U.S. patent application number 16/800377 was filed with the patent office on 2020-06-18 for histone deacetylase as a modulator of pdli expression and activity.
The applicant listed for this patent is H. LEE MOFFITT CANCER CENTER AND RESEARCH INSTITUTE, INC.. Invention is credited to Eduardo M. Sotomayor, Alejandro V. Villagra.
Application Number | 20200188366 16/800377 |
Document ID | / |
Family ID | 54288290 |
Filed Date | 2020-06-18 |
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United States Patent
Application |
20200188366 |
Kind Code |
A1 |
Villagra; Alejandro V. ; et
al. |
June 18, 2020 |
HISTONE DEACETYLASE AS A MODULATOR OF PDLI EXPRESSION AND
ACTIVITY
Abstract
Disclosed herein is a method for modulating Program Death
Receptor Ligand 1 (PDL1) in a cancer cell, comprising contacting
the cell with a composition comprising a histone deacetylase (HDAC)
inhibitor. Also disclosed is a method for treating a tumor in a
subject, comprising administering to the subject a thereapeutically
effective amount of a composition comprising a histone deacetylase
(HDAC) inhibitor and a composition comprising a thereapeutically
effective amount of a Program Death Receptor Ligand 1 (PDL1)
inhibitor, a Programmed Death 1 receptor (PD1) inhibitor, or a
combination thereof.
Inventors: |
Villagra; Alejandro V.;
(Tampa, FL) ; Sotomayor; Eduardo M.; (Tampa,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
H. LEE MOFFITT CANCER CENTER AND RESEARCH INSTITUTE, INC. |
Tampa |
FL |
US |
|
|
Family ID: |
54288290 |
Appl. No.: |
16/800377 |
Filed: |
February 25, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16000678 |
Jun 5, 2018 |
10576066 |
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16800377 |
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15120065 |
Aug 18, 2016 |
9987258 |
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PCT/US15/24485 |
Apr 6, 2015 |
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16000678 |
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61977003 |
Apr 8, 2014 |
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61975858 |
Apr 6, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/17 20130101;
A61K 31/416 20130101; A61K 45/06 20130101; A61K 31/422 20130101;
A61K 31/437 20130101; A61K 31/4406 20130101; A61K 31/505 20130101;
A61K 31/4045 20130101; A61K 31/506 20130101 |
International
Class: |
A61K 31/437 20060101
A61K031/437; A61K 31/506 20060101 A61K031/506; A61K 31/4406
20060101 A61K031/4406; A61K 31/4045 20060101 A61K031/4045; A61K
31/505 20060101 A61K031/505; A61K 31/422 20060101 A61K031/422; A61K
31/416 20060101 A61K031/416; A61K 31/17 20060101 A61K031/17; A61K
45/06 20060101 A61K045/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government Support under Grant
No. CA153246 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method for modulating Program Death Receptor Ligand 1 (PDL1)
in a cancer cell, comprising contacting the cell with a composition
comprising a histone deacetylase (HDAC) inhibitor.
2. The method claim 1, wherein the HDAC inhibitor comprises a
selective inhibitor of histone deacetylase 6 (HDAC6).
3. The method claim 2, wherein the HDAC6 inhibitor is selected from
the group consisting of ACY-1215, Tubacin, Tubastatin A, ST-3-06,
ST-2-92, Nexturastat A, and Nexturastat B.
4. The method of any one of claims 1 to 3, wherein the HDAC
inhibitor comprises a pan inhibitor, a class I HDAC inhibitor, or a
combination thereof.
5. A method for treating a tumor in a subject, comprising
administering to the subject a thereapeutically effective amount of
a histone deacetylase (HDAC) inhibitor and a thereapeutically
effective amount of a Program Death Receptor Ligand 1 (PDL1)
inhibitor, a Programmed Death 1 receptor (PD1) inhibitor, or a
combination thereof.
6. The method of claim 5, wherein the HDAC inhibitor is a class I
HDAC inhibitor.
7. The method of claim 6, wherein the tumor comprises low PDL1
expression.
8. The method of claim 5, wherein the HDAC inhibitor is a selective
HDAC6 inhibitor.
9. The method of claim 8, wherein the selective HDAC6 inhibitor is
selected from the group consisting of ACY-1215, Tubacin, Tubastatin
A, ST-3-06, ST-2-92, Nexturastat A, and Nexturastat B.
10. The method of any one of claims 5 to 9, wherein the tumor
comprises a melanoma, renal cancer, or non-small cell lung
cancer.
11. The method of any preceding claim, wherein the PDL1 inhibitor
comprises an antibody that specifically binds PDL1.
12. The method of any preceding claim, wherein the PD1 inhibitor
comprises an antibody that specifically binds PD1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 61/975,858, filed Apr. 6, 2014, and Application
Ser. No. 61/977,003, filed Apr. 8, 2014, which are hereby
incorporated herein by reference in their entirety.
BACKGROUND
[0003] According to the World Health Organization (WHO), the
incidence of melanoma is increasing faster than any other cancer
(Lens, M. B. & Dawes, M. British Journal of Dermatology
150:179-185 (2004)). With the advent of new therapies like BRAF
inhibitors and ipilimumab, the median overall survival for
metastatic melanoma is 11-14 months, and currently there are no
other therapies which offer any additional improvement in overall
survival (Hassel, J. C., et al. Br J Cancer (2010); Korn, E. L., et
al. Journal of clinical oncology: official journal of the American
Society of Clinical Oncology 26:527-534 (2008)). There is a high
level of interest in defining environmental, genetic and host
factors which might be therapeutic targets.
SUMMARY
[0004] Histone deacetylases (HDACs), originally described as
histone modifiers, have more recently been demonstrated to modify a
variety of other proteins involved in diverse cellular processes
unrelated to the chromatin environment. This includes deacetylation
of multiple non-histone targets, such as proteins involved in cell
cycle/apoptosis and immune regulation. This expanded role raises
the possibility that the effects of HDACs and HDAC inhibitors
(HDACi) may affect non-epigenetic regulatory pathways. In contrast
to the well-documented effects of HDACi in the control of cell
cycle and apoptosis, their role in immunobiology is still not
completely understood, and the reported immunological outcomes when
using HDACi are heterogeneous. Disclosed herein is evidence showing
that the pharmacological or genetic abrogation of a single HDAC,
HDAC6, modifies the immunogenicity and proliferation of melanoma
cells. Additionally, HDAC6 interacts with and modulates the
activity of STAT3 to control downstream target genes. Among these
genes, the Program Death Receptor Ligand 1 (PDL1) is highly
susceptible to this regulatory mechanism involving HDAC6 and STAT3.
The expression of PDL1 has been shown to be induced in almost every
type of cancer, including solid tumors such as melanoma, and it has
been proposed that this could be one of the main mechanisms used by
cancer cells to acquire resistance to T-cell killing, by activating
the negative regulatory pathway PD-1 in T-cells. Thus, this
particular regulatory mechanism could be explored to design more
efficient and tailored therapies to improve the cancer immune
response.
[0005] Disclosed herein is a method for modulating Program Death
Receptor Ligand 1 (PDL1) in a cancer cell, comprising contacting
the cell with a composition comprising a histone deacetylase (HDAC)
inhibitor. Also disclosed is a method for treating a tumor in a
subject, comprising administering to the subject a thereapeutically
effective amount of a composition comprising a histone deacetylase
(HDAC) inhibitor and a composition comprising a therapeutically
effective amount of a Program Death Receptor Ligand 1 (PDL1)
inhibitor, a Programmed Death 1 receptor (PD1) inhibitor, or a
combination thereof. In some cases, the composition is administered
in an amount effective to treat or prevent the cancer cells from
becoming resistant to T-cell killing.
[0006] In some embodiments, the HDAC inhibitor is a selective
inhibitor of histone deacetylase 6 (HDAC6). Selective HDAC6
inhibitors are shown herein to inactivate the STAT3 pathway and
down-regulate its target genes, including the expression of PDL1.
Non-limiting examples of HDAC6 inhibitors include ACY-1215,
Tubacin, Tubastatin A, ST-3-06, ST-2-92, Nexturastat A, and
Nexturastat B.
[0007] In some embodiments, the HDAC inhibitor is a pan class I
HDAC inhibitor. HDAC inhibitors with potency against class I HDACs
are shown herein to upregulate the expression of PDL1 in melanoma
cell lines. Therefore, in some embodiments, a pan class I HDAC
inhibitor can be used when the tumor comprise low PDL1 expression.
Non-limiting examples of class I HDAC inhibitors include
Vorinostat, LBH589, ITF2357, PXD-101, Depsipeptide, MS-275, and
MGCD0103.
[0008] In some embodiments, the PDL1 inhibitor comprises an
antibody that specifically binds PDL1, such as BMS-936559
(Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments,
the PD1 inhibitor comprises an antibody that specifically binds
PD1, such as lambrolizumab (Merck), nivolumab (Bristol-Myers
Squibb), or MEDI4736 (AstraZeneca).
