U.S. patent application number 17/533065 was filed with the patent office on 2022-05-19 for methods and compositions for treating cancer and infectious diseases.
The applicant listed for this patent is Cedars-Sinai Medical Center. Invention is credited to Hyung Kim, Yanping Wang.
Application Number | 20220152198 17/533065 |
Document ID | / |
Family ID | 1000006114240 |
Filed Date | 2022-05-19 |
United States Patent
Application |
20220152198 |
Kind Code |
A1 |
Kim; Hyung ; et al. |
May 19, 2022 |
METHODS AND COMPOSITIONS FOR TREATING CANCER AND INFECTIOUS
DISEASES
Abstract
The invention relates to compositions comprising a CD4
lymphocyte depleting agent; and methods of using the compositions
to treat, prevent, reduce the severity of and/or slow the
progression of a condition in a subject. The invention also relates
to use of combinations of a CD4 lymphocyte depleting agent and at
least one additional agent to treat, prevent, reduce the severity
of and/or slow the progression of a condition in a subject. The
additional agent may be an immune check point inhibitor, an
adoptive immune therapeutic, an immune adjuvant, or an immune
modulating agent, or their combinations.
Inventors: |
Kim; Hyung; (Los Angeles,
CA) ; Wang; Yanping; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cedars-Sinai Medical Center |
Los Angeles |
CA |
US |
|
|
Family ID: |
1000006114240 |
Appl. No.: |
17/533065 |
Filed: |
November 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15116485 |
Aug 3, 2016 |
11213583 |
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PCT/US2015/014687 |
Feb 5, 2015 |
|
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17533065 |
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61936168 |
Feb 5, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/545 20130101;
A61K 45/06 20130101; A61K 39/3955 20130101; A61K 39/39 20130101;
A61K 2039/505 20130101; A61K 2039/507 20130101; A61K 2039/55544
20130101; A61K 2039/5154 20130101; A61K 31/436 20130101; A61K
39/0011 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; A61K 31/436 20060101
A61K031/436; A61K 39/39 20060101 A61K039/39; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method of treating, preventing, reducing the severity of
and/or slowing the progression of a condition in a subject in need
thereof, comprising: administering a therapeutically effective
amount of an anti-CD4 antibody to deplete CD4+ regulatory T cells
(Tregs) in the subject; and administering a therapeutically
effective amount of an immune checkpoint inhibitor to the subject,
thereby treating, reducing the severity of and/or slowing the
progression of the cancer in the subject, wherein the immune
checkpoint inhibitor is selected from the group consisting of an
antibody against PD-1, an antibody against PD-L1, or a combination
thereof, wherein the subject has had the cancer for an amount of
time long enough to prime the immune system prior to administration
of the anti-CD4 antibody.
2. The method of claim 1, wherein the cancer is kidney cancer,
melanoma, prostate cancer, breast cancer, cervical cancer, liver
cancer, ovarian cancer, glioblastoma, renal cancer, lung cancer,
pancreatic cancer, gastric cancer, head and neck cancer, brain
cancer, colon cancer, or bladder cancer.
3. The method of claim 1, wherein the subject is a human.
4. The method of claim 1, wherein the anti-CD4 antibody is a
monoclonal antibody or a fragment thereof, a polyclonal antibody or
a fragment thereof, a chimeric antibody, a humanized antibody, a
human antibody or a fragment thereof, or a single chain
antibody.
5. The method of claim 1, wherein the anti-CD4 antibody depleting
agent is a humanized anti-CD4 antibody.
6. The method of claim 1, wherein the anti-CD4 antibody is
zanolimumab, keliximab or OKT4.
7. The method of claim 1, wherein the anti-CD4 antibody is
administered at 100-200 mg/day, 200-300 mg/day, 300-400 mg/day,
400-500 mg/day, 500-600 mg/day, 600-700 mg/day, 700-800 mg/day,
800-900 mg/day, 900-1000 mg/day, 1000-1100 mg/day, 1100-1200
mg/day, 1200-1300 mg/day, 1300-1400 mg/day, 1400-1500 mg/day,
1500-1600 mg/day, 1600-1700 mg/day, 1700-1800 mg/day, 1800-1900
mg/day or 1900-2000 mg/day.
8. The method of claim 1, wherein the anti-CD4 antibody is
administered intravenously, intramuscularly, subcutaneously, or
intraperitoneally.
9. The method of claim 1, wherein the immune checkpoint inhibitor
is an antibody against PD-1 selected from Lambrolizumab, Nivolumab
and Pidilizumab.
10. The method of claim 1, wherein the immune checkpoint inhibitor
is an antibody against PD-L1 selected from MPDL3280A, MEDI4736 and
BMS-936559.
11. The method of claim 1, wherein the immune checkpoint inhibitor
is administered at 0.1-0.5 mg/day, 0.5-1.0 mg/day, 1.0-1.5 mg/day,
1.5-2.0 mg/day, 2.0-2.5 mg/day, 2.5-5 mg/day, 5-10 mg/day, 10-15
mg/day, 15-20 mg/day, 20-25 mg/day, 25-30 mg/day, 30-35 mg/day,
35-40 mg/day, 40-45 mg/day, 45-50 mg/day, 50-55 mg/day, 55-60
mg/day, 60-65 mg/day, 65-70 mg/day, 70-75 mg/day, 75-80 mg/day,
80-85 mg/day, 85-90 mg/day, 90-95 mg/day or 95-100 mg/day.
12. The method of claim 1, wherein the immune checkpoint inhibitor
is administered intravenously, intramuscularly, subcutaneously, or
intraperitoneally.
13. The method of claim 1, wherein the anti-CD4 antibody and the
immune checkpoint inhibitor are administered concurrently.
14. The method of claim 1, wherein the anti-CD4 antibody is
administered before or after administering the immune checkpoint
inhibitor.
15. The method of claim 1, wherein the administration of the
anti-CD4 antibody induces interferon-gamma (IFN-.gamma.) response
in the subject.
16. The method of claim 1, further comprising administering a
therapeutically effective amount of an immune modulating agent to
the subject.
17. The method of claim 16, wherein the immune modulating agent is
selected from an mTOR inhibitor, a STAT inhibitor, a TGF.beta.
receptor inhibitor, and a tyrosine kinase inhibitor.
18. The method of claim 17, wherein the mTOR inhibitor is
temsirolimus, evirolimus, or sirolimus.
19. The method of claim 17, wherein the tyrosine kinase inhibitor
is selected from sunitinib, erlotinib, vandetanib, cediranib,
brivanib, foretinib, and dovitinib.
20. The method of claim 1, further comprising administering a
therapeutically effective amount of a chemotherapeutic agent to the
subject.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation application of U.S. Ser.
No. 15/116,485, which is the national phase stage of
PCT/US2015/014687 filed on Feb. 5, 2015, which claims the priority
to U.S. provisional patent application No. 61/936,168, filed Feb.
5, 2014,the disclosure of which is incorporated herein by reference
in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to molecular immunology and
cell biology. Specifically described herein are compositions and
methods for treating cancer or infectious diseases using CD4
lymphocyte depleting agents alone or in a combination with any one
or more of an immune check point inhibitor, an adoptive immune
therapeutic, an immune adjuvant, or an immune modulating agent.
BACKGROUND
[0003] All publications cited herein are incorporated by reference
in their entirety to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference. The following
description includes information that may be useful in
understanding the present invention. It is not an admission that
any of the information provided herein is prior art or relevant to
the presently claimed invention, or that any publication
specifically or implicitly referenced is prior art.
[0004] The immune system can provide protection against cancers.
Effective immune stimulation produces long-lasting memory
lymphocytes, capable of rapidly responding to repeat antigen
challenge. CD4 expressing lymphocytes include both helper T cells
and regulatory T cells. T helper cells are critical to mounting an
adoptive immune response. However, regulatory T cells (Tregs)
inhibit the function of cytotoxic T cells and normally function to
limit an immune response. Therefore, CD4 depletion was evaluated as
a strategy for removing Treg activity. Although only a small
fraction of CD4 lymphocytes are Treg cells, CD4 depletion remains
an effective approach for depleting Treg activity, and importantly,
it has the potential for rapid translation to clinical use.
Humanized CD4 depleting antibodies have been evaluated as a
strategy to inhibit the immune system in clinical trials for
autoimmune disorder. However, described herein is the use of CD4
lymphocyte depleting antibody to stimulate the immune system.
Provided herein are therapies against cancer and infectious
diseases in subjects by combining depletion of CD4+ lymphocytes
with any one or more of an immune check point inhibitor, an
adoptive immune therapeutic, an immune adjuvant, and an immune
modulating agent, or a combination thereof.
SUMMARY OF THE INVENTION
[0005] Various embodiments of the present invention provide methods
for treating, preventing, reducing the severity of and/or slowing
the progression of a condition in a subject. The method includes
providing a composition comprising a CD4 lymphocyte depleting agent
and administering a therapeutically effective amount of the
composition to the subject, thereby treating, preventing, reducing
the severity of and/or slowing the progression of the condition in
the subject.
[0006] Various embodiments of the present invention provide methods
for treating, preventing, reducing the severity of and/or slowing
the progression of a condition in a subject. The method includes
providing a CD4 lymphocyte depleting agent and at least one
additional agent selected from the group consisting of an immune
check point inhibitor, an adoptive immune therapeutic, an immune
adjuvant, and an immune modulating agent; and administering a
therapeutically effective amount of the CD4 lymphocyte depleting
agent and a therapeutically effective amount of the at least one of
an immune check point inhibitor, an adoptive immune therapeutic, an
immune adjuvant, and an immune modulating agent to the subject,
thereby treating, preventing, reducing the severity of and/or
slowing the progression of the condition in the subject. In some
embodiments, the methods further comprise administering a
therapeutically effective amount of mTOR inhibitor.
[0007] Various embodiments of the present invention provide methods
for treating, preventing, reducing the severity of and/or slowing
the progression of a condition in a subject. The methods include
providing a CD4 lymphocyte depleting agent and an adoptive immune
therapeutic agent (for example, a dendritic cell (DC) vaccine) and
administering a therapeutically effective amount of the CD4
lymphocyte depleting agent and a therapeutically effective amount
of the adoptive immune therapeutic agent thereby treating,
preventing, reducing the severity of and/or slowing the progression
of the condition in the subject.
[0008] Various embodiments of the present invention provide methods
for treating, preventing, reducing the severity of and/or slowing
the progression of a condition in a subject. The methods include
providing a CD4 lymphocyte depleting agent and a checkpoint
inhibitor (for example, an anti-PD-1 antibody) and administering a
therapeutically effective amount of the CD4 lymphocyte depleting
agent and a therapeutically effective amount of the checkpoint
inhibitor thereby treating, preventing, reducing the severity of
and/or slowing the progression of the condition in the subject.
[0009] Various embodiments of the present invention provide methods
for treating, preventing, reducing the severity of and/or slowing
the progression of a condition in a subject. The methods include
providing a CD4 lymphocyte depleting agent, a dendritic cell (DC)
vaccine and an mTOR inhibitor and administering an effective amount
each of the CD4 lymphocyte depleting agent, dendritic cell vaccine
and an mTOR inhibitor thereby treating, preventing, reducing the
severity of and/or slowing the progression of the condition in the
subject.
[0010] Various embodiments of the present invention provide a
pharmaceutical composition comprising a CD4 lymphocyte depleting
agent. Further embodiments provide pharmaceutical compositions
comprising any one of an immune check point inhibitor, an adoptive
immune therapeutic, an immune adjuvant, and an immune modulating
agent.
[0011] Various embodiments of the present invention provide a kit
for treating, preventing, reducing the severity of and/or slowing
the progression of a condition in a subject. The kit includes a CD4
lymphocyte depleting agent and instructions for using the
composition to treat, prevent, reduce the severity of and/or slow
the progression of the condition in the subject.
[0012] Various embodiments of the present invention provide a kit
for treating, preventing, reducing the severity of and/or slowing
the progression of a condition in a subject. The kit includes a CD4
lymphocyte depleting agent and at least one of an immune check
point inhibitor, an adoptive immune therapeutic, an immune
adjuvant, and an immune modulating agent. The kit further comprises
instructions for using the CD4 lymphocyte depleting agent and the
at least one of the immune check point inhibitor, the adoptive
immune therapeutic, the immune adjuvant, and the immune modulating
agent to treat, prevent, reduce the severity of and/or slow the
progression of the condition in the subject.
[0013] In various embodiments, the condition is cancer. In
exemplary embodiments, the cancer may be any of kidney cancer,
melanoma, prostate cancer, breast cancer, glioblastoma, lung
cancer, colon cancer, or bladder cancer. In some embodiments, the
condition is an infectious disease.
[0014] Various methods, compositions and kits of the present
invention find utility in the treatment of cancer or infectious
diseases. In exemplary embodiments, the methods, compositions and
kits of the present invention find utility in the treatment of
certain subsets of malignant neoplastic cell proliferative
disorders or diseases, including but not limited to carcinomas and
melanomas. In exemplary embodiments, carcinomas include renal cell
carcinomas.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Exemplary embodiments are illustrated in referenced figures.
It is intended that the embodiments and figures disclosed herein
are to be considered illustrative rather than restrictive.
