U.S. patent application number 14/540867 was filed with the patent office on 2015-05-21 for low, immune enhancing, dose mtor inhibitors and uses thereof.
This patent application is currently assigned to NOVARTIS INSTITUTES FOR BIOMEDICAL RESEARCH, INC.. The applicant listed for this patent is David Glass, Joan Mannick, Leon Murphy. Invention is credited to David Glass, Joan Mannick, Leon Murphy.
Application Number | 20150140036 14/540867 |
Document ID | / |
Family ID | 53057999 |
Filed Date | 2015-05-21 |
United States Patent
Application |
20150140036 |
Kind Code |
A1 |
Mannick; Joan ; et
al. |
May 21, 2015 |
LOW, IMMUNE ENHANCING, DOSE MTOR INHIBITORS AND USES THEREOF
Abstract
The present invention relates, in part, to compositions and
methods for enhancement of an immune response by partial mTOR
inhibition, e.g., with low, immune enhancing, doses of an mTOR
inhibitor, such as RAD001.
Inventors: |
Mannick; Joan; (Cambridge,
MA) ; Glass; David; (Cambridge, MA) ; Murphy;
Leon; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mannick; Joan
Glass; David
Murphy; Leon |
Cambridge
Cambridge
Cambridge |
MA
MA
MA |
US
US
US |
|
|
Assignee: |
NOVARTIS INSTITUTES FOR BIOMEDICAL
RESEARCH, INC.
Cambridge
MA
|
Family ID: |
53057999 |
Appl. No.: |
14/540867 |
Filed: |
November 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62052629 |
Sep 19, 2014 |
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62027121 |
Jul 21, 2014 |
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61903636 |
Nov 13, 2013 |
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62076142 |
Nov 6, 2014 |
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Current U.S.
Class: |
424/209.1 ;
424/184.1; 424/244.1; 424/278.1; 435/5 |
Current CPC
Class: |
C12N 7/00 20130101; A61P
21/00 20180101; A61P 9/10 20180101; A61P 13/08 20180101; A61P 25/28
20180101; A61P 31/16 20180101; A61P 25/14 20180101; A61P 9/00
20180101; A61P 27/16 20180101; A61P 37/04 20180101; A61K 31/436
20130101; A61P 11/00 20180101; A61K 45/06 20130101; A61P 13/12
20180101; A61P 35/02 20180101; A61P 3/10 20180101; A61P 27/12
20180101; A61K 31/4745 20130101; A61P 9/12 20180101; C12N
2760/16034 20130101; A61K 2039/55511 20130101; A61P 25/00 20180101;
A61P 31/04 20180101; A61K 31/519 20130101; A61K 39/09 20130101;
A61P 15/00 20180101; G01N 33/6854 20130101; A61K 39/092 20130101;
A61K 39/145 20130101; A61K 39/00 20130101; A61P 3/04 20180101; A61P
19/02 20180101; A61P 19/10 20180101; A61P 43/00 20180101; A61K
39/39 20130101; A61P 35/00 20180101; A61P 17/00 20180101; A61K
39/12 20130101 |
Class at
Publication: |
424/209.1 ;
424/278.1; 424/184.1; 424/244.1; 435/5 |
International
Class: |
A61K 39/39 20060101
A61K039/39; G01N 33/68 20060101 G01N033/68; A61K 39/09 20060101
A61K039/09; C12N 7/00 20060101 C12N007/00; A61K 45/06 20060101
A61K045/06; A61K 39/145 20060101 A61K039/145 |
Claims
1. A method of promoting an immune response in a subject,
comprising, administering to the subject a low, immune enhancing,
dose of an mTOR inhibitor, thereby enhancing or promoting an immune
response in the subject.
2. The method of claim 1, wherein the mTOR inhibitor is an
allosteric mTOR inhibitor or a catalytic inhibitor.
3. The method of claim 1, wherein the mTOR inhibitor is RAD001 or
rapamycin.
4. The method of claim 2, wherein the catalytic inhibitor is a
kinase inhibitor.
5. The method of claim 4, wherein the kinase inhibitor is selective
for mTOR or is selected from BEZ235 and CCG168.
6. The method of claim 1, wherein the dose comprises an allosteric
and a catalytic mTOR inhibitor.
7. The method of claim 1, wherein the mTOR inhibitor is
administered for an amount of time sufficient one or more of the
following to occur: i) a decrease in the number of PD-1 positive
immune effector cells; ii) an increase in the number of PD-1
negative immune effector cells; iii) an increase in the ratio of
PD-1 negative immune effector cells/PD-1 positive immune effector
cells; iv) an increase in the number of naive T cells; v) an
increase in the expression of one or more of the following markers:
CD62L.sup.high, CD127.sup.high, CD27.sup.+, and BCL2, e.g., on
memory T cells, e.g., memory T cell precursors; vi) a decrease in
the expression of KLRG1, e.g., on memory T cells, e.g., memory T
cell precursors; or vii) an increase in the number of memory T cell
precursors, e.g., cells with any one or combination of the
following characteristics: increased CD62L.sup.high increased
CD127.sup.high, increased CD27.sup.+, decreased KLRG1, and
increased BCL2; and wherein i), ii), iii), iv), v), vi), or vii)
occurs at least transiently, as compared to a non-treated
subject.
8. The method of claim 1, wherein the method comprises inhibiting a
negative immune response mediated by the engagement of PD-1 with
PD-L1 or PD-L2.
9. The method of claim 1, which comprises increasing the number of
T cells capable of proliferation, cytotoxic function, secreting
cytokines, or activation.
10. The method of claim 1, wherein the administering results in the
partial, but not total, inhibition of mTOR for at least 1, 5, 10,
20, 30, or 60 days.
11. The method of claim 1, wherein the dose of an mTOR inhibitor is
associated with mTOR inhibition of at least 5% but no more than
90%, as measured by p70 S6K inhibition.
12. The method of claim 11, wherein the mTOR inhibitor comprises
RAD001.
13. The method of claim 1, wherein administering comprises
administering, once per week, in an immediate release dosage form,
0.1 to 20, 0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5 mgs of RAD001,
or a bioequivalent dose of a different mTOR inhibitor.
14. The method of claim 1, wherein administering comprises
administering, once per week, in an immediate release dosage form,
about 5 mgs of RAD001, or a bioequivalent dose of a different mTOR
inhibitor.
15. The method of claim 1, wherein administering comprises
administering, once per week, in a sustained release dosage form,
0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9 to 18, or about 15 mgs of
RAD001, or a bioequivalent dose of a different mTOR inhibitor.
16. The method of claim 1, wherein administering comprises
administering, once per week, in a sustained release dosage form,
about 15 mgs of RAD001 or a bioequivalent dose of a different mTOR
inhibitor.
17. The method of claim 1, wherein administering comprises
administering, once per day, in an immediate release dosage form,
0.005 to 1.5, 0.01 to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4
to 1.5, 0.5 to 1.5, 0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5,
0.3 to 0.6, or about 0.5 mgs of RAD001, or a bioequivalent dose of
a different mTOR inhibitor.
18. The method of claim 1, wherein administering comprises
administering once per day, in an immediate release dosage form,
about 0.5 mgs of RAD001, or a bioequivalent dose of a different
mTOR inhibitor.
19. The method of claim 1, wherein administering comprises
administering, once per day, in a sustained release dosage form,
0.015 to 4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 to 4.5, 0.9 to 4.5, 1.2
to 4.5, 1.5 to 4.5, 1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5,
0.9 to 1.8, or about 1.5 mgs mgs of RAD001, or a bioequivalent dose
of a different mTOR inhibitor.
20. The method of claim 1, wherein administering comprises
administering, once per week, in a sustained release dosage form,
0.1 to 30, 0.2 to 30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30,
1.2 to 30, 14 to 30, 16 to 30, 20 to 30, 6 to 12, or about 10 mgs
of RAD001 or a bioequivalent dose of a different mTOR
inhibitor.
21. The method of claim 1, wherein the mTOR inhibitor is RAD001 and
the dose provides for a trough level of RAD001 in a range of
between about 0.1 and 3 ng/ml, between 0.3 or less and 3 ng/ml, or
between 0.3 or less and 1 ng/ml, or a bioequivalent dose of a
different mTOR inhibitor.
22. The method of claim 1, wherein the subject has cancer and the
method comprises promoting the subject's immune response to the
cancer.
23. The method of claim 22, wherein the subject was selected on the
basis of having cancer.
24. The method of claim 22, wherein a cell of the cancer expresses
PD-L1 or PD-L2.
25. The method of claim 22, wherein a cell in the cancer
microenvironment expresses PD-L1 or PD-L2.
26. The method of claim 22, wherein the cancer comprises a solid
tumor.
27. The method of claim 22, wherein the cancer is a hematological
cancer.
28. The method of claim 22, wherein the cancer is selected from
Table 1.
29. The method of claim 22, wherein the cancer is melanoma.
30. The method of claim 22, further comprising administering a
second treatment to the subject.
31. The method of claim 30, wherein the second treatment is a
chemotherapeutic, radiation, a cellular therapy, or bone marrow
transplant.
32. The method of claim 30, comprising administering a second
treatment that kills T cells.
33. The method of claim 32, wherein the second treatment is
radiation or cytotoxic chemotherapy.
34. The method of claim 30, wherein the mTOR inhibitor is
administered prior to, simultaneously with, or after the initiation
of the second treatment.
35. The method of any of claim 22, comprising treating a cancer
from Table 1, and administering a treatment listed in Table 1 for
that cancer.
36. The method of claim 1, wherein the subject is
immunocompromised.
37. The method of claim 1, wherein the subject is HIV+ or has
AIDs.
38. The method of claim 1, wherein the subject has an infectious
disease.
39. The method of claim 1, wherein the subject has an impaired
immune response.
40. The method of claim 1, wherein the subject is
immunoscenescent.
41. The method of claim 1, comprising treating the subject for an
age related condition.
42. The method of claim 41, wherein the age related condition is
selected from the group consisting of sarcopenia, skin atrophy,
muscle wasting, brain atrophy, atherosclerosis, arteriosclerosis,
pulmonary emphysema, osteoporosis, osteoarthritis, high blood
pressure, erectile dysfunction, dementia, Huntington's disease,
Alzheimer's disease, cataracts, age-related macular degeneration,
prostate cancer, stroke, diminished life expectancy, impaired
kidney function, and age-related hearing loss, aging-related
mobility disability (e.g., frailty), cognitive decline, age-related
dementia, memory impairment, tendon stiffness, heart dysfunction
such as cardiac hypertrophy and systolic and diastolic dysfunction,
immunosenescence, cancer, obesity, and diabetes.
43. The method of claim 1, comprising, enhancing an immune response
to an antigen in the subject.
44. The method of claim 43, further comprising administering the
antigen or a vaccine to the subject.
45. The method of claim 44, wherein prior to the step of
administering, the method comprises a step of identifying a subject
having an impaired immune response to an antigen.
46. The method of claim 44, wherein the antigen is an influenza
antigen.
47. The method of claim 46, wherein the antigen is selected from
the influenza subgroup consisting of H1N1, H2N3, and B influenza
subtypes.
48. The method of claim 44, wherein the antigen is a pneumococcal
antigen.
49. The method of claim 44, wherein the antigen and the mTOR
inhibitor are co-administered.
50. The method of any of claim 44, wherein the antigen and the mTOR
inhibitor are administered sequentially.
51. The method of claim 1, wherein the subject is less than 65
years old.
52. The method of claim 1, wherein the subject does not receive a
vaccine while the mTOR inhibitor is present at levels which promote
the immune response.
53. The method of claim 52, wherein the vaccine is an anti-cancer
vaccine or a vaccine against an infectious agent.
54. The method of claim 52, wherein the vaccine is a therapeutic
vaccine for a neurological disorder or Alzheimer's disease.
55. The method of claim 1, wherein the subject does not receive a
vaccine within 10, 20, 30, 40, 50, 60, 70, 80, or 90 days prior to
or after initiation of the low, immune enhancing, dose of the mTOR
inhibitor.
56. The method of claim 1, wherein the low, mTOR inhibitor is
administered at the time of, or after vaccination.
57. A method of evaluating a subject for treatment with a low,
immune enhancing, dose of mTOR inhibitor, to promote or enhance an
immune response to an influenza vaccine or antigen, comprising:
determining a baseline or pre-immunization level of anti-influenza
antibody, wherein a relatively low baseline or pre-immunization
level of anti-influenza antibody is predictive of a greater mTOR
inhibitor-associated increase in antibody titer for the influenza
antigen, thereby evaluating the subject.
58. The method of claim 57, further comprising comparing the
determined level with a reference value, wherein a value less than
or equal to the reference value is indicative of a greater mTOR
inhibitor-associated increase in antibody titer.
59. The method of claim 57, wherein responsive to a determined
level of antibody titer the subject is classified as to likelihood
of benefiting from a low, immune enhancing, dose of mTOR
inhibitor.
60. The method of claim 57, wherein responsive to a determined
level of antibody titer the subject is administered a low, immune
enhancing, dose of mTOR inhibitor.
61. The method of claim 57, wherein the determining step comprises
determining if the baseline or pre-immunization titer of
anti-influenza antibody of the subject is equal to or less than
1:40; and responsive to said determination, classifying the subject
as to the likelihood of benefiting from a low, immune enhancing,
dose of an mTOR inhibitor, or selecting a course of therapy for
said subject.
62. The method of claim 57, wherein the mTOR inhibitor is
RAD001.
63. The method of claim 1, wherein the subject is a human.
64. A vaccine adjuvant comprising about 0.005 mg to 1.5 mg of the
mTOR inhibitor RAD001, or a bioequivalent dose of a different mTOR
inhibitor.
65. The vaccine adjuvant of claim 64, comprising an amount of an
mTOR inhibitor sufficient to inhibit P70 S6 kinase activity in a
cell by no greater than 80%.
66. The vaccine adjuvant of claim 64, wherein said mTOR inhibitor
is a rapamycin or a rapalog.
67. The vaccine adjuvant of any of claim 64, comprising a 0.01-1
mg, 0.01-0.7 mg, 0.01-0.5 mg, or 0.1-0.5 mg of RAD001 or a
bioequivalent dose of a different mTOR inhibitor.
68. The vaccine adjuvant of any of claim 64, comprising 0.5 mg of
RAD001 or a bioequivalent dose of a different mTOR inhibitor.
69. A composition comprising (a) a vaccine antigen; and (b) about
0.005 mg to 1.5 mg of the mTOR inhibitor RAD001, or a bioequivalent
dose of a different mTOR inhibitor
70. The composition of claim 69, wherein said composition comprises
about 0.01-1 mg, about 0.01-0.7 mg, about 0.01-0.5 mg, or about
0.1-0.5 mg of RAD001, or a bioequivalent dose of a different mTOR
inhibitor.
71. The composition of claim 69, comprising about 0.5 mg of RAD001
or a bioequivalent dose of a different mTOR inhibitor.
72. The composition of claim 69, wherein said composition comprises
an amount of an mTOR inhibitor sufficient to inhibit P70 S6 kinase
activity by no greater than 80% in a subject to which said
composition is administered.
73. The composition of claim 69, wherein said composition produces
at least a 1.2 fold increase in immune response as compared to
placebo in a subject to which said composition is administered.
74. The composition of claim 69, wherein said mTOR inhibitor is a
rapamycin or a rapalog.
75. The composition of claim 69, wherein said vaccine antigen is
derived from influenza.
76. The composition of claim 69, wherein said vaccine antigen is
selected from the group consisting of H1N1, H2N3, and B influenza
subtypes.
77. The composition of claim 69, wherein said vaccine antigen is
derived from pneumococcus.
Description
[0001] This application claims priority to U.S. Ser. No.
61/903,636, filed Nov. 13, 2013, U.S. Ser. No. 62/027,121, filed
Jul. 21, 2014, U.S. Ser. No. 62/052,629, filed Sep. 19, 2014, and
U.S. Ser. No. 62/076,142, filed Nov. 6, 2014, the entire contents
of each of these applications are incorporated herein by
reference.
BACKGROUND
[0002] Functional and effective T-cell responses play an important
role in effective immune responses, for example, against infectious
diseases and cancer. However, under certain conditions, such as
chronic infection or cancer, effector T cells can be suppressed by
various immunosuppressive mechanisms, including programmed death
ligand-1 (PD-L1)/programmed death-1 (PD-1) interaction, leading to
T-cell exhaustion (Pen et al. Gene Therapy 21, 262-271, 2014). It
is thought that PD-L1 is normally expressed by most cell types,
while its receptor PD-1 is only present on certain immune cells,
such as activated T cells and regulatory T (Treg) cells. It is also
thought that PD-L1/PD-1 binding is important in the maintenance of
peripheral T-cell tolerance, preventing auto immune responses. On
the other hand, high levels of PD-1 expression generally correlate
with loss of T cell function, leading to increased viral load in
cases of viral infection (Pen et al. Gene Therapy 21, 262-271,
2014).
SUMMARY OF THE INVENTION
[0003] Methods and compositions disclosed herein are based, at
least in part, on the discovery that partial mTOR inhibition, e.g.,
with low, immune enhancing, doses of an mTOR inhibitor, e.g., an
allosteric mTOR inhibitor, such as RAD001, is effective to improve
immune function in a subject. While not wishing to be bound by
theory, it is believed that treatment with a low, immune enhancing,
dose (e.g., a dose that is insufficient to completely suppress the
immune system but sufficient to improve immune function) of an mTOR
inhibitor is accompanied by a decrease in PD-1 positive immune
effector cells, e.g., T cells, an increase in PD-1 negative immune
effector cells, e.g., T cells, or an increase in the ratio of in
PD-1 negative immune effector cells, e.g., T cells/PD-1 positive
immune effector cells, e.g., T cells. PD-1 positive T cells, but
not PD-1 negative T cells, can be exhausted by engagement with
cells which express a PD-1 ligand, e.g., PD-L1 or PD-L2. Thus,
embodiments of the invention are based, at least in part, on the
recognition that partial mTOR inhibition, e.g., with low, immune
enhancing, dose of an mTOR inhibitor, is associated with a
reduction in the percentage of programmed death (PD)-1 positive CD4
and CD8 T lymphocytes.
[0004] Accordingly, in one aspect, the present invention relates to
a method of promoting an immune response in a subject, e.g., a
human subject, comprising,
[0005] administering to the subject a low, immune enhancing, dose
of an mTOR inhibitor, e.g., RAD001 or rapamycin,
[0006] thereby enhancing or promoting an immune response in the
subject.
[0007] In an embodiment, a low, immune enhancing, dose of an mTOR
inhibitor, e.g., RAD001 or rapamycin and an antigen are
administered as a vaccine.
[0008] In an embodiment, a low, immune enhancing, dose of an mTOR
inhibitor, e.g., RAD001 or rapamycin is administered as an adjuvant
composition or compound.
[0009] Exemplary mTOR inhibitors are described herein, e.g., in the
section below entitled "mTOR INHIBITORS."
[0010] In an embodiment, the mTOR inhibitor is an allosteric mTOR
inhibitor. In an embodiment, the mTOR inhibitor is a RAD001. In an
embodiment, the mTOR inhibitor is rapamycin.
[0011] In an embodiment, the mTOR inhibitor is a catalytic
inhibitor, e.g., a kinase inhibitor. In an embodiment, the kinase
inhibitor is selective for mTOR. In an embodiment, the kinase
inhibitor is selected from BEZ235 and CCG168.
[0012] In an embodiment, the low, immune enhancing, dose comprises
a plurality of mTOR inhibitors. In an embodiment, the low, immune
enhancing, dose comprises an allosteric and a catalytic mTOR
inhibitor.
[0013] In an embodiment, the low, immune enhancing, dose of an mTOR
inhibitor is administered for an amount of time sufficient for one
or more of the following to occur:
[0014] i) a decrease in the number of PD-1 positive immune effector
cells;
[0015] ii) an increase in the number of PD-1 negative immune
effector cells;
[0016] iii) an increase in the ratio of PD-1 negative immune
effector cells/PD-1 positive immune effector cells;
[0017] iv) an increase in the number of naive T cells;
[0018] v) an increase in the expression of one or more of the
following markers: CD62L.sup.high, CD127.sup.high, CD27.sup.+, and
BCL2, e.g., on memory T cells, e.g., memory T cell precursors;
[0019] vi) a decrease in the expression of KLRG1, e.g., on memory T
cells, e.g., memory T cell precursors; or
[0020] vii) an increase in the number of memory T cell precursors,
e.g., cells with any one or combination of the following
characteristics: increased CD62L.sup.high increased CD127.sup.high,
increased CD27.sup.+, decreased KLRG1, and increased BCL2;
and wherein i), ii), iii), iv), v), vi), or vii) occurs e.g., at
least transiently, e.g., as compared to a non-treated subject.
[0021] In an embodiment, the method of treating, e.g., promoting an
immune response in, a subject, e.g., a human subject, comprises
inhibiting a negative immune response mediated by the engagement of
PD-1 with PD-L1 or PD-L2.
[0022] In an embodiment, the method of treating, e.g., promoting an
immune response in, a subject, e.g., a human subject, comprises
increasing the number of T cells capable of proliferation.
[0023] In an embodiment, the method of treating, e.g., promoting an
immune response in, a subject, e.g., a human subject, comprises
increasing the number of T cells capable of cytotoxic function,
secreting cytokines, or activation.
[0024] In an embodiment, the method of treating, e.g., promoting an
immune response in, a subject, e.g., a human subject, comprises
increasing the number of T cells capable of providing T cell help
to B cells.
[0025] In an embodiment, the administering of the low, immune
enhancing, dose of an mTOR inhibitor results in the partial, but
not total, inhibition of mTOR for at least 1, 5, 10, 20, 30, or 60
days.
[0026] In an embodiment, the administering of the low, immune
enhancing, dose of an mTOR inhibitor results in the partial, but
not total, inhibition of mTOR as long as enhancement of the immune
response is needed.
[0027] In an embodiment, the low, immune enhancing, dose of an mTOR
inhibitor is associated with mTOR inhibition of at least 5% but no
more than 90%, e.g., as measured by p70 S6K inhibition. In an
embodiment, the mTOR inhibitor comprises RAD001. (Methods for
evaluation of the level of inhibition of mTOR are described herein,
e.g., in the section below entitled "EVALUATION OF mTOR
INHIBITION.")
[0028] In an embodiment, the low, immune enhancing, dose of an mTOR
inhibitor is associated with mTOR inhibition of at least 10% but no
more than 80%, e.g., as measured by p70 S6K inhibition. In an
embodiment, the mTOR inhibitor comprises RAD001.
[0029] In an embodiment, the low, immune enhancing, dose of an mTOR
inhibitor is associated with mTOR inhibition of at least 10% but no
more than 40%, e.g., as measured by p70 S6K inhibition. In an
embodiment, the mTOR inhibitor comprises RAD001.
[0030] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in an immediate release dosage form, 0.1 to
20, 0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5, mgs of RAD001.
[0031] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per week, in an immediate release dosage form, about 5 mgs of
RAD001.
[0032] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in an immediate release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
one per week, immediate release dosage form of 0.1 to 20, 0.5 to
10, 2.5 to 7.5, 3 to 6, or about 5 mgs of RAD001.
[0033] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per week, in an immediate release dosage form, an amount of an mTOR
inhibitor other than RAD001, that is bioequivalent to a once per
week, immediate release dosage form of about 5 mgs of RAD001.
[0034] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in a sustained release dosage form, 0.3 to 60,
1.5 to 30, 7.5 to 22.5, 9 to 18, or about 15 mgs of RAD001.
[0035] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per week, in a sustained release dosage form, about 15 mgs of
RAD001.
[0036] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in a sustained release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
once per week, sustained release dosage form of 0.3 to 60, 1.5 to
30, 7.5 to 22.5, 9 to 18, or about 15 mgs of RAD001.
[0037] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per week, in a sustained release dosage form, an amount of an mTOR
inhibitor other than RAD001, that is bioequivalent to a once per
week sustained release dosage form of about 15 mgs of RAD001.
[0038] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per day, e.g., in an immediate release dosage form, 0.005 to
1.5, 0.01 to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to 1.5,
0.5 to 1.5, 0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to
0.6, or about 0.5 mgs of RAD001.
[0039] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering once
per day, in an immediate release dosage form, about 0.5 mgs of
RAD001.
[0040] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per day, e.g., in an immediate release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
once per day, immediate release dosage form of 0.005 to 1.5, 0.01
to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to 1.5, 0.5 to 1.5,
0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to 0.6, or
about 0.5 mgs of RAD001.
[0041] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per day, in an immediate release dosage form, an amount of an mTOR
inhibitor other than RAD001, that is bioequivalent to a once per
day, immediate release dosage form of about 0.5 mgs of RAD001.
[0042] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per day, e.g., in a sustained release dosage form, 0.015 to
4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 to 4.5, 0.9 to 4.5, 1.2 to 4.5,
1.5 to 4.5, 1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5, 0.9 to
1.8, or about 1.5 mgs of RAD001.
[0043] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per day, e.g., in a sustained release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
once per day, sustained release dosage form of 0.015 to 4.5, 0.03
to 4.5, 0.3 to 4.5, 0.6 to 4.5, 0.9 to 4.5, 1.2 to 4.5, 1.5 to 4.5,
1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5, 0.9 to 1.8, or
about 1.5 mgs of RAD001.
[0044] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in a sustained release dosage form, 0.1 to 30,
0.2 to 30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30,
14 to 30, 16 to 30, 20 to 30, 6 to 12, or about 10 mgs of
RAD001.
[0045] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in a sustained release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
once per week, sustained release dosage form of 0.1 to 30, 0.2 to
30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30, 14 to
30, 16 to 30, 20 to 30, 6 to 12, or about 10 mgs of RAD001.
[0046] In an embodiment, the mTOR inhibitor is RAD001 and the dose
provides for a trough level of RAD001 in a range of between about
0.1 and 3 ng/ml, between 0.3 or less and 3 ng/ml, or between 0.3 or
less and 1 ng/ml.
[0047] In an embodiment, the mTOR inhibitor is other than RAD001
and the dose is bioequivalent to a dose of RAD001 that provides for
a trough level of RAD001 in a range of between about 0.1 and 3
ng/ml, between 0.3 or less and 3 ng/ml, or between 0.3 or less and
1 ng/ml.
[0048] In an embodiment the subject has cancer. Exemplary cancers
are described herein, e.g., in the section below entitled
"DISORDERS Cancer." In an embodiment, the subject has cancer, but
is not otherwise immunocompromised, e.g, is not HIV+, does not have
AIDS, or is not immunoscenescent. In an embodiment, the subject has
cancer, but, except for that due to any anti-cancer treatment, is
not otherwise immunocompromised, e.g, is not HIV+, does not have
AIDS, or is not immunoscenescent.
[0049] In an embodiment, the subject has cancer and the method
comprises promoting the subject's immune response to the cancer. In
an embodiment, the subject was selected on the basis of having
cancer. In an embodiment, the subject was selected on the basis of
being in need of, or likely to benefit from, promotion of the
immune response. In an embodiment, a cell of the cancer expresses
PD-L1 or PD-L2. In an embodiment, a cell in the cancer
microenvironment expresses PD-L1 or PD-L2.
[0050] In an embodiment, the cancer comprises a solid tumor. In an
embodiment, the cancer is a hematological cancer. In an embodiment,
the cancer is a leukemia. In an embodiment, the cancer is
melanoma.
[0051] In an embodiment, promoting an immune response in a subject
comprises preparing the subject, e.g., a subject having cancer, for
an additional treatment that suppresses the immune system or kills
T cells, e.g., administration of a drug, e.g., a chemotherapeutic,
or radiation. In an embodiment, the low, immune enhancing dose, of
an mTOR inhibitor, e.g., RAD001, reduces immune suppression
associated with the additional treatment.
[0052] In an embodiment, the method further comprises administering
an additional treatment, e.g., a chemotherapeutic, radiation, a
cellular therapy, bone marrow transplant to the subject. In an
embodiment the additional treatment comprises a combination of
drugs or treatments as described herein, see, e.g., the section
below entitled "COMBINATION TREATMENTS." In an embodiment, the
method further comprises administering an additional treatment that
kills T cells, e.g., radiation or cytotoxic chemotherapy. In an
embodiment, the low, immune enhancing, dose of mTOR inhibitor is
administered prior to, with, or after the initiation of the
additional treatment. In an embodiment, the method further
comprises administering an additional treatment for the cancer.
[0053] In an embodiment, the method further comprises administering
an additional treatment that suppresses the immune system, e.g.,
administration of a drug, e.g., a chemotherapeutic, or radiation.
In an embodiment, the low, immune enhancing, dose of mTOR
inhibitor, e.g., RAD001, is administered prior to, with, or after
the initiation of the additional treatment that suppresses the
immune system. While not wishing to be bound by theory, it is
believed that the low, immune enhancing dose of an mTOR inhibitor,
allows for a broader range of therapeutic options. Without wishing
to be bound by theory, it is believed that this is due to the
improvement in the subject's immune responsiveness. In an
embodiment, the low, immune enhancing dose of an mTOR inhibitor,
can allow for more aggressive administration of the additional
treatment. Thus, in an embodiment, the unit dosage, total dosage,
frequency of administration, or number of administrations, is
increased. In an embodiment, the increase is relative to a
reference administration, e.g., the standard of care that is
provided in the absence of a low, immune enhancing, dose of mTOR
inhibitor. In an embodiment, the increase is relative to an
administration that would give the maximum tolerable or acceptable
levels of immune suppression, in the absence of a low, immune
enhancing, dose of mTOR inhibitor. In another embodiment, the
immune enhancing dose of an mTOR inhibitor, can allow for less
aggressive administration of the additional treatment. Thus, in an
embodiment, the unit dosage, total dosage, frequency of
administration, or number of administrations, is decreased. In an
embodiment, the decrease is relative to a reference administration,
e.g., the standard of care that is provided in the absence of a
low, immune enhancing, dose of mTOR inhibitor. In an embodiment,
the decrease is relative to an administration that would give the
maximum tolerable or acceptable levels of immune suppression, in
the absence of a low, immune enhancing, dose of mTOR inhibitor.
[0054] In an embodiment, the subject is immunocompromised. In an
embodiment, the subject is HIV+ or has AIDs.
[0055] Thus, in an embodiment, promoting an immune response in a
subject comprises promoting the immune response of an
immunocompromised subject, e.g., a subject having an
immunodeficiency, e.g., a hereditary or acquired immunodeficiency,
e.g., a virally-mediated immunodeficiency, e.g., a subject that is
HIV+, or a subject having AIDS. In an embodiment, the method
further comprises administering an additional treatment for the
immunodeficiency, e.g., an antiviral agent. In an embodiment, the
subject is HIV+ or has AIDS and the additional treatment comprises
administering an anti-viral agent, e.g., a nucleoside reverse
transcriptase inhibitor, e.g., abacavir, didanosine, emtricitabine,
lamivudine, stavudine, tenofovir, zalcitabine, or zidovudine, or
combinations thereof, e.g. combivir (zidovudine and lamivudine),
trizivir (zidovudine, lamivudine and abacavir), epzicom (abacavir
and lamivudine) and truvada (tenofovir and lamivudine). In an
embodiment, the additional treatment comprises administering a
protease inhibitor, e.g., amprenavir, agenerase, atazanavir,
fosamprenavir, indinavir, lopinavir, ritonavir, or saquinavir, or a
combination thereof. In an embodiment, the low, immune enhancing,
dose of mTOR inhibitor, e.g., RAD001, is administered prior to,
with, or after the initiation of the additional treatment. While
not wishing to be bound by theory, it is believed that the low,
immune enhancing dose of an mTOR inhibitor, allows for a broader
range of therapeutic options. Without wishing to be bound by
theory, it is believed that this is due to the improvement in the
subject's immune responsiveness. In an embodiment, the low, immune
enhancing dose of an mTOR inhibitor, can allow for more aggressive
administration of the additional treatment. Thus, in an embodiment,
the unit dosage, total dosage, frequency of administration, or
number of administrations, is increased. In an embodiment, the
increase is relative to a reference administration, e.g., the
standard of care that is provided in the absence of a low, immune
enhancing, dose of mTOR inhibitor. In an embodiment, the increase
is relative to an administration that would give the maximum
tolerable or acceptable levels of a side effect, in the absence of
a low, immune enhancing, dose of mTOR inhibitor. In another
embodiment, the immune enhancing dose of an mTOR inhibitor, can
allow for less aggressive administration of the additional
treatment. Thus, in an embodiment, the unit dosage, total dosage,
frequency of administration, or number of administrations, is
decreased. In an embodiment, the decrease is relative to a
reference administration, e.g., the standard of care that is
provided in the absence of a low, immune enhancing, dose of mTOR
inhibitor. In an embodiment, the decrease is relative to an
administration that would give the maximum tolerable or acceptable
levels of a side effect, in the absence of a low, immune enhancing,
dose of mTOR inhibitor.
[0056] In an embodiment, the subject has an infectious disease,
e.g., hepatitis, e.g., hepatitis A, B or C, or other pathogenic
infection. Exemplary pathogenic infections are described herein,
e.g., in the section below entitled "DISORDERS Pathogenic
Infections." In an embodiment, the subject has an infectious
disease or has a pathogenic infection, but is not otherwise
immunocompromised, e.g, is not immunosenescent.
[0057] In an embodiment, the subject has an impaired immune
response. In an embodiment, the subject is immunosenescent.
[0058] In an embodiment, the subject has an age related condition.
In an embodiment, the age related condition is selected from the
group consisting of sarcopenia, skin atrophy, muscle wasting, brain
atrophy, atherosclerosis, arteriosclerosis, pulmonary emphysema,
osteoporosis, osteoarthritis, high blood pressure, erectile
dysfunction, dementia, Huntington's disease, Alzheimer's disease,
cataracts, age-related macular degeneration, prostate cancer,
stroke, diminished life expectancy, impaired kidney function, and
age-related hearing loss, aging-related mobility disability (e.g.,
frailty), cognitive decline, age-related dementia, memory
impairment, tendon stiffness, heart dysfunction such as cardiac
hypertrophy and systolic and diastolic dysfunction,
immunosenescence, cancer, obesity, and diabetes.
[0059] In an embodiment, the method comprises enhancing an immune
response to an antigen in the subject. In an embodiment the method
comprises providing or administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor, e.g., RAD001 or rapamycin as
an adjuvant composition or compound. In an embodiment the method
comprises providing or administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor, e.g., RAD001 or rapamycin,
and the antigen, as a, or in combination with a vaccine. In an
embodiment the antigen is a cancer antigen. In an embodiment the
antigen is an infectous disease-, viral, bacterial, protozoan,
microbial, pathogen-, or parasite-, antigen. In an embodiment, the
method further comprises administering an antigen or a vaccine to
the subject. In an embodiment, prior to the step of administering,
the method comprises a step of identifying a subject having an
impaired immune response to an antigen.
[0060] In an embodiment, a relatively low baseline or
pre-immunization level or titer of antibody to the antigen is
predictive of a greater mTOR inhibitor-, e.g., RAD001-, associated
increase in antibody titer for an antigen. In an embodiment, the
subject is evaluated for level or titer of antibody to the antigen
prior to administration of an antigen or vaccine. In an embodiment
evaluation comprises acquiring, e.g., directly or indirectly
acquiring, a measurement of titer or level of antibody. The titer
or level of antibody can be compared with a reference value.
Relatively low titer, e.g., titer below or equal to a reference
value, is indicative of a greater mTOR inhibitor-, e.g., RAD001-,
associated increase in antibody titer. Thus, baseline or
pre-immunization titer can be used to select patients for low,
immune enhancing, dose of mTOR inhibitor, e.g., in combination with
vaccination or administration of antigen to stimulate an immune
response. In an embodiment, responsive to a determined level or
titer of antibody, a subject is classified as to the likelihood of
benefiting from administration of a low, immune enhancing, dose of
mTOR inhibitor, e.g., prior to or with administration of a vaccine
or antigen. In an embodiment, responsive to a determined level or
titer of antibody, e.g., a level or titer that is at or below a
reference value, a subject is selected for, or administered, a low,
immune enhancing, dose of mTOR inhibitor, prior to or with
administration of a vaccine or antigen. In an embodiment,
responsive to a determined level or titer of antibody, e.g., a
level or titer that is above a reference value, a subject is
selected for, or administered an alternative therapy, e.g.,
administration of a vaccine or antigen, without the administration
of a low, immune enhancing, dose of mTOR inhibitor.
[0061] In an embodiment, the subject is infected with, or at risk
for infection with, an influenza virus, e.g., an influenza A or B
virus.
[0062] In an embodiment, the method comprises enhancing an immune
response to an influenza virus, e.g., an influenza A or B virus.
Influenza A viruses are characterized by one or both of two
glycoproteins, a hemagglutinin (HA) polypeptide and a neuraminidase
(NA) polypeptide, which are are displayed on the surface of the
virus. There are 17 HA antigens, denoted H1-17, and nine different
NA antigens, denoted N1-9.