[0009] The disclosed composition can be used in combination with
other cancer treatments. For example, the disclosed inhibitors of
HDAC, PDL1, PD1, or combinations thereof can be administered alone
or in combination with a cancer immunotherapy agent. For example,
the cancer immunotherapy agent can be an antibody that specifically
binds CLTA-4, such as ipilimumab (Bristol-Myers Squibb).
[0010] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0011] FIGS. 1A and 1B show that HDAC6 knock-down decreases STAT3
activation. FIG. 1A is a western blot of melanoma cell lines
knocked-down for HDAC6 (HDAC6KD). In parallel was evaluated the
proliferation of these cells and compared to their homologous
non-target (NT) shRNA controls. FIG. 1B is a western blot of
HDAC6KD and NT melanoma cells stimulated with IL-6 (30 ng/mL).
[0012] FIG. 2A is a Heat Map of the gene expression profiles
obtained by microarray. Genes were clustered according to their
properties using the software GeneCluster 3.0. The values for gene
expression obtained from two clones for each cell line and compared
with their respective controls. Shown are the genes with an over
two-fold increase or decrease in both clones. FIG. 2B is an
ontology distribution of genes affected in HDAC6KD cells. Ontology
report generated by "The database for Annotation, Visualization and
Integrated Discovery (DAVID)". Quantitative real time PCR
validation. FIG. 2C contains bar graphs showing gene expression in
RAW264.7 NT and HDAC6KD cells (2.times.10.sup.6/well) untreated or
stimulated with LPS (1 .mu.g/ml) for 2 hrs, then total RNA was
isolated to analyze the expression of genes affected by HDAC6KD.
GAPDH was used as control. The results are expressed as a percent
over control cells and calculated by the Pfaffl equation. Three
experiments were performed with similar results. Error bars
represent standard deviation from triplicates.
[0013] FIGS. 3A to 3B show HDAC6 knock-down decreases STAT3
activation. FIG. 3A shows the presence of HDAC6, ac-tubulin, and
tubulin in melanoma cell lines knocked-down for HDAC6 (HDAC6KD). In
parallel was evaluated the proliferation of these cells and
compared to their homologous non-target (NT) shRNA controls. FIG.
3B shows the protein expression in HDAC6KD and NT melanoma cells
stimulated with IL-6 (30 ng/mL).
[0014] FIG. 4A shows expression of STAT3 target genes in HDAC6KD
and NT WM164 cells stimulated with IL-6 by qRT-PCR. FIG. 4B shows
the expression of STAT3, PDL1, and GAPDH in STAT3KD and NT melanoma
cells by western blot.
[0015] FIGS. 5A and 5C show the expression of PDL1 evaluated in
HDAC6KD and NT melanoma cells by qRT-PCR (FIG. 5A) or Flow
cytometry (FIG. 5C). FIG. 5B shows the expression of HDAC6, PDL1,
STAT3, pSTAT3, and GAPDH in HDAC6KD and NT melanoma cells by
western blot.
[0016] FIG. 6A shows either Flag-STAT3 (top) or Flag-HDAC6
expressed in WM164 cells and subjected to immunoprecipitation.
HDAC6 and STAT3 were evaluated in the immunoprecipitated fraction.
FIG. 6B shows the expression of HDAC6, MAPK, pMAPK, c-JUN, p c-JUN,
and GAPDH evaluated in HDAC6KD and NT melanoma cells by western
blot. FIG. 6C shows non-target and HDAC6KD cells subjected to
immunoprecipitation using an anti-total acetyl-lysine antibody and
evaluated for the presence of c-JUN in the immunoprecipitated
fraction.
[0017] FIG. 7A shows the in vivo growth of HDAC6KD melanoma cells
(and control non-target and WT melanoma cells) in immune competent
C57BL/6 mice. FIG. 7B shows in vivo growth of B16 cells in C57BL/6
mice treated with Nexturastat. FIG. 7C shows protein levels of
STAT3, pSTAT3, PDL1, and GAPDH in tumor samples treated with
Nexturastat. FIG. 7D shows protein expression in melanoma cells
treated with the HDAC6inh Tubastatin A for 48 hours.
[0018] FIGS. 8A, 8B and 8C show that HDAC inhibitors decrease cell
proliferation of melanoma cells. FIG. 8A shows the structure of
HDAC inhibitors tested. FIG. 8B shows cell viability of melanoma
cells incubated with LBH589, TSA, Tubastatin A, or Nexturastat A at
different concentrations for 24 hrs. Error bars represent standard
deviation from triplicates. This figure is representative of two
independent experiments. FIG. 8C shows tubulin, acetyl-tubulin and
acetyl-histone3 expression in BRAF-mutated melanoma cell lines
treated with HDAC6 inhibitors.
[0019] FIGS. 9A and 9B show HDAC6 profile of melanoma cell lines.
FIG. 9A shows HDAC6 expression in human melanocytes, BRAF mutant,
and NRAS mutant melanoma cell lines. FIG. 9B shows HDAC6 expression
in 9 primary human melanomas.
[0020] FIGS. 10A and 10B show characterization of human (FIG. 10A)
and B16 murine (FIG. 10B) HDAC6KD melanoma cell lines. Cell lines
were transduced with shRNA either coding for HDAC6 or a non-target
sequence. Cells were immunoblotted using specific antibodies to
HDAC6, tubulin and acetylated tubulin. Two HDAC6KD clones and two
NT controls were analyzed and then subjected to MTS assay. Data is
representative of three experiments with similar results. FIG. 10C
shows expression of full length and cleaved protein fragments of
PARP, BAX, cleaved caspase 8, and cleaved caspase 3 in HDAC6KD and
NT melanoma cells. FIG. 10D shows cell cycle analysis of NT and
HDAC6KD human melanoma cell lines stained with propidium iodide.
Data is representative of three experiments with similar
results.
[0021] FIGS. 11A to 11C show expression of tumor antigens in
Melanoma cell lines. FIG. 11A shows expression of tumor antigens in
different melanoma cells incubated with HDAC6i for 48 hours
measured by qRT-PCR. FIG. 11B shows expression of tumor antigens
measured in WM164 non-target and HDAC6KD cells. FIG. 11C shows
expression of tumor antigens measured by western blot.
[0022] FIGS. 12A to 12B show increased MHC1 expression after HDAC6
inhibition in melanoma cell lines. FIG. 12A shows MHC I expression
in NT and HDAC6KD melanoma cell lines. FIG. 12B shows MHC I
expression in wild type melanoma cell lines treated in vitro with
Tubastatin A (3 .mu.M) for 48 hours.
[0023] FIGS. 13A, 13B, and 13C show that HDAC6 modulates tumor
growth in vivo. FIG. 13A shows in vivo tumor growth of C57BL/6 mice
injected subcutaneously with B16 WT cells. Mice were either
untreated or treated with the HDAC6 inhibitor Nexturastat B or
Tubastatin A via daily intraperitoneal injection. FIG. 13B shows in
vivo growth of C57BL6/mice injected with B16 HDAC6KD or NT melanoma
cells. FIG. 13C shows B16 growth in HDAC6 KO C57BL/6 mice and WT
control.
[0024] FIGS. 14A and 14B show differences in growth of melanoma
cells after HDAC6 inhibition in altered immune systems. FIG. 14A
shows in vivo growth of B16 WT melanoma injected into SCID mice was
not significantly different despite treatment with HDAC6 inhibitor
Nexturastat B. FIG. 14B shows in vivo growth of B16 HDAC6KD
melanoma cells and control non-target cells in C57BL/6mice. Mice
were treated with antibodies to deplete CD4, CD8, and NK cells.
These findings suggest that changes in tumor growth after HDAC6
inhibition are in part, due to the immune recognition of the
tumor.
[0025] FIGS. 15A and 15B show characterization of HDAC6KD melanoma
cells. FIG. 15A shows generation of melanoma monoclonal cell lines
with or without HDAC6 expression. Melanoma cells were transduced
with either shRNA coding for HDAC6 or a non-target sequence. Cells
were immunoblotted using specific antibodies to HDAC6, tubulin and
acetylated tubulin. FIG. 15B shows phosphorylation of JAK2 and
STAT3 measured in different human melanoma NT or HDAC6KD cell lines
after stimulation with IL-6. Cells were lysed and immunoblotted
using the specific antibodies above.
[0026] FIG. 16 shows quantitative RT-PCR of STAT3 target genes.
Total RNA was isolated from melanoma cell lines NT and HDAC6KD
before and after treatment with IL-6, and the expressions of STAT3
target genes were analyzed by quantitative RT-PCR. The results are
expressed as a percent over control cells, and data was normalized
by GAPDH expression. This experiment was performed three times with
similar results. Error bars represent standard deviation from
triplicates.
[0027] FIGS. 17A to 17C show PDL-1 expression in melanoma HDAC6KD.