[0016] FIG. 1 depicts, in accordance with various embodiments of
the invention, that there are both immune stimulating and
inhibiting effects of mTOR inhibition; however the net effect is
enhanced anti-tumor immunity. (a) Experimental scheme for a
melanoma tumor prevention model: mice (n=5per group) received tumor
lysate-pulsed DC vaccine on days -30 and -23, and temsirolimus was
injected intraperitoneally daily on days -23 to -13. B16 tumor
cells were injected subcutaneously in the flank on day 0. B16 tumor
growth (left) and survival (right) curves are shown. Results are
representative of duplicate experiments. (b) Experimental scheme to
characterize lymphocytes following treatment with DC vaccine and
temsirolimus: Thy1.1 Pmel-1 lymphocytes were adoptively transfer
into Thy1.2 B6 mice, which received tumor lysate-pulsed DC vaccine
on day -6, and daily temsirolimus for 5 days. Splenocytes were
harvested on day 0, and stained for CD8, Thy1.1, Tbet, Eomes and
CD4/FoxP3 and analyzed by flow cytometry. Representative result
(left) and summary data (right) are provided. Results are
representative of duplicate experiments. (c) Lymphocytes were
characterized with in vitro mixed cultures using pmel-1 lymphocytes
and tumor lysate-pulsed, CpG activated DCs treated with
temsirolimus for 48 hours. Lymphocytes were stained for CD8,
Thy1.1, Tbet, Eomes and CD4/FoxP3 and analyzed by flow cytometry.
Representative result (left) and summary data (right) are provided.
Results are representative of duplicate experiments. *p<0.05,
**p<0.01,***p<0.005.
[0017] FIG. 2 depicts, in accordance with various embodiments of
the invention, that CD4 depletion enhanced the antitumor effect of
mTor inhibition. (a) RENCA-CA9 tumor cells were implanted into
Balb/C mice (n=5 per group) on day 0. CD4 lymphocytes were depleted
with .alpha.CD4 antibody on days 6 and 10. Mice were treated with
daily temsirolimus on days 14 to 34. Tumor growth was monitored.
Results are representative of triplicate experiments. (b) In the
same experiment, lymphocytes were harvested on day 45, restimulated
with CA9 peptide, and stained for CD8 and IFN.gamma.. (c) Following
CD4 depletion, spleen, lymph node and blood were collected on days
0, 1, 10. Lymphocytes were stained for CD4, CD8 and FoxP3 and
analyzed by flow cytometry. (c-e) The percentages of CD4 cells in
the spleen, lymph node and blood on days 0, 1, and 10 following CD4
depletion are reported. (d) Following CD4 depletion, percentages of
splenocytes that are CD4 or CD8 positive are reported, and percent
of CD4 cells that are FoxP3 positive is reported. (e) In the same
experiment, the absolute numbers of splenocytes positive for CD4,
CD8, and CD4/FoxP3 are reported. *p<0.05,
**p<0.01,***p<0.005.
[0018] FIG. 3 depicts, in accordance with various embodiments of
the invention, that combination of CD4 depletion and temsirolimus
generated anti-tumor immunity that was dependent on memory CD8
cells. (a) Tumor-bearing mice (n=8) were treated with daily
temsirolimus and then rechallenged with RENCA-CA9 35 days after
primary tumor implantation. CD8 cells were depleted by injecting
.alpha.CD8 antibody on day 36. (b) Tumor-bearing mice (n=8) treated
with temsirolimus and CD4 depletion, and then rechallenged with
RENCA-CA9 35 days after primary tumor implantation. CD8 cells were
depleted by injecting .alpha.CD8 antibody on day 36. (c-e)
Lymphocytes were harvested from mice treated with temsirolimus and
CD4 depletion. The lymphoctes were cultured in vitro with CA9
peptide and IL2 (10 u/ml) for 3 days and then adoptively
transferred into naive B6 mice, which were challenged 24 hrs later
with 2.times.10.sup.5 RENCA tumor cells injected i.v. Lungs were
collected 30 days after the i.v. tumor challenge. Lung weight (d)
and number of lung tumor deposits (e) were determined. IL2,
interleukin-2, *p<0.05, **p<0.01,***p<0.005.
[0019] FIG. 4 depicts, in accordance with various embodiments of
the invention, that combination of CD4 depletion and temsirolimus
treatment enhanced function of CD8 memory cells. (a) Experimental
scheme: Lymphocytes from Thy1.1 Pmel-1 mice were adoptively
transferred .alpha.CD4 antibody on days 7 and 10, and daily
temsirolimus on days 10 to 24.Results are representative of
triplicate experiments. (b) Splenocytes (n=3 per group) were
harvested one day prior to rechallenging with tumor lysate-pulsed
DC vaccine, stained with antibodies, and analyzed by flow
cytometry. The percent of CD8 cells positive for the indicated
marker is shown. (c) Splenocytes (n=3 per group) were harvested 4
days after rechallenging with tumor lysate-pulsed DC vaccine,
stained with antibodies, and analyzed by flow cytometry. The
percent of CD8 cells positive for the indicated marker is shown.
*p<0.05, **p<0.01,***p<0.005.
[0020] FIG. 5 depicts, in accordance with various embodiments of
the invention, that the combination of FoxP3+ Treg depletion and
temsirolimus enhanced CTL function in vivo. (a) Experimental
scheme: DEREG mice received tumor lysate-pulsed DC vaccine, and
were treated intraperitoneally with diphtheria toxin on days 6 and
10, and daily temsirolimus on days 10 to 20. In vivo CTL assay was
performed on day 35. (b) CD4+FoxP3+ cells were assessed by flow
cytometry using peripheral lymphocytes obtained before and after
treating mice with diphtheria toxin. (c) The in vivo CTL results
were analyzed by flow cytometry using splenocytes harvested 14 hrs
following injection of target cells. CTL, cytotoxic T lymphocyte,
*p<0.05, **p<0.01,***p<0.005.
[0021] FIG. 6 depicts, in accordance with various embodiments of
the invention, that adoptive transfer of FoxP3+ Tregs reduced CTL
function in vivo in mice treated with the combination of CD4
depletion and temsirolimus. (a) Experimental scheme: B6 received
tumor lysate-pulsed DC vaccine, and were treated intraperitoneally
with .alpha.CD4 antibody on days 6 and 10, and daily temsirolimus
on days 14 to 24. Tregs, sorted from lymphocytes from GFP-FoxP3
mice that received tumor lysate-pulsed DC vaccine, were adoptively
transferred on day 20 and in vivo CTL assay was performed on day
35. (b) The in vivo CTL results were analyzed by flow cytometry
using splenocytes harvested 14 hrs following injection of target
cells (n=3 per group). *p<0.05, **p<0.01,***p<0.005.
[0022] FIG. 7 depicts, in accordance with various embodiments of
the invention, that after treatment with CD4 depletion, Treg
population that recovers is less immunosuppressive. (a) Mice were
treated using the experimental scheme outlined in FIG. 4a. On day
45, splenocytes were examined by flow cytometry for CD4+FoxP3+
cells. (b) The splenocytes that recovered after CD4 depletion were
used to enrich for CD4+ cells, which were then sorted by CD25
status. For the resulting groups, representative flow cytometry for
CD4+CD25+ status and CD4+CD25- status are shown. (c) CD4+CD25-
cells were co-cultured with DCs pulsed with B16 tumor lysate. The
CD4+CD25- cells were analyzed by flow cytometry for IFN.gamma. or
IL4 expression. (d) CD4+CD25+ cells were co-cultured with DCs
pulsed with B16 tumor lysate and CD8 cells from mice immunized with
DCs pulsed with B16 tumor lysate. CD8 cell proliferation was
monitored by flow cytometry using a CFSE dilution assay.
*p<0.05, **p<0.01,***p<0.005.
[0023] FIG. 8 depicts, in accordance with various embodiments of
the invention, that tumor increases Tregs. The percent of CD4 cells
expressing FoxP3 were examined from lymph nodes, spleen, bone
marrow and peripheral blood lymphocytes (PBL) from tumor bearing
mice (.about.1 cm in diameter) and control mice without tumor. Flow
cytometry results are shown.
[0024] FIG. 9 depicts, in accordance with various embodiments of
the invention, that .alpha.CD4 antibody depletes effector CD4
subtypes. (a) Splenocytes were collected before (day 0) and 1 or 10
days after administering .alpha.CD4 antibody, and analyzed by flow
cytometry. The number of total splenocytes staining for CD4 and
IL-2, IL-4 or IL-17 is shown. (b) Percent of CD4 cells staining for
IL-2, IL-4 or IL-17 is shown.
[0025] FIG. 10 depicts, in accordance with various embodiments of
the invention, that CD4 depletion prior to immune stimulation
prevents formation of CD8 memory cells. The experimental scheme is
similar to FIG. 4. An additional group is included where .alpha.CD4
antibody is administered on day -1 and +1 (Group A). (a) The
percent of CD8 cells expressing Thy1.1 is shown from splenocytes
harvested 5 days following rechallenge of memory cells. (b) The
percent of CD8 cells expressing Eomes is shown. *p<0.05,
**p<0.01,***p<0.005.
[0026] FIG. 11 depicts, in accordance with various embodiments of
the invention, that Depletion of Tregs or CD4 cells enhance
antitumor effect.
[0027] FIG. 12 depicts, in accordance with various embodiments of
the invention, that CD4 depletion enhances PD-1 blockade antitumor
effect.
[0028] FIG. 13 depicts, in accordance with various embodiments of
the invention, that CD4 depletion enhances to tumor-specific
cellular immunity. A. Experimental schematic. B. CD4 depletion
stimulated tumor-specific interferon-gamma secretion from CD8
cells. Therefore, CD4 depletion led to educating of CD8 cells that
were capable of being activated in response to tumor. C. These same
CD8 cells were capable of killing RENCA tumor cells. D. B16 tumor
growth curve.
[0029] FIG. 14 depicts, in accordance with various embodiments of
the invention, that combination of anti-CD4 antibody, Temsirolimus
and dendritic cell vaccine exhibited enhanced anti-tumor activity.
A. Experimental schematic. B. B16 tumor growth curves are shown
with standard error of mean (SEM). **p<0.001.
DETAILED DESCRIPTION OF THE INVENTION
[0030] All references cited herein are incorporated by reference in
their entirety as though fully set forth. Unless defined otherwise,
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention belongs. Allen et al., Remington: The Science and
Practice of Pharmacy 22.sup.nd ed., Pharmaceutical Press (Sep. 15,
2012); Hornyak et al., Introduction to Nanoscience and
Nanotechnology, CRC Press (2008); Singleton and Sainsbury,
Dictionary of Microbiology and Molecular Biology 3.sup.rd ed.,
revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith,
March's Advanced Organic Chemistry Reactions, Mechanisms and
Structure 7.sup.th ed., J. Wiley & Sons (New York, N.Y. 2013);
Singleton, Dictionary of DNA and Genome Technology 3.sup.rd ed.,
Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular
Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory
Press (Cold Spring Harbor, N.Y. 2012), provide one skilled in the
art with a general guide to many of the terms used in the present
application. For references on how to prepare antibodies, see
Greenfield, Antibodies A Laboratory Manual 2.sup.nd ed., Cold
Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Kohler and
Milstein, Derivation of specific antibody-producing tissue culture
and tumor lines by cell fusion, Eur. J. Immunol. 1976 July,
6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat.
No. 5,585,089 (1996 December); and Riechmann et al., Reshaping
human antibodies for therapy, Nature 1988 Mar. 24,
332(6162):323-7.
[0031] For references on pediatrics, see Schwartz et al., The
5-Minute Pediatric Consult 4.sup.th ed., Lippincott Williams &
Wilkins, (Jun. 16, 2005); Robertson et al., The Harriet Lane
Handbook: A Manual for Pediatric House Officers 17.sup.th ed.,
Mosby (Jun. 24, 2005); and Hay et al., Current Diagnosis and
Treatment in Pediatrics (Current Pediatrics Diagnosis &
Treatment) 18.sup.th ed., McGraw-Hill Medical (Sep. 25, 2006).
[0032] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Other
features and advantages of the invention will become apparent from
the following detailed description, taken in conjunction with the
accompanying drawings, which illustrate, by way of example, various
features of embodiments of the invention. Indeed, the present
invention is in no way limited to the methods and materials
described. For convenience, certain terms employed herein, in the
specification, examples and appended claims are collected here.
[0033] Unless stated otherwise, or implicit from context, the
following terms and phrases include the meanings provided below.
Unless explicitly stated otherwise, or apparent from context, the
terms and phrases below do not exclude the meaning that the term or
phrase has acquired in the art to which it pertains. The
definitions are provided to aid in describing particular
embodiments, and are not intended to limit the claimed invention,
because the scope of the invention is limited only by the claims.
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0034] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are useful to an embodiment, yet open to the
inclusion of unspecified elements, whether useful or not. It will
be understood by those within the art that, in general, terms used
herein are generally intended as "open" terms (e.g., the term
"including" should be interpreted as "including but not limited
to," the term "having" should be interpreted as "having at least,"
the term "includes" should be interpreted as "includes but is not
limited to," etc.).
[0035] Unless stated otherwise, the terms "a" and "an" and "the"
and similar references used in the context of describing a
particular embodiment of the application (especially in the context
of claims) can be construed to cover both the singular and the
plural. The recitation of ranges of values herein is merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range. Unless otherwise
indicated herein, each individual value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(for example, "such as") provided with respect to certain
embodiments herein is intended merely to better illuminate the
application and does not pose a limitation on the scope of the
application otherwise claimed. The abbreviation, "e.g." is derived
from the Latin exempli gratia, and is used herein to indicate a
non-limiting example. Thus, the abbreviation "e.g." is synonymous
with the term "for example." No language in the specification
should be construed as indicating any non-claimed element essential
to the practice of the application.
