice that received the vaccine
only, there was a large increase in MDSC. However, in the LNs of
vaccinated mice also treated with LC, the increase in MDSC was also
completely blocked (FIG. 22).
Example 7
LC Administration Generates Significant Increase in Antibody
Response and T Cell Response to Vaccination Augmented with an MDSC
Inhibiting Agent
[0129] The present example is provided to demonstrate the utility
of the inventor for enhancing antibody response and T cell response
to a vaccine in an animal.
[0130] Antibody Response:
[0131] Mice were vaccinated and treated with LC at various time
points before or after vaccination. Only LC treatment at the time
of vaccination or shortly thereafter was effective in improving
vaccine responses (antibody titers), consistent with the idea that
depletion of the MDSC population is critical for enhancing vaccine
efficacy (FIG. 23).
[0132] T Cell Response: Draining LN cells were collected from
control and vaccinated mice, as well as from vaccinated mice also
treated with LC at the time of vaccination. The LN cells were
incubated in vitro with the vaccine antigen (Ova) and IFN-g
production by the T cells was assessed 3 days later. Vaccinated
mice treated with LC generated significantly higher amount of IFN
than T cells from vaccinated only mice, indicative of increased T
cell responses to the vaccine antigen.
Example 8
T Cell Proliferation is Inhibited after Vaccination In Vivo, and
not Inhibited after Vaccination Together with an MDSC Inhibiting
Agent
[0133] The present example demonstrates that T cell proliferation
after vaccination may be improved in the presence of the MDSC
inhibitory agents after invention, and thus, enhance immune
response to the vaccine.
[0134] Mice were vaccinated, then 24 h later Lymph nodes (LN) were
collected and the LN cells were labeled with the dye CFSE. Flow
cytometry was then used to determine how many CD3+ T cells
underwent cell division during a 72 h in vitro incubation period.
In LNs depleted of MDSC by LC treatment, T cell proliferation was
significantly higher than in the LNs from vaccinated only (no LC)
mice. This data is shown in FIG. 25.
Example 9
Production of Chemokines MCP-1 (CLL2)
[0135] The present example demonstrates that the presence of the
MDSC inhibiting agents also induces the production of chemokines,
compounds that regulate monocyte recruitment.
[0136] FIG. 26 presents the data from this study. Vaccine draining
LNs were harvested 3 h after vaccination and release of CCL2 (the
primary chemokine regulating monocyte recruitment) was measured.
Chemokine (C--C motif), 2 (CCL2) is a small cytokine belonging to
the CC chemokine family that is known as monocyte chemotactic
protein-1(MCP-1) and small inducible cytokine AZ. CCL2 recruits
monocytes, memory T cells, and dendritic cells to sites of tissue
injury, infection and inflammation.
[0137] Vaccination triggered a significant increase in CCL2 release
from the draining LN, which could serve as an important signal for
MDSC recruitment. Therefore, it is anticipated that blocking CCL2
release with a CCL2 inhibitory agent will inhibit recruitment of
myeloid derived suppressor cells (MDSC). As a result, it is
expected that CCL2 inhibitory and/or blocking agents with therefore
also be useful as an additive and/or adjuvant in enhancing immune
response in an animal to a vaccine.
Example 10
The MDS Cell Inhibiting Agents Impair Mobilization of Myeloid
Derived Suppressor Cells
[0138] The present example establishes that the invention may be
used to immobilize populations of myeloid derived suppressor cells
in vivo.
[0139] Studies were done to compare the ability of mice unable to
mobilize MDS cells (MDSC) due to a lack of expression of the CCL2
receptor (ie, CCR2-/- mice). These mice do not generate monocyte
infiltration in response to inflammatory stimuli. The ability of
CCR2 mice to make antibody responses to vaccination was compared to
that of wild type (WT) mice, and the CCR2- mice were found to be
significantly better (FIG. 27). This inability of these CCR2-
animals to mobilize monocytes is demonstrated in this data to
contribute to enhanced immunity to vaccination.
[0140] In addition, the CCR2-/- mice did not respond to LC
treatment. This establishes that inhibition of monocyte migration
has the same effects on vaccination as actually eliminating
monocytes with an MDSC inhibitory agent. These results are
important because they indicate that interfering with monocyte
migration, as for example by administering an MDSC inhibitory
agent, e.g., small molecule such as a CCR2 inhibitor (such as, for
example, RS1028595, PF-04178903, or those listed in Higgins et al.,
(2007, Table 1) drug, can improve vaccine responses as effectively
as eliminating monocytes outright with liposomal clodronate (LC) or
other bisphosphonate drug.