FIG. 17A shows PD-L1 expression in HDAC6KO cells versus wild type
cells measured by qRT-PC Rafter IL-6 or DMSO. FIG. 17B shows PD-L1
expression in melanoma NT and HDAC6KD cell lines analyzed by
qRT-PCR after IL-6 (30 ng/ml), IFN-g (100 ng/ml), or DMSO. FIG. 17C
shows PDL1 expression in HDAC6KD cells analyzed by western
blot.
[0028] FIGS. 18A and 18B show PDL-1 expression in melanoma STAT3KD
and HDAC6KD. FIG. 18A shows STAT3, PDL-1 and GAPDH expression in
melanoma monoclonal cell lines with or without STAT3 expression.
FIG. 18B shows flow cytometric analysis for PDL-1 in HDACKD,
STAT3KD, and non-target melanoma before and after IFN-.gamma.
stimulation.
[0029] FIG. 19 shows characterization of melanoma cell lines after
pharmacologic HDAC6 inhibition. Different melanoma cells lines were
incubated with the HDAC6 inhibitor Tubastatin A (12.5 .mu.M) for 24
hours, followed with stimulation by IL-6 (30 ng/ml). Cells were
lysed and immunoblotted for HDAC6, acTUBULIN, STAT3, pSTAT3-Y705,
PD-L1, and GAPDH.
[0030] FIG. 20 shows melanoma xenograft analysis. Tumors collected
from C57BL mice injected either with B16 NT cells or B16 HDAC6KD
were immunoblotted for HDAC6, acTUBULIN, STAT3, pSTAT3-Y705, PD-L1,
and GAPDH. Decreased STAT3 phosphorylation and PDL-1 expression are
maintained in these tumors.
[0031] FIG. 21 shows selectivity of HDAC inhibitors. The murine
melanoma cell line B16 was treated with indicated HDACi at
indicated doses for 24 hours. Cells were lysed and analyzed for
acetylated histone 3, acetylated alpha-tubulin and total alpha
tubulin protein.
[0032] FIG. 22 shows HDAC inhibitors with potency against class I
HDACs upregulate PDL1 expression in vitro. B16, WM983A, Sk-Mel21,
WM1366, WM35, and WM793 melanoma cells lines were treated with DMSO
("D"), 10 nM LBH589 ("LB"), 500 nM MGCD0103 ("MG), or 500 nM MS275
("MS") for 72 hours. PDL1 expression was assessed by flow
cytometry. Histograms shown are for PDL1 expression or
autofluorescence (solid grey) of 10,000 cells or more.
[0033] FIG. 23 shows PDL1 upregulation by HDAC inhibitors is long
lasting. Melanoma cell lines were treated with DMSO, 10 nM LBH589
(squares), 1.5 .mu.M ACY1215 (triangles), 500 nM MS275 (diamonds),
500 nM CLB66 (circles), or 500 nM MGCD0103 (X). PDL1 expression at
indicated time points was assessed by flow cytometry. Voltages
between time point measurements was standardized using rainbow
beads with standard emissions. Mean fluorescence intensity minus
autofluorescencee is graphed for each time point. DMSO treatment
was plotted as zero hours.
[0034] FIG. 24 shows LBH589 upregulates PDL1 expression in vivo.
C57BL/6 mice were injected subcutaneously with 100,000 B16 cells.
When tumors became palpable, treatment with 15 mg/kg LBH589 via IP
injection began on a Mon, Wed, Fri schedule. After one week of
treatment, mice were sacrificed, tumors harvest and analyzed by
flow cytometry for PDL1 expression.
[0035] FIG. 25 shows HDAC inhibitor-induced PDL1 expression is
enhanced by IFN-.gamma. exposure. SKMel21 cells were treated for 72
hours with DMSO, 10 nM LBH589, 10 ng/mL IFN-.gamma., or LBH589 and
IFN-.gamma.. PDL1 expression was assessed by flow cytometry.
[0036] FIG. 26 shows knockdowns of individual HDACs does not
recapitulate the enhanced PDL1 expression seen in HDAC inhibitor
treated melanoma. Single HDAC knockdowns of class I HDACs were
generated as well as a NT control in the melanoma cell line
SKMel21. Cells were assessed by flow cytometry for PDL1
expression.
[0037] FIG. 27 shows combining LBH589 with PDL1 blockade can
enhance survival. C57BL/6 mice were injected subcutaneously with
100,000 B16 cells. On day 10, they were treated with 15 mg/kg
LBH589 three times weekly, anti-PDL1 twice weekly, combination of
LBH589 and anti-PDL1, or dextrose control injections. Treatment
continued for three weeks and mice were monitored for survival.
DETAILED DESCRIPTION
[0038] A major challenge to turning on the immune system to attack
cancer is that the immune system consists of an elaborate network
of checks and balances to avoid over-activation which could harm
healthy tissue. For cancer to develop, tumor cells need to hide
from the immune systems. One mechanism tumor cells use to hide is
exploiting the checks and balances that are in place for
down-regulation, by hijacking so called "immune check points" that
regulate T-cell activation. Several co-stimulatory pathways have
been characterized, including both, activating and inhibiting
pathways that determine T-cell activation.
[0039] Among several co-stimulatory pathways required for T-cell
regulation, the CTLA-4 (cytotoxic T-lymphocyte-associated antigen
4)/B7 inhibitory pathway has been the first target for a
pharmaceutical intervention. This pathway is one potential
checkpoint that has been hijacked by some tumors to avoid T-cell
activation. It predominantly regulates T-cells at the stage of
initial T-cell activation. CTLA-4 is expressed within 48 hours
after T-cell activation and provides negative signaling that
de-activates the T-cell. Inhibition of CTLA-4 by antibodies such as
ipilumimab (BMS' Yervoy) or AZN's tremelimumab has resulted in
response rate in the 10-15% range in melanoma patients.
[0040] Programmed death 1 (PD-1) receptor and PD ligand (PD-L) is
another inhibitory pathway that down regulates T-cell activation.
PD-1 activities include the inhibition on T-cells during long-term
antigen exposure, as happens in chronic viral infections and
cancers. T-cell down-regulation is mediated by the interaction of
two cell surface molecules (1) PD1 that resides on the T-cell and
(2) its ligand PDL1 that sits on the tumor cell. To overcome this
down-regulation or T-cell blockade, the PD1/PDL1 interactions needs
to be blocked. Such a reversal of the down regulation can be
achieved using antibodies, either against the PD1 receptor that
blocks the inhibition of the T-cell side or against the ligand PDL1
that blocks the inhibitor on the tumor side.
[0041] Histone deacetylases (HDACs) are attractive targets due to
the availability of a broad spectrum of inhibitors targeting their
enzymatic activity (HDACi). HDACs, originally described as histone
modifiers, have recently been demonstrated to modify a variety of
other proteins involved in diverse cellular processes unrelated to
the chromatin environment. This includes deacetylation of multiple
non-histone targets, such as proteins involved in cell
cycle/apoptosis and immune regulation (Woan, K. V., et al. Immunol
Cell Biol 90:55-65 (2012); Villagra, A., et al. Oncogene 29:157-173
(2010)). This expanded role suggests the possibility that the
effects of HDACs and HDACi may include non-epigenetic regulatory
pathways.
[0042] Selective HDAC6 inhibitors are shown herein to inactivate
the STAT3 pathway and down-regulate its target genes, including the
expression of PDL1. However, HDAC inhibitors with potency against
class I HDACs are shown herein to upregulate the expression of PDL1
in melanoma cell lines. Therefore, in some embodiments, a pan class
I HDAC inhibitor can be used when the tumor comprise low PDL1
expression.
[0043] A variety of HDAC6 inhibitors have been investigated (Butler
et al., "Rational Design and Simple Chemistry Yield a Superior,
Neuroprotective HDAC6 Inhibitor, Tubastatin A," J Am Chem Soc 2010,
132(31):10842-10846; Kalin et al., "Second-Generation Histone
Deacetylase 6 Inhibitors Enhance the Immunosuppressive Effects of
Foxp3+ T-Regulatory Cells," J Med Chem 2012, 55(2):639-651).
Non-limiting examples include ACY-1215, Tubacin, Tubastatin A,
ST-3-06, ST-2-92, Nexturastat A, and Nexturastat B.
[0044] Non-limiting examples of class I HDAC inhibitors include
Vorinostat, LBH589, ITF2357, PXD-101, Depsipeptide, MS-275, and
MGCD0103.
[0045] The disclosed HDAC inhibitors (pan or specific) can be used
alone or in combination with a PD1 or PDL1 inhibitor to treat a
tumor in a subject. In some embodiments, the PDL1 inhibitor
comprises an antibody that specifically binds PDL1, such as
BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some
embodiments, the PD1 inhibitor comprises an antibody that
specifically binds PD1, such as lambrolizumab (Merck), nivolumab
(Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Human monoclonal
antibodies to PD-1 and methods for treating cancer using anti-PD-1
antibodies alone or in combination with other immunotherapeutics
are described in U.S. Pat. No. 8,008,449, which is incorporated by
reference for these antibodies. Anti-PD-L1 antibodies and uses
therefor are described in U.S. Pat. No. 8,552,154, which is
incorporated by reference for these antibodies. Anticancer agent
comprising anti-PD-1 antibody or anti-PD-L1 antibody are described
in U.S. Pat. No. 8,617,546, which is incorporated by reference for
these antibodies.