[0036] As used herein, the terms "treat," "treatment," "treating,"
or "amelioration" when used in reference to a disease, disorder or
medical condition, refer to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent, reverse, alleviate, ameliorate, inhibit, lessen, slow down
or stop the progression or severity of a symptom or condition. The
term "treating" includes reducing or alleviating at least one
adverse effect or symptom of a condition. Treatment is generally
"effective" if one or more symptoms or clinical markers are
reduced. Alternatively, treatment is "effective" if the progression
of a disease-state is reduced or halted. That is, "treatment"
includes not just the improvement of symptoms or markers, but also
a cessation or at least slowing of progress or worsening of
symptoms that would be expected in the absence of treatment. Also,
"treatment" may mean to pursue or obtain beneficial results, or
lower the chances of the individual developing the condition even
if the treatment is ultimately unsuccessful. Those in need of
treatment include those already with the condition as well as those
prone to have the condition or those in whom the condition is to be
prevented.
[0037] "Beneficial results" or "desired results" may include, but
are in no way limited to, lessening or alleviating the severity of
the disease condition, preventing the disease condition from
worsening, curing the disease condition, preventing the disease
condition from developing, lowering the chances of a patient
developing the disease condition, decreasing morbidity and
mortality, and prolonging a patient's life or life expectancy. As
non-limiting examples, "beneficial results" or "desired results"
may be alleviation of one or more symptom(s), diminishment of
extent of the deficit, stabilized (i.e., not worsening) state of
cancer progression, delay or slowing of metastasis or invasiveness,
and amelioration or palliation of symptoms associated with the
cancer.
[0038] As used herein, the term "administering," refers to the
placement an agent as disclosed herein into a subject by a method
or route which results in at least partial localization of the
agents at a desired site.
[0039] A "cancer" or "tumor" as used herein refers to an
uncontrolled growth of cells which interferes with the normal
functioning of the bodily organs and systems, and/or all neoplastic
cell growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. Included in this
definition are benign and malignant cancers, as well as dormant
tumors or micro-metastases. As used herein, the term "carcinoma"
refers to a cancer arising from epithelial cells. As used herein,
the term "invasive" refers to the ability to infiltrate and destroy
surrounding tissue. Melanoma is an invasive form of skin tumor.
Examples of cancer include, but are not limited to B-cell lymphomas
(Hodgkin's lymphomas and/or non-Hodgkins lymphomas), brain tumor,
breast cancer, colon cancer, lung cancer, hepatocellular cancer,
gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, cancer of the urinary tract, thyroid
cancer, renal cancer, carcinoma, melanoma, head and neck cancer,
brain cancer, and prostate cancer, including but not limited to
androgen-dependent prostate cancer and androgen-independent
prostate cancer.
[0040] "Conditions" and "disease conditions," as used herein may
include, cancers, tumors or infectious diseases. In exemplary
embodiments, the conditions include but are in no way limited to
any form of malignant neoplastic cell proliferative disorders or
diseases. In exemplary embodiments, conditions include any one or
more of kidney cancer, melanoma, prostate cancer, breast cancer,
glioblastoma, lung cancer, colon cancer, or bladder cancer.
[0041] "Immune cell" as used herein refers to the cells of the
mammalian immune system including but not limited to antigen
presenting cells, B-cells, basophils, cytotoxic T-cells, dendritic
cells, eosinophils, granulocytes, helper T-cells, leukocytes,
lymphocytes, macrophages, mast cells, memory cells, monocytes,
natural killer cells, neutrophils, phagocytes, plasma cells and
T-cells.
[0042] "Immune response" as used herein refers to immunities
including but not limited to innate immunity, humoral immunity,
cellular immunity, immunity, inflammatory response, acquired
(adaptive) immunity, autoimmunity and/or overactive immunity
[0043] The term "sample" or "biological sample" as used herein
denotes a sample taken or isolated from a biological organism,
e.g., a tumor sample from a subject. Exemplary biological samples
include, but are not limited to, a biofluid sample; serum; plasma;
urine; saliva; a tumor sample; a tumor biopsy and/or tissue sample
etc. The term also includes a mixture of the above-mentioned
samples. The term "sample" also includes untreated or pretreated
(or pre-processed) biological samples. In some embodiments, a
sample can comprise one or more cells from the subject. In some
embodiments, a sample can be a tumor cell sample, e.g. the sample
can comprise cancerous cells, cells from a tumor, and/or a tumor
biopsy.
[0044] The term "functional" when used in conjunction with
"derivative" or "variant" or "fragment" refers to a polypeptide
which possess a biological activity that is substantially similar
to a biological activity of the entity or molecule of which it is a
derivative or variant or fragment thereof.
[0045] As used herein, a "subject" means a human or animal. Usually
the animal is a vertebrate such as a primate, rodent, domestic
animal or game animal. Primates include chimpanzees, cynomologous
monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents
include mice, rats, woodchucks, ferrets, rabbits and hamsters.
Domestic and game animals include cows, horses, pigs, deer, bison,
buffalo, feline species, e.g., domestic cat, and canine species,
e.g., dog, fox, wolf. The terms, "patient", "individual" and
"subject" are used interchangeably herein. In an embodiment, the
subject is mammal. The mammal can be a human, non-human primate,
mouse, rat, dog, cat, horse, or cow, but are not limited to these
examples. In addition, the methods described herein can be used to
treat domesticated animals and/or pets.
[0046] A subject can be one who has been previously diagnosed with
or identified as suffering from or having a disease-state in need
of monitoring (e.g., cancer or infectious disease) or one or more
complications related to such a disease-state, and optionally, have
already undergone treatment for the disease-state or the one or
more complications related to the disease/condition. Alternatively,
a subject can also be one who has not been previously diagnosed as
having a disease-state or one or more complications related to the
disease/condition. For example, a subject can be one who exhibits
one or more risk factors for a disease-state or one or more
complications related to a disease-state or a subject who does not
exhibit risk factors. A "subject in need" of treatment for a
particular disease-state can be a subject having that
disease/condition, diagnosed as having that condition, or at risk
of developing that disease.
[0047] The term "statistically significant" or "significantly"
refers to statistical evidence that there is a difference. It is
defined as the probability of making a decision to reject the null
hypothesis when the null hypothesis is actually true. The decision
is often made using the p-value.
[0048] As used herein, "CD4 lymphocytes" refer to lymphocytes that
express CD4, i.e., lymphocytes that are CD4+. CD4 lymphocytes may
be T cells that express CD4.
[0049] The terms "T-cell" and "T-lymphocyte" are interchangeable
and used synonymously herein. Examples include but are not limited
to naive T cells, central memory T cells, effector memory T cells
or combinations thereof.
[0050] "Therapeutic agents" as used herein refers to agents that
are used to, for example, treat, inhibit, prevent, mitigate the
effects of, reduce the severity of, reduce the likelihood of
developing, slow the progression of and/or cure, a disease.
Diseases targeted by the therapeutic agents include but are not
limited to infectious diseases, carcinomas, sarcomas, lymphomas,
leukemia, germ cell tumors, blastomas, antigens expressed on
various immune cells, and antigens expressed on cells associated
with various hematologic diseases, and/or inflammatory
diseases.
[0051] Unless otherwise defined herein, scientific and technical
terms used in connection with the present application shall have
the meanings that are commonly understood by those of ordinary
skill in the art to which this disclosure belongs. It should be
understood that this invention is not limited to the particular
methodology, protocols, and reagents, etc., described herein and as
such can vary. The terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention, which is defined solely
by the claims.
[0052] Provided herein are methods, compositions and kits, in which
a CD4 lymphocyte depleting agent is used alone or in combination
with various other therapies and agents to target the immune system
to treat cancer and/or infectious diseases. Non-limiting examples
of therapies and agents that can be used in combination with a CD4
lymphocyte depleting agent include but are not limited to immune
check point inhibitors, adoptive immune therapies, immune
adjuvants, and immune modulating agents.
Treatment Methods
[0053] In various embodiments, the present invention provides a
method of treating, preventing, reducing the severity of and/or
slowing the progression of a condition in a subject. The method
includes providing a composition comprising a CD4 lymphocyte
depleting agent and administering a therapeutically effective
amount of the composition to the subject, thereby treating,
preventing, reducing the severity of and/or slowing the progression
of the condition in the subject. In some embodiment, the condition
is cancer or an infectious disease. In some embodiments, the
subject has been diagnosed with cancer. In some embodiments, the
subject has been diagnosed with an infectious disease. In various
embodiments, the subject has had the disease for an amount of time
long enough to prime the immune system prior to administration of
the composition comprising a CD4 lymphocyte depleting agent. In
various embodiments, the amount of time for which the subject has
had the condition is 1 day or more, 2 days or more, 3 days or more,
4 days or more, 5days or more, 6 days or more, 7 days or more, 8
days or more, 9 days or more, 10 days or more, 15 days or more, 20
days or more, 1 month or more, 2months or more, 3months or more, 4
months or more, 5 months or more, 6 months or more, 1 year or more,
or combinations thereof.
[0054] In various embodiments, the present invention provides
methods for treating, preventing, reducing the severity of and/or
slowing the progression of a condition in a subject. The methods
include providing a CD4 lymphocyte depleting agent and at least one
additional agent selected from the group consisting of an immune
check point inhibitor, an adoptive immune therapeutic agent, an
immune adjuvant, and an immune modulating agent; and administering
a therapeutically effective amount of the CD4 lymphocyte depleting
agent and a therapeutically effective amount of at least one of an
immune check point inhibitor, an adoptive immune therapeutic agent,
an immune adjuvant, and an immune modulating agent to the subject,
thereby treating, preventing, reducing the severity of and/or
slowing the progression of the condition in the subject. In some
embodiment, the methods further comprise administering an mTOR
inhibitor to the subject. In various embodiments, the CD4
lymphocyte depleting agent and the additional agent may be
administered sequentially or simultaneously. In some embodiments,
the condition is cancer. In some embodiments, the condition is an
infectious disease.
[0055] In various embodiments, the present invention provides
methods for treating, preventing, reducing the severity of and/or
slowing the progression of a condition in a subject. The methods
include providing a CD4 lymphocyte depleting agent and an adoptive
immune therapeutic agent and administering a therapeutically
effective amount of the CD4 lymphocyte depleting agent and a
therapeutically effect amount of the adoptive immune therapeutic
agent to the subject so as to treat, inhibit, prevent, reduce the
severity and/or slow the progression of the condition in the
subject. In an embodiment, the CD4 lymphocyte depleting agent is an
anti-CD4 antibody or a fragment thereof or a variant thereof and
the adoptive immune therapeutic agent is a dendritic cell vaccine.
In various embodiments, the condition is cancer or an infectious
disease. In some embodiments, anti-CD4 antibody and dendritic cell
vaccine are administered simultaneously. In some embodiments, the
anti-CD4 antibody and the dendritic cell vaccine are administered
sequentially. In some embodiments, the methods may further comprise
administering an effective amount of an mTOR inhibitor,
sequentially or simultaneously with the CD4 lymphocyte depleting
agent and the adoptive immune therapeutic agent.
[0056] In various embodiments, the present invention provides
methods for treating, preventing, reducing the severity of and/or
slowing the progression of a condition in a subject. The methods
include providing a CD4 lymphocyte depleting agent and an immune
checkpoint inhibitor and administering a therapeutically effective
amount of the CD4 lymphocyte depleting agent and a therapeutically
effective amount of the immune checkpoint inhibitor to the subject
so as to treat, inhibit, prevent, reduce the severity and/or slow
the progression of the condition in the subject. In an embodiment,
the CD4 lymphocyte depleting agent is an anti-CD4 antibody or a
fragment thereof or a variant thereof and the checkpoint inhibitor
is an anti-PD-1 antibody or a fragment thereof or a variant thereof
In various embodiments, the condition is cancer or an infectious
disease. In some embodiments, anti-CD4 antibody and anti-PD-1
antibody are administered simultaneously. In some embodiments, the
anti-CD4 antibody and the anti-PD-1 antibody are administered
sequentially. The methods may further comprise administering an
mTOR inhibitor. In some embodiments, the methods may further
comprise administering an effective amount of an mTOR inhibitor,
sequentially or simultaneously with the CD4 lymphocyte depleting
agent and the checkpoint inhibitor.
[0057] In various embodiments, the present invention provides
methods for treating, preventing, reducing the severity of and/or
slowing the progression of a condition in a subject. The methods
include providing a CD4 lymphocyte depleting agent, an adoptive
immune therapeutic agent and an mTOR inhibitor and administering a
therapeutically effective amount of the CD4 lymphocyte depleting
agent, a therapeutically effective amount of the adoptive immune
therapeutic agent and a therapeutically effective amount of the
mTOR inhibitor to the subject so as to treat, inhibit, prevent,
reduce the severity and/or slow the progression of the condition in
the subject. In an embodiment, the CD4 lymphocyte depleting agent
is an anti-CD4 antibody or a fragment thereof or a variant thereof
In an embodiment, the adoptive immune therapeutic agent is a
dendritic cell vaccine. In an embodiment, the mTOR inhibitor is
temsirolimus. In various embodiments, the condition is cancer or an
infectious disease. In some embodiments, anti-CD4 antibody,
dendritic cell vaccine and the mTOR inhibitor (for example
temsirolimus) are administered simultaneously. In some embodiments,
anti-CD4 antibody, dendritic cell vaccine and the mTOR inhibitor
(for example temsirolimus) are administered sequentially.
[0058] In various embodiments, the methods described herein may be
used to prevent metastasis of cancer or prevent recurrence of
cancer in a subject in need thereof.