[0141] By way of example, such small molecule drugs may include
RS1028595, Sigma Aldrich.
Example 11
MDSC Inhibiting Agents with Cancer Vaccines
[0142] The present example demonstrates that the MDSC inhibiting
agents used in combination with cancer vaccines will improve the
anti-cancer activity of the cancer vaccine.
[0143] Studies were conducted to determine whether
co-administration of LC with a tumor vaccine could improve
responses to vaccination with an autologous tumor vaccine prepared
with A20 cell membrane proteins. Mice with established A20 lymphoma
tumors were vaccinated once weekly, with or without LC treatment,
and tumor growth rates were monitored. The combined treatment with
tumor vaccine and LC significantly slowed tumor growth, compared to
treatment with either vaccine or LC alone (FIG. 28).
Example 12
IFN-Gamma Response in MDSC Depleted Animals Vaccinated with HA
Peptide Vaccine
[0144] The effects of MDSC depletion and vaccination on the ability
of spleen CD8 T cells to mount a recall response against tumor
antigens is demonstrated. FIG. 29 presents the results of the
effects of MDSC depletion and vaccination on the ability of spleen
CD4 T cells to mount a recall response against soluble tumor
antigens. The A20-HA tumor used in these studies was transfected
with the influenze HA gene, which permitted the use of this antigen
as a surrogate tumor antigen. The ability of vaccinated mice to
mount an IFN-.gamma. recall response against the MHC class II
restricted HA peptide (SFERFEIFPKE) was assessed.
[0145] A significant increase in INF-.gamma. production in the
vaccinated mice was observed, and also a significant further
enhancement in this response in the vaccine+LC treatment group.
These data demonstrate that vaccination plus MDSC depletion with LC
significantly augments CD4 T cell responses to a tumor antigen
(FIG. 29).
[0146] In another study, the effects of MDSC depletion and
vaccination on inability to spleen CD8 T cells to mount a recall
response agent tumor analysis was assessed. The A20-HA tumor used
in these studies was transfected with the influenza HA gene, which
permitted the use of this antigen as a surrogate tumor antigen. The
ability of vaccinated mice to mount an IFN-.gamma. recall response
against the MHC class I restricted HA peptide (IYSTVASSL) was also
examined. A modest increase in IFN-.gamma. production in vaccine
only mice following restimulation with the MHC I peptide was
observed (See FIG. 29, 950+/-40 pg/ml IFNg release). However, there
was a significant enhancement in response in the vaccine+LC
treatment group (1,750 pg/ml IFNg release+/-50 pg/ml) (FIG.
30).
[0147] These data indicate that vaccination plus MDSC depletion
with LC significantly augments CD8 T cell responses to a tumor
antigen. (FIG. 30).
Example 13
MDSC Depletion Augments Generation of Tumor Specific Antibodies
Following Tumor Vaccination
[0148] The present example is provided to demonstrate the use of
the present MDSC depleting agents in augmenting production of
anti-tumor antibodies.
[0149] The impact of MDSC depletion using LC on the magnitude of
anti-tumor antibody responses is demonstrated. Serum from
vaccinated and control mice was evaluated for tumor surface binding
to fixed A20 tumor cells using flow cytometry. Vaccination+LC
administration significantly increased the generation of antibodies
directed to cell surface epitopes on the A20 tumor cells, compared
to vaccination alone. (FIG. 31).
[0150] Experiments were also done to determine whether MDSC
depletion using LC could augment tumor vaccine responses in a
different strain of mice (MDSC) and against a different tumor type
(MCA-205). Mice were vaccinated with an autologous MCA vaccine and
treated with or without LC. After 2 immunizations, spleens were
collected and restimulated in vitro with MCA tumor cells and
IFN-.gamma., indicative of enhanced T cell immunity following MDSC
depletion. These results also indicate that the effectiveness of
MDSC depletion with LC is not limited to a certain strain of mouse
or a certain tumor type (FIG. 32).
[0151] This HA molecule is not a tumor antigen. It is from
influenza. It is actually used as a surrogate for a tumor antigen
in the A20 model used herein. The peptides are HA peptides that are
used here to distinguish CD4 from CD8 T cell responses.