[0063] In such embodiments the antigen or vaccine comprises an
influenza antigen, e.g., an influenza A or B antigen. In an
embodiment the antigen comprises an HA antigen, e.g., any of H1-17.
In an embodiment the antigen is selected from H1N1, H2N2, H3N2,
H5N1, H7N7, H1N2, H9N2, H7N2 H7N3, H10N7, or H7N9.
[0064] In an embodiment, the antigen is selected from H1N1, H2N3,
and B influenza subtypes. In an embodiment, the antigen is a
pneumococcal antigen.
[0065] In an embodiment, the antigen and the mTOR inhibitor are
co-administered. In an embodiment, the antigen and the mTOR
inhibitor are administered sequentially. In an embodiment, the
subject is less than 65 years old.
[0066] In an embodiment, a relatively low baseline or
pre-immunization level or titer of influenza antibody is predictive
of a greater mTOR inhibitor-, e.g., RAD001-, associated increase in
antibody titer for the influenza virus, e.g., an influenza A virus.
In an embodiment, the subject is evaluated for anti-influenza
antibody titer prior to administration of an antigen or vaccine. In
an embodiment, evaluation comprises acquiring, e.g., directly or
indirectly acquiring, a measurement of anti-influenza antibody
titer. The titer of antibody can be compared with a reference
value. Relatively low titer, e.g., titer at or below a reference
value, e.g., less than or equal to a titer of 1:40 (e.g., as
measured herein), is indicative of a greater mTOR inhibitor-, e.g.,
RAD001-, associated increase in antibody titer. Thus, baseline or
pre-immunization titer can be used to select patients for low,
immune enhancing, dose of mTOR inhibitor, e.g., in combination with
vaccination or administration of antigen to protect against
influenza, e.g., influenza A. In an embodiment, responsive to a
determined antibody titer, a subject is classified as to the
likelihood of benefiting from administration of a low, immune
enhancing, dose of mTOR inhibitor, e.g., prior to or with
administration of a vaccine or antigen. In an embodiment,
responsive to a determined antibody titer, e.g., a titer that is at
or below a reference value, a subject is selected for, or
administered, a low, immune enhancing, dose of mTOR inhibitor,
prior to or with administration of a vaccine or antigen. In an
embodiment, responsive to a determined antibody titer, e.g., a
titer that is above a reference value, a subject is selected for,
or administered an alternative therapy, e.g., administration of a
vaccine or antigen without the administration of a low, immune
enhancing, dose of mTOR inhibitor.
[0067] In an embodiment, the subject does not receive a vaccine,
e.g., does not receive a vaccine while the mTOR inhibitor is
present at levels which promote the immune response. In an
embodiment, the vaccine is an anti-cancer vaccine or a vaccine
against an infectious agent. In an embodiment the vaccine is a
therapeutic vaccine for a neurological disorder, e.g.,
Alzheimers.
[0068] In an embodiment, the subject does not receive a vaccine,
e.g., a cancer vaccine, within 10, 20, 30, 40, 50, 60, 70, 80, or
90 days prior to initiation of the low, immune enhancing, dose of
the mTOR inhibitor.
[0069] In an embodiment, the subject does not receive a vaccine,
e.g., a cancer vaccine, within 10, 20, 30, 40, 50, 60, 70, 80, or
90 days after initiation of the low, immune enhancing, dose of the
mTOR inhibitor.
[0070] In an embodiment, the low, immune enhancing, dose of a mTOR
inhibitor is administered at the time of, or after vaccination. In
an embodiment, the low, immune enhancing, dose of a mTOR inhibitor
is administered within 24, 10, 5, 4, 3, 2, or 1 hour, prior to, at
the time of, or after the vaccination.
[0071] In another aspect, the invention features, a method of
evaluating a subject for treatment with a low, immune enhancing,
dose of mTOR inhibitor, e.g., to promote or enhance an immune
response to an influenza vaccine or antigen, comprising:
[0072] determining if the baseline or pre-immunization titer of
anti-influenza antibody of the subject is equal to or less than
1:40; and
[0073] responsive to the determination, classifying the subject,
e.g., as to the likelihood of benefiting from a low, immune
enhancing, dose of RAD001, or selecting a course of therapy for
said subject.
[0074] In an embodiment, determining comprises directly acquiring
the antibody titer.
[0075] In an embodiment, determining comprises indirectly acquiring
the antibody titer.
[0076] In an embodiment, the antibody titer is equal to or less
than 1:40 and the subject is classified as likely to benefit from a
low, immune enhancing, dose of RAD001.
[0077] In an embodiment the antibody titer is equal to or less than
1:40 and the subject is administered a low, immune enhancing, dose
of RAD001.
[0078] In an embodiment the subject is administered an influenza
vaccine or antigen.
[0079] In an embodiment the antibody titer is greater than 1:40 and
the subject is classified as not likely to benefit from a low,
immune enhancing, dose of RAD001.
[0080] In another aspect, the invention features a vaccine or
vaccine composition comprising a low, immune enhancing, dose of an
mTOR inhibitor described herein, e.g., RAD001 or rapamycin, and an
antigen.
[0081] In an embodiment, the vaccine or vaccine composition
comprises a vaccine antigen, and about 0.005 mg to 1.5 mg of the
mTOR inhibitor RAD001, or a bioequivalent dose of a different mTOR
inhibitor.
[0082] In an embodiment, the vaccine or vaccine composition
comprises about 0.01-1 mg, about 0.01-0.7 mg, about 0.01-0.5 mg, or
about 0.1-0.5 mg of RAD001, or a bioequivalent dose of a different
mTOR inhibitor.
[0083] In an embodiment, the composition comprises about 0.5 mg of
RAD001 or a bioequivalent dose of a different mTOR inhibitor.
[0084] In an embodiment, the composition comprises an amount of an
mTOR inhibitor sufficient to inhibit P70 S6 kinase activity by no
greater than 80% in a subject to which said composition is
administered.
[0085] In an embodiment, the composition comprises an amount of an
mTOR inhibitor sufficient to inhibit P70 S6 kinase activity by no
greater than 38% in a subject to which said composition is
administered.
[0086] In an embodiment, the composition produces at least a 1.2
fold increase in immune response as compared to placebo in a
subject to which said composition is administered.
[0087] In an embodiment, the mTOR inhibitor is a rapamycin.
[0088] In an embodiment, the mTOR inhibitor is a rapalog.
[0089] In an embodiment, the vaccine antigen is derived from
influenza.
[0090] In an embodiment, the vaccine antigen is selected from the
group consisting of H1N1, H2N3, and B influenza subtypes.
[0091] In an embodiment, the vaccine antigen is derived from
pneumococcus.
[0092] In another aspect, the invention features, an adjuvant, or
adjuvant composition or compound, comprising a low, immune
enhancing, dose of an mTOR inhibitor described herein, e.g., RAD001
or rapamycin.
[0093] In an embodiment, a vaccine adjuvant comprises about 0.005
mg to 1.5 mg of the mTOR inhibitor RAD001, or a bioequivalent dose
of a different mTOR inhibitor.
[0094] In an embodiment, a vaccine adjuvant comprises an amount of
an mTOR inhibitor sufficient to inhibit P70 S6 kinase activity in a
cell by no greater than 80%. In another embodiment, a vaccine
adjuvant comprises an amount of an mTOR inhibitor sufficient to
inhibit P70 S6 kinase activity in a cell by no greater than
38%.
[0095] In an embodiment, the vaccine adjuvant comprises an mTOR
inhibitor, wherein the mTOR inhibitor is a rapamycin.
[0096] In an embodiment, the vaccine adjuvant comprises an mTOR
inhibitor, wherein the mTOR inhibitor is a rapalog.
[0097] In an embodiment, the vaccine adjuvant comprises about
0.01-1 mg, 0.01-0.7 mg, 0.01-0.5 mg, or 0.1-0.5 mg of RAD001 or a
bioequivalent dose of a different mTOR inhibitor.
[0098] In an embodiment, the vaccine adjuvant comprises about 0.5
mg of RAD001 or a bioequivalent dose of a different mTOR
inhibitor.
[0099] In an aspect, the invention features a method of collecting
immune effector cells, e.g., T cells, or preparing a mammal, e.g.,
a primate, e.g., a human, for collection of T cells to form a
preparation of immune effector cells, T cells, wherein the method
comprises: administering to the subject a low, immune enhancing
dose, of an mTOR inhibitor, e.g., RAD001, or rapamycin, for an
amount of time sufficient to decrease the proportion of PD-1
positive immune effector cells, e.g., T cells or increase the
proportion of PD-1 negative immune effector cells, e.g., T cells,
in the mammal or in a preparation of immune effector cells, e.g., T
cells, collected from the mammal.
[0100] In an embodiment the method comprises collecting the immune
effector cells, e.g., T cells. In an embodiment the method
comprises forming an immune effector cell preparation, e.g., a T
cell preparation.
[0101] In an embodiment, the immune effector cells are T cells. In
an embodiment, the T cells are CD4-expressing (CD4+ or CD4) T
cells. In an embodiment, the T cells are CD8-expressing (CD8+ or
CD8) T cells. In an embodiment, the T cells comprise a plurality of
CD4+ T cells and CD8+ T cells.
[0102] In an embodiment, the method of collecting immune effector
cells further comprises evaluating the level of PD1 negative or PD1
positive immune effector cells, e.g., T cells, in the subject or in
T cells taken from the subject.
[0103] In an embodiment, the method of collecting immune effector
cells further comprises collecting T cells to form the preparation
of T cells.
[0104] In an embodiment, the method of collecting immune effector
cells further comprises providing a preparation of T cells.
[0105] In an embodiment, the administering to the subject a low,
immune enhancing dose, of an mTOR inhibitor is initiated at least
1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 days prior to collection of T
cells.
[0106] In an embodiment, the administering to the subject a low,
immune enhancing dose, of an mTOR inhibitor is initiated at least
30, 60, 90 or 120 days prior to collection of T cells.
[0107] In an embodiment, collection of the T cells is performed
within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, or 90, days after
the last administration of a low, immune enhancing dose, of an mTOR
inhibitor.
[0108] In an embodiment, the administering to the subject a low,
immune enhancing dose, of an mTOR inhibitor results in the partial,
but not total, inhibition of mTOR for at least at least 1, 2, 3, 4,
5, 10, 15, 20, 25, or 30 days prior to collection of T cells to
form a preparation of T cells from the mammal.
[0109] In an embodiment, collection of the T cells is performed
within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, or 90, days after
a determination has been made that there is partial inhibition of
mTOR in the subject.
[0110] In an embodiment, collection of the T cells is performed
within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, or 90, days after
onset of partial mTOR inhibition in the subject.
[0111] In an embodiment, the preparation of T cells comprises a
PD-1 negative T cell.
[0112] In an embodiment, at least 80-90% of the T cells collected
are PD-1 negative.
[0113] In an embodiment, no more than 10-20% of the T cells
collected are PD-1 positive.
[0114] In an embodiment, the mTOR inhibitor is an allosteric mTOR
inhibitor. In an embodiment, the mTOR inhibitor is a RAD001. In an
embodiment, the mTOR inhibitor is rapamycin.
[0115] In an embodiment, the mTOR inhibitor is a catalytic
inhibitor, e.g., a kinase inhibitor. In an embodiment, the kinase
inhibitor is selective for mTOR. In an embodiment, the kinase
inhibitor is selected from BEZ235 and CCG168.
[0116] In an embodiment, the low, immune enhancing, dose comprises
a plurality of mTOR inhibitors. In an embodiment, the dose
comprises an allosteric and a catalytic mTOR inhibitor.
[0117] In an embodiment, the low, immune enhancing, dose of an mTOR
inhibitor is administered for an amount of time sufficient for one
or more of the following to occur: [0118] i) a decrease in the
number of PD-1 positive immune effector cells; [0119] ii) an
increase in the number of PD-1 negative immune effector cells;
[0120] iii) an increase in the ratio of PD-1 negative immune
effector cells/PD-1 positive immune effector cells; [0121] iv) an
increase in the number of naive T cells; [0122] v) an increase in
the expression of one or more of the following markers:
CD62L.sup.high, CD127.sup.high, CD27.sup.+, and BCL2, e.g., on
memory T cells, e.g., memory T cell precursors; [0123] vi) a
decrease in the expression of KLRG1, e.g., on memory T cells, e.g.,
memory T cell precursors; or [0124] vii) an increase in the number
of memory T cell precursors, e.g., cells with any one or
combination of the following characteristics: increased
CD62L.sup.high increased CD127.sup.high, increased CD27.sup.+,
decreased KLRG1, and increased BCL2; [0125] and wherein i), ii),
iii), iv), v), vi), or vii) occurs e.g., at least transiently,
e.g., as compared to a non-treated subject.
[0126] In an embodiment, the method of treating, e.g., promoting an
immune response in, a subject, e.g., a human subject, comprises
inhibiting a negative immune response mediated by the engagement of
PD-1 with PD-L1 or PD-L2.
[0127] In an embodiment, the method of treating, e.g., promoting an
immune response in, a subject, e.g., a human subject, comprises
increasing the number of T cells capable of proliferation.
[0128] In an embodiment, the method of treating, e.g., promoting an
immune response in, a subject, e.g., a human subject, comprises
increasing the number of T cells capable of cytotoxic function,
secreting cytokines, or activation.
[0129] In an embodiment, the method of treating, e.g., promoting an
immune response in, a subject, e.g., a human subject, comprises
increasing the number of T cells capable of providing T cell help
to B cells.
[0130] In an embodiment, the administering of the low, immune
enhancing, dose of an mTOR inhibitor results in the partial, but
not total, inhibition of mTOR for at least 1, 5, 10, 20, 30, or 60
days.
[0131] In an embodiment, the administering of the low, immune
enhancing, dose of an mTOR inhibitor results in the partial, but
not total, inhibition of mTOR as long as enhancement of the immune
response is needed.
[0132] In an embodiment, the low, immune enhancing, dose of an mTOR
inhibitor is associated with mTOR inhibition of at least 5 but no
more than 90%, e.g., as measured by p70 S6K inhibition. In an
embodiment, the mTOR inhibitor comprises RAD001.
[0133] In an embodiment, the low, immune enhancing, dose of an mTOR
inhibitor is associated with mTOR inhibition of at least 10% but no
more than 80%, e.g., as measured by p70 S6K inhibition. In an
embodiment, the mTOR inhibitor comprises RAD001.
[0134] In an embodiment, the low, immune enhancing, dose of an mTOR
inhibitor is associated with mTOR inhibition of at least 10% but no
more than 40%, e.g., as measured by p70 S6K inhibition. In an
embodiment, the mTOR inhibitor comprises RAD001.
[0135] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in an immediate release dosage form, 0.1 to
20, 0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5, mgs of RAD001.
[0136] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per week, in an immediate release dosage form, about 5 mgs of
RAD001.
[0137] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in an immediate release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
one per week, immediate release dosage form of 0.1 to 20, 0.5 to
10, 2.5 to 7.5, 3 to 6, or about 5 mgs of RAD001.
[0138] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per week, in an immediate release dosage form, an amount of an mTOR
inhibitor other than RAD001, that is bioequivalent to a once per
week, immediate release dosage form of about 5 mgs of RAD001.
[0139] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in a sustained release dosage form, 0.3 to 60,
1.5 to 30, 7.5 to 22.5, 9 to 18, or about 15 mgs of RAD001.
[0140] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per week, in a sustained release dosage form, about 15 mgs of
RAD001.
[0141] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in a sustained release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
once per week, sustained release dosage form of 0.3 to 60, 1.5 to
30, 7.5 to 22.5, 9 to 18, or about 15 mgs of RAD001.
[0142] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per week, in a sustained release dosage form, an amount of an mTOR
inhibitor other than RAD001, that is bioequivalent to a once per
week sustained release dosage form of about 15 mgs of RAD001.
[0143] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per day, e.g., in an immediate release dosage form, 0.005 to
1.5, 0.01 to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to 1.5,
0.5 to 1.5, 0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to
0.6, or about 0.5 mgs of RAD001.
[0144] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering once
per day, in an immediate release dosage form, about 0.5 mgs of
RAD001.
[0145] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per day, e.g., in an immediate release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
once per day, immediate release dosage form of 0.005 to 1.5, 0.01
to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to 1.5, 0.5 to 1.5,
0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to 0.6, or
about 0.5 mgs of RAD001.
[0146] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, once
per day, in an immediate release dosage form, an amount of an mTOR
inhibitor other than RAD001, that is bioequivalent to a once per
day, immediate release dosage form of about 0.5 mgs of RAD001.
[0147] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per day, e.g., in a sustained release dosage form, 0.015 to
4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 to 4.5, 0.9 to 4.5, 1.2 to 4.5,
1.5 to 4.5, 1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5, 0.9 to
1.8, or about 1.5 mgs of RAD001.
[0148] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per day, e.g., in a sustained release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
once per day, sustained release dosage form of 0.015 to 4.5, 0.03
to 4.5, 0.3 to 4.5, 0.6 to 4.5, 0.9 to 4.5, 1.2 to 4.5, 1.5 to 4.5,
1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5, 0.9 to 1.8, or
about 1.5 mgs of RAD001.
[0149] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in a sustained release dosage form, 0.1 to 30,
0.2 to 30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30,
14 to 30, 16 to 30, 20 to 30, 6 to 12, or about 10 mgs of
RAD001.
[0150] In an embodiment, administering to the subject a low, immune
enhancing, dose of an mTOR inhibitor comprises administering, e.g.,
once per week, e.g., in a sustained release dosage form, an amount
of an mTOR inhibitor other than RAD001, that is bioequivalent to a
once per week, sustained release dosage form of 0.1 to 30, 0.2 to
30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30, 14 to
30, 16 to 30, 20 to 30, 6 to 12, or about 10 mgs of RAD001.
[0151] In an embodiment, the mTOR inhibitor is RAD001 and the dose
provides for a trough level of RAD001 in a range of between about
between 0.3 or less and 3 ng/ml, or between 0.3 or less and 1
ng/ml.
[0152] In an embodiment, the mTOR inhibitor is other than RAD001
and the dose is bioequivalent to a dose of RAD001 that provides for
a trough level of RAD001 in a range of between about between 0.3 or
less and 3 ng/ml, or between 0.3 or less and 1 ng/ml.
[0153] In an embodiment, the subject has cancer and the method
comprises promoting the subject's immune response to the cancer. In
an embodiment, the subject was selected on the basis of having
cancer. In an embodiment, a cell of the cancer expresses PD-L1 or
PD-L2. In an embodiment, a cell in the cancer microenvironment
expresses PD-L1 or PD-L2.
[0154] In an embodiment, the cancer comprises a solid tumor. In an
embodiment, the cancer is a hematological cancer. In an embodiment,
the cancer is a leukemia. In an embodiment, the cancer is melanoma.
In an embodiment, the cancer is selected from Table 1.
[0155] In an embodiment, the subject is immunocompromised. In an
embodiment, the subject is HIV+ or has AIDs. In an embodiment, the
subject has an infectious disease.
[0156] In an embodiment, the subject has an infectious disease,
e.g., hepatitis, e.g., hepatitis A, B or C. In an embodiment, the
subject has an infectious disease, but is not otherwise
immunocompromised, e.g, is not immunosenescent.
[0157] In an embodiment, the subject has an impaired immune
response. In an embodiment, the subject is immunoscenescent.
[0158] In an embodiment, the subject is infected with a virus,
bacteria, protozoan, microbe, pathogen, or parasite.
[0159] In an embodiment, the subject has an age related
condition.
[0160] In an embodiment, the subject is less than 65 years old.
[0161] In an embodiment, the subject does not receive a vaccine,
e.g., does not receive a vaccine while the mTOR inhibitor is
present at levels which promote the immune response. In an
embodiment, the vaccine is an anti-cancer vaccine or a vaccine
against an infectious agent. In an embodiment the vaccine is a
therapeutic vaccine for a neurological disorder, e.g., Alzheimers
disease.
[0162] In an embodiment, the subject does not receive a vaccine,
e.g., a cancer vaccine, within 10, 20, 30, 40, 50, 60, 70, 80, or
90 days prior to initiation of the low, immune enhancing, dose of
the mTOR inhibitor.
[0163] In an embodiment, the subject does not receive a vaccine,
e.g., a cancer vaccine, within 10, 20, 30, 40, 50, 60, 70, 80, or
90 days after initiation of the low, immune enhancing, dose of the
mTOR inhibitor.
[0164] In an embodiment, the low, immune enhancing, dose of an mTOR
inhibitor is administered at the time of, or after vaccination.
[0165] In an aspect, a preparation of human T cells, e.g., as made
by a method described herein, enriched for PD-1 negative T cells is
provided herein. In an embodiment, the subject has cancer or is
immunocompromised.
[0166] In another aspect, the invention features, a preparation of
T cells, e.g., human T cells, achievable by, or which could be made
by, practice of a method described herein.
[0167] In another aspect, the invention features a unit dosage
form, composition, or formulation, of an mTOR inhibitor, e.g.,
RAD001, e.g., a dosage form suitable for oral administration.
Embodiments are described herein, e.g., in the section below
entitled "Low-DOSE mTOR INHIBITORS". Unit dosage forms or
compositions can be provided as immediate or sustained release
formulations, se, e.g., the sections below entitled "PHARMACEUTICAL
COMPOSITIONS" and "SUSTAINED RELEASE."
BRIEF DESCRIPTION OF THE DRAWINGS
[0168] FIGS. 1A and 1B are graphs showing an increase in titers to
influenza vaccine strains as compared to placebo. In FIG. 1A, the
increase above baseline in influenza geometric mean titers to each
of the 3 influenza vaccine strains (H1N1 A/California/07/2009, H3N2
A/Victoria/210/2009, B/Brisbane/60/2008) relative to the increase
in the placebo cohort 4 weeks after vaccination is shown for each
of the RAD001 dosing cohorts in the intention to treat population.
The bold black line indicates the 1.2 fold increase in titers
relative to placebo that is required to be met for 2 out of 3
influenza vaccine strains to meet the primary endpoint of the
study. The star "*" indicates that the increase in GMT titer
relative to placebo exceeds 1 with posterior probability of at
least 80%. FIG. 1B is a graph of the same data as in FIG. 1A for
the subset of subjects with baseline influenza titers
<=1:40.
[0169] FIG. 2 shows a scatter plot of RAD001 concentration versus
fold increase in geometric mean titer to each influenza vaccine
strain 4 weeks after vaccination. RAD001 concentrations (1 hour
post dose) were measured after subjects had been dosed for 4 weeks.
All subjects who had pharmacokinetic measurements were included in
the analysis set. The fold increase in geometric mean titers at 4
weeks post vaccination relative to baseline is shown on the y
axis.
[0170] FIG. 3 is a graphic representation showing increase in
titers to heterologous influenza strains as compared to placebo.
The increase above baseline in influenza geometric mean titers to 2
heterologous influenza strains (A/H1N1 strain A/New Jersey/8/76 and
A/H3N2 strain A/Victoria/361/11) not contained in the influenza
vaccine relative to the increase in the placebo cohort 4 weeks
after vaccination is shown for each of the RAD001 dosing cohorts in
the intention to treat population. * indicates increase in titer
relative to placebo exceeds 1 with a posterior probability of at
least 80%.
[0171] FIGS. 4A and 4B are graphic representations of IgG and IgM
levels before and after influenza vaccination. Levels of
anti-A/H1N1/California/07/2009 influenza IgG and IgM were measured
in serum obtained from subjects before and 4 weeks post influenza
vaccination. No significant difference in the change from baseline
to 4 weeks post vaccination in anti-H1N1 influenza IgG and IgM
levels were detected between the RAD001 and placebo cohorts (all p
values >0.05 by Kruskal-Wallis rank sum test).
[0172] FIGS. 5A, 5B, and 5C are graphic representations of the
decrease in percent of PD-1-positive CD4 and CD8 and increase in
PD-1-negative CD4 T cells after RAD001 treatment. The percent of
PD-1-positive CD4, CD8 and PD-1-negative CD4 T cells was determined
by FACS analysis of PBMC samples at baseline, after 6 weeks of
study drug treatment (Week 6) and 6 weeks after study drug
discontinuation and 4 weeks after influenza vaccination (Week 12).
FIG. 5A shows there was a significant decrease (-37.1--28.5%) in
PD-1-positive CD4 T cells at week 12 in cohorts receiving RAD001 at
dose levels 0.5 mg/Day (n=25), 5 mg/Week (n=29) and 20 mg/Week
(n=30) as compared to the placebo cohort (n=25) with p=0.002
(0.02), p=0.003 (q=0.03), and p=0.01 (q=0.05) respectively. FIG. 5B
shows there was a significant decrease (-43.3--38.5%) in
PD-1-positive CD8 T cells at week 12 in cohorts receiving RAD001
(n=109) at dose levels 0.5 mg/Day (n=25), 5 mg/Week (n=29) and 20
mg/Week (n=30) as compared to the placebo cohort (n=25) with p=0.01
(0.05), p=0.007 (q=0.04), and p=0.01 (q=0.05) respectively. FIG. 5C
shows was a significant increase (3.0-4.9%) in PD-1-negative CD4 T
cells at week 12 in cohorts receiving RAD001 (n=109) at dose levels
0.5 mg/Day (n=25), 5 mg/Week (n=29) and 20 mg/Week (n=30) as
compared to the placebo cohort (n=25) with p=0.0007 (0.02), p=0.03
(q=0.07), and p=0.03 (q=0.08) respectively.
[0173] FIGS. 6A and 6B are graphic representations of the decrease
in percent of PD-1-positive CD4 and CD8 and increase in
PD-1-negative CD4 T cells after RAD001 treatment adjusted for
differences in baseline PD-1 expression. The percent of
PD-1-positive CD4, CD8 and PD-1-negative CD4 T cells was determined
by FACS analysis of PBMC samples at baseline, after 6 weeks of
study drug treatment (Week 6) and 6 weeks after study drug
discontinuation and 4 weeks after influenza vaccination (Week 12).
FIG. 6A shows a significant decrease of 30.2% in PD-1+CD4 T cells
at week 6 in the pooled RAD cohort (n=84) compared to placebo
cohort (n=25) with p=0.03 (q=0.13). The decrease in PD-1-positive
CD4 T cells at week 12 in the pooled RAD as compared to the placebo
cohort is 32.7% with p=0.05 (q=0.19). FIG. 6B shows a significant
decrease of 37.4% in PD-1-positive CD8 T cells at week 6 in the
pooled RAD001 cohort (n=84) compared to placebo cohort (n=25) with
p=0.008 (q=0.07). The decrease in PD-1-positive CD8 T cells at week
12 in the pooled RAD001 as compared to the placebo cohort is 41.4%
with p=0.066 (q=0.21). FIGS. 6A and 6B represent the data in FIGS.
5A, 5B, and 5C but with the different RAD001 dosage groups of FIGS.
5A, 5B, and 5C pooled into the single RAD001-treated group in FIGS.
6A and 6B.
[0174] FIG. 7 depicts increases in exercise and energy in elderly
subjects in response to RAD001.
[0175] FIGS. 8A and 8B depict the predicted effect of RAD001 on P70
S6K activity in cells.
[0176] FIG. 8A depicts P70 S6 kinase inhibition with higher doses
of weekly and daily RAD001; FIG. 8B depicts P70 S6 kinase
inhibition with lower doses of weekly RAD001.
DETAILED DESCRIPTION
Definitions
[0177] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains.
[0178] The term "a" and "an" refers to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
By way of example, "an element" means one element or more than one
element.
[0179] The term "about" when referring to a measurable value such
as an amount, a temporal duration, and the like, refers to
variations of .+-.20% or in some instances .+-.10%, or in some
instances .+-.5%, or in some instances .+-.1%, or in some instances
.+-.0.1% from the specified value, as such variations are
appropriate to perform the disclosed methods.
[0180] The term "adjuvant" refers to a compound that, when used in
combination with a specific immunogen, e.g., a vaccine immunogen,
in a formulation, augments or otherwise alters, modifies or
enhances the resultant immune responses.
[0181] The term "anti-tumor effect" refers to a biological effect
which can be manifested by various means, including but not limited
to, e.g., a decrease in tumor volume, a decrease in the number of
tumor cells, a decrease in the number of metastases, an increase in
life expectancy, decrease in tumor cell proliferation, decrease in
tumor cell survival, or amelioration of various physiological
symptoms associated with the cancerous condition. An "anti-tumor
effect" can also be manifested by the ability of the compounds
(e.g., mTOR inhibitors), peptides, polynucleotides, cells and
antibodies of the invention in prevention of the occurrence of
tumor in the first place.
[0182] The term "antibody," refers to a protein, or polypeptide
sequence derived from an immunoglobulin molecule which specifically
binds with an antigen. Antibodies can be polyclonal or monoclonal,
multiple or single chain, or intact immunoglobulins, and may be
derived from natural sources or from recombinant sources.
Antibodies can be tetramers of immunoglobulin molecules.
[0183] The term "antibody fragment" refers to at least one portion
of an intact antibody, or recombinant variants thereof, and refers
to the antigen binding domain, e.g., an antigenic determining
variable region of an intact antibody, that is sufficient to confer
recognition and specific binding of the antibody fragment to a
target, such as an antigen. Examples of antibody fragments include,
but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv
antibody fragments, linear antibodies, single domain antibodies
such as sdAb (either VL or VH), camelid VHH domains, and
multi-specific antibodies formed from antibody fragments. The term
"scFv" refers to a fusion protein comprising at least one antibody
fragment comprising a variable region of a light chain and at least
one antibody fragment comprising a variable region of a heavy
chain, wherein the light and heavy chain variable regions are
contiguously linked via a short flexible polypeptide linker, and
capable of being expressed as a single chain polypeptide, and
wherein the scFv retains the specificity of the intact antibody
from which it is derived. Unless specified, as used herein an scFv
may have the VL and VH variable regions in either order, e.g., with
respect to the N-terminal and C-terminal ends of the polypeptide,
the scFv may comprise VL-linker-VH or may comprise
VH-linker-VL.
[0184] The term "antibody heavy chain," refers to the larger of the
two types of polypeptide chains present in antibody molecules in
their naturally occurring conformations, and which normally
determines the class to which the antibody belongs.
[0185] The term "antibody light chain," refers to the smaller of
the two types of polypeptide chains present in antibody molecules
in their naturally occurring conformations. Kappa (.kappa.) and
lambda (.lamda.) light chains refer to the two major antibody light
chain isotypes.
[0186] The term "antigen" or "Ag" refers to a molecule that
provokes an immune response. This immune response may involve
either antibody production, or the activation of specific
immunologically-competent cells, or both. The skilled artisan will
understand that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen. Furthermore, antigens can be
derived from recombinant or genomic DNA. A skilled artisan will
understand that any DNA, which comprises a nucleotide sequences or
a partial nucleotide sequence encoding a protein that elicits an
immune response therefore encodes an "antigen" as that term is used
herein. Furthermore, one skilled in the art will understand that an
antigen need not be encoded solely by a full length nucleotide
sequence of a gene. It is readily apparent that the present
invention includes, but is not limited to, the use of partial
nucleotide sequences of more than one gene and that these
nucleotide sequences are arranged in various combinations to encode
polypeptides that elicit the desired immune response. Moreover, a
skilled artisan will understand that an antigen need not be encoded
by a "gene" at all. It is readily apparent that an antigen can be
generated synthesized or can be derived from a biological sample,
or might be macromolecule besides a polypeptide. Such a biological
sample can include, but is not limited to a tissue sample, a tumor
sample, a cell or a fluid with other biological components.
[0187] The term "antigen presenting cell" or "APC" refers to an
immune system cell such as an accessory cell (e.g., a B-cell, a
dendritic cell (DC), and the like) that displays a foreign antigen
complexed with major histocompatibility complexes (MHC's) on its
surface. T-cells may recognize these complexes using their T-cell
receptors (TCRs). APCs process antigens and present them to
T-cells.
[0188] The term "bioequivalent" refers to an amount of an agent
other than the reference compound (e.g., RAD001), required to
produce an effect equivalent to the effect produced by the
reference dose or reference amount of the reference compound (e.g.,
RAD001). In an embodiment the effect is the level of mTOR
inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as
evaluated in an in vivo or in vitro assay, e.g., as measured by an
assay described herein, e.g., the Boulay assay, or measurement of
phosphorylated S6 levels by western blot. In an embodiment, the
effect is alteration of the ratio of PD-1 positive/PD-1 negative T
cells, as measured by cell sorting. In an embodiment a
bioequivalent amount or dose of an mTOR inhibitor is the amount or
dose that achieves the same level of P70 S6 kinase inhibition as
does the reference dose or reference amount of a reference
compound. In an embodiment, a bioequivalent amount or dose of an
mTOR inhibitor is the amount or dose that achieves the same level
of alteration in the ratio of PD-1 positive/PD-1 negative T cells
as does the reference dose or reference amount of a reference
compound.
[0189] The term "cancer" refers to a disease characterized by the
rapid and uncontrolled growth of aberrant cells. Cancer cells can
spread locally or through the bloodstream and lymphatic system to
other parts of the body. Examples of various cancers are described
herein and include but are not limited to, breast cancer, prostate
cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic
cancer, colorectal cancer, renal cancer, liver cancer, brain
cancer, lymphoma, leukemia, lung cancer and the like. In an
embodiment, a cancer is characterized by expression of a PD-1
ligand, e.g., PD-L1 or PD-L2, on a cancer cell or in a tumor
microenvironment. The term "cancer" is refers to all types of
cancerous growths or oncogenic processes, metastatic tissues or
malignantly transformed cells, tissues, or organs, irrespective of
histopathologic type or stage of invasiveness.
[0190] The terms "co-administration" or "combined administration"
or the like as utilized herein are meant to encompass
administration of the selected therapeutic agents to a single
patient, and are intended to include treatment regimens in which
the agents are not necessarily administered by the same route of
administration or at the same time. In one aspect of the methods
described herein, an mTOR inhibitor and an antigen may be
co-administered.
[0191] The term "effective amount" or "therapeutically effective
amount" are used interchangeably herein, and refer to an amount of
a compound, formulation, material, or composition, as described
herein effective to achieve a particular biological result.
[0192] The term "encoding" refers to the inherent property of
specific sequences of nucleotides in a polynucleotide, such as a
gene, a cDNA, or an mRNA, to serve as templates for synthesis of
other polymers and macromolecules in biological processes having
either a defined sequence of nucleotides (e.g., rRNA, tRNA and
mRNA) or a defined sequence of amino acids and the biological
properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes
a protein if transcription and translation of mRNA corresponding to
that gene produces the protein in a cell or other biological
system. Both the coding strand, the nucleotide sequence of which is
identical to the mRNA sequence and is usually provided in sequence
listings, and the non-coding strand, used as the template for
transcription of a gene or cDNA, can be referred to as encoding the
protein or other product of that gene or cDNA.
[0193] The term "endogenous" refers to any material from or
produced inside an organism, cell, tissue or system.
[0194] The term "exogenous" refers to any material introduced from
or produced outside an organism, cell, tissue or system.
[0195] The term "expression" refers to the transcription and/or
translation of a particular nucleotide sequence driven by a
promoter.
[0196] The term "fully human" refers to an immunoglobulin, such as
an antibody or antibody fragment, where the whole molecule is of
human origin or consists of an amino acid sequence identical to a
human form of the antibody or immunoglobulin.
[0197] The term "homologous" or "identity" refers to the subunit
sequence identity between two polymeric molecules, e.g., between
two nucleic acid molecules, such as, two DNA molecules or two RNA
molecules, or between two polypeptide molecules. When a subunit
position in both of the two molecules being compared is occupied by
the same monomeric subunit; e.g., if a position in each of two DNA
molecules is occupied by adenine, then they are homologous or
identical at that position. The homology between two sequences is a
direct function of the number of matching or homologous positions;
e.g., if half (e.g., five positions in a polymer ten subunits in
length) of the positions in two sequences are homologous, the two
sequences are 50% homologous; if 90% of the positions (e.g., 9 of
10), are matched or homologous, the two sequences are 90%
homologous.
[0198] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. For the most part, humanized
antibodies and antibody fragments thereof are human immunoglobulins
(recipient antibody or antibody fragment) in which residues from a
complementary-determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, a
humanized antibody/antibody fragment can comprise residues which
are found neither in the recipient antibody nor in the imported CDR
or framework sequences. These modifications can further refine and
optimize antibody or antibody fragment performance. In general, the
humanized antibody or antibody fragment thereof will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or a
significant portion of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody or antibody
fragment can also comprise at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. For
further details, see Jones et al., Nature, 321: 522-525, 1986;
Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op.
Struct. Biol., 2: 593-596, 1992.
[0199] The terms "immunosenescence or immunosenescent" refer to a
decrease in immune function resulting in impaired immune response,
e.g., to cancer, vaccination, infectious pathogens, among others.
