U.S. patent application number 16/545916 was filed with the patent office on 2020-06-25 for profiling peptides and methods for sensitivity profiling.
This patent application is currently assigned to Tolero Pharmaceuticals, Inc.. The applicant listed for this patent is Tolero Pharmaceuticals, Inc.. Invention is credited to David J. BEARSS, Lars MOURITSEN, Peter W. PETERSON, Adam SIDDIQUI-JAIN, Steven L. WARNER, Clifford J. WHATCOTT.
Application Number | 20200200737 16/545916 |
Document ID | / |
Family ID | 61006345 |
Filed Date | 2020-06-25 |
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United States Patent
Application |
20200200737 |
Kind Code |
A1 |
BEARSS; David J. ; et
al. |
June 25, 2020 |
PROFILING PEPTIDES AND METHODS FOR SENSITIVITY PROFILING
Abstract
The present disclosure is generally directed to profiling
peptides, compositions, and kits, as well as methods of use
thereof. The profiling peptides comprise an Mcl-1 binding domain,
and optionally a cellular uptake moiety. The methods of using such
profiling peptides include predicting sensitivity of a cancer,
selecting a treatment, treating a cancer, producing a sensitivity
profile, and the like.
Inventors: |
BEARSS; David J.; (Alpine,
UT) ; SIDDIQUI-JAIN; Adam; (South Jordan, UT)
; WHATCOTT; Clifford J.; (West Jordan, UT) ;
PETERSON; Peter W.; (Salt Lake City, UT) ; WARNER;
Steven L.; (Sandy, UT) ; MOURITSEN; Lars;
(South Jordan, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tolero Pharmaceuticals, Inc. |
Lehi |
UT |
US |
|
|
Assignee: |
Tolero Pharmaceuticals,
Inc.
Lehi
UT
|
Family ID: |
61006345 |
Appl. No.: |
16/545916 |
Filed: |
August 20, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16279623 |
Feb 19, 2019 |
10422788 |
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16545916 |
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16023360 |
Jun 29, 2018 |
10267787 |
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16279623 |
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15847697 |
Dec 19, 2017 |
10132797 |
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16023360 |
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62436221 |
Dec 19, 2016 |
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62562990 |
Sep 25, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/56 20130101;
A61K 31/7068 20130101; G01N 33/5079 20130101; C07K 2319/10
20130101; C12N 2740/16322 20130101; A61K 31/136 20130101; A61P
35/00 20180101; G01N 2333/47 20130101; A61K 31/4545 20130101; G01N
33/5011 20130101; A61P 35/02 20180101; G01N 33/5091 20130101; C12N
7/00 20130101; C12Q 1/6886 20130101; A61K 45/06 20130101; C07K
14/4747 20130101; A61K 31/704 20130101; C07K 14/00 20130101; A61K
31/496 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; C12Q 1/6886 20060101 C12Q001/6886; C07K 14/47 20060101
C07K014/47; A61P 35/00 20060101 A61P035/00; C12N 7/00 20060101
C12N007/00; C07K 14/00 20060101 C07K014/00; A61P 35/02 20060101
A61P035/02; A61K 45/06 20060101 A61K045/06; A61K 31/704 20060101
A61K031/704; A61K 31/496 20060101 A61K031/496; A61K 31/136 20060101
A61K031/136; A61K 31/7068 20060101 A61K031/7068; A61K 31/4545
20060101 A61K031/4545 |
Claims
1. A profiling peptide comprising a cellular uptake moiety and an
Mcl-1 binding domain, the Mcl-1 binding domain having the sequence
of SEQ ID NO: 1 with 0-8 modifications.
2. The profiling peptide of claim 1, wherein the Mcl-1 binding
domain has the sequence of SEQ ID NO: 1 with 0-6 modifications.
3. (canceled)
4. The profiling peptide of claim 1, wherein the Mcl-1 binding
domain has the sequence of SEQ ID NO: 1 with 0-3 modifications.
5-9. (canceled)
10. The profiling peptide of claim 1, wherein each modification is
independently a conservative amino acid substitution, addition, or
deletion.
11-14. (canceled)
15. The profiling peptide of claim 1, wherein the cellular uptake
moiety comprises at least five contiguous amino acids from a
transduction domain of a human immunodeficiency virus (HIV)
Trans-Activator of Transcription (TAT) peptide.
16. The profiling peptide of claim 1, wherein the cellular uptake
moiety has at least 90% identity with SEQ ID NO: 12.
17. (canceled)
18. The profiling peptide of claim 1, wherein the cellular uptake
moiety comprises at least five contiguous amino acids from an
Atennapedia plasma membrane (ANT) translocation domain; or wherein
the cellular uptake moiety comprises 3-10 contiguous arginine
residues.
19-24. (canceled)
25. The profiling peptide of claim 1, wherein the cellular uptake
moiety is conjugated via a linker to the Mcl-1 binding domain.
26-28. (canceled)
29. The profiling peptide of claim 1, comprising the sequence of
SEQ ID NO: 14 or SEQ ID NO: 15.
30. (canceled)
31. A composition comprising the profiling peptide of claim 1 and a
carrier.
32-40. (canceled)
41. A method for treating a cancer in a subject in need thereof,
the method comprising administering a treatment regimen comprising
a therapeutic agent to a subject having an Mcl-1 dependency
percentage of at least 15%, the Mcl-1 dependency percentage having
been obtained by an in vitro method comprising contacting a first
portion of a plurality of cancer cells with a profiling peptide of
claim 1.
42. The method of claim 41, wherein the in vitro method further
comprises detecting a change in mitochondrial integrity of the
plurality of cancer cells.
43. (canceled)
44. The method of claim 41, wherein the subject has an Mcl-1
dependency percentage of at least 20%.
45. The method of claim 41, wherein the subject has an Mcl-1
dependency percentage of at least 30%.
46. The method of claim 41, wherein the subject has an Mcl-1
dependency percentage of at least 40%.
47. The method of claim 41, wherein the therapeutic agent is a
Cyclin-dependent kinase 9 (CDK9) inhibitor.
48. The method of claim 47, wherein the CDK9 inhibitor is
Alvocidib.
49. The method of claim 41, further comprising administering Ara-C
or Mitoxanthrone, or both.
50. The method of claim 41, further comprising administering a
Bcl-2 inhibitor.
51. The method of claim 50, wherein the Bcl-2 inhibitor is
Venetoclax.
52. The method of claim 41, further comprising administering Ara-C
or Daunorubicin, or both.
53. The method claim 41, further comprising administering a Brd4
inhibitor or a DNA methyltransferase inhibitor, or both.
54. The method of claim 41, wherein the in vitro method further
comprises contacting at least the first portion of the plurality of
cancer cells with a diterpenoid.
55-59. (canceled)
60. The method of claim 42, wherein the change in mitochondrial
integrity is measured using a detecting agent.
61-64. (canceled)
65. The method of claim 60, wherein the detecting agent is a
3,3'-Dihexyloxacarbocyanine Iodide (DiOC6).
66. The method of claim 60, wherein the detecting agent is a
potentiometric dye.
67. (canceled)
68. The method of claim 41, wherein the plurality of cancer cells
is from a hematologic cancer.
69. The method of claim 68, wherein the hematologic cancer is
multiple myeloma, myelodysplastic syndrome (MDS), acute myeloid
leukemia (AML), acute lymphoblastic leukemia (ALL), acute
lymphocytic leukemia, chronic lymphogenous leukemia, chronic
lymphocytic leukemia (CLL), mantle cell lymphoma, diffuse large
B-cell lymphoma, follicular lymphoma, or non-Hodgkin's
lymphoma.
70. The method of claim 69, wherein the hematologic cancer is acute
myeloid leukemia (AML).
71-121. (canceled)
Description
STATEMENT REGARDING SEQUENCE LISTING
[0001] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
910208_428C1_SEQUENCELISTING.txt. The text file is 6 KB, was
created on Jun. 28, 2018, and is being submitted electronically via
EFS-Web.
FIELD
[0002] The present disclosure is generally directed to profiling
peptides, compositions, and kits, as well as methods for predicting
sensitivity of a cancer to a treatment, selecting a treatment for a
cancer, producing a sensitivity profile for a cancer, and treating
a cancer. In some embodiments, the treatment includes
administration of a therapeutic agent that is a cyclin-dependent
kinase 9 (CDK9) inhibitor.
BACKGROUND
[0003] While there have been advances in cancer treatment,
chemotherapy remains largely inefficient and ineffective. One
reason for the generally poor performance of chemotherapy is that
the selected treatment is often not closely matched to the genetic
and molecular dependencies of an individual's disease.
[0004] For example, cancer cells exhibit abnormalities, such as DNA
damage, genetic instability, abnormal growth factor signaling, and
abnormal or missing matrix interactions, any of which should
typically induce apoptosis through the intrinsic (mitochondrial)
apoptosis pathway. As a result of these aberrant phenotypes, cancer
cells develop blocks in apoptosis pathways that allow the cells to
survive rather than respond to the apoptosis signals. As many
cancer therapies rely on apoptosis to be effective, modulation of
apoptosis by a specific anti-apoptotic protein may relate to
responsiveness to particular therapy.
[0005] The concept of "oncogene addiction" describes the phenomena
of the acquired dependence of cancer cells on, or addiction to,
particular proteins for survival. These dependencies make some
cancer cells both resistant to particular therapies, and,
surprisingly, sensitive to other therapies.
[0006] Dependence by cancer cells on the anti-apoptotic Bcl-2
family proteins frequently relates to their otherwise unintended
survival. Cancer cells generally rely on one Bcl-2 family member or
another (e.g. Bcl-2, Bcl-xL, Ml-1) to suppress cell death signals
and resist apoptosis. Bcl-2 family proteins are regulated by
distinct protein-protein interactions between pro-survival (i.e.,
anti-apoptotic) and pro-apoptotic members. These interactions occur
primarily through Bcl-2 homology domain-3 (BH3) mediated binding
and can have various outcomes, including homeostasis, cell death,
sensitization to apoptosis, and blockade of apoptosis. Many cancer
cells in which apoptotic signaling is blocked have an accumulation
of the BH3 only activator proteins at the mitochondrial surface, a
result of these proteins being sequestered by the anti-apoptotic
proteins. This accumulation and proximity to their effector target
proteins accounts for increased sensitivity to antagonism of Bcl-2
family proteins in the "primed" state. Accordingly, measurement of
the functionality of anti-apoptotic Bcl-2 family proteins have
proven to provide sound predictions for the dependency cancer cells
have on a given Bcl-2 family member and how a cancer subject will
respond to a treatment.
[0007] There are two main profiling assays currently used for BH3
profiling. The primary difference in the two commonly used assays
is the use of flow cytometry to measure the response versus a
fluorescence microplate reader. Using flow cytometry requires
fluorescently labeling the cells with antibodies directed toward
various cell surface markers and using the gating functions on the
flow cytometer to only measure the response in the malignant
population of cells. In short, after isolating leukocytes from a
sample, the cells are labeled, the outer membrane is permeabilized,
contacted with a BH3 peptide (e.g., NOXA), and stained with JC-1
fluorescent dye. Then, flow cytometry is used to quantify the
response.
[0008] Alternatively, a microplate reader uses cell surface marker
antibodies and cell separation techniques to isolate the malignant
population of cells. After isolating leukocytes from a sample, the
cells of interest are purified, the outer membrane is
permeabilized, contacted with a BH3 peptide (e.g., NOXA), and
stained with JC-1 fluorescent dye. Then, a microplate reader is
used to quantify the response.
[0009] Both approaches generally require the cancer cells to be
permeabilized by digitonin, which allows fragments of Bcl-2 family
peptides (such as NOXA, BIM, etc.) to enter the cell and interact
with mitochondrial proteins. In most assays using the standard NOXA
peptide, this step is essential. However, cell permeabilization
adds complexity, introduces significant variation to the assay, and
increases the overall assay run time, all of which introduce
technical challenges to providing accurate profiling results that
are cost effective. As digitonin non-selectively permeabilizes
biological membranes, including the mitochondrial membrane (Hoppel,
C, and Cooper, C. Biochem J. 1968 April; 107(3): 367-375), the
assays that use digitonin generally require precise titration of
the digitonin such that the concentration used is within the window
that permeabilizes the outer cellular membrane with minimal effects
on the mitochondrial membrane. This window of digitonin
concentration and treatment time is narrow, may vary between
different cell types, and is directly related to having a robust
assay that produces accurate results. This challenge is
traditionally overcome currently by performing the assay at a
single central laboratory that has the experience and the
appropriate controls to ensure the assay is performed correctly.
Thus, the cell permeabilization step is a challenge for
decentralizing the use of such assays, for example, in producing an
in vitro diagnostic kit that may be used in clinical
laboratories.
[0010] Accordingly, improved methods of measuring the functionality
of anti-apoptotic Bcl-2 proteins that are more accurate,
reproducible, and cost-effective are needed.
BRIEF SUMMARY
[0011] In one aspect, the present disclosure provides a profiling
peptide comprising a cellular uptake moiety and an Mcl-1 binding
domain, the Mcl-1 binding domain having the sequence of SEQ ID NO:1
with 0-8 modifications.
[0012] In another aspect, the present disclosure provides a
composition comprising a profiling peptide comprising a cellular
uptake moiety and an Mcl-1 binding domain, the Mcl-1 binding domain
having the sequence of SEQ ID NO:1 with 0-8 modifications, and a
carrier.
[0013] In further aspects, the present disclosure provides a kit
comprising: a profiling peptide comprising a cellular uptake moiety
and an Mcl-1 binding domain, the Mcl-1 binding domain having the
sequence of SEQ ID NO:1 with 0-8 modifications; and a detecting
agent.
[0014] In aspects, the present disclosure provides a method for
treating a cancer in a subject in need thereof, the method
comprising administering a treatment regimen comprising a
therapeutic agent to a subject having an Mcl-1 dependency
percentage above a predetermined value, the Mcl-1 dependency
percentage having been obtained by an in vitro method comprising
contacting a first portion of a plurality of cancer cells with a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain, the Mcl-1 binding domain having the sequence of SEQ
ID NO:1 with 0-8 modifications, or a composition comprising a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain, the Mcl-1 binding domain having the sequence of SEQ
ID NO:1 with 0-8 modifications and a carrier.
[0015] In other embodiments, the present disclosure provides a
method of predicting sensitivity of a cancer cell from a subject to
a therapeutic agent, the method comprising: contacting the cancer
cell with a profiling peptide comprising a cellular uptake moiety
and an Mcl-1 binding domain, the Mcl-1 binding domain having the
sequence of SEQ ID NO:1 with 0-8 modifications; and detecting a
change in mitochondrial integrity of the cancer cell; wherein a
decrease in mitochondrial integrity indicates that the cancer cell
is sensitive to the therapeutic agent.
[0016] In further embodiments, the present disclosure provides a
method of treating a cancer in a subject in need thereof, the
method comprising: contacting a cancer cell from the subject with a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain, the Mcl-1 binding domain having the sequence of SEQ
ID NO:1 with 0-8 modifications; detecting a change in mitochondrial
integrity of the cancer cell; and administering an effective amount
of a therapeutic agent to the subject if a decrease in
mitochondrial integrity is detected, thereby treating the cancer in
the subject.
[0017] In another aspect, the present disclosure provides a method
of producing a sensitivity profile for a plurality of cancer cells
from a subject, the method comprising: contacting a first portion
of the plurality of cancer cells with a profiling peptide
comprising a cellular uptake moiety and an Mcl-1 binding domain,
the Mcl-1 binding domain having the sequence of SEQ ID NO:1 with
0-8 modifications, or a composition comprising a profiling peptide
comprising a cellular uptake moiety and an Mcl-1 binding domain,
the Mcl-1 binding domain having the sequence of SEQ ID NO:1 with
0-8 modifications and a carrier; and detecting a change in
mitochondrial integrity of the first portion of the plurality of
cancer cells.
[0018] In yet further embodiments, the present disclosure provides
a method of selecting a therapeutic agent for treating a cancer in
a subject, the method comprising: receiving a sensitivity profile
for a cancer cell of the subject, the sensitivity profile
comprising mitochondrial integrity data of the cancer cell when
contacted with a profiling peptide comprising a cellular uptake
moiety and an Mcl-1 binding domain, the Mcl-1 binding domain having
the sequence of SEQ ID NO:1 with 0-8 modifications; and selecting
the therapeutic agent to treat the subject if the mitochondrial
integrity data shows a decrease in mitochondrial integrity.
[0019] In further embodiments, the present disclosure provides a
method of treating a cancer in a subject in need thereof, the
method comprising: receiving a sensitivity profile for a cancer
cell of the subject, the sensitivity profile comprising
mitochondrial integrity data of the cancer cell when contacted with
a profiling peptide comprising a cellular uptake moiety and an
Mcl-1 binding domain, the Mcl-1 binding domain having the sequence
of SEQ ID NO:1 with 0-8 modifications; and administering an
effective amount of a therapeutic agent to the subject if the
mitochondrial integrity data shows a decrease in mitochondrial
integrity, thereby treating the cancer in the subject.
[0020] In still further embodiments, the present disclosure
provides a method of predicting sensitivity of a cancer cell from a
subject to a therapeutic agent, the method comprising: contacting
the cancer cell with a profiling peptide comprising a cellular
uptake moiety and an Mcl-1 binding domain, the Mcl-1 binding domain
having the sequence of SEQ ID NO:1 with 0-8 modifications;
detecting a change in mitochondrial integrity of the cancer cell;
and determining an Mcl-1 dependency percentage for the cancer cell
based at least on the change in mitochondrial integrity, wherein an
Mcl-1 dependency percentage above a predetermined value indicates
that the cancer cell is sensitive to the therapeutic agent.
[0021] In other embodiments, the present disclosure provides a
method of treating a cancer in a subject in need thereof, the
method comprising: contacting a cancer cell from the subject with a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain, the Mcl-1 binding domain having the sequence of SEQ
ID NO:1 with 0-8 modifications; detecting a change in mitochondrial
integrity of the cancer cell; determining an Mcl-1 dependency
percentage for the cancer cell based at least on the change in
mitochondrial integrity; and administering an effective amount of a
therapeutic agent to the subject if the Mcl-1 dependency percentage
is above a predetermined value, thereby treating the cancer in the
subject.
[0022] In other embodiments, the present disclosure provides a
method of producing a sensitivity profile for a cancer cell from a
subject, the method comprising: contacting the cancer cell with a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain, the Mcl-1 binding domain having the sequence of SEQ
ID NO:1 with 0-8 modifications; detecting a change in mitochondrial
integrity of the cancer cell; and determining an Mcl-1 dependency
percentage for the cancer cell based at least on the change in
mitochondrial integrity.
[0023] In yet other embodiments, the present disclosure provides a
method of selecting a therapeutic agent for treating a cancer in a
subject, the method comprising: receiving a sensitivity profile for
a cancer cell of the subject, the sensitivity profile comprising
Mcl-1 dependency data for the cancer cell, the Mcl-1 dependency
data determined based at least on a change in mitochondrial
integrity of the cancer cell when contacted with a profiling
peptide comprising a cellular uptake moiety and an Mcl-1 binding
domain, the Mcl-1 binding domain having the sequence of SEQ ID NO:1
with 0-8 modifications; and selecting the therapeutic agent to
treat the subject if the Mcl-1 dependency data shows an Mcl-1
dependency percentage above a predetermined value.
[0024] In still other embodiments, the present disclosure provides
a method of treating a cancer in a subject in need thereof, the
method comprising: receiving a sensitivity profile for cancer cells
of the subject, the sensitivity profile comprising Mcl-1 dependency
data for the cancer cell, the Mcl-1 dependency data being
determined based at least on a change in mitochondrial integrity of
the cancer cell when contacted with a profiling peptide comprising
a cellular uptake moiety and an Mcl-1 binding domain, the Mcl-1
binding domain having the sequence of SEQ ID NO:1 with 0-8
modifications; and administering an effective amount of a
therapeutic agent to the subject if the Mcl-1 dependency data shows
an Mcl-1 dependency percentage above a predetermined value, thereby
treating the cancer in the subject.
[0025] In another aspect, the present disclosure provides a method
of producing a sensitivity profile for a plurality of cancer cells
from a subject, the method comprising isolating the plurality of
cancer cells from a sample, contacting the plurality of cancer
cells with a stain, treating a first portion of the plurality of
cancer cells with a negative control, treating a second portion of
the plurality of cancer cells with a positive control, treating a
third portion of the plurality of cancer cells with a profiling
peptide comprising a cellular uptake moiety and an Mcl-1 binding
domain, the Mcl-1 binding domain having the sequence of SEQ ID NO:1
with 0-8 modifications, or a composition comprising a profiling
peptide comprising a cellular uptake moiety and an Mcl-1 binding
domain, the Mcl-1 binding domain having the sequence of SEQ ID NO:1
with 0-8 modifications and a carrier, contacting the first portion,
the second portion, and the third portion of the plurality of
cancer cells with a dye, and analyzing the first portion, the
second portion, and the third portion of the plurality of cancer
cells by flow cytometry.
[0026] In a further aspect, the present disclosure provides a
therapeutic composition for use in the treatment of cancer in a
subject with a Mel-1 dependency percentage of at least 15%, the
Mcl-1 dependency percentage having been obtained by an in vitro
method comprising: contacting a first portion of a plurality of
cancer cells with a profiling peptide comprising a cellular uptake
moiety and an Mcl-1 binding domain, the Mcl-1 binding domain having
the sequence of SEQ ID NO:1 with 0-8 modifications, or a
composition comprising a profiling peptide comprising a cellular
uptake moiety and an Mcl-1 binding domain, the Mcl-1 binding domain
having the sequence of SEQ ID NO:1 with 0-8 modifications and a
carrier.
[0027] In yet further aspects, the present disclosure provides a
therapeutic composition for cancer comprising a therapeutic agent,
which is administered to a subject having a Mcl-1 dependency
percentage of at least 15%, wherein, the Mcl-1 dependency
percentage is obtained by an in vitro method comprising: contacting
a first position of plurality of cancer cells with a profiling
peptide comprising a cellular uptake moiety and an Mcl-1 binding
domain, the Mcl-1 binding domain having the sequence of SEQ ID NO:1
with 0-8 modifications, or a composition comprising a profiling
peptide comprising a cellular uptake moiety and an Mcl-1 binding
domain, the Mcl-1 binding domain having the sequence of SEQ ID NO:1
with 0-8 modifications and a carrier.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIGS. 1A-1D show the results of a whole cell assay of the
profiling peptides described herein compared to NOXA.