[0059] In various embodiments of the invention, the CD4 lymphocyte
depleting agent may be any one or more of small molecule, a
peptide, an antibody or a fragment thereof, and a nucleic acid
molecule. In an embodiment, the antibody specifically binds CD4 on
CD4-expressing T cells such as regulatory T cells (Treg cells).
[0060] An antibody (for example, anti-CD4 antibody or anti-PD-1
antibody) may be any one or more of a monoclonal antibody or
fragment thereof, a polyclonal antibody or a fragment thereof, a
chimeric antibody, a humanized antibody, a human antibody or a
fragment thereof, or a single chain antibody. These antibodies can
be from any source, e.g., rat, mouse, guinea pig, dog, cat, rabbit,
pig, cow, horse, goat, donkey or human. Fragments of antibodies may
be any one or more of Fab, F(a')2, Fv fragments or their fusion
proteins.
[0061] In various embodiments, the CD4 lymphocyte depleting agent
is a humanized anti-CD4 antibody or a fragment thereof. In various
embodiments, the CD4 lymphocyte depleting agents are zanolimumab,
keliximab, and OKT4.
[0062] In some embodiments of the invention, the CD4 lymphocyte
depleting agent and the additional agent are administered
concurrently. In other embodiments, the CD4 lymphocyte depleting
agent and the additional agent are administered sequentially. For
example, the CD4 lymphocyte depleting agent is administered before,
during or after administering the additional agent. In further
embodiments, the the CD4 lymphocyte depleting agent and the
additional agent are administered with food or without food. In
accordance with the invention, the CD4 lymphocyte depleting agent
and/or the additional agent may be used in combination with other
agents, including but not limited to cancer vaccines and
chemotherapeutic agents. As described herein, the additional agent
is any one or more of an immune check point inhibitor, an adoptive
immune therapeutic agent, an immune adjuvant, and an immune
modulating agent.
[0063] In various embodiments, the additional agent is an immune
checkpoint inhibitor. Examples of immune checkpoint inhibitors
include but are not limited to anti-PD-1 antibodies such as
Lambrolizumab (MK-3475), Nivolumab (BMS-936558) and Pidilizumab
(CT-011), anti-PD-L1 antibodies such as MPDL3280A(RG7446), MEDI4736
and BMS-936559, anti-PD-L2 antibodies, B7-DC-Fc fusion proteins
such as AMP-224, anti-CTLA-4 antibodies such as tremelimumab
(CP-675,206) and ipilimumab (MDX-00), antibodies against the
B7/CD28 receptor superfamily, anti-Indoleamine (2,3)-dioxygenase
(IDO) antibodies, anti-IDO1 antibodies, anti-IDO2 antibodies,
tryptophan, tryptophan mimetic, 1-methyl tryptophan (1-MT)),
Indoximod (D-1-methyl tryptophan (D-1-MT)), L-1-methyl tryptophan
(L-1-MT), TX-2274, hydroxyamidine inhibitors such as INCB024360,
anti-TIM-3 antibodies, anti-LAG-3 antibodies such as BMS-986016,
recombinant soluble LAG-31 g fusion proteins that agonize MHC class
II-driven dendritic cell activation such as IMP321,
anti-KIR2DL1/2/3 or anti-KIR) antibodies such lirilumab (IPH2102),
urelumab (BMS-663513), anti-phosphatidylserine (anti-PS) antibodies
such as Bavituximab, anti-idiotype murine monoclonal antibodies
against the human monoclonal antibody for N-glycolil-GM3
ganglioside such as Racotumomab (formerly known as 1E10),
anti-OX4OR antibodies such as IgG CD134 mAb, anti-B7-H3 antibodies
such as MGA271, and small interfering (si) RNA-based cancer
vaccines designed to treat cancer by silencing immune checkpoint
genes. Additional information can be found in Creelan BC (Update on
immune checkpoint inhibitors in lung cancer, Cancer Control. 2014
January;21(1):80-9) and Jane de Lartigue (Another Immune Checkpoint
Emerges as Anticancer Target, Published online by onclive.com,
Tuesday, Sep. 24, 2013), which are incorporated herein by reference
in their entirety as though fully set forth. In some embodiments,
the immune checkpoint inhibitor is selected from the group
consisting of an antibody against PD-1, an antibody against PD-L1,
an antibody against PD-L2, an antibody against CTLA-4, an antibody
against KIR, an antibody against IDO1, an antibody against IDO2, an
antibody against TIM-3, an antibody against LAG-3, an antibody
against OX4OR, and an antibody against PS, or a combination
thereof.
[0064] In various embodiments, the additional agent is an adoptive
immune therapeutic. In some embodiments, the adoptive immune
therapeutic is selected from the group consisting of a dendritic
cell vaccine, a peptide vaccine, a chimeric T cell antigen-based
therapy, T cell-based therapy, an immune cytokine, a heat shock
protein-based vaccine, a tumor lysate-based vaccine, a viral vector
carrying a tumor antigen, a viral vaccine, a bacterial vaccine, and
a fungal vaccine, or a combination thereof. Additional information
can be found in Radvanyi et al. Clin Cancer Res. 2012 Dec.
15;18(24):6758-70; Epub 2012 Oct. 2, which is incorporated herein
by reference in its entirety as though fully set forth.
[0065] In various embodiments, the additional agent is an immune
adjuvant. Examples of adjuvants include but are not limited to
cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine
adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium
sulfate adjuvant, Alhydrogel, ISCOM(s).TM., Freund's Complete
Adjuvant, Freund's Incomplete Adjuvant, CpG DNA Vaccine Adjuvant,
Cholera toxin, Cholera toxin B subunit, Liposomes, Saponin Vaccine
Adjuvant, DDA Adjuvant, Squalene-based Adjuvants, Etx B subunit
Adjuvant, IL-12 Vaccine Adjuvant, LTK63 Vaccine Mutant Adjuvant,
TiterMax Gold Adjuvant, Ribi Vaccine Adjuvant, Montanide ISA 720
Adjuvant, Corynebacterium-derived P40 Vaccine Adjuvant, MPL.TM.
Adjuvant, AS04, AS02, Lipopolysaccharide Vaccine Adjuvant, Muramyl
Dipeptide Adjuvant, CRL1005, Killed Corynebacterium parvum Vaccine
Adjuvant, Montanide ISA 51, Bordetella pertussis component Vaccine
Adjuvant, Cationic Liposomal Vaccine Adjuvant, Adamantylamide
Dipeptide Vaccine Adjuvant, Arlacel A, VSA-3 Adjuvant, Aluminum
vaccine adjuvant, Polygen Vaccine Adjuvant, Adjumer.TM., Algal
Glucan, Bay R1005, Theramide.RTM., Stearyl Tyrosine, Specol,
Algammulin, Avridine.RTM., Calcium Phosphate Gel, CTA1-DD gene
fusion protein, DOC/Alum Complex, Gamma Inulin, Gerbu Adjuvant,
GM-CSF, GMDP, Recombinant hIFN-gamma/Interferon-g,
Interleukin-1.beta., Interleukin-2, Interleukin-7, Sclavo peptide,
Rehydragel LV, Rehydragel HPA, Loxoribine, MF59, MTP-PE Liposomes,
Murametide, Murapalmitine, D-Murapalmitine, NAGO, Non-Ionic
Surfactant Vesicles, PMMA, Protein Cochleates, QS-21, SPT (Antigen
Formulation), nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine
adjuvant, RC529 vaccine adjuvant, LTR192G Vaccine Adjuvant, E. coli
heat-labile toxin, LT, amorphous aluminum hydroxyphosphate sulfate
adjuvant, Calcium phosphate vaccine adjuvant, Montanide Incomplete
Seppic Adjuvant, Imiquimod, Resiquimod, AF03, Flagellin, Poly(I:C),
ISCOMATRIX.RTM., Abisco-100 vaccine adjuvant, Albumin-heparin
microparticles vaccine adjuvant, AS-2 vaccine adjuvant, B7-2
vaccine adjuvant, DHEA vaccine adjuvant, Immunoliposomes Containing
Antibodies to Costimulatory Molecules, SAF-1, Sendai
Proteoliposomes, Sendai-containing Lipid Matrices, Threonyl muramyl
dipeptide (TMDP), Ty Particles vaccine adjuvant, Bupivacaine
vaccine adjuvant, DL-PGL (Polyester poly (DL-lactide-co-glycolide))
vaccine adjuvant, IL-15 vaccine adjuvant, LTK72 vaccine adjuvant,
MPL-SE vaccine adjuvant, non-toxic mutant E112K of Cholera Toxin
mCT-E112K, Matrix-S, and water soluble triterpene glucoside
compounds. Additional information can be found in Sayers et al.,
(VAXJO: A WEB-BASED VACCINE ADJUVANT DATABASE AND ITS APPLICATION
FOR ANALYSIS OF VACCINE ADJUVANTS AND THEIR USES IN VACCINE
DEVELOPMENT, 2012;2012:831486; Epub 2012 Mar. 13), which is
incorporated herein by reference in its entirety as though fully
set forth. In some embodiments, the immune adjuvant is selected
from the group consisting of an aluminum salt, a virosome and an
oil-based adjuvant, or a combination thereof.
[0066] In various embodiments, the additional agent is an immune
modulating agent. In some embodiments, the immune modulating agent
is selected from the group consisting of an mTOR inhibitor, a STAT
inhibitor, a TGF.beta. receptor inhibitor, and a tyrosine kinase
inhibitor, or a combination thereof.
[0067] In various embodiments, the methods may further include
administering an mTOR inhibitor to the subject. In accordance with
the invention, the mTOR inhibitor may be any one or more of a small
molecule, a peptide, an antibody or a fragment thereof, a nucleic
acid molecule and/or a macrolide compound. In an embodiment, the
antibody specifically binds mTOR so as to inhibit mTOR. The
antibody may be any one or more of a monoclonal antibody or
fragment thereof, a polyclonal antibody or a fragment thereof, a
chimeric antibody, a humanized antibody, a human antibody or a
fragment thereof, or a single chain antibody. These antibodies can
be from any source, e.g., rat, mouse, guinea pig, dog, cat, rabbit,
pig, cow, horse, goat, donkey or human. Fragments of antibodies may
be any one or more of Fab, F(ab')2, Fv fragments or their fusion
proteins.
[0068] In an embodiment of the invention, the mTOR inhibitor is a
macrolide compound. Examples of macrolide compounds that may be
used with the claimed invention include but are not limited to
temsirolimus (CCI-779) or a pharmaceutical equivalent, analog,
derivative or a salt thereof, evirolimus (RAD-001) or a
pharmaceutical equivalent, analog, derivative or a salt thereof,
and/or sirolimus (rapamycin) or a pharmaceutical equivalent,
analog, derivative or a salt thereof.
[0069] In various embodiments, the additional agent is a STAT
inhibitor. Examples of STAT inhibitors include but are not limited
to SOCS (supressors of cytokine signaling), PIAS (protein
inhibitors of activated stats) including PIAS1, PIAS2, PIAS3,
PIAS4, PIASxa, PIASxb, and PIASy, Nifuroxazide
(5-Nitro-2-furaldehyde-p-hydroxybenzoylhydrazone), N-[2-(1, 3,
4-oxadiazolyl)]-4-Quinolinecarboxamide, non-peptidic small molecule
inhibitors, Stattic, STA-21, LLL-3, LLL12, XZH-5, S31-201, SF-1066,
SF-1087, 17o, Cryptotanshinon, FLL32, C188-9, LY5, BP-1108,
BP-1075, Galiellalactone, JQ1, STX-0119, FLLL11, FLLL12, FLLL32,
FLLL62, hormone-derived nicotinyl hydrazine, IS3 295,
oligonucleotides targeting STAT pathway, antisense oligonucleotide
(ASO) targeting STAT pathway, AZD9150 (ISIS-STAT3Rx or ISIS 481464,
a synthetic ASO against STAT3, STAT3 decoy oligonucleotide (ODN),
STAT3-siRNA, STAT3-G-Quartet, STAT5-ODN, STAT5-siRNA, OPB-31121,
peptides and peptidomimetics inhibitors, XpYL, Ac-pYLPQTV-NH3,
ISS610, S31-M2001, and CJ-1383. Additional information can be found
in Furqan et al. (STAT inhibitors for cancer therapy, J Hematol
Oncol. 2013 Dec. 5;6:90), which is incorporated herein by reference
in its entirety as though fully set forth.
[0070] In various embodiments, the additional agent is a JAK
inhibitor. Examples of JAK inhibitors include but are not limited
to Tyrphostin AG490, CP-690550, ruxolitinib (INCB018424), TG101348
(SAR 30253), lestaurtinib (CEP701), CYT387, pacritinib (SB1518),
AZD1480, XL019, and LY2784544. Additional information can be found
in Mascarenhas et al. (Biology and clinical management of
myeloproliferative neoplasms and development of the JAK inhibitor
ruxolitinib, Curr Med Chem. 2012; 19(26):4399-413), which is
incorporated herein by reference in its entirety as though fully
set forth.