[0046] The disclosed compositions and methods can be used in
combination with other cancer immunotherapies. There are two
distinct types of immunotherapy: passive immunotherapy uses
components of the immune system to direct targeted cytotoxic
activity against cancer cells, without necessarily initiating an
immune response in the patient, while active immunotherapy actively
triggers an endogenous immune response. Passive strategies include
the use of the monoclonal antibodies (mAbs) produced by B cells in
response to a specific antigen. The development of hybridoma
technology in the 1970s and the identification of tumor-specific
antigens permitted the pharmaceutical development of mAbs that
could specifically target tumor cells for destruction by the immune
system. Thus far, mAbs have been the biggest success story for
immunotherapy; the top three best-selling anticancer drugs in 2012
were mAbs. Among them is rituximab (Rituxan, Genentech), which
binds to the CD20 protein that is highly expressed on the surface
of B cell malignancies such as non-Hodgkin's lymphoma (NHL).
Rituximab is approved by the FDA for the treatment of NHL and
chronic lymphocytic leukemia (CLL) in combination with
chemotherapy. Another important mAb is trastuzumab (Herceptin;
Genentech), which revolutionized the treatment of HER2 (human
epidermal growth factor receptor 2)-positive breast cancer by
targeting the expression of HER2.
[0047] In order to actively drive an antitumor immune response,
therapeutic cancer vaccines have been developed. Unlike the
prophylactic vaccines that are used preventatively to treat
infectious diseases, therapeutic vaccines are designed to treat
established cancer by stimulating an immune response against a
specific tumor-associated antigen. In 2010, sipuleucel-T (Provenge;
Dendreon Corporation) was approved by the FDA for the treatment of
metastatic, castration-resistant prostate cancer based on the
results of the IMPACT (Immunotherapy Prostate Adenocarcinoma
Treatment) trial in which it improved OS by 4.1 months and reduced
the risk of death by 22% versus placebo. The advantage of active
immunotherapies is that they have the potential to provide
long-lasting anticancer activity by engaging both the innate and
adaptive arms of the immune response. While mAbs are typically
considered passive immunotherapies, there is increasing evidence
that they also induce an adaptive immune response via a
"vaccination-like" effect.
[0048] Generating optimal "killer" CD8 T cell responses also
requires T cell receptor activation plus co-stimulation, which can
be provided through ligation of tumor necrosis factor receptor
family members, including OX40 (CD134) and 4-1BB (CD137). OX40 is
of particular interest as treatment with an activating (agonist)
anti-OX40 mAb augments T cell differentiation and cytolytic
function leading to enhanced anti-tumor immunity against a variety
of tumors.
[0049] Numerous anti-cancer drugs are available for combination
with the present method and compositions. The following is a
non-exhaustive lists of anti-cancer (anti-neoplastic) drugs that
can be used in conjunction with irradiation: Acivicin; Aclarubicin;
Acodazole Hydrochloride; AcrQnine; Adozelesin; Aldesleukin;
Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;
Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;
Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;
Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate;
Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine;
Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer;
Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin;
Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine;
Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine;
Dactinomycin; Daunorubicin Hydrochloride; Decitabine;
Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone;
Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene;
Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;
Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin;
Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;
Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate
Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide
Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;
Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;
Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine;
Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin
Hydrochloride; Ifosfamide; Ilmofosine; Iproplatin; Irinotecan
Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate;
Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone
Hydrochloride; Masoprocol; Maytansine; Mechlorethamine
Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;
Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;
Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;
Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone
Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;
Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;
Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman;
Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane;
Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine
Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin;
Riboprine; Rogletimide; Safmgol; Safingol Hydrochloride; Semustine;
Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium
Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin;
Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin;
Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone
Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;
Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine;
Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate;
Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate;
Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa;
Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate;
Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate
Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine
Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin;
Zorubicin Hydrochloride.
[0050] The cancer of the disclosed methods can be any cell in a
subject undergoing unregulated growth, invasion, or metastasis. In
some aspects, the cancer can be any neoplasm or tumor for which
radiotherapy is currently used. Alternatively, the cancer can be a
neoplasm or tumor that is not sufficiently sensitive to
radiotherapy using standard methods. Thus, the cancer can be a
sarcoma, lymphoma, leukemia, carcinoma, blastoma, or germ cell
tumor. A representative but non-limiting list of cancers that the
disclosed compositions can be used to treat include lymphoma, B
cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's
Disease, myeloid leukemia, bladder cancer, brain cancer, nervous
system cancer, head and neck cancer, squamous cell carcinoma of
head and neck, kidney cancer, lung cancers such as small cell lung
cancer and non-small cell lung cancer, neuroblastoma/glioblastoma,
ovarian cancer, pancreatic cancer, prostate cancer, skin cancer,
liver cancer, melanoma, squamous cell carcinomas of the mouth,
throat, larynx, and lung, colon cancer, cervical cancer, cervical
carcinoma, breast cancer, epithelial cancer, renal cancer,
genitourinary cancer, pulmonary cancer, esophageal carcinoma, head
and neck carcinoma, large bowel cancer, hematopoietic cancers;
testicular cancer; colon and rectal cancers, prostatic cancer, and
pancreatic cancer.
[0051] Compositions, Formulations and Methods of Administration
[0052] In vivo application of the disclosed compounds, and
compositions containing them, can be accomplished by any suitable
method and technique presently or prospectively known to those
skilled in the art. For example, the disclosed compounds can be
formulated in a physiologically- or pharmaceutically-acceptable
form and administered by any suitable route known in the art
including, for example, oral, nasal, rectal, topical, and
parenteral routes of administration. As used herein, the term
parenteral includes subcutaneous, intradermal, intravenous,
intramuscular, intraperitoneal, and intrasternal administration,
such as by injection. Administration of the disclosed compounds or
compositions can be a single administration, or at continuous or
distinct intervals as can be readily determined by a person skilled
in the art.
[0053] The compounds disclosed herein, and compositions comprising
them, can also be administered utilizing liposome technology, slow
release capsules, implantable pumps, and biodegradable containers.
These delivery methods can, advantageously, provide a uniform
dosage over an extended period of time. The compounds can also be
administered in their salt derivative forms or crystalline
forms.
[0054] The compounds disclosed herein can be formulated according
to known methods for preparing pharmaceutically acceptable
compositions. Formulations are described in detail in a number of
sources which are well known and readily available to those skilled
in the art. For example, Remington's Pharmaceutical Science by E.
W. Martin (1995) describes formulations that can be used in
connection with the disclosed methods. In general, the compounds
disclosed herein can be formulated such that an effective amount of
the compound is combined with a suitable carrier in order to
facilitate effective administration of the compound. The
compositions used can also be in a variety of forms. These include,
for example, solid, semi-solid, and liquid dosage forms, such as
tablets, pills, powders, liquid solutions or suspension,
suppositories, injectable and infusible solutions, and sprays. The
preferred form depends on the intended mode of administration and
therapeutic application. The compositions also preferably include
conventional pharmaceutically-acceptable carriers and diluents
which are known to those skilled in the art. Examples of carriers
or diluents for use with the compounds include ethanol, dimethyl
sulfoxide, glycerol, alumina, starch, saline, and equivalent
carriers and diluents. To provide for the administration of such
dosages for the desired therapeutic treatment, compositions
disclosed herein can advantageously comprise between about 0.1% and
99%, and especially, 1 and 15% by weight of the total of one or
more of the subject compounds based on the weight of the total
composition including carrier or diluent.
[0055] Formulations suitable for administration include, for
example, aqueous sterile injection solutions, which can contain
antioxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient; and
aqueous and nonaqueous sterile suspensions, which can include
suspending agents and thickening agents. The formulations can be
presented in unit-dose or multi-dose containers, for example sealed
ampoules and vials, and can be stored in a freeze dried
(lyophilized) condition requiring only the condition of the sterile
liquid carrier, for example, water for injections, prior to use.
Extemporaneous injection solutions and suspensions can be prepared
from sterile powder, granules, tablets, etc. It should be
understood that in addition to the ingredients particularly
mentioned above, the compositions disclosed herein can include
other agents conventional in the art having regard to the type of
formulation in question.
[0056] Compounds disclosed herein, and compositions comprising
them, can be delivered to a cell either through direct contact with
the cell or via a carrier means. Carrier means for delivering
compounds and compositions to cells are known in the art and
include, for example, encapsulating the composition in a liposome
moiety. Another means for delivery of compounds and compositions
disclosed herein to a cell comprises attaching the compounds to a
protein or nucleic acid that is targeted for delivery to the target
cell. U.S. Pat. No. 6,960,648 and U.S. Application Publication Nos.