Prophetic Example 14
Myeloid Suppressor Cell Inhibitory Agents for Use with Vaccine
Regimens
[0152] Any variety of small molecules that are capable of
inhibiting the action of myeloid suppressor cell migration,
accumulation and viability is anticipated to be useful as the
adjuvant additive of the present invention. By way of example, the
following MDSC agents may include drugs that block monocyte release
from bone marrow (CCL2 or CCR2 inhibitors or competitors, M-CSF
inhibitors, GM-CSF inhibitors). Further examples include compounds
that block chemokines that mobilize neutrophils from the bone
marrow, including inhibitors of IL-8, KC, and G-CSF.
[0153] Vaccine Regimens: It is anticipated that the MDSC agents
described herein may be used to boost immune response to virtually
any vaccine regimen in an animal. By way of example, these vaccine
include vaccines for: 1.) any infectious agent (bacterial, viral,
fungal, protozoal); 2.) vaccines for allergy, 3.) vaccines for
autoimmune disorders; 4.) vaccines for toxins; 5.) vaccines for
addictive substances (eg., nicotine, alcohol, caffeine, etc.).
[0154] While not intended to be exhaustive, the following presents
exemplary vaccines for this purpose.
[0155] Exemplary Infectious Pathogens
[0156] The present example demonstrates the utility of the present
invention with disease associated with a wide variety of infectious
pathogens and biological toxins, including by way of example and
not exclusion, tetanus, influenza, rabies, viral hepatitis,
diphtheria, anthrax, Streptococcus pneumoniae infection, malaria,
leishmaniasis, ricin toxicosis, and Staphylococcal enterotoxin B
toxicosis.
[0157] TABLE 2 Classification of Common Vaccines for Humans Disease
or Pathogen Type of Vaccine Whole Organisms:
[0158] Bacterial cells: Cholera Inactivated Plague Inactivated
Tuberculosis Attenuated BCG+Salmonella typhi Attenuated Viral
Particles: Influenza Inactivated Measles Attenuated Mumps
Attenuated Rubella Attenuated Polio (Sabin/OPV) Attenuated Polio
(Salk/IPV) Inactivated V. zoster Attenuated Yellow fever Attenuated
Type of Vaccine (Purified) Macromolecules Toxoids: Diphtheria
Inactivated exotoxin Tetanus Inactivated exotoxin acellular
Pertussis Inactivated exotoxins Capsular polysaccharide:
Haemophilus influenzae b polysaccharide+protein carrier Neisseria
meningidis Polysaccaride Streptococcus pneumoniae 23 distinct
capsular polysaccharides Surface antigen: Hepatitis B Recombinant
surface antigen (HbsAg)+Bacillus Calmette-Guerin (BCG) is an
antiviral strain of Mycobacterium bovis.
[0159] Vaccines for Disease Associated with Viral Infections
[0160] Influenza--Influenza is an acute febrile respiratory disease
resulting from infection with the influenza virus. Current
influenza vaccines use aluminum adjuvants. To enhance the efficacy
of vaccines, several adjuvants have been examined. For example, the
oil-in-water emulsion MF59 has been reported to improve vaccine
immunity (Higgins (1996); Martin (1997), though it does not
completely solve the low efficiency of the influenza vaccine in the
elderly (Banzhoff (2003). A synthetic peptide, GK1, derived from
Taenia crassiceps cysticerci was reported to enhance the immune
response accompanying influenza vaccination in both young and aged
mice (Segura-Velasquez (2006).
[0161] As part of the present invention, an influenza vaccine may
be provided that comprises the MDS inhibiting agent combined with
an immunologically effective amount of an influenza antigen with an
adjuvant. By way of example, such an influenza antigen may comprise
a current influenza virus combination of antigens of an H5N1
(hemagglutinin [HA] subtype 1; neuraminidase [NA] subtype 1), and
H3N2 influenza A virus, and an influenza B virus. This preparation
and other influenza antigen preparations are described in Palese
(2006). This article and all of its teachings are incorporated
herein by reference.
[0162] Rabies--Rabies is a devastating neurological disease that is
caused by infection with the rabies virus. Vaccination against
rabies typically utilizes inactivated virus and an aluminum
adjuvant. A lipoid adjuvant of the oil-in-water type, based on
squalene, significantly increased the immunologic response of mice
to vaccination with an inactivated virus vaccine when compared to
vaccination using an aluminum salt adjuvant (Suli, 2004). An
adjuvant based on glycopeptidolipids extracted from Mycobacterium
cheloniae enhanced the immune response of mice to vaccination with
an inactivated rabies virus vaccine (de Souza Matos (2000).