[0071] In various embodiments, the additional agent is a TGF.beta.
receptor inhibitor. Examples of TGF.beta. receptor inhibitors
include but are not limited to dominant negative TGF-.beta. Type II
receptors (T.beta.RII), ligand traps, the soluble T.beta.RII
ectodomain, the soluble betaglycan ectodomain, recombinant
Fc-fusion proteins containing the soluble ectodomain of either
T.beta.RII (T.beta.RII-Fc) or the type III receptor/betaglycan,
soluble human .alpha.2-macroglobulin plasma protein, fully
humanized pan-TGF-.beta. monoclonal neutralizing antibodies
including Lerdelimumab (CAT-152), Metelimumab (CAT-192) and GC-1008
(Fresolimumab), and 1D11; antisense oligonucleotides (ASO) designed
to hybridize to their complementary RNA sequence and accelerate
mRNA degradation, AP12009 (Trabedersen) and AP-11014; receptor
kinase inhibitors, SB505124, SB-431542, LY550410, LY580276,
LY215729, LY364937, LY2109761, Ki26894, SD-093 and SD-208; peptide
aptamers, the Trx-SARA aptamer; vectors coding for small hairpin
RNA which silence TGF-.beta. receptor Type II gene expression by
RNA interference, and shRNA with lentiviral or adeno-associated
vectors. Additional information can be found in Connolly et al.
(Complexities of TGF-.beta. targeted cancer therapy, Int J Biol
Sci. 2012;8(7):964-78; Epub 2012 Jul. 12) and Kaminska et al. (TGF
beta signalling and its role in tumour pathogenesis; Acta Biochim
Pol. 2005;52(2):329-37. Epub 2005 Jun. 25), which are incorporated
herein by reference in their entirety as though fully set
forth.
[0072] In various embodiments, the additional agent is a tyrosine
kinase inhibitor. Examples of tyrosine kinase inhibitors include
but are not limited to sunitinib, erlotinib, vandetanib, cediranib,
brivanib, foretinib, and dovitinib. Additional information can be
found in Huynh H. (Molecularly targeted therapy in hepatocellular
carcinoma, Biochem Pharmacol. 2010 Sep. 1;80(5):550-60. Epub 2010
Apr. 4), which is incorporated herein by reference in its entirety
as though fully set forth.
[0073] In some embodiments, the methods described herein further
comprise administering a chemotherapeutic agent to the subject
being administered a composition comprising CD4 lymphocyte
depleting agent or a composition comprising a CD4 lymphocyte
depleting agent and an additional agent selected from the group
consisting of an immune check point inhibitor, an adoptive immune
therapeutic, an immune adjuvant, and an immune modulating agent.
Non-limiting examples of chemotherapeutic agents can 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 gammaII and calicheamicin omegall (see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, 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; elformithine; 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;
sizofuran; 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; 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); lapatinib (Tykerb); 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. In addition, the methods of
treatment can further include the use of radiation therapy.
[0074] Examples of cancer include but are not limited to,
carcinoma, blastoma, and sarcoma. More particular examples of such
cancers include, but are not limited to, basal cell carcinoma,
biliary tract cancer; bladder cancer; bone cancer; brain and CNS
cancer; breast cancer; cancer of the peritoneum; cervical cancer;
choriocarcinoma; colon and rectum cancer; connective tissue cancer;
cancer of the digestive system; endometrial cancer; esophageal
cancer; eye cancer; cancer of the head and neck; gastric cancer
(including gastrointestinal cancer); glioblastoma; hepatic
carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal
cancer; larynx cancer; liver cancer; lung cancer (e.g., small-cell
lung cancer, non-small cell lung cancer, adenocarcinoma of the
lung, and squamous carcinoma of the lung); lymphoma including
Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma;
neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and
pharynx); ovarian cancer; pancreatic cancer; prostate cancer;
retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the
respiratory system; salivary gland carcinoma; sarcoma; skin cancer;
squamous cell cancer; stomach cancer; testicular cancer; thyroid
cancer; uterine or endometrial cancer; cancer of the urinary
system; vulval cancer; as well as other carcinomas and
sarcomas.
[0075] In accordance with the invention, the condition may be a
malignant neoplastic cell proliferative disorder or disease. Still
in accordance with the invention, the condition may be renal cell
carcinoma or melanoma. Diseases targeted by the therapeutic agents
include carcinomas, sarcomas, germ cell tumors and/or
blastomas.
[0076] In various embodiments, infectious diseases are caused by
infectious bacteria. Examples of infectious bacteria include:
Helicobacterpyloris, Borelia burgdorferi, Legionella pneumophilia,
Mycobacteria sps (such as M. tuberculosis, M. avium, M.
intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,
Neisseria gonorrhoeae, Neisseria meningitidis, Listeria
monocytogenes, Streptococcus pyogenes (Group A Streptococcus),
Streptococcus agalactiae (Group B Streptococcus), Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic sps.), Streptococcus pneumoniae,
pathogenic Campylobacter sp., Enterococcus sp., Haemophilus
influenzae, Bacillus anthracis, corynebacterium diphtherias,
corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium
perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella
pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium
nucleatum, Streptobacillus moniliformis, Treponema pallidium,
Treponema pertenue, Leptospira, and Actinomyces israelli. The
compositions and methods described herein are contemplated for use
in treating infections with these bacterial agents. Other
infectious organisms (such as protists) include: Plasmodium
falciparum and Toxoplasma gondii. The compositions and methods
described herein are contemplated for use in treating infections
with these agents.
[0077] In various embodiments, viral antigens may be any antigens
present in infectious viruses and that induce an immune response in
a subject. Examples of infectious viruses include: Retroviridae
(for example, HIV); Picornaviridae (for example, polio viruses,
hepatitis A virus; enteroviruses, human coxsackie viruses,
rhinoviruses, echoviruses); Calciviridae (such as strains that
cause gastroenteritis); Togaviridae (for example, equine
encephalitis viruses, rubella viruses); Flaviridae (for example,
dengue viruses, encephalitis viruses, yellow fever viruses);
Coronaviridae (for example, coronaviruses); Rhabdoviridae (for
example, vesicular stomatitis viruses, rabies viruses); Filoviridae
(for example, ebola viruses); Paramyxoviridae (for example,
parainfluenza viruses, mumps virus, measles virus, respiratory
syncytial virus); Orthomyxoviridae (for example, influenza
viruses); Bungaviridae (for example, Hantaan viruses, bunga
viruses, phleboviruses and Nairo viruses); Arena viridae
(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses,
orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae
(Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae
(papilloma viruses, polyoma viruses); Adenoviridae (most
adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and
HSV-2, varicella zoster virus, cytomegalovirus (CMV), herpes
viruses); Poxviridae (variola viruses, vaccinia viruses, pox
viruses); and Iridoviridae (such as African swine fever virus); and
unclassified viruses (for example, the etiological agents of
Spongiform encephalopathies, the agent of delta hepatitis (thought
to be a defective satellite of hepatitis B virus), the agents of
non-A, non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related
viruses, and astroviruses). The compositions and methods described
herein are contemplated for use in treating infections with these
viral agents.
[0078] Examples of fungal infections that may be treated with the
compositions and methods described herein include but are not
limited to: aspergillosis; thrush (caused by Candida albicans);
cryptococcosis (caused by Cryptococcus); and histoplasmosis. Thus,
examples of infectious fungi include, but are not limited to,
Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida
albicans. The compositions and methods described herein are
contemplated for use in treating infections with these fungal
agents.
[0079] In various embodiments, the subject is a human. In various
embodiments, the subject is a a mammalian including but not limited
to human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit,
mouse and rat.
[0080] In various embodiments, the composition is administered
intravenously, intramuscularly, subcutaneously, intraperitoneally,
orally or via inhalation. In accordance with the invention, various
routes may be utilized to administer the composition of the claimed
methods, including but not limited to aerosol, nasal, oral,
transmucosal, transdermal, parenteral, implantable pump, continuous
infusion, topical application, capsules and/or injections.
[0081] In various embodiments, the CD4 lymphocyte depleting agent
is administered at 100-200 mg/day, 200-300 mg/day, 300-400 mg/day,
400-500 mg/day, 500-600 mg/day, 600-700 mg/day, 700-800 mg/day,
800-900 mg/day, 900-1000 mg/day, 1000-1100 mg/day, 1100-1200
mg/day, 1200-1300 mg/day, 1300-1400 mg/day, 1400-1500 mg/day,
1500-1600 mg/day, 1600-1700 mg/day, 1700-1800 mg/day, 1800-1900
mg/day or 1900-2000 mg/day. In various embodiments, the additional
agent is administered at 0.1-0.5 mg/day, 0.5-1.0 mg/day, 1.0-1.5
mg/day, 1.5-2.0 mg/day, 2.0-2.5 mg/day, 2.5-5 mg/day, 5-10 mg/day,
10-15 mg/day, 15-20 mg/day, 20-25 mg/day, 25-30 mg/day, 30-35
mg/day, 35-40 mg/day, 40-45 mg/day, 45-50 mg/day, 50-55 mg/day,
55-60 mg/day, 60-65 mg/day, 65-70 mg/day, 70-75 mg/day, 75-80
mg/day, 80-85 mg/day, 85-90 mg/day, 90-95 mg/day or 95-100
mg/day.
Pharmaceutical Compositions
[0082] In various embodiments, the present invention provides a
composition comprising a CD4 lymphocyte depleting agent. In various
embodiments, the present invention provides compositions comprising
at least one of an immune check point inhibitor, an adoptive immune
therapeutic, an immune adjuvant, and an immune modulating agent.
Examples of immune check point inhibitor, an adoptive immune
therapeutic, an immune adjuvant, and an immune modulating agent are
described herein. In various embodiments, a composition comprising
a CD4 lymphocyte depleting agent and a composition comprising an
additional agent are two separate compositions.
[0083] In accordance with the invention, the CD4 lymphocyte
depleting agents and/or the additional agents useful in the
treatment of disease in mammals will often be prepared
substantially free of naturally-occurring immunoglobulins or other
biological molecules. Preferred CD4 lymphocyte depleting agents
and/or the additional agents will also exhibit minimal toxicity
when administered to a mammal.
[0084] The pharmaceutical compositions according to the invention
can contain any pharmaceutically acceptable excipient.
"Pharmaceutically acceptable excipient" means an excipient that is
useful in preparing a pharmaceutical composition that is generally
safe, non-toxic, and desirable, and includes excipients that are
acceptable for veterinary use as well as for human pharmaceutical
use. Such excipients may be solid, liquid, semisolid, or, in the
case of an aerosol composition, gaseous. Examples of excipients
include but are not limited to starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants, binders,
disintegrating agents, wetting agents, emulsifiers, coloring
agents, release agents, coating agents, sweetening agents,
flavoring agents, perfuming agents, preservatives, antioxidants,
plasticizers, gelling agents, thickeners, hardeners, setting
agents, suspending agents, surfactants, humectants, carriers,
stabilizers, and combinations thereof.
[0085] In various embodiments, the pharmaceutical compositions
according to the invention may be formulated for delivery via any
route of administration. "Route of administration" may refer to any
administration pathway known in the art, including but not limited
to aerosol, nasal, oral, transmucosal, transdermal, parenteral,
enteral, topical or local. "Parenteral" refers to a route of
administration that is generally associated with injection,
including intraorbital, infusion, intraarterial, intracapsular,
intracardiac, intradermal, intramuscular, intraperitoneal,
intrapulmonary, intraspinal, intrasternal, intrathecal,
intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,
transmucosal, or transtracheal. Via the parenteral route, the
compositions may be in the form of solutions or suspensions for
infusion or for injection, or as lyophilized powders. Via the
parenteral route, the compositions may be in the form of solutions
or suspensions for infusion or for injection. Via the enteral
route, the pharmaceutical compositions can be in the form of
tablets, gel capsules, sugar-coated tablets, syrups, suspensions,
solutions, powders, granules, emulsions, microspheres or
nanospheres or lipid vesicles or polymer vesicles allowing
controlled release. Typically, the compositions are administered by
injection. Methods for these administrations are known to one
skilled in the art.
[0086] The pharmaceutical compositions according to the invention
can contain any pharmaceutically acceptable carrier.
"Pharmaceutically acceptable carrier" as used herein refers to a
pharmaceutically acceptable material, composition, or vehicle that
is involved in carrying or transporting a compound of interest from
one tissue, organ, or portion of the body to another tissue, organ,
or portion of the body. For example, the carrier may be a liquid or
solid filler, diluent, excipient, solvent, or encapsulating
material, or a combination thereof. Each component of the carrier
must be "pharmaceutically acceptable" in that it must be compatible
with the other ingredients of the formulation. It must also be
suitable for use in contact with any tissues or organs with which
it may come in contact, meaning that it must not carry a risk of
toxicity, irritation, allergic response, immunogenicity, or any
other complication that excessively outweighs its therapeutic
benefits.
[0087] The pharmaceutical compositions according to the invention
can also be encapsulated, tableted or prepared in an emulsion or
syrup for oral administration. Pharmaceutically acceptable solid or
liquid carriers may be added to enhance or stabilize the
composition, or to facilitate preparation of the composition.
Liquid carriers include syrup, peanut oil, olive oil, glycerin,
saline, alcohols and water. Solid carriers include starch, lactose,
calcium sulfate, dihydrate, terra alba, magnesium stearate or
stearic acid, talc, pectin, acacia, agar or gelatin. The carrier
may also include a sustained release material such as glyceryl
monostearate or glyceryl distearate, alone or with a wax.
[0088] The pharmaceutical preparations are made following the
conventional techniques of pharmacy involving milling, mixing,
granulation, and compressing, when necessary, for tablet forms; or
milling, mixing and filling for hard gelatin capsule forms. When a
liquid carrier is used, the preparation will be in the form of a
syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
Such a liquid formulation may be administered directly p.o. or
filled into a soft gelatin capsule.