20030032594 and 20020120100 disclose amino acid sequences that can
be coupled to another composition and that allows the composition
to be translocated across biological membranes. U.S. Application
Publiation No. 20020035243 also describes compositions for
transporting biological moieties across cell membranes for
intracellular delivery. Compounds can also be incorporated into
polymers, examples of which include poly (D-L lactide-co-glycolide)
polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)
propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL);
chondroitin; chitin; and chitosan.
[0057] For the treatment of oncological disorders, the compounds
disclosed herein can be administered to a patient in need of
treatment in combination with other antitumor or anticancer
substances and/or with radiation and/or photodynamic therapy and/or
with surgical treatment to remove a tumor. These other substances
or treatments can be given at the same as or at different times
from the compounds disclosed herein. For example, the compounds
disclosed herein can be used in combination with mitotic inhibitors
such as taxol or vinblastine, alkylating agents such as
cyclophosamide or ifosfamide, antimetabolites such as
5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin
or bleomycin, topoisomerase inhibitors such as etoposide or
camptothecin, antiangiogenic agents such as angiostatin,
antiestrogens such as tamoxifen, and/or other anti-cancer drugs or
antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals
Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or an
immunotherapeutic such as ipilimumab and bortezomib.
[0058] In certain examples, compounds and compositions disclosed
herein can be locally administered at one or more anatomical sites,
such as sites of unwanted cell growth (such as a tumor site or
benign skin growth, e.g., injected or topically applied to the
tumor or skin growth), optionally in combination with a
pharmaceutically acceptable carrier such as an inert diluent.
Compounds and compositions disclosed herein can be systemically
administered, such as intravenously or orally, optionally in
combination with a pharmaceutically acceptable carrier such as an
inert diluent, or an assimilable edible carrier for oral delivery.
They can be enclosed in hard or soft shell gelatin capsules, can be
compressed into tablets, or can be incorporated directly with the
food of the patient's diet. For oral therapeutic administration,
the active compound can be combined with one or more excipients and
used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and
the like.
[0059] The tablets, troches, pills, capsules, and the like can also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring can be added. When the unit dosage form is a capsule, it
can contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials can be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules can be coated with gelatin, wax,
shellac, or sugar and the like. A syrup or elixir can contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
active compound can be incorporated into sustained-release
preparations and devices.
[0060] Compounds and compositions disclosed herein, including
pharmaceutically acceptable salts, or hydrates thereof, can be
administered intravenously, intramuscularly, or intraperitoneally
by infusion or injection. Solutions of the active agent or its
salts can be prepared in water, optionally mixed with a nontoxic
surfactant. Dispersions can also be prepared in glycerol, liquid
polyethylene glycols, triacetin, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations
can contain a preservative to prevent the growth of
microorganisms.
[0061] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient, which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. The ultimate dosage form should be sterile, fluid and
stable under the conditions of manufacture and storage. The liquid
carrier or vehicle can be a solvent or liquid dispersion medium
comprising, for example, water, ethanol, a polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycols, and the
like), vegetable oils, nontoxic glyceryl esters, and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the formation of liposomes, by the maintenance of the
required particle size in the case of dispersions or by the use of
surfactants. Optionally, the prevention of the action of
microorganisms can be brought about by various other antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
buffers or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the inclusion of agents that
delay absorption, for example, aluminum monostearate and
gelatin.
[0062] Sterile injectable solutions are prepared by incorporating a
compound and/or agent disclosed herein in the required amount in
the appropriate solvent with various other ingredients enumerated
above, as required, followed by filter sterilization. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and the freeze drying techniques, which yield a powder of the
active ingredient plus any additional desired ingredient present in
the previously sterile-filtered solutions.
[0063] For topical administration, compounds and agents disclosed
herein can be applied in as a liquid or solid. However, it will
generally be desirable to administer them topically to the skin as
compositions, in combination with a dermatologically acceptable
carrier, which can be a solid or a liquid. Compounds and agents and
compositions disclosed herein can be applied topically to a
subject's skin to reduce the size (and can include complete
removal) of malignant or benign growths, or to treat an infection
site. Compounds and agents disclosed herein can be applied directly
to the growth or infection site. Preferably, the compounds and
agents are applied to the growth or infection site in a formulation
such as an ointment, cream, lotion, solution, tincture, or the
like.
[0064] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers, for example.
[0065] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0066] Useful dosages of the compounds and agents and
pharmaceutical compositions disclosed herein can be determined by
comparing their in vitro activity, and in vivo activity in animal
models. Methods for the extrapolation of effective dosages in mice,
and other animals, to humans are known to the art.
[0067] Also disclosed are pharmaceutical compositions that comprise
a compound disclosed herein in combination with a pharmaceutically
acceptable carrier. Pharmaceutical compositions adapted for oral,
topical or parenteral administration, comprising an amount of a
compound constitute a preferred aspect. The dose administered to a
patient, particularly a human, should be sufficient to achieve a
therapeutic response in the patient over a reasonable time frame,
without lethal toxicity, and preferably causing no more than an
acceptable level of side effects or morbidity. One skilled in the
art will recognize that dosage will depend upon a variety of
factors including the condition (health) of the subject, the body
weight of the subject, kind of concurrent treatment, if any,
frequency of treatment, therapeutic ratio, as well as the severity
and stage of the pathological condition.
[0068] Definitions
[0069] The term "subject" refers to any individual who is the
target of administration or treatment. The subject can be a
vertebrate, for example, a mammal. Thus, the subject can be a human
or veterinary patient. The term "patient" refers to a subject under
the treatment of a clinician, e.g., physician.
[0070] The term "therapeutically effective" refers to the amount of
the composition used is of sufficient quantity to ameliorate one or
more causes or symptoms of a disease or disorder. Such amelioration
only requires a reduction or alteration, not necessarily
elimination.
[0071] The term "treatment" refers to the medical management of a
patient with the intent to cure, ameliorate, stabilize, or prevent
a disease, pathological condition, or disorder. This term includes
active treatment, that is, treatment directed specifically toward
the improvement of a disease, pathological condition, or disorder,
and also includes causal treatment, that is, treatment directed
toward removal of the cause of the associated disease, pathological
condition, or disorder. In addition, this term includes palliative
treatment, that is, treatment designed for the relief of symptoms
rather than the curing of the disease, pathological condition, or
disorder; preventative treatment, that is, treatment directed to
minimizing or partially or completely inhibiting the development of
the associated disease, pathological condition, or disorder; and
supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the
associated disease, pathological condition, or disorder.
[0072] The term "prevent" refers to a treatment that forestalls or
slows the onset of a disease or condition or reduced the severity
of the disease or condition. Thus, if a treatment can treat a
disease in a subject having symptoms of the disease, it can also
prevent that disease in a subject who has yet to suffer some or all
of the symptoms.
[0073] The term "inhibit" refers to a decrease in an activity,
response, condition, disease, or other biological parameter. This
can include but is not limited to the complete ablation of the
activity, response, condition, or disease. This may also include,
for example, a 10% reduction in the activity, response, condition,
or disease as compared to the native or control level. Thus, the
reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any
amount of reduction in between as compared to native or control
levels.
[0074] The term "antibody" refers to natural or synthetic
antibodies that selectively bind a target antigen. The term
includes polyclonal and monoclonal antibodies. In addition to
intact immunoglobulin molecules, also included in the term
"antibodies" are fragments or polymers of those immunoglobulin
molecules, and human or humanized versions of immunoglobulin
molecules that selectively bind the target antigen.
[0075] Antibodies that can be used in the disclosed compositions
and methods include whole immunoglobulin (i.e., an intact antibody)
of any class, fragments thereof, and synthetic proteins containing
at least the antigen binding variable domain of an antibody. The
variable domains differ in sequence among antibodies and are used
in the binding and specificity of each particular antibody for its
particular antigen. However, the variability is not usually evenly
distributed through the variable domains of antibodies. It is
typically concentrated in three segments called complementarity
determining regions (CDRs) or hypervariable regions both in the
light chain and the heavy chain variable domains. The more highly
conserved portions of the variable domains are called the framework
(FR). The variable domains of native heavy and light chains each
comprise four FR regions, largely adopting a beta-sheet
configuration, connected by three CDRs, which form loops
connecting, and in some cases forming part of, the beta-sheet
structure. The CDRs in each chain are held together in close
proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen binding site of
antibodies.
[0076] Also disclosed are fragments of antibodies which have
bioactivity. The fragments, whether attached to other sequences or
not, include insertions, deletions, substitutions, or other
selected modifications of particular regions or specific amino
acids residues, provided the activity of the fragment is not
significantly altered or impaired compared to the nonmodified
antibody or antibody fragment.