[0163] As part of the present invention, a rabies vaccine may be
provided that comprises the MDSC inhibiting agent combined with an
immunologically effective amount of a rabies antigen. By way of
example, a rabies antigen may comprise an inactivated rabies virus.
One example of an inactivated rabies virus vaccine antigen that may
be used in the present formulations is described in de Souza Matos
(2000).
[0164] 3. Viral Hepatitis--Viral hepatitis, particularly that
caused by Hepatitis B virus, is a serious health problem with over
300 million people affected worldwide. Vaccination offers hope for
effective prophylaxis. Peptide epitopes of the virus stimulated a
significant immune response when fused with heat shock protein 70
from Mycobacterium tuberculosis as an adjuvant (Peng (2006).
Unmethylated CpG dinucleotides were effective as an adjuvant with
hepatitis B antigen in aged mice (Qin (2004); and a vaccine
consisting of hepatitis B virus antigens and an immunostimulatory
DNA sequence is in human clinical trials (Sung (2006). In
development of an intranasal vaccine, it was shown that
DL-lactide/glycolide copolymer microspheres with chitosan were an
effective adjuvant for a vaccine based on recombinant Hepatitis B
surface protein (Jaganathan (2006).
[0165] As part of the present invention, a viral hepatitis vaccine
may be provided that comprises the MDSC inhibiting agent combined
with an immunologically effective amount of a viral hepatitis
antigen. By way of example, such a hepatitis antigen may comprise
recombinant hepatitis B surface protein. By way of example, such a
hepatitis B surface protein antigen is described in Jaganathan,
(2006), which reference is specifically incorporated herein by
reference.
[0166] Vaccines for Disease Associated with Bacterial
Infections:
[0167] Diphtheria--A respiratory disease characterized by dysnepea,
weakness, and pyrexia, diphtheria is the result of infection with
Corynebacterium diphtheriae, bacteria which produces a toxin that
is carried hematogenously through the body. Immunization against
diphtheria is frequently combined with immunization against tetanus
and pertussis; these vaccines typically contain aluminum salt
adjuvants (Sugai (2005). Unmethylated CpG dinucleotides were
effective as an adjuvant in a diphtheria-tetanus-pertussis vaccine
and shifted the immune response toward cell-mediated immunity in
mice immunized intraperitoneally (Sugai (2005). Trials to reduce
adverse side-effects related to the aluminum salt adjuvant of a
vaccine consisting of diphtheria toxoid, tetanus toxoid, and
purified Bordetella pertussis antigens including pertussis toxoid
showed that reduction of the aluminum salt content of the vaccine
resulted in reduced geometric mean antibody concentrations to the
relevant antigens, but did not result in reduction of local or
general side effects (Theeten (2005). Monophosphoryl lipid A was
shown in mice to effectively serve as an adjuvant for diphtheria
toxin in mice (Caglar (2005).
[0168] As part of the present invention, a diphtheria vaccine may
be provided that comprises the MDSC inhibiting agent combined with
an immunologically effective amount of a diphtheria antigen. By way
of example, a diphtheria antigen may comprise a diphtheria toxoid.
One example of a diphtheria toxoid that may be used in the practice
of the present invention is described in Theeten (2005).
[0169] 2. Anthrax--Anthrax is a disease caused by the bacterium,
Bacillus anthracis. Specifically, the bacterium produces a toxin
which results in hemorrhagic necrosis of lymph nodes, hematogenous
spread, shock, and death. A vaccine consisting of one subunit
(protective antigen) of this toxin was shown to protect mice when
combined with a microparticle adjuvant administered by either the
intramuscular or intranasal routes (Flick-Smith (2002). Further,
vaccination protected mice against infection with B. anthracis
spores. While the aluminum salt-adjuvanted anthrax-vaccine-adsorbed
is the only anthrax vaccine licensed in the United States, major
drawbacks exist, including a very lengthy and complicated dosing
schedule, followed by annual booster injections. Further, the
aluminum adjuvant of anthrax vaccine has been linked to Gulf War
Illness among veterans of the 1991 conflict (Petrik (2007)).