It involves both the host's capacity to respond to infections and
the development of long-term immune memory, especially by
vaccination. This immune deficiency is ubiquitous and found in both
long- and short-lived species as a function of their age relative
to life expectancy rather than chronological time. It is considered
a major contributory factor to the increased frequency of morbidity
and mortality among the elderly. Immunosenescence is not a random
deteriorative phenomenon, rather it appears to inversely repeat an
evolutionary pattern and most of the parameters affected by
immunosenescence appear to be under genetic control.
Immunosenescence can also be sometimes envisaged as the result of
the continuous challenge of the unavoidable exposure to a variety
of antigens such as viruses and bacteria. Immunosenescence is a
multifactorial condition leading to many pathologically significant
health problems, e.g., in the aged population. Age-dependent
biological changes such as depletion of hematopoietic stem cells,
an increase in PD1+ lymphocytes, a decline in the total number of
phagocytes and NK cells and a decline in humoral immunity
contribute to the onset of immunosenescence. In one aspect,
immunosenescence can be measured in an individual by measuring
telomere length in immune cells (See, e.g., U.S. Pat. No.
5,741,677). Immunosenescence can also be determined by documenting
in an individual a lower than normal number of naive CD4 and/or CD8
T cells, T cell repertoire, the number of PD1-expressing T cells,
e.g., a lower than normal number of PD-1 negative T cells, or
response to vaccination in a subject greater than or equal to 65
years of age.
[0200] The term "impaired immune response" refers to a state in
which a subject does not have an appropriate immune response, e.g.,
to cancer, vaccination, pathogen infection, among others. In some
embodiments, a subject having an impaired immune response is
predicted not to get protective antibody titer levels following
prophylactic vaccination, or in which a subject does not have a
decrease in disease burden after therapeutic vaccination. A subject
can also have an impaired immune response if the subject is a
member of a population known to have decreased immune function or
that has a history of decreased immune function such as the
elderly, subjects undergoing chemotherapy treatment, asplenic
subjects, immunocompromised subjects, or subjects having HIV/AIDS.
Methods described herein allow for the treatment of an impaired
immune response by administration of a low, immune enhancing, dose
of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, such as
RAD001.
[0201] The term "isolated" refers to altered or removed from the
natural state. For example, a nucleic acid or a peptide naturally
present in a living animal is not "isolated," but the same nucleic
acid or peptide partially or completely separated from the
coexisting materials of its natural state is "isolated." An
isolated nucleic acid or protein can exist in substantially
purified form, or can exist in a non-native environment such as,
for example, a host cell.
[0202] The term "low, immune enhancing, dose" when used in
conjunction with an mTOR inhibitor, e.g., an allosteric mTOR
inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR
inhibitor, refers to a dose of mTOR inhibitor that partially, but
not fully, inhibits mTOR activity, e.g., as measured by the
inhibition of P70 S6 kinase activity. Methods for evaluating mTOR
activity, e.g., by inhibition of P70 S6 kinase, are discussed
herein. The dose is insufficient to result in complete immune
suppression but is sufficient to enhance the immune response. In an
embodiment, the low, immune enhancing, dose of mTOR inhibitor
results in a decrease in the number of PD-1 positive T cells and/or
an increase in the number of PD-1 negative T cells, or an increase
in the ratio of PD-1 negative T cells/PD-1 positive T cells. In an
embodiment, the low, immune enhancing, dose of mTOR inhibitor
results in an increase in the number of naive T cells. In an
embodiment, the low, immune enhancing, dose of mTOR inhibitor
results in one or more of the following:
[0203] an increase in the expression of one or more of the
following markers: CD62L.sup.high, CD127.sup.high, CD27.sup.+, and
BCL2, e.g., on memory T cells, e.g., memory T cell precursors;
[0204] a decrease in the expression of KLRG1, e.g., on memory T
cells, e.g., memory T cell precursors; and
[0205] an increase in the number of memory T cell precursors, e.g.,
cells with any one or combination of the following characteristics:
increased CD62L.sup.high increased CD127.sup.high, increased
CD27.sup.+, decreased KLRG1, and increased BCL2;
wherein any of the changes described above occurs, e.g., at least
transiently, e.g., as compared to a non-treated subject.
[0206] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
90%, at least 10 but no more than 90%, at least 15, but no more
than 90%, at least 20 but no more than 90%, at least 30 but no more
than 90%, at least 40 but no more than 90%, at least 50 but no more
than 90%, at least 60 but no more than 90%, or at least 70 but no
more than 90%.
[0207] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
80%, at least 10 but no more than 80%, at least 15, but no more
than 80%, at least 20 but no more than 80%, at least 30 but no more
than 80%, at least 40 but no more than 80%, at least 50 but no more
than 80%, or at least 60 but no more than 80%.
[0208] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
70%, at least 10 but no more than 70%, at least 15, but no more
than 70%, at least 20 but no more than 70%, at least 30 but no more
than 70%, at least 40 but no more than 70%, or at least 50 but no
more than 70%.
[0209] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
60%, at least 10 but no more than 60%, at least 15, but no more
than 60%, at least 20 but no more than 60%, at least 30 but no more
than 60%, or at least 40 but no more than 60%.
[0210] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
50%, at least 10 but no more than 50%, at least 15, but no more
than 50%, at least 20 but no more than 50%, at least 30 but no more
than 50%, or at least 40 but no more than 50%.
[0211] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
40%, at least 10 but no more than 40%, at least 15, but no more
than 40%, at least 20 but no more than 40%, at least 30 but no more
than 40%, or at least 35 but no more than 40%.
[0212] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 5 but no more than
30%, at least 10 but no more than 30%, at least 15, but no more
than 30%, at least 20 but no more than 30%, or at least 25 but no
more than 30%.
[0213] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but
no more than 20%, at least 1, 2, 3, 4 or 5 but no more than 30%, at
least 1, 2, 3, 4 or 5, but no more than 35, at least 1, 2, 3, 4 or
5 but no more than 40%, or at least 1, 2, 3, 4 or 5 but no more
than 45%.
[0214] In an embodiment, a dose of an mTOR inhibitor is associated
with, or provides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but
no more than 90%.
[0215] As is discussed herein, the extent of mTOR inhibition can be
expressed as the extent of P70 S6K inhibition, e.g., the extent of
mTOR inhibition can be determined by the level of decrease in P70
S6K activity, e.g., by the decrease in phosphorylation of a P70 S6K
substrate. The level of mTOR inhibition can be evaluated by a
method described herein, e.g. by the Boulay assay.
[0216] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The phrase nucleotide sequence that encodes a
protein or a RNA may also include introns to the extent that the
nucleotide sequence encoding the protein may in some version
contain an intron(s).
[0217] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0218] The terms "acquire" or "acquiring" as used herein, refer to
obtaining possession of a physical entity (e.g., a sample), or a
value, e.g., a numerical value, or image, by "directly acquiring"
or "indirectly acquiring" the physical entity or value. "Directly
acquiring" means performing a process (e.g., performing a synthetic
or analytical method, contacting a sample with a detection reagent,
or capturing a signal from a sample) to obtain the physical entity
or value. "Indirectly acquiring" refers to receiving the physical
entity or value from another party or source (e.g., a third party
laboratory that directly acquired the physical entity or value).
Directly acquiring a physical entity includes performing a process
that includes a physical change in a physical substance. Exemplary
changes include making a physical entity from two or more starting
materials, shearing or fragmenting a substance, separating or
purifying a substance, combining two or more separate entities into
a mixture, performing a chemical reaction that includes breaking or
forming a covalent or non-covalent bond. Directly acquiring a value
includes performing a process that includes a physical change in a
sample or another substance, e.g., performing an analytical process
which includes a physical change in a substance, e.g., a sample,
analyte, or reagent (sometimes referred to herein as "physical
analysis"), performing an analytical method, e.g., a method which
includes one or more of the following: separating or purifying a
substance, e.g., an analyte, or a fragment or other derivative
thereof, from another substance; combining an analyte, or fragment
or other derivative thereof, with another substance, e.g., a
buffer, solvent, or reactant; or changing the structure of an
analyte, or a fragment or other derivative thereof, e.g., by
breaking or forming a covalent or non-covalent bond, between a
first and a second atom of the analyte; inducing or collecting a
signal, e.g., a light based signal, e.g., a fluorescent signal, or
by changing the structure of a reagent, or a fragment or other
derivative thereof, e.g., by breaking or forming a covalent or
non-covalent bond, between a first and a second atom of the
reagent. Directly acquiring a value includes methods in which a
computer or detection device, e.g, a scanner is used, e.g., when a
change in electronic state responsive to impingement of a photon on
a detector. Directly acquiring a value includes capturing a signal
from a sample.
[0219] The term "nucleic acid" or "polynucleotide" refers to
deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing
known analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions), alleles, orthologs, SNPs, and
complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98
(1994)).
[0220] The term "parenteral" administration of an immunogenic
composition includes, e.g., subcutaneous (s.c.), intravenous
(i.v.), intramuscular (i.m.), or intrasternal injection,
intratumoral, or infusion techniques.
[0221] The terms "peptide," "polypeptide," and "protein" are used
interchangeably, and refer to a compound comprised of amino acid
residues covalently linked by peptide bonds. A protein or peptide
must contain at least two amino acids, and no limitation is placed
on the maximum number of amino acids that can comprise a protein's
or peptide's sequence. Polypeptides include any peptide or protein
comprising two or more amino acids joined to each other by peptide
bonds. As used herein, the term refers to both short chains, which
also commonly are referred to in the art as peptides, oligopeptides
and oligomers, for example, and to longer chains, which generally
are referred to in the art as proteins, of which there are many
types. "Polypeptides" include, for example, biologically active
fragments, substantially homologous polypeptides, oligopeptides,
homodimers, heterodimers, variants of polypeptides, modified
polypeptides, derivatives, analogs, fusion proteins, among others.
A polypeptide includes a natural peptide, a recombinant peptide, or
a combination thereof.
[0222] "Prodrug", or "pro-drug" refers to a compound that is
processed, in the body of a subject, into a drug. In an embodiment
the processing comprises the breaking or formation of a bond, e.g.,
a covalent bond. Typically, breakage of a covalent bond releases
the drug.
[0223] The term "promote" or "enhance" in the context of an immune
response refers to an increase in immune response, such as an
increase in the ability of immune cells to target and/or kill
cancer cells, to target and/or kill pathogens and pathogen infected
cells, and protective immunity following vaccination, among others.
In some embodiments, protective immunity refers to the presence of
sufficient immune response (such as antibody titers) to protect
against subsequent infection by a pathogen expressing the same
antigen.
[0224] The term "prophylaxis" refers to the prevention of or
protective treatment for a disease or disease state. Prevention may
be complete, e.g., the total absence of a disease or disease state.
The prevention may also be partial, such that the likelihood of the
occurrence of the disease or disease state in a subject is less
likely to occur than had the subject not received the prophylactic
treatment.
[0225] As used herein, the term "rapalog" refers to a small
molecule analog of rapamycin.
[0226] The term "recombinant antibody" refers to an antibody which
is generated using recombinant DNA technology, such as, for
example, an antibody expressed by a bacteriophage or yeast
expression system. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using recombinant DNA or amino acid sequence technology which is
available and well known in the art.
[0227] The term "signal transduction pathway" refers to the
biochemical relationship between a variety of signal transduction
molecules that play a role in the transmission of a signal from one
portion of a cell to another portion of a cell. The phrase "cell
surface receptor" includes molecules and complexes of molecules
capable of receiving a signal and transmitting signal across the
membrane of a cell.
[0228] The term "specifically binds," refers to an antibody, or a
ligand, which recognizes and binds with a cognate binding partner
(e.g., a molecule present on a T cell) protein present in a sample,
but the antibody or ligand does not substantially recognize or bind
other molecules in the sample.
[0229] The term "subject", refers to any living organisms in which
an immune response can be elicited (e.g., mammals, human). In an
embodiment the subject is a human. A subject may be of any age. In
an embodiment the subject is an elderly human subject, e.g., 65
years of age or older. In an embodiment, a subject is a human
subject who is not an elderly, e.g., less than 65 years of age. In
an embodiment, a subject is a human pediatric subject, e.g., 18
years of age or less. In an embodiment, a subject is an adult
subject, e.g., older than 18 years of age.
[0230] The term "therapeutic" refers to a treatment. A therapeutic
effect is obtained by reduction, suppression, remission, or
eradication of a disease state.
[0231] The term "tumor antigen" or "hyperproliferative disorder
antigen" or "antigen associated with a hyperproliferative disorder"
refers to antigens that are common to specific hyperproliferative
disorders. In certain aspects, the hyperproliferative disorder
antigens of the present invention are derived from, cancers
including but not limited to primary or metastatic melanoma,
thymoma, lymphoma, sarcoma, lung cancer, liver cancer,
non-Hodgkin's lymphoma, non-Hodgkins lymphoma, leukemias, uterine
cancer, cervical cancer, bladder cancer, kidney cancer and
adenocarcinomas such as breast cancer, prostate cancer, ovarian
cancer, pancreatic cancer, and the like.
[0232] "Unit dosage form" as the term is used herein refers to a
dosage suitable for one administration. By way of example a unit
dosage form can be a tablet, a capsule, or an amount of therapeutic
disposed in a delivery device, e.g., a syringe or intravenous drip
bag. In an embodiment a unit dosage form is administered in a
single administration. In an embodiment more than one unit dosage
form, e.g., two tablets, can be administered simultaneously. The
term "vaccine" refers to a composition, such as a suspension or
solution of antigen or antigenic moieties, usually containing an
antigen (e.g., an inactivated infectious agent, or some part of the
infectious agent, a tumor antigen, among others) that is injected
or otherwise introduced into the body to produce active immunity.
The antigen or antigenic moiety making up the vaccine can be a live
or killed microorganism, or a natural product purified from a
microorganism or other cell including, but not limited to tumor
cells, a synthetic product, a genetically engineered protein,
peptide, polysaccharide or similar product or an allergen. The
antigen or antigenic moiety can also be a subunit of a protein,
peptide, polysaccharide or similar product.
[0233] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as
95-99% identity, includes something with 95%, 96%, 97%, 98% or 99%
identity, and includes subranges such as 96-99%, 96-98%, 96-97%,
97-99%, 97-98% and 98-99% identity. This applies regardless of the
breadth of the range.
[0234] The term "preparation of T cells," refers to a preparation
that comprises at least one T cell. In an embodiment it is enriched
for T cell as compared to peripheral blood.
[0235] The term a "substantially purified" cell refers to a cell
that is essentially free of other cell types. A substantially
purified cell also refers to a cell which has been separated from
other cell types with which it is normally associated in its
naturally occurring state. In some instances, a population of
substantially purified cells refers to a homogenous population of
cells. In other instances, this term refers simply to cell that
have been separated from the cells with which they are naturally
associated in their natural state. In some aspects, the cells are
cultured in vitro. In other aspects, the cells are not cultured in
vitro.
mTOR Inhibitors
[0236] As used herein, the term "mTOR inhibitor" refers to a
compound or ligand, or a pharmaceutically acceptable salt thereof,
which inhibits the mTOR kinase in a cell. In an embodiment an mTOR
inhibitor is an allosteric inhibitor. In an embodiment an mTOR
inhibitor is a catalytic inhibitor.
[0237] Allosteric mTOR inhibitors include the neutral tricyclic
compound rapamycin (sirolimus), rapamycin-related compounds, that
is compounds having structural and functional similarity to
rapamycin including, e.g., rapamycin derivatives, rapamycin analogs
(also referred to as rapalogs) and other macrolide compounds that
inhibit mTOR activity.
[0238] Rapamycin is a known macrolide antibiotic produced by
Streptomyces hygroscopicus having the structure shown in Formula
A.
##STR00001##
[0239] See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991)
44: 688; Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113:
7433; U.S. Pat. No. 3,929,992. There are various numbering schemes
proposed for rapamycin. To avoid confusion, when specific rapamycin
analogs are named herein, the names are given with reference to
rapamycin using the numbering scheme of formula A.
[0240] Rapamycin analogs useful in the invention are, for example,
O-substituted analogs in which the hydroxyl group on the cyclohexyl
ring of rapamycin is replaced by OR.sub.1 in which R.sub.1 is
hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl;
e.g. RAD001, also known as, everolimus as described in U.S. Pat.
No. 5,665,772 and WO94/09010 the contents of which are incorporated
by reference. Other suitable rapamycin analogs include those
substituted at the 26- or 28-position. The rapamycin analog may be
an epimer of an analog mentioned above, particularly an epimer of
an analog substituted in position 40, 28 or 26, and may optionally
be further hydrogenated, e.g. as described in U.S. Pat. No.
6,015,815, WO95/14023 and WO99/15530 the contents of which are
incorporated by reference, e.g. ABT578 also known as zotarolimus or
a rapamycin analog described in U.S. Pat. No. 7,091,213, WO98/02441
and WO01/14387 the contents of which are incorporated by reference,
e.g. AP23573 also known as ridaforolimus.
[0241] Examples of rapamycin analogs suitable for use in the
present invention from U.S. Pat. No. 5,665,772 include, but are not
limited to, 40-O-benzyl-rapamycin,
40-O-(4'-hydroxymethyl)benzyl-rapamycin,
40-O-[4'-(1,2-dihydroxyethyl)]benzyl-rapamycin,
40-O-allyl-rapamycin,
40-O-[3'-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2'-en-1'-yl]-rapamycin,
(2'E,4'S)-40-O-(4',5'-dihydroxypent-2'-en-1'-yl)-rapamycin,
40-O-(2-hydroxyl)ethoxycarbonylmethyl-rapamycin,
40-O-(2-hydroxyl)ethyl-rapamycin,
40-O-(3-hydroxyl)propyl-rapamycin,
40-O-(6-hydroxyl)hexyl-rapamycin,
40-O-[2-(2-hydroxyl)ethoxy]ethyl-rapamycin,
40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,
40-O-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin,
40-O-(2-acetoxyl)ethyl-rapamycin,
40-O-(2-nicotinoyloxy)ethyl-rapamycin,
40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin,
40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin,
40-O-[2-(N-methyl-N'-piperazinyl)acetoxy]ethyl-rapamycin,
39-O-desmethyl-39,40-O,O-ethylene-rapamycin,
(26R)-26-dihydro-40-O-(2-hydroxyl)ethyl-rapamycin,
40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin,
40-O-(2-nicotinamidoethyl)-rapamycin,
40-O-(2-(N-methyl-imidazo-2'-ylcarbethoxamido)ethyl)-rapamycin,
40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,
40-O-(2-tolylsulfonamidoethyl)-rapamycin and
40-O-[2-(4',5'-dicarboethoxy-1',2',3'-triazol-1'-yl)-ethyl]-rapamycin.
[0242] Other rapamycin analogs useful in the present invention are
analogs where the hydroxyl group on the cyclohexyl ring of
rapamycin and/or the hydroxy group at the 28 position is replaced
with an hydroxyester group are known, for example, rapamycin
analogs found in U.S. RE44,768, e.g. temsirolimus.
[0243] Other rapamycin analogs useful in the preset invention
include those wherein the methoxy group at the 16 position is
replaced with another substituent, preferably (optionally
hydroxy-substituted) alkynyloxy, benzyl, orthomethoxybenzyl or
chlorobenzyl and/or wherein the mexthoxy group at the 39 position
is deleted together with the 39 carbon so that the cyclohexyl ring
of rapamycin becomes a cyclopentyl ring lacking the 39 position
methyoxy group; e.g. as described in WO95/16691 and WO96/41807 the
contents of which are incorporated by reference. The analogs can be
further modified such that the hydroxy at the 40-position of
rapamycin is alkylated and/or the 32-carbonyl is reduced.
[0244] Rapamycin analogs from WO95/16691 include, but are not
limited to, 16-demthoxy-16-(pent-2-ynyl)oxy-rapamycin,
16-demthoxy-16-(but-2-ynyl)oxy-rapamycin,
16-demthoxy-16-(propargyl)oxy-rapamycin,
16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin,
16-demthoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin,
16-demthoxy-16-benzyloxy-rapamycin,
16-demethoxy-16-ortho-methoxybenzyl-rapamycin,
16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin,
39-demethoxy-40-desoxy-39-formyl-42-nor-rapamycin,
39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin,
39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin,
39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamy-
cin,
39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin,
39-demethoxy-40-desoxy-39-[N-methyl,
N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and
39-demethoxy-40-desoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapam-
ycin.
[0245] Rapamycin analogs from WO96/41807 include, but are not
limited to, 32-deoxo-rapamycin,
16-O-pent-2-ynyl-32-deoxo-rapamycin,
16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin,
16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,
32(S)-dihydro-40-O-(2-methoxyl)ethyl-rapamycin and
32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.
[0246] Another suitable rapamycin analog is umirolimus as described
in US2005/0101624 the contents of which are incorporated by
reference.
[0247] In mammalian cells, the target of rapamycin (mTOR) kinase
exists as a multiprotein complex described as the mTORC1 complex or
mTORC2 complex, which senses the availability of nutrients and
energy and integrates inputs from growth factors and stress
signaling. The mTORC1 complex is sensitive to allosteric mTOR
inhibitors such as rapamycin, is composed of mTOR, G.beta.L, and
regulatory associated proteins of mTOR (raptor), and binds to the
peptidyl-prolyl isomerase FKBP12 protein (a FK506-binding protein
1A, 12 kDa). In contrast, the mTORC2 complex is composed of mTOR,
G.beta.L, and rapamycin-insensitive companion proteins of mTOR
(rictor), and does not bind to the FKBP12 protein in vitro.
[0248] The mTORC1 complex has been shown to be involved in protein
translational control, operating as a growth factor and nutrient
sensitive apparatus for growth and proliferation regulation. mTORC1
regulates protein translation via two key downstream substrates:
P70 S6 kinase, which in turn phosphorylates ribosomal protein P70
S6, and eukaryotic translation initiation factor 4E binding protein
1 (4EBP1), which plays a key role in modulating eIF4E regulated
cap-dependent translation. The mTORC1 complex regulates cell growth
in response to the energy and nutrient homeostasis of the cell, and
the deregulation of mTORC1 is common in a wide variety of human
cancers. The function of mTORC2 involves the regulation of cell
survival via phosphorylation of Akt and the modulation of actin
cytoskeleton dynamics.
[0249] The mTORC1 complex is sensitive to allosteric mTOR
inhibitors such as rapamycin and derivatives in large part due to
rapamycin's mode of action, which involves the formation of an
intracellular complex with the FKBP12 and binding to the
FKBP12-rapamycin binding (FRB) domain of mTOR. This results in a
conformational change in mTORC1 which is believed to alter and
weaken the interaction with its scaffolding protein raptor, in turn
impeding substrates such as P70 S6K1 from accessing mTOR and being
phosphorylated. Rapamycin and rapalogues such as RAD001 have gained
clinical relevance by inhibiting hyperactivation of mTOR associated
with both benign and malignant proliferation disorders.
[0250] RAD001, otherwise known as everolimus (Afinitor.RTM.), has
the chemical name
(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydrox-
y-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methyl-
ethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tric-
yclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone
and the following chemical structure
##STR00002##
[0251] Everolimus is an FDA approved drug for the treatment of
advanced kidney cancer and is being investigated in several other
phase III clinical trials in oncology. Preclinical studies have
shown that Everolimus is able to inhibit the proliferation of a
wide variety of tumor cell lines both in vitro and in vivo,
presumably through the suppression of rapamycin sensitive mTORC1
function. Everolimus, as a derivative of rapamycin, is an
allosteric mTOR inhibitor that is highly potent at inhibiting part
of the mTORC1 function, namely P70 S6 kinase (P70 S6K) and the
downstream P70 S6K substrate P70 S6. Allosteric mTOR inhibitors
like everolimus (and other rapamycin analogs) have little or no
effect at inhibiting the mTORC2 pathway, or its resulting
activation of Akt signaling. Further examples of allosteric mTOR
inhibitors include sirolimus (rapamycin, AY-22989),
40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (also
called temsirolimus or CCI-779) and ridaforolimus
(AP-23573/MK-8669). Other examples of allosteric mTOR inhibitors
include zotarolimus (ABT578) and umirolimus.
[0252] Alternatively or additionally, catalytic, ATP-competitive
mTOR inhibitors have been found to target the mTOR kinase domain
directly and target both mTORC1 and mTORC2. These are also more
complete inhibitors of mTORC1 than such allosteric mTOR inhibitors
as rapamycin, because they modulate rapamycin-resistant mTORC1
outputs such as 4EBP1-T37/46 phosphorylation and cap-dependent
translation.
[0253] BEZ235 is a catalytic mTOR inhibitor, having the chemical
name
2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]q-
uinolin-1-yl)-phenyl]-propionitrile and the following chemical
structure
##STR00003##
[0254] BEZ235 may also be used in its monotosylate salt form. The
synthesis of BEZ235 is described in WO2006/122806.
[0255] As a catalytic mTOR inhibitor BEZ235 is capable of shutting
down the complete function of mTORC1 complex, including both the
rapamycin sensitive (phosphorylation of P70 S6K, and subsequently
phosphorylation of P70 S6) and rapamycin insensitive
(phosphorylation of 4EBP1) functions. BEZ235 has a differential
effect according to the drug concentration used, whereby mTOR
inhibition predominates at a low concentration (less than 100
nmol/L) but dual PI3K/mTOR inhibition at relatively higher
concentrations (approximately 500 nmol/L), Serra et al., 2008.
[0256] Another catalytic mTOR inhibitor described in the literature
is CCG168 (otherwise known as AZD-8055, Chresta, C. M., et al.,
Cancer Res, 2010, 70(1), 288-298) which has the chemical name
{5-[2,4-bis-((S)-3-methyl-morpholin-4-yl)-pyrido[2,3d]pyrimidin-7-yl]-2-m-
ethoxy-phenyl}-methanol and the following chemical structure
##STR00004##
[0257] Another catalytic mTOR inhibitor described in the literature
is
3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-met-
hylbenzamide (WO09104019) having the following chemical
structure:
##STR00005##
[0258] Another catalytic mTOR inhibitor described in the literature
is
3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4--
amine (WO10051043 and WO2013023184) having following chemical
structure:
##STR00006##
[0259] Another catalytic mTOR inhibitor described in the literature
is
N-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxaline-2-yl)sulfamoyl)phenyl)-
-3-methoxy-4-methylbenzamide (WO07044729 and WO12006552) having the
following chemical structure:
##STR00007##
[0260] Another catalytic mTOR inhibitor described in the literature
is PKI-587 (Venkatesan, A. M., J. Med. Chem., 2010, 53, 2636-2645)
which has the chemical name
1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholi-
no-1,3,5-triazin-2-yl)phenyl]urea and having the following chemical
structure:
##STR00008##
[0261] Another catalytic mTOR inhibitor described in the literature
is GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43) which has the
chemical name
2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyrid-
inyl}benzenesulfonamide and having the following chemical
structure:
##STR00009##
[0262] Another catalytic mTOR inhibitor described in the literature
is
5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine
(WO10114484) having the following chemical structure:
##STR00010##
[0263] Another catalytic mTOR inhibitor described in the literature
is
(E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2--
yl)pyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-ylidene)cyanamid-
e (WO12007926) having the following chemical structure:
##STR00011##
[0264] Further examples of catalytic mTOR inhibitors include
8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-
-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (WO2006/122806)
and Ku-0063794 (Garcia-Martinez J M, et al., Biochem J., 2009,
421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammalian
target of rapamycin (mTOR).) WYE-354 is another example of a
catalytic mTor inhibitor (Yu K, et al. (2009). Biochemical,
Cellular, and In vivo Activity of Novel ATP-Competitive and
Selective Inhibitors of the Mammalian Target of Rapamycin. Cancer
Res. 69(15): 6232-6240).
[0265] mTOR inhibitors useful according to the present invention
also include prodrugs, derivatives, pharmaceutically acceptable
salts, or analogs thereof of any of the foregoing.
[0266] mTOR inhibitors, such as RAD001, may be formulated for
delivery based on well-established methods in the art based on the
particular dosages described herein. In particular, U.S. Pat. No.
6,004,973 (incorporated herein by reference) provides examples of
formulations useable with the mTOR inhibitors described herein.
[0267] Downstream Inhibitors
[0268] Many of the methods described herein rely on the use of a
low, immune enhancing, dose of an mTOR inhibitors, e.g., to
increase the level of PD1 negative immune effector cells, e.g., T
cells, to decrease the level of PD1 positive immune effector cells,
e.g., T cells, to increase the ratio of PD1 negative immune
effector cells, e.g., T cells/PD1 positive immune effector cells,
e.g., T cells, to increase the level of naive T cells, or to
increase the number of memory T cell precursors or the expression
level of memory T cell precursor markers. Any of these methods can
also be practiced with, in place of the low, immune enhancing, dose
of an mTOR inhibitors, the administration of an inhibitor of a
downstream element in the pathway, e.g., P70 S6K or mTORC1.
Examples of inhibitors of P70 S6K include PF-4708671 (Pfizer) or
LY2584702 tosylate (Eli Lilly). Examples of inhibitors of mTORC1
include allosteric mTOR inhibitors that specifically inhibit
mTORC1, but do not inhibit mTORC2. In an embodiment, a downstream
inhibitor is adminered at a dose effective to increase the level of
PD1 negative immune effector cells, e.g., T cells, to decrease the
level of PD1 positive immune effector cells, e.g., T cells, to
increase the ratio of PD1 negative immune effector cells, e.g., T
cells/PD1 positive immune effector cells, e.g., T cells, to
increase the level of naive T cells, or to increase the number of
memory T cell precursors or the expression level of memory T cell
precursor markers.
[0269] Evaluation of mTOR Inhibition
[0270] mTOR phosphorylates the kinase P70 S6, thereby activating
P70 S6K and allowing it to phosphorylate its substrate. The extent
of mTOR inhibition can be expressed as the extent of P70 S6K
inhibition, e.g., the extent of mTOR inhibition can be determined
by the level of decrease in P70 S6K activity, e.g., by the decrease
in phosphorylation of a P70 S6K substrate. One can determine the
level of mTOR inhibition, by measuring P70 S6K activity (the
ability of P70 S6K to phsophorylate a substrate), in the absence of
inhibitor, e.g., prior to administration of inhibitor, and in the
presences of inhibitor, or after the administration of inhibitor.
The level of inhibition of P70 S6K gives the level of mTOR
inhibition. Thus, if P70 S6K is inhibited by 40%, mTOR activity, as
measured by P70 S6K activity, is inhibited by 40%. The extent or
level of inhibition referred to herein is the average level of
inhibition over the dosage interval. By way of example, if the
inhibitor is given once per week, the level of inhibition is given
by the average level of inhibition over that interval, namely a
week.
[0271] Boulay et al., Cancer Res, 2004, 64:252-61, hereby
incorporated by reference, teaches an assay that can be used to
assess the level of mTOR inhibition (referred to herein as the
Boulay assay). In an embodiment, the assay relies on the
measurement of P70 S6 kinase activity from biological samples
before and after administration of an mTOR inhibitor, e.g., RAD001.
Samples can be taken at preselected times after treatment with an
mTOR inhibitor, e.g., 24, 48, and 72 hours after treatment.
Biological samples, e.g., from skin or peripheral blood mononuclear
cells (PBMCs) can be used. Total protein extracts are prepared from
the samples. P70 S6 kinase is isolated from the protein extracts by
immunoprecipitation using an antibody that specifically recognizes
the P70 S6 kinase. Activity of the isolated P70 S6 kinase can be
measured in an in vitro kinase assay. The isolated kinase can be
incubated with 40S ribosomal subunit substrates (which is an
endogenous substrate of P70 S6K) and gamma-.sup.32P under
conditions that allow phosphorylation of the substrate. Then the
reaction mixture can be resolved on an SDS-PAGE gel, and .sup.32P
signal analyzed using a PhosphorImager. A .sup.32P signal
corresponding to the size of the 40S ribosomal subunit indicates
phosphorylated substrate and the activity of P70 S6K. Increases and
decreases in kinase activity can be calculated by quantifying the
area and intensity of the .sup.32P signal of the phosphorylated
substrate (e.g., using ImageQuant, Molecular Dynamics), assigning
arbitrary unit values to the quantified signal, and comparing the
values from after administration with values from before
administration or with a reference value. For example, percent
inhibition of kinase activity can be calculated with the following
formula: 1-(value obtained after administration/value obtained
before administration).times.100. As described above, the extent or
level of inhibition referred to herein is the average level of
inhibition over the dosage interval.
[0272] Methods for the evaluation of kinase activity, e.g., P70 S6
kinase activity, are also provided in U.S. Pat. No. 7,727,950,
hereby incorporated by reference.
[0273] The level of mTOR inhibition can also be evaluated by a
change in the ration of PD1 negative to PD1 positive T cells. T
cells from peripheral blood can be identified as PD1 negative or
positive by art-known methods.
Low-Dose mTOR Inhibitors
[0274] Methods described herein use low, immune enhancing, dose
mTOR inhibitors, doses of mTOR inhibitors, e.g., allosteric mTOR
inhibitors, including rapalogs such as RAD001. In contrast, levels
of inhibitor that fully or near fully inhibit the mTOR pathway are
immunosuppressive and are used, e.g., to prevent organ transplant
rejection. In addition, high doses of rapalogs that fully inhibit
mTOR also inhibit tumor cell growth and are used to treat a variety
of cancers (See, e.g., Antineoplastic effects of mammalian target
of rapamycine inhibitors. Salvadori M. World J Transplant. 2012
Oct. 24; 2(5):74-83; Current and Future Treatment Strategies for
Patients with Advanced Hepatocellular Carcinoma: Role of mTOR
Inhibition. Finn R S. Liver Cancer. 2012 November; 1(3-4):247-256;
Emerging Signaling Pathways in Hepatocellular Carcinoma. Moeini A,
Cornelia H, Villanueva A. Liver Cancer. 2012 September; 1(2):83-93;
Targeted cancer therapy--Are the days of systemic chemotherapy
numbered? Joo W D, Visintin I, Mor G. Maturitas. 2013 Sep. 20.;
Role of natural and adaptive immunity in renal cell carcinoma
response to VEGFR-TKIs and mTOR inhibitor. Santoni M, Berardi R,
Amantini C, Burattini L, Santini D, Santoni G, Cascinu S. Int J
Cancer. 2013 Oct. 2).
[0275] The present invention is based, at least in part, on the
surprising finding that doses of mTOR inhibitors well below those
used in current clinical settings had a superior effect in
increasing an immune response in a subject and increasing the ratio
of PD-1 negative T cells/PD-1 positive T cells. It was surprising
that low doses of mTOR inhibitors, producing only partial
inhibition of mTOR activity, were able to effectively improve
immune responses in human subjects and increase the ratio of PD-1
negative T cells/PD-1 positive T cells.
[0276] Alternatively, or in addition, without wishing to be bound
by any theory, it is believed that low, a low, immune enhancing,
dose of an mTOR inhibitor can increase naive T cell numbers, e.g.,
at least transiently, e.g., as compared to a non-treated subject.
Alternatively or additionally, again while not wishing to be bound
by theory, it is believed that treatment with an mTOR inhibitor
after a sufficient amount of time or sufficient dosing results in
one or more of the following:
[0277] an increase in the expression of one or more of the
following markers: CD62L.sup.high, CD127.sup.high CD27.sup.+, and
BCL2, e.g., on memory T cells, e.g., memory T cell precursors;
[0278] a decrease in the expression of KLRG1, e.g., on memory T
cells, e.g., memory T cell precursors; and
[0279] an increase in the number of memory T cell precursors, e.g.,
cells with any one or combination of the following characteristics:
increased CD62L.sup.high, increased CD127.sup.high, increased
CD27.sup.+, decreased KLRG1, and increased BCL2;
[0280] and wherein any of the changes described above occurs, e.g.,
at least transiently, e.g., as compared to a non-treated subject
(Araki, K et al. (2009) Nature 460:108-112). Memory T cell
precursors are memory T cells that are early in the differentiation
program. For example, memory T cells have one or more of the
following characteristics: increased CD62L.sup.high, increased
CD127.sup.high increased CD27.sup.+, decreased KLRG1, and/or
increased BCL2.
[0281] Accordingly, in one aspect, the present invention provides
compositions, e.g., provides as a unit dosage form, comprising an
mTOR inhibitor, e.g., a allosteric mTOR inhibitor, e.g., RAD001, at
a concentration of about 0.005-1.5 mg, about 0.005-1.5 mg, about
0.01-1 mg, about 0.01-0.7 mg, about 0.01-0.5 mg, or about 0.1-0.5
mg. In a further aspect the present invention provides compositions
comprising an mTOR inhibitor, e.g., RAD001, at a concentration of
0.005-1.5 mg, 0.005-1.5 mg, 0.01-1 mg, 0.01-0.7 mg, 0.01-0.5 mg, or
0.1-0.5 mg. More particularly, in one aspect, the invention
provides compositions comprising an mTOR inhibitor, e.g., RAD001,
at a dose of about 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009
mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg,
0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg,
0.7 mg, 0.8 mg, 0.9 mg, or 1.0 mg. In one aspect, the mTOR
inhibitor, e.g., RAD001, is at a dose of 0.5 mg or less. In a still
further aspect, the mTOR inhibitor, e.g., RAD001, is at a dose of
about 0.5 mg. In a further aspect, the invention provides
compositions comprising an mTOR inhibitor, e.g., RAD001, at a dose
of 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02
mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg,
0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9
mg, or 1.0 mg. In one aspect, the mTOR inhibitor, e.g., RAD001, is
at a dose of 0.5 mg or less. In a still further aspect, the mTOR
inhibitor, e.g., RAD001, is at a dose of 0.5 mg.