[0077] Techniques can also be adapted for the production of
single-chain antibodies specific to an antigenic protein of the
present disclosure. Methods for the production of single-chain
antibodies are well known to those of skill in the art. A single
chain antibody can be created by fusing together the variable
domains of the heavy and light chains using a short peptide linker,
thereby reconstituting an antigen binding site on a single
molecule. Single-chain antibody variable fragments (scFvs) in which
the C-terminus of one variable domain is tethered to the N-terminus
of the other variable domain via a 15 to 25 amino acid peptide or
linker have been developed without significantly disrupting antigen
binding or specificity of the binding. The linker is chosen to
permit the heavy chain and light chain to bind together in their
proper conformational orientation.
[0078] Divalent single-chain variable fragments (di-scFvs) can be
engineered by linking two scFvs. This can be done by producing a
single peptide chain with two VH and two VL regions, yielding
tandem scFvs. ScFvs can also be designed with linker peptides that
are too short for the two variable regions to fold together (about
five amino acids), forcing scFvs to dimerize. This type is known as
diabodies. Diabodies have been shown to have dissociation constants
up to 40-fold lower than corresponding scFvs, meaning that they
have a much higher affinity to their target. Still shorter linkers
(one or two amino acids) lead to the formation of trimers
(triabodies or tribodies). Tetrabodies have also been produced.
They exhibit an even higher affinity to their targets than
diabodies.
[0079] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a substantially homogeneous population of
antibodies, i.e., the individual antibodies within the population
are identical except for possible naturally occurring mutations
that may be present in a small subset of the antibody molecules.
The monoclonal antibodies herein specifically include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, as long as they exhibit the desired antagonistic
activity.
[0080] The term "specifically binds", as used herein, when
referring to a polypeptide (including antibodies) or receptor,
refers to a binding reaction which is determinative of the presence
of the protein or polypeptide or receptor in a heterogeneous
population of proteins and other biologics. Thus, under designated
conditions (e.g. immunoassay conditions in the case of an
antibody), a specified ligand or antibody "specifically binds" to
its particular "target" (e.g. an antibody specifically binds to an
endothelial antigen) when it does not bind in a significant amount
to other proteins present in the sample or to other proteins to
which the ligand or antibody may come in contact in an organism.
Generally, a first molecule that "specifically binds" a second
molecule has an affinity constant (Ka) greater than about 10.sup.5
M.sup.-1 (e.g., 10.sup.6 M.sup.-1, 10.sup.7 M.sup.-1, 10.sup.8
M.sup.-1, 10.sup.9 M.sup.-1, 10.sup.10 M.sup.-1, 10.sup.11
M.sup.-1, and 10.sup.12 M.sup.-1 or more) with that second
molecule.
[0081] The term "cancer" or "malignant neoplasm" refers to a cell
that displays uncontrolled growth, invasion upon adjacent tissues,
and often metastasis to other locations of the body.
[0082] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
EXAMPLES
Example 1
HDAC6 as a Modulator of PDL1 Expression and Activity
[0083] Results
[0084] HDAC6 is a 131 KDa protein considered to be a key regulator
of cytoskeleton dynamics and cell-cell interactions (Hubbert, C.,
et al. Nature 417:455-458 (2002); Valenzuela-Fernandez, A., et al.
Trends in Cell Biology 18:291-297 (2008)). Although this HDAC is
predominantly cytoplasmic, studies have demonstrated its presence
in nuclear extracts and its recruitment to gene promoter regions
(Toropainen, S., et al. J Mol Biol. 400:284-294 (2010)). HDAC6 has
been reported to be over-expressed in several cancer types,
including ovarian cancer, prostate cancer and acute myeloid
leukemia (AML) (Aldana-Masangkay, G. I., et al. J Biomed Biotechnol
2011:875824 (2010)). As shown in FIG. 1, HDAC6 is also
over-expressed in several melanoma tumors. Recently, HDAC6 has been
implicated in other cellular processes, including the modulation of
immune responses (Serrador, J. M., et al. Immunity 20:417-428
(2004); Kalin, J. H., et al. J Med Chem. (2012)). This is
consistent with icroarray data analyzing the gene expression
profile (GEP) of untreated and LPS-treated RAW264.7 macrophages in
which HDAC6 was knocked down using specific shRNA (HDAC6KD) or
treated with control shRNA non-coding for any mouse mRNA
(non-target, NT). 1542 genes were down-regulated and 775 were
up-regulated in HDAC6KD cells (FIG. 2A). Their ontology
distribution revealed important changes in both immune-related and
apoptosis/cell cycle control genes (FIG. 2B). An interesting
finding gathered from the GEP analysis was the down-regulation of
almost every STAT3 target gene in HDAC6KD cells, suggesting the
potential participation of STAT3 in the outcome that is observed in
the absence of HDAC6 (FIG. 2C). Similarly, several previously
described c-Jun target genes were down-regulated, suggesting that
the inhibition of HDAC6 affected the MAPK pathway as well.
[0085] STAT3 activation can be achieved by different stimuli and is
often the point of convergence for many signaling pathways
triggered by cytokines, growth factors and other stimuli, being
considered by itself an oncogene. Hyperactivation and/or
constitutive activation of STAT3 has been found in a wide range of
tumors and transformed cell lines. In particular, constitutively
active STAT3 has been reported in more than 70% of solid and
hematological tumors, including melanoma and lung cancer
(Kortylewski, M., et al. Cancer Metastasis Rev 24:315-327 (2005);
Yu, H., et al. Nat Rev Immunol. 7:41-51 (2007)). There are numerous
reports describing the effect of STAT3 manipulation upon tumor
growth, survival, invasiveness, metastatic potential, angiogenesis,
and immune-escape. In fact, the over-expression of constitutive
active STAT3 (STAT3c) leads to the immortalization of non-malignant
cell lines (Regis, G., et al. Seminars in Cell & Developmental
Biology 19:351-359 (2008)). Hyperactivity of STAT3 also deregulates
the expression of several important cytokines such as IL-6 and
IL-10. Interestingly, HDAC6 interacts with STAT3 and is recruited
to and regulates the expression of IL10 and IL6 genes.
[0086] Given these findings, as well as the well known role of
STAT3 deregulation in the pathogenesis of melanoma, a study was
conducted to determine whether the absence of HDAC6 affected the
activation of the JAK/STAT3 pathway in melanoma cells. By using
lentiviral HDAC6 shRNA, stable HDAC6KD cell lines were generated in
several melanoma cell lines carrying either NRAS (SKMEL21,
SKMEL103, IPC298) or BRAF (WM164, WM35, WM983, WM795) mutations. Of
note, all HDAC6KD cell lines demonstrated slower proliferation
rates when compared with their respective non-target shRNA control
stable cell lines (FIG. 3A, representative from all cell lines
analyzed). When the activation of the Jak2/STAT3 pathway was
analyzed in these HDAC6KD cells, there was diminished
phosphorylation of the Ser-727 and Tyr-705 residues of STAT3 upon
IL-6 stimulation (similar results obtained upon IL-10 and
IFN.gamma. stimulation) (FIG. 3B, lines 4 and 5). Recent reports
have assigned the key role of acetylation over the activation of
STAT3, a process mediated mainly by CBP/p300 acetylation and class
I HDAC deacetylation (Togi, S., et al. Biochem Biophys Res Commun.
379:616-620 (2009); Lee, H., et al. Proc Natl Acad Sci USA
109:7765-7769 (2012). Taking this antecedent into consideration,
the acetylation status of STAT3 was analyzed. However, major
differences in its acetylation status in the absence of HDAC6 (FIG.
1B, line 6) was did not detected, suggesting that the effect of
HDAC6 on its activation does not depend on a direct deacetylation
of STAT3.
[0087] STAT3 must be phosphorylated in order to be translocated to
the nucleus and properly exert its function over target genes
(Ihle, J. N. Current opinion in cell biology 13:211-217 (2001)).
Therefore, the diminished phosphorylation of STAT3 observed in the
absence of HDAC6 might interfere with the activation of STAT3
target genes. To answer this question well defined STAT3 target
genes were selected and their expression measured upon IL-6
stimulation. An important reduction in the mRNA was observed for
all tested genes, including CDKN1A, SOCS1, SOCS3, IL10, FOS and MYC
(FIG. 4A).
[0088] The GEP microarray data in HDAC6KD macrophages revealed
changes in immune related genes. Among these genes, an 8-fold
decrease in the expression of PDL1 (CD274) was observed. This
finding was validated by qRT-PCR in primary macrophages isolated
from wild type and HDAC6KO mice stimulated with IL-6. PDL1 and
PD-L2 are ligands for PD-1, a co-stimulatory molecule that plays an
inhibitory role in regulating T-cell activation. Specifically, the
interaction between PDL1 (from cancer cells) and the PD-1 present
on T-cells inhibits T-cell activation, proliferation, and promotes
T-cell apoptosis. The importance of the interaction of PDL1 and
PD-1 has been extensively described in in vitro and in vivo models,
as well as in clinical studies (Topalian, S. L., et al. Curr Opin
Immunol 24:207-212 (2012)), with promising antitumor results in
several preclinical and clinical studies involving PDL1 blocking
antibodies (Pardoll, D. M. Nat Rev Cancer 12:252-264 (2012)).