[0170] As part of the present invention, an anthrax vaccine may be
provided that comprises the MDSC inhibiting agent combined with an
immunologically effective amount of an anthrax antigen and an
adjuvant. By way of example, such an anthrax antigen may comprise
the one subunit (protective antigen) of the Bacillus anthracis
bacterium. One such particular antigenic subunit is described in
Flick-Smith (2002).
[0171] 3. Streptococcus pneumoniae--A bacterial pathogen of
particular importance to the elderly and young adults,
Streptococcus pneumoniae causes disease including sepsis and
pneumonia, otitis media and meningitis. Vaccines typically involve
adsorption of S. pneumoniae antigens to aluminum salt adjuvants,
and reduced aluminum salt content led to reduced immunogenicity of
S. pneumoniae vaccines (Levesque (2006). In human trials, IL-12
failed to improve the immune response to a pneumococcal
polysaccharide vaccine; and IL-12 was associated with a high
incidence of local and systemic side effects in humans (Hedlund
(2002). Intranasal immunization against S. pneumoniae has been
shown to be an effective method for preventing infection and
disease, with unmethylated CpG dinucleotides serving as an
effective adjuvant for an intranasal polysaccharide-protein
conjugate vaccine (Sen (2006). Likewise, IL-12 and the B-subunit of
cholera toxin were both shown to enhance efficacy of
intranasally-administered preparations of S. pneumoniae antigens
(Sabirov (2006); Pimenta (2006)).
[0172] As part of the present invention, a pneumonia vaccine may be
provided that comprises the MDSC inhibiting agent described herein
together with a vaccine adjuvant combined with an immunologically
effective amount of a pneumococcal antigen. By way of example, such
a pneumococcal antigen may comprise a pneumococcal polysaccharide
antigen. One form of a pneumococcal polysaccharide antigen is
described in Hedlund (2002). This pneumococcal antigen may used as
part in combination with the herein described MDSC inhibiting agent
with an adjuvant containing vaccine preparation.
[0173] Vaccines for Diseases Associated with Parasitic
Infections
[0174] Malaria--Malaria affects millions of people worldwide and
each year, 1-2 million people die from the disease caused by
Plasmodium falciparum. Thus, the need for prophylactic measures has
led to great interest in anti-malaria vaccines. The apical membrane
antigen, a malaria vaccine candidate, was reported to have an
enhanced immunogenicity by the aluminum salt adjuvant Alhydrogel
(HCl Biosector, Denmark); and this adjuvant effect was further
enhanced, and shifted from a Th1 response to a mixed Th1/Th2
response, by inclusion of the adjuvant CpG oligodeoxynucleotide
(Mullen (2006). Alhydrogel and Montanide ISA 720 (Seppic, France)
were compared in rhesus monkeys as adjuvants for a vaccine based on
protective epitopes from the circumsporozoite protein of P.
falciparum. Though Montanide ISA 720 induced superior immune
responses, the formation of sterile abscesses at injection sites
were noted as a significant disadvantage (Langermans (2005). Other
studies with a circumsporozoite protein vaccine conducted in rhesus
monkeys showed that some novel oil-in-water adjuvants with
components of immunostimulants 3-deacetylated monophosphoryl lipid
A (3D-MPL) and the saponin Quillaja saponaria 21 (QS21) were safe
and stimulated improved antibody responses (Stewart (2006). Some of
these same oil-in-water adjuvants improved the immune response to a
vaccine constructed of the P. falciparum antigen, Liver Stage
Antigen-1 (Brando (2006).
[0175] As part of the present invention, a malarial vaccine may be
provided that comprises the MDSC inhibiting agent together with a
vaccine adjuvant combined with an immunologically effective amount
of a malarial antigen. By way of example, such a malarial antigen
may comprise a P. falciparum antigen Liver Stage Antigen-1. This
antigen is described in detail in Brando (2006), this article being
specifically incorporated herein by reference. This antigen may be
combined with the myeloid derived suppressor cell inhibiting agent
material described herein as an adjuvant to provide an
anti-malarial vaccine as described herein.