[0282] In a further aspect, the invention relates to compositions
comprising an mTOR inhibitor that is not RAD001, in an amount that
is bioequivalent to the specific amounts or doses specified for
RAD001.
[0283] In a further aspect, the invention relates to compositions
comprising an mTOR inhibitor in an amount sufficient to inhibit P70
S6 kinase by no greater than 80%. In a further aspect the
compositions described herein comprise an mTOR inhibitor in an
amount sufficient to inhibit P70 S6 kinase by no greater than
38%.
[0284] In an embodiment, the invention relates to a composition, or
dosage form, of an mTOR inhibitor, e.g., an allosteric mTOR
inhibitor, e.g., a rapalog, rapamycin, or RAD001, or a catalytic
mTOR inhibitor, which, when administered on a selected dosing
regimen, e.g., once daily or once weekly, is associated with: a
level of mTOR inhibition that is not associated with complete, or
significant immune suppression, but is associated with enhancement
of the immune response.
[0285] In a further aspect, the invention provides methods for
enhancing immune response, e.g., treating immunosenescence,
comprising a step of administering to a subject an mTOR inhibitor.
In some embodiments, an mTOR inhibitor, e.g., an allosteric mTOR
inhibitor, e.g., RAD001, can be administered at a dose of about
0.005-1.5 mg daily, about 0.01-1 mg daily, about 0.01-0.7 mg daily,
about 0.01-0.5 mg daily, or about 0.1-0.5 mg daily. In a further
aspect, an mTOR inhibitor, e.g., RAD001, can be administered at a
dose of about 0.1-20 mg weekly, about 0.5-15 mg weekly, about 1-10
mg weekly, or about 3-7 mg weekly. In some embodiments, an mTOR
inhibitor, e.g., RAD001, can be administered at a dose of 0.005-1.5
mg daily, 0.01-1 mg daily, 0.01-0.7 mg daily, 0.01-0.5 mg daily, or
0.1-0.5 mg daily. In some embodiments, an mTOR inhibitor, e.g.,
RAD001, can be administered at a dose of about 0.1-20 mg weekly,
0.5-15 mg weekly, 1-10 mg weekly, 3-7 mg weekly, or 5 mg
weekly.
[0286] In a further aspect, the invention relates to methods for
enhancing immune response, e.g., treating immunosenescence,
comprising the step of administering an mTOR inhibitor that is not
RAD001, in an amount that is bioequivalent to the specific amounts
or doses described herein for RAD001.
[0287] In some embodiments, an mTOR inhibitor, e.g., a allosteric
mTOR inhibitor, eg., e.g., RAD001, can be administered at a dose of
about 0.005 mg daily, 0.006 mg daily, 0.007 mg daily, 0.008 mg
daily, 0.009 mg daily, 0.01 mg daily, 0.02 mg daily, 0.03 mg daily,
0.04 mg daily, 0.05 mg daily, 0.06 mg daily, 0.07 mg daily, 0.08 mg
daily, 0.09 mg daily, 0.1 mg daily, 0.2 mg daily, 0.3 mg daily, 0.4
mg daily, 0.5 mg daily, 0.6 mg daily, 0.7 mg daily, 0.8 mg daily,
0.9 mg daily, or 1.0 mg daily. In some embodiments, RAD001 can be
administered at a dose of no greater than about 0.7 mg in a 24 hour
period. In some embodiments, an mTOR inhibitor, e.g., an allosteric
mTOR inhibitor, e.g., RAD001, can be administered at a dose of no
greater than about 0.5 mg in a 24 hour period. In some embodiments,
RAD001 can be administered at a dose of 0.5 mg or less daily. In
some embodiments, RAD001 can be administered at a dose of 0.5 mg
daily.
[0288] In a further aspect, the invention can utilize an mTOR
inhibitor other than RAD001 in an amount that is bioequivalent to
the specific amounts or doses specified for RAD001.
[0289] In some embodiments, an mTOR inhibitor, e.g., an allosteric
mTOR inhibitor, e.g., RAD001, can be administered at a dose of 0.1
mg weekly, 0.2 mg weekly, 0.3 mg weekly, 0.4 mg weekly, 0.5 mg
weekly, 0.6 mg weekly, 0.7 mg weekly, 0.8 mg weekly, 0.9 mg weekly,
1 mg weekly, 2 mg weekly, 3 mg weekly, 4 mg weekly, 5 mg weekly, 6
mg weekly, 7 mg weekly, 8 mg weekly, 9 mg weekly, 10 mg weekly, 11
mg weekly, 12 mg weekly, 13 mg weekly, 14 mg weekly, 15 mg weekly,
16 mg weekly, 17 mg weekly, 18 mg weekly, 19 mg weekly, or 20 mg
weekly. In some embodiments, an mTOR inhibitor, e.g., an allosteric
mTOR inhibitor, e.g., RAD001, is administered at a dose of 5 mg or
less weekly. In some embodiments, an mTOR inhibitor, e.g., an
allosteric mTOR inhibitor, e.g., RAD001, is administered at a dose
of 5 mg weekly.
[0290] In some embodiments, the invention can utilize an mTOR
inhibitor other than RAD001 in an amount that is bioequivalent to
the specific amounts or doses specified for RAD001.
[0291] An mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g.,
a rapalog, rapamycin, or RAD001, or a catalytic mTOR inhibitor, can
be provided in a sustained release formulation. Any of the
compositions or unit dosage forms described herein can be provided
in a sustained release formulation. In some embodiments, a
sustained release formulation will have lower bioavailability than
an immediate release formulation. E.g., in embodiments, to attain a
similar therapeutic effect of an immediate release formulation a
sustained release formulation will have from about 2 to about 5,
about 2.5 to about 3.5, or about 3 times the amount of inhibitor
provided in the immediate release formulation.
[0292] In an embodiment, immediate release forms, e.g., of RAD001,
typically used for one administration per week, having 0.1 to 20,
0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5, mgs per unit dosage
form, are provided. For once per week administrations, these
immediate release formulations correspond to sustained release
forms, having, respectively, 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9
to 18, or about 15 mgs of an mTOR inhibitor, e.g., an allosteric
mTOR inhibitor, e.g., rapamycin or RAD001. In embodiments both
forms are administered on a once/week basis.
[0293] In an embodiment, immediate release forms, e.g., of RAD001,
typically used for one administration per day, having having 0.005
to 1.5, 0.01 to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to
1.5, 0.5 to 1.5, 0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5,
0.3 to 0.6, or about 0.5 mgs per unit dosage form, are provided.
For once per day administrations, these immediate release forms
correspond to sustained release forms, having, respectively, 0.015
to 4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 to 4.5, 0.9 to 4.5, 1.2 to
4.5, 1.5 to 4.5, 1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5,
0.9 to 1.8, or about 1.5 mgs of an mTOR inhibitor, e.g., an
allosteric mTOR inhibitor, e.g., rapamycin or RAD001. For once per
week administrations, these immediate release forms correspond to
sustained release forms, having, respectively, 0.1 to 30, 0.2 to
30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30, 14 to
30, 16 to 30, 20 to 30, 6 to 12, or about 10 mgs of an mTOR
inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or
RAD001.
[0294] In an embodiment, immediate release forms, e.g., of RAD001,
typically used for one administration per day, having having 0.01
to 1.0 mgs per unit dosage form, are provided. For once per day
administrations, these immediate release forms correspond to
sustained release forms, having, respectively, 0.03 to 3 mgs of an
mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin
or RAD001. For once per week administrations, these immediate
release forms correspond to sustained release forms, having,
respectively, 0.2 to 20 mgs of an mTOR inhibitor, e.g., an
allosteric mTOR inhibitor, e.g., rapamycin or RAD001.
[0295] In an embodiment, immediate release forms, e.g., of RAD001,
typically used for one administration per week, having having 0.5
to 5.0 mgs per unit dosage form, are provided. For once per week
administrations, these immediate release forms correspond to
sustained release forms, having, respectively, 1.5 to 15 mgs of an
mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin
or RAD001.
[0296] As described above, one target of the mTOR pathway is the
P70 S6 kinase. Thus, doses of mTOR inhibitors which are useful in
the methods and compositions described herein are those which are
sufficient to achieve no greater than 80% inhibition of P70 S6
kinase activity relative to the activity of the P70 S6 kinase in
the absence of an mTOR inhibitor, e.g., as measured by an assay
described herein, e.g., the Boulay assay. In a further aspect, the
invention provides an amount of an mTOR inhibitor sufficient to
achieve no greater than 38% inhibition of P70 S6 kinase activity
relative to P70 S6 kinase activity in the absence of an mTOR
inhibitor, e.g., as measured by an assay described herein, e.g.,
the Boulay assay. In one aspect the dose of mTOR inhibitor useful
in the methods and compositions of the invention is sufficient to
achieve, e.g., when administered to a human subject, 90%, 89%, 88%,
87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%,
74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%,
61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 54%, 53%, 52%, 51%, 50%,
49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%,
36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%,
23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or
10% or less inhibition of P70 S6 kinase activity, e.g., as measured
by an assay described herein, e.g., the Boulay assay.
[0297] In one aspect the dose of mTOR inhibitor useful in the
methods and compositions of the invention is sufficient to achieve,
e.g., when administered to a human subject, 90+/-5% (i.e., 85-95%),
89+/-5%, 88+/-5%, 87+/-5%, 86+/-5%, 85+/-5%, 84+/-5%, 83+/-5%,
82+/-5%, 81+/-5%, 80+/-5%, 79+/-5%, 78+/-5%, 77+1-5%, 76+/-5%,
75+/-5%, 74+/-5%, 73+/-5%, 72+/-5%, 71+/-5%, 70+/-5%, 69+/-5%,
68+/-5%, 67+/-5%, 66+/-5%, 65+/-5%, 64+/-5%, 63+/-5%, 62+/-5%,
61+/-5%, 60+/-5%, 59+/-5%, 58+/-5%, 57+/-5%, 56+/-5%, 55+/-5%,
54+/-5%, 54+/-5%, 53+/-5%, 52+/-5%, 51+/-5%, 50+/-5%, 49+/-5%,
48+/-5%, 47+/-5%, 46+/-5%, 45+/-5%, 44+/-5%, 43+/-5%, 42+/-5%,
41+/-5%, 40+/-5%, 39+/-5%, 38+/-5%, 37+/-5%, 36+/-5%, 35+/-5%,
34+/-5%, 33+/-5%, 32+/-5%, 31+/-5%, 30+/-5%, 29+/-5%, 28+/-5%,
27+/-5%, 26+/-5%, 25+/-5%, 24+/-5%, 23+/-5%, 22+/-5%, 21+/-5%,
20+/-5%, 19+/-5%, 18+/-5%, 17+/-5%, 16+/-5%, 15+/-5%, 14+/-5%,
13+/-5%, 12+/-5%, 11+/-5%, or 10+/-5%, inhibition of P70 S6 kinase
activity, e.g., as measured by an assay described herein, e.g., the
Boulay assay.
[0298] P70 S6 kinase activity in a subject may be measured using
methods known in the art, such as, for example, according to the
methods described in U.S. Pat. No. 7,727,950, by immunoblot
analysis of phosphoP70 S6K levels and/or phosphoP70 S6 levels or by
in vitro kinase activity assays.
[0299] In a further aspect, the invention relates to compositions
comprising an mTOR inhibitor such as an mTOR inhibitor, e.g., an
allosteric mTOR inhibitor, e.g., RAD001. Doses of an mTOR
inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001, in
such compositions can be in the range of about 30 pM to 4 nM. In
one aspect, the dose of an mTOR inhibitor, e.g., an allosteric mTOR
inhibitor, e.g., RAD001, is in the range of about 50 pM to 2 nM,
about 100 pM to 1.5 nM, about 200 pM to 1 nM, or about 300 pM to
500 pM. In one aspect, the dose of RAD001 is in the range of 50 pM
to 2 nM, 100 pM to 1.5 nM, 200 pM to 1 nM, or 300 pM to 500 pM. In
a further aspect the dose of RAD001 is about 30 pM, 40 pM, 50 pM,
60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 150 pM, 200 pM, 250 pM, 300 pM,
350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750
pM, 800 pM, 850 pM, 900 pM, 950 pM, 1 nM, 1.5 nM, 2 nM, 2.5 nM, 3
nM, 3.5 nM, or 4 nM.
[0300] In a further aspect, the invention can utilize an mTOR
inhibitor other than RAD001 in an amount that is bioequivalent to
the specific amounts or doses specified for RAD001.
[0301] The invention further relates to methods comprising the
administration of an mTOR inhibitor to a subject. Such methods may
employ doses of the mTOR inhibitor RAD001 in the range of about 30
pM to 4 nM. In a further aspect, the dose of RAD001 can be in the
range of about 50 pM to 2 nM, about 100 pM to 1.5 nM, about 200 pM
to 1 nM, or about 300 pM to 500 pM. In one aspect, the dose of
RAD001 is in the range of 50 pM to 2 nM, 100 pM to 1.5 nM, 200 pM
to 1 nM, or 300 pM to 500 pM. In a further aspect the dose of
RAD001 is about 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM,
100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500
pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 900 pM,
950 pM, 1 nM, 1.5 nM, 2 nM, 2.5 nM, 3 nM, 3.5 nM, or 4 nM.
[0302] In a further aspect, the methods of the invention can
utilize an mTOR inhibitor other than RAD001 in an amount that is
bioequivalent to the specific amounts or doses specified for
RAD001.
[0303] As used herein, the term "about" in reference to a dose of
mTOR inhibitor refers to up to a +/-10% variability in the amount
of mTOR inhibitor, but can include no variability around the stated
dose.
[0304] In some embodiments, the invention provides methods
comprising administering to a subject an mTOR inhibitor, e.g., an
allosteric inhibitor, e.g., RAD001, at a dosage within a target
trough level. In some embodiments, the trough level is
significantly lower than trough levels associated with dosing
regimens used in organ transplant and cancer patients. In an
embodiment mTOR inhibitor, e.g., RAD001, or rapamycin, is
administered to result in a trough level that is less than 1/2,
1/4, 1/10, or 1/20 of the trough level that results in
immunosuppression or an anticancer effect. In an embodiment mTOR
inhibitor, e.g., RAD001, or rapamycin, is administered to result in
a trough level that is less than 1/2, 1/4, 1/10, or 1/20 of the
trough level provided on the FDA approved packaging insert for use
in immunosuppression or an anticancer indications.
[0305] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 0.1 to 3 ng/ml, 0.1 to 2 ng/ml, or 0.1 to 1 ng/ml.
[0306] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 00.2 to 3 ng/ml, 0.2 to 2 ng/ml, or 0.2 to 1 ng/ml.
[0307] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g. an, allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 0.3 to 3 ng/ml, 0.3 to 2 ng/ml, or 0.3 to 1 ng/ml.
[0308] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 0.4 to 3 ng/ml, 0.4 to 2 ng/ml, or 0.4 to 1 ng/ml.
[0309] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 0.5 to 3 ng/ml, 0.5 to 2 ng/ml, or 0.5 to 1 ng/ml.
[0310] In an embodiment a method disclosed herein comprises
administering to a subject an mTOR inhibitor, e.g., an allosteric
inhibitor, e.g., RAD001, at a dosage that provides a target trough
level of 1 to 3 ng/ml, or 1 to 2 ng/ml.
[0311] As used herein, the term "level" refers to the concentration
of a drug in plasma just before the next dose, or the minimum drug
concentration between two doses.
[0312] In some embodiments, a target trough level of RAD001 is in a
range of between about 0.1 and 3 ng/ml. In an embodiment, the
target trough level is below 3 ng/ml, e.g., is between 0.3 or less
and 3 ng/ml. In an embodiment, the target trough level is below 3
ng/ml, e.g., is between 0.3 or less and 1 ng/ml. In some
embodiments, a target trough level of RAD001 is in a range of
between about 2.4 and 3. In some embodiments, a target trough level
of RAD001 is in a range of between about 0.1 and 2.4 ng/ml. In some
embodiments, a target trough level of RAD001 is in a range of
between about 0.1 and 1.5 ng/ml. In some embodiments, a target
trough level of RAD001 is in a range of between 0.1 and 3 ng/ml. In
some embodiments, a target trough level of RAD001 is in a range of
between 2.4 and 3 ng/ml. In some embodiments, a target trough level
of RAD001 is in a range of between 0.1 and 2.4 ng/ml. In some
embodiments, a target trough level of RAD001 is in a range of
between 0.1 and 1.5 ng/ml. In some embodiments, a target trough
level of RAD001 is 0.1 ng/ml. In some embodiments, a target trough
level of RAD001 is 0.2 ng/ml. In some embodiments, a target trough
level of RAD001 is 0.3 ng/ml. In some embodiments, a target trough
level of RAD001 is 0.4 ng/ml. In some embodiments, a target trough
level of RAD001 is 0.5 ng/ml. In some embodiments, a target trough
level of RAD001 is 0.6 ng/ml. In some embodiments, a target trough
level of RAD001 is 0.7 ng/ml. In some embodiments, a target trough
level of RAD001 is 0.8 ng/ml. In some embodiments, a target trough
level of RAD001 is 0.9 ng/ml. In some embodiments, a target trough
level of RAD001 is 1.0 ng/ml. In some embodiments, a target trough
level of RAD001 is 1.1 ng/ml. In some embodiments, a target trough
level of RAD001 is 1.2 ng/ml. In some embodiments, a target trough
level of RAD001 is 1.3 ng/ml. In some embodiments, a target trough
level of RAD001 is 1.4 ng/ml. In some embodiments, a target trough
level of RAD001 is 1.5 ng/ml. In some embodiments, a target trough
level of RAD001 is less than 3 ng/ml. In some embodiments, a target
trough level of RAD001 is less than 2.5 ng/ml. In some embodiments,
a target trough level of RAD001 is less than 3 ng/ml, 2 ng/ml, 1.9
ng/ml, 1.8 ng/ml, 1.7 ng/ml, 1.6 ng/ml, 1.5 ng/ml, 1.4 ng/ml, 1.3
ng/ml, 1.2 ng/ml, 1.1 ng/ml, 1.0 ng/ml, 0.9 ng/ml, 0.8 ng/ml, 0.7
ng/ml, 0.6 ng/ml, 0.5 ng/ml, 0.4 ng/ml, 0.3 ng/ml, 0.2 ng/ml, or
0.1 ng/ml.
[0313] In a further aspect, the invention can utilize an mTOR
inhibitor other than RAD001 in an amount that is associated with a
target trough level that is bioequivalent to the specified target
trough level for RAD001. In an embodiment, the target trough level
for an mTOR inhibitor other than RAD001, is a level that gives the
same level of mTOR inhibition (e.g., as measured by a method
described herein, e.g., the inhibition of P70 S6K) as does a trough
level of RAD001 described herein.
Disorders
Cancer
[0314] The methods described herein can be used with any cancer. In
an embodiment, the cancer comprises a solid tumor. In an
embodiment, the cancer is a hematological cancer. The cancer can be
a carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma or a
mixed type.
[0315] In some embodiments, the cancer is associated with elevated
percentages of PD1+ T cells in the subject. In certain embodiments,
the cancer is a cancer that generally responds to PD-1 targeted
drugs, such as melanoma. In certain embodiments, the cancer is a
cancer that generally responds to T-cell directed immunotherapies,
such as renal cell carcinoma. In an embodiment the cancer is one in
which can be treated by increasing the ration of PD-1 negative to
PD-1 positive T cells.
[0316] Examples of cancers that can be treated with methods
disclosed herein include bone cancer, pancreatic cancer, skin
cancer, cancer of the head or neck, cutaneous or intraocular
malignant melanoma, uterine cancer, ovarian cancer, rectal cancer,
cancer of the anal region, stomach cancer, testicular cancer,
uterine cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma,
cancer of the esophagus, cancer of the small intestine, cancer of
the endocrine system, cancer of the thyroid gland, cancer of the
parathyroid gland, cancer of the adrenal gland, sarcoma of soft
tissue, cancer of the urethra, cancer of the penis, chronic or
acute leukemias including acute myeloid leukemia, chronic myeloid
leukemia, acute lymphoblastic leukemia, chronic lymphocytic
leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer
of the bladder, cancer of the kidney or ureter, carcinoma of the
renal pelvis, neoplasm of the central nervous system (CNS), primary
CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem
glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer,
squamous cell cancer, T-cell lymphoma, environmentally induced
cancers including those induced by asbestos, and combinations of
said cancers.
[0317] Examples of solid tumors that can be treated with methods
disclosed herein include malignancies, e.g., sarcomas,
adenocarcinomas, and carcinomas, of the various organ systems, such
as those affecting liver, lung, breast, lymphoid, gastrointestinal
(e.g., colon), genitourinary tract (e.g., renal, urothelial cells),
prostate and pharynx. Adenocarcinomas include malignancies such as
most colon cancers, rectal cancer, renal-cell carcinoma, liver
cancer, non-small cell carcinoma of the lung, cancer of the small
intestine and cancer of the esophagus. In one embodiment, the
cancer is a melanoma, e.g., an advanced stage melanoma. Metastatic
lesions of the aforementioned cancers can also be treated or
prevented using the methods and compositions of the invention.
[0318] Methods described herein can be used to treat any of the
following cancers:
[0319] Digestive/gastrointestinal cancers such as anal cancer; bile
duct cancer; extrahepatic bile duct cancer; appendix cancer;
carcinoid tumor, gastrointestinal cancer; colon cancer; colorectal
cancer including childhood colorectal cancer; esophageal cancer
including childhood esophageal cancer; gallbladder cancer; gastric
(stomach) cancer including childhood gastric (stomach) cancer;
hepatocellular (liver) cancer including adult (primary)
hepatocellular (liver) cancer and childhood (primary)
hepatocellular (liver) cancer; pancreatic cancer including
childhood pancreatic cancer; sarcoma, rhabdomyo sarcoma; islet cell
pancreatic cancer; rectal cancer; and small intestine cancer;
[0320] Endocrine cancers such as islet cell carcinoma (endocrine
pancreas); adrenocortical carcinoma including childhood
adrenocortical carcinoma; gastrointestinal carcinoid tumor;
parathyroid cancer; pheochromocytoma; pituitary tumor; thyroid
cancer including childhood thyroid cancer; childhood multiple
endocrine neoplasia syndrome; and childhood carcinoid tumor;
[0321] Eye cancers such as intraocular melanoma; and
retinoblastoma;
[0322] Musculoskeletal cancers such as Ewing's family of tumors;
osteosarcoma/malignant fibrous histiocytoma of the bone; childhood
rhabdomyosarcoma; soft tissue sarcoma including adult and childhood
soft tissue sarcoma; clear cell sarcoma of tendon sheaths; and
uterine sarcoma;
[0323] Breast cancer such as breast cancer including childhood and
male breast cancer and pregnancy;
[0324] Neurologic cancers such as childhood brain stem glioma;
brain tumor; childhood cerebellar astrocytoma; childhood cerebral
astrocytoma/malignant glioma; childhood ependymoma; childhood
medulloblastoma; childhood pineal and supratentorial primitive
neuroectodermal tumors; childhood visual pathway and hypothalamic
glioma; other childhood brain cancers; adrenocortical carcinoma;
central nervous system lymphoma, primary; childhood cerebellar
astrocytoma; neuroblastoma; craniopharyngioma; spinal cord tumors;
central nervous system atypical teratoid/rhabdoid tumor; central
nervous system embryonal tumors; and childhood supratentorial
primitive neuroectodermal tumors and pituitary tumor;
[0325] Genitourinary cancers such as bladder cancer including
childhood bladder cancer; renal cell (kidney) cancer; ovarian
cancer including childhood ovarian cancer; ovarian epithelial
cancer; ovarian low malignant potential tumor; penile cancer;
prostate cancer; renal cell cancer including childhood renal cell
cancer; renal pelvis and ureter, transitional cell cancer;
testicular cancer; urethral cancer; vaginal cancer; vulvar cancer;
cervical cancer; Wilms tumor and other childhood kidney tumors;
endometrial cancer; and gestational trophoblastic tumor;
[0326] Germ cell cancers such as childhood extracranial germ cell
tumor; extragonadal germ cell tumor; ovarian germ cell tumor; and
testicular cancer;
[0327] Head and neck cancers such as lip and oral cavity cancer;
oral cancer including childhood oral cancer; hypopharyngeal cancer;
laryngeal cancer including childhood laryngeal cancer; metastatic
squamous neck cancer with occult primary; mouth cancer; nasal
cavity and paranasal sinus cancer; nasopharyngeal cancer including
childhood nasopharyngeal cancer; oropharyngeal cancer; parathyroid
cancer; pharyngeal cancer; salivary gland cancer including
childhood salivary gland cancer; throat cancer; and thyroid
cancer;
[0328] Lung cancer such as non-small cell lung cancer; and small
cell lung cancer;
[0329] Respiratory cancers such as malignant mesothelioma, adult;
malignant mesothelioma, childhood; malignant thymoma; childhood
thymoma; thymic carcinoma; bronchial adenomas/carcinoids including
childhood bronchial adenomas/carcinoids; pleuropulmonary blastoma;
non-small cell lung cancer; and small cell lung cancer;
[0330] Skin cancers such as Kaposi's sarcoma; Merkel cell
carcinoma; melanoma; and childhood skin cancer;
[0331] AIDS-related malignancies;
[0332] Other childhood cancers, unusual cancers of childhood and
cancers of unknown primary site;
[0333] and metastases of the aforementioned cancers can also be
treated or prevented in accordance with the methods described
herein.
[0334] Methods described herein can be used to treat a
hematological cancer or malignancy or precancerous condition, e.g.,
a leukemia or a lymphoma. The cancer can be one associated with
expression of a cancer associated antigen as described herein.
Hematological cancers and malignancies include, one or more acute
leukemias including, e.g., B-cell acute Lymphoid Leukemia ("BALL"),
T-cell acute Lymphoid Leukemia ("TALL"), acute lymphoid leukemia
(or acute lymphoblastic leukemia) (ALL), including adult and
childhood acute lymphoid leukemia; acute myeloid leukemia,
including adult and childhood acute myeloid leukemia; one or more
chronic leukemias, e.g., chronic myelogenous leukemia (CML),
Chronic Lymphoid Leukemia (or chronic lymphocytic leukemia) (CLL).
Additional cancers or hematologic conditions that can be treated
with methods disclosed herein include, e.g., AIDS-related lymphoma,
B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell
neoplasm, Burkitt's lymphoma, chronic myeloproliferative disorders;
cutaneous T-cell lymphoma, diffuse large B cell lymphoma,
Follicular lymphoma, Hairy cell leukemia, Hodgkin's lymphoma
(including adult and childhood Hogkin's lymphoma and Hodgkin's
lymphoma during pregnancy), small cell- or a large cell-follicular
lymphoma, malignant lymphoproliferative conditions, MALT lymphoma,
mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma,
multiple myeloma/plasma cell neoplasm, myelodysplasia and
myelodysplastic syndrome, myelodysplastic/myeloproliferative
disorders, mycosis fungoides, non-Hodgkin's lymphoma (including
adult and childhood non-Hodgkin's lymphoma and non-Hodkin's
lymphoma during pregnancy), plasmablastic lymphoma, plasmacytoid
dendritic cell neoplasm, Sezary syndrome, Waldenstrom
macroglobulinemia, primary central system lymphoma, and
"preleukemia" which are a diverse collection of hematological
conditions united by ineffective production (or dysplasia) of
myeloid blood cells, and the like. Further a disease associated
with a cancer associated antigen as described herein expression
include, but not limited to, e.g., atypical and/or non-classical
cancers, malignancies, precancerous conditions or proliferative
diseases associated with expression of a cancer associated antigen
as described herein.
Pathogenic Infections
[0335] In another aspect, the methods provided herein can be used
to treat infection by a pathogen in a subject. In some embodiments,
the pathogen is a viral pathogen, e.g., a viral pathogen e.g. HIV,
meningitis causing viruses, encephalitis causing viruses, Hepatitis
A, Hepatitis B, Hepatitis C, rabies virus, polio virus, influenza
virus, parainfluenza virus, adenovirus, rhinovirus, measles virus,
mumps virus, rubella, pertussis, papilloma virus, yellow fever
virus, respiratory syncytial virus, parvovirus, Norwalk virus,
chikungunya virus, haemorrhagic fever viruses, dengue virus, and
Herpes viruses, e.g., varicella, cytomegalovirus and Epstein-Barr
virus. In some embodiments, the infection is a viral infection,
such as a chronic viral infection. In some embodiments, a chronic
viral infection is selected from Hepatitis A, Hepatitis B,
Hepatitis C, Epstein Barr Virus, HIV, Cytomegalovirus, Herpes
Simplex Virus 1, Herpes Simplex Virus 2, Human Papillomavirus,
Adenovirus, and Kaposi's Sarcoma-Associated Herpesvirus. In some
embodiments, a chronic viral infection comprises HIV.
[0336] For example, Lichterfeld and colleagues observed that
HIV-specific CD8+ T-cells showed reduced telomere length and an
increase in telomere length and telomerase activity upon inhibition
of PD-1(see e.g., Lichterfeld, M et al. (2008) Blood
112(9):3679-3687). In another example, PD-1 was significantly
upregulated in hepatitis C (HVC)-specific CD8+ cytotoxic T
lymphocytes (see e.g., Golden-Mason, L (2007) J. Virol. 81(17):
9249-9258).
[0337] In some embodiments, a viral infection comprises a viral
acute lower respiratory tract infection. In some embodiments viral
acute lower respiratory tract infection is caused by a rhinovirus,
coronavirus, influenza virus, respiratory syncytial virus (RSV),
adenovirus, and/or parainfluenza. In some embodiments, a viral
acute lower respiratory tract infection is pneumonia. In some
embodiments, a viral acute lower respiratory tract infection
includes a lung abscess. In some embodiments, a viral acute lower
respiratory tract infection includes bronchitis.
[0338] In some embodiments, the pathogen is a bacterial pathogen,
e.g., a bacterial pathogen selected from Meningococcus,
Haemophilus, Pneumococcus, Staphylococcus, Streptococcus,
Neisseria, Moraxella, Escherichia coli, Klebsiella, Pseudomonas,
Enterobacter, Proteus, Serratia, Legionella, Salmonella, Shigella,
Acinetobacer, Listeria, Chlamydia, Mycobacterium among others.
[0339] In some embodiments, the pathogen is a parasitic pathogen,
e.g., Toxoplasma, Leishmania and malaria, T. cruzii, Helminth,
e.g., Schistosoma.
[0340] In some embodiments, the pathogen is a yeast or fungal
pathogen, e.g., Candida, Cryptococcus or Coccidioides.
Senescence and Other Disorders
[0341] In another aspect, the methods provided herein can be used
to treat senescence in a subject. As used herein, the term
"senescence" is meant to include all types of aging. In some
embodiments, senescence comprises immunosenescence.
Immunosenescence includes reduced immune response to infection with
age and results from thymic involution in T-cell lineages,
resulting in decreased T cell production and export (see e.g.,
Shimatani, K et al. (2009) PNAS 106 (37):15807-15812). In some
embodiments, there is an increase in population of a bona fide
age-dependent CD4+ T cell population defined by a constitutive
expression of PD-1, which is induced only transiently on activation
in regular T cells and, therefore, reduced immune response to
infection (see e.g., Shimatani, K et al. (2009) PNAS 106
(37):15807-15812). In some embodiments, there is in increase in
population of CD8+ T cell population defined by increased
expression of PD-1 upon receptor-mediated activation of CD8+ T
cells (see e.g., Nunes, C et al. (2012) Clinical Cancer Research
18(3):678-687). In some embodiments, senescence comprises cellular
senescence, in which a cell no longer divides. In some embodiments,
age-related immunosenescence comprises decreased production of
naive lymphocytes by hematopoietic stem cells (Chen, Science
Signaling, ra75, 2009). Cellular senescence is correlated with the
progressive shortening of telomeres that occurs with each cell
division.
[0342] The term "age-related condition" refers to any disease,
disorder, or pathology whose incidence in a population or severity
in an individual correlates with the progression of age. More
specifically, an age-related condition is a disease, disorder, or
pathology whose incidence is at least 1.5 fold higher among human
individuals greater than 60 years of age relative to human
individuals between the ages of 30-40 and in a selected population
of greater than 100,000 individuals. In one aspect, the invention
relates to the treatment of conditions including, but not limited
to sarcopenia, skin atrophy, muscle wasting, brain atrophy,
atherosclerosis, arteriosclerosis, pulmonary emphysema,
osteoporosis, osteoarthritis, high blood pressure, erectile
dysfunction, dementia, Huntington's disease, Alzheimer's disease,
cataracts, age-related macular degeneration, prostate cancer,
stroke, diminished life expectancy, impaired kidney function, and
age-related hearing loss, aging-related mobility disability (e.g.,
frailty), cognitive decline, age-related dementia, memory
impairment, tendon stiffness, heart dysfunction such as cardiac
hypertrophy and systolic and diastolic dysfunction,
immunosenescence, cancer, obesity, and diabetes.
Antigens and Vaccines
[0343] The mTOR inhibitors, such as RAD001, described herein can be
used in combination with an antigen to enhance an immune response
to the antigen in a subject. The antigens selected for the methods
and compositions of the invention are not a limitation on this
invention. The antigen may be, without limitation, a whole cell, a
virus, a protein, a protein subunit or fragment. Examples of viral
antigens which may be enhanced by administration with an mTOR
inhibitor, include, without limitation, those derived from and/or
useful in treatment or prevention of HIV, meningitis and
encephalitis-causing viruses, Hepatitis A, Hepatitis B, Hepatitis
C, rabies virus, polio virus, influenza virus, measles virus, mumps
virus, rubella, pertussis, papilloma virus, yellow fever virus,
respiratory syncytial virus, parvovirus, chikungunya virus,
haemorrhagic fever viruses, and Herpes viruses, particularly,
varicella, cytomegalovirus and Epstein-Ban virus. Examples of
bacterial and mycobacterial antigens include those derived from
and/or useful against meningococcus, haemophilus, pneumococcus,
staphylococcus, leprosy and tuberculosis among others. Examples of
parasitic antigens include those derived from and/or useful against
such infections as toxoplasmosis, leishmaniasis and malaria. Still
other composition antigens include those derived from a protozoan,
e.g., T. cruzii, or against a helminth, e.g., Schistosoma. Still
other antigens useful in the methods described herein include those
derived from yeast or fungus such as Cryptococcus or Coccidioides.
Still other antigens useful in the methods described herein include
those derived from pathologic tissues such as tumors.
[0344] In particular an mTOR inhibitor such as RAD001 can be used
in combination with a vaccine against a viral or pathogenic agent,
such as an influenza vaccine, pneumococcal vaccine, or HIV vaccine.
More specifically, an mTOR inhibitor can be used as described
herein to enhance the immune response to, or adjuvant a vaccine for
any influenza strain, such as H1N1, H2N3, and B influenza
subtypes.
[0345] It is further anticipated that an mTOR inhibitor can be used
as an adjuvant in therapeutic vaccines for certain cancers and
solid tumors, and infectious diseases including, without
limitation, malaria, HIV, and influenza. Such a therapeutic vaccine
is used in a manner similar to that disclosed above for its use as
an adjuvant for vaccines containing antigens of a pathogenic
microorganism or virus. Particularly where the tumor antigen by
itself has been unsuccessful in activating a response to a
particular cancer, the use of an mTOR inhibitor as an adjuvant in a
cancer vaccine or therapeutic is encompassed by the present
invention. Cancer vaccines typically include an antigen expressed
on and isolated from a cancer cell or a cancer cell transfected
with, and capable of expressing, a selected antigen. For example,
any purified tumor antigen may be co-administered with an mTOR
inhibitor such as RAD001 as described above for pathogenic
vaccines. Identification of relevant cancer antigens will permit
the development of such vaccines. Alternatively, other cancer
therapeutics are designed using an antigen normally not expressed
on a cancer cell. For example, a selected antigen may be
transfected into the cancer cell and the transfected cell itself,
expressing the antigen, is used as the vaccine or therapeutic.
[0346] The ability of an mTOR inhibitor to provide an adjuvant
effect in a vaccine or to enhance an immune response to an antigen,
such as a vaccine antigen (e.g., influenza) can be measured using
methods well known in the art, such as, but not limited to an ELISA
assay and a hemagglutination inhibition assay (See, e.g., Lee et
al. Pediatr Infect Dis J. 2004 September; 23(9):852-6). Typically,
the enhancement of an immune response to an antigen by an mTOR
inhibitor can be determined by measuring titers of antibodies
against the antigen in the subject, wherein an increase in the
titer of antibodies directed against the particular antigen is
indicative of the mTOR inhibitor having enhanced the immune
response to the antigen.