Furthermore, PDL1 expression is correlated with poor clinical
prognosis for a number of cancers including renal, breast, and
esophageal cancers. As a result, increased PDL1 expression by
cancer cells remains a fundamental escape mechanism from host
immunity, and the understanding of molecular mechanisms modulating
PDL1 expression could lead to improved treatments for cancer
patients. The expression of PDL1 is controlled by several pathways,
including those activated by IL-6, IL-10, GM-CSF, TLRs, interferons
and TNF.alpha. (Francisco, L. M., et al. Immunol Rev 236:219-242
(2010)). In addition, recent reports have described STAT3 as one of
the main regulators of PDL1 expression (Wolfle, S. J., et al. Eur J
Immunol 41:413-424 (2011)). This finding was also verified in by
evaluating the expression of PDL1 in human and mouse melanoma cells
lacking STAT3 (STAT3KD) (FIG. 4B). Therefore, HDAC6 could be an
indirect regulator of the expression of PDL1 in melanoma via STAT3
modulation. Taking this observation into consideration, the
expression of PDL1 in HDAC6KD human melanoma cells stimulated with
IL-6 was evaluated by qRT-PCR (FIG. 5A). When compared to
non-target controls, decreased expression of PDL1 was observed.
This result was also validated by measuring the PDL1 protein by
western blot (FIG. 5B) and flow cytometry (FIG. 5C).
[0089] HDAC6 is recruited to regulatory sequences in gene promoters
such as MYC (Toropainen, S., et al. J Mol Biol. 400:284-294
(2010)), glucocorticoid receptor (Govindan, M. V. J Biol Chem.
285:4489-4510 (2010)) and estrogen receptor .alpha.-inducible genes
(Palijan, A., et al. J Biol Chem. 284:30264-30274 (2009)). However,
there is no evidence showing that HDAC6 is directly affecting the
acetylation status of chromatin. In fact, the deacetylation of
histones by HDAC6 has only been demonstrated by in vitro assays
(Todd, P. K., et al. PLoS Genet 6:e1001240 (2010)). Thus, the
transcriptional regulatory effects observed for HDAC6 could be
mediated by other regulatory factors recruited along with this
deacetylase to specific DNA sequences. This hypothesis suggests
that HDAC6 may be a regulator of the activation status of these
transcription factors, perhaps by modulating their acetylation
and/or phosphorylation. HDAC6 and STAT3 are recruited to the same
region of the IL-10 promoter, and the recruitment of HDAC6 is
impaired when cells are treated with the STAT3 inhibitor CPA-7.
Moreover, the recruitment of STAT3 to the IL-10 promoter diminishes
considerably in HDAC6KD cells, suggesting that the down-regulation
of PDL1 expression might be a consequence of the effect of HDAC6 on
STAT3 activation.
[0090] Another potential regulator of the transcriptional
regulation of PDL1 is c-Jun. The inhibition of the MEK cascade and
the subsequent c-Jun inactivation may lead to the down-regulation
of PDL1. This phenomena is also observed in BRAF
inhibitor-resistant melanoma cells (Jiang, X., et al. Clin Cancer
Res 19:598-609 (2013)). HDAC6 interacts with STAT3 and c-Jun to
form stable protein complexes, as detected by
co-immunoprecipitation (FIG. 6A). Additionally, HDAC6 does not
interfere with the phosphorylation of Erk or c-Jun in melanoma
cells (FIG. 6B), suggesting that its effect over the activation of
c-Jun target genes could involve another molecular mechanism. In
this regard, it has been proposed that the acetylation of c-Jun
modulates its transcriptional activity over target genes.
Specifically, the acetylation of Lys271 of c-Jun facilitates its
interaction with co-repressors and the subsequent repression of its
target genes (Vries, R. G., et al. EMBO J 20:6095-6103 (2001)). To
further explore this possibility, the acetylation status of c-Jun
in the absence of HDAC6 was evaluated, demonstrating an important
increase in its acetylation, suggesting the participation of HDAC6
in this process (FIG. 6C).
[0091] Besides STAT3 and c-Jun, there are no known transcriptional
regulators or chromatin modifiers affecting the PDL1 promoter.
Therefore, it is highly desirable to perform a more comprehensive
analysis of the transcriptional regulation of this gene. Further
understanding of the PDL1 promoter could identify targets to
control its expression, which in turn could be used as a
therapeutic option to ameliorate cancer immune evasion mediated by
PDL1.
[0092] Highly selective HDAC6 inhibitors (HDAC6inh) are currently
available, which make this deacetylase a very attractive target to
pursue as a therapeutic option. In this context, selective HDAC6
inhibitors, alone or in combination with other agents, are
currently under evaluation in clinical trials, including the
ongoing Phase 2 Multiple Myeloma clinical trial using the HDAC6inh
ACY1215, which has shown important anti-tumor activity in
preliminary studies (Santo, L., et al. Blood 119:2579-2589 (2012)).
Pan-HDAC inhibitors (pan-HDACi) slows the proliferation and
improves the immunogenicity of melanoma cells (Woods, D. M., et al.
Melanoma Res (2013)). However, the non-selective nature of
pan-HDACi makes the assumption of the specific participation of
HDACs on these processes impossible. As shown in FIG. 2A, HDAC6KD
melanoma cells have a slower rate of proliferation when compared to
their respective controls. This result was mirrored in melanoma
cell lines treated with selective HDAC6 inhibitors. The next step
was identifying if HDAC6KD would affect the growth of melanoma
cells in vivo. Thus, a delayed tumor growth of HDAC6KD B16 murine
melanoma cells was observed when compared to wild type or
non-target controls (FIG. 7A). A similar result was obtained in
another experiment injecting wild type B16 melanoma cells into
C57BL/6 mice treated daily with 20 mg/kg of the HDAC6inh
Nexturastat A or Nexturastat B (FIG. 7B). The amount of PDL1 and
activation of STAT3 was decreased in tumors isolated after the in
vivo treatment with HDAC6inh (FIG. 7C). This observation was also
made in melanoma cell lines treated with HDAC6inh (FIG. 7D),
suggesting the potential role of HDAC6 in this process, and
evidence that its deacetylase activity is necessary to mediate this
effect.
[0093] This delay in tumor growth in HDAC6KD melanoma cells and
melanoma cells treated with HDAC6inh could be a reflection of their
diminished proliferation (as evidenced in in vitro studies) and/or
an increase in their immunogenicity leading to improved immune
recognition and clearance.
[0094] Conclusions
[0095] The expression of PDL1 has been shown to be induced in
almost every type of cancer, including solid tumors such as
melanoma, and it has been proposed that this could be one of the
main mechanisms used by cancer cells to acquire resistance to
T-cell killing, by activating the negative regulatory pathway PD-1
in T-cells. This is particularly important in the resistance to
BRAF inhibitors, phenomena frequently associated with an
up-regulation of the expression of PDL1 (Jiang, X., et al. Clin
Cancer Res 19:598-609 (2013)). Therefore, the inhibition of PDL1
expression could offer new therapeutic options to prevent or revert
the resistance to current therapies aiming to improve the immune
recognition of cancer cells (i.e. PDL1, PD-1, and CTLA-4 blocking
antibodies).
Example 2
Histone Deacetylase 6 (HDAC6) as a New Target Modulating the
Proliferation and Immune-Related Pathways in Melanoma
[0096] Histone deacetylases (HDACs), originally described as
histone modifiers, have more recently been demonstrated to modify a
variety of other proteins involved in diverse cellular processes
unrelated to the chromatin environment. This includes the
deacetylation of multiple non-histone targets, such as proteins
involved in cell cycle/apoptosis and immune regulation.
Specifically, HDACs have garnered significant interest due to the
availability of drugs that selectively inhibits HDACs. The
pharmacological or genetic abrogation of a single HDAC, HDAC6,
modifies the immunogenicity and proliferation of melanoma in both
in vitro and in vivo models.
[0097] Using specific HDAC6 inhibitors (HDAC6i), decreased
proliferation and G1 cell cycle arrest was observed in all melanoma
cell lines measured by MTS assay and flow cytometry (FIG. 8). These
results were also observed in stable HDAC6 knockdown melanoma cell
lines (HDAC6KD) generated by specific lentiviral shRNA for HDAC6
(FIG. 10D). In addition to the effects observed in proliferation
and apoptosis after inhibiting HDAC6, also shown are important
changes in the expression of immune-related pathways, including
increased expression of MHC (FIG. 12), co-stimulatory molecules,
and specific melanoma tumor associated antigens such as gp100,
MART-1, Tyrp1 and Tyrp2 (FIG. 11A-11C).