[0176] 2. Leishmaniasis--Leishmaniasis is a parasitic disease
associated with infection by a species of parasites from the
Leishmania genus. A large spectrum of clinical disease forms can
result from infection, ranging from cutaneous lesions to fatal
visceral forms. In the absence of effective, non-toxic treatments,
great effort has been given to vaccine development. Vaccines based
on DNA of the parasite have been shown to induce partial
protection; aluminum phosphate adjuvant has no effect on the
humoral response to this vaccine, but has been reported to slightly
increase the cellular immune response and protection against
infection in a mouse model (Rosado-Vallado (2005). In evaluations
in rhesus monkeys using a soluble Leishmania antigen and alum with
IL-12 as adjuvants, it was shown that the adjuvants improved
protective immunity, though transient nodules developed at the site
of subcutaneous injection (Kenney (1999). CpG oligodeoxynucleotides
served as an effective adjuvant for a vaccine consisting of live,
nonattenuated L. major organisms alone or in combination with
lysates of heat-killed L. major promastigotes, either without or
bound to alum (Mendez (2003). Partial protective immunity was
stimulated, but mice receiving alum-containing vaccines developed
large dermal lesions that required up to 10 weeks to heal.
[0177] As part of the present invention, an anti-parasitic
infection associated disease vaccine may be provided that comprises
the MDSC inhibiting agent together with a vaccine adjuvant combined
with an immunologically effective amount of a Leishmaniasis
antigen, or any of the other antigenic species described above. By
way of example, a Leishmaniasis antigen may comprise the
Leishmaniasis antigen described in detail in Kenny (1999), which
article is specifically incorporated herein by reference.
[0178] Vaccines for Disease Associated with Biological Toxins
[0179] 1. Ricin--Ricin is a toxin produced naturally by the seeds
of the castor bean plant, Ricinus communis. When humans or animals
are exposed to the toxin, severe respiratory distress and death may
result. Because of its potency and ability to be administered via
aerosol, ingestion, or injection, ricin is considered a powerful
bioweapon. Though there is presently no approved commercial vaccine
for ricin, pilot trials in humans have examined the use of
recombinant, non-toxic forms of one of the subunits of ricin
(Vitetta (2006). This preparation was administered without an
adjuvant and elicited ricin-neutralizing antibodies in some of
those tested, particularly at higher doses. However, all dose
groups were found to result in significant side-effects, including
myalgia and headache. Ricin toxoid adjuvantized by liposomal
encapsulation was found to induce a stronger immune response when
administered intra-tracheally than the vaccine adjuvantized with an
aluminum salt adjuvant (Griffiths, 1997)..sup.29 A vaccine
consisting of a deglycosylated chain A ricin (DCAR) and the
adjuvant LTR72, a mutant of the heat-labile enterotoxin of
Escherichia coli, resulted in a stronger antibody response of
vaccinated mice to ricin, but did not result in improved protection
against lung injury when challenged with ricin (Kende (2006).
[0180] As part of the present invention, an anti-ricin vaccine may
be provided that comprises the MDSC inhibiting agent together with
a vaccine adjuvant as described herein combined with an
immunologically effective amount of a ricin toxoid antigen. By way
of example, such a ricin toxoid antigen is described in detail in
Griffiths (1997), which article is specifically incorporated herein
by reference.
[0181] 2. Staphylococcal enterotoxin B (SEB)--SEB is produced by
the bacteria, Staphylococcus aureus and is associated with food
poisoning. Incorporation of SEB toxoid into biodegradable
poly(DL-lactide-co-glycolide) microspheres enhanced the immune
response of mice to a degree similar to SEB toxoid adsorbed to alum
and combined with complete Freund adjuvant (Eldridge, 1991).
Similarly, SEB toxoid was effectively adjuvantized by incorporation
into polylactic polyglycolic acid copolymer nanospheres; the
resulting immune response was comparable to that achieved by using
alum as an adjuvant (Desai (2000).
[0182] As part of the present invention, an anti-toxin-associated
disease vaccine may be provided that comprises the MDSC inhibiting
agent together with a vaccine adjuvant combined with an
immunologically effective amount of an antigen such as ricin toxoid
or SEB toxoid as antigen. By way of example, such antigens are
described in detail in Vitetta (2006) and Eldridge (1991), the
teachings of which are specifically incorporated herein by
reference.
[0183] Vaccines for Diseases Associated with Prions:
[0184] In some embodiments, the invention provides an adjuvant
preparation that is suitable for use in combination with a
prion-associated disease. By way of example, such prion associated
diseases include, all of which are classified as transmissible
spongiform encephalopathies, bovine spongiform encephalopathy,
scrapie, cervix chronic wasting disease and Creutzfeldt-Jakob
disease.