[0347] When used as a vaccine adjuvant for a selected antigen, or
when used according to the methods described herein, an mTOR
inhibitor may be admixed as part of the antigen-containing
composition itself. Such a composition is desirably a vaccine
composition which contains a suitable carrier and, optionally,
other desired components. Selection of appropriate carriers, e.g.,
phosphate buffered saline and the like, are well within the skill
of those in the art. Similarly, one skilled in the art may readily
select appropriate stabilizers, preservatives, and the like for
inclusion in the composition. Any route of administration known in
the art may be employed for the administration of an antigen or
vaccine, e.g., subcutaneous, intraperitoneal, oral, intramuscular,
intranasal and the like.
[0348] Alternatively, the immunostimulatory effect of an mTOR
inhibitor may be obtained by administering the mTOR inhibitor
separately from the vaccine composition. When separately
administered, the mTOR inhibitor can be administered in a
formulation as described hereinabove. The mTOR inhibitor may be
administered contemporaneously with the vaccine composition, either
simultaneously therewith, or before or after the vaccine antigen
administration. If the mTOR inhibitor is administered before the
vaccine composition, it is desirable to administer it one or more
days before the vaccine. In one aspect, the mTOR inhibitor can be
administered for a period of time prior to administration of the
antigen. For example, the mTOR inhibitor can be administered for
1-7 days prior to administration of the vaccine, one week, two
weeks, three weeks, four weeks, five weeks, or six weeks or more
prior to administration of the antigen. In one aspect, the antigen
is administered immediately following administration of the mTOR
inhibitor. In another aspect, there can be a period of time between
administration of the mTOR inhibitor and administration of the
antigen. For example, the antigen may be administered 1-7 days
following administration of the mTOR inhibitor, or can be
administered one week, two weeks, three weeks or more following
administration of the mTOR inhibitor. In one aspect, the mTOR
inhibitor is administered to a subject for six weeks, followed by a
two week period in which the subject is given neither mTOR
inhibitor or antigen, followed by administration of the antigen.
When the mTOR inhibitor is administered as a separate component
from the vaccine, it is can be administered by the same route of
administration as the vaccine antigen, or it may be administered by
a different route, or any other route as selected by a physician.
In a further aspect of the foregoing dosing schedules,
administration of the mTOR inhibitor can continue after
administration of the antigen. For example, whether administered
prior to, or at the same time as the antigen, the mTOR inhibitor
can continue to be administered on a weekly or daily dosing
schedule as described herein for 1, 2, 3, 4, 5, 6, or 7 or more
days following administration of the antigen. The mTOR inhibitor
can continue to be administered for 1, 2, 3, 4, 5, or 6 weeks or
more following administration of the antigen.
Other Methods Utilizing mTOR Inhibitors
[0349] In one aspect, the present invention relates to the use of
low doses of an mTOR inhibitor in a method of enhancing an immune
response to an antigen in a subject. In one aspect, the immune
response to the antigen is enhanced by 1.2 fold when antigen
exposure is combined with a low dose of an mTOR inhibitor. In a
further aspect, the immune response to the antigen is enhanced by
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 or greater
when antigen exposure is combined with a low, immune enhancing,
dose of an mTOR inhibitor as described herein. In a further aspect
the mTOR inhibitor is an mTOR inhibitor described herein, e.g.,
RAD001, and is administered at a dose described herein, e.g., a
dose of 0.005-1.5 mg daily, 0.01-1 mg daily, 0.01-0.7 mg daily,
0.01-0.5 mg daily, or 0.1-0.5 mg daily or 0.1-20 mg weekly, 0.5-15
mg weekly, 1-10 mg weekly, or 3-7 mg weekly. In one aspect, the
mTOR inhibitor RAD001 is administered at a dose of 0.5 mg daily or
5 mg weekly. In each of the foregoing aspects, an mTOR inhibitor
other than RAD001 can be administered at a bioequivalent dose.
[0350] In a further aspect, the invention relates to methods for
enhancing the immune response to an antigen by administering an
amount of an mTOR inhibitor sufficient to inhibit P70 S6 kinase by
an amount described herein, e.g., by no greater than 80%. In a
further aspect, the mTOR inhibitor is sufficient to inhibit P70 S6
kinase by no greater than 38%. In one aspect, the mTOR inhibitor is
RAD001, rapamycin, a rapalog, or other mTOR inhibitor known in the
art, such as Ridaforolimus, emsirolimus. In a further aspect the
mTOR inhibitor may be a combination of two or more mTOR inhibitors.
The method includes the steps of administering to a subject an
antigen, such as, for example, a vaccine (e.g., influenza vaccine)
and an mTOR inhibitor such as RAD001.
[0351] In one aspect, the antigen is a vaccine antigen, and can
include, for example, influenza, pneumococcus, HIV, or other
vaccine antigens. In particular, the vaccine antigen can be an
influenza antigen such as H1N1, H2N3, and B influenza subtypes.
[0352] The present method of enhancing an immune response to an
antigen encompasses a method in which the immune response to an
antigen in a subject is increased. That is, where as a result of
the inclusion of an mTOR inhibitor with administration of the
antigen, there is an increase in protective immunity following
exposure to the antigen, where protective immunity refers to the
presence of sufficient antibody titers to protect against
subsequent infection by the same antigen. In addition, an enhanced
immune response to an antigen in response to treatment with an mTOR
inhibitor can mean that in a population, there is an increase in
the percentage of individuals that have protective immunity after
exposure to an antigen such as a vaccine.
[0353] In one aspect, an indicator of a suppressed or impaired
immune function/response is a reduced number of lymphocytes or
reduced lymphocyte function, such as a reduced response to
mitogenic stimulation. A human immune system can also be considered
to be impaired when (1) the ratio of Th/Ts is less than about 1.0,
(2) when the stimulation index to ConA is approximately 50% less
than "normal" or (2) when the stimulation index to PHA is
approximately 50% less than "normal" (See, e.g., EP0507872). A
human immune system can also be considered impaired when antigen
presentation and/or lymphocyte activation by macrophages and
dendritic cells is below what is seen in cells derived from a
healthy person less than 40 years of age, when the response of
lymphocytes to activating signals is less than what is seen in
lymphoctyes derived from a healthy person less than 40 years of
age, when the secretion of inflammatory cytokines is above what is
seen in a healthy person less than 40 years of age, when
lymphopoiesis by hematopoietic stem cells is below that of
hematopoietic stem cells from a person less than 40 years of age,
or when the percentage of PD1+CD4+ and/or CD8+ T cells is above the
percentage of PD1+CD4+ and/or CD8+ T cells in a person less than 40
years of age.
[0354] An impaired human immune response is frequently observed as
a secondary effect of such conditions as trauma, for example, from
an accident or from undergoing a major surgical procedure, from a
debilitating disease, such as cancer or infection with the HIV
virus (AIDS), or from malnutrition or old age. As a result of an
impaired immune response, patients are unable to respond to and
eliminate infectious agents, such as bacteria, viruses, and fungi,
from their bodies.
[0355] In one aspect the method of enhancing an immune response in
a subject also includes the step of first identifying a subject
with an impaired immune response. A subject with an impaired immune
response refers to a subject that is predicted not to get
protective antibody titer levels following prophylactic
vaccination, or in which a subject does not have a decrease in
disease burden after therapeutic vaccination. Methods for
determining antibody titers following vaccination and/or measuring
disease burden are well known in the art and may be routinely
performed by a physician or other medical professional.
[0356] For example, titer of an anti-influenza virus antibody can
be measured by hemagglutination inhibition (HI) assay. The HI assay
can be performed as described in Kendal, A P et al. (1982)
Concenpts and procedures for laboratory-based influenza
surveillance. Atlanta: Centers for Disease Control and Prevention
B17-B35 and below. A constant amount of hemagglutinating antigen
(HA) is added to each well of a microtiter plate. A test sample,
e.g., serum of a patient, is added to the first well and serially
diluted, e.g., two-fold, to desired dilution or number of wells.
RBCs are added to each well. The plate is incubated for an amount
of time sufficient for hemagglutination to occur, e.g., 1 hour. The
plate is then observed for wells with agglutinated RBCs (indicating
that there is insufficient antibody present to prevent
hemagglutination) or unagglutinated RBCs (indicating that there is
sufficient antibody present to prevent hemagglutination). The
highest dilution of test sample required to prevent
hemagglutination indicates the HI titer.
[0357] A subject can also be said to have an impaired immune
response if the subject is a member of a population known to have
decreased immune function such as the elderly, subjects undergoing
immunosuppressive or chemotherapy treatment, asplenic subjects,
immunocompromised subjects, or subjects having HIV/AIDS. That is, a
subject can be predicted to have an impaired immune response based
on their inclusion in a class of subjects typically associated with
impaired immune function. Such individuals may be deemed to have
impaired immune response without specific testing, or following
confirmation of an impaired immune response using methods routine
in the art. In addition a subject may be deemed to have an impaired
immune response if that subject has a history of decreased immune
function, such as a history of an inability to establish protective
immunity after vaccination or exposure to an antigen.
[0358] Once a subject is identified as having an impaired immune
response, the subject can be treated with an mTOR inhibitor in the
context of vaccination and/or exposure to antigen as described
herein.
[0359] In addition, in a further aspect, the invention relates to
methods for treating immunosenescence in a subject by administering
to the subject an amount of an mTOR inhibitor effective to increase
the immune response to an antigen (e.g., a vaccine antigen) so that
protective antibody titers or T cell response to the antigen are
achieved. In one aspect, the invention provides a method for
treating immunosenescence in a subject by administering low doses
of an mTOR inhibitor such as RAD001. The mTOR inhibitor RAD001 can
administered at a dose described herein, e.g., a dose of about
0.005-1.5 mg daily, about 0.01-1 mg daily, about 0.01-0.7 mg daily,
about 0.01-0.5 mg daily, or about 0.1-0.5 mg daily or about 0.1-20
mg weekly, about 0.5-15 mg weekly, about 1-10 mg weekly, or about
3-7 mg weekly. In one aspect, RAD001 is administered at a dose of
0.5 mg daily or 5 mg weekly. In a further embodiment of the
foregoing, the mTOR inhibitor can be an inhibitor other than RAD001
administered at a dose that is bioequivalent to the doses of RAD001
indicated above. In a further aspect, the invention relates to
methods of treating immunosenescence in a subject by administering
an amount of an mTOR inhibitor sufficient to inhibit P70 S6 kinase
by no greater than 80%. In a further aspect, the mTOR inhibitor is
sufficient to inhibit P70 S6 kinase by no greater than 38%. In one
aspect, the mTOR inhibitor is RAD001, rapamycin, a rapalog, or
other mTOR inhibitor known in the art. In a further aspect the mTOR
inhibitor may be a combination of two or more mTOR inhibitors.
[0360] Immunosenescence refers to a decrease in immune function
associated with age resulting in impaired response to vaccination
and infectious pathogens. It involves both the host's capacity to
respond to infections and the development of long-term immune
memory, especially by vaccination. This age-associated immune
deficiency is ubiquitous and found in both long- and short-lived
species as a function of their age relative to life expectancy
rather than chronological time. It is considered a major
contributory factor to the increased frequency of morbidity and
mortality among the elderly. Immunosenescence is not a random
deteriorative phenomenon, rather it appears to inversely repeat an
evolutionary pattern and most of the parameters affected by
immunosenescence appear to be under genetic control.
Immunosenescence can also be sometimes envisaged as the result of
the continuous challenge of the unavoidable exposure to a variety
of antigens such as viruses and bacteria. Immunosenescence is a
multifactorial condition leading to many pathologically significant
health problems in the aged population. Age-dependent biological
changes such as depletion of hematopoietic stem cells, decline in
the total number of phagocytes and NK cells and a decline in
humoral immunity contribute to the onset of immunosenescence and
may be used as indicators of the onset or presence of
immunosenescence. In one aspect, immunosenescence can be measured
in an individual by measuring telomere length in immune cells (See,
e.g., U.S. Pat. No. 5,741,677). Immunosenescence can also be
measured in an individual by measuring the number of naive CD4
and/or CD8 T cells, by measuring T cell repertoire, by measuring
percentage of PD1+CD4 and CD8 T cells, or by measuring the response
to vaccination in a subject over the age of 65. In a further
aspect, the invention relates to methods for the treatment of an
age related condition in a subject by administering to the subject
the mTOR inhibitor RAD001 at a dose of about 0.005-1.5 mg daily,
about 0.01-1 mg daily, about 0.01-0.7 mg daily, about 0.01-0.5 mg
daily, or about 0.1-0.5 mg daily or about 0.1-20 mg weekly, about
0.5-15 mg weekly, about 1-10 mg weekly, or about 3-7 mg weekly. In
one aspect, the mTOR inhibitor is administered at a dose of about
0.5 mg daily or about 5 mg weekly. In one aspect the mTOR inhibitor
can be an mTOR inhibitor other than RAD001 administered at a dose
that is bioequivalent to the specified doses of RAD001. In a
further aspect, the invention relates to a method of treating an
age related condition in a subject by administering an amount of an
mTOR inhibitor sufficient to inhibit P70 S6 kinase by no greater
than 80%. In a further aspect, the mTOR inhibitor is sufficient to
inhibit P70 S6 kinase by no greater than 38%. In one aspect, the
mTOR inhibitor is RAD001, rapamycin, a rapalog, or other mTOR
inhibitor known in the art. In a further aspect the mTOR inhibitor
may be a combination of two or more mTOR inhibitors.
[0361] An age-related condition can be any disease, disorder, or
pathology whose incidence in a population or severity in an
individual correlates with the progression of age. More
specifically, an age-related condition is a disease, disorder, or
pathology whose incidence is at least 1.5 fold higher among human
individuals greater than 60 years of age relative to human
individuals between the ages of 30-40 and in a selected population
of greater than 100,000 individuals. Age-related conditions
relevant to the present invention include, but are not limited to
sarcopenia, skin atrophy, muscle wasting, brain atrophy,
atherosclerosis, arteriosclerosis, pulmonary emphysema,
osteoporosis, osteoarthritis, high blood pressure, erectile
dysfunction, dementia, Huntington's disease, Alzheimer's disease,
cataracts, age-related macular degeneration, prostate cancer,
stroke, diminished life expectancy, impaired kidney function, and
age-related hearing loss, aging-related mobility disability (e.g.,
frailty), cognitive decline, age-related dementia, memory
impairment, tendon stiffness, heart dysfunction such as cardiac
hypertrophy and systolic and diastolic dysfunction,
immunosenescence, cancer, and diabetes.
[0362] The treatment of an age-related condition using the mTOR
inhibitors described herein may be complete, e.g., the total
absence of an age-related condition or metabolic disorder. The
prevention may also be partial, such that the likelihood of the
occurrence of the age-related condition or metabolic disorder in a
subject is less likely to occur than had the subject not received
an mTOR inhibitor of the present disclosure. Methods for measuring
the effectiveness of an mTOR inhibitor in the treatment of an
age-related condition described herein are known in the art and
examples of such methods may be found in U.S. Pat. No.
8,420,088.
Combination Treatments
[0363] In some embodiments, it may be advantageous to administer an
mTOR inhibitor, e.g., an mTOR inhibitor described herein, at a low,
immune enhancing, dose with one or more therapeutic agents
(pharmaceutical combinations). For example, synergistic effects can
occur with other immunostimulatory, anti-infective, anti-tumor or
anti-proliferative agents, for example, mitotic inhibitors,
alkylating agents, anti-metabolites, intercalating antibiotics,
growth factor inhibitors (e.g., trastuzumab, panitumumab,
cetuximab, gefitinib, erlotinib, lapatinib, sorafenib, etc.), cell
cycle inhibitors, enzymes, topoisomerase inhibitors, biological
response modifiers, antibodies, cytotoxics, bronchodilators,
anti-hormones, anti-androgens, an anti-angiogenesis agent, kinase
inhibitor, pan kinase inhibitor or growth factor inhibitor. Other
suitable therapeutic agents are listed in the Physicians' Desk
Reference. Where the compounds of the invention are administered in
conjunction with other therapies, dosages of the co-administered
compounds will of course vary depending on the type of co-drug
employed, on the specific drug employed, on the condition being
treated and so forth.
[0364] Accordingly, an mTOR inhibitor, e.g., an mTOR inhibitor
described herein, may be used at low, immune enhancing, dose in
combination with other known agents and therapies. Administered "in
combination", as used herein, means that two (or more) different
treatments are delivered to the subject during the course of the
subject's affliction with the disorder, e.g., the two or more
treatments are delivered after the subject has been diagnosed with
the disorder and before the disorder has been cured or eliminated
or treatment has ceased for other reasons. In some embodiments, the
delivery of one treatment is still occurring when the delivery of
the second begins, so that there is overlap in terms of
administration. This is sometimes referred to herein as
"simultaneous" or "concurrent delivery". In other embodiments, the
delivery of one treatment ends before the delivery of the other
treatment begins. In some embodiments of either case, the treatment
is more effective because of combined administration. For example,
the second treatment is more effective, e.g., an equivalent effect
is seen with less of the second treatment, or the second treatment
reduces symptoms to a greater extent, than would be seen if the
second treatment were administered in the absence of the first
treatment, or the analogous situation is seen with the first
treatment. In some embodiments, delivery is such that the reduction
in a symptom, or other parameter related to the disorder is greater
than what would be observed with one treatment delivered in the
absence of the other. The effect of the two treatments can be
partially additive, wholly additive, or greater than additive. The
delivery can be such that an effect of the first treatment
delivered is still detectable when the second is delivered.
[0365] An mTOR inhibitor, e.g., an mTOR inhibitor described herein,
at low, immune enhancing, dose, and the at least one additional
therapeutic agent can be administered simultaneously, in the same
or in separate compositions, or sequentially. For sequential
administration, the mTOR inhibitor can be administered first, and
the additional agent can be administered second, or the order of
administration can be reversed. In some embodiments, the mTOR
inhibitor is administered as a pretreatment, e.g., 1 week, 2 weeks,
3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10
weeks, 11 weeks, 12 weeks or more, before treatment with the at
least one additional therapeutic agent.
[0366] In some embodiments, an mTOR inhibitor, e.g., an mTOR
inhibitor described herein, is administered at low, immune
enhancing, dose to a subject who has cancer, e.g., a cancer
described herein. The subject may receive treatment with an
additional therapeutic agent, such as an approved drug for that
type of cancer, in combination with the mTOR inhibitor. For
example, Table 1 below provides a list of various cancers and their
approved treatments.
TABLE-US-00001 TABLE 1 Cancers and Approved Treatment(s) Cancer
Treatment(s) Acute Lymphoblastic Abitrexate (Methotrexate);
Adriamycin Leukemia PFS (Doxorubicin Hydrochloride); Adriamycin RDF
(Doxorubicin Hydrochloride); Arranon (Nelarabine); Asparaginase
Erwinia chrysanthemi; Cerubidine (Daunorubicin Hydrochloride);
Clafen (Cyclophosphamide); Clofarabine; Clofarex (Clofarabine);
Clolar (Clofarabine); Cyclophosphamide; Cytarabine; Cytosar-U
(Cytarabine); Cytoxan (Cyclophosphamide); Dasatinib; Daunorubicin
Hydrochloride; Doxorubicin Hydrochloride; Erwinaze (Asparaginase
Erwinia Chrysanthemi); Folex (Methotrexate); Folex PFS
(Methotrexate); Gleevec (Imatinib Mesylate); Iclusig (Ponatinib
Hydrochloride); Imatinib Mesylate; Marqibo (Vincristine Sulfate
Liposome); Mercaptopurine; Methotrexate; Methotrexate LPF
(Methorexate); Mexate (Methotrexate); Mexate-AQ (Methotrexate);
Nelarabine; Neosar (Cyclophosphamide); Oncaspar (Pegaspargase);
Pegaspargase; Purinethol (Mercaptopurine); Purixan
(Mercaptopurine); Rubidomycin (Daunorubicin Hydrochloride); Sprycel
(Dasatinib); Tarabine PFS (Cytarabine); Vincasar PFS (Vincristine
Sulfate); Vincristine Sulfate; or Vincristine Sulfate Liposome.
DRUG COMBINATIONS hyper-CVAD: Cyclophosphamide; Vincristine
Sulfate; Doxorubicin Hydrochloride (Adriamycin); Dexamethasone.
Acute Myeloid Adriamycin PFS (Doxorubicin Hydrochloride); Leukemia
Adriamycin RDF (Doxorubicin Hydrochloride); Arsenic Trioxide;
Cerubidine (Daunorubicin Hydrochloride); Clafen (Cyclophosphamide);
Cyclophosphamide; Cytarabine; Cytosar-U (Cytarabine); Cytoxan
(Cyclophosphamide); Daunorubicin Hydrochloride; Doxorubicin
Hydrochloride; Neosar (Cyclophosphamide); Rubidomycin (Daunorubicin
Hydrochloride); Tarabine PFS (Cytarabine); Trisenox (Arsenic
Trioxide); Vincasar PFS (Vincristine Sulfate); or Vincristine
Sulfate. DRUG COMBINATIONS ADE: Cytarabine; Daunorubicin
Hydrochloride; and Etoposide. AIDS-Related Kaposi Dox-SL
(Doxorubicin Hydrochloride Sarcoma Liposome); Doxil (Doxorubicin
Hydrochloride Liposome); Doxorubicin Hydrochloride Liposome; Evacet
(Doxorubicin Hydrochloride Liposome); Intron A (Recombinant
Interferon Alfa-2b); LipoDox (Doxorubicin Hydrochloride Liposome);
Paclitaxel; Recombinant Interferon Alfa- 2b; Taxol (Paclitaxel);
Velban (Vinblastine Sulfate); Velsar (Vinblastine Sulfate); or
Vinblastine Sulfate. Basal Cell Carcinoma Adrucil (Fluorouracil);
Aldara (Imiquimod); Efudex (Fluorouracil); Erivedge (Vismodegib);
Fluoroplex (Fluorouracil); Fluorouracil; Imiquimod; or Vismodegib.
Bladder Cancer Adriamycin PFS (Doxorubicin Hydrochloride);
Adriamycin RDF (Doxorubicin Hydrochloride); Cisplatin; Doxorubicin
Hydrochloride; Platinol (Cisplatin); or Platinol-AQ (Cisplatin).
Bone Cancer Abitrexate (Methotrexate); Adriamycin PFS (Doxorubicin
Hydrochloride); Adriamycin RDF (Doxorubicin Hydrochloride);
Doxorubicin Hydrochloride; Folex (Methotrexate); Folex PFS
(Methotrexate); Methotrexate; Methotrexate LPF (Methotrexate);
Mexate (Methotrexate); or Mexate-AQ (Methotrexate). Brain Tumor
Afinitor (Everolimus); Afinitor Disperz (Everolimus); Avastin
(Bevacizumab); Bevacizumab; CeeNu (Lomustine); Everolimus;
Lomustine; Methazolastone (Temozolomide); Temodar (Temozolomide);
or Temozolomide. Breast Cancer Abitrexate (Methotrexate); Abraxane
(Paclitaxel Albumin-stabilized Nanoparticle Formulation);
Ado-Trastuzumab Emtansine; Adriamycin PFS (Doxorubicin
Hydrochloride); Adriamycin RDF (Doxorubicin Hydrochloride); Adrucil
(Fluorouracil); Afinitor (Everolimus); Anastrozole; Aredia
(Pamidronate Disodium); Arimidex (Anastrozole); Aromasin
(Exemestane); Capecitabine; Clafen (Cyclophosphamide);
Cyclophosphamide; Cytoxan (Cyclophosphamide); Docetaxel;
Doxorubicin Hydrochloride; Efudex (Fluorouracil); Ellence
(Epirubicin Hydrochloride); Epirubicin Hydrochloride; Everolimus;
Exemestane; Fareston (Toremifene); Faslodex (Fulvestrant); Femara
(Letrozole); Fluoroplex (Fluorouracil); Fluorouracil; Folex
(Methotrexate); Folex PFS (Methotrexate); Fulvestrant; Gemcitabine
Hydrochloride; Gemzar (Gemcitabine Hydrochloride); Goserelin
Acetate; Herceptin (Trastuzumab); Ixabepilone; Ixempra
(Ixabepilone); Kadcyla (Ado- Trastuzumab Emtansine); Lapatinib
Ditosylate; Letrozole; Megace (Megestrol Acetate); Megestrol
Acetate; Methotrexate; Methotrexate LPF (Methotrexate); Mexate
(Methotrexate); Mexate-AQ (Methotrexate); Neosar
(Cyclophosphamide); Nolvadex (Tamoxifen Citrate); Novaldex
(Tamoxifen Citrate); Paclitaxel; Paclitaxel Albumin-stabilized
Nanoparticle Formulation; Pamidronate Disodium; Perjeta
(Pertuzumab); Pertuzumab; Tamoxifen Citrate; Taxol (Paclitaxel);
Taxotere (Docetaxel); Trastuzumab; Toremifene; Tykerb (Lapatinib
Ditosylate); Xeloda (Capecitabine); or Zoladex (Goserelin Acetate).
DRUG COMBINATIONS AC: Doxorubicin Hydrochloride (Adriamycin) and
Cyclophosphamide. AC-T: Doxorubicin Hydrochloride (Adriamycin);
Cyclophosphamide; and Paclitaxel (Taxol). CAF: Cyclophosphamide;
Doxorubicin Hydrochloride (Adriamycin); and Fluorouracil. CMF:
Cyclophosphamide; Methotrexate; and Fluorouracil. FEC:
Fluorouracil; Epirubicin Hydrochloride; and Cyclophosphamide. TAC:
Docetaxel (Taxotere); Doxorubicin Hydrochloride (Adriamycin); and
Cyclophosphamide. Cervical Cancer Blenoxane (Bleomycin); Bleomycin;
Cisplatin; Hycamtin (Topotecan Hydrochloride); Platinol
(Cisplatin); Platinol-AQ (Cisplatin); or Topotecan Hydrochloride.
DRUG COMBINATIONS Gemcitabine-Cisplatin: Gemcitabine Hydrochloride
and Cisplatin. Chronic Lymphocytic Alemtuzumab; Ambochlorin
Leukemia (Chlorambucil); Amboclorin (Chlorambucil); Arzerra
(Ofatumumab); Bendamustine Hydrochloride; Campath (Alemtuzumab);
Chlorambucil; Clafen (Cyclophosphamide); Cyclophosphamide; Cytoxan
(Cyclophosphamide); Fludara (Fludarabine Phosphate); Fludarabine
Phosphate; Gazyva (Obinutuzumab); Ibrutinib; Imbruvica (Ibrutinib);
Leukeran (Chlorambucil); Linfolizin (Chlorambucil); Neosar
(Cyclophosphamide); Obinutuzumab; Ofatumumab; or Treanda
(Bendamustine Hydrochloride). DRUG COMBINATIONS
CHLORAMBUCIL-PREDNISONE: Chlorambucil and Prednisone. CVP:
Cyclophosphamide; Vincristine Sulfate; and Prednisone. Chronic
Myelogenous Bosulif (Bosutinib); Bosutinib; Leukemia Busulfan;
Busulfex (Busulfan); Clafen; Cyclophosphamide); Cyclophosphamide;
Cytarabine; Cytosar-U (Cytarabine); Cytoxan (Cyclophosphamide);
Dasatinib; Gleevec (Imatinib Mesylate); Iclusig (Ponatinib
Hydrochloride); Imatinib Mesylate; Myleran (Busulfan); Neosar
(Cyclophosphamide); Nilotinib; Omacetaxine Mepesuccinate; Ponatinib
Hydrochloride; Sprycel (Dasatinib); Synribo (Omacetaxine
Mepesuccinate); Tarabine PFS (Cytarabine); or Tasigna (Nilotinib).
Colon Cancer Adrucil (Fluorouracil); Avastin (Bevacizumab);
Bevacizumab; Camptosar (Irinotecan Hydrochloride); Capecitabine;
Cetuximab; Efudex (Fluorouracil); Eloxatin (Oxaliplatin); Erbitux
(Cetuximab); Fluoroplex (Fluorouracil); Fluorouracil; Irinotecan
Hydrochloride; Leucovorin Calcium; Oxaliplatin; Panitumumab;
Regorafenib; Stivarga (Regorafenib); Vectibix (Panitumumab);
Wellcovorin (Leucovorin Calcium); Xeloda (Capecitabine); Zaltrap
(Ziv- Aflibercept); or Ziv-Aflibercept. DRUG COMBINATIONS CAPDX:
Capecitabine and Oxaliplatin. FOLFIRI: Leucovorin Calcium (Folinic
Acid); Fluorouracil; and Irinotecan Hydrochloride.
FOLFIRI-BEVACIZUMAB: Leucovorin Calcium (Folinic Acid);
Fluorouracil; Irinotecan Hydrochloride; and Bevacizumab.
FOLFIRI-CETUXIMAB: Leucovorin Calcium (Folinic Acid); Fluorouracil;
Irinotecan Hydrochloride; and Cetuximab. FOLFOX: Leucovorin Calcium
(Folinic Acid); Fluorouracil; and Oxaliplatin. XELOX:
Capecitabine
(Xeloda) and Oxaliplatin. Endometrial Cancer Megace (Megestrol
Acetate) or Megestrol Acetate. Gastric (Stomach) Adriamycin PFS
(Doxorubicin Cancer Hydrochloride); Adriamycin RDF (Doxorubicin
Hydrochloride); Adrucil (Fluorouracil); Cyramza (Ramucirumab);
Docetaxel; Doxorubicin Hydrochloride; Efudex (Fluorouracil);
Fluoroplex (Fluorouracil); Fluorouracil; Herceptin (Trastuzumab);
Mitomycin C; Mitozytrex (Mitomycin C); Mutamycin (Mitomycin C);
Ramucirumab; Taxotere (Docetaxel); or Trastuzumab. Gastrointestinal
stromal Gleevec (Imatinib Mesylate); Imatinib tumors Mesylate;
Regorafenib; Stivarga (Regorafenib); Sunitinib Malate; Sutent
(Sunitinib Malate) Head and neck cancer Abitrexate (Methotrexate);
Adrucil (Fluorouracil); Blenoxane (Bleomycin); Bleomycin;
Cetuximab; Cisplatin; Docetaxel; Efudex (Fluorouracil); Erbitux
(Cetuximab); Fluoroplex (Fluorouracil); Fluorouracil; Folex
(Methotrexate); Folex PFS (Methotrexate); Methotrexate;
Methotrexate LPF (Methotrexate); Mexate (Methotrexate); Mexate-AQ
(Methotrexate); Platinol (Cisplatin); Platinol-AQ (Cisplatin); or
Taxotere (Docetaxel). Hodkin Lymphoma Adcetris (Brentuximab
Vedotin); Adriamycin PFS (Doxorubicin Hydrochloride); Adriamycin
RDF (Doxorubicin Hydrochloride); Ambochlorin (Chlorambucil);
Amboclorin (Chlorambucil); Blenoxane (Bleomycin); Bleomycin;
Brentuximab Vedotin; Chlorambucil; Clafen (Cyclophosphamide);
Cyclophosphamide; Cytoxan (Cyclophosphamide); Dacarbazine;
Doxorubicin Hydrochloride; DTIC-Dome (Dacarbazine); Leukeran
(Chlorambucil); Linfolizin (Chlorambucil); Lomustine; Matulane
(Procarbazine Hydrochloride); Neosar (Cyclophosphamide);
Procarbazine Hydrochloride; Velban (Vinblastine Sulfate); Velsar
(Vinblastine Sulfate); Vinblastine Sulfate; Vincasar PFS
(Vincristine Sulfate); or Vincristine Sulfate. DRUG COMBINATIONS:
ABVD: Doxorubicin Hydrochloride (Adriamycin); Bleomycin;
Vinblastine Sulfate; and Dacarbazine. ABVE: Doxorubicin
Hydrochloride (Adriamycin); Bleomycin; Vinblastine Sulfate; and
Etoposide. ABVE-PC: Doxorubicin Hydrochloride (Adriamycin);
Bleomycin; Vinblastine Sulfate; Etoposide; Prednisone; and
Cyclophosphamide. BEACOPP: Bleomycin; Etoposide; Doxorubicin
Hydrochloride (Adriamycin); Cyclophosphamide; Vincristine Sulfate
(Oncovin); Procarbazine Hydrochloride; and Prednisone. COPP:
Cyclophosphamide; Vincristine Sulfate (Oncovin); Procarbazine
Hydrochloride; and Prednisone. COPP-ABV: Cyclophosphamide;
Vincristine Sulfate (Oncovin); Procarbazine Hydrochloride;
Prednisone; Doxorubicin Hydrochloride (Adriamycin); Bleomycin; and
Vinblastine Sulfate. ICE: Ifosfamide; Carboplatin; and Etoposide.
MOPP: Mechlorethamine Hydrochloride; Vincristine Sulfate (Oncovin);
Procarbazine Hydrochloride; and Prednisone. OEPA: Vincristine
Sulfate (Oncovin); Etoposide; Prednisone; and Doxorubicin
Hydrochloride (Adriamycin). OPPA: Vincristine Sulfate (Oncovin);
Procarbazine Hydrochloride; Prednisone; and Doxorubicin
Hydrochloride (Adriamycin). STANFORD V: Mechlorethamine
Hydrochloride; Doxorubicin Hydrochloride; Vinblastine Sulfate;
Vincristine Sulfate; Bleomycin; Etoposide; and Prednisone. VAMP:
Vincristine Sulfate; Doxorubicin Hydrochloride (Adriamycin); and
Methotrexate; and Prednisone. Kidney (Renal Cell) Afinitor
(Everolimus); Aldesleukin; Cancer Avastin (Bevacizumab); Axitinib;
Bevacizumab; Everolimus; Inlyta (Axitinib); Nexavar (Sorafenib
Tosylate); Pazopanib Hydrochloride; Proleukin (Aldesleukin);
Sorafenib Tosylate; Sunitinib Malate; Sutent (Sunitinib Malate);
Temsirolimus; Torisel (Temsirolimus); or Votrient (Pazopanib
Hydrochloride). Liver Cancer Nexavar (Sorafenib Tosylate) or
Sorafenib Tosylate. Melanoma Aldesleukin; Dabrafenib; Dacarbazine;
DTIC-Dome (Dacarbazine); Intron A (Recombinant Interferon Alfa-2b);
Ipilimumab; Mekinist (Trametinib); Peginterferon Alfa-2b;
PEG-Intron (Peginterferon Alfa- 2b); Proleukin (Aldesleukin);
Recombinant Interferon Alfa-2b; Sylatron (Peginterferon Alfa-2b);
Tafinlar (Dabrafenib); Trametinib; Vemurafenib; Yervoy
(Ipilimumab); or Zelboraf (Vemurafenib). Malignant Alimta
(Pemetrexed Disodium); Mesothelioma Cisplatin; Pemetrexed Disodium;
Platinol (Cisplatin); or Platinol-AQ (Cisplatin). Multiple myeloma
Aredia (Pamidronate Disodium); Bortezomib; Carfilzomib; Clafen
(Cyclophosphamide); Cyclophosphamide; Cytoxan (Cyclophosphamide);
Doxil (Doxorubicin Hydrochloride Liposome); Doxorubicin
Hydrochloride Liposome; Dox-SL (Doxorubicin Hydrochloride
Liposome); Evacet (Doxorubicin Hydrochloride Liposome); Kyprolis
(Carfilzomib); Lenalidomide; LipoDox (Doxorubicin Hydrochloride
Liposome); Mozobil (Plerixafor); Neosar (Cyclophosphamide);
Pamidronate Disodium; Plerixafor; Pomalidomide (Pomalyst);
Pomalyst; Revlimid (Lenalidomide); Synovir (Thalidomide);
Thalidomide; Thalomid (Thalidomide); Velcade (Bortezomib);
Zoledronic Acid; Zometa (Zoledronic Acid) Myeloproliferative
Adriamycin PFS (Doxorubicin Disorders Hydrochloride); Adriamycin
RDF (Doxorubicin Hydrochloride); Arsenic Trioxide; Azacitidine;
Cerubidine (Daunorubicin Hydrochloride); Clafen (Cyclophosphamide);
Cyclophosphamide; Cytarabine; Cytosar-U (Cytarabine); Cytarabine;
Cytoxan (Cyclophosphamide); Dacogen (Decitabine); Dasatinib;
Daunorubicin Hydrochloride; Decitabine; Doxorubicin Hydrochloride;
Gleevec (Imatinib Mesylate); Imatinib Mesylate; Jakafi (Ruxolitinib
Phosphate); Lenalidomide; Mylosar (Azacitidine); Neosar
(Cyclophosphamide); Nilotinib; Revlimid (Lenalidomide); Rubidomycin
(Daunorubicin Hydrochloride); Ruxolitinib Phosphate; Sprycel
(Dasatinib); Tarabine PFS (Cytarabine); Tasigna (Nilotinib);
Trisenox (Arsenic Trioxide); Vidaza (Azacitidine); Vincasar PFS
(Vincristine Sulfate); or Vincristine Sulfate. DRUG COMBINATIONS
ADE: Cytarabine; Daunorubicin Hydrochloride; and Etoposide.