[0089] The pharmaceutical compositions according to the invention
may be delivered in a therapeutically effective amount. The precise
therapeutically effective amount is that amount of the composition
that will yield the most effective results in terms of efficacy of
treatment in a given subject. This amount will vary depending upon
a variety of factors, including but not limited to the
characteristics of the therapeutic compound (including activity,
pharmacokinetics, pharmacodynamics, and bioavailability), the
physiological condition of the subject (including age, sex, disease
type and stage, general physical condition, responsiveness to a
given dosage, and type of medication), the nature of the
pharmaceutically acceptable carrier or carriers in the formulation,
and the route of administration. One skilled in the clinical and
pharmacological arts will be able to determine a therapeutically
effective amount through routine experimentation, for instance, by
monitoring a subject's response to administration of a compound and
adjusting the dosage accordingly. For additional guidance, see
Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th
edition, Williams & Wilkins PA, USA) (2000).
[0090] Before administration to patients, formulants may be added
to the composition. A liquid formulation may be preferred. For
example, these formulants may include oils, polymers, vitamins,
carbohydrates, amino acids, salts, buffers, albumin, surfactants,
bulking agents or combinations thereof
[0091] Carbohydrate formulants include sugar or sugar alcohols such
as monosaccharides, disaccharides, or polysaccharides, or water
soluble glucans. The saccharides or glucans can include fructose,
dextrose, lactose, glucose, mannose, sorbose, xylose, maltose,
sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin,
soluble starch, hydroxethyl starch and carboxymethylcellulose, or
mixtures thereof. "Sugar alcohol" is defined as a C4 to C8
hydrocarbon having an --OH group and includes galactitol, inositol,
mannitol, xylitol, sorbitol, glycerol, and arabitol. These sugars
or sugar alcohols mentioned above may be used individually or in
combination. There is no fixed limit to amount used as long as the
sugar or sugar alcohol is soluble in the aqueous preparation. In
one embodiment, the sugar or sugar alcohol concentration is between
1.0 w/v % and 7.0 w/v %, more preferable between 2.0 and 6.0 w/v
%.
[0092] Amino acids formulants include levorotary (L) forms of
carnitine, arginine, and betaine; however, other amino acids may be
added.
[0093] In some embodiments, polymers as formulants include
polyvinylpyrrolidone (PVP) with an average molecular weight between
2,000 and 3,000, or polyethylene glycol (PEG) with an average
molecular weight between 3,000 and 5,000.
[0094] It is also preferred to use a buffer in the composition to
minimize pH changes in the solution before lyophilization or after
reconstitution. Most any physiological buffer may be used including
but not limited to citrate, phosphate, succinate, and glutamate
buffers or mixtures thereof. In some embodiments, the concentration
is from 0.01 to 0.3 molar. Surfactants that can be added to the
formulation are shown in EP Nos. 270,799 and 268,110.
[0095] Another drug delivery system for increasing circulatory
half-life is the liposome. Methods of preparing liposome delivery
systems are discussed in Gabizon et al., Cancer Research (1982)
42:4734; Cafiso, Biochem Biophys Acta (1981) 649:129; and Szoka,
Ann Rev Biophys Eng (1980) 9:467. Other drug delivery systems are
known in the art and are described in, e.g., Poznansky et al., DRUG
DELIVERY SYSTEMS (R. L. Juliano, ed., Oxford, N.Y. 1980), pp.
253-315; M. L. Poznansky, Pharm Revs (1984) 36:277.
[0096] After the liquid pharmaceutical composition is prepared, it
may be lyophilized to prevent degradation and to preserve
sterility. Methods for lyophilizing liquid compositions are known
to those of ordinary skill in the art. Just prior to use, the
composition may be reconstituted with a sterile diluent (Ringer's
solution, distilled water, or sterile saline, for example) which
may include additional ingredients. Upon reconstitution, the
composition is administered to subjects using those methods that
are known to those skilled in the art.
[0097] The compositions of the invention may be sterilized by
conventional, well-known sterilization techniques. The resulting
solutions may be packaged for use or filtered under aseptic
conditions and lyophilized, the lyophilized preparation being
combined with a sterile solution prior to administration. The
compositions may contain pharmaceutically-acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, calcium chloride, and
stabilizers (e.g., 1-20% maltose, etc.).
Kits of the Invention
[0098] In various embodiments, the present invention provides a kit
for treating, preventing, reducing the severity of and/or slowing
the progression of a condition in a subject. The kit comprises a
composition comprising a CD4 lymphocyte depleting agent and
instructions for using the composition to treat, prevent, reduce
the severity of and/or slow the progression of the condition in the
subject.
[0099] In various embodiments, the present invention provides a kit
for treating, preventing, reducing the severity of and/or slowing
the progression of a condition in a subject. The kit comprises a
composition comprising a CD4 lymphocyte depleting agent and a
composition comprising at least one additional agent selected from
the group consisting of an immune check point inhibitor, an
adoptive immune therapeutic, an immune adjuvant, and an immune
modulating agent; and instructions for using the composition to
treat, prevent, reduce the severity of and/or slow the progression
of the condition in the subject.
[0100] The kit is an assemblage of materials or components,
including at least one of the inventive compositions. Thus, in some
embodiments the kit contains a composition including a drug
delivery molecule complexed with a therapeutic agent, as described
above.
[0101] The exact nature of the components configured in the
inventive kit depends on its intended purpose. In one embodiment,
the kit is configured particularly for the purpose of treating
mammalian subjects. In another embodiment, the kit is configured
particularly for the purpose of treating human subjects. In further
embodiments, the kit is configured for veterinary applications,
treating subjects such as, but not limited to, farm animals,
domestic animals, and laboratory animals.
[0102] Instructions for use may be included in the kit.
"Instructions for use" typically include a tangible expression
describing the technique to be employed in using the components of
the kit to affect a desired outcome. Optionally, the kit also
contains other useful components, such as, diluents, buffers,
pharmaceutically acceptable carriers, syringes, catheters,
applicators, pipetting or measuring tools, bandaging materials or
other useful paraphernalia as will be readily recognized by those
of skill in the art.
[0103] The materials or components assembled in the kit can be
provided to the practitioner stored in any convenient and suitable
ways that preserve their operability and utility. For example the
components can be in dissolved, dehydrated, or lyophilized form;
they can be provided at room, refrigerated or frozen temperatures.
The components are typically contained in suitable packaging
material(s). As employed herein, the phrase "packaging material"
refers to one or more physical structures used to house the
contents of the kit, such as inventive compositions and the like.
The packaging material is constructed by well-known methods,
preferably to provide a sterile, contaminant-free environment. As
used herein, the term "package" refers to a suitable solid matrix
or material such as glass, plastic, paper, foil, and the like,
capable of holding the individual kit components. Thus, for
example, a package can be a glass vial used to contain suitable
quantities of a composition containing a CD lymphocyte depleting
agent or a composition containing a CD lymphocyte depleting agent
and at least one additional agent selected from the group
consisting of an immune check point inhibitor, an adoptive immune
therapeutic, an immune adjuvant, and an immune modulating agent.
The packaging material generally has an external label which
indicates the contents and/or purpose of the kit and/or its
components.
Dosages of the Invention
[0104] In some embodiments, the effective amounts of the CD4
lymphocyte depleting agent in the claimed methods, compositions
and/or kits may be in the range of about 100-200 mg/day, 200-300
mg/day, 300-400 mg/day, 400-500 mg/day, 500-600 mg/day, 600-700
mg/day, 700-800 mg/day, 800-900 mg/day, 900-1000 mg/day, 1000-1100
mg/day, 1100-1200 mg/day, 1200-1300 mg/day, 1300-1400 mg/day,
1400-1500 mg/day, 1500-1600 mg/day, 1600-1700 mg/day, 1700-1800
mg/day, 1800-1900 mg/day or 1900-2000 mg/day. In one embodiment of
the invention, the CD4 lymphocyte depleting agent is a humanized
anti-CD4 antibody (for example zanolimumab).
[0105] In other embodiments, the effective amounts of the CD4
lymphocyte depleting agent in the claimed methods, compositions
and/or kits may be in the range of about 100-200 mg/week, 200-300
mg/week, 300-400 mg/week, 400-500 mg/week, 500-600 mg/week, 600-700
mg/week, 700-800 mg/week, 800-900 mg/week, 900-1000 mg/week,
1000-1100 mg/week, 1100-1200 mg/week, 1200-1300 mg/week, 1300-1400
mg/week, 1400-1500 mg/week, 1500-1600 mg/week, 1600-1700 mg/week,
1700-1800 mg/week, 1800-1900 mg/week or 1900-2000 mg/week. In one
embodiment of the invention, the CD4 lymphocyte depleting agent is
a humanized anti-CD4 antibody (for example zanolimumab).
Zanolimumab may be administered at a dose of 980 mg per week.
[0106] In some embodiments, the effective amounts of the additional
agent in the claimed methods, compositions and/or kits may be in
the range of about 0.1-0.5 mg/day, 0.5-1.0 mg/day, 1.0-1.5 mg/day,
1.5-2 mg/day, 2.0-2.5 mg/day, 2.5-5 mg/day, 5-10 mg/day, 10-15
mg/day, 15-20 mg/day, 20-25 mg/day, 25-30 mg/day, 30-35 mg/day,
35-40 mg/day, 40-45 mg/day, 45-50 mg/day, 50-55 mg/day, 55-60
mg/day, 60-65 mg/day, 65-70 mg/day, 70-75 mg/day, 75-80 mg/day,
80-85 mg/day, 85-90 mg/day, 90-95 mg/day, 95-100 mg/day, 0.75-10
mg/day or 2-10 mg/day. In various embodiments, the additional
agents are any of immune check point inhibitor, an adoptive immune
therapeutic, an immune adjuvant or an immune modulating agent.
[0107] In some embodiments, the effective amounts of the additional
agent in the claimed methods, compositions and/or kits may be in
the range of about 1-5 mg/week, 5-10 mg/week, 10-15 mg/week, 15-20
mg/week, 20-25 mg/week, 25-30 mg/week, 30-35 mg/week, 35-40
mg/week, 40-45 mg/week, 45-50 mg/week, 50-55 mg/week, 55-60
mg/week, 60-65 mg/week, 65-70 mg/week, 70-75 mg/day, 75-80 mg/mg,
80-85 mg/mg, 85-90 mg/week, 90-95 mg/week or 95-100 mg/week. In
various embodiments, the additional agents are any of immune check
point inhibitor, an adoptive immune therapeutic, an immune adjuvant
or an immune modulating agent.
[0108] In some embodiments, the at least one additional agent
comprises temsirolimus administered at a dose of 25 mg over 30-60
minutes per week, evirolimus administered at a dose of 0.75-10 mg
per day and/or rapamycin administered at a dose of 2-10 mg per
day.
[0109] In an embodiment of the claimed methods of the invention,
the CD4 lymphocyte depleting agent and the additional agent may be
administered simultaneously at the aforementioned dosages using the
appropriate modes of administration, for instance, the modes of
administration recommended by the manufacturer for each of the CD4
lymphocyte depleting agent and the additional agent.
[0110] Alternately, the CD4 lymphocyte depleting agent and the
additional agent may be administered sequentially at the
aforementioned dosages. For example, the additional agent may be
administered, for example, daily at the aforementioned dosages and
the CD4 lymphocyte depleting agent (for example a humanized
anti-CD4 antibody) may be administered for example, daily, weekly,
biweekly, every fortnight and/or monthly at the aforementioned
dosages. Alternately, the additional agent may be administered, for
example, daily, weekly, biweekly, every fortnight and/or monthly,
at the aforementioned dosages and the CD4 lymphocyte depleting
agent (for example a humanized anti-CD4 antibody) may be
administered, for example, daily at the aforementioned dosages.
Further, each of the additional agent and the CD4 lymphocyte
depleting agent (for example a humanized anti-CD4 antibody) may be
administered daily, weekly, biweekly, every fortnight and/or
monthly, wherein the additional agent is administered at the
aforementioned dosages on a day different than the day on which the
CD4 lymphocyte depleting agent is administered at the
aforementioned dosages.
[0111] The cancer vaccine dose would depend on the vaccine being
used. The effective dose of the cancer vaccine may be determined by
one skilled in the art (such as the physician) or it may be
administered per the manufacturers' recommendation. In one
embodiment, the first dose of the cancer vaccine is administered on
day 0 and the second dose is administered on day 7. The additional
agent may be administered on days 2-32, at the aforementioned
dosages. Further, 2-3 weekly doses of anti-CD4 depleting agent may
be administered starting on day 10, at the aforementioned dosages.
For example, if a heat shock protein-based vaccine is used, a heat
shock protein (for example, hsp110 or grp170) may be complexed with
a tumor antigen (such as gp100) and subsequently administered. In
an embodiment, for a melanoma vaccine, a complex of hsp110 and
gp100 at 2.5 mg/kg, may be administered intradermally.
[0112] Typical dosages of an effective amount of the CD4 lymphocyte
depleting agent or the additional agent can be in the ranges
recommended by the manufacturer where known therapeutic compounds
are used, and also as indicated to the skilled artisan by the in
vitro responses in cells or in vivo responses in animal models.
[0113] For example, the FDA approved dosage for Temsirolimus is 25
mg administered intravenously over 30-60 min every week, for
Evirolimus is 0.75 mg-10 mg per day administered orally, for
Rapamycin is about 2-10 mg per day administered orally and for
Zanolimumab is about 980 mg per week administered intravenously.
The same or similar dosing can be used in accordance with various
embodiments of the present invention, or an alternate dosage may be
used in connection with alternate embodiments of the invention. The
actual dosage can depend upon the judgment of the physician, the
condition of the patient, and the effectiveness of the therapeutic
method based, for example, on the in vitro responsiveness of
relevant cultured cells or histocultured tissue sample, or the
responses observed in the appropriate animal models.