[0185] Although prions use immune and lymphoreticular cells to gain
access to the brain (Aguzzi, 2003), existing evidence suggests that
humoral immune responses can suppress infection. In particular,
antibodies to the cellular prion protein (PrPc) are known to
inhibit prion propagation (Petetz, 2001; Enari, 2001). Still, host
tolerance to endogenous PrPc remains a major obstacle to active
vaccination. In mice, vaccination with recombinant PrPc antigens
such as peptides and polypeptides stimulated only weak immune
responses. Co-administration of prion antigens with adjuvants such
as Freund's (Polymenidou, 2004; Koller, 2002; Sigurddson, 2002;
Gilch, 2003; Hanan, 2001; Hanan, 2001; Souan, 2001; Arbel, 2003);
Montanide IMS-1313 (Schwartz, 2003); TiterMax.RTM., a combination
of a proprietary block copolymer CRL-8941, squalene, a
metabolizable oil, and a unique microparticulate stabilizer (Gilch,
2003); and CpG oligonucleotides (Rosset, 2004) all failed to induce
strong immune responses.
[0186] It is anticipated that the presently described MDSC
inhibitory agents may included with an adjuvant preparation
together with the prion protein (PrPc) to provide an improved
vaccine against prion-associated infections.
Prophetic Example 15
CCR2 Inhibitors and/or CCR2 Antagonists as Vaccine/Adjuvant
Additives
[0187] The present example demonstrates the utility of the
invention for providing the use of CCR2 inhibitors and/or CCR2
antagonists as an adjuvant or as an additive to a vaccine treatment
regimen. The present disclosure outlines the use of the small
molecule myeloid derived suppressor inhibiting agent, CCR2
inhibitor RS1028595 (Sigma Aldrich), as a vaccine/adjuvant additive
or adjuvant. It is expected that virtually any CCR2 inhibitor
and/or antagonist that demonstrates the myeloid derived suppressor
cell inhibiting activity described herein, and especially an
activity for blocking the migration of myeloid derived suppressor
cells to draining lymph nodes, or interferes with inflammatory
monocytes trafficking, would be useful and within the reasonable
scope of the present preparations and methods.
[0188] Applicants incorporate specifically herein by reference the
disclosure of Higgins et al. (2007) (Chemokine Biology--Basic
Research an Clinical Application, Vol. II, Birkhauser Verlag Basel
Switzerland), "Small molecule CCR2 antagonists", pg. 115-123).
While not intending to be limited in any way by this exemplary set
of CCR2 molecules, Table 1 below presents a number of CCR2
antagonists that are considered to be within the scope of the
present adjuvant/vaccine additives and adjuvants described as part
of the present vaccine treatment regimens.
TABLE-US-00001 TABLE 1 Company CCR2 Antagonists Roche/Iconix
##STR00001## CCR2 IC(50) - 89 nM bind, 210 nM taxis
Millennium/Pfizer ##STR00002## Benzimidazoles CCR2 IC(50) - 200-300
nM bind SmithKline ##STR00003## SB-380732 50 nM bind AstraZeneca
AZD-6942 29 nM bind; 60 nM taxis Merck ##STR00004## 41 nM bind; 59
nM taxis Teijn/BMS ##STR00005## 3-Aminopyrrolidines 3 nM Telik
##STR00006## Incyte ##STR00007## INCB-003284 Tocris RS 102895
hydrochloride (Catalog #2089) Biosciences IC.sub.59 values are 0.36
and 17.8 .mu.M for inhibition of human recombinant CCR2b and CCR1
receptors respectively. Blocks MCP-1-stimulated calcium influx and
chemotaxis with IC.sub.50 values of 32 nM and 1.7 .mu.M
respectively. Also inhibits .alpha..sub.1As .alpha..sub.1D and
5-HT.sub.1A receptors.
[0189] In addition, many other CCR2 inhibitors and/or CCR2
antagonists in development are considered to be useful together
with the present vaccine, adjuvant, and vaccine treatment regimens.
For example, it is anticipated in the present application that the
CCR2 inhibitor PF-04178903, would be useful to enhance vaccine
response (i.e., enhance antibody titer level production in response
to a vaccine containing an adjuvant, or as an adjuvant alone),
administered at the same time, before, or after the vaccine is
administered to an animal. For example, the present vaccine
treatment regimen may comprise administering to an animal an amount
of the PF-04178903 as part of (at the same time) a vaccine, after
the vaccine, or before the vaccine treatment.