Neuroblastoma Adriamycin PFS (Doxorubicin Hydrochloride);
Adriamycin RDF (Doxorubicin Hydrochloride); Clafen
(Cyclophosphamide); Cyclophosphamide; Cytoxan (Cyclophosphamide);
Doxorubicin Hydrochloride; Neosar (Cyclophosphamide); Vincasar PFS
(Vincristine Sulfate); or Vincristine Sulfate. Non-Hodkin Lymphoma
Abitrexate (Methotrexate); Adcetris (Brentuximab Vedotin);
Adriamycin PFS (Doxorubicin Hydrochloride); Adriamycin RDF
(Doxorubicin Hydrochloride); Ambochlorin (Chlorambucil); Amboclorin
(Chlorambucil); Arranon (Nelarabine); Bendamustine Hydrochloride;
Bexxar (Tositumomab and Iodine I 131 Tositumomab); Blenoxane
(Bleomycin); Bleomycin; Bortezomib; Brentuximab Vedotin;
Chlorambucil; Clafen (Cyclophosphamide); Cyclophosphamide; Cytoxan
(Cyclophosphamide); Denileukin Diftitox; DepoCyt (Liposomal
Cytarabine); Doxorubicin Hydrochloride; DTIC-Dome (Dacarbazine);
Folex (Methotrexate); Folex PFS (Methotrexate); Folotyn
(Pralatrexate); Ibritumomab Tiuxetan; Ibrutinib; Imbruvica
(Ibrutinib); Intron A (Recombinant Interferon Alfa-2b); Istodax
(Romidepsin); Lenalidomide; Leukeran (Chlorambucil); Linfolizin
(Chlorambucil); Liposomal Cytarabine; Matulane (Procarbazine
Hydrochloride); Methotrexate; Methotrexate LPF (Methotrexate);
Mexate (Methotrexate); Mexate-AQ (Methotrexate); Mozobil
(Plerixafor); Nelarabine; Neosar (Cyclophosphamide); Ontak
(Denileukin Diftitox); Plerixafor; Pralatrexate; Recombinant
Interferon Alfa-2b; Revlimid (Lenalidomide); Rituxan (Rituximab);
Rituximab; Romidepsin; Tositumomab and Iodine I 131 Tositumomab;
Treanda (Bendamustine Hydrochloride); Velban (Vinblastine Sulfate);
Velcade (Bortezomib); Velsar (Vinblastine Sulfate); Vinblastine
Sulfate; Vincasar PFS (Vincristine Sulfate); Vincristine Sulfate;
Vorinostat; Zevalin (Ibritumomab Tiuxetan); or Zolinza
(Vorinostat). DRUG COMBINATIONS CHOP: Cyclophosphamide; Doxorubicin
Hydrochloride
(Hydroxydaunomycin); Vincristine Sulfate (Oncovin); and Prednisone.
COPP: Cyclophosphamide; Vincristine Sulfate (Oncovin); Procarbazine
Hydrochloride; and Prednisone. CVP: Cyclophosphamide; Vincristine
Sulfate; and Prednisone. EPOCH: Etoposide; Prednisone; Vincristine
Sulfate (Oncovin); Cyclophosphamide; and Doxorubicin Hydrochloride
(Hydroxydaunomycin). Hyper-CVAD: Cyclophosphamide; Vincristine
Sulfate; Doxorubicin Hydrochloride (Adriamycin); and Dexamethasone.
ICE: Ifosfamide; Carboplatin; and Etoposide. R-CHOP: Rituximab;
Cyclophosphamide; Doxorubicin Hydrochloride (Hydroxydaunomycin);
Vincristine Sulfate (Oncovin); and Prednisone. Non-Small Cell Lung
Abitrexate (Methotrexate); Abraxane Cancer (Paclitaxel
Albumin-stabilized Nanoparticle Formulation); Afatinib Dimaleate;
Alimta (Pemetrexed Disodium); Avastin (Bevacizumab); Bevacizumab;
Carboplatin; Ceritinib; Cisplatin; Crizotinib; Docetaxel; Erlotinib
Hydrochloride; Folex (Methotrexate); Folex PFS (Methotrexate);
Gefitinib; Gilotrif (Afatinib Dimaleate); Gemcitabine
Hydrochloride; Gemzar (Gemcitabine Hydrochloride); Iressa
(Gefitinib); Methotrexate; Methotrexate LPF (Methotrexate); Mexate
(Methotrexate); Mexate- AQ (Methotrexate); Paclitaxel; Paclitaxel
Albumin-stabilized Nanoparticle Formulation; Paraplat
(Carboplatin); Paraplatin (Carboplatin); Pemetrexed Disodium;
Platinol (Cisplatin); Platinol- AQ (Cisplatin); Tarceva (Erlotinib
Hydrochloride); Taxol (Paclitaxel); Taxotere (Docetaxel); Xalkori
(Crizotinib); or Zykadia (Ceritinib). DRUG COMBINATIONS
CARBOPLATIN-TAXOL; Carboplatin and Paclitaxel (Taxol).
Gemcitabine-Cisplatin: Gemcitabine Hydrochloride and Cisplatin.
Ovarian Cancer Adriamycin PFS (Doxorubicin Hydrochloride);
Adriamycin RDF (Doxorubicin Hydrochloride); Carboplatin; Clafen
(Cyclophosphamide); Cisplatin; Cyclophosphamide; Cytoxan
(Cyclophosphamide); Doxorubicin Hydrochloride; Dox-SL (Doxorubicin
Hydrochloride Liposome); DOXIL (Doxorubicin Hydrochloride
Liposome); Doxorubicin Hydrochloride Liposome; Evacet (Doxorubicin
Hydrochloride Liposome); Gemcitabine Hydrochloride; Gemzar
(Gemcitabine Hydrochloride); Hycamtin (Topotecan Hydrochloride);
LipoDox (Doxorubicin Hydrochloride Liposome); Neosar
(Cyclophosphamide); Paclitaxel; Paraplat (Carboplatin); Paraplatin
(Carboplatin); Platinol (Cisplatin); Platinol-AQ (Cisplatin); Taxol
(Paclitaxel); or Topotecan Hydrochloride. DRUG COMBINATIONS BEP:
Bleomycin; Etoposide; and Cisplatin (Platinol). CARBOPLATIN-TAXOL:
Carboplatin and Paclitaxel (Taxol). Gemcitabine-Cisplatin:
Gemcitabine Hydrochloride and Cisplatin. Pancreatic cancer Adrucil
(Fluorouracil); Afinitor (Everolimus); Efudex (Fluorouracil);
Erlotinib Hydrochloride; Everolimus; Fluoroplex (Fluorouracil);
Fluorouracil; Gemcitabine Hydrochloride; Gemzar (Gemcitabine
Hydrochloride); Mitomycin C; Mitozytrex (Mitomycin C); Mutamycin
(Mitomycin C); Sunitinib Malate; Sutent (Sunitinib Malate); or
Tarceva (Erlotinib Hydrochloride). DRUG COMBINATIONS
GEMCITABINE-OXALIPLATIN: Gemcitabine Hydrochloride and Oxaliplatin.
Penile cancer Blenoxane (Bleomycin); Bleomycin Rectal Cancer
Adrucil (Fluorouracil); Avastin (Bevacizumab); Bevacizumab;
Camptosar (Irinotecan Hydrochloride); Cetuximab; Efudex
(Fluorouracil); Erbitux (Cetuximab); Fluoroplex (Fluorouracil);
Fluorouracil; Irinotecan Hydrochloride; Panitumumab; Regorafenib;
Stivarga (Regorafenib); Vectibix (Panitumumab); Zaltrap (Ziv-
Aflibercept); or Ziv-Aflibercept. DRUG COMBINATIONS CAPOX:
Capecitabine and Oxaliplatin. FOLFIRI: Leucovorin Calcium (Folinic
Acid); FluorouracilL; Irinotecan Hydrochloride.
FOLFIRI-BEVACIZUMAB: Leucovorin Calcium (Folinic Acid);
Fluorouracil; Irinotecan Hydrochloride; and Bevacizumab.
FOLFIRI-CETUXIMAB: Leucovorin Calcium (Folinic Acid); Fluorouracil;
Irinotecan Hydrochloride; and Cetuximab. FOLFOX: Leucovorin Calcium
(Folinic Acid); Fluorouracil; and Oxaliplatin. XELOX: Capecitabine
(Xeloda) and Oxaliplatin. Renal Cell Carcinoma Afinitor
(Everolimus); Aldesleukin, Avastin (Bevacimub); Axitinib;
Bevacizumab; Everolimus, Inlyta (Axitinib); Nexavar (Sorafenib
Tosylate); Sunitinib Malate; Sutent (Sunitinib Malate);
Temsirolimus; Torisel (Temsirolimus); Votrient (Pazopanib
Hydrochloride) Retinoblastoma Clafen (Cyclophosphamide);
Cyclophosphamide; Cytoxan (Cyclophosphamide); or Neosar
(Cyclophosphamide). Rhabdomyosarcoma Cosmegen (Dactinomycin);
Dactinomycin; Vincasar PFS (Vincristine Sulfate); or Vincristine
Sulfate. Skin cancer (basal cell Adrucil (Fluorouracil); Aldara
(Imiquimod); carcinoma) Efudex (Fluorouracil); Erivedge
(Vismodegib); Fluoroplex (Fluorouracil); Fluorouracil; Imiquimod;
or Vismodegib. Skin cancer Aldesleukin; Dacarbazine; DTIC-Dome
(melanoma) (Dacarbazine); Ipilimumab; Proleukin (Aldesleukin);
Vemurafenib; Yervoy (Ipilimumab); or Zelboraf (Vemurafenib). Small
cell lung cancer Abitrexate (Methotrexate); Etopophos (Etoposide
Phosphate); Etoposide; Etoposide Phosphate; Folex (Methotrexate);
Folex PFS (Methotrexate); Hycamtin (Topotecan Hydrochloride);
Methotrexate; Methotrexate LPF (Methotrexate); Mexate
(Methotrexate); Mexate- AQ (Methotrexate); Toposar (Etoposide);
Topotecan Hydrochloride; or VePesid (Etoposide). Soft tissue
sarcoma Adriamycin PFS (Doxorubicin Hydrochloride); Adriamycin RDF
(Doxorubicin Hydrochloride); Cosmegen (Dactinomycin); Dactinomycin;
orDoxorubicin Hydrochloride. Testicular cancer Blenoxane
(Bleomycin); Bleomycin; Cisplatin; Cosmegen (Dactinomycin); Cyfos
(Ifosfamide); Dactinomycin; Etopophos (Etoposide Phosphate);
Etoposide; Etoposide Phosphate; Ifex (Ifosfamide); Ifosfamide;
Ifosfamidum (Ifosfamide); Platinol (Cisplatin); Platinol-AQ
(Cisplatin); Toposar (Etoposide; ; Velban (Vinblastine Sulfate);
Velsar (Vinblastine Sulfate); or VePesid (Etoposide); Vinblastine
Sulfate. Thyroid cancer Adriamycin PFS (Doxorubicin Hydrochloride);
Adriamycin RDF (Doxorubicin Hydrochloride); Cabozantinib-S-Malate;
Caprelsa (Vandetanib); Cometriq (Cabozantinib- S-Malate);
Doxorubicin Hydrochloride; Nexavar (Sorafenib Tosylate); or
Sorafenib Tosylate; Vandetanib. Vaginal cancer Gardasil
(Recombinant HPV Quadrivalent Vaccine); or Recombinant Human
Papillomavirus (HPV) Quadrivalent Vaccine. Vulvar cancer Blenoxane
(Bleomycin); Bleomycin; Gardasil (Recombinant HPV Quadrivalent
Vaccine); or Recombinant Human Papillomavirus (HPV) Quadrivalent
Vaccine. Wilms Tumor or other Adriamycin PFS (Doxorubicin childhood
Hydrochloride); Adriamycin RDF kidney (Doxorubicin Hydrochloride);
cancers Cosmegen (Dactinomycin); Dactinomycin; Doxorubicin
Hydrochloride; Vincasar PFS (Vincristine Sulfate); or Vincristine
Sulfate.
[0367] In further aspects, an mTOR inhibitor, e.g., an mTOR
inhibitor described herein, may be used in a treatment regimen in
combination with surgery, chemotherapy, radiation,
immunosuppressive agents, such as cyclosporin, azathioprine,
methotrexate, mycophenolate, and FK506, antibodies, or other
immunoablative agents such as CAMPATH, anti-CD3 antibodies or other
antibody therapies, cytoxin, fludarabine, cyclosporin, FK506,
rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and
irradiation. peptide vaccine, such as that described in Izumoto et
al. 2008 J Neurosurg 108:963-971.
[0368] In one embodiment, an mTOR inhibitor described herein can be
used in combination with a chemotherapeutic agent. Exemplary
chemotherapeutic agents include an anthracycline (e.g., doxorubicin
(e.g., liposomal doxorubicin)). a vinca alkaloid (e.g.,
vinblastine, vincristine, vindesine, vinorelbine), an alkylating
agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide,
temozolomide), an immune cell antibody (e.g., alemtuzamab,
gemtuzumab, rituximab, tositumomab), an antimetabolite (including,
e.g., folic acid antagonists, pyrimidine analogs, purine analogs
and adenosine deaminase inhibitors (e.g., fludarabine)), an mTOR
inhibitor, a TNFR glucocorticoid induced TNFR related protein
(GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A,
gliotoxin or bortezomib), an immunomodulator such as thalidomide or
a thalidomide derivative (e.g., lenalidomide).
[0369] General Chemotherapeutic agents considered for use in
combination therapies include anastrozole (Arimidex.RTM.),
bicalutamide (Casodex.RTM.), bleomycin sulfate (Blenoxane.RTM.),
busulfan (Myleran.RTM.), busulfan injection (Busulfex.RTM.),
capecitabine (Xeloda.RTM.),
N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin
(Paraplatin.RTM.), carmustine (BiCNU.RTM.), chlorambucil
(Leukeran.RTM.), cisplatin (Platinol.RTM.), cladribine
(Leustatin.RTM.), cyclophosphamide (Cytoxan.RTM. or Neosar.RTM.),
cytarabine, cytosine arabinoside (Cytosar-U.RTM.), cytarabine
liposome injection (DepoCyt.RTM.), dacarbazine (DTIC-Dome.RTM.),
dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride
(Cerubidine.RTM.), daunorubicin citrate liposome injection
(DaunoXome.RTM.), dexamethasone, docetaxel (Taxotere.RTM.),
doxorubicin hydrochloride (Adriamycin.RTM., Rubex.RTM.), etoposide
(Vepesid.RTM.), fludarabine phosphate (Fludara.RTM.),
5-fluorouracil (Adrucil.RTM., Efudex.RTM.), flutamide
(Eulexin.RTM.), tezacitibine, Gemcitabine (difluorodeoxycitidine),
hydroxyurea (Hydrea.RTM.), Idarubicin (Idamycin.RTM.), ifosfamide
(IFEX.RTM.), irinotecan (Camptosar.RTM.), L-asparaginase
(ELSPAR.RTM.), leucovorin calcium, melphalan (Alkeran.RTM.),
6-mercaptopurine (Purinethol.RTM.), methotrexate (Folex.RTM.),
mitoxantrone (Novantrone.RTM.), mylotarg, paclitaxel (Taxol.RTM.),
phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with
carmustine implant (Gliadel.RTM.), tamoxifen citrate
(Nolvadex.RTM.), teniposide (Vumon.RTM.), 6-thioguanine, thiotepa,
tirapazamine (Tirazone.RTM.), topotecan hydrochloride for injection
(Hycamptin.RTM.), vinblastine (Velban.RTM.), vincristine
(Oncovin.RTM.), and vinorelbine (Navelbine.RTM.).
[0370] Exemplary alkylating agents include, without limitation,
nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,
nitrosoureas and triazenes): uracil mustard (Aminouracil
Mustard.RTM., Chlorethaminacil.RTM., Demethyldopan.RTM.,
Desmethyldopan.RTM., Haemanthamine.RTM., Nordopan.RTM., Uracil
nitrogen Mustard.RTM., Uracillost.RTM., Uracilmostaza.RTM.,
Uramustin.RTM., Uramustine.RTM.), chlormethine (Mustargen.RTM.),
cyclophosphamide (Cytoxan.RTM., Neosar.RTM., Clafen.RTM.,
Endoxan.RTM., Procytox.RTM., Revimmune.TM.), ifosfamide
(Mitoxana.RTM.), melphalan (Alkeran.RTM.), Chlorambucil
(Leukeran.RTM.), pipobroman (Amedel.RTM., Vercyte.RTM.),
triethylenemelamine (Hemel.RTM., Hexalen.RTM., Hexastat.RTM.),
triethylenethiophosphoramine, Temozolomide (Temodar.RTM.), thiotepa
(Thioplex.RTM.), busulfan (Busilvex.RTM., Myleran.RTM.), carmustine
(BiCNU.RTM.), lomustine (CeeNU.RTM.), streptozocin (Zanosar.RTM.),
and Dacarbazine (DTIC-Dome.RTM.). Additional exemplary alkylating
agents include, without limitation, Oxaliplatin (Eloxatin.RTM.);
Temozolomide (Temodar.RTM. and Temodal.RTM.); Dactinomycin (also
known as actinomycin-D, Cosmegen.RTM.); Melphalan (also known as
L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran.RTM.);
Altretamine (also known as hexamethylmelamine (HMM), Hexalen.RTM.);
Carmustine (BiCNU.RTM.); Bendamustine (Treanda.RTM.); Busulfan
(Busulfex.RTM. and Myleran.RTM.); Carboplatin (Paraplatin.RTM.);
Lomustine (also known as CCNU, CeeNU.RTM.); Cisplatin (also known
as CDDP, Platinol.RTM. and Platinol.RTM.-AQ); Chlorambucil
(Leukeran.RTM.); Cyclophosphamide (Cytoxan.RTM. and Neosar.RTM.);
Dacarbazine (also known as DTIC, DIC and imidazole carboxamide,
DTIC-Dome.RTM.); Altretamine (also known as hexamethylmelamine
(HMM), Hexalen.RTM.); Ifosfamide (Ifex.RTM.); Prednumustine;
Procarbazine (Matulane.RTM.); Mechlorethamine (also known as
nitrogen mustard, mustine and mechloroethamine hydrochloride,
Mustargen.RTM.); Streptozocin (Zanosar.RTM.); Thiotepa (also known
as thiophosphoamide, TESPA and TSPA, Thioplex.RTM.);
Cyclophosphamide (Endoxan.RTM., Cytoxan.RTM., Neosar.RTM.,
Procytox.RTM., Revimmune.RTM.); and Bendamustine HCl
(Treanda.RTM.).
[0371] Exemplary immunomodulators include, e.g., afutuzumab
(available from Roche.RTM.); pegfilgrastim (Neulasta.RTM.);
lenalidomide (CC-5013, Revlimid.RTM.); thalidomide (Thalomid.RTM.),
actimid (CC4047); and IRX-2 (mixture of human cytokines including
interleukin 1, interleukin 2, and interferon .gamma., CAS
951209-71-5, available from IRX Therapeutics).
[0372] Exemplary anthracyclines include, e.g., doxorubicin
(Adriamycin.RTM. and Rubex.RTM.); bleomycin (Lenoxane.RTM.);
daunorubicin (dauorubicin hydrochloride, daunomycin, and
rubidomycin hydrochloride, Cerubidine.RTM.); daunorubicin liposomal
(daunorubicin citrate liposome, DaunoXome.RTM.); mitoxantrone
(DHAD, Novantrone.RTM.); epirubicin (Ellence.TM.); idarubicin
(Idamycin.RTM., Idamycin PFS.RTM.); mitomycin C (Mutamycin.RTM.);
geldanamycin; herbimycin; ravidomycin; and
desacetylravidomycin.
[0373] Exemplary vinca alkaloids include, e.g., vinorelbine
tartrate (Navelbine.RTM.), Vincristine (Oncovin.RTM.), and
Vindesine (Eldisine.RTM.)); vinblastine (also known as vinblastine
sulfate, vincaleukoblastine and VLB, Alkaban-AQ.RTM. and
Velban.RTM.); and vinorelbine (Navelbine.RTM.).
[0374] Exemplary proteosome inhibitors include bortezomib
(Velcade.RTM.); carfilzomib (PX-171-007,
(S)-4-Methyl-N--((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxope-
ntan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamid-
o)-4-phenylbutanamido)-pentanamide); marizomib (NPI-0052); ixazomib
citrate (MLN-9708); delanzomib (CEP-18770); and
O-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(-
2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide
(ONX-0912).
[0375] Exemplary GITR agonists include, e.g., GITR fusion proteins
and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such
as, e.g., a GITR fusion protein described in U.S. Pat. No.
6,111,090, European Patent No.: 090505B1, U.S. Pat. No. 8,586,023,
PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an
anti-GITR antibody described, e.g., in U.S. Pat. No. 7,025,962,
European Patent No.: 1947183B1, U.S. Pat. No. 7,812,135, U.S. Pat.
No. 8,388,967, U.S. Pat. No. 8,591,886, European Patent No.: EP
1866339, PCT Publication No.: WO 2011/028683, PCT Publication No.:
WO 2013/039954, PCT Publication No.: WO2005/007190, PCT Publication
No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCT
Publication No.: WO 99/40196, PCT Publication No.: WO 2001/03720,
PCT Publication No.: WO99/20758, PCT Publication No.:
WO2006/083289, PCT Publication No.: WO 2005/115451, U.S. Pat. No.
7,618,632, and PCT Publication No.: WO 2011/051726.
[0376] In one embodiment, an mTOR inhibitor described herein is
administered at a low, immune enhancing, dose to a subject in
combination with a protein tyrosine phosphatase inhibitor, e.g., a
protein tyrosine phosphatase inhibitor described herein. In one
embodiment, the protein tyrosine phosphatase inhibitor is an SHP-1
inhibitor, e.g., an SHP-1 inhibitor described herein, such as,
e.g., sodium stibogluconate. In one embodiment, the protein
tyrosine phosphatase inhibitor is an SHP-2 inhibitor, e.g., an
SHP-2 inhibitor described herein.
[0377] In one embodiment, a low, immune enhancing, dose, of an mTOR
inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a
catalytic inhibitor is administered in combination with a kinase
inhibitor.
[0378] In one embodiment, the kinase inhibitor is an MNK inhibitor,
e.g., a MNK inhibitor selected from CGP052088;
4-amino-3-(p-fluorophenylamino)-pyrazolo[3,4-d]pyrimidine
(CGP57380); cercosporamide; ETC-1780445-2; and
4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine. The MNK
inhibitor can be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b
inhibitor. In one embodiment, the kinase inhibitor is
4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.
[0379] In one embodiment, the kinase inhibitor is a CDK4 inhibitor
selected from
7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridine-2-yl)amino)-7H--
pyrrolo[2,3-d]pyrimidine-6-carboxamide (also referred to as
LEE011); aloisine A; flavopiridol or HMR-1275,
2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidi-
nyl]-4-chromenone; crizotinib (PF-02341066;
2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3--
pyrrolidinyl]-4H-1-benzopyran-4-one, hydrochloride (P276-00);
1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N--
[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265);
indisulam (E7070); roscovitine (CYC202); palbociclib (PD0332991);
dinaciclib (SCH727965);
N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-car-
boxamide (BMS 387032);
4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]-
amino]-benzoic acid (MLN8054);
5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methy-
l-3-pyridinemethanamine (AG-024322);
4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid
N-(piperidin-4-yl)amide (AT7519);
4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phen-
yl]-2-pyrimidinamine (AZD5438); and XL281 (BMS908662).
[0380] In one embodiment, the kinase inhibitor is a CDK4 inhibitor,
e.g., a CDK4 inhibitor described herein, e.g., a CDK4/6 inhibitor,
such as, e.g.,
7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridine-2-yl)amin-
o)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (also referred to as
LEE011) or
6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-
-8H-pyrido[2,3-d]pyrimidin-7-one, hydrochloride (also referred to
as palbociclib or PD0332991).
[0381] In one embodiment, the kinase inhibitor is a CDK4 inhibitor,
e.g., palbociclib (PD0332991), and the palbociclib is administered
at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100
mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g.,
75 mg, 100 mg or 125 mg) daily for a period of time, e.g., daily
for 14-21 days of a 28 day cycle, or daily for 7-12 days of a 21
day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
or more cycles of palbociclib are administered.
[0382] In one embodiment, the kinase inhibitor is a BTK inhibitor,
e.g., selected from ibrutinib (PCI-32765); GDC-0834; RN-486;
CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and
LFM-A13.
[0383] In one embodiment, the kinase inhibitor is a BTK inhibitor,
e.g., ibrutinib (PCI-32765), and the ibrutinib is administered at a
dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460
mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g.,
250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily
for 21 day cycle cycle, or daily for 28 day cycle. In one
embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of
ibrutinib are administered.
[0384] In one embodiment, the kinase inhibitor is an mTOR
inhibitor. MTOR inhibitors can be selected from the section
elsewhere herein entitled mTOR Inhibitors. The dose referred to
here is not the low, immune enhancing, dose of an mTOR inhibitor,
but rather a dose sufficient to give an anti-cancer effect, and is
higher than the low, immune enhancing, dose, described herein,
e.g., a dose. Thus, in an embodiment, two different administrations
of an mTOR inhibitor are given, a low, immune enhancing dose, e.g.,
to optimize immune effector cell function, and a higher dose given
for an anticancer effect.
[0385] In one embodiment, the kinase inhibitor is an mTOR
inhibitor, e.g., rapamycin, and the rapamycin is administered at a
dose sufficient to give an anti-cancer effect, and higher than the
low, immune enhancing, dose, described herein, e.g., a dose of
about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg)
daily for a period of time, e.g., daily for 21 day cycle cycle, or
daily for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12 or more cycles of rapamycin are administered.
[0386] In one embodiment, the kinase inhibitor is an mTOR
inhibitor, e.g., everolimus and the everolimus is administered at a
dose sufficient to give an anti-cancer effect, and higher than the
low, immune enhancing, dose, described herein, e.g., a dose of
about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10
mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for a
period of time, e.g., daily for 28 day cycle. In one embodiment, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus are
administered.
[0387] In another aspect, an mTOR inhibitor, e.g., an mTOR
inhibitor described herein, can be administered at low, immune
enhancing, dose in combination with an additional agent which
inhibits one or more inhibitory molecules, e.g., PD1, PD-L1, CTLA4,
TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
Inhibition of an inhibitory molecule, e.g., by inhibition at the
DNA, RNA or protein level, can lead to increased immune function,
as described herein. In embodiments, an inhibitory nucleic acid,
e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or
shRNA, can be used to inhibit expression of an inhibitory molecule.
In an embodiment the inhibitor is an shRNA. In one embodiment, the
inhibitor of an inhibitory signal can be, e.g., an antibody or
antibody fragment that binds to an inhibitory molecule. For
example, the agent can be an antibody or antibody fragment that
binds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also
referred to as MDX-010 and MDX-101, and marketed as Yervoy.RTM.;
Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibody
available from Pfizer, formerly known as ticilimumab,
CP-675,206).). In an embodiment, the agent is an antibody or
antibody fragment that binds to TIM3. In an embodiment, the agent
is an antibody or antibody fragment that binds to LAG3.
[0388] In an embodiment, an mTOR inhibitor can be used in low,
immune enhancing, dose in combination with an inhibitor of PD1,
e.g., an inhibitor of the interaction of PD1 and one of its natural
ligands. In an embodiment, the mTOR inhibitor is administered
first, e.g., the PD1 inhibitor is not administered until the level
of PD1 positive T cells is reduced. In an embodiment, the mTOR
inhibitor is administered at the same time as or after the PD1
inhibitor is administered.
[0389] PD1 is an inhibitory member of the CD28 family of receptors
that also includes CD28, CTLA-4, ICOS, and BTLA. PD1 is expressed
on activated B cells, T cells and myeloid cells (Agata et al. 1996
Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2 have
been shown to downregulate T cell activation upon binding to PD1
(Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat
Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1
is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7;
Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et
al. 2004 Clin Cancer Res 10:5094). Immune suppression can be
reversed by inhibiting the local interaction of PD1 with PD-L1.
Antibodies, antibody fragments, and other inhibitors of PD1, PD-L1
and PD-L2 are available in the art and may be used combination with
an mTOR inhibitor described herein. For example, nivolumab (also
referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a
fully human IgG4 monoclonal antibody which specifically blocks PD1.
Nivolumab (clone 5C4) and other human monoclonal antibodies that
specifically bind to PD1 are disclosed in U.S. Pat. No. 8,008,449
and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized
IgG1k monoclonal antibody that binds to PD1Pidilizumab and other
humanized anti-PD1 monoclonal antibodies are disclosed in
WO2009/101611. Lambrolizumab (also referred to as MK03475; Merck)
is a humanized IgG4 monoclonal antibody that binds to PD1.
Lambrolizumab and other humanized anti-PD1 antibodies are disclosed
in U.S. Pat. No. 8,354,509 and WO2009/114335. MDPL3280A
(Genentech/Roche) is a human Fc optimized IgG1 monoclonal antibody
that binds to PD-L1. MDPL3280A and other human monoclonal
antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and
U.S Publication No.: 20120039906. Other anti-PD-L1 binding agents
include YW243.55.570 (heavy and light chain variable regions are
shown in SEQ ID NOs 20 and 21 in WO2010/077634) and MDX-1 105 (also
referred to as BMS-936559, and, e.g., anti-PD-L1 binding agents
disclosed in WO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g.,
disclosed in WO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion
soluble receptor that blocks the interaction between PD1 and B7-H1.
Other anti-PD1 antibodies include AMP 514 (Amplimmune), among
others, e.g., anti-PD1 antibodies disclosed in U.S. Pat. No.
8,609,089, US 2010028330, and/or US 20120114649.
[0390] In some embodiments, an mTOR inhibitor, e.g., an mTOR
inhibitor described herein, is administered at low, immune
enhancing, dose to a subject who has a viral infection, e.g., a
viral infection described herein. The subject may receive treatment
with an additional therapeutic agent, such as an approved drug for
that type of viral infection, in combination with the mTOR
inhibitor. Exemplary antiviral agents that may be used in the
compositions and methods of the invention include, but are not
limited to, Abacavir; Acyclovir; Adefovir; Amantadine; Amprenavir;
Ampligen; Arbidol; Atazanavir; Atripla; Balavir; Boceprevirertet;
Cidofovir; Combivir; Dolutegravir; Darunavir; Delavirdine;
Didanosine; Docosanol; Edoxudine; Efavirenz; Emtricitabine;
Enfuvirtide; Entecavir; Ecoliever; Famciclovir; Fomivirsen;
Fosamprenavir; Foscarnet; Fosfonet; Ganciclovir; Ibacitabine;
Imunovir; Idoxuridine; Imiquimod; Indinavir; Inosine; Interferon;
Interferon type I; Interferon type II; Interferon type III;
Lamivudine; Lopinavir; Loviride; Maraviroc; Moroxydine;
Methisazone; Nelfinavir; Nevirapine; Nexavir; Nucleoside analogues;
Oseltamivir (Tamiflu); Peginterferon alfa-2a; Penciclovir;
Peramivir; Pleconaril; Podophyllotoxin; Raltegravir; Ribavirin;
Rimantadine; Ritonavir; Pyramidine; Saquinavir; Sofosbuvir;
Stavudine; Telaprevir; Tenofovir; Tenofovir disoproxil; Tipranavir;
Trifluridine; Trizivir; Tromantadine; Truvada; traporved;
Valaciclovir; Valganciclovir; Vicriviroc; Vidarabine; Viramidine;
Zalcitabine; Zanamivir; and Zidovudine.
[0391] In an embodiment the method further comprises the
administration of a low, immune enhancing, dose of an mTOR
inhibitor in combination with anti-bacterial, anti-mycobacterial,
anti-fungal or anti-parasitic or protozoal agents.
Pharmaceutical Compositions
[0392] In one aspect, the present invention relates to
pharmaceutical compositions comprising an mTOR inhibitor, e.g., an
mTOR inhibitor as described herein. In some embodiments, the mTOR
inhibitor is formulated for administration in combination with
another agent, e.g., as described herein.
[0393] In one aspect, the present invention relates to
pharmaceutical compositions comprising an mTOR inhibitor as
described herein, potentially in combination with an antigen such
as a vaccine or vaccine antigen.
[0394] In general, compounds of the invention will be administered
in therapeutically effective amounts as described above via any of
the usual and acceptable modes known in the art, either singly or
in combination with one or more therapeutic agents (e.g., a vaccine
or other antigen).
[0395] The pharmaceutical formulations may be prepared using
conventional dissolution and mixing procedures. For example, the
bulk drug substance (e.g., an mTOR inhibitor or stabilized form of
the compound (e.g., complex with a cyclodextrin derivative or other
known complexation agent) is dissolved in a suitable solvent in the
presence of one or more of the excipients described herein. The
mTOR inhibitor is typically formulated into pharmaceutical dosage
forms to provide an easily controllable dosage of the drug and to
give the patient an elegant and easily handleable product.
[0396] Compounds of the invention can be administered as
pharmaceutical compositions by any conventional route, in
particular enterally, e.g., orally, e.g., in the form of tablets or
capsules, or parenterally, e.g., in the form of injectable
solutions or suspensions, topically, e.g., in the form of lotions,
gels, ointments or creams, or in a nasal or suppository form. Where
an mTOR inhibitor is administered in combination with (either
simultaneously with or separately from) another agent as described
herein, in one aspect, both components can be administered by the
same route (e.g., parenterally). Alternatively, another agent may
be administered by a different route relative to the mTOR
inhibitor. For example, an mTOR inhibitor may be administered
orally and the other agent may be administered parenterally.
Pharmaceutical compositions comprising an mTOR inhibitor in free
form or in a pharmaceutically acceptable salt form in association
with at least one pharmaceutically acceptable carrier or diluent
can be manufactured in a conventional manner by mixing, granulating
or coating methods. For example, oral compositions can be tablets
or gelatin capsules comprising the active ingredient together with
a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,
cellulose and/or glycine; b) lubricants, e.g., silica, talcum,
stearic acid, its magnesium or calcium salt and/or
polyethyleneglycol; for tablets also c) binders, e.g., magnesium
aluminum silicate, starch paste, gelatin, tragacanth,
methylcellulose, sodium carboxymethylcellulose and or
polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches,
agar, alginic acid or its sodium salt, or effervescent mixtures;
and/or e) absorbents, colorants, flavors and sweeteners. Oral
formulations can also comprise the active ingredient along with
20-60% Eudragit EPO, Hydroxypropyl cellulose EF, Hydroxypropyl
methylcellulose, or Kollidon VA64, and up to 5% of pluronic F68,
Cremophor EL, or Gelucire 44/14. Injectable compositions can be
aqueous isotonic solutions or suspensions, and suppositories can be
prepared from fatty emulsions or suspensions. The compositions may
be sterilized and/or contain adjuvants, such as preserving,
stabilizing, wetting or emulsifying agents, solution promoters,
salts for regulating the osmotic pressure and/or buffers. In
addition, they may also contain other therapeutically valuable
substances. Suitable formulations for transdermal applications
include an effective amount of a compound of the present invention
with a carrier. A carrier can include absorbable pharmacologically
acceptable solvents to assist passage through the skin of the host.
For example, transdermal devices are in the form of a bandage
comprising a backing member, a reservoir containing the compound
optionally with carriers, optionally a rate controlling barrier to
deliver the compound to the skin of the host at a controlled and
predetermined rate over a prolonged period of time, and means to
secure the device to the skin. Matrix transdermal formulations may
also be used. In a further aspect, the mTOR inhibitors described
herein may be administered via a microneedle patch. Microneedle
based drug delivery is well known in the art (See, e.g., U.S. Pat.
No. 8,162,901) and these technologies and methods may be adapted by
one of skill in the art for administration of an mTOR inhibitor as
described herein. Suitable formulations for topical application,
e.g., to the skin and eyes, are preferably aqueous solutions,
ointments, creams or gels well-known in the art. Such formulations
may contain solubilizers, stabilizers, tonicity enhancing agents,
buffers and preservatives.
[0397] The pharmaceutical composition (or formulation) for
application may be packaged in a variety of ways depending upon the
method used for administering the drug. Generally, an article for
distribution includes a container having deposited therein the
pharmaceutical formulation in an appropriate form. Suitable
containers are well-known to those skilled in the art and include
materials such as bottles (plastic and glass), sachets, ampoules,
plastic bags, metal cylinders, and the like. The container may also
include a tamper-proof assemblage to prevent indiscreet access to
the contents of the package. In addition, the container has
deposited thereon a label that describes the contents of the
container. The label may also include appropriate warnings. The
invention also provides for a pharmaceutical combinations, e.g. a
kit, comprising a) a first agent which is an mTOR inhibitor as
disclosed herein, in free form or in pharmaceutically acceptable
salt form, and b) at least one additional agent. The kit can
comprise instructions for its administration.