EXAMPLES
[0114] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
Example 1: Experimental Methods
Mice and Tumor Cells
[0115] Female C57BL/6J, BALB/c mice and Pmel-1 mice, 6-8 week old,
were purchased from Jackson laboratory (Bar Harbor, Me.) and housed
under pathogen-free conditions. FoxP3-GFP transgenic mice express
Green Fluorescent Protein under the control of the mouse Foxp3
(forkhead box P3) promoter. DEREG (DEpletion of REGulatory T cells)
transgenic mice was generated and described by T. S. Lahl K,
Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, et al. J
Exp Med. 2007;204:57-63. All experiments involving animals were in
compliance with federal and state standards, which include the
federal Animal Welfare Act and the NIH guide for the care and use
of laboratory animals.
[0116] B16 cells (mouse melanoma cell line) were transduced with
human gp100 (human melanoma antigen) (B16-gp100). RENCA is a murine
renal cell carcinoma (RCC) cell line. The cells were maintained in
DMEM or RPMI 1640 medium supplemented with 10% heat-inactivated
fetal bovine serum (FBS; Life Technologies, Grand Island, N.Y.), 2
mmol/L of L-glutamine, 100 units/mL of penicillin, and 100 .mu.g/mL
of streptomycin.
[0117] Mouse tumors were generated by subcutaneously injecting
2.times.10.sup.5 cells into the flank: B16 tumors into C57BL/6 mice
and RENCA tumors into Balb/C mice. Tumor diameter was measured with
calipers twice a week and tumor volume was calculated (shortest
diameter.sup.2.times.longest diameter/2). In the lung metastasis
model, tumor cells were injected intravenously through the tail
vein. Lung metastases were counted using a dissection
microscope.
Antibodies and Reagents
[0118] The following monoclonal antibodies (mAb), with or without a
fluorescent conjugate, were obtained from Biolegend (San Diego):
anti-CD4 (GK 1.5 and RM4-5), anti-CD8 (53-6.7),
anti-CD16/CD32(9.3), anti-CD90.1 (OX-7), anti-CD11c (N418),
anti-Bcl2 (BCL/10C4), anti-T-bet (4B10), anti-CD62L (MEL-14),
anti-CD279 (PD-1,29F.1A12), anti-FoxP3 (FJK-16s), anti-IFN-.gamma.
(XMG1.2), anti-IL-2 (JES6.5H4), anti-IL-4 (11B11), IL-17A
(eBio1787). CellTrace 5-(and 6-)carboxyfluorescein diacetate
succinimidyl ester (CFSE) cell proliferation kit was purchased from
Molecular Probes (Eugene, Oreg.). Temsirolimus was purchased from
LC Laboratory (Woburn, Mass.).
T Cell Enrichment and Treg Sorting
[0119] Mouse spleen and lymph nodes were collected and processed
into single-cell suspensions. CD8 and CD4 T-cells were negatively
enriched using mouse CD8 or CD4 recovery column kits (Cedarlane
Labs, Burlington, N.C.). Purity of CD8 and CD4 cells after negative
selection was greater than 85%. FoxP3-GFP cells or antibody stained
CD4+CD25+ cells were sorted by MoFlo Cell Sorter (Fort Collins,
Co.).
Preparation of DCs and T Cell Stimulation
[0120] DC preparation has been described (Wang Y, Wang X Y, Subjeck
J R, Shrikant P A, Kim H L. Br J Cancer. 2011;104:643-52). Briefly,
mouse bone marrows were harvested from femurs and tibias and then
placed in 12 well plates at a density of 1.times.10.sup.6 cells per
well with 10% FBS and 10 ng/ml mouse GM-CSF. The cells were fed
every 2 days and harvested 7-9 days later. 75-90% of cells were
CD11c positive. To prepare DC vaccine for treatment of mice, DCs
were pulsed with tumor cell lysate and activated with 10 ug/ml CpG.
DCs were subcutaneously injected into mouse. For in vitro
activation of Pmel-1 cells, DC was pulsed with 10 ng/ml mouse gp100
peptide (amino acids 25-33, which is presented by H2-Db class I
molecules, Alpha Diagnostic International, San Antonio, Tex.) and
activated with 10 ug/ml CpG for 2 hours. DC was washed with PBS,
and co-cultured with CFSE labeled Pmel-1 cells. Pmel-1 cells
proliferation was analyzed by FACscan.
Adoptive Transfer, CD4 Cells Depletion, and mTOR Inhibition
[0121] Pmel-1 lymphocytes were isolated from lymph nodes and spleen
of naive Pmel-1 mice. CD8 lymphocytes were enriched by negative
selection using Cedarlane purification column. At least 85% of the
resulting cells were CD8+. 5.times.10.sup.5 cells were transferred
into B57BL/6 mice. The day after adoptive transfer, mice received
tumor lysate pulsed DC vaccine. To deplete CD4 cells, .alpha.CD4
was administered approximately 7 and 9 days later; mice were inject
ip with 250 ug of CD4 mAb (clone GK1.5). To deplete CD8 cells, mice
received 250 ug of CD8 mAb (clone 2.43). To deplete FoxP3 cells in
DEREG mice, 5 .mu.g DT was injected. Flow cytometry was used to
confirm depletion of target cells. For mTor inhibitor treatment, 15
.mu.g is temsirolimus was injected i.p. each day for 2 weeks. Flow
cytometry was used to analyze memory cells and Treg cells.
In Vivo CTL Assay
[0122] The in vivo CTL assay has been described (Wang Y, Wang X Y,
Subjeck J R, Shrikant P A, Kim H L. Br J Cancer. 2011;104:643-52).
Briefly, single-cell suspensions of splenocytes (1.times.10.sup.7
cells/ml) were obtained from naive mice and pulsed with or without
10 .mu.M peptide in DMEM containing 10% FBS for 30 min at
37.degree. C. Each cell population was then labeled with a
different concentration of CFSE (0.5 or 12.5 .mu.M) at
2.times.10.sup.7 cells/ml in PBS/0.1% BSA. CFSE labeling was
stopped by addition of an equal volume of FBS for 1 min, and washed
3 times with RPMI complete medium. 5>10.sup.6 cells from each
peptide-pulsed or unpulsed population were mixed and injected i.v.
into immunized and unimmunized mice. Sixteen hours following
transfer, mice were sacrificed, and splenocytes were harvested.
Single-cell suspensions of splenocytes were prepared, and analyzed
by flow cytometry. Percent-specific-lysis of fluorescent donor
splenocytes was calculated as follows: [(number of unpulsed
targets.times.A-number of pulsed targets)/number of unpulsed
targets.times.A].times.100, where A=[number of pulsed
targets/number of unpulsed targets] in unimmunized recipient
mice.
Statistics
[0123] Differences in tumor growth were assessed using repeating
measure ANOVA. Statistical differences between numbers of lung
metastasis, in vivo CTL killing rates and mean percentages from
flow cytometry were evaluated by two-tailed student's T test. All
statistical analyzes were performed using Stata 8.0 (StataCorp,
College Station, Tex.). P values<0.05 were considered
significant.
Example 2: mTOR Inhibition Enhances Anti-Tumor Immunity
[0124] In animal models, pharmacologic mTOR inhibition can enhance
the formation of immune memory, which can help clear infections and
decrease tumor growth. This was a surprising finding because mTOR
inhibitors are used to suppress the immune system in patients who
have had solid organ transplants. Temsirolimus is a rapamycin
analog and one of the first mTOR inhibitors approved by the US,
Food and Drug Administration (FDA) as a cancer treatment. In our
preclinical model, mTOR inhibition with temsirolimus enhanced the
antitumor immunity of tumor lysate-pulsed DCs (referred to here as
DC vaccine) (FIG. 1a). Temsirolimus can directly inhibit the growth
of some tumors, therefore a tumor prevention study was performed to
assess the immune effects of temsirolimus. By administering DC
vaccine and temsirolimus 13 days prior to tumor challenge, there is
no possibility for a direct antitumor effect, and any decrease in
tumor growth can be attributed to immune stimulation. Administering
DC vaccine alone decreased growth of B16 tumor cells in mice,
however, most mice eventually died due to tumor growth. In
contrast, the combination of DC vaccine and temsirolimus resulted
in 100% survival and completely prevented the growth of B16 tumor
cells.
[0125] To assess the immune effect of temsirolimus on specific CD8
lymphocytes, Thy1.1 Pmel-1 lymphocytes were adoptively transferred
into Thy1.2 B6 mice (FIG. 1b). Pmel-1 transgenic mice carry a
rearranged T cell receptor that recognizes a gp100 epitope (amino
acids 25-33) presented by H2-Db MHC class I molecules. Lymphocytes
were harvested from B6 mice after they were treated with DC vaccine
and temsirolimus. Temsirolimus had both immune stimulating and
immune suppressing effects when administered with the DC vaccine.
Temsirolimus slightly decreased the percent of CD8 cells that were
Pmel-1 lymphocytes (p-value 0.08), however, Pmel-1 lymphocytes had
increased expression of Eomes, which is an early marker for memory
cell formation. Potentially immune suppressive effects included a
decrease in Tbet expression in Pmel-1 lymphocytes and increase in
Tregs. These observations were largely mirrored by in vitro mixed
lymphocyte culture studies (FIG. 1c). In the in vitro studies,
temsirolimus significantly decreased the proliferation of Pmel-1
lymphocytes induced by the DC vaccine.
Example 3: CD4 Depletion Enhanced the Antitumor Effect of mTor
Inhibition
[0126] Temsirolimus produced a net antitumor immune response
despite an increase in Tregs. Furthermore, the presence of tumor
itself increased Tregs (FIG. 8). Therefore, we hypothesized that
the antitumor immunity induced by mTOR inhibition can be further
enhanced by targeting Tregs. Currently there is no clinical
strategy to selectively remove Tregs; however, it is feasible to
deplete all CD4 lymphocytes. However, CD4 effector cells are
required for immune activation. Therefore, in our mouse model, CD4
lymphocytes were depleted with .alpha.CD4 antibody (.alpha.CD4)
starting 6 days after immune stimulation by the implanted tumor
(FIG. 2). FIG. 1 shows the results in a mouse model of melanoma. We
tested this approach in a second model of renal cell carcinoma,
another classically immune sensitive tumor. In a tumor treatment
model, palpable RENCA tumors were established in Balb/c mice.
Temsirolimus has been shown to directly inhibit the growth of RENCA
tumor cells in vitro (Wang Y, Wang X Y, Subjeck J R, Shrikant P A,
Kim H L. Br J Cancer. 2011;104:643-52) and was effective in
decreasing tumor growth in our mouse model (FIG. 2a). Addition of
.alpha.CD4 to temsirolimus treatment further decreased tumor growth
while .alpha.CD4 alone had no effect. It is interesting to note
that the combination of .alpha.CD4 and temsirolimus decreased tumor
growth even when no cancer vaccine was used and the implanted tumor
was the only source of specific immune stimulation.
[0127] In the same experiment, lymphocytes were harvested on day 45
and assessed for tumor-specific IFN-.gamma. response (FIG. 2b). In
tumor-bearing mice that received no treatment, there was no
IFN-.gamma. response. Treatment with either .alpha.CD4 or
temsirolimus produced some IFN-.gamma. response; however, the
combination treatment produced the largest IFN-.gamma. response. To
characterize the CD4 lymphocyte depletion in response to
.alpha.CD4, naive mice were treated with a single dose of
.alpha.CD4. Nearly all CD4 cells were depleted from the peripheral
blood, spleen and lymph nodes by the next day (FIG. 2c) while CD8
cells were preserved (FIG. 2d). Importantly, FoxP3+CD4+ cells were
depleted and remained low even 10 days following administration of
.alpha.CD4 (FIG. 2e). A single dose of .alpha.CD4 reduced the
population of all CD4 subsets (FIG. 9).
Example 4: Combination of CD4 Depletion and Temsirolimus Generated
Anti-Tumor Immunity that was Dependent on Memory CD8 Cells
[0128] Since temsirolimus can have a direct antitumor effect, it
was important to establish that the combination of .alpha.CD4 and
temsirolimus was generating an effective antitumor immunity
dependent on CD8 lymphocytes. Balb/c mice bearing RENCA tumors were
treated with temsirolimus alone for 10 days and then challenged
with a second RENCA tumor (FIG. 3a). Mice injected with .alpha.CD8
antibody (.alpha.CD8) to deplete CD8 effectors cells had increased
growth of the second RENCA tumor, indicating that even temsirolimus
alone works at least in part by stimulating an immune response. The
combination of temsirolimus and .alpha.CD4 completely prevented the
growth of second RENCA tumors; however, .alpha.CD8 removed the
antitumor effect on the second tumors, indicating the importance of
cellular immunity to tumor control (FIG. 3b).
[0129] To further establish the role of the immune system and test
our proposed treatment in a more aggressive tumor model, we
assessed whether antitumor immunity can be transferred to prevent
growth of metastatic lung deposits. The combination of .alpha.CD4
and temsirolimus was used to treat established, subcutaneous RENCA
tumors (FIG. 3c). Lymphocytes from these mice were adoptively
transferred to naive mice, which were challenged intravenously with
RENCA cells. The combination treatment significantly decreased the
establishment and growth of lung deposits (FIG. 3c) as quantified
by comparing lung weights (FIG. 3d) and counting lung deposits
(FIG. 3e). Therefore, memory cells were successfully transferred
into naive mice, where they helped control tumor growth. By
depleting CD8 cells, anti-tumor activity was shown to be dependent
on CD8 cells. Further confirmation of immune stimulation was
provided by transferring CD8 lymphocytes from treated mice to naive
mice. In a very aggressive tumor model, the transferred lymphocytes
were effective in controlling growth of metastatic deposits.