[0029] FIG. 2 shows the Mel-1 dependency percentages for varying
concentrations of a profiling peptide described herein compared to
NOXA.
[0030] FIG. 3 shows gating for the acute myeloid leukemia (AML)
blast cell population using CD45 dim and CD13, CD33 and CD34 high
as described in Example 5.
[0031] FIG. 4 shows the results of ryanodine testing. Calcium
release in MOLM-13 cells treated with the peptide of SEQ ID NO: 14
are shown.
[0032] FIG. 5 shows the complete remission (CR) rate in AML
subjects with MCL-1 Dependence .gtoreq.40%.
DETAILED DESCRIPTION
[0033] The present disclosure relates to profiling peptides
comprising an optionally modified Mel-1 binding domain having the
sequence of any one of SEQ ID NOS: 1-11, as well as compositions,
methods of use, and kits. More specifically, the profiling peptides
optionally include a cellular uptake moiety and an Mcl-1 binding
domain, the Mcl-1 binding domain having the sequence of SEQ ID NO:1
with 0-8 modifications. In some embodiments, the profiling peptides
comprise a cellular uptake moiety and an Mcl-1 binding domain
having the sequence of any one of SEQ ID NO:1-11. The methods of
using such profiling peptides include predicting sensitivity of a
cancer cell, selecting a therapeutic agent, treating a cancer,
producing a sensitivity profile, and the like.
[0034] Prior to setting forth this disclosure in more detail, it
may be helpful to an understanding thereof to provide definitions
of certain terms to be used herein. Additional definitions are set
forth throughout this disclosure.
[0035] "Optional" or "optionally" means that the subsequently
described element, component, event, or circumstance may or may not
occur, and that the description includes instances in which the
element, component, event, or circumstance occurs and instances in
which they do not.
[0036] "Peptide" refers to a polymer of amino acid residues.
Peptides include naturally occurring amino acid polymers and
non-naturally occurring amino acid polymers, as well as amino acid
polymers in which one or more amino acid residues is an artificial
chemical mimetic of a corresponding naturally occurring amino
acid.
[0037] As used herein, "amino acid" refers to naturally occurring
amino acids and synthetic amino acids, as well as amino acid
analogs and amino acid mimetics that function in a manner similar
to the naturally occurring amino acids. Naturally occurring amino
acids are those encoded by the genetic code, as well as those amino
acids that are later modified, e.g., hydroxyproline,
.gamma.-carboxyglutamate, and O-phosphoserine. Amino acid analogs
refer to compounds that have the same basic chemical structure as a
naturally occurring amino acid, i.e., an .alpha.-carbon that is
bound to a hydrogen, a carboxyl group, an amino group, and an R
group (e.g., homoserine, norleucine, methionine sulfoxide, and
methionine methyl sulfonium). Such analogs have modified R groups
(e.g., norleucine) or modified peptide backbones, but retain the
same basic chemical structure as a naturally occurring amino acid.
Amino acid mimetics refer to chemical compounds that have a
structure that is different from the general chemical structure of
an amino acid, but that function in a manner similar to a naturally
occurring amino acid.
[0038] A "cancer," including a "tumor," refers to an uncontrolled
growth of cells and/or abnormal increased cell survival and/or
inhibition of apoptosis which interferes with the normal
functioning of the bodily organs and systems. "Cancer" (e.g., a
tumor) includes solid and non-solid cancers. A subject that has a
cancer or a tumor has an objectively measurable number of cancer
cells present in the subject's body. "Cancers" include benign and
malignant cancers (e.g., benign and malignant tumors,
respectively), as well as dormant tumors or micrometastases.
"Cancers" include acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), adrenocortical carcinoma, AIDS-related cancers,
anal cancer, appendix cancer, astrocytoma (e.g. childhood
cerebellar or cerebral), basal-cell carcinoma, bile duct cancer,
bladder cancer, bone tumor (e.g. osteosarcoma, malignant fibrous
histiocytoma), brainstem glioma, brain cancer, brain tumors (e.g.
cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,
ependymoma, medulloblastoma, supratentorial primitive
neuroectodermal tumors, visual pathway and hypothalamic glioma),
breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma,
carcinoid tumors, central nervous system lymphomas, cerebellar
astrocytoma, cervical cancer, chronic lymphocytic leukemia (CLL),
chronic myelogenous leukemia (CML), chronic myeloproliferative
disorders, colon cancer, cutaneous t-cell lymphoma, desmoplastic
small round cell tumor, endometrial cancer, ependymoma, esophageal
cancer, Ewing's sarcoma, extracranial germ cell tumor, extragonadal
germ cell tumor, extrahepatic bile duct cancer, eye cancer,
gallbladder cancer, gastric (stomach) cancer, gastrointestinal
stromal tumor (GIST), germ cell tumor (e.g. extracranial,
extragonadal, ovarian), gestational trophoblastic tumor, gliomas
(e.g. brain stem, cerebral astrocytoma, visual pathway and
hypothalamic), gastric carcinoid, head and neck cancer, heart
cancer, hepatocellular (liver) cancer, hypopharyngeal cancer,
hypothalamic and visual pathway glioma, intraocular melanoma, islet
cell carcinoma (endocrine pancreas), kidney cancer (renal cell
cancer), laryngeal cancer, leukemias (e.g. acute lymphocytic
leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia,
chronic myeloid leukemia, hairy cell), lip and oral cavity cancer,
liposarcoma, liver cancer, lung cancer (e.g. non-small cell, small
cell), lymphoma (e.g. AIDS-related, Burkitt, cutaneous T-cell
Hodgkin, non-Hodgkin, primary central nervous system),
medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma,
metastatic squamous neck cancer, mouth cancer, multiple endocrine
neoplasia syndrome, multiple myeloma, mycosis fungoides,
myelodysplastic syndromes, myelodysplastic/myeloproliferative
diseases, myelogenous leukemia, myeloid leukemia, myeloid leukemia,
myeloproliferative disorders, chronic, nasal cavity and paranasal
sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin
lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal
cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic
cancer, paranasal sinus and nasal cavity cancer, parathyroid
cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal
astrocytoma and/or germinoma, pineoblastoma and supratentorial
primitive neuroectodermal tumors, pituitary adenoma, plasma cell
neoplasia/multiple myeloma, pleuropulmonary blastoma, primary
central nervous system lymphoma, prostate cancer, rectal cancer,
renal cell carcinoma (kidney cancer), renal pelvis and ureter,
retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma
(e.g. Ewing family, Kaposi, soft tissue, uterine), Sezary syndrome,
skin cancer (e.g. nonmelanoma, melanoma, merkel cell), small cell
lung cancer, small intestine cancer, soft tissue sarcoma, squamous
cell carcinoma, squamous neck cancer, stomach cancer,
supratentorial primitive neuroectodermal tumor, t-cell lymphoma,
testicular cancer, throat cancer, thymoma and thymic carcinoma,
thyroid cancer, trophoblastic tumors, ureter and renal pelvis
cancers, urethral cancer, uterine cancer, uterine sarcoma, vaginal
cancer, visual pathway and hypothalamic glioma, vulvar cancer,
Waldenstrom macroglobulinemia, or Wilms tumor.
[0039] "Metastasis" refers to the spread of cancer from its primary
site to other places in the body. "Metastases" are cancers which
migrate from their original location and seed vital organs, which
can eventually lead to the death of the subject through the
functional deterioration of the affected organs. Metastasis is a
sequential process, where cancer cells can break away from a
primary tumor, penetrate into lymphatic and blood vessels,
circulate through the bloodstream, and grow in a distant focus
(metastasize) in normal tissues elsewhere in the body. At the new
site, the cells establish a blood supply and can grow to form a
life-threatening mass. Metastasis can be local or distant. Both
stimulatory and inhibitory molecular pathways within the tumor cell
regulate this behavior, and interactions between the tumor cell and
host cells in the new site are also significant.
[0040] "Subject" includes humans, domestic animals, such as
laboratory animals (e.g. dogs, monkeys, rats, mice, etc.),
household pets (e.g., cats, dogs, rabbits, etc.), and livestock
(e.g., pigs, cattle, sheep, goats, horses, etc.), and non-domestic
animals (e.g., bears, elephants, porcupines, etc.). In embodiments,
a subject is a human.
[0041] "Treating" or "treatment" as used herein refers to the
administration of a medication or medical care to a subject, such
as a human, having a disease or condition of interest, e.g., a
cancer, including: (i) preventing the disease or condition from
occurring in a subject, in particular, when such subject is
predisposed to the condition but has not yet been diagnosed as
having it; (ii) inhibiting the disease or condition, i.e.,
arresting its development; (iii) relieving the disease or
condition, i.e., causing regression of the disease or condition; or
(iv) relieving the symptoms resulting from the disease or
condition, (e.g., pain, weight loss, cough, fatigue, weakness,
etc.) without addressing the underlying disease or condition. As
used herein, the terms "disease" and "condition" may be used
interchangeably or may be different in that the particular malady
or condition may not have a known causative agent (so that etiology
has not yet been confirmed) and it is therefore not yet recognized
as a disease but only as an undesirable condition or syndrome,
wherein a more or less specific set of symptoms have been
identified by clinicians.
[0042] "Effective amount" refers to the amount of a compound or
composition which, when administered to a subject, such as a human,
is sufficient to effect treatment of the subject's cancer. The
amount of a compound or composition that constitutes an "effective
amount" will vary depending on the compound or composition, the
condition being treated and its severity, the manner of
administration, the duration of treatment, and/or the age of the
subject to be treated, but can be determined routinely by one of
ordinary skill in the art based on his own knowledge and this
disclosure. In embodiments, an "effective amount" effects treatment
(e.g., treats, prevents, inhibits, relieves, promotes, improves,
increases, reduces, and the like) as measured by a statistically
significant change in one or more indications, symptoms, signs,
diagnostic tests, vital signs, and the like. In other embodiments,
an "effective amount" suppresses, manages, or prevents a condition
as measured by a lack of a statistically significant change in one
or more indications, symptoms, signs, diagnostic tests, vital
signs, and the like.
[0043] As used herein, "statistically significant" refers to a p
value of 0.050 or less when calculated using the Students t-test
and indicates that it is unlikely that a particular event or result
being measured has arisen by chance.
[0044] In the present description, any concentration range,
percentage range, ratio range, or integer range is to be understood
to include the value of any integer within the recited range and,
when appropriate, fractions thereof (such as one tenth and one
hundredth of an integer), unless otherwise indicated. Also, any
number range recited herein relating to any physical feature, such
as polymer subunits, size, or thickness, are to be understood to
include any integer within the recited range, unless otherwise
indicated. As used herein, the term "about" means .+-.20%, .+-.10%,
.+-.5% or .+-.1% of the indicated range, value, or structure,
unless otherwise indicated. It should be understood that the terms
"a" and "an" as used herein refer to "one or more" of the
enumerated components. The use of the alternative (e.g., "or")
should be understood to mean either one, both, or any combination
thereof of the alternatives. Unless the context requires otherwise,
throughout the present specification and claims, the word
"comprise" and variations thereof, such as, "comprises" and
"comprising," as well as synonymous terms like "include" and "have"
and variants thereof, are to be construed in an open, inclusive
sense; that is, as "including, but not limited to," such that
recitation of items in a list is not to the exclusion of other like
items that may also be useful in the materials, compositions,
devices, and methods of this technology. Although the open-ended
term "comprising," as a synonym of terms such as including,
containing, or having, is used herein to describe and claim the
invention, the present technology, or embodiments thereof, may
alternatively be described using more limiting terms such as
"consisting of" or "consisting essentially of" the recited
ingredients.
[0045] Unless defined otherwise, all technical and scientific terms
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0046] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure, or
characteristic described in connection with the embodiment is
included in at least one embodiment of the present disclosure.
Thus, the appearances of the phrases "in one embodiment" or "in an
embodiment" in various places throughout this specification are not
necessarily all referring to the same embodiment. Similarly, the
terms "can" and "may" and their variants are intended to be
non-limiting, such that recitation that an embodiment can or may
comprise certain elements or features does not exclude other
embodiments of the present technology that do not contain those
elements or features. Furthermore, the particular features,
structures, or characteristics may be combined in any suitable
manner in one or more embodiments.
[0047] In the following description, certain specific details are
set forth in order to provide a thorough understanding of various
embodiments of this disclosure. However, one skilled in the art
will understand that the disclosure may be practiced without these
details.
Profiling Peptides
[0048] As noted herein, the present disclosure provides profiling
peptides. Generally, profiling peptides comprise an Mcl-1 binding
domain having a sequence shown in Table 1, which may be optionally
modified.
TABLE-US-00001 TABLE 1 Exemplary Mcl-1 Binding Domains. SEQ ID NO:
Sequence 1 RPEIWMTQGLRRLGDEINAYYAR 2 RPEIWLTQSLQRLGDEINAYYAR 3
RPEIWLTQHLQRLGDEINAYYAR 4 RPEIWMGQGLRRLGDEINAYYAR 5
RPEIWLGQSLQRLGDEINAYYAR 6 RPEIWLGQHLQRLGDEINAYYAR 7
RPEIWITQELRRIGDEFNAYYAR 8 RPEIWMTQELRRIGDEFNAYYAR 9
RPEIWITQGLRRIGDEFNAYYAR 10 RPEIWITQELRRLGDEFNAYYAR 11
RPEIWITQELRRIGDEINAYYAR
[0049] In some embodiments, a profiling peptide comprises an Mcl-1
binding domain having the sequence of any one of SEQ ID NOS:1-1
with 0-8 modifications. In some embodiments, a profiling peptide
comprises an Mcl-1 binding domain having the sequence of any one of
SEQ ID NOS:1-11 with 1-8 modifications.
[0050] "Modified" peptides include peptides having one or more
amino acid substitutions as compared to a sequence disclosed
herein. The substitution can be a conservative or a
non-conservative substitution. Modified peptides also include
peptides having additions of amino acids to, or deletions of amino
acids from, the original peptide sequence. Therefore, modified
peptides include fragments of the original peptide sequence. In
some embodiments, the modifications comprise one or more
conservative amino acid substitutions, additions, deletions, or
combinations thereof.
[0051] As used herein, a "modification" refers to a substitution,
addition, or deletion of a single amino acid. Accordingly, when a
number of modifications is referenced (e.g., an Mcl-1 binding
domain having the sequence of SEQ ID NO:1 with two modifications),
the number refers to the number of amino acids of the sequence that
may be substituted, added, or deleted. In other words, each
"substitution," "addition," or "deletion" replaces, adds, or
removes a single amino acid, respectively, and does not refer to a
single instance that replaces, adds, or removes more than one amino
acid.
[0052] Modifications may be introduced by altering a polynucleotide
encoding a profiling peptide, and may be performed by a variety of
methods, including site-specific or site-directed mutagenesis. For
example, mutations may be introduced at a particular location by
synthesizing oligonucleotides containing a mutant sequence flanked
by restriction sites enabling ligation to fragments of the
unmodified sequence. Following ligation, the resulting sequence
would encode a modified peptide having the desired amino acid
addition, substitution, or deletion.
[0053] A "conservative substitution" includes a substitution found
in one of the following conservative substitutions groups: Group 1:
Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S),
Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic
acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or
Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His
or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L),
Methionine (Met or M), Valine (Val or V); and Group 6:
Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or
W). Additionally or alternatively, amino acids can be grouped into
conservative substitution groups by similar function or chemical
structure or composition (e.g., acidic, basic, aliphatic, aromatic,
or sulfur-containing). For example, an aliphatic grouping may
include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile.
Other conservative substitutions groups include: sulfur-containing:
Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small
aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr,
Proline (Pro or P), and Gly; polar, negatively charged residues and
their amides: Asp, Asn, Glu, and Gln; polar, positively charged
residues: His, Arg, and Lys; large aliphatic, nonpolar residues:
Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr,
and Trp. Additional information can be found in Creighton (1984)
Proteins, W.H. Freeman and Company.
[0054] In embodiments, the modifications described herein may
include the substitution of a naturally-occurring amino acid with a
synthetic amino acid, amino acid analog, or amino acid mimetic, or
the addition of a synthetic amino acid, amino acid analog, or amino
acid mimetic. In such embodiments, modifications can include the
substitution of one more L-amino acids with D-amino acids. The
D-amino acid can be the same amino acid type as that found in the
natural sequence or can be a different amino acid.
[0055] "Modification" also includes the substitution of a
naturally-occurring amino acid with an amino acid that has been
conjugated to, or otherwise associated with, a functional group.
Such an amino acid may be, e.g., a glycosylated amino acid, a
PEGylated amino acid, a farnesylated amino acid, an acetylated
amino acid, a biotinylated amino acid, an amino acid conjugated to
a lipid moiety, or an amino acid conjugated to an organic
derivatizing agent. The presence of such amino acids may be
preferred to, for example, increase polypeptide storage stability,
and/or increase peptide solubility. Such modifications can be
performed co-translationally or post-translationally during
recombinant production, or by synthetic means.
[0056] In embodiments, the profiling peptides described herein
comprise an Mcl-1 binding domain (e.g., any one of SEQ ID NOS:
1-11) with 0 to 1 modifications. In some embodiments, the profiling
peptides described herein comprise an Mcl-1 binding domain with 0
to 2 modifications. In some embodiments, the profiling peptides
described herein comprise an Mcl-1 binding domain with 0 to 3
modifications. In some embodiments, the profiling peptides
described herein comprise an Mcl-1 binding domain with 0 to 4
modifications. In some embodiments, the profiling peptides
described herein comprise an Mcl-1 binding domain with 0 to 5
modifications. In some embodiments, the profiling peptides
described herein comprise an Mcl-1 binding domain with 0 to 6
modifications. In some embodiments, the profiling peptides
described herein comprise an Mcl-1 binding domain with 0 to 7
modifications. In some embodiments, the profiling peptides
described herein comprise an Mcl-1 binding domain with 0 to 8
modifications. In some embodiments, the profiling peptides
described herein comprise an Mcl-1 binding domain with 0 to 9
modifications, 0 to 10 modifications, 0 to 12 modifications, 0 to
15 modifications, or 0 to 20 modifications.
[0057] In some embodiments, the profiling peptides described herein
comprise an Mcl-1 binding domain with 1 to 2 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 1 to 3 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 1 to 4 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 1 to 5 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 1 to 6 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 1 to 7 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 1 to 8 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 1 to 9 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 1 to 10 modifications, 1 to 12
modifications, 1 to 15 modifications, or 1 to 20 modifications. In
some embodiments, the profiling peptides described herein comprise
an Mcl-1 binding domain with 2 to 3 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 2 to 4 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 2 to 5 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 2 to 6 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 2 to 7 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 2 to 8 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 2 to 9 modifications, 2 to 10
modifications, 2 to 12 modifications, 2 to 15 modifications, or 2
to 20 modifications.
[0058] In some embodiments, the profiling peptides described herein
comprise an Mcl-1 binding domain with 3 to 4 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 3 to 5 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 3 to 6 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 3 to 7 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 3 to 8 modifications. In some
embodiments, the profiling peptides described herein comprise an
Mcl-1 binding domain with 3 to 9 modifications, 3 to 10
modifications, 3 to 12 modifications, 3 to 15 modifications, 3 to
20 modifications, 4 to 5 modifications, 4 to 6 modifications, 4 to
7 modifications, 4 to 8 modifications, 4 to 9 modifications, 4 to
10 modifications, 4 to 12 modifications, 4 to 15 modifications, 4
to 20 modifications, 5 to 6 modifications, 5 to 7 modifications, 5
to 8 modifications, 5 to 9 modifications, 5 to 10 modifications, 5
to 12 modifications, 5 to 15 modifications, 5 to 20 modifications,
6 to 7 modifications, 6 to 8 modifications, 6 to 9 modifications, 6
to 10 modifications, 7 to 8 modifications, 7 to 9 modifications, 7
to 10 modifications, 8 to 9 modifications, 8 to 10 modifications,
or 9 to 10 modifications. In some embodiments, the profiling
peptides described herein comprise a modified Mcl-1 binding domain
with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 modifications. The Mcl-1 binding domain sequence included
in a profiling peptide of the present disclosure may include a
modification at any position.
[0059] In embodiments where the Mcl-1 binding domain is a fragment
of any one of SEQ ID NOS:1-11, the amino acid sequence can have a
minimum length of 5 amino acids, 6 amino acids, 7 amino acids, 8
amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12
amino acids, 13 amino acids, or 14 amino acids. In some embodiments
where the Mcl-1 binding domain is a fragment of any one of SEQ ID
NOS:1-11, the amino acid sequence can have a minimum length of 15
amino acids. In some embodiments where the Mcl-1 binding domain is
a fragment of any one of SEQ ID NOS:1-11, the amino acid sequence
can have a minimum length of 16 amino acids. In some embodiments
where the Mcl-1 binding domain is a fragment of any one of SEQ ID
NOS:1-11, the amino acid sequence can have a minimum length of 17
amino acids. In some embodiments where the Mcl-1 binding domain is
a fragment of any one of SEQ ID NOS:1-11, the amino acid sequence
can have a minimum length of 18 amino acids. In some embodiments
where the Mcl-1 binding domain is a fragment of any one of SEQ ID
NOS:1-11, the amino acid sequence can have a minimum length of 19
amino acids. In some embodiments where the Mcl-1 binding domain is
a fragment of any one of SEQ ID NOS:1-11, the amino acid sequence
can have a minimum length of 20 amino acids. In some embodiments
where the Mcl-1 binding domain is a fragment of any one of SEQ ID
NOS:1-11, the amino acid sequence can have a minimum length of 21
amino acids.