[0190] By way of example, the vaccine may be a vaccine for
influenza. Standard influenza vaccine preparations are commercially
available, and may be used in the practice of this particular
example of the vaccine treatment regimen. It is expected that
antibody titer levels in an animal treated with this type of
vaccine together with the CCR2 antagonist, PF-04178903 or
RS1028595, would be 2-fold or higher than a vaccine treatment
regimen that did not include the CCR2 antagonist PF-04178903, or
RS1028595.
[0191] The invention may also be used in the vaccination of an
animal for Staphylococcus. In particular, the CCR2 inhibitor
RS1028595 or PF-04178903, may be included before, at the same time,
or after administration of a vaccine containing an immuno-provoking
amount of the vaccine.
[0192] It is expected that only a routine amount of trial and error
would be required in providing such a vaccine treatment regimen by
one of ordinary skill in the art of vaccine therapeutics.
Example 16
Small Molecule CCR2 Inhibitor RS 102895 Hydrochloride Enhances
Antibody Response
[0193] The present example is presented to demonstrate the utility
of the present invention for enhancing antibody titer levels in
vivo in response to vaccination where the treatment includes the
small molecule myeloid cell inhibitor, RS 102895 (Obtained from
Tocris, Catalog number 2089).
[0194] FIG. 33 presents the results obtained with the small
molecule inhibitor RS 102895 on antibody response in an animal upon
vaccination with a representative conventional agent, ova.
[0195] Mice were vaccinated subcutaneous (s.c.) with standard
vaccine adjuvant (CLDC) (Cationic liposome-DNA complexes) and 5 ug
ovalbumin and boosted 10 days later. One group of mice (n=4 per
group) received the vaccine only. A second group of mice was
vaccinated and also treated 1 day before, on the same day, and 1
day after with 5 mg/kg of the CCR2 antagonist RS102895,
administered intra peu i.p. A third group of mice was vaccinated
and also treated 1 day before, on the same day, and 1 day after
with 5 mg/kg of the CCR2 antagonist RS102895, administered s.c. at
the site of vaccination. Antibody titers to ova were determined 2
weeks after the boost and plotted as endpoint dilution titers. Mice
that were vaccinated and treated with RS102895 by either route
developed significantly higher antibody titers (at least double
(2.times.)) than mice that received the vaccine alone without an
adjuvant additive. In additional, the data shows that the enhanced
immune response may be achieved through administration of the
additive by either IP or SQ administration. (See FIG. 33).
Example 17
Small Molecule CCR2 Inhibitor RS 102895 and T Cell Response
[0196] The present example demonstrates the effect of the small
molecule inhibitor RS 102895 on T cell response in vivo.
[0197] Mice were vaccinated s.c. with standard vaccine adjuvant
(CLDC) and 5 ug ovalbumin and boosted 10 days later. One group of
mice (n=4 per group) received the vaccine only. A second group of
mice was vaccinated and also treated 1 day before, on the same day,
and 1 day after with 5 mg/kg of the CCR2 antagonist RS 102895,
administered i.p. A third group of mice was vaccinated and also
treated 1 day before, on the same day, and 1 day after with 5 mg/kg
of the CCR2 antagonist RS102895, administered s.c. at the site of
vaccination. The mice were sacrificed 2 weeks after the booster
vaccination and spleen cells were incubated in vitro with ovalbumin
(50 ug/ml) for 72 hours and release of IFN-.gamma. into the
supernatants was determined by ELISA. Lymphocytes from mice that
were vaccinated and treated with RS102895 by either route produced
significantly higher amounts of IFN-.gamma. than lymphocytes from
mice that were vaccinated without RS102895, indicating that
co-administration of RS102895 by either route triggered
significantly greater T cell responses to vaccination. These data
are consistent with the physiological premise that recruitment of
inflammatory monocytes during vaccination significantly suppresses
vaccine responses. The data from this study is presented at FIG.
34.
[0198] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims. Tyrosine kinase
inhibitors (eg, sunitinib), MDSC differentiating agents (eg,
all-trans retinoic acid), reactive nitrogen inhibitors (eg,
aminoguanidine or similar drugs); arginase enzyme inhibitors,
indoleamine deoxygenase enzyme inhibitors, reactive oxygen species
inhibitors, TGF-b inhibitors, IL-10 inhibitors, VEGF inhibitors,
and PGE2 synthesis inhibitors.
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