[0398] The term "pharmaceutical combination" as used herein means a
product that results from the mixing or combining of more than one
active ingredient and includes both fixed and non-fixed
combinations of the active ingredients. The term "fixed
combination" means that the active ingredients, e.g. an mTOR
inhibitor and other agent, are both administered to a patient
simultaneously in the form of a single entity or dosage. The term
"non-fixed combination" means that the active ingredients, e.g. an
mTOR inhibitor and other agent, are both administered to a patient
as separate entities either simultaneously, concurrently or
sequentially with no specific time limits, wherein such
administration provides therapeutically effective levels of the 2
compounds in the body of the patient. The latter also applies to
cocktail therapy, e.g. the administration of 3 or more active
ingredients.
Sustained Release
[0399] mTOR inhibitors, e.g., allosteric mTOR inhibitors or
catalytic mTOR inhibitors, disclosed herein can be provided as
pharmaceutical formulations in form of oral solid dosage forms
comprising an mTOR inhibitor disclosed herein, e.g., rapamycin or
RAD001, which satisfy product stability requirements and/or have
favorable pharmacokinetic properties over the immediate release
(IR) tablets, such as reduced average plasma peak concentrations,
reduced inter- and intra-patient variability in the extent of drug
absorption and in the plasma peak concentration, reduced
C.sub.max/C.sub.min ratio and/or reduced food effects. Provided
pharmaceutical formulations may allow for more precise dose
adjustment and/or reduce frequency of adverse events thus providing
safer treatments for patients with an mTOR inhibitor disclosed
herein, e.g., rapamycin or RAD001.
[0400] In some embodiments, the present disclosure provides stable
extended release formulations of an mTOR inhibitor disclosed
herein, e.g., rapamycin or RAD001, which are multi-particulate
systems and may have functional layers and coatings.
[0401] The term "extended release, multi-particulate formulation as
used herein refers to a formulation which enables release of an
mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, over an
extended period of time e.g. over at least 1, 2, 3, 4, 5 or 6
hours. The extended release formulation may contain matrices and
coatings made of special excipients, e.g., as described herein,
which are formulated in a manner as to make the active ingredient
available over an extended period of time following ingestion.
[0402] The term "extended release" can be interchangeably used with
the terms "sustained release" (SR) or "prolonged release". The term
"extended release" relates to a pharmaceutical formulation that
does not release active drug substance immediately after oral
dosing but over an extended in accordance with the definition in
the pharmacopoeias Ph. Eur. (7.sup.th edition) mongraph for tablets
and capsules and USP general chapter <1151> for
pharmaceutical dosage forms. The term "Immediate Release" (IR) as
used herein refers to a pharmaceutical formulation which releases
85% of the active drug substance within less than 60 minutes in
accordance with the definition of "Guidance for Industry:
"Dissolution Testing of Immediate Release Solid Oral Dosage Forms"
(FDA CDER, 1997). In some embodiments, the term "immediate release"
means release of everolismus from tablets within the time of 30
minutes, e.g., as measured in the dissolution assay described
herein.
[0403] Stable extended release formulations of an mTOR inhibitor
disclosed herein, e.g., rapamycin or RAD001, can be characterized
by an in-vitro release profile using assays known in the art, such
as a dissolution assay as described herein: a dissolution vessel
filled with 900 mL phosphate buffer pH 6.8 containing sodium
dodecyl sulfate 0.2% at 37.degree. C. and the dissolution is
performed using a paddle method at 75 rpm according to USP by
according to USP testing monograph 711, and Ph.Eur. testing
monograph 2.9.3. respectively.
[0404] In some embodiments, stable extended release formulations of
an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001,
release the mTOR inhibitor in the in-vitro release assay according
to following release specifications:
[0405] 0.5 h: <45%, or <40, e.g., <30%
[0406] 1 h: 20-80%, e.g., 30-60%
[0407] 2 h: >50%, or >70%, e.g., >75%
[0408] 3 h: >60%, or >65%, e.g., >85%, e.g., >90%.
[0409] In some embodiments, stable extended release formulations of
an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001,
release 50% of the mTOR inhibitor not earlier than 45, 60, 75, 90,
105 min or 120 min in the in-vitro dissolution assay.
[0410] In one embodiment, stable extended release formulations of
an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001,
comprise an mTOR inhibitor in a fast dissolving or disintegrating
carrier matrix in combination with coatings wherein at least one of
the coatings is an extended release coating. In another embodiment,
stable extended release formulations of an mTOR inhibitor disclosed
herein, e.g., rapamycin or RAD001, comprise an mTOR inhibitor in a
non-disintegrating carrier matrix with extended release properties,
which can be combined optionally with additional coatings.
[0411] In some embodiments, a carrier matrix comprises matrix
formers, typically matrix forming polymers, and may contain
additional excipients, such as fillers, e.g., lactose, mannitol,
maltodextrine, pregelatinized starch, calcium phosphate, or
microcrystallline cellulose, and disintegrants, e.g., corn starch,
croscamellose, sodium starch glycolate, or crospovidone,
antioxidants, e.g., butylhydroxy anisol, butylhydroxy toluol,
ascorbyl palmitate, tocopherol, vitamin E polyethylene glycol
succinate, and process enhancing agents, such as lubricants and
glidants, e.g., colloidal silicon dioxide, talc, glyceryl
monostearate, magnesium stearate, calcium stearate, or sodium
stearyl fumarate. The term "matrix former" typically relates to a
pharmaceutically inert material which provides physical stability,
such as e.g., mechanical or binding stability.
[0412] Suitable matrix forming polymers used for fast dissolving or
disintegrating carrier matrices are known in the art include for
instance cellulose or starch, for instance microcrystalline
cellulose ("MCC"), for example Avicel PH 101 (FMC BioPolymer),
acacia, sodium alginate, gelatine, starch, pregeliatinised starch,
methylcellulose, hydroxypropyl methylcellulose ("HPMC"),
hydroxypropylcellulose, hydroxyethylcellulose, polyethylene glycol
or polyvinylpyrrolidone ("PVP"), carrageenan, such as Gelcarin GP
812 or combinations thereof.
[0413] Suitable matrix forming excipients for non-disintegrating
carrier matrices with extended release properties are known in the
art include for instance acacia, sodium alginate, gelatine,
carboxmethylcellulose sodium, (or "CMC sodium"), methylcellulose,
ethylcellulose and cellulose acetate or polyacrylates, e.g.,
ammonio methacrylate copolymers (Eudragit RS/RL), hydroxypropyl
methylcellulose ("HPMC"), hydroxypropylcellulose,
hydroxyethylcellulose, polyvinylacetate, polyethylene glycol or
polyvinylpyrrolidone ("PVP"), e.g., carrageenan, such as Gelcarin
GP 812, glyceryl monostearate, stearylalcohol, stearic acid,
glyceryl behenate, Vitamin E polyethylen glycol succinate, or
combinations thereof.
[0414] In one embodiment, the extended release coating is a layer
formed with water insoluble, non-disintegrating polymers,
controlling the release by permeation of the drug through this
layer.
[0415] The extended release coating may also contain one or more of
pore formers, plasticizers, and processing enhancing agents, such
as lubricants and anti tacking agents. Suitable extended release
coating forming polymers which enable diffusion controlled release
are known in the art include for instance ethylcellulose and
cellulose acetate or polyacrylates, e.g., ammonio methacrylate
copolymers (Eudragit RS/RL), polyvinylacetate or combinations
thereof. In a particular embodiment, the extended release coating
forming polymer is ethylcellulose or cellulose acetate or
polyacrylates, e.g., ammoniomethacrylate copolymer Type A (Eudragit
RS) or ammonio-methacrylate copolymer Type B (Eudragit RL) or
combinations thereof. Moreover, the extended release coating may
include plasticizer, such as triacetine, triethyl citrate,
dibutylsebacate, diethylsebacate, polyethylene glycol 3000, 4000 or
6000, acetyltriethylcitrate, acetyltributylcitrate, or
diethylphthalate, and/or anti-tacking agents such Syloid 244 FP,
talc, glyceryl monostearate, or titanium dioxide. In some
embodiments, the amount of plasticizer may be between 5 to 40%,
preferably 10 to 25%, relative to the amount of sustained release
polymer.
[0416] In an embodiment, an extended release coating is a pore
forming system which comprises a water insoluble coating forming
polymer and a pore former. The term "pore former" relates to a
readily soluble excipient which allows pores to be introduced or
permeability of the coating to be increased, and a diffusion
controlled release of the active ingredient. Suitable pore formers
are known in the art include for instance hydroxypropylcellulose
(HPC (e.g., Klucel.TM. EF, EXF, LF), or hydroxypropyl
methylcellulose (HPMC, e.g., Methocel.TM. E3/E5, Pharmacoat
603.TM.), polyethylen glycol (e.g., Macrogol 1500, 3500, 4000,
6000), poloxamer 188 (Pluronic F68.TM.) or povidone (PVP, e.g.,
Kollidon K25/K30), a saccharide, e.g., a monosaccharide, such as
dextrose, mannose, fructose, a disaccharide, such as sucrose or
glucodifructose or combinations thereof. Preferably the pore former
is hydroxypropylcellulose (HPC (Klucel.TM. EF, EXF, LF), or
hydroxypropyl methylcellulose (HPMC, Methocel.TM. E3/E5, Pharmacoat
603.TM.) polyethylen glycol (Macrogol 1500, 3500, 4000, 6000),
poloxamer 188 (Pluronic F68.TM.) or povidone (PVP, Kollidon
K25/K30) or combinations thereof. In some embodiments, suitable
amounts of pore formers included in coating are equal to ratios of
coating polymer to pore former of e.g. 100:20 to 100:50, or 100:20
to 100:100, preferably ratios of 100:35 to 100:45, particularly
ratios of 100:35 to 100:50 relative to the amount of coating
forming polymer. In some embodiments, suitable amounts of coating
forming polymers included are equal to percentages of polymer
weight increase of e.g., 4% to 15%, 5% to 15%, preferably 5% to
12%, more preferably 6% to 12% weight of total weight of
pharmaceutical formulation.
[0417] In another embodiment, a non-disintegrating extended release
carrier matrix comprises matrix forming polymers which enable
diffusion controlled release of the active ingredient by hydration
of the polymer. The extended carrier matrix may contain further
excipients, such as binders and or fillers and process enhancing
agents, such as lubricants and glidants, etc.
[0418] The following exemplary matrix forming polymers may be used
for diffusion controlled release: sodium alginate, polyacrylic
acids (or "carbomers"), carboxmethylcellulose sodium, (or "CMC
sodium"), methylcellulose, ethylcellulose and cellulose acetate or
polyacrylates, e.g., ammonio methacrylate copolymers (Eudragit
RS/RL), hydroxypropyl methylcellulose ("HPMC") of different
viscosity grades (i.e., average polymer chain lengths) and
combinations thereof, e.g., Methocel.TM. CR grades, hydroxypropyl
cellulose, e.g. Klucel.TM. HF/MF, polyoxyethylene, e.g., Polyox.TM.
or polyvinylpyrrolidone ("PVP"), e.g., PVP K60, K90, carrageenan,
such as Viscarin.TM. GP-209/GP-379, or combinations thereof.
Combining of matrix forming polymers allows adjusting the
dissolution rate of the active ingredient according to the
need.
[0419] In some embodiments, a non-disintegrating extended release
matrix is formed with excipients, which enable release of the
active ingredient by a controlled erosion. The erosion controlled
matrices may contain lipophilic matrix formers, and also further
excipients, such as fillers, disintegrants and process enhancing
agents, such as lubricants and glidants. Exemplary lipophilic
matrix forming excipients related to this matrix type include
lipophilic excipients, such as glyceryl monostearate, e.g., Cutina
GMS, glyceryl behenate, e.g., Compritol 888 ATO, stearyl alcohol,
stearic acid, hart fat, e.g., Gelucire.TM., or Vitamin E
polyethylen glycol succinate, e.g., Speziol TPGS or combinations
thereof.
[0420] Exemplary suitable binders, fillers or further excipients
include, but are not limited to, mannitol, pregelatinized starch,
microcrystalline cellulose, lactose, calcium phosphate, talc,
titanium dioxide, triethylcitrate, Aerosil, antioxidants such as
e.g., BHT, desiccants and disintegrant such as e.g., crospovidone
or sodium starch glycolate, starch, or croscarmellose.
[0421] In an embodiment, a stable extended release formulation
comprises an mTOR inhibitor disclosed herein, e.g., rapamycin or
RAD001, in a fast dissolving/disintegrating matrix, e.g., in form
of a solid dispersion as described herein, in combination with
functional layers or coatings wherein at least one of the
functional layer(s) or coating(s) has release controlling behavior
enabling extended release of the active ingredient. In another
embodiment, a stable extended release formulation comprises an mTOR
inhibitor disclosed herein, e.g., rapamycin or RAD001, in the
extended release matrix which, optionally, can further contain
functional layers or coatings, such as protective or sustained
release layers or coatings. In some embodiments, the coating, e.g.,
the extended release coating may have a thickness in the range of
10 to 100 .mu.m, e.g., 10 to 50 .mu.m (assessed by confocal RAMAN
spectroscopy).
[0422] In some embodiments, the formulation of an mTOR inhibitor
disclosed herein, e.g., rapamycin or RAD001, is in form of a
multi-particulate delivery system. In some embodiments, a
multi-particulate drug delivery system is an oral dosage form
consisting of multiple, small discrete dose units. In such systems,
the dosage form of the drug substances such as capsule, tablets,
sachet or stickpack, may contain a plurality of subunits, typically
consisting of tens to hundreds or even up to thousands of spherical
particles with diameter of 0.05-2.00 mm. Formulations of the size
1.5-3 mm, e.g., minitablets, present another alternative. The
dosage form may be designed to disintegrate rapidly in the stomach
releasing the multi-particulates. Without wishing to be bound by a
particular theory, it is thought that the multi-particulates are
spread in the gastro-intestinal lumen and will be emptied gradually
from the stomach releasing the drug substance in a controlled
manner.
[0423] In one embodiment, the formulation of an mTOR inhibitor
disclosed herein, e.g., rapamycin or RAD001, e.g., in form of
multi-particulate delivery system, comprises an mTOR inhibitor as
active ingredient, e.g., dissolved or dispersed in the core of the
particle, (e.g., a bead, pellet, granule or minitablet), or in a
layer surrounding an inert core of the particle. The active
ingredient can be for instance be embedded in an extended release
matrix, preferably comprising a hydrophilic or lipophilic matrix
forming excipients, or embedded in a fast disintegrating and/or
dissolving matrix in combination with functional layer(s) and top
coating(s) wherein at least one of the functional layer(s) or top
coating(s) comprises a coating forming polymer enabling diffusion
controlled extended release of the active ingredient. Optionally, a
protection layer for improving stability of the active ingredient
separates the matrix containing the active substance from
functional layers or top coatings, to ensure stability of the drug
product.
[0424] In a another embodiment, the formulation of an mTOR
inhibitor disclosed herein, e.g., rapamycin or RAD001, e.g., in
form of a multi-particulate delivery system, comprises an mTOR
inhibitor as active ingredient and an outer coating layer
comprising an insoluble polymer and a soluble component as pore
former, and optionally further functional layers. For the purpose
of the present invention the terms "outer layer" is a layer located
towards to the outside of a particle and may be coated with a
further layer(s) or may be a top coating. The terms "outer layer",
"coating layer" or "top coat" may be used interchangeably depending
on the context in which the terms are used.
[0425] In one embodiment, the particles comprise one or several top
coats enabling extended release of the active ingredient. Top coats
typically are final layers with release controlling behavior, which
are enclosing each particle of the multi-particulates
separately.
[0426] In an embodiment, the formulation of an mTOR inhibitor
disclosed herein, e.g., rapamycin or RAD001, comprises an outer
layer or a top coating that controls the release by the diffusion
of the drug through the coating layer which is permeable,
optionally by the formation of pores in the insoluble polymer
layer, or alternatively solely by the hydration of the insoluble
polymer, or that controls the release by a combination of a pore
former and hydration of the insoluble polymer. The polymer is
insoluble independently from pH, and optionally contains water
soluble pore former. The release rate is affected by the extent of
pore formation after the pore former is dissolved. The insoluble
coating polymer can be cellulose ethers such as ethylcellulose and
cellulose acetate or polyacrylates, e.g., ammonio methacrylate
copolymers (Eudragit RS/RL). Suitable pore formers include water
soluble cellulose ethers, for instance hydroxypropylcellulose (HPC
(Klucel.TM. EF, EXF, LF) or hydroxypropyl methylcellulose (HPMC,
Methocel.TM. E3/E5, Pharmacoat 603.TM.), polyethylen glycol
(Macrogol 1500, 3500, 4000, 6000), poloxamer 188 (Pluronic F68.TM.)
or povidone (PVP, Kollidon K12, K25, K30). For instance, water
soluble pore former can be mixed with insoluble polymer in a ratio
of 2:1 to 1:10, e.g. 1:1 to 1:5, 1:3 or 1:5. In an embodiment, the
pore former to insoluble polymer ratio is HPC, e.g Klucel.TM. EF,
EXF, LF or HMPC 3 cP, e.g., Methocel.TM. E3, in a ratio of 1:1 to
1:4, e.g., about 1:1, 1:1.2, 1:1.5 or 1:2. Exemplary insoluble
polymers include, but are not limited to ethylcellulose (EC,
Aqualon EC N10.TM.) in combination with a pore former. In some
embodiments, without the use of a pore former, the combination of
the insoluble polymers ammoniomethacrylate copolymer Type A
(Eudragit RS) and ammonio-methacrylate copolymer Type B (Eudragit
RL) may be at ratios of 1:2 to 9:1, preferably 1:1 to 4:1.
[0427] A sustained release top coat(s) may achieve release of
majority of the active substance into the small intestine and
allows protection of the active substance from stomach fluids and
minimizes the exposure of the active substance to the mouth,
esophagus and stomach.
[0428] In one embodiment, the formulation of an mTOR inhibitor
disclosed herein, e.g., rapamycin or RAD001, comprise a drug
substance containing matrix, e.g., fast disintegrating and/or
dissolving matrix layer or in an extended release matrix layer,
e.g., on a starter core such as beads, pellets or granules, which
can consist of one or more components, and in which the active
ingredient is dispersed or dissolved. For instance, amorphous or
crystalline mTOR inhibitor, e.g., rapamycin or RAD001, can be
dispersed or dissolved in the matrix in a ratio from 1:100 to 100:1
in the matrix, e.g., 1:50 to 5:1; or 1:50 to 1:1 by weight, or 1:5
to 2:3, or 1:10 to 1:5 by weight (as to the matrix former).
[0429] In an embodiment, the drug substance containing matrix is
layered onto the surface of starter cores. The layer may be built
by spraying a dispersion or solution of the matrix components and
the drug substance on to particles of uniform, regular size and
shape in a fluid bed process. Alternatively, powder mixtures of the
matrix components can be layered using a rotating disk processor.
Starter cores have an average particle size 0.1 to 2.5 mm. They can
be single crystals, e.g., sucrose, or granular agglomerates
manufactured by fluid bed granulation, a rotorgranulation,
extrusion and spheronization, or a compaction process. In some
embodiments, minitablets can be used as starter cores. In
particular embodiments, the starter cores have a spherical shape
and consist of inert material such as sucrose and starch (Sugar
Spheres, Suglets.TM., Non-pareils), mannitol (e.g. MCells.TM.),
lactose (e.g., spray dried lactose) or microcrystalline cellulose
(e.g., Cellets.TM.)
[0430] In another embodiment, the drug substance containing matrix
is incorporated in the cores of the particles. The matrix forming
excipients, fillers, and other ingredients for enhancing the
process are mixed together with the drug substance. The powder
mixtures obtained can be formulated as particles by using wet
extrusion or melt extrusion and subsequent spheronization, or by
compacting the mixtures to minitablets. The matrices formed could
be either fast disintegrating/dissolving matrices, or
non-disintegrating matrices with extended release properties built
with hydrophilic or lipophilic matrix forming excipients.
[0431] In an embodiment, multi-particulates consisting of a
hydrophilic, non-disintegrating matrix which contains the drug
substance or a solid dispersion thereof, are prepared by mixing the
active ingredient, a filler, e.g., lactose, together with
hydrophilic, hydrogel forming polymers with different viscosities,
a glidant, and a lubricant. In some embodiments, the hydrophilic,
hydrogel forming polymer may be, for example hydroxypropyl
methylcellulose, with low viscosity grade of less than 20 mPas for
a 2% by weight aqueous solution, e.g., Methocel E5, combined with
hydroxypropyl methylcellulose grade with high viscosity of more
than 100 mPas for a 2% by weight aqueous solution, e.g., Methocel
K100. The powder mixture is then compressed on the tabletting
machine to obtain minitablets. Alternatively, the powder mixture
can be wetted with organic solvent, e.g., ethanol, and then
extruded and spheronized for obtaining multi-particulates.
[0432] In another embodiment, multi-particulates consisting of a
lipophilic, non-disintegrating matrix which contains the drug
substance or a solid dispersion thereof are prepared by mixing the
active ingredient, lipophilic, meltable, matrix forming excipients,
and fillers. The mixture is processed by melting and mixing in an
extruder. The obtained extudate strands are cut into particles and
are optionally spheronized. The lipophilic excipients used are for
example Vitamin E polyethylen glycol succinate (Vit E TPGS, e.g.,
Kolliphor TPGS Pharma from BASF) solely, or in combination with
glycerol monostearate (GMS, e.g., Kolliwax GMS from BASF) at ratios
of 9:1 to 1:9.
[0433] In some embodiments, an extended release formulation of an
mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, reduces
the peak concentration (C.sub.max) to concentration at 24 hours
post-dose (C.sub.24h) ratio after a single dose administration in
24 healthy subjects, as compared to an immediate release tablet,
e.g., a rapamycin or RAD001 immediate release tablet available to
patients (Final Market Image or "FMI" tablets). In some
embodiments, the C.sub.max/C.sub.24h ratio is decreased, e.g., as
measured by pharmacokinetic model simulations. An advantage of a
reduced C.sub.max/C.sub.min ratio is that, with the appropriate
dose based on the bioavailability of the mTOR inhibitor relative to
an FMI formulation, the concentration of mTOR inhibitor may be
maintained above the lower therapeutic range of drug (for
sufficient efficacy) and at the same time distance away from the
upper therapeutic range of drug (concentration region of toxicity).
Thus, in some embodiments, an extended release formulation of an
mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, is able
to improve the safety profile of the mTOR inhibitor without
affecting its efficacy. In an embodiment, a C.sub.max/C.sub.24h
(thus C.sub.max/C.sub.min) ratio in patients having been
administered an extended release formulation of an mTOR inhibitor
disclosed herein, e.g., rapamycin or RAD001, is <5 or <4,
e.g. 3.5.+-.1 or 3.+-.0.5.
[0434] In an embodiment, an mTOR inhibitor disclosed herein, e.g.,
rapamycin or RAD001, is contained in a layer separate from the
functional layer or top coat controlling the extended release
properties of the formulation. Such layer may be made of any
substance which is suitable for dispersing or dissolving the mTOR
inhibitor. In an embodiment, the layer comprising the mTOR
inhibitor is made of a hydrophilic carrier matrix. The carrier
matrix may be embedding the active ingredient and protecting it
against degradation. Suitable matrix formers include, but are not
limited to, hydrophilic polymers, e.g. HPMC type 2910 or type 2280,
HPC, HEC, MEC, MHEC, povidone, which can be dissolved or rapidly
dispersed in water. In one embodiment, the matrix layer is in form
of a solid dispersion, for instance as described in WO97/03654 or
WO03/028705, the entire contents of each of which are incorporated
herein by reference.
[0435] In an embodiment, the fast dissolving/disintegrating carrier
matrix for an mTOR inhibitor disclosed herein, e.g., rapamycin or
RAD001, is in form of a solid dispersion. In some embodiments, the
solid dispersion comprises a carrier, e.g., a water-soluble
polymer, for example one or a mixture of the following polymers may
be used: [0436] hydroxypropylmethylcellulose (HPMC), e.g.,
Hypromellose type 2910, which is available as Methocel.TM. E from
Dow Chemicals or Pharmacoat.TM. from Shin Etsu. Good results may be
obtained using HPMC with a low apparent viscosity, e.g., below 100
cps as measured at 20.degree. C. for a 2% by weight aqueous
solution, e.g. below 50 cps, preferably below 20 cps, for example
HPMC 3 cps; [0437] polyvinylpyrrolidone (povidone, PVP), e.g., PVP
K25, K30 or PVP K12. PVP is available commercially, for example, as
Kollidon.RTM. from the BASF company or as Plasdone.RTM. from ISP
company. A PVP having an average molecular weight between about
8,000 and about 50,000 Daltons is preferred, e.g., PVP K30; [0438]
hydroxypropylcellulose (HPC), e.g., Klucel EF/LF/J For a derivative
thereof. Examples of HPC derivatives include those having low
dynamic viscosity in aqueous media, e.g., water, e.g. below about
400 cps as measured in a 5% aqueous solution at 25.degree. C.
Preferred HPC derivatives an average molecular weight below about
200,000 Daltons, e.g., between 80,000 and 140,000 Daltons. Examples
of HPC available commercially include Klucel.RTM. LF, Klucel.RTM.
EF and Klucel.RTM. JF from the Hercules Aqualon company; and
Nisso.RTM. HPC-L available from Nippon Soda Ltd; [0439] a
polyethylene glycol (PEG). Examples include PEGs having an average
molecular weight between 1000 and 9000 Daltons, e.g. between about
1800 and 7000, for example PEG 2000, PEG 4000, or PEG 6000
(Handbook of Pharmaceutical Excipients, p. 355-361); [0440] a
saturated polyglycolised glyceride, available for example, as
Gelucire.RTM., e.g., Gelucire.RTM. 44/14, 53/10, 50/13, 42/12, or
35/10 from the Gattefosse company; or [0441] a cyclodextrin, for
example a .beta.-cyclodextrin or an .alpha.-cyclodextrin. Examples
of suitable .beta.-cyclodextrins include, but are not limited to,
methyl-.beta.-cyclodextrin; dimethyl-.beta.-cyclodextrin;
hydroxyproypl-.beta.-cyclodextrin; glycosyl-.beta.-cyclodextrin;
maltosyl-.beta.-cyclodextrin; sulfo-.beta.-cyclodextrin; a
sulfo-alkylethers of .beta.-cyclodextrin, e.g.
sulfo-C.sub.1-4-alkyl ethers. Examples of .alpha.-cyclodextrins
include, but are not limited to, glucosyl-.alpha.-cyclodextrin and
maltosyl-.alpha.-cyclodextrin.
[0442] In one embodiment, an mTOR inhibitor-containing layer
contains antioxidant in a ratio of 1:1000 to 1:1 related to the
amount of drug substance. The antioxidant may also be present in
other functional layers, e.g., at concentration of 0.1 to 10%,
preferably 0.1 to 1%. Suitable antioxidants include, but are not
limited to, butyl hydroxyl toluol, butyl hydroxy anisol, ascorbyl
palmitate, tocopherol, vitamin E polyethylene glycol succinate. In
a particular embodiment, the antioxidant is butyl hydroxyl
toluol.
[0443] In one embodiment, a protection layer separates the layer
containing the active substance from other functional layers, such
as e.g., the top coating, to enhance stability of the of the drug
product. The drug substance is stabilized by excluding any direct
contact with the top coating. The protection layer also acts as
diffusion barrier preventing any components in the top coating,
e.g., polymer by-products or plasticizers, which can migrate
through the layers, from getting in direct contact with the active.
Beside the polymers, which are used also as matrix formers (e.g.,
the matrix formers described above), high content, of inorganic
pigments or anti-tacking agents such as talc and/or titanium
dioxide, e.g., 10 to 100%, e.g., 20 to 50%, relative to the applied
amount of polymer, contribute to the barrier function. The
protection layer thickness can be adjusted to gain optimized drug
product stability.
[0444] In another embodiment, the mTOR inhibitor, e.g., rapamycin
or RAD001, is directly embedded in the extended release carrier
matrix.
[0445] In some embodiments, a formulation comprising an mTOR
inhibitor disclosed herein, e.g., rapamycin or RAD001, contains
strongly hygroscopic excipients, which are able to bind water
moisture enclosed in the formulation working as an internal
desiccant. Adsorbents such as e.g., crospovidone, croscarmellose
sodium, sodium starch glycolate, or starch can be used. For
example, in some embodiments, crospovidone is used as tablet
disintegrant, e.g., at 2% to 25% crospovidone. The adsorbent, e.g.,
crospovidone, may be part of the powder mixtures used for wet and
melt extrusion, part of the powder blend for compressing the
minitablets, part of powder blend for tabletting the
multi-particulates, and/or directly added to the multi-particulates
in a sachet or capsule filling process.
[0446] In one aspect, an mTOR inhibitor disclosed herein, e.g.,
rapamycin or RAD001, is present in a particle (e.g., 0.1 to 0.5
mm), bead, pellet (e.g., 0.2 to 2 mm) or mini-tablet (e.g., 1.5 to
3 mm), with a low water moisture content of less than 5% in total,
e.g., less than 3% or less than 2.5% in total.
[0447] In some embodiments, a pharmaceutical compositions, e.g., a
multi-particulate delivery system of an mTOR inhibitor disclosed
herein, e.g., rapamycin or RAD001, can be formulated into a drug
product such as e.g., capsules (e.g., HPMC or Hart Gelatine
capsules), or filled into sachets or stick-packs, or formulated as
tablets which release the particles upon disintegration.
[0448] In some embodiments, the primary packaging, such as sachets,
stickpacks, blisters or bottles may include an water sorbing
ingredient, e.g., silica gel, which reduces or stabilizes the water
moisture content of the drug product during shelf life storage
and/or in during in-use time.
[0449] Provided formulations may comprise and/or release multiple
pellets, granules or minitablets.
[0450] In some embodiments, provided formulations, e.g.,
multi-particulates formulations, can be prepared by extruding and
spheronizing a mixture of the matrix forming excipients together
with the drug substance with the aid of heat or wetting liquids, or
by compacting minitablets with drug containing mixtures, or by
layering the drug containing matrix layer onto cores in a fluid bed
or rotogranulation process.
[0451] In some embodiments, the layer containing the active
substance can be prepared by spraying a spray dispersion with
organic solvents in which the hydrophilic components and the active
substance are dispersed or dissolved onto the core material, while
concurrently the solvents are continuously removed by the aid of
heated, dry air. By this process a matrix layer surrounding the
cores is formed, e.g., the layer formed is a solid dispersion of
the active in polymers such as e.g., HPMC, HPC, HEC.
[0452] In one aspect, a provided pharmaceutical formulation may be
prepared as follows: An organic feed mixture for spraying in which
the hydrophilic polymer is dispersed in colloidal manner and an
mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, is
dispersed or dissolved, which precipitate together as a uniform,
smooth layer of solid dispersion upon removal of the solvent in
such a way that they can be coated with modified release coats. In
some embodiments, the obtained drug containing multi-particulates
can be coated with additional functional layers and top coatings. A
spray dispersion containing coating polymers, lubricants, anti tack
agents, pore formers and plastisizers, which are dissolved,
dispersed and suspended in organic solvents and mixtures thereof,
is sprayed onto the drug containing multi-particulates. During
processing the multi-particulates are kept continuously in a
controlled motion or fluidization, while dry, heated process gas is
applied to the product bed for evaporating the solvents from the
surface of the multi-particulates, where the film layer is formed
at a defined temperature. The film layer thickness can be
controlled by the amount of coating dispersion sprayed. Final
drying is applied for minimizing the residual solvent content in
the layered and coated multi-particulates.
[0453] In another aspect, an mTOR inhibitor disclosed herein, e.g.,
rapamycin or RAD001, may be formulated as part of a high drug load
part of an extended release formulation. In some embodiments, the
formulation further comprises a surfactant. The term "surfactant"
can be used interchangeably with a "wetting agent" or "detergent"
and refers to a non-ionic, ionic, anionic, cationic or amphoteric
surfactant, e.g., a non-ionic, ionic, anionic, or amphoteric
surfactant. Examples of suitable surfactants/wetting agents
include, but are not limited to, polyoxyethylene-polyoxypropylene
co-polymers and block co-polymers known, for example, under the
trademarks Pluronic or Poloxamer (e.g. poloxamer 188 (Pluronic
F68), polyoxyethylene, sorbitan fatty acid esters including mono
and tri lauryl, palmityl, stearyl and oleyl esters of the type
known under the trade name Tween, polyoxyethylene fatty acid esters
including polyoxyethylene stearic acid esters of the type known
under the trade name Myrj, polyoxyethylene alkyl ethers known under
the trade mark Brij, sodium alkyl sulfates like Soldium lauryl
sulphate (SDS) and sulfonates, and sodium alkyl aryl sulfonates,
water soluble tocopheryl polyethylene glycol succinic acid esters
(TPGS), polyglycerol fatty acid esters, alkylene polyol ethers or
esters, polyethylene glycol glyceryl fatty acid esters, sterols and
derivatives thereof, transesterified, polyoxyethylated
caprylic-capric acid glycerides, sugar fatty acid esters, PEG
sterol ethers, phospholipids, salts of fatty acids, fatty acid
sulfates and sulfonates, salts of fatty acids, fatty acid sulfates
and sulfonates, medium or long-chain alkyl, e.g., C.sub.6-C.sub.18,
ammonium salts, bile acid or salt thereof; for example cholic acid,
glycolic acid or a salt, e.g., sodium cholate and polyoxyethylene
mono esters of a saturated C.sub.10 to C.sub.22 fatty acid. In a
particular embodiment the surfactant is
polyoxyethylene-polyoxypropylene co-polymer or block co-polymer, or
a water soluble tocopheryl polyethylene glycol succinic acid ester,
e.g., a water soluble tocopheryl polyethylene glycol succinic acid
ester, e.g., Vitamin E polyethylene glycol 1000 succinate (TPGS).
In another embodiment the surfactant in the present pharmaceutical
formulation is polyoxyethylene-polyoxypropylene co-polymer, e.g.,
poloxamer 188. In yet another embodiment, the pharmaceutical
formulation comprises the surfactant sodium alkyl sulfate, e.g.,
sodium lauryl sulfate.
[0454] The surfactant or wetting agent may be present in a
formulation in a ratio to mTOR inhibitor, e.g., rapamycin or
RAD001, from 10:1 to 1:200 by weight, e.g., 1:1 to 1:100 by weight,
1:2 to 1:8 by weight, 1:4 to 1:6 by weight.
[0455] In some embodiments, the mTOR inhibitor, e.g., rapamycin or
RAD001, is in a high drug load containing first layer, and a
surfactant in a second layer, wherein the second layer is beneath
the first layer, optionally with additional extended release
coating. In some such embodiments, the surfactant is not poloxamer
188 and TPGS. In some embodiments, the surfactant or wetting agent
in a second layer can form a protection layer which separates the
active ingredient containing layer from the coating covering the
formulation. The coating covering the formulation may be an
extended release coating.
Other Embodiments
[0456] The invention further provides T cell preparations of T
cells treated with a low, immune enhancing, dose of mTOR inhibitor,
as described herein, e.g., for use in treating a subject with a
disease, e.g., a lymphoproliferative disease. In some embodiments,
the T cells are recovered from a subject that has been administered
a low, immune enhancing, dose of mTOR inhibitor, as described
herein. Suitable methods of recovering T cells from a subject are
known in the art, and include isolation from peripheral blood or
bone marrow by filtration, affinity chromatography, or magnetic
labelling and separation. In other embodiments, the T cells
recovered from a subject are treated with a low, immune enhancing,
dose of mTOR inhibitor as described herein in vitro, e.g., in cell
culture. In one embodiment, the T cell preparation is obtained from
a subject with a lymphoproliferative disease before the subject
receives a bone marrow or stem cell transplant, and the T cell
preparation is delivered to the subject after the bone marrow or
stem cell transplant. The T cell preparation can increase or
improve the effect of the bone marrow or stem cell transplant,
e.g., increasing anti-cancer cell immune function and recovery of
the immune system. The lymphoproliferative disease can be a
leukemia or a lymphoma, e.g., chronic myelogenous leukemia (CML),
acute myeloid leukemia (AML), Burkitt's lymphoma, diffuse large
cell lymphoma, follicular lymphoma, hairy cell lymphoma, mantle
cell lymphoma, myelodysplastic syndromes, and non-Hodgkin's
lymphoma.
EXAMPLES
[0457] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0458] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples specifically point out various aspects
of the present invention, and are not to be construed as limiting
in any way the remainder of the disclosure.
Example 1
Effects of mTOR Inhibition on Immunosenescence in the Elderly
[0459] One of the pathways most clearly linked to aging is the mTOR
pathway. The mTOR inhibitor rapamycin has been shown to extend
lifespan in mice and improve a variety of aging-related conditions
in old mice (Harrison, D E et al. (2009) Nature 460:392-395;
Wilkinson J E et al. (2012) Aging Cell 11:675-682; and Flynn, J M
et al. (2013) Aging Cell 12:851-862). Thus, these findings indicate
that mTOR inhibitors may have beneficial effects on aging and
aging-related conditions in humans.
[0460] An age-related phenotype that can be studied in a short
clinical trial timeframe is immunosenescence. Immunosenescence is
the decline in immune function that occurs in the elderly, leading
to an increased susceptibility to infection and a decreased
response to vaccination, including influenza vaccination. The
decline in immune function with age is due to an accumulation of
immune defects, including a decrease in the ability of
hematopoietic stem cells (HSCs) to generate naive lymphocytes, and
an increase in the numbers of exhausted PD-1 positive lymphocytes
that have defective responses to antigenic stimulation (Boraschi, D
et al. (2013) Sci. Transl. Med. 5:185ps8; Lages, C S et al. (2010)
Aging Cell 9:785-798; and Shimatani, K et al., (2009) Proc. Natl.
Acad. Sci. USA 106:15807-15812). Studies in elderly mice showed
that 6 weeks of treatment with the mTOR inhibitor rapamycin
rejuvenated HSC function leading to increased production of naive
lymphocytes, improved response to influenza vaccination, and
extended lifespan (Chen, C et al. (2009) Sci. Signal. 2:ra75).
[0461] To assess the effects of mTOR inhibition on human
aging-related phenotypes and whether the mTOR inhibitor RAD001
ameliorates immunosenescence, the response to influenza vaccine in
elderly volunteers receiving RAD001 or placebo was evaluated. The
findings presented herein suggest that RAD001 enhanced the response
to influenza vaccine in elderly volunteers at doses that were well
tolerated. RAD001 also reduced the percentage of programmed death
(PD)-1 positive CD4 and CD8 T lymphocytes that accumulate with age.
These results show that mTOR inhibition has beneficial effects on
immunosenescence in elderly volunteers.
[0462] As described herein, a 6 week treatment with the mTOR
inhibitor RAD001, an analog of rapamycin, improved the response to
influenza vaccination in elderly human volunteers.
Methods
Study Population
[0463] Elderly volunteers >=65 years of age without unstable
underlying medical diseases were enrolled at 9 sites in New Zealand
and Australia. Exclusion criteria at screening included hemoglobin
<9.0 g/dL, white blood cell count <3,500/mm.sup.3, neutrophil
count <2,000/mm.sup.3, or platelet count <125,000/mm.sup.3,
uncontrolled diabetes, unstable ischemic heart disease, clinically
significant underlying pulmonary disease, history of an
immunodeficiency or receiving immunosuppressive therapy, history of
coagulopathy or medical condition requiring long-term
anticoagulation, estimated glomerular filtration rate <30
ml/min, presence of severe uncontrolled hypercholesterolemia
(>350 mg/dL, 9.1 mmol/L) or hypertriglyceridemia (>500 mg/dL,
5.6 mmol/L).
[0464] Baseline demographics between the treatment arms were
similar (Table 2). Of the 218 subjects enrolled, 211 completed the
study. Seven subjects withdrew from the study. Five subjects
withdrew due to adverse events (AEs), one subject withdrew consent,
and one subject left the study as a result of a protocol
violation.
TABLE-US-00002 TABLE 2 Demographic and Baseline characteristics of
the Study Patients RAD001 RAD001 RAD001 Placebo 0.5 mg daily 5 mg
weekly 20 mg weekly pooled Total Population N = 53 N = 53 N = 53 N
= 59 N = 218 Age (Years) Mean (SD) 70.8 (5.0) .sup. 72.0 (5.3)
.sup. 71.4 (5.2) .sup. 71.1 (5.1) .sup. 71.3 (5.2).sup. Gender
Male- n (%) 34 (64%) 27 (51%) 32 (60%) 31 (53%) 124 (57%) BMI*
(kg/m2) Mean (SD) 27.4 (4.2) .sup. 28.8 (5.0) .sup. 28.0 (4.1)
.sup. 28.0 (4.2) .sup. 28.0 (4.4).sup. Race - n (%) Caucasian 48
(91%) 50 (94%) 46 (87%) 54 (92%) 198 (91%) Other 5 (9%) 3 (6%) 7
(13%) 5 (8%) 20 (9%) *The body-mass index is weight in kilograms
divided by the square of the height in meters
Study Design and Conduct
[0465] From December 2011 to April 2012, 218 elderly volunteers
were enrolled in a randomized, observer-blind, placebo-controlled
trial. The subjects were randomized to treatment arms using a
validated automated randomization system with a ratio of RAD001 to
placebo of 5:2 in each treatment arm. The treatment arms were:
[0466] RAD001 0.5 mg daily or placebo
[0467] RAD001 5 mg weekly or placebo
[0468] RAD001 20 mg weekly or placebo
[0469] The trial was observer-blind because the placebo in the
RAD001 0.5 mg daily and 20 mg weekly cohorts differed slightly from
the RAD001 tablets in those cohorts. The study personnel evaluating
the subjects did not see the study medication and therefore were
fully blinded. The treatment duration for all cohorts was 6 weeks
during which time subjects underwent safety evaluations in the
clinic every 2 weeks. After subjects had been dosed for 4 weeks,
RAD001 steady state levels were measured pre-dose and at one hour
post dose. After completing the 6 week course of study drug,
subjects were given a 2 week drug free break to reverse any
possible RAD001-induced immunosuppression, and then were given a
2012 seasonal influenza vaccination (Agrippal.RTM., Novartis
Vaccines and Diagnostics, Siena, Italy) containing the strains H1N1
A/California/07/2009, H3N2 A/Victoria/210/2009, B/Brisbane/60/2008.
Four weeks after influenza vaccination, subjects had serum
collected for influenza titer measurements. Antibody titers to the
3 influenza vaccine strains as well as to 2 heterologous strains
(A/H1N1 strain A/New Jersey/8/76 and A/H3N2 strain
A/Victoria/361/11) were measured by standard hemagglutination
inhibition assay (Kendal, A P et al. (1982) Concepts and procedures
for laboratory-based influenza surveillance. Atlanta: Centers for
Disease Control and Prevention B17-B35). Levels of IgG and IgM
specific for the A/H1N1/California/07/2009 were measured in serum
samples taken before and 4 weeks after influenza vaccination as
described previously (Spensieri, F. et al. (2013) Proc. Natl. Acad.
Sci. USA 110:14330-14335). Results were expressed as fluorescence
intensity.
[0470] All subjects provided written informed consent. The study
was conducted in accordance with the principals of Good Clinical
Practice and was approved by the appropriate ethics committees and
regulatory agencies.
Safety
[0471] Adverse event assessment and blood collection for
hematologic and biochemical safety assessments were performed
during study visits. Adverse event information was also collected
in diaries that subjects filled out at home during the 6 weeks they
were on study drug. Data on all adverse events were collected from
the time of informed consent until 30 days after the last study
visit. Events were classified by the investigators as mild,
moderate or severe.
Statistical Analysis
[0472] The primary analysis of geometric mean titer ratios was done
using a normal Bayesian regression model with non-informative
priors. This model was fitted to each antibody titer on the log
scale. The primary outcome in each model was the Day 84
measurement. The Day 63 measurement was included in the outcome
vector. The model fitted using SAS 9.2 proc mixed with the prior
statement. The covariance structure of the matrix was considered as
unstructured (option type=UN). A flat prior was used. For the
secondary analysis of seroconversion rates, logistic regression was
used.
[0473] The intention to treat population was defined as all
subjects who received at least one full dose of study drug and who
had no major protocol deviations impacting efficacy data. 199 out
of the total of 218 subjects enrolled in the study were in the
intention to treat population.
Immunophenotyping
[0474] Peripheral blood mononuclear cells were isolated from whole
blood collected at 3 time points: baseline; after 6 weeks of study
drug treatment; and at the end of study when subjects had been off
study drug for 6 weeks and 4 weeks after influenza vaccination.
Seventy-six PBMC subsets were analyzed by flow cytometry using
8-color immunophenotyping panels at the Human Immune Monitoring
Center at Stanford University, CA, USA as described previously
(Maecker, H T et al. (2012) Nat Rev Immunol. 12:191-200).
Seventy-six PBMC subsets were analyzed by flow cytometry using
8-color lyophilized immunophenotyping panels (BD Lyoplate, BD
Biosciences, San Diego, Calif.). PBMC samples with viability
>80% and yield of 2.times.10.sup.6 cells or greater were
included in the analysis.
[0475] Relative changes of the immunophenotypes from baseline to
Week 6 of study drug treatment and from baseline to the end of
study (Week 12) were calculated for each of the RAD001 dosing
cohorts. Student T test was conducted to examine if the relative
change of the immunophenotypes from baseline to the two blood
sampling time points was significantly different from zero,
respectively, within each dosing group after adjusting for placebo
effect. Missing data imputation in treatment effect analysis was
not conducted. Therefore if a patient has a missing phenotype data
at baseline, this patient was not be included in the analysis for
this phenotype. If a patient had a missing phenotype data at 6 or
12 weeks, then this patient did not contribute to the analysis of
this phenotype for the affected timepoint.
[0476] 608 tests in 76 phenotypes under 3 dosing groups were
conducted to compare the treatment effect against the placebo
effect. Stratified false discovery rate (FDR) control methodology
was implemented to control the occurrence of false positives
associated with multiple testing yet provide considerably better
power. The cell type group was taken as the stratification factor
and conducted FDR (q-value) calculation within each stratum
respectively. All null-hypotheses were rejected at 0.05
significance level with corresponding q-value <0.1. The multiple
testing adjustment strategy with rejecting at 0.05 significance
level and corresponding q<0.1 ensured that less than 10% of the
findings are false.
[0477] In a second analysis, the immunophenotype changes between
pooled treatment and placebo groups, where all three RAD001 dosing
groups were combined. To determine which immunophenotype changes
differed between the treated and placebo groups, within-patient
cell count ratios for each measured phenotype were calculated
between baseline and Week 6 of study drug treatment and between
baseline and the end of study (Week 12). The ratios were log
transformed, and analyzed by analysis of covariance at each time
point in order to detect a difference between the pooled treatment
and placebo groups. 152 tests in 76 phenotypes were performed to
compare the pooled treatment effect against the placebo effect.
Stratified false discovery rate (FDR) control methodology was
implemented to control the occurrence of false positives associated
with multiple testing yet provide considerably better power
(Benjamini, Y. et al. (1995) J. Roy. Statist. 57:289-300; and Sun,
L. et al. (2006) Genet. Epidemiol. 30:519-530). The cell type group
was taken as the stratification factor and FDR (q-value)
calculation was conducted within each stratum respectively. All
null-hypotheses at 0.05 significance level and q-value less than
20% were rejected. This can be interpreted as rejecting only those
hypotheses with P values less than 0.05 and less than 20%
probability that the each observed significant result is due to
multiple testing.
Results
[0478] In general, RAD001 was well tolerated, particularly the 0.5
mg daily and 5 mg weekly dosing regimens. No deaths occurred during
the study. Three subjects experienced four serious adverse events
(SAEs) that were assessed as unrelated to RAD001. The 4 SAEs were
retinal hemorrhage of the left eye with subsequent blindness in a
subject with normal platelet counts who had completed a 6 week
course of 5 mg weekly RAD001 6 weeks previously; severe back pain
in a subject treated with placebo and severe gastroenteritis in a
subject treated with placebo. A list of treatment-related adverse
events (AEs) with an incidence >2% in any treatment group is
provided in Table 3. The most common RAD001-related AE was mouth
ulcer that, in the majority of cases, was of mild severity.
Overall, subjects who received RAD001 had a similar incidence of
severe AEs as those treated with placebo. Only one severe AE was
assessed as related to RAD001 mouth ulcers in a subject treated
with 20 mg weekly RAD001.
TABLE-US-00003 TABLE 3 Incidence of treatment-related AEs > 2%
in any treatment group by preferred term RAD001 RAD001 RAD001 0.5
mg 5 mg 20 mg Placebo, daily weekly weekly pooled Total N = 53 N =
53 N = 53 N = 59 N = 218 n (%) n (%) n (%) n (%) n (%) Total AE(s)
35 46 109 21 211 Patients 22 20 27 12 81 with AE(s) (41.5%) (37.7%)
(50.9%) (20.3%) (37.2%) Mouth 6 2 9 3 20 ulceration (11.3%) (3.8%)
(17.0%) (5.1%) (9.2%) Headache 0 2 9 1 12 (3.8%) (17.0%) (1.7%)
(5.5%) Blood 2 2 2 0 6 cholesterol (3.8%) (3.8%) (3.8%) (2.8%)
increased Diarrhea 1 4 1 0 6 (1.9%) (7.5%) (1.9%) (2.8%) Dyspepsia
0 3 2 1 6 (5.7%) (3.8%) (1.7%) (2.8%) Fatigue 0 2 4 0 6 (3.8%)
(7.5%) (2.8%) Low 2 1 2 0 5 density (3.8%) (1.9%) (3.8%) (2.3%)
lipoprotein increased Tongue 3 1 0 1 5 ulceration (5.7%) (1.9%)
(1.7%) (2.3%) Insomnia 1 2 1 0 4 (1.9%) (3.8%) (1.9%) (1.8%) Dry
mouth 0 0 2 1 3 (3.8%) (1.7%) (1.4%) Neutropenia 0 0 3 0 3 (5.7%)
(1.4%) Oral pain 0 2 1 0 3 (3.8%) (1.9%) (1.4%) Pruritus 0 2 1 0 3
(3.8%) (1.9%) (1.4%) Conjunctivitis 0 2 0 0 2 (3.8%) (0.9%)
Erythema 0 2 0 0 2 (3.8%) (0.9%) Limb 0 2 0 0 2 discomfort (3.8%)
(0.9%) Mucosal 0 0 2 0 2 inflammation (3.8%) (0.9%) Paresthesia 2 0
0 0 2 oral (3.8%) (0.9%) Stomatitis 0 0 2 0 2 (3.8%) (0.9%)
Thrombocytopenia 0 0 2 0 2 (3.8%) (0.9%) Urinary tract 0 0 2 0 2
infection (3.8%) (0.9%)
[0479] The ability of RAD001 to improve immune function in elderly
volunteers was evaluated by measuring the serologic response to the
2012 seasonal influenza vaccine. The hemagglutination inhibition
(HI) geometric mean titers (GMT) to each of the 3 influenza vaccine
strains at baseline and 4 weeks after influenza vaccination are
provided in Table 4. The primary analysis variable was the HI GMT
ratio (4 weeks post vaccination/baseline). The study was powered to
be able to demonstrate that in at least 2 out of 3 influenza
vaccine strains there was 1) a >1.2-fold GMT increase relative
to placebo; and 2) a posterior probability no lower than 80% that
the placebo-corrected GMT ratio exceeded 1. This endpoint was
chosen because a 1.2-fold increase in the influenza GMT ratio
induced by the MF-59 vaccine adjuvant was associated with a
decrease in influenza illness (Iob, A et al. (2005) Epidemiol
Infect 133:687-693).
TABLE-US-00004 TABLE 4 HI GMTs for each influenza vaccine strain at
baseline and at 4 weeks after influenza vaccination Influenza
RAD001 RAD001 RAD001 Vaccine 0.5 mg dally 5 mg weekly 20 mg weekly
Placebo Strain Time N = 50 N = 49 N = 49 N = 55 A/H1N1 GMT (CV %)
Baseline 102.8 (186.9) 84.2 (236.4) 90.1 (188.4) 103.2 (219.7) Week
4 190.2 (236.9) 198.73 (195.6) 129.7 (175.9) 169.4 (259.8) GMT
ratio (CV %) 2.6 (302.5) 2.5 (214.3) 1.8 (201.5) 2.0 (132.7) A/H3N2
GMT (CV %) Baseline 106.8 (168.2) 126.04 (162.6) 137.1 (211.5)
131.7 (162.3) Week 4 194.4 (129.1) 223.0 (118.8) 223.0 (163.6)
184.3 (153.2) GMT ratio (CV %) 2.1 (152.6) 2.0 (189.2) 2.1 (277.3)
1.6 (153.6) B GMT (CV %) Baseline 44.2 (96.6) 64.8 (87.3) 58.0
(156.0) 57.0 (112.6) Week 4 98.4 (94.8) 117.3 (99.9) 99.2 (124.1)
114.6 (136.7) GMT ratio (CV %) 2.5 (111.2) 2.2 (112.8) 2.1 (126.5)
2.2 (109.2) Baseline indicates 2 weeks prior to influenza
vaccination Week 4 indicates 4 weeks after influenza vaccination N
is number of subjects per cohort GMT is geometric mean titer GMT
ratio is the GMT at week 4 post vaccination/GMT at baseline CV %
indicates coefficient of variation
[0480] In the intent-to-treat (ITT) population, the low, immune
enhancing, dose RAD001 (0.5 mg daily or 5 mg weekly) cohorts but
not higher dose (20 mg weekly) cohort met the primary endpoint of
the study (FIG. 1A). This demonstrates that there is a distinct
immunomodulatory mechanism of RAD001 at the lower doses, and that
at the higher dose the known immunosuppressive effects of mTOR
inhibition may come into play. Furthermore, the results suggest a
trend toward improved immune function in the elderly after low,
immune enhancing, dose RAD001 treatment.
[0481] In a subgroup analysis, the subset of subjects with low
baseline influenza titers (<1:40) experienced a greater
RAD001-associated increase in titers than did the ITT population
(FIG. 1B). These data show that RAD001 is particularly effective at
enhancing the influenza vaccine response of subjects who did not
have protective (>1:40) titers at baseline, and therefore were
at highest risk of influenza illness.
[0482] Scatter plots of RAD001 concentration versus increase in
titer to each influenza vaccine strain show an inverse
exposure/response relationship (FIG. 2). Modeling and simulation
based on mTOR mediated phosphorylation of S6 kinase (S6K) predicts
that the 20 mg weekly dosing regimen inhibits mTOR-mediated S6K
activity almost completely, the 5 mg weekly dosing regimen inhibits
S6K activity by over 50%, and the 0.5 mg daily dosing regiment
inhibits S6K phosphorylation by approximately 38% during the dosing
interval (Tanaka, C et al. (2008) J. Clin. Oncol 26:1596-1602).
Thus, partial mTOR inhibition, e.g., mTOR-mediated S6K
phosphorylation, with low, immune enhancing, dose RAD001 may be as,
if not more effective, than near complete mTOR inhibition with high
dose RAD001 at enhancing the immune response of the elderly.
[0483] Rates of seroconversion 4 weeks after influenza vaccination
were also evaluated. Seroconversion was defined as the change from
a negative pre-vaccination titer (i.e., HI titer <1:10) to
post-vaccination HI titer >1:40 or at least 4-fold increase from
a non-negative (>1:10) pre-vaccination HI titer. In the
intention-to-treat population, seroconversion rates for the H3N2
and B strains were increased in the RAD001 as compared to the
placebo cohorts although the increases did not meet statistical
significance (Table 5). In the subpopulation of subjects with
baseline influenza titers <=1:40, RAD001 treatment also
increased the rates of seroconversion to the H3N2 and B strains,
and these results reached statistical significance for the B strain
in the 0.5 mg daily dosing cohort. These data further show that
RAD001 enhanced the serologic response to influenza vaccination in
the elderly.
TABLE-US-00005 TABLE 5 Percent of subjects with seroconversion to
influenza 4 weeks after vaccination Placebo 0.5 mg 5 mg 20 mg N =
54 N = 48 N = 49 N = 48 Intention to Treat Population H1N1 24 27 27
17 H3N2 17 27 24 25 B 17 27 22 19 Subjects with Baseline Titers
<=40 H1N1 40 42 45 36 H3N2 42 64 53 71 B 16 40* 33 28 *Odds
ratio for seroconversion between RAD001 and Placebo significantly
different than 1 (two-sided p-value < 0.05 obtained by logistic
regression with treatment as fixed effect)
[0484] Current seasonal influenza vaccines often provide inadequate
protection against continuously emerging strains of influenza that
present as variants of previously circulating viruses. However,
mice vaccinated against influenza in the presence of the mTOR
inhibitor rapamycin, as compared to placebo, developed a broader
serologic response to influenza. The broader serologic response
included antibodies to conserved epitopes expressed by multiple
subtypes of influenza that provided protection against infection
with heterologous strains of influenza not contained in the vaccine
(Keating, R et al. (2013) Nat Immunology 14:2166-2178). To
determine if RAD001 broadened the serologic response to influenza
in the elderly volunteers, HI titers to 2 heterologous strains of
influenza not contained in the influenza vaccine (A/H1N1 strain
A/New Jersey/8/76 and A/H3N2 strain A/Victoria/361/11) were
measured. The increase in the HI GMT ratios for the heterologous
strains was higher in the RAD001 as compared to placebo cohorts
(FIG. 3). In addition, seroconversion rates for the heterologous
strains were higher in the RAD001 as compared to placebo cohorts.
The increase in seroconversion rates in the 5 and 20 mg weekly
RAD001 dosing cohorts was statistically significant for the H3N2
heterologous strain (Table 6). The H3N2 seroconversion rate for the
pooled RAD001 cohorts was 39% versus 20% for the placebo cohort
(p=0.007). The results presented herein suggest that mTOR
inhibition broadens the serologic response of elderly volunteers to
influenza vaccination, and increases antibody titers to
heterologous strains of influenza not contained in the seasonal
influenza vaccine.
[0485] Broadened serologic response to heterologous strains of
influenza in mice treated with rapamycin has been associated with
an inhibition of class switching in B cells and an increase in
anti-influenza IgM levels (Keating, R. et al. (2013) Nat Immunol
14:2166-2178). However, inhibition of class switching may not be
involved in the broadened serologic response in humans treated with
RAD001 because the post-vaccination anti-influenza IgM and IgG
levels did not differ between RAD001 and placebo treated cohorts
(FIGS. 4A and 4B, respectively).
TABLE-US-00006 TABLE 6 Percentage of subjects who seroconvert to
heterologous strains of influenza 4 weeks after seasonal influenza
vaccination Placebo, RAD001 RAD001 RAD001 pooled 0.5 mg daily 5 mg
weekly 20 mg weekly A/H1N1 strain: 7% 17% 16% 8% A/NewJersey/8/76
A/H3N2 strain: 20% 38% 39%* 40%* A/Victoria/361/11 *Odds ratio for
seroconversion between RAD001 and Placebo significantly different
than 1 (two-sided p-value < 0.05 obtained by logistic regression
with treatment as fixed effect)
[0486] To address the mechanism by which RAD001 enhanced immune
function in elderly volunteers, immunophenotyping was performed on
PBMC samples obtained from subjects at baseline, after 6 weeks of
study drug treatment and 4 weeks after influenza vaccination (6
weeks after study drug discontinuation). Although the percentage of
most PBMC subsets did not differ between the RAD001 and placebo
cohorts, the percentage of PD-1 positive CD4 and CD8 cells was
lower in the RAD001 as compared to placebo cohorts (FIGS. 5A, 5B,
and 5C). PD-1 positive CD4 and CD8 cells accumulate with age and
have defective responses to antigen stimulation because PD-1
inhibits T cell receptor-induced T cell proliferation, cytokine
production and cytolytic function (Lages, C S et al. (2010) Aging
Cell 9:785-798). There was an increase in percentage of PD-1
positive T cells over time in the placebo cohort. At week 12 (4
weeks post-vaccination) this increase may have been due to
influenza vaccination since influenza virus has been shown to
increase PD-1 positive T cells (Erikson, J J et al. (2012) JCI
122:2967-2982). However the percentage of CD4 PD-1 positive T cells
decreased from baseline at week 6 and 12 in all RAD001 cohorts
(FIG. 5A). The percentage of CD8 PD-1 positive cells also decreased
from baseline at both week 6 and 12 in the two lower dose RAD001
cohorts (FIG. 5B). The percentage of PD-1 negative CD4 T cells was
evaluated and increased in the RAD001 cohorts as compared to the
placebo cohorts (FIG. 5C).
[0487] Under more stringent statistical analysis, where the results
from the RAD001 cohorts were pooled and adjusted for differences in
baseline PD-1 expression, there was a statistically significant
decrease of 30.2% in PD-1 positive CD4 T cells at week 6 in the
pooled RAD cohort (n=84) compared to placebo cohort (n=25) with
p=0.03 (q=0.13) (FIG. 6A). The decrease in PD-1 positive CD4 T
cells at week 12 in the pooled RAD as compared to the placebo
cohort is 32.7% with p=0.05 (q=0.19). FIG. 6B shows a statistically
significant decrease of 37.4% in PD-1 positive CD8 T cells at week
6 in the pooled RAD001 cohort (n=84) compared to placebo cohort
(n=25) with p=0.008 (q=0.07). The decrease in PD-1 positive CD8 T
cells at week 12 in the pooled RAD001 as compared to the placebo
cohort is 41.4% with p=0.066 (q=0.21). Thus, the results from FIGS.
5 and 6 together suggest that the RAD001-associated decrease in the
percentage of PD-1 positive CD4 and CD8 T cells may contribute to
enhanced immune function.
Conclusion
[0488] In conclusion, the data presented herein show that the mTOR
inhibitor RAD001 ameliorates the age-related decline in
immunological function of the human elderly as assessed by response
to influenza vaccination, and that this amelioration is obtained
with an acceptable risk/benefit balance. In a study of elderly
mice, 6 weeks treatment with the mTOR inhibitor rapamycin not only
enhanced the response to influenza vaccination but also extended
lifespan, suggesting that amelioration of immunosenescence may be a
marker of a more broad effect on aging-related phenotypes.
[0489] Since RAD001 dosing was discontinued 2 weeks prior to
vaccination, the immune enhancing effects of RAD001 may be mediated
by changes in a relevant cell population that persists after
discontinuation of drug treatment. The results presented herein
show that RAD001 decreased the percentage of exhausted PD-1
positive CD4 and CD8 T cells as compared to placebo. PD-1
expression is induced by TCR signaling and remains high in the
setting of persistent antigen stimulation including chronic viral
infection. While not wishing to be bound by theory, is possible
that RAD001 reduced chronic immune activation in elderly volunteers
and thereby led to a decrease in PD-1 expression. RAD001 may also
directly inhibit PD-1 expression as has been reported for the
immunophilin cyclosporine A (Oestreich, K J et al. (2008) J
Immunol. 181:4832-4839). A RAD001-induced reduction in the
percentage of PD-1 positive T cells is likely to improve the
quality of T cell responses. This is consistent with previous
studies showing that mTOR inhibition improved the quality of memory
CD8 T cell response to vaccination in mice and primates (Araki, K
et al. (2009) Nature 460:108-112). In aged mice, mTOR inhibition
has also been shown to increase the number of hematopoietic stem
cells, leading to increased production of naive lymphocytes (Chen,
C et al. (2009) Sci Signal 2:ra75). Although significant
differences in the percentages of naive lymphocytes in the RAD001
versus placebo cohorts were not detected in this example, this
possible mechanism may be further investigated.
[0490] The mechanism by which RAD001 broadened the serologic
response to heterologous strains of influenza may be further
investigated. Rapamycin has also been shown to inhibit class
switching in B cells after influenza vaccination. As a result, a
unique repertoire of anti-influenza antibodies was generated that
promoted cross-strain protection against lethal infection with
influenza virus subtypes not contained in the influenza vaccine
(Keating, R et al. (2013) Nat Immunol. 14:2166-2178). The results
described herein did not show that RAD001 altered B cell class
switching in the elderly subjects who had discontinued RAD001 2
weeks prior to influenza vaccination. Although the underlying
mechanism requires further elucidation, the increased serologic
response to heterologous influenza strains described herein may
confer enhanced protection to influenza illness in years when there
is a poor match between the seasonal vaccine and circulating
strains of influenza in the community.
[0491] The effect of RAD001 on influenza antibody titers was
comparable to the effect of the MF59 vaccine adjuvant that is
approved to enhance the response of the elderly to influenza
vaccination (Podda, A (2001) Vaccine 19:2673-2680). Therefore,
RAD001-driven enhancement of the antibody response to influenza
vaccination may translate into clinical benefit as demonstrated
with MF59-adjuvanted influenza vaccine in the elderly (Iob, A et
al. (2005) Epidemiol Infect. 133:687-693). However, RAD001 is also
used to suppress the immune response of organ transplant patients.
These seemingly paradoxical findings raise the possibility that the
immunomodulatory effects of mTOR inhibitors may be dose and/or
antigen-dependent (Ferrer, I R et al. (2010) J Immunol.
185:2004-2008). A trend toward an inverse RAD001
exposure/vaccination response relationship was seen herein. It is
possible that complete mTOR inhibition suppresses immune function
through the normal cyclophilin-rapamycin mechanism, whereas partial
mTOR inhibition, at least in the elderly, enhances immune function
due to a distinct aging-related phenotype inhibition. Of interest,
mTOR activity is increased in a variety of tissues including
hematopoietic stem cells in aging animal models (Chen C. et al.
(2009) Sci Signal 2:ra75 and Barns, M. et al. (2014) Int J Biochem
Cell Biol. 53:174-185). Thus, turning down mTOR activity to levels
seen in young tissue, as opposed to more complete suppression of
mTOR activity, may be of clinical benefit in aging indications.
[0492] The safety profile of mTOR inhibitors such as RAD001 in the
treatment of aging-related indications has been of concern. The
toxicity of RAD001 at doses used in oncology or organ transplant
indications includes rates of stomatitis, diarrhea, nausea,
cytopenias, hyperlipidemia, and hyperglycemia that would be
unacceptable for many aging-related indications. However, these AEs
are related to the trough levels of RAD001 in blood. Therefore the
RAD001 dosing regimens used in this study were chosen to minimize
trough levels. The average RAD001 trough levels of the 0.5 mg
daily, 5 mg weekly and 20 mg weekly dosing cohorts were 0.9 ng/ml,
below 0.3 ng/ml (the lower limit of quantification), and 0.7 ng/ml,
respectively. These trough levels are significantly lower than the
trough levels associated with dosing regimens used in organ
transplant and cancer patients. In addition, the limited 6 week
course of treatment decreased the risk of adverse events. These
findings suggest that the dosing regimens used in this study may
have an acceptable risk/benefit for some conditions of the elderly.
Nonetheless, significant numbers of subjects in the experiments
described herein developed mouth ulcers even when dosed as low as
0.5 mg daily. Therefore the safety profile of low, immune
enhancing, dose RAD001 warrants further study. Development of mTOR
inhibitors with cleaner safety profiles than currently available
rapalogs may provide better therapeutic options in the future for
aging-associated conditions.
Example 2
Enhancement of Immune Response to Vaccine in Elderly Subjects
[0493] Immune function declines in the elderly, leading to an
increase incidence of infection and a decreased response to
vaccination. As a first step in determining if mTOR inhibition has
anti-aging effects in humans, a randomized placebo-controlled trial
was conducted to determine if the mTOR inhibitor RAD001 reverses
the aging-related decline in immune function as assessed by
response to vaccination in elderly volunteers. In all cases,
appropriate patent consents were obtained and the study was
approved by national health authorities.
[0494] The following 3 dosing regimens of RAD001 were used in the
study:
[0495] 20 mg weekly (trough level: 0.7 ng/ml)
[0496] 5 mg weekly (trough level was below detection limits)
[0497] 0.5 mg daily (trough level: 0.9 ng/ml)
[0498] These dosing regimens were chosen because they have lower
trough levels than the doses of RAD001 approved for transplant and
oncology indications. Trough level is the lowest level of a drug in
the body. The trough level of RAD001 associated with the 10 mg
daily oncology dosing regimen is approximately 20 ng/ml. The trough
level associated with the 0.75-1.5 mg bid transplant dosing regimen
is approximately 3 ng/ml. In contrast, the trough level associated
with the dosing regimens used in our immunization study were 3-20
fold lower.
[0499] Since RAD001-related AEs are associated with trough levels,
the 3 dosing regimens were predicted to have adequate safety for
normal volunteers. In addition, the 3 doses were predicted to give
a range of mTOR inhibition. P70 S6 Kinase (P70 S6K) is a downstream
target that is phosphorylated by mTOR. Levels of P70 S6K
phosphorylation serve as a measure of mTOR activity. Based on
modeling and simulation of P70 S6K phosphorylation data obtained in
preclinical and clinical studies of RAD001, 20 mg weekly was
predicted to almost fully inhibit mTOR activity for a full week,
whereas 5 mg weekly and 0.5 mg daily were predicted to partially
inhibit mTOR activity.
[0500] Elderly volunteers >=65 years of age were randomized to
one of the 3 RAD001 treatment groups (50 subjects per arm) or
placebo (20 subjects per arm). Subjects were treated with study
drug for 6 weeks, given a 2 week break, and then received influenza
(Aggrippal, Novartis) and pneumoccal (Pneumovax 23, Merck),
vaccinations. Response to influenza vaccination was assessed by
measuring the geometric mean titers (GMTs) by hemagglutination
inhibition assay to the 3 influenza strains (H1N1, H3N2 and B
influenza subtypes) in the influenza vaccine 4 weeks after
vaccination. The primary endpoints of the study were (1) safety and
tolerability and (2) a 1.2 fold increase in influenza titers as
compared to placebo in 2/3 of the influenza vaccine strains 4 weeks
after vaccination. This endpoint was chosen because a 1.2 fold
increase in influenza titers is associated with a decrease in
influenza illness post vaccination, and therefore is clinically
relevant. The 5 mg weekly and 0.5 mg daily doses were well
tolerated and unlike the 20 mg weekly dose, met the GMT primary
endpoint (FIG. 1A). Not only did RAD001 improve the response to
influenza vaccination, it also improved the response to
pneumococcal vaccination as compared to placebo in elderly
volunteers. The pneumococcal vaccine contains antigens from 23
pneumococcal serotypes. Antibody titers to 7 of the serotypes were
measured in our subjects. Antibody titers to 6/7 serotypes were
increased in all 3 RAD cohorts compared to placebo.
[0501] The combined influenza and pneumococcal titer data suggest
that partial (less than 80-100%) mTOR inhibition is more effective
at reversing the aging-related decline in immune function than more
complete mTOR inhibition.
Example 3
Low Dose mTOR Inhibition Increases Energy and Exercise
[0502] In preclinical models, mTOR inhibition with the rapalog
rapamycin increases spontaneous physical activity in old mice
(Wilkinson et al. Rapamycin slows aging in mice. (2012) Aging Cell;
11:675-82). Of interest, subjects in the 0.5 mg daily dosing cohort
described in Example 2 also reported increased energy and exercise
ability as compared to placebo in questionnaires administered one
year after dosing (FIG. 7). These data suggest that partial mTOR
inhibition with rapalogs may have beneficial effects on
aging-related morbidity beyond just immune function.
Example 4
P70 S6 Kinase Inhibition with RAD001
[0503] Modeling and simulation were performed to predict daily and
weekly dose ranges of RAD001 that are predicted to partially
inhibit mTOR activity. As noted above, P70 S6K is phosphorylated by
mTOR and is the downstream target of mTOR that is most closely
linked to aging because knockout of P70 S6K increases lifespan.
Therefore modeling was done of doses of RAD001 that partially
inhibit P70 S6K activity. Weekly dosing in the range of >=0.1 mg
and <20 mg are predicted to achieve partial inhibition of P70
S6K activity (FIGS. 8A and 8B).
[0504] For daily dosing, concentrations of RAD001 from 30 pM to 4
nM partially inhibited P70 S6K activity in cell lines (Table 7).
These serum concentrations are predicted to be achieved with doses
of RAD001 >=0.005 mg to <1.5 mg daily.
TABLE-US-00007 TABLE 7 Percent inhibition of P70 S6K activity in
HeLa cells in vitro RAD001 concentration 0 6 pM 32 pM 160 pM 800 pM
4 nM 20 nM % P70 S6K 0 0 18 16 62 90 95 inhibition
CONCLUSION
[0505] Methods of treating aging-related morbidity, or generally
enhancing an immune response, with doses of mTOR inhibitors that
only partially inhibit P70 S6K. The efficacy of partial mTOR
inhibition with low doses of RAD001 in aging indications is an
unexpected finding. RAD001 dose ranges between >=0.1 mg to
<20 mg weekly and >=0.005 mg to <1.5 mg daily will achieve
partial mTOR inhibition and therefore are expected to have efficacy
in aging-related morbidity or in the enhancement of the immune
response.
EQUIVALENTS
[0506] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific aspects, it is apparent
that other aspects and variations of this invention may be devised
by others skilled in the art without departing from the true spirit
and scope of the invention. The appended claims are intended to be
construed to include all such aspects and equivalent
variations.
[0507] Other embodiments are within the following claims.
* * * * *