[0060] In some embodiments, the Mcl-1 binding domain is a fragment
of SEQ ID NO: 1, and the amino acid sequence has a minimum length
of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9
amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13
amino acids, or 14 amino acids. In some embodiments, the Mcl-1
binding domain is a fragment of SEQ ID NO:1, and the amino acid
sequence has a minimum length of 15 amino acids. In some
embodiments, the Mcl-1 binding domain is a fragment of SEQ ID NO:1,
and the amino acid sequence has a minimum length of 16 amino acids.
In some embodiments, the Mcl-1 binding domain is a fragment of SEQ
ID NO:1, and the amino acid sequence has a minimum length of 17
amino acids. In some embodiments, the Mcl-1 binding domain is a
fragment of SEQ ID NO:1, and the amino acid sequence has a minimum
length of 18 amino acids. In some embodiments, the Mcl-1 binding
domain is a fragment of SEQ ID NO:1, and the amino acid sequence
has a minimum length of 19 amino acids. In some embodiments, the
Mcl-1 binding domain is a fragment of SEQ ID NO:1, and the amino
acid sequence has a minimum length of 20 amino acids. In some
embodiments, the Mcl-1 binding domain is a fragment of SEQ ID NO:1,
and the amino acid sequence has a minimum length of 21 amino
acids.
[0061] In further embodiments, the Mcl-1 binding domain comprises
at least 10 contiguous amino acids of any one of SEQ ID NOS:1-11.
In some embodiments, the Mcl-1 binding domain comprises at least 11
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 12
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 13
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 14
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 15
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 16
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 17
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 18
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 19
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 20
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least 21
contiguous amino acids of any one of SEQ ID NOS:1-11. In some
embodiments, the Mcl-1 binding domain comprises at least at least
10 contiguous amino acids of SEQ ID NO:1, at least 11 contiguous
amino acids of SEQ ID NO:1, at least 12 contiguous amino acids of
SEQ ID NO:1, at least 13 contiguous amino acids of SEQ ID NO: 1, at
least 14 contiguous amino acids of SEQ ID NO: 1, at least 15
contiguous amino acids of SEQ ID NO:1, at least 16 contiguous amino
acids of SEQ ID NO:1, at least 17 contiguous amino acids of SEQ ID
NO:1, at least 18 contiguous amino acids of SEQ ID NO: 1, at least
19 contiguous amino acids of SEQ ID NO: 1, at least 20 contiguous
amino acids of SEQ ID NO:1, or at least 21 contiguous amino acids
of SEQ ID NO:1.
[0062] In some embodiments, the Mcl-1 binding domain comprises no
more than 10 contiguous amino acids of any one of SEQ ID NOS:1-11,
no more than 11 contiguous amino acids of any one of SEQ ID
NOS:1-11, no more than 12 contiguous amino acids of any one of SEQ
ID NOS:1-11, no more than 13 contiguous amino acids of any one of
SEQ ID NOS:1-11, no more than 14 contiguous amino acids of any one
of SEQ ID NOS:1-11, no more than 15 contiguous amino acids of any
one of SEQ ID NOS:1-11, no more than 16 contiguous amino acids of
any one of SEQ ID NOS:1-11, no more than 17 contiguous amino acids
of any one of SEQ ID NOS:1-11, no more than 18 contiguous amino
acids of any one of SEQ ID NOS: 1-11, no more than 19 contiguous
amino acids of any one of SEQ ID NOS:1-11, no more than 20
contiguous amino acids of any one of SEQ ID NOS:1-11, or no more
than 21 contiguous amino acids of any one of SEQ ID NOS:1-11. In
some embodiments, the Mcl-1 binding domain comprises no more than
10 contiguous amino acids of SEQ ID NO:1, no more than 11
contiguous amino acids of SEQ ID NO:1, no more than 12 contiguous
amino acids of SEQ ID NO:1, no more than 13 contiguous amino acids
of SEQ ID NO: 1, no more than 14 contiguous amino acids of SEQ ID
NO: 1, no more than 15 contiguous amino acids of SEQ ID NO:1, no
more than 16 contiguous amino acids of SEQ ID NO:1, no more than 17
contiguous amino acids of SEQ ID NO:1, no more than 18 contiguous
amino acids of SEQ ID NO:1, no more than 19 contiguous amino acids
of SEQ ID NO:1, no more than 20 contiguous amino acids of SEQ ID
NO:1, or no more than 21 contiguous amino acids of SEQ ID NO:
1.
[0063] Embodiments of the Mcl-1 binding domains disclosed herein
include amino acid sequences with at least 70% sequence identity to
the sequence of any one of SEQ ID NOS:1-11. In some embodiments,
the Mcl-1 binding domain has at least 75% sequence identity with
the sequence of any one of SEQ ID NOS:1-11. In some embodiments,
the Mcl-1 binding domain has at least 80% sequence identity with
the sequence of any one of SEQ ID NOS:1-11. In some embodiments,
the Mcl-1 binding domain has at least 85% sequence with the
sequence of any one of SEQ ID NOS:1-11. In some embodiments, the
Mcl-1 binding domain has at least 90% sequence identity with the
sequence of any one of SEQ ID NOS:1-11. In some embodiments, the
Mcl-1 binding domain has at least 95% sequence identity with the
sequence of any one of SEQ ID NOS:1-11. In some embodiments, the
Mcl-1 binding domain has at least 96% sequence identity with the
sequence of any one of SEQ ID NOS:1-11. In some embodiments, the
Mcl-1 binding domain has at least 97% sequence identity with the
sequence of any one of SEQ ID NOS: 1-11. In some embodiments, the
Mcl-1 binding domain has at least 98% sequence identity with the
sequence of any one of SEQ ID NOS:1-11. In some embodiments, the
Mcl-1 binding domain has at least 99% sequence identity with the
sequence of any one of SEQ ID NOS:1-11.
[0064] In some embodiments, the Mcl-1 binding domain has a sequence
with at least 70% sequence identity to the sequence of SEQ ID NO:1.
In some embodiments, the Mcl-1 binding domain has a sequence with
at least 75% sequence identity to the sequence of SEQ ID NO:1. In
some embodiments, the Mcl-1 binding domain has a sequence with at
least 80% sequence identity to the sequence of SEQ ID NO:1. In some
embodiments, the Mcl-1 binding domain has a sequence with at least
85% sequence to the sequence of SEQ ID NO:1. In some embodiments,
the Mcl-1 binding domain has a sequence with at least 90% sequence
identity to the sequence of SEQ ID NO:1. In some embodiments, the
Mcl-1 binding domain has a sequence with at least 95% sequence
identity to the sequence of SEQ ID NO:1. In some embodiments, the
Mcl-1 binding domain has a sequence with at least 96% sequence
identity to the sequence of SEQ ID NO:1. In some embodiments, the
Mcl-1 binding domain has a sequence with at least 97% sequence
identity to the sequence of SEQ ID NO:1. In some embodiments, the
Mcl-1 binding domain has a sequence with at least 98% sequence
identity to the sequence of SEQ ID NO: 1. In some embodiments, the
Mcl-1 binding domain has a sequence with at least 99% sequence
identity to the sequence of SEQ ID NO:1.
[0065] "Percent sequence identity" refers to a relationship between
two or more sequences, as determined by comparing the sequences.
Preferred methods to determine sequence identity are designed to
give the best match between the sequences tested. For example, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in one or both of a first and a second amino acid
or nucleic acid sequence for optimal alignment). Further,
non-homologous sequences may be disregarded for comparison
purposes. In embodiments, the length of a sequence aligned for
comparison purposes is at least 70%, 80%, 90%, or 100% of the
length of the reference sequence. In embodiments, the percent
sequence identity referenced herein is calculated over the length
of the reference sequence. Methods to determine sequence identity
and similarity can be found in publicly available computer
programs. Sequence alignments and percent identity calculations may
be performed using a BLAST program (e.g., BLAST 2.0, BLASTP,
BLASTN, or BLASTX). The mathematical algorithm used in the BLAST
programs can be found in Altschul et al., Nucleic Acids Res.
25:3389-3402, 1997. Within the context of this disclosure it will
be understood that where sequence analysis software is used for
analysis, the results of the analysis are based on the "default
values" of the program referenced. "Default values" mean any set of
values or parameters which originally load with the software when
first initialized.
[0066] In embodiments, a modified Mcl-1 binding domain retains the
specificity and affinity for binding to Mcl-1 of the unmodified
sequence (i.e., the modifications to the Mcl-1 binding domain do
not alter the specificity or affinity for binding to Mcl-1 in a
statistically significant, clinically significant, or biologically
significant manner). In some embodiments, a modified Mcl-1 binding
domain retains the specificity and affinity for binding to Mcl-1 of
the unmodified sequence if the specificity and affinity of the
modified Mcl-1 binding domain are at least 70%, 80%, 85%, 90%, 95%,
97%, or 99% of the specificity and affinity of the unmodified
sequence. For example, a modified Mcl-1 binding domain may retain
the specificity and affinity for binding to Mcl-1 of the unmodified
sequence if the specificity and affinity of the modified Mcl-1
binding domain are at least at least 70%, 80%, 85%, 90%, 95%, 97%,
or 99% of the specificity and affinity of any one of SEQ ID NOS:
1-11.
[0067] In embodiments, the Mcl-1 binding domain binds to Mcl-1 with
at least a 20-fold increased affinity over NOXA. In some
embodiments, the Mcl-1 binding domain binds to Mcl-1 with at least
a 22-fold increased affinity over NOXA. In some embodiments, the
Mcl-1 binding domain binds to Mcl-1 with at least a 24-fold
increased affinity over NOXA. In particular embodiments, the Mcl-1
binding domain binds to Mcl-1 with at least a 28-fold increased
affinity over NOXA.
[0068] The effect of any amino acid modification to an Mcl-1
binding domain may be determined empirically by testing the
resulting modified Mcl-1 binding domain for the ability to function
in a biological assay, or to bind to a target molecule, such as a
monoclonal or polyclonal antibody. For example, the ability of the
modified Mcl-1 binding domain to fold into a conformation
comparable to the unmodified sequence can be tested using assays
known in the art, including reacting with monoclonal or polyclonal
antibodies that are specific for the native or unfolded peptides,
testing the retention of binding functions, and testing the
sensitivity or resistance of the modified Mcl-1 binding domain to
digestion with proteases.
[0069] Analysis and/or computer modeling of the primary and
secondary amino acid structure of the Mcl-1 binding domain to
analyze the tertiary structure of the peptide may aid in
identifying specific amino acid residues that can be substituted,
added, or deleted without significantly altering the structure and
as a consequence, potentially significantly reducing the binding
specificity and affinity of the Mcl-1 binding domain.
[0070] In embodiments, profiling peptides of the present disclosure
further comprise a cellular uptake moiety, which is optionally
joined to the Mcl-1 binding domain by a linker. A "cellular uptake
moiety" refers to an amino acid sequence or chemical compound that,
when conjugated to a peptide, allows the peptide and the cellular
uptake moiety to cross the outer cell membrane, thereby
transferring the peptide into the cell. Additionally, in some
embodiments, the cellular uptake moiety may act as a targeting
moiety, such that it directs the peptide to a desired cellular
location (e.g., the mitochondria).
[0071] In embodiments, the cellular uptake moiety is a peptide
sequence. In such embodiments, the cellular uptake moiety peptide
is at least four amino acids in length, at least five amino acids
in length, at least six amino acids in length, at least seven amino
acids in length, at least eight amino acids in length, or at least
nine amino acids in length. In some embodiments, the cellular
uptake moiety comprises an amino acid sequence of 1 to 20 amino
acids, 5 to 20 amino acids, 6 to 20 amino acids, 7 to 20 amino
acids, 8 to 20 amino acids, 9 to 20 amino acids, 10 to 20 amino
acids, 11 to 20 amino acids, 12 to 20 amino acids, 15 to 20 amino
acids, 1 to 15 amino acids, 5 to 15 amino acids, 6 to 15 amino
acids, 7 to 15 amino acids, 8 to 15 amino acids, 9 to 15 amino
acids, 10 to 15 amino acids, 11 to 15 amino acids, 12 to 15 amino
acids, 1 to 12 amino acids, 5 to 12 amino acids, 6 to 12 amino
acids, 7 to 12 amino acids, 8 to 12 amino acids, 9 to 12 amino
acids, 10 to 12 amino acids, 1 to 10 amino acids, 5 to 10 amino
acids, 6 to 10 amino acids, or 7 to 10 amino acids.
[0072] In embodiments, the cellular uptake moiety peptide is a
transduction domain isolated from a known peptide sequence.
Peptides with transduction domains are well known in the art and
include, for example, human immunodeficiency virus (HIV)
Trans-Activator of Transcription (TAT; described in Vives et al. J
Biol Chem. 1997 Jun. 20; 272(25):16010-7), Herpes simplex virus
tegument protein VP22, Atennapedia plasma membrane (ANT)
translocation domain, a poly-Arg sequence, and the like. In
embodiments, the cellular uptake moiety peptide is a continuous
amino acid sequence from transduction domain. In other embodiments,
the cellular uptake moiety peptide is two or more amino acid
sequences from one or more known transduction domains that are not
naturally present in a contiguous amino acid sequence, for example,
a cellular uptake domain comprising two amino acid sequences would
be separated by a third amino acid sequence in nature.
[0073] In embodiments, the cellular uptake moiety peptide is an
optionally modified transduction domain from a known peptide. The
modifications may be made using known techniques.
[0074] In embodiments, the cellular uptake moiety peptide is an
optionally modified TAT translocation domain. The optionally
modified TAT translocation domain can have 0 to 1 modifications, 0
to 2 modifications, 0 to 3 modifications, 0 to 4 modifications, 0
to 5 modifications, 0 to 6 modifications, 0 to 7 modifications, 0
to 8 modifications, 0 to 9 modifications, 1 to 2 modifications, 1
to 3 modifications, 1 to 4 modifications, 1 to 5 modifications, 1
to 6 modifications, 1 to 7 modifications, 1 to 8 modifications, 1
to 9 modifications, 2 to 3 modifications, 2 to 4 modifications, 2
to 5 modifications, 2 to 6 modifications, 2 to 7 modifications, 2
to 8 modifications, 2 to 9 modifications, 3 to 4 modifications, 3
to 5 modifications, 3 to 6 modifications, 3 to 7 modifications, 3
to 8 modifications, 3 to 9 modifications, 4 to 5 modifications, 4
to 6 modifications, 4 to 7 modifications, 4 to 8 modifications, or
4 to 9 modifications. In embodiments where the cellular uptake
moiety peptide is a fragment of the TAT translocation domain, the
cellular uptake moiety peptide sequence can have a minimum length
of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9
amino acids, or 10 amino acids. In some embodiments where the
cellular uptake moiety peptide is a fragment of the TAT
translocation domain, the cellular uptake moiety peptide sequence
can have a minimum of 5 contiguous amino acids, 6 contiguous amino
acids, 7 contiguous amino acids, 8 contiguous amino acids, 9
contiguous amino acids, or 10 contiguous amino acids of a TAT
translocation domain. Modified TAT translocation domains disclosed
herein include amino acid sequences with at least 70% sequence
identity, at least 75% sequence identity, at least 80% sequence
identity, at least 85% sequence, at least 90% sequence identity, at
least 95% sequence identity, at least 96% sequence identity, at
least 97% sequence identity, at least 98% sequence identity, or at
least 99% sequence identity to the sequence of YGRKKRRQRRR (SEQ ID
NO:12). In some embodiments, the cellular uptake moiety is a
modified TAT translocation domain. In other embodiments, the
cellular uptake moiety peptide is a TAT translocation domain having
the sequence SEQ ID NO: 12.
[0075] In embodiments, the profiling peptide of the present
disclosure comprises a TAT translocation domain and an Mcl-1
binding domain having a sequence of SEQ ID NOS:1-11 with 0-8
modifications. In some embodiments, the profiling peptide of the
present disclosure comprises a TAT translocation domain and an
Mcl-1 binding domain having a sequence of SEQ ID NOS:1-11 with 1-8
modifications. In embodiments, the profiling peptide of the present
disclosure comprises a TAT translocation domain and an Mcl-1
binding domain having SEQ ID NO:1 with 0-8 modifications. In some
embodiments, the profiling peptide of the present disclosure
comprises a TAT translocation domain and an Mcl-1 binding domain
having any one of SEQ ID NOS:1-11. In some embodiments, the
profiling peptide of the present disclosure comprises a TAT
translocation domain having SEQ ID NO:12 and an Mcl-1 binding
domain having any one of SEQ ID NOS:1-11 with 0-8 modifications. In
some embodiments, the profiling peptide of the present disclosure
comprises a TAT translocation domain having SEQ ID NO:12 and an
Mcl-1 binding domain having any one of SEQ ID NOS:1-11 with 1-8
modifications. In embodiments, the profiling peptide of the present
disclosure comprises a TAT translocation domain having SEQ ID NO:12
and an Mcl-1 binding domain having SEQ ID NO:1 with 0-8
modifications. In some embodiments, the profiling peptide of the
present disclosure comprises a TAT translocation domain having SEQ
ID NO:12 and an Mcl-1 binding domain having any one of SEQ ID
NOS:1-11.
[0076] In embodiments, the cellular uptake moiety peptide is an
optionally modified ANT translocation domain. The optionally
modified ANT translocation domain can have 0 to 1 modifications, 0
to 2 modifications, 0 to 3 modifications, 0 to 4 modifications, 0
to 5 modifications, 0 to 6 modifications, 0 to 7 modifications, 0
to 8 modifications, 0 to 9 modifications, 1 to 2 modifications, 1
to 3 modifications, 1 to 4 modifications, 1 to 5 modifications, 1
to 6 modifications, 1 to 7 modifications, 1 to 8 modifications, 1
to 9 modifications, 2 to 3 modifications, 2 to 4 modifications, 2
to 5 modifications, 2 to 6 modifications, 2 to 7 modifications, 2
to 8 modifications, 2 to 9 modifications, 3 to 4 modifications, 3
to 5 modifications, 3 to 6 modifications, 3 to 7 modifications, 3
to 8 modifications, 3 to 9 modifications, 4 to 5 modifications, 4
to 6 modifications, 4 to 7 modifications, 4 to 8 modifications, or
4 to 9 modifications. In embodiments where the cellular uptake
moiety peptide is a fragment of the ANT translocation domain, the
cellular uptake moiety peptide sequence can have a minimum length
of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9
amino acids, or 10 amino acids. In some embodiments where the
cellular uptake moiety peptide is a fragment of the ANT
translocation domain, the cellular uptake moiety peptide sequence
can have a minimum of 5 contiguous amino acids, 6 contiguous amino
acids, 7 contiguous amino acids, 8 contiguous amino acids, 9
contiguous amino acids, or 10 contiguous amino acids of an ANT
translocation domain. Modified ANT translocation domains disclosed
herein include amino acid sequences with at least 70% sequence
identity, at least 75% sequence identity, at least 80% sequence
identity, at least 85% sequence, at least 90% sequence identity, at
least 95% sequence identity, at least 96% sequence identity, at
least 97% sequence identity, at least 98% sequence identity, or at
least 99% sequence identity to the sequence of RQIKIWFQNRRMKWKK
(SEQ ID NO:13). In some embodiments, the cellular uptake moiety
peptide is a modified ANT translocation domain. In other
embodiments, the cellular uptake moiety peptide is an ANT
translocation domain having the sequence SEQ ID NO: 13.
[0077] In embodiments, the profiling peptide of the present
disclosure comprises an ANT translocation domain and an Mcl-1
binding domain having any one of SEQ ID NOS:1-11 with 0-8
modifications. In some embodiments, the profiling peptide of the
present disclosure comprises an ANT translocation domain and an
Mcl-1 binding domain having any one of SEQ ID NOS:1-11 with 1-8
modifications. In embodiments, the profiling peptide of the present
disclosure comprises an ANT translocation domain and an Mcl-1
binding domain having SEQ ID NO:1 with 0-8 modifications. In
embodiments, the profiling peptide of the present disclosure
comprises an ANT translocation domain and an Mcl-1 binding domain
having any one of SEQ ID NOS:1-11. In some embodiments, the
profiling peptide of the present disclosure comprises an ANT
translocation domain having SEQ ID NO:13 and an Mcl-1 binding
domain having any one of SEQ ID NOS:1-11 with 0-8 modifications. In
some embodiments, the profiling peptide of the present disclosure
comprises an ANT translocation domain having SEQ ID NO:13 and an
Mcl-1 binding domain having any one of SEQ ID NOS:1-11 with 1-8
modifications. In embodiments, the profiling peptide of the present
disclosure comprises an ANT translocation domain having SEQ ID
NO:13 and an Mcl-1 binding domain having SEQ ID NO:1 with 0-8
modifications. In some embodiments, the profiling peptide of the
present disclosure comprises an ANT translocation domain having SEQ
ID NO:13 and an Mcl-1 binding domain having any one of SEQ ID NOS:
1-11.
[0078] In embodiments, the cellular uptake moiety is an arginine
rich amino acid sequence, such as a poly-Arg sequence. In some
embodiments, the arginine rich amino acid sequence includes 3 to 9
Arg residues, 3 to 10 Arg residues, 3 to 11 Arg residues, 3 to 12
Arg residues, 4 to 9 Arg residues, 4 to 10 Arg residues, 4 to 11
Arg residues, 4 to 12 Arg residues, 5 to 9 Arg residues, 5 to 10
Arg residues, 5 to 11 Arg residues, 5 to 12 Arg residues, 6 to 9
Arg residues, 6 to 10 Arg residues, 6 to 11 Arg residues, 6 to 12
Arg residues, 7 to 9 Arg residues, 7 to 10 Arg residues, 7 to 11
Arg residues, 7 to 12 Arg residues, 8 to 9 Arg residues, 8 to 10
Arg residues, 8 to 11 Arg residues, 8 to 12 Arg residues, 9 to 10
Arg residues, 9 to 11 Arg residues, or 9 to 12 Arg residues. In
some embodiments, the poly-Arg sequence includes 3 Arg residues, 4
Arg residues, 5 Arg residues, 6 Arg residues, 7 Arg residues, 8 Arg
residues, 9 Arg residues, 10 Arg residues, 11 Arg residues, or 12
Arg residues. In some embodiments, the poly-Arg sequence includes 3
to 9 contiguous Arg residues, 3 to 10 contiguous Arg residues, 3 to
11 contiguous Arg residues, 3 to 12 contiguous Arg residues, 4 to 9
contiguous Arg residues, 4 to 10 contiguous Arg residues, 4 to 11
contiguous Arg residues, 4 to 12 contiguous Arg residues, 5 to 9
contiguous Arg residues, 5 to 10 contiguous Arg residues, 5 to 11
contiguous Arg residues, 5 to 12 contiguous Arg residues, 6 to 9
contiguous Arg residues, 6 to 10 contiguous Arg residues, 6 to 11
contiguous Arg residues, 6 to 12 contiguous Arg residues, 7 to 9
contiguous Arg residues, 7 to 10 contiguous Arg residues, 7 to 11
contiguous Arg residues, 7 to 12 contiguous Arg residues, 8 to 9
contiguous Arg residues, 8 to 10 contiguous Arg residues, 8 to 11
contiguous Arg residues, 8 to 12 contiguous Arg residues, 9 to 10
contiguous Arg residues, 9 to 11 contiguous Arg residues, or 9 to
12 contiguous Arg residues.
[0079] In embodiments, the profiling peptide of the present
disclosure comprises an arginine rich amino sequence and an Mcl-1
binding domain having any one of SEQ ID NOS:1-11 with 0-8
modifications. In some embodiments, the profiling peptide of the
present disclosure comprises an arginine rich amino sequence and an
Mcl-1 binding domain having any one of SEQ ID NOS:1-11 with 1-8
modifications. In embodiments, the profiling peptide of the present
disclosure comprises an arginine rich amino acid sequence and an
Mcl-1 binding domain having SEQ ID NO:1 with 0-8 modifications. In
embodiments, the profiling peptide of the present disclosure
comprises an arginine rich amino sequence and an Mcl-1 binding
domain having any one of SEQ ID NOS:1-11. In some embodiments, the
profiling peptide of the present disclosure comprises a poly-Arg
sequence having 3 to 10 contiguous Arg residues and an Mcl-1
binding domain having any one of SEQ ID NOS:1-11 with 0-8
modifications. In some embodiments, the profiling peptide of the
present disclosure comprises a poly-Arg sequence having 3 to 10
contiguous Arg residues and an Mcl-1 binding domain having any one
of SEQ ID NOS:1-11 with 1-8 modifications. In embodiments, the
profiling peptide of the present disclosure comprises a poly-Arg
sequence having 3 to 10 Arg residues and an Mcl-1 binding domain
having SEQ ID NO: 1 with 0-8 modifications. In some embodiments,
the profiling peptide of the present disclosure comprises a
poly-Arg sequence having 3 to 10 contiguous Arg residues and an
Mcl-1 binding domain having any one of SEQ ID NOS:1-11.
[0080] In embodiments, a modified cellular uptake moiety peptide
retains the ability of the unmodified sequence to cross the cell
membrane when conjugated to a peptide (i.e., the modifications to
the cellular uptake moiety peptide do not alter the ability to
cross the cell membrane when conjugated to a peptide in a
statistically significant, clinically significant, or biologically
significant manner). In some embodiments, a modified cellular
uptake moiety peptide retains the ability of the unmodified
sequence to cross the cell membrane when conjugated to a peptide if
the internalization efficiency of the modified cellular uptake
moiety peptide is at least 70%, 80%, 85%, 90%, 95%, 97%, or 99% of
the internalization efficiency of the unmodified sequence.
[0081] Alternatively, the cellular uptake moiety can be a chemical
compound. Chemical compounds that facilitate cellular
internalization are understood by one of skill in the art, and
include, for example, cholesterol moieties, octanoic acid,
lithocholic acid, oleyl alcohol, lithocholic acid oleylamide, and
decanoic acid.
[0082] The Mcl-1 binding domain and the cellular uptake moiety can
be linked by chemical coupling in any suitable manner known in the
art. The cellular uptake moiety may be linked to the Mcl-1 binding
domain at any suitable location, for example the N-terminus or the
C-terminus of the Mcl-1 binding domain, either directly or via a
linker. In embodiments, the cellular uptake moiety is conjugated to
the Mcl-1 binding domain via a linker. In some embodiments, the
cellular uptake moiety is conjugated to the N-terminus of the Mcl-1
binding domain. In further embodiments, the cellular uptake moiety
is conjugated via a linker to the N-terminus of the Mcl-1 binding
domain. In other embodiments, the cellular uptake moiety is
conjugated to the C-terminus of the Mcl-1 binding domain. In still
further embodiments, the cellular uptake moiety is conjugated via a
linker to the C-terminus of the Mcl-1 binding domain.
[0083] Suitable linkers include peptide sequences of any length and
other chemical linkers as would be understood by one of ordinary
skill. Short peptide sequences are employed in certain embodiments,
for example peptide sequences including uncharged amino acids,
non-polar amino acids and/or small amino acids. In some
embodiments, a linker is an amino acid sequence of 1-5 amino acids.
For example some exemplary linkers include Gly, Pro, Ala, Val, Leu,
Met, Ile, and/or Phe amino acids. Other examples of suitable
peptide sequences include two Pro residues, three Gly residues, and
the like. In some embodiments, the cellular uptake moiety is linked
to the Mcl-1 binding domain in such a way that the cellular uptake
moiety is cleaved upon or after entry into the cell. In certain
embodiments, the linker comprises three Gly residues, for example
GGG.
[0084] Embodiments of the profiling peptides of the present
disclosure may be 20 to 40 amino acids in length, 20 to 45 amino
acids in length, 20 to 50 amino acids in length, 25 to 40 amino
acids in length, 25 to 45 amino acids in length, 25 to 50 amino
acids in length, 30 to 36 amino acids in length, 30 to 37 amino
acids in length, 30 to 38 amino acids in length, 30 to 39 amino
acids in length, 30 to 40 amino acids in length, 30 to 45 amino
acids in length, 30 to 50 amino acids in length, 31 to 36 amino
acids in length, 31 to 37 amino acids in length, 31 to 38 amino
acids in length, 31 to 39 amino acids in length, 31 to 40 amino
acids in length, 32 to 36 amino acids in length, 32 to 37 amino
acids in length, 32 to 38 amino acids in length, 32 to 39 amino
acids in length, 32 to 40 amino acids in length, 33 to 36 amino
acids in length, 33 to 37 amino acids in length, 33 to 38 amino
acids in length, 33 to 39 amino acids in length, 33 to 40 amino
acids in length, 34 to 36 amino acids in length, 34 to 37 amino
acids in length, 34 to 38 amino acids in length, 34 to 39 amino
acids in length, 34 to 40 amino acids in length, 35 to 36 amino
acids in length, 35 to 37 amino acids in length, 35 to 38 amino
acids in length, 35 to 39 amino acids in length, 35 to 40 amino
acids in length, 35 to 45 amino acids in length, 35 to 50 amino
acids in length, 36 to 37 amino acids in length, 36 to 38 amino
acids in length, 36 to 39 amino acids in length, 36 to 40 amino
acids in length, 37 to 38 amino acids in length, 37 to 39 amino
acids in length, 37 to 40 amino acids in length, 38 to 39 amino
acids in length, 38 to 40 amino acids in length, or 39 to 40 amino
acids in length.
[0085] In embodiments, a profiling peptide comprises a cellular
uptake moiety, and an Mcl-1 binding domain having any one of SEQ ID
NOS:1-11 with 0-8 modifications. In embodiments, a profiling
peptide comprises a cellular uptake moiety, and an Mcl-1 binding
domain having any one of SEQ ID NOS:1-11 with 1-8 modifications. In
embodiments, a profiling peptide comprises a cellular uptake
moiety, and an Mcl-1 binding domain having SEQ ID NO:1 with 0-8
modifications. In embodiments, a profiling peptide comprises a
cellular uptake moiety, and an Mcl-1 binding domain having any one
of SEQ ID NOS:1-11. In some embodiments, a profiling peptide
comprises a cellular uptake moiety, and an Mcl-1 binding domain
having SEQ ID NO:1.
[0086] In embodiments, a profiling peptide comprises a cellular
uptake moiety having SEQ ID NO:12 conjugated to an Mcl-1 binding
domain having any one of SEQ ID NOS:1-11 with 0-8 modifications by
a linker. In embodiments, a profiling peptide comprises a cellular
uptake moiety having SEQ ID NO: 12 conjugated to an Mcl-1 binding
domain having any one of SEQ ID NOS:1-11 with 1-8 modifications by
a linker. In embodiments, a profiling peptide comprises a cellular
uptake moiety of SEQ ID NO:12 conjugated to an Mcl-1 binding domain
having SEQ ID NO:1 with 0-8 modifications by a linker. In
embodiments, a profiling peptide comprises a cellular uptake moiety
having SEQ ID NO:12 conjugated to an Mcl-1 binding domain having
any one of SEQ ID NOS: 1-11 by a linker.
[0087] In certain embodiments, the profiling peptide has the
sequence of YGRKKRRQRRRGGGRPEIWMTQGLRRLGDEINAYYAR (SEQ ID NO:14).
In other embodiments, the profiling peptide has the sequence of
RPEIWMTQGLRRLGDEINAYYARGGGYGRKKRRQRRR (SEQ ID NO: 15).
[0088] Modified profiling peptides may be synthesized and purified
by standard chemical methods. Peptides may be chemically
synthesized by manual techniques or by automated procedures.
Equipment for automated synthesis of peptides is commercially
available from suppliers such as Perkin-Elmer, Inc. (Waltham,
Mass.) and may be operated according to the manufacturer's
instructions. Additionally, synthesized profiling peptides may be
obtained from any number of different custom peptide synthesizing
manufacturers. If required, synthesized peptides may be purified
using preparative reverse phase chromatography, partition
chromatography, gel filtration, gel electrophoresis, ion-exchange
chromatography, or other methods used in the art.
[0089] Alternatively, modified profiling peptides may be readily
prepared by genetic engineering and recombinant molecular biology
methods and techniques. For example, polynucleotides encoding
modified profiling peptides, or fragments thereof, may be
constructed by recombinant methods or chemically synthesized (using
such devices as an automatic synthesizer). Methods for purifying
polynucleotides after either chemical synthesis or recombinant
synthesis are known to persons skilled in the art. The constructed
or synthesized polynucleotides may be incorporated into expression
vectors (e.g., a plasmid, a viral particle, or a phage) for
production of the profiling peptide in a host cell into which the
expression vector has been introduced. Polynucleotides that encode
a profiling peptide described herein may be recombinantly expressed
in a variety of different host cells. Host cells may then be
genetically engineered (transduced, transformed, or transfected)
with the expression vectors. Selection and maintenance of culture
conditions for particular host cells, such as temperature, pH and
the like, will be readily apparent to the ordinarily skilled
artisan. The produced peptides may then be harvested and purified
using methods known in the art.
Compositions
[0090] Also disclosed herein are compositions comprising a
profiling peptide as described herein and a carrier. Suitable
carriers include those that maintain the stability and integrity of
the profiling peptide. Carriers may be a diluent, excipient,
preservative, or solvent.
[0091] In embodiments, more than one profiling peptide may be
included in a composition. In such embodiments, at least two,
three, four, five, six, seven, eight, nine, or ten profiling
peptides are included.
[0092] In further embodiments, the compositions disclosed herein
further comprise a detecting agent. The detecting agent can be any
suitable agent, such as a fluorescent dye, a non-fluorescent dye
(e.g., a non-fluorescent dye that can be converted to a fluorescent
dye), an antibody, and the like. Fluorescent dyes include, for
example,
5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide
(JC-1), propidium iodide (PI),
1,1',3,3,3',3'-hexamethylindodicarbo-cyanine iodide (DilC1), and
3,3'-Dihexyloxacarbocyanine Iodide (DiOC.sub.6). In various
embodiments, the fluorescent dye is a potentiometric dye.
"Potentiometric dyes" are dyes that change properties, for example,
fluoresce, in response to voltage changes. Suitable potentiometric
dyes include, for example, DilC1, JC-1, and rhodamine 123. In
embodiments, the potentiometric dye included is JC-1 or rhodamine
123. In other embodiments, the dye is dihydrorhodamine 123, a
non-fluorescent dye that can be converted via oxidation to
rhodamine 123, a fluorescent dye.
[0093] In certain embodiments, the compositions described herein do
not include a cell permeabilization agent, such as digitonin. A
"cell permeabilization agent" is any reagent that breaks down the
outer cell membrane, such that access is provided to the
intracellular area, including the organelles. Two types of reagents
are commonly used as cell permeabilization agents: (1) organic
solvents, such as methanol and acetone, and (2) detergents such as
a saponin, Triton X-100, and Tween-20. Generally, organic solvents
permeabilize the outer cell membrane by dissolving lipids in the
membranes leaving holes. Detergents generally create pores in the
outer membrane, such as by interacting with and selectively
removing membrane cholesterol.
[0094] In some embodiments, the compositions described herein
further comprise a whole cell. In embodiments, the whole cell is a
cancer cell. In certain embodiments, the cancer cell is from a
human tumor-derived cell line. In certain embodiments, the cancer
cell is a cancer stem cell. In some embodiments, the cancer cell is
isolated from a tumor. In certain embodiments, the cancer cell is
derived from the biopsy of a non-solid tumor. In embodiments, the
cancer cell is obtained from peripheral blood from the subject. In
other embodiments the cancer cell is obtained from bone marrow of
the subject.
[0095] In specific embodiments, the cancer cell is derived from the
biopsy of a subject with multiple myeloma, AML, acute lymphocytic
leukemia, chronic lymphogenous leukemia, mantle cell lymphoma,
diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma. In some
embodiments, the cancer cell is derived from a hematologic cancer,
including, for example, multiple myeloma, myelodysplastic syndrome
(MDS), AML, ALL, acute lymphocytic leukemia, chronic lymphogenous
leukemia, CLL, mantle cell lymphoma, diffuse large B-cell lymphoma,
follicular lymphoma, or non-Hodgkin's lymphoma. In certain
embodiments, the cancer is AML.
[0096] In some embodiments, the cancer cell is derived from a solid
tumor. In embodiments, the cancer cell is derived from the biopsy
of a solid tumor, such as, for example, a biopsy of a colorectal,
breast, prostate, lung, pancreatic, renal, or ovarian primary
tumor. In various embodiments, the cancer cell is isolated from a
pre-metastatic cancer, or a metastatic cancer.
[0097] Compositions of the present disclosure include a therapeutic
composition for use in the treatment of cancer in a subject with a
Mcl-1 dependency percentage of at least 15%, the Mcl-1 dependency
percentage having been obtained by an in vitro method comprising:
contacting a first portion of a plurality of cancer cells with a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain, the Mcl-1 binding domain having the sequence of SEQ
ID NO:1 with 0-8 modifications, or a composition comprising a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain, the Mcl-1 binding domain having the sequence of SEQ
ID NO:1 with 0-8 modifications and a carrier. Compositions of the
present disclosure also include a therapeutic composition for
cancer comprising a therapeutic agent, which is administered to a
subject having a Mcl-1 dependency percentage of at least 15%,
wherein, the Mcl-1 dependency percentage is obtained by an in vitro
method comprising: contacting a first position of plurality of
cancer cells with a profiling peptide comprising a cellular uptake
moiety and an Mcl-1 binding domain, the Mcl-1 binding domain having
the sequence of SEQ ID NO:1 with 0-8 modifications, or a
composition comprising a profiling peptide comprising a cellular
uptake moiety and an Mcl-1 binding domain, the Mcl-1 binding domain
having the sequence of SEQ ID NO: 1 with 0-8 modifications and a
carrier.
Kits
[0098] The present disclosure further provides for kits comprising
a profiling peptide as described herein, and a detecting agent. The
detecting agent included in a kit of the disclosure can be any
suitable agent, such as a fluorescent dye, a non-fluorescent dye
that can be converted to a fluorescent dye, an antibody, and the
like. Fluorescent dyes include, for example, 5,5',
6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide
(JC-1), propidium iodide (PI),
1,1',3,3,3',3'-hexamethylindodicarbo-cyanine iodide (DilC1), and
3,3'-Dihexyloxacarbocyanine Iodide (DiOC.sub.6). In various
embodiments, the fluorescent dye is a potentiometric dye. Suitable
potentiometric dyes include, for example, DilC1, JC-1, and
rhodamine 123. In embodiments, the potentiometric dye included is
JC-1 or rhodamine 123. In other embodiments, the dye is
dihydrorhodamine 123, a non-fluorescent dye that can be converted
via oxidation to rhodamine 123, a fluorescent dye.
[0099] In embodiments, more than one profiling peptide may be
included in a kit. In such embodiments, at least two, three, four,
five, six, seven, eight, nine, or ten profiling peptides are
included. In various embodiments in which more than one profiling
peptide is provided, the sequencing of use and/or instructions for
use of combinations of the profiling peptides can be included in
the kit.
[0100] The kits can further comprise written instructions for using
the kit in the methods disclosed herein. In various embodiments,
the written instructions may include instructions regarding
preparation of the profiling peptide and/or detecting agent;
appropriate reference levels to interpret results associated with
using the kit; proper disposal of the related waste; and the like.
The written instructions can be in the form of printed instructions
provided within the kit, or the written instructions can be printed
on a portion of the container housing the kit. Written instructions
may be in the form of a sheet, pamphlet, brochure, CD-Rom, or
computer-readable device, or can provide directions to locate
instructions at a remote location, such as a website. The written
instructions may be in English and/or in a national or regional
language.
[0101] Such kits can further comprise one or more reagents, assay
controls, or other supplies necessary for evaluation of a sample,
such as welled plates, syringes, ampules, vials, tubes, tubing,
facemask, a needleless fluid transfer device, an injection cap,
sponges, sterile adhesive strips, Chloraprep, gloves, and the like.
In certain embodiments, the kits described herein do not include a
cell permeabilization agent, such as digitonin. Variations in
contents of any of the kits described herein can be made. In
various embodiments, the profiling peptide and detecting agent,
optionally with one or more reagents or supplies, are combined into
a compact container, optionally with written instructions for
use.
[0102] In some embodiments, a kit of the present disclosure
comprises a detecting agent and a profiling peptide comprising a
cellular uptake moiety and an Mcl-1 binding domain having any one
of SEQ ID NOS:1-11 with 0-8 modifications. In some embodiments, a
kit of the present disclosure comprises a detecting agent and a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain having any one of SEQ ID NOS:1-11 with 1-8
modifications. In some embodiments, a kit of the present disclosure
comprises a detecting agent and a profiling peptide comprising a
cellular uptake moiety, and an Mcl-1 binding domain having SEQ ID
NO:1 with 0-8 modifications. In some embodiments, a kit of the
present disclosure comprises a detecting agent and a profiling
peptide comprising a cellular uptake moiety and an Mcl-1 binding
domain having any one of SEQ ID NOS:1-11. In any of the above
embodiments, the cellular uptake moiety may be a TAT translocation
domain or an ANT translocation domain. In some embodiments, the
cellular uptake moiety is conjugated to the Mcl-1 binding domain
via a linker. In some embodiments, a kit of the present disclosure
comprises a detecting agent and a profiling peptide with the
sequence of (SEQ ID NO: 14). In some embodiments, a kit of the
present disclosure comprises a detecting agent and a profiling
peptide with the sequence of (SEQ ID NO: 15). In any of the above
embodiments, the kit may not include a cell permeabilization agent.
In any of the above embodiments, the detecting agent may be a
potentiometric dye.
Methods of Use
[0103] Also described herein are methods of profiling a cancer cell
from a subject. Some embodiments comprise contacting a cancer cell
with a profiling peptide. The profiling peptide may be any of those
known in the art (e.g., NOXA) or any of the profiling peptides
disclosed herein. Such methods include methods of producing a
sensitivity profile for a cancer cell or a plurality of cancer
cells. In some embodiments, methods of producing a sensitivity
profile for a cancer cell from a subject includes isolating a
cancer cell or a plurality of cancer cells from a subject. In
certain embodiments, the cancer cell is from a human tumor-derived
cell line. In certain embodiments, the cancer cell is a cancer stem
cell. In some embodiments, the cancer cell is isolated from a
tumor. In certain embodiments, the cancer cell is derived from the
biopsy of a non-solid tumor. In embodiments, the cancer cell is
obtained from peripheral blood from the subject. In other
embodiments the cancer cell is obtained from bone marrow of the
subject.
[0104] In specific embodiments, the cancer cell is derived from the
biopsy of a subject with multiple myeloma, AML, acute lymphocytic
leukemia, chronic lymphogenous leukemia, mantle cell lymphoma,
diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma. In some
embodiments, the cancer cell is derived from a hematologic cancer,
including, for example, multiple myeloma, MDS, AML, ALL, acute
lymphocytic leukemia, chronic lymphogenous leukemia, CLL, mantle
cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma,
or non-Hodgkin's lymphoma. In certain embodiments, the cancer is
AML.
[0105] In some embodiments, the cancer cell is derived from a solid
tumor. In embodiments, the cancer cell is derived from the biopsy
of a solid tumor, such as, for example, a biopsy of a colorectal,
breast, prostate, lung, pancreatic, renal, or ovarian primary
tumor. In various embodiments, the cancer cell is isolated from a
pre-metastatic cancer, or a metastatic cancer. In some embodiments,
the cancer cell is a circulating tumor cell.
[0106] In a specific embodiment, the cancer cell is a multiple
myeloma cell that is enriched by selection from a biopsy sample
with an anti-CD138 antibody bound to a solid matrix or bead. In a
specific embodiment, the cancer cell is an AML cell that is
enriched by binding to a CD45-directed antibody. In a specific
embodiment, the cancer cell is from a chronic lymphogenous leukemia
or diffuse large B-cell lymphoma that is enriched by non-B cell
depletion.
[0107] In various embodiments, the plurality of cancer cells is
from a sample that has been frozen. In other embodiments, the
plurality of cancer cells is from a sample that has not been
frozen, i.e., that has been freshly collected.
[0108] Methods of profiling a cancer cell or a plurality of cancer
cells may include contacting the plurality of cancer cells with one
or more labels. In some embodiments that use flow cytometry, the
labels are fluorophores attached to antibodies or a chemical entity
with affinity for a cell membrane feature or other cellular
structure. In other embodiments that use flow cytometry, the labels
are quantum dots attached to antibodies or a chemical entity with
affinity for a cell membrane feature or other cellular structure.
In any of these embodiments, the antibodies or chemical entities
may recognize any suitable cell surface marker, such as CD3, CD13,
CD20, CD33, CD34, or CD45. In various embodiments, a combination of
labels is used.
[0109] Methods of profiling the cancer cell from the subject may
comprise contacting the cancer cell with one or more profiling
peptides disclosed herein and detecting a change in mitochondrial
integrity of the cancer cell. In various embodiments, at least two,
three, four, five, six, seven, eight, or nine profiling peptides
may be used at once. In such embodiments, a panel of profiling
peptides may be screened on a single subject specimen.
[0110] A change in mitochondrial integrity can be detected in any
suitable manner, such as, for example, a change in mitochondrial
membrane potential, chromatin condensation, loss of viability,
Cytochrome C translocation from the mitochondrial intermembrane
space to the cytosol, swelling of the mitochondria, mitochondrial
fission, morphological changes (e.g., cell shrinkage, membrane
blebbing, etc.), phosphatidyl serine externalization (e.g., as
measured by annexin V staining) or the increase in reactive oxygen
intermediates. As is understood by one of skill in the art, various
methods of detection for each of the indications of a change in
mitochondrial integrity may be employed. For example, a change in
mitochondrial membrane potential may be measured using
potentiometric dyes, such as, for example, DilC1, JC-1, or
rhodamine 123. In one embodiment, the potentiometric dye is JC-1 or
rhodamine 123. In another example, Cytochrome C translocation can
be measured using immunofluorescence staining. In a further
example, an increase in reactive oxygen intermediates can be
measured flow cytometric analysis after staining with
carboxy-dichlorofluorescin diacetate.
[0111] In embodiments, the change in mitochondrial integrity will
be a decrease in mitochondrial integrity. In some embodiments, the
decrease in mitochondrial integrity is measured by a decrease in
mitochondrial membrane potential. The decrease in mitochondrial
potential may be determined using any suitable method known in the
art, such as using a potentiometric dye. (e.g., JC-1 or rhodamine
123). In some embodiments, the decrease in mitochondrial integrity
is measured by Cytochrome C leakage. In some embodiments, the
decrease will be a statistically significant, clinically
significant, or biologically significant decrease. In some
embodiments, the decrease is a 2%, 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% difference in a
measurement of mitochondrial integrity, as described herein, as
compared to a control.
[0112] In various embodiments, the plurality of cancer cells is
divided into three portions for the purposes of profiling. In such
embodiments, one portion may be treated with a negative control,
one may be contacted with a positive control, and one may be
contacted with one or more profiling peptides or a composition
comprising one or more profiling peptides disclosed herein.
[0113] Any suitable positive control may be used. Examples of
positive controls include Carbonyl
cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), Carbonyl
cyanide m-chlorophenyl hydrazone (CCCP),
N5,N6-bis(2-fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine
(BAM-15), and the like. In particular embodiments, the positive
control used is CCCP. Any suitable negative control may be used.
Examples of negative controls include water and water soluble
organic solvents, such as DMSO, ethanol, and methanol.
[0114] In some embodiments, the plurality of cancer cells are then
contacted with a fluorescent dye, as described above. In particular
embodiments, the dye is JC-1 or DiOC.sub.6. In such embodiments,
the plurality of cancer cells may then be analyzed using flow
cytometry. Any suitable gating may be used in flow cytometry
analysis. In some embodiments, such gating is CD45 dim, CD13, CD33,
and CD34 high population. In other embodiments, such gating is the
CD34 dim, CD3 and CD20 high population. Accordingly, embodiments of
the present disclosure include a method of producing a sensitivity
profile for a plurality of cancer cells from a subject, the method
comprising: isolating the plurality of cancer cells from a sample,
contacting the plurality of cancer cells with a label, treating a
first portion of the plurality of cancer cells with a negative
control, treating a second portion of the plurality of cancer cells
with a positive control, treating a third portion of the plurality
of cancer cells with a profiling peptide comprising a cellular
uptake moiety and an Mcl-1 binding domain, the Mcl-1 binding domain
having the sequence of SEQ ID NO:1 with 0-8 modifications, or a
composition comprising a profiling peptide comprising a cellular
uptake moiety and an Mcl-1 binding domain, the Mcl-1 binding domain
having the sequence of SEQ ID NO:1 with 0-8 modifications and a
carrier, contacting the first portion, the second portion, and the
third portion of the plurality of cancer cells with a dye and
analyzing the first portion, the second portion, and the third
portion of the plurality of cancer cells by flow cytometry.
[0115] In some embodiments, an additive with a high affinity for
calcium channels is added to the plurality of cancer cells. In some
such embodiments, the additive is a diterpenoid. In particular
embodiments, the additive is ryanodine. In embodiments, the
additive is added in a concentration that is sufficient to
significantly reduce or prevent nonspecific dye uptake. In some
embodiments, the additive is added in a concentration of at least
20 nM. In some embodiments, the additive is added in a
concentration of at least 30 nM.
[0116] Methods of the disclosure include isolating a plurality of
cancer cells from a subject sample; the cells are then labeled;
treated with a negative control, a positive control, or a profiling
peptide of the disclosure; contacted with a dye; and analyzed with
flow cytometry.
[0117] In an illustrative method of the disclosure, a plurality of
cancer cells are isolated from a subject sample, and sample quality
is confirmed. The cells are then pelleted, blocked in BSA, and
labeled. After staining, cells are pelleted and separated into
three portions and treated with either water or dimethyl sulfoxide
(DMSO) (negative control), CCCP (positive control) or a profiling
peptide of the disclosure (subject dependency). DiOC.sub.6, a
cationic mitochondrial dye is added. Later, the cells are analyzed
via flow cytometry.
[0118] In some embodiments, a plurality of cancer cells are
isolated from primary bone marrow aspirates and sample quality is
determined. Cells are then pelleted, blocked in BSA and labeled for
markers specific to B and T cells, as well as monocyte
differentiation markers and blast-specific markers. After staining,
cells are pelleted and separated into three portions and treated
with either water (negative control), CCCP (positive control) or
SEQ ID NO: 14 (subject dependency). DiOC.sub.6, a cationic
mitochondrial dye is added. The cells are analyzed via flow
cytometry. Blast cells are isolated by gating on the CD45 dim,
CD13, CD33, and CD34 high population of each sample.
[0119] In particular embodiments, a plurality of cancer cells are
isolated from primary bone marrow aspirates using density-gradient
centrifugation. Sample quality is determined using trypan blue
exclusion. Cells are then pelleted, blocked in BSA and labeled for
markers specific to B and T cells, as well as monocyte
differentiation markers and blast-specific markers. After staining,
cells are pelleted and separated into fluorescent-activated cell
sorting (FACS) tubes and treated with either water (negative
control), CCCP (positive control) or SEQ ID NO: 14 (subject
dependency). DiOC.sub.6, a cationic mitochondrial dye is added. The
cells are then analyzed via flow cytometry. Blast cells are
isolated by gating on the CD45 dim, CD13, CD33 and CD34 high
population of each sample.
[0120] Some methods described herein further comprise determining
an Mcl-1 dependency percentage for the first portion of the
plurality of cancer cells based at least on the change in
mitochondrial integrity.
[0121] In embodiments, the Mcl-1 dependency percentage (also
referred to as Mcl-1 priming percentage; PP) is defined by the
following equation:
PP = [ 1 - ( Pep - PC NC - PC ) ] * 100 ##EQU00001##
[0122] Where PC is the AUC of the positive control, NC is the AUC
of the negative control, and Pep is the AUC of the profiling
peptide. Unless otherwise noted, the Mcl-1 dependency percentages
calculated herein correspond to a profiling peptide concentration
of 1 M with CCCP as the positive control and water or DMSO as the
negative control. The AUC is either area under the curve or signal
intensity. In embodiments, the AUC is the median fluorescent
intensity (MFI). In some embodiments, the area under the curve is
established by homogenous time-resolved fluorescence (HTRF).
[0123] In some embodiments, the time occurs over a window from
between about 0 to about 300 min to about 0 to about 30 min. In
some embodiments, the area under the curve is established by
fluorescence activated cell sorting (FACS) or microplate assay as
known in the art or described herein. In some embodiments, the
signal intensity is a single time point measurement that occurs
between about 5 min and about 300 min.
[0124] In embodiments where more than one profiling peptide is
used, the Mcl-1 dependency percentage (PP) is defined by the
following equation:
PP = [ 100 * ( NCAUC - Pep 1 AUC NCAUC - PC avg AUC ) ] Pep 1 + [
100 * ( NCAUC - Pep 2 AUC NC AUC - PC avg AUC ) ] Pep 2 + [ 100 * (
NCAUC - Pep n AUC NCAUC - PC avg AUC ) ] Pep n ##EQU00002##
[0125] In embodiments, a decrease in mitochondrial integrity
indicates that the cancer cell is sensitive to a therapeutic agent.
As used herein, "therapeutic agent" refers to any anti-cancer
compound that is administered as a part of an anti-cancer therapy
regimen. In embodiments, the therapeutic agent is a
cyclin-dependent kinase 9 (CDK9) inhibitor. In some embodiments,
the therapeutic agent is alvocidib.
[0126] In embodiments, methods of profiling a cancer cell from a
subject include methods of predicting sensitivity of a cancer cell
from a subject to a therapeutic agent. Therefore, methods of the
present disclosure include a method of predicting sensitivity of a
cancer cell from a subject to a therapeutic agent, comprising:
contacting the cancer cell with a profiling peptide comprising a
cellular uptake moiety, and an Mcl-1 binding domain having SEQ ID
NO:1 with 0-8 modifications; and detecting a change in
mitochondrial integrity of the cancer cell; wherein a decrease in
mitochondrial integrity indicates that the cancer cell is sensitive
to the therapeutic agent. In further embodiments, a method of
predicting sensitivity of a cancer cell from a subject to a
therapeutic agent, comprising: contacting the cancer cell with a
profiling peptide comprising a cellular uptake moiety, and an Mcl-1
binding domain having SEQ ID NO:1 with 0-8 modifications; detecting
a change in mitochondrial integrity of the cancer cell; and
determining an Mcl-1 dependency percentage for the cancer cell
based at least on the change in mitochondrial integrity, wherein an
Mcl-1 dependency percentage above a predetermined value indicates
that the cancer cell is sensitive to the therapeutic agent. In any
of the above embodiments, the Mcl-1 binding domain has any one of
SEQ ID NOS:1-11 with 0-8 modifications. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 1-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS:1-11. In any of
the above embodiments, the cellular uptake moiety may be a TAT
translocation domain or an ANT translocation domain. In any of the
above embodiments, the cellular uptake moiety is conjugated to the
Mcl-1 binding domain via a linker. In any of the above embodiments,
the profiling peptide has the sequence of (SEQ ID NO: 14). In any
of the above embodiments, the profiling peptide has the sequence of
(SEQ ID NO:15). In any of the above embodiments, the cancer cell
may not be permeabilized.
[0127] Further embodiments provide methods of predicting
sensitivity of a cancer cell from a subject to a therapeutic agent,
comprising: contacting the cancer cell with a profiling peptide
comprising an Mcl-1 binding domain having SEQ ID NO:1 with 0-8
modifications, the cancer cell not being permeabilized; and
detecting a change in mitochondrial integrity of the cancer cell;
wherein a decrease in mitochondrial integrity indicates that the
cancer cell is sensitive to the therapeutic agent. Still further
embodiments provide a method of predicting sensitivity of a cancer
cell from a subject to a therapeutic agent, comprising: contacting
the cancer cell with a profiling peptide comprising an Mcl-1
binding domain having SEQ ID NO:1 with 0-8 modifications, the
cancer cell not being permeabilized; detecting a change in
mitochondrial integrity of the cancer cell; and determining an
Mcl-1 dependency percentage for the cancer cell based at least on
the change in mitochondrial integrity, wherein an Mcl-1 dependency
percentage above a predetermined value indicates that the cancer
cell is sensitive to the therapeutic agent. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID NOS:
1-11 with 0-8 modifications. In any of the above embodiments, the
Mcl-1 binding domain has any one of SEQ ID NOS:1-11 with 1-8
modifications. In any of the above embodiments, the Mcl-1 binding
domain has SEQ ID NO:1-11.
[0128] Further embodiments provide use of a profiling peptide
comprising an Mcl-1 binding domain having SEQ ID NO:1 with 0-8
modifications. Accordingly, embodiments of the present disclosure
include use of a profiling peptide comprising an Mcl-1 binding
domain having SEQ ID NO:1 with 0-8 modifications in a method to
predict a patient response to a therapeutic agent, the method
comprising: contacting the cancer cell with a profiling peptide
comprising a cellular uptake moiety, and an Mcl-1 binding domain
having SEQ ID NO:1 with 0-8 modifications. In some embodiments, the
method further comprises detecting a change in mitochondrial
integrity of the cancer cell; wherein a decrease in mitochondrial
integrity indicates that the cancer cell is sensitive to the
therapeutic agent. In any of the above embodiments, the Mcl-1
binding domain has any one of SEQ ID NOS:1-11 with 0-8
modifications. In any of the above embodiments, the Mcl-1 binding
domain has any one of SEQ ID NOS:1-11 with 1-8 modifications. In
any of the above embodiments, the Mcl-1 binding domain has any one
of SEQ ID NOS:1-11. In any of the above embodiments, the cellular
uptake moiety may be a TAT translocation domain or an ANT
translocation domain. In any of the above embodiments, the cellular
uptake moiety is conjugated to the Mcl-1 binding domain via a
linker. In any of the above embodiments, the profiling peptide has
the sequence of (SEQ ID NO: 14). In any of the above embodiments,
the profiling peptide has the sequence of (SEQ ID NO:15). In any of
the above embodiments, the cancer cell may not be
permeabilized.
[0129] In embodiments, the Mcl-1 dependency percentage being over a
predetermined value of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, or 75% indicates that the cancer cell is
sensitive to the therapeutic agent, such as alvocidib, as a single
agent, or combinations of alvocidib with other therapeutic agents,
such as Ara-C, Mitoxantrone, Venetoclax, Daunorubicin, Brd4
inhibitors (e.g., JQ1), and/or DNA methyltransferase inhibitors
(e.g., azacitidine or decitabine). In some embodiments, the Mcl-1
dependency percentage being over 15% indicates that the cancer cell
is sensitive to the therapeutic agent, such as alvocidib, as a
single agent, or combinations of alvocidib with other therapeutic
agents. In some embodiments, the Mcl-1 dependency percentage being
over 20% indicates that the cancer cell is sensitive to the
therapeutic agent, as a single agent, or in combination with other
therapeutic agents. In some embodiments, the Mcl-1 dependency
percentage being over 25% indicates that the cancer cell is
sensitive to the therapeutic agent, as a single agent, or in
combination with other therapeutic agents. In some embodiments, the
Mcl-1 dependency percentage being over 30% indicates that the
cancer cell is sensitive to the therapeutic agent, as a single
agent, or in combination with other therapeutic agents. In some
embodiments, the Mcl-1 dependency percentage being over 35%
indicates that the cancer cell is sensitive to the therapeutic
agent, as a single agent, or in combination with other therapeutic
agents. In some embodiments, the Mcl-1 dependency percentage being
over 40% indicates that the cancer cell is sensitive to the
therapeutic agent, as a single agent, or in combination with other
therapeutic agents. In some embodiments, the Mcl-1 dependency
percentage being over 45% indicates that the cancer cell is
sensitive to the therapeutic agent, as a single agent, or in
combination with other therapeutic agents. In some embodiments, the
Mcl-1 dependency percentage being over 50% indicates that the
cancer cell is sensitive to the therapeutic agent, as a single
agent, or in combination with other therapeutic agents. In certain
of the foregoing embodiments, the therapeutic agent is
alvocidib.
[0130] In some embodiments, the methods of profiling a cancer
described herein are useful in the evaluation of a subject, for
example, for evaluating diagnosis, prognosis, and response to
treatment. Diagnosis refers to the process of attempting to
determine or identify a possible disease or disorder, such as, for
example, cancer. Prognosis refers to predicting a likely outcome of
a disease or disorder. A complete prognosis often includes the
expected duration, the function, and a description of the course of
the disease, such as progressive decline, intermittent crisis, or
sudden, unpredictable crisis. Response to treatment is a prediction
of a subject's medical outcome when receiving a treatment.
Responses to treatment can be, by way of example, pathological
complete response, survival, and progression free survival.
[0131] In embodiments, methods of profiling a cancer cell from a
subject include methods of producing a sensitivity profile for a
cancer cell from a subject. Therefore, methods of the disclosure
further include a method of producing a sensitivity profile for a
cancer cell from a subject, comprising: contacting the cancer cell
with a profiling peptide comprising a cellular uptake moiety, and
an Mcl-1 binding domain having SEQ ID NO: 1 with 0-8 modifications;
and detecting a change in mitochondrial integrity of the cancer
cell. Additional methods of the disclosure include a method of
producing a sensitivity profile for a cancer cell from a subject,
comprising: contacting the cancer cell with a profiling peptide
comprising a cellular uptake moiety, and an Mcl-1 binding domain
having SEQ ID NO:1 with 0-8 modifications; detecting a change in
mitochondrial integrity of the cancer cell; and determining an
Mcl-1 dependency percentage for the cancer cell based at least on
the change in mitochondrial integrity. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 0-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS:1-11 with 1-8
modifications. In any of the above embodiments, the Mcl-1 binding
domain has any one of SEQ ID NOS:1-11. In any of the above
embodiments, the cellular uptake moiety may be a TAT translocation
domain or an ANT translocation domain. In any of the above
embodiments, the cellular uptake moiety is conjugated to the Mcl-1
binding domain via a linker. In any of the above embodiments, the
profiling peptide has the sequence of (SEQ ID NO: 14). In any of
the above embodiments, the profiling peptide has the sequence of
(SEQ ID NO:15). In any of the above embodiments, the cancer cell
may not be permeabilized.
[0132] Further methods of the disclosure include a method of
producing a sensitivity profile for a cancer cell from a subject,
comprising: contacting the cancer cell with a profiling peptide
comprising an Mcl-1 binding domain having SEQ ID NO:1 with 0-8
modifications, the cancer cell not being permeabilized; and
detecting a change in mitochondrial integrity of the cancer cell.
In further embodiments, methods of the disclosure include a method
of producing a sensitivity profile for a cancer cell from a
subject, comprising: contacting the cancer cell with a profiling
peptide comprising an Mcl-1 binding domain having SEQ ID NO:1 with
0-8 modifications, the cancer cell not being permeabilized;
detecting a change in mitochondrial integrity of the cancer cell;
and determining an Mcl-1 dependency percentage for the cancer cell
based at least on the change in mitochondrial integrity. In any of
the above embodiments, the Mcl-1 binding domain has any one of SEQ
ID NOS:1-11 with 0-8 modifications. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 1-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS:1-11.
[0133] In various embodiments, the methods of profiling a cancer
from a subject disclosed herein direct a clinical decision
regarding whether a subject is to receive a specific treatment. In
various embodiments, the present methods direct the treatment of a
cancer subject, including, for example, what type of treatment
should be administered or withheld. In various embodiments, a
cancer treatment is administered or withheld based on the methods
described herein. Examples of treatments include surgical
resection, radiation therapy, chemotherapy, pharmacodynamic
therapy, targeted therapy, immunotherapy, and supportive therapy
(e.g., painkillers, diuretics, antidiuretics, antivirals,
antibiotics, nutritional supplements, anemia therapeutics, blood
clotting therapeutics, bone therapeutics, and psychiatric and
psychological therapeutics). In various embodiments, the treatments
include those described in US Patent Publication No. US
2012-0225851 and International Patent Publication No. WO
2012/122370.
[0134] In some embodiments, the present methods provide information
about the likely response that a subject is to have to a particular
treatment. In some embodiments, the present methods provide a high
likelihood of response and may direct treatment, including
aggressive treatment. In some embodiments, the present methods
provide a low likelihood of response and may direct cessation of
treatment, including aggressive treatment, and the use of
palliative care, to avoid unnecessary toxicity from ineffective
chemotherapies for a better quality of life.
[0135] In some embodiments, the present methods indicate a high or
low likelihood of response to a pro-apoptotic agent and/or an agent
that operates via apoptosis and/or an agent that operates via
apoptosis driven by direct protein modulation. In various
embodiments, exemplary pro-apoptotic agents and/or agents that
operate via apoptosis and/or an agent that operates via apoptosis
driven by direct protein modulation include ABT-263 (navitoclax),
and obatoclax, WEP, bortezomib, and carfilzomib. In some
embodiments, the present methods indicate a high or low likelihood
of response to an agent that does not operate via apoptosis and/or
an agent that does not operate via apoptosis driven by direct
protein modulation. In various embodiments, exemplary agents that
do not operate via apoptosis include kinesin spindle protein
inhibitors, cyclin-dependent kinase inhibitors (e.g., alvocidib),
Arsenic Trioxide (TRISENOX), MEK inhibitors, pomalidomide,
azacitidine, decitibine, vorinostat, entinostat, dinaciclib,
gemtuzumab, BTK inhibitors, PI3 kinase delta inhibitors,
lenalidomide, anthracyclines, cytarabine, melphalam, Akt
inhibitors, mTOR inhibitors. In a specific embodiment, the present
methods are useful in predicting a subject's response to any of the
treatments (including agents) described herein.
[0136] In embodiments, the present methods are predictive of a
positive response to a pro-apoptotic agent or an agent that
operates via apoptosis. In embodiments, the present methods are
predictive of a positive response to an agent that does not operate
via apoptosis. In further embodiments, the present methods are
predictive of non-responsiveness to an apoptotic effector agent
and/or an agent that does not operate via apoptosis.
[0137] In certain embodiments, the methods described herein predict
a subject's response to a treatment regimen comprising one or more
therapeutic agents. In some embodiments, the methods described
herein direct the selection of a therapeutic agent for treating a
cancer in a subject. In embodiments, the therapeutic agent is a
cyclin-dependent kinase 9 (CDK9) inhibitor. In some embodiments,
the CDK9 inhibitor is alvocidib.
[0138] In embodiments, the methods described herein predict a
subject's response to a treatment regimen comprising a combination
of two or more therapeutic agents. In some embodiments, the two or
more therapeutic agents comprise a CDK9 inhibitor. In some
embodiments, the two or more therapeutic agents comprise alvocidib.
In some embodiments, the two or more therapeutic agents comprise
alvocidib, cytarabine, mitoxantrone, daunorubicin, decitabine,
azacitidine, venetoclax, bortezomib, dacogen, ibrutinib,
lenalidomide, thalidomide, or a combination thereof. In some
embodiments, the two or more therapeutic agents comprise alvocidib
and cytarabine, mitoxantrone, daunorubicin, decitabine,
azacitidine, venetoclax, bortezomib, dacogen, ibrutinib,
lenalidomide, thalidomide, or a combination thereof.
[0139] In embodiments, methods of profiling a cancer cell from a
subject include methods of selecting a therapeutic agent for
treating a cancer in a subject. Therefore, methods of the
disclosure further include a method of selecting a therapeutic
agent for treating a cancer in a subject, comprising: contacting
the cancer cell with a profiling peptide comprising a cellular
uptake moiety, and an Mcl-1 binding domain having SEQ ID NO:1 with
0-8 modifications; detecting a change in mitochondrial integrity of
the cancer cell; and selecting the therapeutic agent to treat the
subject if the change in mitochondrial integrity is a decrease in
mitochondrial integrity. Additional methods of the disclosure
include a method of producing a sensitivity profile for a cancer
cell from a subject, comprising: contacting the cancer cell with a
profiling peptide comprising a cellular uptake moiety, and an Mcl-1
binding domain having SEQ ID NO:1 with 0-8 modifications; detecting
a change in mitochondrial integrity of the cancer cell; determining
an Mcl-1 dependency percentage for the cancer cell based at least
on the change in mitochondrial integrity; and selecting the
therapeutic agent to treat the subject if the Mcl-1 dependency
percentage is above a predetermined value. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 0-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS: 1-11 with 1-8
modifications. In any of the above embodiments, the Mcl-1 binding
domain has any one of SEQ ID NOS:1-11. In any of the above
embodiments, the cellular uptake moiety may be a TAT translocation
domain or an ANT translocation domain. In any of the above
embodiments, the cellular uptake moiety is conjugated to the Mcl-1
binding domain via a linker. In any of the above embodiments, the
profiling peptide has the sequence of (SEQ ID NO: 14). In any of
the above embodiments, the profiling peptide has the sequence of
(SEQ ID NO:15). In any of the above embodiments, the cancer cell
may not be permeabilized, for example with a cell permeabilization
agent such as digitonin.
[0140] Further methods of the disclosure include a method of
selecting a therapeutic agent for treating a cancer in a subject,
comprising: receiving a sensitivity profile for a cancer cell of
the subject, the sensitivity profile comprising mitochondrial
integrity data of the cancer cell when contacted with a profiling
peptide comprising a cellular uptake moiety, and an Mcl-1 binding
domain having SEQ ID NO:1 with 0-8 modifications; and selecting the
therapeutic agent to treat the subject if the mitochondrial
integrity data shows a decrease in mitochondrial integrity. Yet
further methods of the disclosure include a method of selecting a
therapeutic agent for treating a cancer in a subject, comprising:
receiving a sensitivity profile for a cancer cell of the subject,
the sensitivity profile comprising Mcl-1 dependency data for the
cancer cell, the Mcl-1 dependency data determined based at least on
a change in mitochondrial integrity of the cancer cell when
contacted with a profiling peptide comprising a cellular uptake
moiety, and an Mcl-1 binding domain having SEQ ID NO:1 with 0-8
modifications; and selecting the therapeutic agent to treat the
subject if the Mcl-1 dependency data shows an Mcl-1 dependency
percentage above a predetermined value. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 0-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS: 1-11 with 1-8
modifications. In any of the above embodiments, the Mcl-1 binding
domain has any one of SEQ ID NOS: 1-11. In any of the above
embodiments, the cellular uptake moiety may be a TAT translocation
domain or an ANT translocation domain. In any of the above
embodiments, the cellular uptake moiety is conjugated to the Mcl-1
binding domain via a linker. In any of the above embodiments, the
profiling peptide has the sequence of (SEQ ID NO:14). In any of the
above embodiments, the profiling peptide has the sequence of (SEQ
ID NO:15). In any of the above embodiments, the cancer cell may not
be permeabilized, for example with a cell permeabilization agent
such as digitonin.
[0141] Still further methods of the disclosure include a method of
selecting a therapeutic agent for treating a cancer in a subject,
comprising: contacting the cancer cell with a profiling peptide
comprising an Mcl-1 binding domain having SEQ ID NO:1 with 0-8
modifications, the cancer cell not being permeabilized; detecting a
change in mitochondrial integrity of the cancer cell; and selecting
the therapeutic agent to treat the subject if the change in
mitochondrial integrity is a decrease in mitochondrial integrity.
Yet further methods of the disclosure include a method of selecting
a therapeutic agent for treating a cancer in a subject, comprising:
contacting the cancer cell with a profiling peptide comprising an
Mcl-1 binding domain having SEQ ID NO: 1 with 0-8 modifications,
the cancer cell not being permeabilized; detecting a change in
mitochondrial integrity of the cancer cell; determining an Mcl-1
dependency percentage for the cancer cell based at least on the
change in mitochondrial integrity; and selecting the therapeutic
agent to treat the subject if the Mcl-1 dependency percentage is
above a predetermined value. In any of the above embodiments, the
Mcl-1 binding domain has any one of SEQ ID NOS:1-11 with 0-8
modifications. In any of the above embodiments, the Mcl-1 binding
domain has any one of SEQ ID NOS: 1-11 with 1-8 modifications. In
any of the above embodiments, the Mcl-1 binding domain has SEQ ID
NO:1-11.
[0142] In further embodiments, methods of the disclosure include a
method of selecting a therapeutic agent for treating a cancer in a
subject, comprising: receiving a sensitivity profile for a cancer
cell of the subject, the sensitivity profile comprising
mitochondrial integrity data of the cancer cell when contacted with
a profiling peptide comprising an Mcl-1 binding domain having SEQ
ID NO:1 with 0-8 modifications, the cancer cell not being
permeabilized; and selecting the therapeutic agent to treat the
subject if the mitochondrial integrity data shows a decrease in
mitochondrial integrity. In additional embodiments, methods of the
disclosure include a method of selecting a therapeutic agent for
treating a cancer in a subject, comprising: receiving a sensitivity
profile for a cancer cell of the subject, the sensitivity profile
comprising Mcl-1 dependency data for the cancer cell, the Mcl-1
dependency data determined based at least on a change in
mitochondrial integrity of the cancer cell when contacted with a
profiling peptide comprising an Mcl-1 binding domain having SEQ ID
NO:1 with 0-8 modifications, the cancer cell not being
permeabilized; and selecting the therapeutic agent to treat the
subject if the Mcl-1 dependency data shows an Mcl-1 dependency
percentage above a predetermined value. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 0-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS:1-11 with 1-8
modifications. In any of the above embodiments, the Mcl-1 binding
domain has any one of SEQ ID NOS:1-11.
[0143] In embodiments, methods of the present disclosure include
administering a therapeutic agent described herein to the subject
based on mitochondrial integrity and/or Mcl-1 dependency data
obtained by contacting a subject's cancer cell with any one or more
of the profiling peptides disclosed herein. In one embodiment, the
therapeutic agent is one or more of a BH3 mimetic, epigenetic
modifying agent, topoisomerase inhibitor, cyclin-dependent kinase
inhibitor (e.g., alvocidib), and/or kinesin-spindle protein
stabilizing agent. In another embodiment, the therapeutic agent is
a proteasome inhibitor; and/or a modulator of cell cycle regulation
(by way of example, a cyclin dependent kinase inhibitor); and/or a
modulator of cellular epigenetic mechanistic (by way of example,
one or more of a histone deacetylase (HDAC) (e.g. one or more of
vorinostat or entinostat), azacitidine, decitabine); and/or an
anthracycline or anthracenedione (by way of example, one or more of
epirubicin, doxorubicin, mitoxantrone, daunorubicin, idarubicin);
and/or a platinum-based therapeutic (by way of example, one or more
of carboplatin, cisplatin, and oxaliplatin); cytarabine or a
cytarabine-based chemotherapy; a BH3 mimetic (by way of example,
one or more of BCL2, BCLXL, or MCL1); and an inhibitor of MCL1.
[0144] In various embodiments, the chemotherapeutic agent is
selected from: one or more of alkylating agents such as thiotepa
and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (e.g., bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin
doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy
doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,
mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,
rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, zorubicin; anti-metabolites such as methotrexate and
5-fluorouracil (5-FU); folic acid analogues such as denopterin,
methotrexate, pteropterin, trimetrexate; purine analogs such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine
analogs such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as minoglutethimide, mitotane, trilostane; folic acid
replenisher such as folinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; demecolcine; diaziquone; elformithine;
elliptinium acetate; an epothilone; etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidainine; maytansinoids such as
maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;
nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;
podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (e.g.,
T-2 toxin, verracurin A, roridin A and anguidine); urethan;
vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide;
thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, 111), and TAXOTERE doxetaxel
(Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin, oxaliplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; NAVELBINE. vinorelbine; novantrone; teniposide;
edatrexate; daunomycin; aminopterin; xeloda; ibandronate;
irinotecan (Camptosar, CPT-11) (including the treatment regimen of
irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS
2000; difluoromethylornithine (DMFO); retinoids such as retinoic
acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin,
including the oxaliplatin treatment regimen (FOLFOX); lapatinib
(Tykerb); inhibitors of PKC-J, Raf, H-Ras, EGFR (e.g., erlotinib
(Tarceva)) and VEGF-A that reduce cell proliferation, dacogen,
velcade, and pharmaceutically acceptable salts, acids or
derivatives of any of the agents listed herein. Exemplary
therapeutic agents include alvocidib, cytarabine, mitoxantrone,
daunorubicin, decitabine, azacitidine, venetoclax, bortezomib,
dacogen, ibrutinib, lenalidomide, thalidomide, and pharmaceutically
acceptable salts, acids or derivatives thereof.
[0145] Suitable methods may include administration of a
pro-apoptotic agent and/or an agent that operates via apoptosis
and/or an agent that operates via apoptosis driven by direct
protein modulation. Examples of such agents include ABT-263
(Navitoclax), and obatoclax, WEP, bortezomib, and carfilzomib.
Other suitable treatments may include an agent that does not
operate via apoptosis and/or an agent that does not operate via
apoptosis driven by direct protein modulation. Examples of such
agents include kinesin spindle protein inhibitors, cyclin-dependent
kinase (CDK) inhibitors, Arsenic Trioxide (TRISENOX), MEK
inhibitors, pomalidomide, azacitidine, decitibine, vorinostat,
entinostat, dinaciclib, gemtuzumab, BTK inhibitors, PI3 kinase
delta inhibitors, lenalidomide, anthracyclines, cytarabine,
melphalan, Akt inhibitors, mTOR inhibitors. In embodiments, the CDK
inhibitor is a CDK9 inhibitor. In some embodiments, the CDK9
inhibitor is alvocidib.
[0146] In embodiments, the methods of treatment disclosed herein
comprise administering an effective amount of a therapeutic agent
to the subject, thereby treating their cancer. In various
embodiments, effective amounts of a therapeutic agent can decrease
the number of tumor cells, decrease the number of metastases,
decrease tumor volume, induce apoptosis of cancer cells, induce
cancer cell death, induce--or radio-sensitivity in cancer cells,
inhibit angiogenesis near cancer cells, inhibit cancer cell
proliferation, inhibit tumor growth, prevent metastasis, reduce the
number of metastases, increase life expectancy, prolong a subject's
life, reduce cancer-associated pain, and/or reduce relapse or
re-occurrence of the cancer following treatment.
[0147] For administration, effective amounts (also referred to as
doses) can be initially estimated based on results from in vitro
assays and/or animal model studies. For example, a dose can be
formulated in animal models to achieve a circulating concentration
range that includes an IC50 as determined in cell culture against a
particular target. Such information can be used to more accurately
determine useful doses in subjects of interest.
[0148] The actual dose amount administered to a particular subject
can be determined by a physician, veterinarian, or researcher
taking into account parameters such as physical and physiological
factors including target, body weight, severity of condition, type
of cancer, previous or concurrent therapeutic interventions,
idiopathy of the subject, and route of administration.
[0149] In embodiments, methods of profiling a cancer cell from a
subject include methods of treating a cancer in a subject in need
thereof. Accordingly, methods of the present disclosure include a
method for treating a cancer in a subject in need thereof, the
method comprising administering a treatment regimen comprising a
therapeutic agent to a subject having an Mcl-1 dependency
percentage above a predetermined value, the Mcl-1 dependency
percentage having been obtained by an in vitro method comprising
contacting a first portion of a plurality of cancer cells with a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain, the Mcl-1 binding domain having the sequence of SEQ
ID NO: 1 with 0-8 modifications, or a composition comprising a
profiling peptide comprising a cellular uptake moiety and an Mcl-1
binding domain, the Mcl-1 binding domain having the sequence of SEQ
ID NO:1 with 0-8 modifications and a carrier. Additionally, methods
of the present disclosure include a method of treating a cancer in
a subject in need thereof, comprising: contacting a cancer cell
from the subject with a profiling peptide comprising a cellular
uptake moiety, and an Mcl-1 binding domain having SEQ ID NO:1 with
0-8 modifications; detecting a change in mitochondrial integrity of
the cancer cell; and administering an effective amount of a
therapeutic agent to the subject if a decrease in mitochondrial
integrity is detected, thereby treating the cancer in the subject.
Further methods of the present disclosure include a method of
treating a cancer in a subject in need thereof, comprising:
contacting a cancer cell from the subject with a profiling peptide
comprising a cellular uptake moiety, and an Mcl-1 binding domain
having SEQ ID NO: 1 with 0-8 modifications; detecting a change in
mitochondrial integrity of the cancer cell; determining an Mcl-1
dependency percentage for the cancer cell based at least on the
change in mitochondrial integrity; and administering an effective
amount of a therapeutic agent to the subject if the Mcl-1
dependency percentage is above a predetermined value, thereby
treating the cancer in the subject. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 0-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS:1-11 with 1-8
modifications. In any of the above embodiments, the Mcl-1 binding
domain has any one of SEQ ID NOS:1-11. In any of the above
embodiments, the cellular uptake moiety may be a TAT translocation
domain or an ANT translocation domain. In any of the above
embodiments, the cellular uptake moiety is conjugated to the Mcl-1
binding domain via a linker. In any of the above embodiments, the
profiling peptide has the sequence of (SEQ ID NO: 14). In any of
the above embodiments, the profiling peptide has the sequence of
(SEQ ID NO:15). In any of the above embodiments, the cancer cell
may not be permeabilized.
[0150] In some embodiments, methods of the present disclosure
include a method of treating a cancer in a subject in need thereof,
comprising: receiving a sensitivity profile for a cancer cell of
the subject, the sensitivity profile comprising mitochondrial
integrity data of the cancer cell when contacted with a profiling
peptide comprising a cellular uptake moiety, and an Mcl-1 binding
domain having SEQ ID NO: 1 with 0-8 modifications; and
administering an effective amount of a therapeutic agent to the
subject if the mitochondrial integrity data shows a decrease in
mitochondrial integrity, thereby treating the cancer in the
subject. In further embodiments, methods of the present disclosure
include a method of treating a cancer in a subject in need thereof,
comprising: receiving a sensitivity profile for a cancer cell of
the subject, the sensitivity profile comprising Mcl-1 dependency
data for the cancer cell, the Mcl-1 dependency data being
determined based at least on a change in mitochondrial integrity of
the cancer cell when contacted with a profiling peptide comprising
a cellular uptake moiety, and an Mcl-1 binding domain having SEQ ID
NO: 1 with 0-8 modifications; and administering an effective amount
of a therapeutic agent to the subject if the Mcl-1 dependency data
shows an Mcl-1 dependency percentage above a predetermined value,
thereby treating the cancer in the subject. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 0-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS:1-11 with 1-8
modifications. In any of the above embodiments, the Mcl-1 binding
domain has any one of SEQ ID NOS:1-11. In any of the above
embodiments, the cellular uptake moiety may be a TAT translocation
domain or an ANT translocation domain. In any of the above
embodiments, the cellular uptake moiety is conjugated to the Mcl-1
binding domain via a linker. In any of the above embodiments, the
profiling peptide has the sequence of (SEQ ID NO:14). In any of the
above embodiments, the profiling peptide has the sequence of (SEQ
ID NO: 15). In any of the above embodiments, the cancer cell may
not be permeabilized.
[0151] Further embodiments of the present disclosure include a
method of treating a cancer in a subject in need thereof,
comprising: contacting a cancer cell from the subject with a
profiling peptide comprising an Mcl-1 binding domain having SEQ ID
NO:1 with 0-8 modifications, the cancer cell not being
permeabilized; detecting a change in mitochondrial integrity of the
cancer cell; and administering an effective amount of a therapeutic
agent to the subject if a decrease in mitochondrial integrity is
detected, thereby treating the cancer in the subject. In still
further embodiments, methods of the present disclosure include a
method of treating a cancer in a subject in need thereof,
comprising: contacting a cancer cell from the subject with a
profiling peptide comprising an Mcl-1 binding domain having SEQ ID
NO:1 with 0-8 modifications, the cancer cell not being
permeabilized; detecting a change in mitochondrial integrity of the
cancer cell; determining an Mcl-1 dependency percentage for the
cancer cell based at least on the change in mitochondrial
integrity; and administering an effective amount of a therapeutic
agent to the subject if the Mcl-1 dependency percentage is above a
predetermined value, thereby treating the cancer in the subject. In
any of the above embodiments, the Mcl-1 binding domain has any one
of SEQ ID NOS:1-11 with 0-8 modifications. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 1-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS:1-11.
[0152] Additional embodiments of the present disclosure include a
method of treating a cancer in a subject in need thereof,
comprising: receiving a sensitivity profile for a cancer cell of
the subject, the sensitivity profile comprising mitochondrial
integrity data of the cancer cell when contacted with a profiling
peptide comprising an Mcl-1 binding domain having SEQ ID NO:1 with
0-8 modifications, the cancer cell not being permeabilized; and
administering an effective amount of a therapeutic agent to the
subject if the mitochondrial integrity data shows a decrease in
mitochondrial integrity, thereby treating the cancer in the
subject. In some embodiments, methods of the present disclosure
include a method of treating a cancer in a subject in need thereof,
comprising: receiving a sensitivity profile for cancer cells of the
subject, the sensitivity profile comprising Mcl-1 dependency data
for the cancer cell, the Mcl-1 dependency data being determined
based at least on a change in mitochondrial integrity of the cancer
cell when contacted with a profiling peptide comprising an Mcl-1
binding domain having SEQ ID NO:1 with 0-8 modifications, the
cancer cell not being permeabilized; and administering an effective
amount of a therapeutic agent to the subject if the Mcl-1
dependency data shows an Mcl-1 dependency percentage above a
predetermined value, thereby treating the cancer in the subject. In
any of the above embodiments, the Mcl-1 binding domain has any one
of SEQ ID NOS:1-11 with 0-8 modifications. In any of the above
embodiments, the Mcl-1 binding domain has any one of SEQ ID
NOS:1-11 with 1-8 modifications. In any of the above embodiments,
the Mcl-1 binding domain has any one of SEQ ID NOS:1-11.
[0153] In any of the above embodiments, an effective amount of one
or more of the following therapeutic agents may be administered to
the subject: (i) a CDK inhibitor (e.g. a CDK4 inhibitor, a CDK6
inhibitor, a CDK7 inhibitor, a CDK8 inhibitor, a CDK9 inhibitor, a
CDK10 inhibitor, and/or a CDK11 inhibitor); (ii) a bromodomain
inhibitor (e.g., a Brd2 inhibitor, a Brd3 inhibitor, a Brd4
inhibitor and/or a BrdT inhibitor); (iii) a histone
methyltransferase inhibitor (e.g., a DOT1-like histone
methyltransferase (Dot1L) inhibitor); (iv) a histone deacetylase
(HDAC) inhibitor (e.g., a Class I HDAC (e.g., HDAC1, HDAC2, HDAC3
and HDAC8) inhibitor, a Class IIa HDAC (e.g., HDAC4, HDAC5, HDAC7,
and HDAC9) inhibitor; a Class IIb HDAC (e.g., HDAC6 and HDAC10)
inhibitor; and a Class IV HDAC (e.g., HDAC11) inhibitor); and (v) a
histone demethylase inhibitor (e.g. an inhibitor of a
lysine-specific demethylase, such as lysine-specific demethylase 1A
(Lsd1)).
[0154] In some embodiments, the CDK inhibitor is a CDK7, CDK9
inhibitor, or both. In some embodiments, the CDK inhibitor is a
CDK9-specific siRNA, alvocidib, or dinaciclib. In some embodiments,
the bromodomain inhibitor is a Brd4 inhibitor. In some embodiments,
the bromodomain inhibitor is JQ-1 (Nature 2010 Dec. 23;
468(7327):1067-73), BI2536 (ACS Chem. Biol. 2014 May 16;
9(5):1160-71; Boehringer Ingelheim), TG101209 (ACS Chem. Biol. 2014
May 16; 9(5):1160-71), OTX015 (Mol. Cancer Ther. November 201312;
C244; Oncoethix), IBET762 (J Med Chem. 2013 Oct. 10;
56(19):7498-500; GlaxoSmithKline), IBET151 (Bioorg. Med. Chem.
Lett. 2012 Apr. 15; 22(8):2968-72; GlaxoSmithKline), PFI-1 (J. Med.
Chem. 2012 Nov. 26; 55(22):9831-7; Cancer Res. 2013 Jun. 1;
73(11):3336-46; Structural Genomics Consortium), or CPI-0610
(Constellation Pharmaceuticals). In some embodiments, the histone
methyltransferase inhibitor is EPZ004777, EPZ-5676 (Blood. 2013
Aug. 8; 122(6):1017-25) or SGC0946 (Nat. Commun. 2012; 3:1288). In
specific embodiments, the histone methyltransferase inhibitor is
EPZ-5676. In some embodiments, the HDAC inhibitor is trichostatin
A, vorinostat (Proc. Natl. Acad. Sci. U.S.A. 1998 Mar. 17;
95(6):3003-7), givinostat, abexinostat (Mol. Cancer Ther. 2006 May;
5(5):1309-17), belinostat (Mol. Cancer Ther. 2003 August;
2(8):721-8), panobinostat (Clin. Cancer Res. 2006 Aug. 1;
12(15):4628-35), resminostat (Clin. Cancer Res. 2013 Oct. 1;
19(19):5494-504), quisinostat (Clin. Cancer Res. 2013 Aug. 1;
19(15):4262-72), depsipeptide (Blood. 2001 Nov. 1; 98(9):2865-8),
entinostat (Proc. Natl. Acad. Sci. U.S.A. 1999 Apr. 13;
96(8):4592-7), mocetinostat (Bioorg. Med. Chem. Lett. 2008 Feb. 1;
18(3):1067-71) or valproic acid (EMBO J. 2001 Dec. 17;
20(24):6969-78). For example, in some embodiments, the HDAC
inhibitor is panobinostat. In some embodiments, the histone
demethylase inhibitor is HCl-2509 (BMC Cancer. 2014 Oct. 9;
14:752), tranylcypromine or ORY-1001 (J. Clin. Oncol 31, 2013
(suppl; abstr e13543).
[0155] In embodiments, an effective amount of two or more of the
following therapeutic agents may be administered to the subject:
(i) a cyclin-dependent kinase inhibitor; (ii) a bromodomain
inhibitor; (iii) a histone methyltransferase inhibitor; (iv) a
histone deacetylase inhibitor; and (v) a histone demethylase
inhibitor.
[0156] In some embodiments, the two or more therapeutic agents are
a CDK inhibitor, and a bromodomain inhibitor. In some embodiments,
the CDK inhibitor is alvocidib or a CDK9-specific siRNA. In
particular embodiments, the CDK inhibitor is alvocidib. In some
embodiments, the CDK inhibitor is alvocidib or dinaciclib, and the
bromodomain inhibitor is JQ1, IBET762, or OTX015. In certain
embodiments, the bromodomain inhibitor is JQ1. In certain
embodiments, the bromodomain inhibitor is IBET762. In certain
embodiments, the bromodomain inhibitor is OTX015. In some
embodiments, the two or more therapeutic agents are alvocidib and
JQ1. In some embodiments, the two or more therapeutic agents are
alvocidib and IBET762. In some embodiments, the two or more
therapeutic agents are alvocidib and OTX015.
[0157] In some embodiments, the therapeutic agent is a CDK
inhibitor, and an effective amount of a histone deacetylase
inhibitor is also administered to the subject. In some embodiments,
the CDK inhibitor is alvocidib. In some embodiments, the histone
deacetylase inhibitor is panobinostat. In some embodiments, the CDK
inhibitor is alvocidib or dinaciclib, and the histone deacetylase
inhibitor is panobinostat. In certain embodiments, the CDK
inhibitor is alvocidib, and the histone deacetylase inhibitor is
panobinostat.
[0158] In any of the above embodiments, the therapeutic agent is a
CDK inhibitor, and an effective amount of a DNA methyltransferase
inhibitor is further administered to the subject. In such
embodiments, the CDK inhibitor can be alvocidib or dinaciclib and
the DNA methyltransferase can be a nucleoside analogue. In some
embodiments, the CDK inhibitor can be alvocidib or dinaciclib and
the DNA methyltransferase can be azacitidine or decitabine.
[0159] In any of the above embodiments, the therapeutic agent is a
CDK9 inhibitor. In such embodiments, the CDK9 inhibitor may be
alvocidib. In any of the above embodiments, the therapeutic agent
is alvocidib, and an effective amount of Ara-C and mitoxantrone are
further administered to the subject. In another embodiment, the
therapeutic agent is alvocidib, and an effective amount of a Bcl-2
inhibitor, such as Venetoclax, is further administered to the
subject. In a specific embodiment, the therapeutic agent is
alvocidib, and the Bcl-2 inhibitor is Venetoclax. In other of the
above embodiments, the therapeutic agent is alvocidib, and an
effective amount of Ara-C and Daunorubicin is further administered
to the subject. In other of the above embodiments, the therapeutic
agent is alvocidib, and an effective amount of a Brd4 inhibitor,
such as JQ1, is further administered to the subject. In other of
the above embodiments, the therapeutic agent is alvocidib, and an
effective amount of a DNA methyltransferase inhibitor, such as
azacitidine or decitabine, is further administered to the subject.
In further of the above embodiments, the therapeutic agent is
alvocidib, and an effective amount of a DNA methyltransferase
inhibitor, such as azacitidine or decitabine, and a Brd4 inhibitor,
such as JQ1, is further administered to the subject.
[0160] In any of the above embodiments, the CDK9 inhibitor is
dinaciclib.
[0161] In any of the above embodiments, the therapeutic agent is
Venetoclax. In any of the above embodiments, the therapeutic agent
is Ara-C. In any of the above embodiments, the therapeutic agent is
Ara-C, and an effective amount of Daunorubicin is further
administered.
[0162] In any of the above embodiments, a combination of two or
more therapeutic agents is administered to a subject. In some
embodiments, the two or more therapeutic agents comprise a CDK9
inhibitor. In some embodiments, the two or more therapeutic agents
comprise alvocidib. In some embodiments, the two or more
therapeutic agents comprise alvocidib, cytarabine, mitoxantrone,
daunorubicin, decitabine, azacitidine, venetoclax, bortezomib,
dacogen, ibrutinib, lenalidomide, thalidomide, or a combination
thereof. In some embodiments, the two or more therapeutic agents
comprise alvocidib and cytarabine, mitoxantrone, daunorubicin,
decitabine, azacitidine, venetoclax, bortezomib, dacogen,
ibrutinib, lenalidomide, thalidomide, or a combination thereof.
[0163] In more embodiments of the foregoing, the cancer cell
specimen is derived from the biopsy of a solid tumor. In still more
embodiments of the foregoing, the cancer cell specimen is derived
from the biopsy of a non-solid tumor. In any of the foregoing
treatment methods, the cancer is a hematologic cancer. For example,
in some embodiments the hematologic cancer the hematologic cancer
is multiple myeloma, MDS, AML, ALL, acute lymphocytic leukemia,
chronic lymphogenous leukemia, CLL, mantle cell lymphoma, diffuse
large B-cell lymphoma, follicular lymphoma, or non-Hodgkin's
lymphoma. In some specific embodiments, the hematological cancer is
AML. In some other embodiments of the foregoing, the hematologic
cancer is MDS. In different embodiments of the foregoing, the
hematologic cancer is CLL.
[0164] In any of the above embodiments, the cancer cell profiled
may not be permeabilized, for example with a cell permeabilization
agent such as digitonin.
[0165] In any of the above embodiments, an additional treatment
agent can be selected and optionally administered. Examples of such
agents include one or more of anti-cancer drugs, therapy, surgery,
adjuvant therapy, and neoadjuvant therapy, such as those specific
agents described herein.
[0166] In one embodiment, the present methods further direct a
clinical decision regarding whether a subject is to receive
adjuvant therapy after primary, main, or initial treatment,
including a single sole adjuvant therapy. Adjuvant therapy, also
called adjuvant care, is treatment that is given in addition to the
primary, main or initial treatment. By way of example, adjuvant
therapy may be an additional treatment usually given after surgery
where all detectable disease has been removed, but where there
remains a statistical risk of relapse due to occult disease.
[0167] In some embodiments, the present methods direct a subject's
treatment to include adjuvant therapy. For example, a subject that
is scored to be responsive to a specific treatment may receive such
treatment as adjuvant therapy. Further, the present methods may
direct the identity of an adjuvant therapy, by way of example, as a
treatment that induces and/or operates in a pro-apoptotic manner or
one that does not. In one embodiment, the present methods may
indicate that a subject will not be or will be less responsive to a
specific treatment and therefore such a subject may not receive
such treatment as adjuvant therapy. Accordingly, in some
embodiments, the present methods provide for providing or
withholding adjuvant therapy according to a subject's likely
response. In this way, a subject's quality of life, and the cost of
care, may be improved.
[0168] In any of the above embodiments, the methods further
comprise evaluating a clinical factor. In various embodiments, the
clinical factor is one or more of age, cytogenetic status,
performance, histological subclass, gender, and disease stage. In
embodiments, the clinical factor is age. In such embodiments, the
subject age profile is classified as over about 10, over about 20,
over about 30, over about 40, over about 50, over about 60, over
about 70, over about 80 years old.
[0169] In some embodiments, the clinical factor is cytogenetic
status. Cytogenetic status can be measured in a variety of manners
known in the art. For example, FISH, traditional karyotyping, and
virtual karyotyping (e.g. comparative genomic hybridization arrays,
CGH and single nucleotide polymorphism arrays) may be used. For
example, FISH may be used to assess chromosome rearrangement at
specific loci and these phenomena are associated with disease risk
status. In some embodiments, the cytogenetic status is favorable,
intermediate, or unfavorable.
[0170] In some embodiments, the clinical factor is performance.
Performance status can be quantified using any system and methods
for scoring a subject's performance status are known in the art.
The measure is often used to determine whether a subject can
receive therapy, adjustment of dose adjustment, and to determine
intensity of palliative care. There are various scoring systems,
including the Kamofsky score and the Zubrod score. Parallel scoring
systems include the Global Assessment of Functioning (GAF) score,
which has been incorporated as the fifth axis of the Diagnostic and
Statistical Manual (DSM) of psychiatry. Higher performance status
(e.g., at least 80%, or at least 70% using the Karnofsky scoring
system) may indicate treatment to prevent progression of the
disease state, and enhance the subject's ability to accept therapy
and/or radiation treatment. For example, in these embodiments, the
subject is ambulatory and capable of self-care. In other
embodiments, the evaluation is indicative of a subject with a low
performance status (e.g., less than 50%, less than 30%, or less
than 20% using the Karnofsky scoring system), so as to allow
conventional radiotherapy and/or therapy to be tolerated. In these
embodiments, the subject is largely confined to bed or chair and is
disabled even for self-care.
[0171] The Karnofsky score runs from 100 to 0, where 100 is
"perfect" health and 0 is death. The score may be employed at
intervals of 10, where: 100% is normal, no complaints, no signs of
disease; 90% is capable of normal activity, few symptoms or signs
of disease, 80% is normal activity with some difficulty, some
symptoms or signs; 70% is caring for self, not capable of normal
activity or work; 60% is requiring some help, can take care of most
personal requirements; 50% requires help often, requires frequent
medical care; 40% is disabled, requires special care and help; 30%
is severely disabled, hospital admission indicated but no risk of
death; 20% is very ill, urgently requiring admission, requires
supportive measures or treatment; and 10% is moribund, rapidly
progressive fatal disease processes.
[0172] The Zubrod scoring system for performance status includes:
0, fully active, able to carry on all pre-disease performance
without restriction; 1, restricted in physically strenuous activity
but ambulatory and able to carry out work of a light or sedentary
nature, e.g., light house work, office work; 2, ambulatory and
capable of all self-care but unable to carry out any work
activities, up and about more than 50% of waking hours; 3, capable
of only limited self-care, confined to bed or chair more than 50%
of waking hours; 4, completely disabled, cannot carry on any
self-care, totally confined to bed or chair; 5, dead.
[0173] In further embodiments, the clinical factor is histological
subclass. In some embodiments, histological samples of tumors are
graded according to Elston & Ellis, Histopathology, 1991,
19:403-10, the contents of which are hereby incorporated by
reference in their entirety.
[0174] In some embodiments, the clinical factor is gender. In one
embodiment, the gender is male. In another embodiment the gender is
female.
[0175] In some embodiments, the clinical factor is disease stage.
By way of example, using the overall stage grouping, Stage I
cancers are localized to one part of the body; Stage II cancers are
locally advanced, as are Stage III cancers. Whether a cancer is
designated as Stage II or Stage III can depend on the specific type
of cancer. In one example, Hodgkin's disease, Stage II indicates
affected lymph nodes on only one side of the diaphragm, whereas
Stage III indicates affected lymph nodes above and below the
diaphragm. The specific criteria for Stages II and III therefore
differ according to diagnosis. Stage IV cancers have often
metastasized, spread to other organs, or spread throughout the
body.
[0176] In some embodiments, the clinical factor is the
French-American-British (FAB) classification system for hematologic
diseases (e.g. indicating the presence of dysmyelopoiesis and the
quantification of myeloblasts and erythroblasts). In one
embodiment, the FAB for acute lymphoblastic leukemias is L1-L3, or
for acute myeloid leukemias is M0-M7. Further, in some embodiments,
the any one of the following clinical factors may be useful in the
methods described herein: gender; genetic risk factors; family
history; personal history; race and ethnicity; features of the
certain tissues; various benign conditions (e.g. non-proliferative
lesions); previous chest radiation; carcinogen exposure and the
like.
[0177] In another embodiment, the method further comprises a
measurement of an additional biomarker selected from mutational
status, single nucleotide polymorphisms, steady state protein
levels, and dynamic protein levels, which can add further
specificity and/or sensitivity. In some embodiments, the
measurement of an additional biomarker can be measurement of one or
more of a cell surface marker CD33, a cell surface marker CD34, a
FLT3 mutation status, a p53 mutation status, a phosphorylation
state of MEK-1 kinase, and phosphorylation of serine at position 70
of Bcl-2. In some embodiments, the biomarker is expression levels
of the cytokines, including, for example, interleukin-6. In another
embodiments, the biomarker is a mutation in one or more of the
genes MLL, AML/ETO, Flt3-ITD, NPM1 (NPMc+), CEBP, IDH1, IDH2,
RUNX1, ras, and WT1 and/or in the epigenetic modifying genes TET2
and ASXL. In further embodiments, the measurement of the biomarker
indicates a change in the cell signaling protein profile.
[0178] In some cancers, such as Wilms tumor and retinoblastoma, for
example, gene deletions or inactivations are responsible for
initiating cancer progression, as chromosomal regions associated
with tumor suppressors are commonly deleted or mutated. For
example, deletions, inversions, and translocations are commonly
detected in chromosome region 9p21 in gliomas, non-small-cell lung
cancers, leukemias, and melanomas. Without wishing to be bound by
theory, these chromosomal changes may inactivate the tumor
suppressor cyclin-dependent kinase inhibitor 2A. Along with these
deletions of specific genes, large portions of chromosomes can also
be lost. For instance, chromosomes 1p and 16q are commonly lost in
solid tumor cells. Gene duplications and increases in gene copy
numbers can also contribute to cancer and can be detected with
transcriptional analysis or copy number variation arrays. For
example, the chromosomal region 12q13-q14 is amplified in many
sarcomas. This chromosomal region encodes a binding protein called
MDM2, which is known to bind to a tumor suppressor called p53. When
MDM2 is amplified, it prevents p53 from regulating cell growth,
which can result in tumor formation. Further, certain breast
cancers are associated with overexpression and increases in copy
number of the human epidermal growth factor receptor 2 (ERBB2)
gene. Also, gains in chromosomal number, such as chromosomes 1q and
3q, are also associated with increased cancer risk.
[0179] In various embodiments, the present methods further comprise
evaluating a presence, absence, or level of a protein and/or a
nucleic acid, such as when measuring a biomarker. In various
embodiments, the present methods further comprise evaluating a
presence, absence, or level of a protein and/or a nucleic acid
which can enhance the specificity and/or sensitivity of the
sensitivity profiling. In some embodiments, the evaluating is of a
marker for subject response. In some embodiments, the present
methods comprise measurement using one or more of
immunohistochemical staining, western blotting, in cell western,
immunofluorescent staining, ELISA, and fluorescent activating cell
sorting (FACS), or any other method described herein or known in
the art. The present methods may comprise contacting an antibody
with a tumor specimen (e.g. biopsy or tissue or body fluid) to
identify an epitope that is specific to the tissue or body fluid
and that is indicative of a state of a cancer.
[0180] In various embodiments, antibodies include whole antibodies
and/or any antigen binding fragment (e.g., an antigen-binding
portion) and/or single chains of these (e.g. an antibody comprising
at least two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds, an Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2
fragment, a bivalent fragment including two Fab fragments linked by
a disulfide bridge at the hinge region; a Fd fragment consisting of
the VH and CH1 domains; a Fv fragment consisting of the VL and VH
domains of a single arm of an antibody; and the like). In various
embodiments, polyclonal and monoclonal antibodies are useful, as
are isolated human or humanized antibodies, or functional fragments
thereof.
[0181] There are generally two strategies used for detection of
epitopes on antigens in body fluids or tissues, direct methods and
indirect methods. The direct method comprises a one-step staining,
and may involve a labeled antibody (e.g. FITC conjugated antiserum)
reacting directly with the antigen in a body fluid or tissue
sample. The indirect method comprises an unlabeled primary antibody
that reacts with the body fluid or tissue antigen, and a labeled
secondary antibody that reacts with the primary antibody. Labels
can include radioactive labels, fluorescent labels, hapten labels
such as, biotin, or an enzyme such as horse radish peroxidase or
alkaline phosphatase. Methods of conducting these assays are well
known in the art. See, e.g., Harlow et al. (Antibodies, Cold Spring
Harbor Laboratory, N Y, 1988), Harlow et al. (Using Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, N Y, 1999),
Virella (Medical Immunology, 6th edition, Informa HealthCare, New
York, 2007), and Diamandis et al. (Immunoassays, Academic Press,
Inc., New York, 1996). Kits for conducting these assays are
commercially available from, for example, Clontech Laboratories,
LLC. (Mountain View, Calif.).
[0182] Standard assays to evaluate the binding ability of the
antibodies toward the target of various species are known in the
art, including for example, ELISAs, western blots and RIAs. The
binding kinetics (e.g., binding affinity) of antibodies also can be
assessed by standard assays known in the art, such as by Biacore
analysis.
[0183] In another embodiment, the measurement comprises evaluating
a presence, absence, or level of a nucleic acid. A person skilled
in the art will appreciate that a number of methods can be used to
detect or quantify the DNA/RNA levels of appropriate markers.
[0184] Gene expression can be measured using, for example,
low-to-mid-plex techniques, including reporter gene assays,
Northern blot, fluorescent in situ hybridization (FISH), and
reverse transcription PCR (RT-PCR). Gene expression can also be
measured using, for example, higher-plex techniques, including
serial analysis of gene expression (SAGE), DNA microarrays. Tiling
array, RNA-Seq/whole transcriptome shotgun sequencing (WTSS),
high-throughput sequencing, multiplex PCR, multiplex
ligation-dependent probe amplification (MLPA), DNA sequencing by
ligation, and Luminex/XMAP. A person skilled in the art will
appreciate that a number of methods can be used to detect or
quantify the level of RNA products of the biomarkers within a
sample, including arrays, such as microarrays, RT-PCR (including
quantitative PCR), nuclease protection assays and Northern blot
analyses.
[0185] In another embodiment, the method further comprises
predicting a clinical response in the subject. In another
embodiment, the clinical response is at least about one, about two,
about three, or about five year progression/event-free
survival.
[0186] In some embodiments, the methods disclosed herein comprise
preventive treatment. For example, administering a treatment to a
subject that is likely to be afflicted by cancer in accordance with
the methods described herein. In some embodiments, a subject is
likely to be afflicted by cancer if the subject is characterized by
one or more of a high risk for a cancer, a genetic predisposition
to a cancer (e.g. genetic risk factors), a previous episode of a
cancer (e.g. new cancers and/or recurrence), a family history of a
cancer, exposure to a cancer-inducing agent (e.g. an environmental
agent), and pharmacogenomic information (the effect of genotype on
the pharmacokinetic, pharmacodynamic or efficacy profile of a
therapeutic).
[0187] In some embodiments, a subject is likely to be afflicted by
cancer if the subject is characterized by a high risk for a cancer.
In some embodiments, a subject is likely to be afflicted by cancer
if the subject is characterized by a genetic predisposition to a
cancer. In some embodiments, a genetic predisposition to a cancer
is a genetic clinical factor, as is known in the art. Such clinical
factors may include, by way of example, HNPCC, MLH1, MSH2, MSH6,
PMS1, PMS2 for at least colon, uterine, small bowel, stomach,
urinary tract cancers. In some embodiments, a subject is likely to
be afflicted by cancer if the subject is characterized by a
previous episode of a cancer. In some embodiments, the subject has
been afflicted with 1, 2, 3, 4, 5, or 6, previous episodes of
cancer. In some embodiments, a subject is likely to be afflicted by
cancer if the subject is characterized by a family history of a
cancer. In some embodiments, a parent and/or grandparent and/or
sibling and/or aunt/uncle and/or great aunt/great uncle, and/or
cousin has been or is afflicted with a cancer. In some embodiments,
a subject is likely to be afflicted by cancer if the subject is
characterized by exposure to a cancer-inducing agent (e.g. an
environmental agent). For example, exposing skin to strong sunlight
is a clinical factor for skin cancer. By way of example, smoking is
a clinical factor for cancers of the lung, mouth, larynx, bladder,
kidney, and several other organs.
[0188] Embodiments of this invention are further illustrated by the
following non-limiting examples.
EXAMPLES
Example 1
Whole Cell Assay Using Profiling Peptides in Permeabilized and
Non-Permeabilized Cells
[0189] Briefly, frozen MOLM-13 and OCI-AML3 cell stocks were
rapidly thawed, and cell viability was determined by Trypan Blue
exclusion. Cells were washed in PBS and resuspended in DTEB (or
MEB) buffer (135 mM Trehalose [or 150 mM Mannitol], 10 mM HEPES, 50
mM KCl, 20 .mu.M EGTA, 20 .mu.M EDTA, 0.1% BSA, and 5 mM
succinate). The profiling peptides and NOXA (AELPPEFAAQLRKIGDKVYC;
SEQ ID NO:16) were reconstituted in water to make working solutions
allowing for final concentrations of: SEQ ID NO:1 (100 .mu.M), SEQ
ID NO:14 (100 .mu.M), SEQ ID NO:15 (100 .mu.M), and NOXA (100
.mu.M). DMSO and FCCP (50 .mu.M) were used as negative and positive
peptide controls. Profiling peptides, controls, or NOXA (for
comparison purposes) were first added to a microplate. Cells
(4.times.10.sup.5) resuspended in DTEB or MEB buffers were then
added to a staining solution (20 mg/mL oligomycin, 50 mg/mL
digitonin, 40 .mu.M JC-1, 1 M 2-mercaptoethanol, DTEB or MEB
buffer), before being added to the wells of the microplate in the
above (for non-digitonin-treated samples, digitonin was not added
to the staining solution above). The mixture was then incubated for
up to 2 hours at 30.degree. C., in order for cell permeabilization
(if needed), delivery of peptides or compounds, and mitochondrial
depolarization to occur. Fluorescence signal of each well was
assessed using an Envision multilabel plate reader at Ex475/Em530
and Ex530/Em595. Additional cells that were not treated with a
peptide, but were treated with digitonin, were stained with
propidium iodide (PI) to assess whether cells were effectively
permeabilized by the digitonin. The normalized mitochondrial
potential of the median JC-1 red fluorescence of the wells was then
compared to DMSO (negative) and FCCP (positive) controls. The
results of this comparison are shown in FIGS. 1A-1D. FIGS. 1A and
1B show the results from the MOLM-13 cells, and FIGS. 1C and 1D
show the results from the OCI-AML3 cells. Additionally, FIGS. 1A
and 1C show the results of cells treated with digitonin, and FIGS.
1B and 1D show the results of cells that were not exposed to
digitonin. The labels for the graphs correspond as follows: (NC)
Negative Control--DMSO; (PC) Positive Control--FCCP; (1) NOXA, (2)
SEQ ID NO:1, (3) SEQ ID NO:14, and (4) SEQ ID NO:15.
[0190] As can be seen in FIGS. 1A-1D, cell-membrane
permeabilization is necessary for the NOXA peptide, commonly used
in similar assays, to induce apoptosis. However, the profiling
peptides of the present disclosure do not require the use of
digitonin. In the absence of digitonin, the profiling peptides of
the present disclosure with the cellular uptake moiety appended at
the amino-terminus of the Mcl-1 binding domain, SEQ ID NO:14, has
superior activity compared to the profiling peptide comprising the
Mcl-1 binding domain alone or the profiling peptide with the
cellular uptake moiety appended at the carboxy-terminus of the
Mcl-1 binding domain, SEQ ID NO: 15.
[0191] Additionally, the results shown in FIGS. 1A-1D show that the
decoupling agent and positive control, FCCP, requires a cell
permeabilizing agent, such as digitonin, to efficiently enter the
cell and fully initiate mitochondrial outer-membrane
permeabilization. Therefore, in the studies in MOLM-13 cells
lacking digitonin (FIG. 1B), SEQ ID NO:14 and SEQ ID NO:15 exceeded
the activity of the positive control.
[0192] The Z-factors for these assays were then assessed. Z-factors
are a valuable method for evaluating the reliability of a
biological assay. The goal for an assay is for a Z-factor to be 0.5
or higher. Evaluating the signal-to-noise ratios for the
sensitivity profiling assays by calculating the Z-factors shows
that using the profiling peptides of the present disclosure results
in a much more reliable assay (Table 2). Additionally, the removal
of the cell permeabilization step markedly improves the
signal-to-noise ratio (Table 3).
TABLE-US-00002 TABLE 2 Z-factors for NOXA and profiling peptides of
the present disclosure. Z' (NOXA) Z' (SEQ ID NO: 1) Z' (SEQ ID NO:
14) -3.4 0.32 0.50
TABLE-US-00003 TABLE 3 Z-factors for permeabilized cells and
non-permeabilized cells. Cell Line Z' (digitonin) Z' (no digitonin)
MOLM13 -1.1 0.6 OCI-AML3 0 0.6
Example 2
Titration of Concentrations of Profiling Peptides
[0193] In an effort to assess the concentration of profiling
peptides of the present disclosure used in the absence of a cell
permeabilization agent such that the Mcl-1 dependency percentage
values are comparable to those achieved using NOXA in the presence
of a cell permeabilization agent, the following experiment was
performed.
[0194] Briefly, frozen OCI-AML3 cell stocks were rapidly thawed,
and cell viability was determined by Trypan Blue exclusion, Cells
were washed in PBS and resuspended in DTEB (or MEB) buffer (135 mM
Trehalose [or 150 mM Mannitol], 10 mM HEPES, 50 mM KCl, 20 .mu.M
EGTA, 20 .mu.M EDTA, 0.1% BSA, 5 mM succinate). The profiling
peptides and NOXA (SEQ ID NO:16) were reconstituted in water to
make working solutions allowing for final concentrations of: SEQ ID
NO:1 (100 .mu.M), SEQ ID NO:1 (30 .mu.M), SEQ ID NO:1 (10 .mu.M),
and NOXA (100 .mu.M). DMSO and FCCP (50 .mu.M) were used as
negative and positive peptide controls. Profiling peptides,
controls, or NOXA (for comparison purposes) were first added to a
microplate. Cells (4.times.10.sup.5) resuspended in DTEB or MEB
buffers were then added to a staining solution (20 mg/mL
oligomycin, 50 mg/mL digitonin, 40 .mu.M JC-1, 1 M
2-mercaptoethanol, DTEB or MEB buffer), before being added to the
wells of the microplate in the above (for non-digitonin-treated
samples, digitonin was not added to the staining solution above).
The mixture is then incubated for up to 2 hours at 30.degree. C.,
in order for cell permeabilization (if needed), delivery of
peptides or compounds, and mitochondrial depolarization to occur.
Fluorescence signal of each well was assessed using an Envision
multilabel plate reader at Ex475/Em530 and Ex530/Em595. The median
JC-1 red fluorescence of the gated population was then was then
used to calculate % dependency as compared to DMSO (negative) and
FCCP (positive) controls.
[0195] Statistical Analysis: For each peptide, the Mcl-1 dependency
percentage was calculated using the following formula that
determines the dependency:
PP = [ 1 - ( Pep - PC NC - PC ) ] * 100 ##EQU00003##
Where PC is the fluorescence intensity of the positive control, NC
is the fluorescence intensity of the negative control, and Pep is
the fluorescence intensity of the peptide at the noted
concentration.
[0196] The results are shown in FIG. 2. The labels for the graphs
correspond as follows: (1) NOXA, (2) SEQ ID NO:1-100 .mu.M, (3) SEQ
ID NO:1-30 .mu.M, and (4) SEQ ID NO:1-10 .mu.M. As can be seen in
FIG. 2, in OCI-AML-3 cells, reducing the concentration of the
profiling peptide to 10 .mu.M brings the Mcl-1 dependency
percentage to a comparable value of the standard NOXA peptide at
100 .mu.M.
Example 3
Studies Using AML Subject-Based Cohorts
[0197] Peripheral blood and bone marrow samples from newly
diagnosed subjects with AML are obtained and analyzed by contacting
cells from the sample with any one or more of the profiling
peptides disclosed herein (e.g., SEQ ID NOS: 1, 14, or 15).
Subjects are treated with an alvocidib-containing regimen (e.g.,
FLAM: alvocidib (Flavopiridol), Ara-C and Mitoxantrone) if Mcl-1
dependency in their sample is above a predetermined amount (e.g.,
above 5%, 10%, 15%, 20%, 25%, or 30%). A statistically significant
percentage of treated subjects (e.g., greater than 75% or even
greater than 95%) have a complete response. Complete response is
characterized by less than 5% myeloblasts with normal maturation of
all cell lines, an ANC 1000/.mu.L and platelet count 100,000/.mu.L,
absence of blast in peripheral blood, absence of leukemic cells in
the marrow, clearance of cytogenetics associated with disease, and
clearance of previous extramedullary disease.
Sensitivity Profiling
[0198] Briefly, frozen, extracted leukocyte samples are rapidly
thawed, and cell viability determined by Trypan Blue exclusion.
Cells are washed in FACS buffer (1.times.PBS with 2% FBS) and
immunophenotyped using fluorescently labeled CD45, CD3, and CD20
monoclonal antibodies. Cells are then resuspended in Newmeyer
buffer (10 mM Trehalose, 10 mM HEPES, 80 mM KCl, 20 .mu.M EGTA, 20
.mu.M EDTA, 5 mM succinate, pH 7.4) for the perturbation step. The
profiling peptides are diluted in Newmeyer buffer to make working
solutions resulting in final concentrations of: SEQ ID NO:1 (100
.mu.M), SEQ ID NO:14 (100 .mu.M), and SEQ ID NO:15 (100 .mu.M).
DMSO and BAM-15 are used as negative and positive peptide controls.
Oligomycin is added to individual FACS tubes, followed by the
profiling peptides. Cells are then added to the FACS tubes and
incubated for 2 hours and 15 minutes at room temperature, in order
for delivery of peptides and mitochondrial depolarization to occur.
After the incubation, JC-1 dye is prepared in Newmeyer buffer and
added to directly to the treated cells. After 45 minutes of
incubation with JC-1, cells are analyzed on a three laser BD
FACSCanto II. AML Blasts will be gated based on three parameters:
1) singlet discrimination based on SSC, 2) CD45 dim and CD3/CD20
negative, and 3) SSC low. The median JC-1 red fluorescence of the
gated blast population is used to calculate % depolarization as
compared to DMSO (negative) and BAM-15 (positive) controls.
Individual Subject cytogenetic risk classification (Favorable,
Intermediate, and Adverse) is determined from the Cancer and
Leukemia Group B (CALGB) guidelines.
[0199] Statistical Analysis: For each peptide, the Mcl-1 dependency
percentage is calculated using the following formula that
determines the dependency based on the DMSO negative control as
completely unprimed and the BAM-15 as a 100% primed reference:
PP = [ 1 - ( Pep - PC NC - PC ) ] * 100 ##EQU00004##
Where PC is the AUC of the positive control, NC is the AUC of the
negative control, and Pep is the AUC of the peptide.
[0200] For analysis, all subjects not classified as CR are treated
as non-responders [Minimal Residual Disease (MRD), Partial
Remission (PR), and TF (treatment failure)]. Student's t-tests,
Mann-Whitney rank-sum non-parametric tests, multi-variate logistic
regression, and ROC curve analyses, between the profiling peptides
(and other tumor characteristics, such as cytogenetics, etc.) and
response, is calculated using GraphPad Prism Version 5.04 and
MedCalc Version 14.8.1.
Mitochondrial Profiling of AML Subject Samples Enrolled on FLAM
Protocols
[0201] The clinical variables obtained from the subjects is
compared to response to determine which, if any, of these factors
influence whether subjects would respond to the therapies or not.
The variable that is expected to have a significant association
with CR is the cytogenetic risk factor, where those with adverse
classifications being less likely to respond to the therapies. The
WBC, history of MDS, and which protocol was followed are
potentially significant.
[0202] It is expected that the addition of sensitivity profiling to
the analysis will greatly increase the ability to identify subjects
who would respond to alvocidib, either alone or in combination
therapy.
Example 4
Illustrative Assay Procedure
[0203] Mononuclear cells are isolated from primary bone marrow
aspirates using density-gradient centrifugation. Sample quality is
determined using trypan blue exclusion. Cells are then pelleted,
blocked in BSA and stained for markers specific to B and T cells,
as well as monocyte differentiation markers and blast-specific
markers. After staining, cells are pelleted and separated into
fluorescent-activated cell sorting (FACS) tubes and treated with
either water (negative control), CCCP (positive control) or SEQ ID
NO:14 (subject dependency). After 1 hour, DiOC.sub.6, a cationic
mitochondrial dye is added. One hour later the cells are analyzed
via flow cytometry. Blast cells are isolated by gating on the CD45
dim, CD13, CD33, and CD34 high population of each sample.
Dependency values are calculated using the median fluorescent
intensity (MFI) of DiOC.sub.6 in each sample according to the
following equation:
PP = [ 1 - ( Peptide MFI - CCCP MFI ) ( H 2 O MFI - CCCP MFI ) ] *
100 ##EQU00005##
Example 5
Illustrative Assay Procedure
[0204] Leukocytes are isolated from primary bone marrow aspirates
using ficoll preparation. Leukocytes may be fresh or frozen. If
frozen, the sample is thawed prior to testing. Incubate the sample
in RPMI with DNase 1 in 37.degree. C. incubator for 60 minutes.
Cells are then pelleted, blocked in PBS with 1% BSA for 15 minutes
on ice, followed by staining for 30 minutes on ice in the dark.
After staining, cells are pelleted and resuspended in DTEB with
Ryanodine (30 nM) and Oligomycin. After incubating in a 37.degree.
C. incubator for 30 minutes, the sample is separated into
fluorescent-activated cell sorting (FACS) tubes and treated with
either water (negative control), CCCP (positive control) or SEQ ID
NO: 14 (subject dependency) and incubated in a 37.degree. incubator
for 60 minutes. DiOC.sub.6, a cationic mitochondrial dye is then
added and incubated for 60 minutes in a 37.degree. C. incubator.
The cells are then analyzed via flow cytometry. Blast cells are
isolated by gating on the CD45 dim, CD13, CD33 and CD34 high
population of each sample. (See FIG. 3). Where indicated, cells may
be further gated using CD3 and CD20. Dependency values are
calculated using the median fluorescent intensity (MFI) of
DiOC.sub.6 in each sample according to the following equation:
PP = [ 1 - ( Peptide MFI - CCCP MFI ) ( H 2 O MFI - CCCP MFI ) ] *
100 ##EQU00006##
Example 6
Addition of Ryanodine to Assay
[0205] The effects of the addition of ryanodine to the assay
described in Example 5, was tested. Ryanodine was added to the
assay, and it was determined that this effect was due to
Tat-mediated calcium release from the ER and that treatment with 20
nM Ryanodine prevented nonspecific dye uptake. As shown in FIG. 4,
the addition of ryanodine reduced non-specificity and allows for an
increase in the profiling peptide concentration thereby bringing
the assay results into full parity with the NOXA assay results.
Example 7
Comparison of NOXA Priming Assay
[0206] NOXA assay results were compared to the results of the assay
described in Example 5. Table 4 shows a comparison of NOXA and SEQ
ID NO: 14 assay results from cell lines for which NOXA test results
have been published (Ishizawa et al. (2015) Mitochondrial Profiling
of Acute Myeloid Leukemia in the Assessment of Response to
Apoptosis Modulating Drugs. PLoS ONE 10(9): e0138377).
TABLE-US-00004 TABLE 4 Comparison of NOXA Assay Results and SEQ ID
NO: 14 Assay Results. SEQ ID NO: 14 Cell Line NOXA Priming %
Dependency % Concordance* MOLM-13 19.24 20.78 Y OCI-AML3 21.96
19.40 Y THP-1 11.28 11.70 Y HL-60 7.60 24.5 Y U-937 24.35 10.1 Y
KG-1 12.39 0.0 Y MV4-11 12.84 5.0 Y *Concordance based on cutoff of
.gtoreq.40% MCL-1 Dependency for both methods.
[0207] Table 5 shows a comparison of NOXA and SEQ ID NO: 14 assay
results from subject samples for which NOXA test results have been
produced. Of note, Samples 17 through 19 in Table 5 were collected
from subjects treated with FLAM and who showed complete response to
therapy.
TABLE-US-00005 TABLE 5 Comparison of NOXA Assay Results and SEQ ID
NO: 14 Assay Results. Reference SEQ ID Reference Lab SEQ ID NO: 14
Lab NOXA NO: 14 Result Con- Sample NOXA Result Dependency (Pos/
cordance* # Priming % (Pos/Neg)* % Neg) (Y/N) 1 14.0 Neg 18.0 Neg Y
2 60.0 Pos 44.9 Pos Y 3 16.0 Neg 53.8 Pos N 4 0.0 Neg 26.7 Neg Y 5
94.4 Pos 70.8 Pos Y 6 48.6 Pos 44.3 Pos Y 7 0.0 Neg 48.1 Pos N 8
23.5 Neg 17.0 Neg Y 9 79.1 Pos 71.8 Pos Y 10 38.0 Neg 0.0 Neg Y 11
43.3 Pos 40.6 Pos Y 12 0.0 Neg 38.3 Neg Y 13 0.0 Neg 35.2 Neg Y 14
0.0 Neg 2.6 Neg Y 15 7.0 Neg 0.0 Neg Y 16 7.0 Neg 0.1 Neg Y 17
10.89 Neg 62.4 Pos N 18 14.43 Neg 55.0 Pos N 19 0.0 Neg 27.8 Neg Y
*Concordance based on cutoff of .gtoreq.40% MCL-1 Dependency for
both methods.
[0208] As seen in Table 4 and Table 5, qualitative agreement has
been observed for 15 of 19 samples, qualitative agreement is
observed for 7 of 7 cell lines with published dependency values,
and concordance was observed for 22 of 26 total samples (subject
samples and cell lines). The overall observed accuracy was 81%
Specificity (17/21 negative samples), and 100% Sensitivity (5/5
positive samples).
[0209] As can be seen in Table 5, four samples which originally
tested negative subsequently tested positive using the SEQ ID NO:
14 assay. It is believed that these four samples were considered
positive due to the improved assay methodology and flow cytometry
gating strategy.
[0210] Results from this study indicate that the SEQ ID NO: 14
assay and the original NOXA assay are greater than 85% concordant
when using a cutoff of >40% dependency. FIG. 5 shows that the
rate of complete response for AML subjects with MCL-1 dependence
.gtoreq.40% is 100%.
[0211] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific embodiments described specifically
herein. Such equivalents are intended to be encompassed in the
scope of certain embodiments.
[0212] All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification or the attached Application Data Sheet are
incorporated herein by reference, in their entirety to the extent
not inconsistent with the present description. Aspects of the
embodiments can be modified, if necessary to employ concepts of the
various patents, applications and publications to provide yet
further embodiments. These and other changes can be made to the
embodiments in light of the above-detailed description.
[0213] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
16123PRTArtificial SequenceExemplary Mcl-1 Binding Domain sequence
1Arg Pro Glu Ile Trp Met Thr Gln Gly Leu Arg Arg Leu Gly Asp Glu1 5
10 15Ile Asn Ala Tyr Tyr Ala Arg 20223PRTArtificial
SequenceExemplary Mcl-1 Binding Domain sequence 2Arg Pro Glu Ile
Trp Leu Thr Gln Ser Leu Gln Arg Leu Gly Asp Glu1 5 10 15Ile Asn Ala
Tyr Tyr Ala Arg 20323PRTArtificial SequenceExemplary Mcl-1 Binding
Domain sequence 3Arg Pro Glu Ile Trp Leu Thr Gln His Leu Gln Arg
Leu Gly Asp Glu1 5 10 15Ile Asn Ala Tyr Tyr Ala Arg
20423PRTArtificial SequenceExemplary Mcl-1 Binding Domain sequence
4Arg Pro Glu Ile Trp Met Gly Gln Gly Leu Arg Arg Leu Gly Asp Glu1 5
10 15Ile Asn Ala Tyr Tyr Ala Arg 20523PRTArtificial
SequenceExemplary Mcl-1 Binding Domain sequence 5Arg Pro Glu Ile
Trp Leu Gly Gln Ser Leu Gln Arg Leu Gly Asp Glu1 5 10 15Ile Asn Ala
Tyr Tyr Ala Arg 20623PRTArtificial SequenceExemplary Mcl-1 Binding
Domain sequence 6Arg Pro Glu Ile Trp Leu Gly Gln His Leu Gln Arg
Leu Gly Asp Glu1 5 10 15Ile Asn Ala Tyr Tyr Ala Arg
20723PRTArtificial SequenceExemplary Mcl-1 Binding Domain sequence
7Arg Pro Glu Ile Trp Ile Thr Gln Glu Leu Arg Arg Ile Gly Asp Glu1 5
10 15Phe Asn Ala Tyr Tyr Ala Arg 20823PRTArtificial
SequenceExemplary Mcl-1 Binding Domain sequence 8Arg Pro Glu Ile
Trp Met Thr Gln Glu Leu Arg Arg Ile Gly Asp Glu1 5 10 15Phe Asn Ala
Tyr Tyr Ala Arg 20923PRTArtificial SequenceExemplary Mcl-1 Binding
Domain sequence 9Arg Pro Glu Ile Trp Ile Thr Gln Gly Leu Arg Arg
Ile Gly Asp Glu1 5 10 15Phe Asn Ala Tyr Tyr Ala Arg
201023PRTArtificial SequenceExemplary Mcl-1 Binding Domain sequence
10Arg Pro Glu Ile Trp Ile Thr Gln Glu Leu Arg Arg Leu Gly Asp Glu1
5 10 15Phe Asn Ala Tyr Tyr Ala Arg 201123PRTArtificial
SequenceExemplary Mcl-1 Binding Domain sequence 11Arg Pro Glu Ile
Trp Ile Thr Gln Glu Leu Arg Arg Ile Gly Asp Glu1 5 10 15Ile Asn Ala
Tyr Tyr Ala Arg 201211PRTArtificial SequenceTAT translocation
domain 12Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5
101316PRTArtificial SequenceANT translocation domain sequence 13Arg
Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10
151437PRTArtificial Sequenceprofiling peptide sequence 14Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Gly Arg Pro1 5 10 15Glu
Ile Trp Met Thr Gln Gly Leu Arg Arg Leu Gly Asp Glu Ile Asn 20 25
30Ala Tyr Tyr Ala Arg 351537PRTArtificial Sequenceprofiling peptide
sequence 15Arg Pro Glu Ile Trp Met Thr Gln Gly Leu Arg Arg Leu Gly
Asp Glu1 5 10 15Ile Asn Ala Tyr Tyr Ala Arg Gly Gly Gly Tyr Gly Arg
Lys Lys Arg 20 25 30Arg Gln Arg Arg Arg 351620PRTArtificial
SequenceNOXA sequence 16Ala Glu Leu Pro Pro Glu Phe Ala Ala Gln Leu
Arg Lys Ile Gly Asp1 5 10 15Lys Val Tyr Cys 20
* * * * *