Example 5: Combination of CD4 Depletion and Temsirolimus Treatment
Enhanced Function of CD8 Memory Cells
[0130] An important mechanism through which temsirolimus inhibits
tumor growth is to enhance the quality of specific CD8 memory.
Therefore, we characterized the quality of CD8 memory cells with
the goal of assessing whether .alpha.CD4 further enhances the
specific CD8 memory formed in the presence of mTOR inhibition. We
used a model where DC vaccines stimulated an immune response rather
than the tumor itself since the duration of experiments with
tumor-bearing mice is limited by rapid tumor growth in the control
groups. By using a DC vaccine, long-term memory can be assessed,
including recall responses. Thy1.1 Pmel-1 lymphocytes were
adoptively transferred into B6 mice, which were then challenged
with B16-DC vaccine and treated with .alpha.CD4 and temsirolimus
(FIG. 4a).
[0131] To assess memory cells, splenocytes were harvest before
(FIG. 4b) or after (FIG. 4c) rechallenging mice with DC vaccine on
day 46. Immediately prior to rechallenge, there was no significant
difference in percent of Pmel-1 lymphocytes in the experimental
groups (FIG. 4b). However, the CD8 lymphocytes from mice treated
with both .alpha.CD4 and temsirolimus had significantly higher
expression of memory markers Eomes and BCL2. The CD8 lymphocytes
from this group had significantly higher expression of CD62L, which
is a marker for highly effective central memory cells. Consistent
with high quality memory cells, following rechallenge with DC
vaccine, the Pmel-1 cells in the combination treatment group had
the greatest expansion and CD8 cells had the highest Tbet and IL2
expression (FIG. 4c). Interestingly, even after rechallenge, the
expanded CD8 cells from the combination treatment group had the
highest expression of Eomes, an early memory marker. The
combination therapy produced CD8 lymphocytes with the strongest
memory phenotype capable of rapidly expanding in response to a
repeat antigen challenge. In our model, Tregs were present during
immune priming since CD4 depletion was initiated at least 6 days
after immune stimulation.
[0132] Others have reported that Tregs are necessary, during immune
priming, for CD8 memory formation. Therefore, in our treatment
models, Tregs were depleted at least 6 days after primary immune
stimulation. However, we verified the importance of having CD4
cells during immune priming. When CD4 depletion was performed prior
to immune priming, CD8 memory formation was poor, as indicated by a
weak tumor-specific CD8 expansion after stimulation of memory cells
and low Eomes expression (FIG. 10).
Example 6: Depleting or Replacing Foxp3 Treg Cells Alter CTL
Function In Vivo
[0133] Our original hypothesis was that depletion of Tregs normally
induced by temsirolimus will enhance antitumor immunity. We
selected CD4 depletion as a strategy for depleting Tregs since CD4
depletion is feasible in patients. However, we wanted to test
whether the effect of CD4 depletion can be directly attributed to
Tregs depletion. Therefore we used DEREG (DEpletion of REGulatory T
cells) transgenic mice, which carry a DTR-eGFP transgene under the
control of Foxp3 promoter, allowing specific depletion of Tregs by
administering diphtheria toxin (DT) (Lahl K, Loddenkemper C, Drouin
C, Freyer J, Arnason J, Eberl G, et al. J Exp Med.
2007;204:57-63.). In an experiment analogous to one shown in FIG.
2, DT was administered on days 6 and 10, in place of .alpha.CD4
(FIG. 5a). The immune system was stimulated with DC vaccine and
specific immune memory was assessed on day 35 by in vivo CTL (FIG.
5c). DT administration removed nearly all CD4+FoxP3+ lymphocytes
(FIG. 5b). Specific killing significantly increased in mice treated
with DT and temsirolimus when compared to control groups (FIG. 5c,
right most panel). Therefore, removing Tregs had a similar immune
effect to CD4 depletion.
[0134] To fully establish Treg depletion as the underlying
mechanism for immune stimulation following CD4 depletion, Tregs
were replaced after CD4 depletion (FIG. 6a). Mice treated with
.alpha.CD4 and temsirolimus developed the best specific immune
memory as assessed by in vivo CTL (FIG. 6b). However, when Tregs
from mice treated with DC vaccine were adoptively transferred,
specific killing decreased to that of control mice that only
received the DC vaccine (FIG. 6b, right most panel). These
experiments confirm that with .alpha.CD4 it is the Treg depletion
that enhances specific immune memory formation.
Example 7: Following CD4 Depletion, Treg Population that Eventually
Recovers is less Immunosuppressive
[0135] Following treatment with DC vaccine, .alpha.CD4, and
temsirolimus, the Treg population eventually recovers (FIG. 7a).
Between experimental groups, the differences in absolute number of
Tregs in the spleen were not statistically significant. However,
the treatments may have had a long-term effect on Treg function.
Therefore, we assessed the immunosuppressive function of the
recovered Tregs. CD4 lymphocytes were sorted based on CD25 status
(FIG. 7b). The vast majority of the CD4+CD25+ cells were FoxP3
positive and were considered Tregs, and the vast majority of
CD4+CD25- cells were FoxP3 negative and were considered CD4
effector cells. In functional studies, control CD4+CD25+ cells
suppressed the proliferation of CD8 lymphocytes. However, CD4+CD25+
that recovered after CD4 depletion were less immunosuppressive,
possibly because they were less likely to be tumor-specific Tregs
(FIG. 7d). Interestingly, following CD4 depletion, the recovered
CD4 effector cells were also less effective as indicated by lower
IFN.gamma. and IL4 secretion (FIG. 7c). It is possible that both
CD4 effector cells and Tregs were less likely to be
tumor-specific.
[0136] Immunotherapeutic approaches have proven effective for the
treatment of solid tumors. The FDA approved sipuleucel-T, which
became the first commercially available cancer vaccine for the
treatment of a solid tumor. Ipilimumab, a monoclonal antibody
targeting CTLA4, was more recently approved for the treatment of
melanoma. Immune checkpoint inhibitors that target CTLA4 and PD-1
are being actively investigated in a large number of clinical
trials for various malignancies. There have always been hints that
immune-based therapies can even be curative in subsets of patients
with metastatic disease, but recent advances in immunotherapy
reaffirm that durable complete responses are possible. Therefore,
immunotherapy is one of the most promising approaches to cancer
therapy.
Example 8: Combination of CD4 Lymphocyte Depleting Agent and
Adoptive Immune Therapeutic Agent Enhances Antitumor Affect
[0137] B16 tumor cells were implanted into DREG mice, which are B6
mice engineered to express the diphtheria toxin receptor behind the
FoxP3 promoter. Mice received tumor lyate-pulsed DC vaccine on day
2. Tregs were specifically depleted with diphtheria toxin and CD4
cells were removed with .alpha.CD4 antibody on day 6 and 10. Tumor
growth was monitored. *p=0.05, **p=0.0009. As shown in FIG. 11,
combination of CD4 lymphocyte depletion and a DC vaccine enhances
the antitumor effect when compared to DC vaccine alone.
Example 9: Combination of CD4 Lymphocyte Depleting Agent and Immune
Checkpoint Inhibitor Enhances Antitumor Affect
[0138] B16 tumor cells were implanted into B6 mice. On day 0. CD4
cells were depleted with .alpha.CD4 antibody on day 6 and 10. Mice
were treated with .alpha.PD-1 antibody on day 10 to 24. Tumor
growth was monitored. *p=0.02. As shown in FIG. 12, combination of
CD4 lymphocyte depletion and anti-PD-1 antibody enhances antitumor
effect when compared to anti-PD-1 antibody alone.
Example 10: anti-CD4 Antibody Exhibits Anti-Tumor Activity
[0139] RENCA-CA9 tumor cells were implanted into Balb/C mice (n=5
per group) on day 0. CD4 lymphocytes were depleted with .alpha.CD4
antibody on days 6 and 10 (FIG. 13A). Lymphocytes were harvested on
day 45, restimulated with CA9 peptide, and stained for CD8 and
IFN.gamma.. Results are representative of duplicate experiments
(FIG. 13B). For the in vitro CTL assay, splenocytes were harvest on
day 45 and cultured with IL2, RENCA lysate and CA9 peptide. Target
cells were prepared by labeling RENCA cells with CFSE. Effector and
target cells were co-cultured at a ratio of 50:1 and analyzed by
FACS for the percent of CFSE+ cells that were 7-AAD positive and
annexin V negative (FIG. 13C). Histograms provide mean+SEM.
*p<0.05. B16 tumor growth curve shows that anti-CD4 antibody has
anti-tumor effect (FIG. 13D). As shown in FIG. 13, CD4 depletion
produces tumor-specific CD8 cells capable of activating and tumor
cell killing in the presence of tumor antigen.
Example 11: Combination of CD4 Lymphocyte Depleting Agent, Adoptive
Immune Therapeutic Agent and mTOR Inhibitor Enhances Anti-Tumor
Affect
[0140] B16 tumor cells were injected subcutaneously in the flank of
B6 mice (n=5 per group) on day 0. Mice received tumor lysate-pulsed
DC vaccine on days 3 and CD4 lymphocyte depletion (using anti-CD4
antibody) was performed on days 6 and 9. Temsirolimus was injected
intraperitoneally daily on days 9 to 20. B16 tumor growth curves
are shown with SEM. **p<0.001. As shown in FIG. 14, the
combination of CD4 lymphocyte depleting agent, dendritic cell
vaccine and mTOR inhibitor (temsirolimus) effectively controlled
growth of tumors.
[0141] The various methods and techniques described above provide a
number of ways to carry out the application. Of course, it is to be
understood that not necessarily all objectives or advantages
described can be achieved in accordance with any particular
embodiment described herein. Thus, for example, those skilled in
the art will recognize that the methods can be performed in a
manner that achieves or optimizes one advantage or group of
advantages as taught herein without necessarily achieving other
objectives or advantages as taught or suggested herein. A variety
of alternatives are mentioned herein. It is to be understood that
some preferred embodiments specifically include one, another, or
several features, while others specifically exclude one, another,
or several features, while still others mitigate a particular
feature by inclusion of one, another, or several advantageous
features.
[0142] Furthermore, the skilled artisan will recognize the
applicability of various features from different embodiments.
Similarly, the various elements, features and steps discussed
above, as well as other known equivalents for each such element,
feature or step, can be employed in various combinations by one of
ordinary skill in this art to perform methods in accordance with
the principles described herein. Among the various elements,
features, and steps some will be specifically included and others
specifically excluded in diverse embodiments.
[0143] Although the application has been disclosed in the context
of certain embodiments and examples, it will be understood by those
skilled in the art that the embodiments of the application extend
beyond the specifically disclosed embodiments to other alternative
embodiments and/or uses and modifications and equivalents
thereof.
[0144] Preferred embodiments of this application are described
herein, including the best mode known to the inventors for carrying
out the application. Variations on those preferred embodiments will
become apparent to those of ordinary skill in the art upon reading
the foregoing description. It is contemplated that skilled artisans
can employ such variations as appropriate, and the application can
be practiced otherwise than specifically described herein.
Accordingly, many embodiments of this application include all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the application unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0145] All patents, patent applications, publications of patent
applications, and other material, such as articles, books,
specifications, publications, documents, things, and/or the like,
referenced herein are hereby incorporated herein by this reference
in their entirety for all purposes, excepting any prosecution file
history associated with same, any of same that is inconsistent with
or in conflict with the present document, or any of same that may
have a limiting affect as to the broadest scope of the claims now
or later associated with the present document. By way of example,
should there be any inconsistency or conflict between the
description, definition, and/or the use of a term associated with
any of the incorporated material and that associated with the
present document, the description, definition, and/or the use of
the term in the present document shall prevail.
[0146] It is to be understood that the embodiments of the
application disclosed herein are illustrative of the principles of
the embodiments of the application. Other modifications that can be
employed can be within the scope of the application. Thus, by way
of example, but not of limitation, alternative configurations of
the embodiments of the application can be utilized in accordance
with the teachings herein. Accordingly, embodiments of the present
application are not limited to that precisely as shown and
described.
[0147] Various embodiments of the invention are described above in
the Detailed Description. While these descriptions directly
describe the above embodiments, it is understood that those skilled
in the art may conceive modifications and/or variations to the
specific embodiments shown and described herein. Any such
modifications or variations that fall within the purview of this
description are intended to be included therein as well. Unless
specifically noted, it is the intention of the inventors that the
words and phrases in the specification and claims be given the
ordinary and accustomed meanings to those of ordinary skill in the
applicable art(s).
[0148] The foregoing description of various embodiments of the
invention known to the applicant at this time of filing the
application has been presented and is intended for the purposes of
illustration and description. The present description is not
intended to be exhaustive nor limit the invention to the precise
form disclosed and many modifications and variations are possible
in the light of the above teachings. The embodiments described
serve to explain the principles of the invention and its practical
application and to enable others skilled in the art to utilize the
invention in various embodiments and with various modifications as
are suited to the particular use contemplated. Therefore, it is
intended that the invention not be limited to the particular
embodiments disclosed for carrying out the invention.
[0149] While particular embodiments of the present invention have
been shown and described, it will be obvious to those skilled in
the art that, based upon the teachings herein, changes and
modifications may be made without departing from this invention and
its broader aspects and, therefore, the appended claims are to
encompass within their scope all such changes and modifications as
are within the true spirit and scope of this invention.
* * * * *