[0098] These in vitro results were further supported by in vivo
tumor growth studies. Delayed tumor growth of inoculated B16
melanoma cells was observed in C57BL/6 mice treated with selective
HDAC6i (FIG. 13). A similar outcome was identified after
inoculation of HDAC6KD B16 melanoma cells in C57BL/6 mice (FIG.
14A). Such an effect was reverted partially in CD4+ and CD8+
depleted C57BL/6 mice challenged with HDAC6KD cells (FIG. 14B),
suggesting that the disruption of HDAC6 enhances immune system
recognition of melanoma cells. This delay in tumor growth could be
a reflection of their diminished proliferation and an increase in
their immunogenicity leading to improved immune recognition and
clearance. These studies provide critical insights into the
molecular pathways that are involved in the regulatory role of
HDAC6 in cell proliferation, survival, and cytokine signaling of
human melanoma cells. Collectively, these data have identified
HDAC6 as an attractive therapeutic target in melanoma.
Example 3
Histone Deacetylase 6 (HDAC6) as a Regulator of PDL-1 Expression
through STAT3 Modulation in Melanoma
[0099] In spite of the progress made in the understanding of the
cell biology, genetics and immunology of melanoma, the outcome for
patients with advanced-stage disease has remained poor with a
median survival ranging from 2-16 months. Some optimism was
recently provided in metastatic melanoma by the improved clinical
outcomes observed in patients receiving PDL-1 blocking
antibodies.
[0100] A better understanding of the environmental, genetic and
epigenetic factors limiting the efficacy of melanoma immunotherapy
will provide appropriate partner(s) for combination with Ipilimumab
or PD1/PDL1 antibodies. Among the epigenetic factors, one member of
the histone deacetylase family, HDAC6, is shown to play a critical
role not only in the regulation of survival/apoptosis of melanoma
cells but also in limiting their immunogenicity and recognition by
immune effector cells. In particular, disclosed is a major role of
HDAC6 as a modulator of the immunosuppresive STAT3/IL-6 pathway,
resulting in the down-regulation of tolerogenic PDL1 molecules in
melanoma cells. By analyzing HDAC6 knock-down melanoma cell lines
(HDAC6KD), shown herein is the inactivation of the STAT3 pathway
and the subsequent down-regulation of its target genes, including
the expression of PDL1. It was also observed that the PDL1
expression and phosphorylation of STAT3 was decreased in melanoma
isolated from xenograph tumor growth models after in vivo treatment
with specific HDAC6 inhibitors.
[0101] FIG. 15 shows the characterization of HDAC6KD melanoma
cells. FIG. 15A shows the generation of melanoma monoclonal cell
lines with or without HDAC6 expression. Melanoma cells were
transduced with either shRNA coding for HDAC6 or a non-target
sequence. Cells were immunoblotted using specific antibodies to
HDAC6, tubulin and acetylated tubulin. FIG. 15B shows
posphorylation of JAK2 and STAT3 measured in different human
melanoma NT or HDAC6KD cell lines after stimulation with IL-6.
Cells were lysed and immunoblotted using the specific antibodies
above.
[0102] FIG. 16 shows quantitative RT-PCR of STAT3 target genes.
Total RNA was isolated from melanoma cell lines NT and HDAC6KD
before and after treatment with IL-6, and the expressions of STAT3
target genes were analyzed by quantitative RT-PCR. The results are
expressed as a percent over control cells, and data was normalized
by GAPDH expression. This experiment was performed three times with
similar results. Error bars represent standard deviation from
triplicates.
[0103] FIG. 17 shows PDL-1 expression in melanoma HDAC6KD: FIG. 17A
shows total RNA was isolated from C57BL/mice cells in the HDAC6KO
cells versus wild type cells and was measured by qRT-PCR. FIG. 17B
shows total RNA isolated from melanoma cell lines NT and HDAC6KD.
The expression of PDL1 was analyzed by qRT-PCR after IL-6 (30
ng/ml), IFN-g (100 ng/ml), and DMSO. FIG. 18C shows a Western blot
demonstrating decreased PDL1 in HDAC6KD cells.
[0104] FIG. 18 shows PDL-1 expression in melanoma STAT3KD and
HDAC6KD. FIG. 18A shows generation of melanoma monoclonal cell
lines with or without STAT3 expression. Cells were immunoblotted
using specific antibodies for STAT3, PDL-1 and GAPDH. FIG. 18B
shows Flow cytometric analysis for PDL-1 in HDACKD, STAT3KD and non
target melanoma demonstrates decreased PDL-1 in HDAC6KD and STAT3KD
compared to NT after IFN-g stimulation.
[0105] FIG. 19 shows characterization of melanoma cell lines after
pharmacologic HDAC6 inhibition. Different melanoma cells lines were
incubated with the HDAC6 inhibitorTubastatin A (12.5 .mu.M) for 24
hours, followed with stimulation by IL-6 (30 ng/ml). Cells were
lysed and immunoblotted using the specific antibodies listed in the
figure.
[0106] FIG. 20 shows Melanoma xenograft analysis. Tumors collected
from C57BL mice injected either with B16 NT cells or B16 HDAC6KD
were lyzed for immunoblotting analysis using the specific
antibodies listed in the figure. Decreased STAT3 phosphorylation
and PDL-1 expression are maintained in these tumors.
Example 4
Inhibition of Class I Histone Deacetylases Promotes Robust and
Durable Enhancement of PDL1 Expression in Melanoma: Rationale for
Combination Therapy
[0107] Histone deacetylase inhibitors (HDACi) have shown remarkable
anti-tumor activity, leading to FDA approval of two HDACi for the
treatment of CTCL and several others currently at various stages of
clinical development for the treatment of both solid and
hematological malignancies. Treatment with HDACi results in
increased expression of pro-inflammatory promoting surface markers
on melanoma cells, promoting enhanced T-cell activation. Recent
clinical trial data has shown that blockade of the PD1/PDL1
interaction is effective in the treatment of melanoma, renal cell
and non-small cell lung cancer. Importantly, responses to PD1
blocking antibodies were preferentially seen in patients with
tumors expressing PDL1.
[0108] In this Example, HDACi targeting class I HDACs, but not
class II, is shown to augment expression of PDL1 in melanoma cells.
Two murine and five human melanoma cell lines were treated for up
to 72 hours with DMSO, LBH589 (pan-HDACi), MS275 (class I
inhibitor), MGCD0103 (class I inhibitor), an HDAC6 specific
inhibitor, or a class IIa inhibitor (FIG. 21). Using flow
cytometry, dose dependent, increases in PDL1 expression were found
in the LBH589, MS275 and MGCD0103 treated groups, but not in those
receiving HDAC6i or class IIa inhibitor, relative to DMSO (FIG.
22). Increased expression was noted as early as 24 hours after
treatment and peaked at 72 to 96 hours post-treatment (FIG.
23).
[0109] As IFN-.gamma. is known to upregulate the expression of PDL1
in both normal and transformed cells, experiments were conducted to
determine whether these results were associated with induction of
IFN-.gamma. expression by the melanoma cells. However, no
detectable levels of IFN-.gamma. were seen in either nontreated,
class I HDACi, or class II HDACi-treated cells. Melanoma cells
treated with HDACi in addition to IFN-.gamma. have enhanced
expression of PDL1 relative to either treatment alone (FIG. 25). To
further gain insight into the specific HDAC regulating the
expression of PDL1, experiments utilizing knockdowns (KD) of
individual class I HDACs were performed. In all KD melanoma cells
no increase in PDL1 expression was seen (FIG. 26), suggesting that
the increased expression of PDL1 is dependent on inhibition of
multiple class I HDACs. Supporting this conclusion, treatment of
class I HDAC-KDs with HDACi recapitulates the increased PDL1
expression seen with WT melanoma. Finally, in in vivo experiments
combining treatment of melanoma bearing mice with anti-PDL1
antibodies, mice receiving the combination treatment had a survival
advantage over those receiving PDL1 blocking antibodies or HDACi
alone (FIG. 27). These results provide a strong rationale for the
evaluation of combination therapies utilizing PDL1 or PD1 blocking
antibodies in combination with HDACi.
[0110] These results demonstrated that HDAC inhibitors with potency
against class I HDACs upregulate the expression of PDL1 in melanoma
cell lines. This upregulation occurs in vitro and in vivo, and is
long lasting. Evaluation of IFN-.gamma. as a mechanism of PDL1
upregualtion reveals that PDL1 upregulation is further increased by
the addition of exogneous IFN-.gamma.. Additionally, HDACi treated
melanomas fail to produce IFN-.gamma., TNF or TGF-b, highlighting
other cytokines or an alternative mechanism of PDL1 upregulation.
Furthermore, data utilizing knock down of individual class I HDACs
does not recapitulate the PDL1 upregulation seen by HDACi. This may
indicate that PDL1 upregulation is dependent on inhibition of a
combination of class I HDACs. Finally, in vivo experiments show
promising results with a combination of the HDACi LBH589 and
anti-PDL1 blockade.
[0111] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
[0112] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *