Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF TREATING CANCER AND PREVENTING DRUG RESISTANCE
FIELD
[0001] Provided herein are therapies for the treatment of pathological
conditions, such as cancer, using
ALDH inhibitors and targeted therapeutics.
BACKGROUND
[0002] The relatively rapid acquisition of resistance to cancer drugs remains
a key obstacle to successful
cancer therapy. Substantial efforts to elucidate the molecular basis for such
drug resistance have
revealed a variety of mechanisms, including drug efflux, acquisition of drug
binding-deficient mutants
of the target, engagement of alternative survival pathways, epigenetic
alterations). Such mechanisms are
generally believed to reflect the existence of rare, stochastic, resistance-
conferring genetic alterations
within a tumor cell population that are selected during drug treatment. Sharma
et al., Cell 141(1):69-80
(2010). An increasingly observed phenomenon in cancer therapy is the so-called
"re-treatment
response." For example, some non-small cell lung cancer (NSCLC) patients who
respond well to
treatment with EGFR (epidermal growth factor receptor) tyrosine kinase
inhibitors (TKIs), and who later
experience therapy failure, demonstrate a second response to EGFR TKI re-
treatment after a "drug
holiday" Kurata et al., Ann. Oncol. 15:173-174 (2004); Yano et al., Oncol.
Res. 15:107-111 (2005).
Similar re-treatment responses are well established for several other anti-
cancer agents. Cara and
Tannock, Ann. Oncol. 12:23-27 (2001). Such findings suggest that acquired
resistance to cancer drugs
may involve a reversible "drug-tolerant" state, whose mechanistic basis
remains to be established.
[0003] The existence of a reversibly "drug-tolerant" cell population within
various human tumor cell
lines has been shown to be maintained via engagement of IGF-1 receptor
signaling and an altered
chromatin state that requires the histone demethylase KDM5A. While some
specific resistance-
conferring mutations have indeed been identified in many cancer patients
demonstrating acquired drug
resistance, the relative contribution of mutational and non-mutational
mechanisms to drug resistance,
and the role of tumor cell subpopulations remain somewhat unclear. New
treatment methods are needed
to successfully address heterogeneity within cancer cell populations and the
emergence of cancer cells
resistant to drug treatments.
SUMMARY
[0004] Provided herein are combinations comprising an ALDH inhibitor (e.g.,
disulfiram and/or
derivatives thereof) and a targeted therapeutic (e.g., TM). Provided herein
are methods of treating
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cancer in an individual comprising concomitantly administering to the
individual an effective amount of
an ALDH inhibitor and an effective amount of a targeted therapeutic. In some
embodiments, the
respective amounts of the ALDH inhibitor (e.g., disulfiram and/or derivatives
thereof) and the targeted
therapeutic (e.g., TKI) are effective to increase efficacy of a cancer
treatment comprising a targeted
therapeutic (e.g., TKI). For example, in some embodiments, the respective
amounts of the ALDH
inhibitor (e.g., disulfiram and/or derivatives thereof) and the targeted
therapeutic (e.g., TKI) are
effective to increased efficacy compared to a standard treatment comprising
administering an effective
amount of the targeted therapeutic (e.g., TKI) without (in the absence of) the
ALDH inhibitor (e.g.,
disulfiram and/or derivatives thereof). In some embodiments, the respective
amounts of the ALDH
inhibitor (e.g., disulfiram and/or derivatives thereof) and the targeted
therapeutic (e.g., TKI) are
effective to increased response (e.g., complete response) compared to a
standard treatment comprising
administering an effective amount of the targeted therapeutic (e.g., TM)
without (in the absence of) the
ALDH inhibitor (e.g., disulfiram and/or derivatives thereof). In some
embodiments of any of the
methods, the ALDH inhibitor is disulfiram and/or derivatives thereof. In some
embodiments, the ALDH
inhibitor is disulfiram. In some embodiments of any of the methods, the ALDH
inhibitor is gossypol
and/or derivatives thereof. In some embodiments, the ALDH inhibitor is
gossypol.
100051 Also provided herein are methods of increasing efficacy of a cancer
treatment comprising a
targeted therapeutic in an individual comprises concomitantly administering to
the individual an
effective amount of the targeted therapeutic and an effective amount of an
ALDH inhibitor.
[0006] Provided herein are methods of treating cancer in an individual wherein
cancer treatment
comprising concomitantly administering to the individual an effective amount
of a targeted therapeutic
and an effective amount of an ALDH inhibitor, wherein the cancer treatment has
increased efficacy
compared to a standard treatment comprising administering an effective amount
of the targeted
therapeutic without (in the absence of) the targeted therapeutic. Further,
provided herein are methods of
delaying and/or preventing development of cancer resistant to a targeted
therapeutic in an individual,
comprising concomitantly administering to the individual an effective amount
of an ALDH inhibitor and
an effective amount of the targeted therapeutic.
[0007] Provided herein are methods of treating an individual with cancer who
has increased likelihood
of developing resistance to a targeted therapeutic comprising concomitantly
administering to the
individual an effective amount of an ALDH inhibitor and an effective amount of
the targeted
therapeutic. In addition, provided herein are methods of increasing
sensitivity to a targeted therapeutic in
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an individual with cancer comprising concomitantly administering to the
individual an effective amount
of an ALDH inhibitor and an effective amount of the targeted therapeutic.
[0008] In another aspect, provided herein are methods of extending the period
of an targeted therapeutic
sensitivity in an individual with cancer comprising concomitantly
administering to the individual an
effective amount of an ALDH inhibitor and an effective amount of targeted
therapeutic. In addition,
provided herein are methods of extending the duration of response to a
targeted therapeutic in an
individual with cancer comprising concomitantly administering to the
individual an effective amount of
an ALDH inhibitor and an effective amount of the targeted therapeutic.
[0010] In some embodiments of any of the methods, the ALDH inhibitor is a
small molecule ALDH
inhibitor. In some embodiments, the small molecule ALDH inhibitor is
disulfiram or an ALDH-
inhibiting derivative or metabolite thereof. In some embodiments, the ALDH
inhibitor is N,N-
diethylfldiethylcarbamothioyl) disulfanyl[carbothioamide or pharmaceutically
acceptable salt thereof. In
some embodiments, the ALDH inhibitor is N,N-diethylfldiethylcarbamothioyl)
disulfanyl[carbothioamide. In some embodiments of any of the methods, the ALDH
inhibitor is gossypol
and/or or an ALDH-inhibiting derivative or metabolite thereof. In some
embodiments, the ALDH
inhibitor is gossypol. In some embodiments, the ALDH inhibitor is 2,2'-bis-
(Formy1-1,6,7-trihydroxy-5-
isopropy1-3-methylnaphthalene) or pharmaceutically acceptable salt thereof. In
some embodiments, the
ALDH inhibitor is 2,2'-bis-(Formy1-1,6,7-trihydroxy-5-isopropy1-3-
methylnaphthalene).
[0011] In some embodiments of any of the methods, the targeted therapeutic is
a tyrosine kinase
inhibitor (TM). In some embodiments, the TKI is an EGFR inhibitor, HER2
inhibitor, MET inhibitor,
ALK inhibitor, BRAF inhibitor, ROS1 inhibitor, and/or MEK inhibitor. In some
embodiments, the TKI
is a receptor tyrosine kinase inhibitor (RTKI). In some embodiments, the RTKI
is an EGFR inhibitor,
HER2 inhibitor, MET inhibitor, and/or ALK inhibitor. In some embodiments, the
inhibitor is an
antibody inhibitor, a small molecule inhibitor, a binding polypeptide
inhibitor, and/or a polynucleotide
antagonist. In some embodiments, the TKI is N-(3-ethynylpheny1)-6,7-bis(2-
methoxyethoxy)quinazolin-
4-amine or a pharmaceutically acceptable salt thereof (e.g., erlotinnib). In
some embodiments, the TM
is N-(4-(3-fluorobenzyloxy)-3-chloropheny1)-6-(54(2-
(methylsulfonyl)ethylamino)nethypfuran-2-
yllquinazolin-4-amine,di4-methylbenzenesulfonate or a pharmaceutically
acceptable salt thereof (e.g.,
lapatinib). In some embodiments, the TM is (S)-N-(2,3-dihydroxypropy1)-3-(2-
fluoro-4-
iodophenylamino)isonicotinamide) or a pharmaceutically acceptable salt thereof
(e.g., AS703026). In
some embodiments, the TM is vemurafenib. In some embodiments, the TM is 3-((R)-
1-(2,6-dichloro-3-
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fluorophenyllethoxy)-5-(1-(piperidin-4-y1)-1H-pyrazol-4-yppyridin-2-amine or a
pharmaceutically
acceptable salt thereof (e.g., crizotinib).
[0012] In some embodiments of any of the methods, the cancer is gastric
cancer, lung cancer (e.g., non-
small cell lung cancer (NSCL)), colorectal cancer (e.g., colon cancer and/or
rectal cancer), or basel cell
carcinoma.
[0013] In one embodiment, there is provided a pharmaceutical product
comprising a) as a first
component an effective amount of an ALDH inhibitor, and b) as a second
component an effective
amount of a targeting agent (targeted therapeutic) for the concomitant or
sequential use for the treatment
of cancer.
[0014] In another embodiment, there is provided the pharmaceutical product as
indicated above,
wherein the ALDH inhibitor is a small molecule ALDH inhibitor. In another
embodiment, there is
provided the pharmaceutical product as indicated above, wherein the ALDH
inhibitor is disulfiram or an
ALDH-inhibiting derivative or metabolite thereof. In another embodiment, there
is provided the
pharmaceutical product as indicated above, wherein the ALDH inhibitor is N,N-
diethylfldiethylcarbamothioyl) disulfanyllcarbothioamide or pharmaceutically
acceptable salt thereof. In
another embodiment, there is provided the pharmaceutical product as indicated
above, wherein the
ALDH inhibitor is N,N-diethylfldiethylcarbamothioyl)
disulfanyllcarbothioamide. In another
embodiment, there is provided the pharmaceutical product as indicated above,
wherein the ALDH
inhibitor is 2,2'-bis-(Formy1-1,6,7-trihydroxy-5-isopropy1-3-
methylnaphthalene) or pharmaceutically
acceptable salt thereof. In another embodiment, there is provided the
pharmaceutical product as
indicated above, wherein the ALDH inhibitor is 2,2'-bis-(Formy1-1,6,7-
trihydroxy-5-isopropy1-3-
methylnaphthalene).
[0015] In another embodiment, there is provided the pharmaceutical product as
indicated above,
wherein the targeted therapeutic is a tyrosine kinase inhibitor (TKI). In
another embodiment, there is
provided the pharmaceutical product as indicated above, wherein the TKI is an
EGFR inhibitor, HER2
inhibitor, MET inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor,
and/or MEK inhibitor. In
another embodiment, there is provided the pharmaceutical product as indicated
above, wherein the TKI
is a receptor tyrosine kinase inhibitor (RTKI). In another embodiment, there
is provided the
pharmaceutical product as indicated above, wherein the RTKI is an EGFR
inhibitor, HER2 inhibitor,
MET inhibitor, and/or ALK inhibitor. In another embodiment, there is provided
the pharmaceutical
product as indicated above, wherein the inhibitor is an antibody inhibitor, a
small molecule inhibitor, a
binding polypeptide inhibitor, and/or a polynucleotide antagonist. In another
embodiment, there is
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provided the pharmaceutical product as indicated above, wherein the TKI is N-
(3-ethynylpheny1)-6,7-
bis(2-methoxyethoxy)quinazolin-4-amine or a pharmaceutically acceptable salt
thereof, in particular
erlotinib. In another embodiment, there is provided the pharmaceutical product
as indicated above,
wherein the TM is N-(4-(3-fluorobenzyloxy)-3-chloropheny1)-6-(5-((2-
(methylsulfonyl)ethylamino)methyllfuran-2-yllquinazolin-4-amine,di4-
methylbenzenesulfonate or a
pharmaceutically acceptable salt thereof, in particular lapatinib. In another
embodiment, there is
provided the pharmaceutical product as indicated above, wherein the TM is (S)-
N-(2,3-
dihydroxypropy1)-3-(2-fluoro-4-iodophenylamino)isonicotinamide) or a
pharmaceutically acceptable
salt thereof, in particular AS703026. In another embodiment, there is provided
the pharmaceutical
product as indicated above, wherein the TKI is vemurafenib. In another
embodiment, there is provided
the pharmaceutical product as indicated above, wherein the TKI is 3-((R)-1-
(2,6-dichloro-3-
fluorophenyl)ethoxy)-5-(1-(piperidin-4-y1)-1H-pyrazol-4-yppyridin-2-amine or a
pharmaceutically
acceptable salt thereof, in particular crizotinib.
[0016] In another embodiment, there is provided the pharmaceutical product as
indicated above,
wherein the cancer is gastric cancer, lung cancer, non-small cell lung cancer
(NSCL), colon cancer
and/or rectal cancer, or basel cell carcinoma.
[0017] In addition to providing improved treatment for cancer, administration
of certain combinations
described herein may improve the quality of life for a patient compared to the
quality of life experienced
by the same patient receiving a different treatment. For example,
administration of a combination of a
targeted therapeutic (e.g., TKI), and a ALDH inhibitor (e.g., disulfiram
and/or derivatives thereof)õ as
described herein to an individual may provide an improved quality of life
compared to the quality of life
the same patient would experience if they received only the targeted
therapeutic as therapy. For
example, the combined therapy with the combination described herein may lower
the dose of targeted
therapeutic needed, thereby lessening the side-effects associated with the
therapeutic (e.g. nausea,
vomiting, hair loss, rash, decreased appetite, weight loss, etc.). The
combination may also cause reduced
tumor burden and the associated adverse events, such as pain, organ
dysfunction, weight loss, etc.
Accordingly, one aspect of the invention provides ALDH inhibitor (e.g.,
disulfiram and/or derivatives
thereof) for therapeutic use for improving the quality of life of a patient
treated for a cancer with a
targeted therapeutic (e.g., TKI). Accordingly, another aspect of the invention
provides ALDH inhibitor
(e.g., disulfiram and/or derivatives thereof) for therapeutic use for
improving the quality of life of an
individual treated for a cancer disorder with a targeted therapeutic, or a
pharmaceutically acceptable salt
thereof.
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BRIEF DESCRIPTION OF THE FIGURES
[0018] Figure 1A-C. I Drug Tolerant Gastric Cancer Cells Express High level of
ALDH1A1. (A) ALDH
activity was measured in Kato II parental and crizotinib (luM) tolerant cells
using Aldefluor assay
(Stem Cell Technology). The bodipy labeled substrate emit fluorescence when
oxidized by ALDH to
corresponding acid. Photographs showing enrichment of ALDHhigh cells following
crizotinib treatment.
Arrows marked the ALDHhigh cells in the parental population. (B) RNA
expression level of 19 ALDH
family members was determined using oligonucleotide based microarrays. Kato II
parental cells were
incubated with Aldefluor substrate at 37 C for 30 and ALDHhigh and ALDH1'
cells were sorted by
flowcytometry. Gene expression analysis was performed using RNAs isolated from
ALDHhigh and
ALDH1' cells. The bar graph illustrates differential expression of only one
ALDH family members,
ALDH1A1 in ALDHhigh cells. (C) Immunoblots illustrating higher expression
level of ALDH1A1
protein in ALDHhigh cells and in crizotinib tolerant Kato II and GTL-16 cells
compared to ALDHlow cells
and the parental cells respectively.
[0019] Figure 2A-C. I ALDH inhibitor Disulfiram eliminates drug tolerant
cells. Parental Kato 11(A)
and GTL-16 (B) cells were treated with luM crizotinib for 25 days and
disulfiram, 200nM, was added
either on dayl (d1) or at different time intervals during crizotinib
treatment. (C) Parental PC9 cells were
treated with erlotinib and disulfiram, 200nM, which was added at different
time points during erlotinib
treatment. The bar graphs representing quantitative measurements made from
triplicate wells per
treatment show lethal effect of disulfiram on drug tolerant cells as measured
by Syto60 viability assay
(Wilson et al., 2011). The data is expressed as fractions of no treatment
control, the error bar reflects
SEM values.
[0020] Figure 3A-B. I Disulfiram kills drug tolerant cells of various cancer
types. (A) Illutrating the
effect of disulfiram and targeted cancer drug combinations on cancer cells of
various tissue origins,
which are addicted to different oncogenes. The cancer cells sensitive to
erlotinib (HCC827 and
HCC4006), lapatinib (HCC1419, SKBR3 and MDA-MB-175 v2), MEK inhibitor A5703026
(A549 and
EBC-1) and BRAF inhibitor vemurafenib (Colo-205) were treated with appropriate
drug, either alone or
in combination with 200nM-300nM disulfiram. The duration of treatment varied
from 11 to 25 days
depending on the time it took for TKI+disulfiram treatment to kill almost all
drug tolerant cells. (B) The
bar graphs representing quantitative measurements (triplicate wells per
treatment) of the effect of
targeted cancer drug, disulfiram and their combination as measured by Syto60
viability assay illustrate
the dependence of drug tolerant cells, in general, on ALDH for their survival
. The data is expressed as
fractions of no treatment control, the error bar reflects SEM values.
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[0021] Figure 4A-C. I Increased Mitochondrial Respiration and ROS Level in
Drug Tolerant Cells. (A)
ROS level was detected using fluorescein based H2DCFDA reagent (Molecular
probes) and measured
by flow cytometry. PC9-derived and GTL-16 derived DTPs were treated with
disulfiram (200nM) and
NAC (5mM) in the presence of TM for 48h and the effect of TKI, disulfiram and
NAC were measured.
The bar graphs representing fold change in ROS level compared to untreated
parental cells illustrates
role of ALDH as ROS scavenger. (B) Oxygen consumption rate (OCR) and
extracellular acidification
rate (ECAR) to measure energy production by mitochondrial respiration and
glycolysis respectively of
GTL-16 and PC9-derived DTPs was determined using Seahorse XF 96. The bar
graphs illustrate
increased use of mitochondrial respiration in the drug tolerant cell. (C)
Immunoblots illustrates
increased double stranded DNA breaks and activation of DNA repair mechanisms
as a result of high
ROS level in GTL-16 and PC9 drug tolerant cells.
[0022] Figure 5A-C. I ROS scavenger N-acetyl cysteine reverses the effect of
disulfiram. (A) PC9 and
(B) GTL-16 parental cells were treated for 15 days with erlotinib and
crizotinib respectively either alone
or in combination with disulfiram, NAC and disulfiram+NAC. The bar graphs
depicting the effect of
these treatments on cell viability illustrate the ability of NAC to rescue the
lethal effect of disulfiram on
cell viability. (C1-2) GTL-16-derived DTPs were treated with disulfiram and
NAC for 48h. Immunoblot
data demonstrating reversal of the effect of disulfiram on yH2A.x, BimEL,
BimS, and cleaved PARP
level by NAC. Disulfiram increases ROS level and induce apoptosis in DTPs.
[0023] Figure 6A-B. I Disulfiram delays Tumor Relapse. (A) PC9 parental cells
were treated with
erlotinib alone or in combination with disulfiram. After 6 days of TKI
treatment the DTPs were allowed
to grow in erlotinib-free growth media with or without disulfiram. The cell
viability data demonstrating
delayed growth of PC9-derived DTPs that received disulfiram first 6 days and
even longer delay in
growth for those which continued to receive disulfiram for four subsequent
days. The bar graph shows
the effect of DS on PC9-derived DTPs measured from triplicate wells and
expressed as mean +/- SD.
(B) In vivo data showing delayed relapse of PC9 derived tumors in xenograft
mice model treated with
erlotinib and disulfiram combo compared to erlotinib alone.
[0024] Figure 7A-C. I Pre-treatment with disulfiram is not sufficient to kill
all DTPs. PC9 (A) and GTL-
16 (B) parental cells were treated with DS first for 3 or 6 days and then with
erlotinib for PC9 cells and
crizotinib for GTL-16 cells in absence of DS. Syto60 cell viability staining
showing brief exposure to
DS reduces the number of DTPs but do not eliminate them. (C) Knock-down of
ALDH1A1 alone has no
significant effect on crizotinib drug sensitivity in GLT16 cells. Graph shows
relative expression of
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ALDH1A1 in GTL16 cells using multiple shRNAs. Table shows relative percentage
of GTL16-derived
DTPs upon treatment with crizotinib in ALDH1A1 knock down cells.
[0025] Figure 8A-C. I Drug treatment induces expression of multiple ALDH
family members in
multiple cell lines using multiple TKI inhibitors. (A) Relative change in RNA
expression of ALDH
family members in GTL16-derived DTPs (parental cells treated with crizotinib)
and PC9-derived DTPs
(parental cells treated with erlotinib). (B) RNA expression levels of ALDH
family members in GTL16
parental cells and GTL16-derived DTPs (parental cells treated with crizotinib)
and PC9 parental cells
and PC9-derived DTPs (parental cells treated with erlotinib). (C) ALDH1A1 is
upregulated GTL-16-
derived DTP (parental cells treated with crizotinib) compared to GTL-16
parental cells. Conversely,
ALDH1A1 expression is not significantly expressed in PC9-derived DTP (parental
cells treated with
erlotinib) or PC9 parental cells, and there is no significant change in
expression levels of ALDH1A1 in
PC9-derived DTP (parental cells treated with erlotinib) compared to PC9
parental cells.
[0026] Figure 9. I ALDH inhibitor Gossypol significantly reduces drug tolerant
cells. (A) Parental PC9
cells were treated with 2uM erlotinib and 1.5uM Gossypol either alone or
combination for 8 days. (B)
Parental GTL-16 cells were treated with luM crizotinib and 1.5uM Gossypol
either alone or
combination for 17 days. The bar graphs representing quantitative measurements
of cell viability by
Syto60 assay from triplicate wells per treatment show similar to DS but weaker
effect of gossypol on
drug tolerant cells.
DETAILED DESCRIPTION
I. Definitions
[0027] As used herein, the term "ALDH" or "aldehyde dehydrogenase" refers to
an enzyme or a class of
enzymes which are capable of oxidizing aldehydes. Aldehyde dehydrogenase
(ALDH) (Enzyme
Commission 1.2.1.3) is an enzyme responsible for oxidizing intracellular
aldehydes and plays a role in
metabolism of ethanol, vitamin A, cyclophosphamide and other
oxazaphosphorines. Examples of ALDH
enzymes in humans include ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1,
ALDH1L2,
ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1, ALDH6A1,
ALDH7A1, ALDH8A1, ALDH9A1, ALDH16A1, ALDH18A1. The term "wild type ALDH"
generally
refers to a polypeptide comprising the amino acid sequence of a naturally
occurring ALDH protein.
[0028] The terms "HER2", "ErbB2" "c-Erb-B2" are used interchangeably. Unless
indicated otherwise,
the terms "ErbB2" "c-Erb-B2" and "HER2" when used herein refer to the human
protein, and "erbB2,"
"c-erb-B2," and "her2" refer to human gene. The human erbB2 gene and ErbB2
protein are, for
example, described in Semba et al., PNAS (USA) 82:6497-6501 (1985) and
Yamamoto et al. Nature
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319:230-234 (1986) (Genebank accession number X03363). ErbB2 comprises four
domains (Domains
1-4). The term "wild type HER2" generally refers to a polypeptide comprising
the amino acid sequence
of a naturally occurring HER2 protein.
[0029] By "EGFR" is meant the receptor tyrosine kinase polypeptide Epidermal
Growth Factor
Receptor which is described in Ullrich et al, Nature (1984) 309:418425,
alternatively referred to as Her-
1 and the c-erbB gene product, as well as variants thereof such as EGFRvIII.
Variants of EGFR also
include deletional, substitutional and insertional variants, for example those
described in Lynch et al.
(NEJM 2004, 350:2129), Paez et al. (Science 2004, 304:1497), Pao et al. (PNAS
2004, 101:13306). The
term "wild type EGFR" generally refers to a polypeptide comprising the amino
acid sequence of a
naturally occurring EGFR protein.
[0030] The term "c-met" or "Met", as used herein, refers, unless indicated
otherwise, to any native or
variant (whether native or synthetic) c-met polypeptide. The term "wild type c-
met" generally refers to
a polypeptide comprising the amino acid sequence of a naturally occurring c-
met protein.
[0031] The term "BRAF", as used herein, refers, unless indicated otherwise, to
any native or variant
(whether native or synthetic) BRAF polypeptide. The term "wild type BRAF"
generally refers to a
polypeptide comprising the amino acid sequence of a naturally occurring BRAF
protein.
[0032] The term "ALK" refers to Anaplastic Lymphoma Kinase. ALK (Anaplastic
Lymphoma Kinase)
(GenBank accession Number: AB209477, UniProt Accession No. Q9UM73) is a
receptor tyrosine
kinase. This protein (which is 1620 amino acids long in humans) has a
transmembrane domain in the
central part and has a carboxyl-terminal tyrosine kinase region and an amino-
terminal extracellular
domain (Oncogene. 1997 Jan. 30; 14 (4): 439-49). See Pulford et al., J. of
Cellular Physiol., 199:330-
358, 2004 for a comprehensive review relating to ALK. The full-length ALK
sequence is disclosed in
U.S. Pat. No. 5,770,421. The term "wild type ALK" generally refers to a
polypeptide comprising the
amino acid sequence of a naturally occurring ALK protein.
[0033] An "antagonist" (interchangeably termed "inhibitor") of a polypeptide
of interest is an agent that
interferes with activation or function of the polypeptide of interest, e.g.,
partially or fully blocks,
inhibits, or neutralizes a biological activity mediated by a polypeptide of
interest. For example, an
antagonist of polypeptide X may refers to any molecule that partially or fully
blocks, inhibits, or
neutralizes a biological activity mediated by polypeptide X. Examples of
inhibitors include antibodies;
ligand antibodies; small molecule antagonists; antisense and inhibitory RNA
(e.g., shRNA) molecules.
Preferably, the inhibitor is an antibody or small molecule which binds to the
polypeptide of interest. In a
particular embodiment, an inhibitor has a binding affinity (dissociation
constant) to the polypeptide of
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interest of about 1,000 nM or less. In another embodiment, inhibitor has a
binding affinity to the
polypeptide of interest of about 100 nM or less. In another embodiment, an
inhibitor has a binding
affinity to the polypeptide of interest of about 50 nM or less. In a
particular embodiment, an inhibitor is
covalently bound to the polypeptide of interest. In a particular embodiment,
an inhibitor inhibits
signaling of the polypeptide of interest with an IC50 of 1,000 nM or less. In
another embodiment, an
inhibitor inhibits signaling of the polypeptide of interest with an IC50 of
500 nM or less. In another
embodiment, an inhibitor inhibits signaling of the polypeptide of interest
with an IC50 of 50 nM or less.
In certain embodiments, the antagonist reduces or inhibits, by at least 10%,
20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or more, the expression level or biological activity of the
polypeptide of interest.
[0034] As used herein, the term "targeted therapeutic" refers to a therapeutic
agent that binds to
polypeptide(s) of interest and inhibits the activity and/or activation of the
specific polypeptide(s) of
interest. Examples of such agents include antibodies and small molecules that
bind to the polypeptide of
interest. In some embodiments, the targeted therapeutic is a TM. In some
embodiments, the TKI is a
RTKI.
[0035] A "tyrosine kinase inhibitor" or "TKI" refers to an agent that
interferes with activation or
function mediated by the tyrosine kinase activity of a tyrosine kinase, e.g.,
partially or fully blocks,
inhibits, or neutralizes a biological activity mediated by the tyrosine kinase
activity of a tyrosine kinase.
[0036] A "receptor tyrosine kinase inhibitor" or "RTKI" refers to an agent
that interferes with activation
or function mediated by the tyrosine kinase activity of a receptor tyrosine
kinase, e.g., partially or fully
blocks, inhibits, or neutralizes a biological activity mediated by the
tyrosine kinase activity of a receptor
tyrosine kinase.
[0037] The term "polypeptide" as used herein, refers to any native polypeptide
of interest from any
vertebrate source, including mammals such as primates (e.g., humans) and
rodents (e.g., mice and rats),
unless otherwise indicated. The term encompasses "full-length," unprocessed
polypeptide as well as
any form of the polypeptide that results from processing in the cell. The term
also encompasses
naturally occurring variants of the polypeptide, e.g., splice variants or
allelic variants.
[0038] "Polynucleotide," or "nucleic acid," as used interchangeably herein,
refer to polymers of
nucleotides of any length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their analogs, or any
substrate that can be
incorporated into a polymer by DNA or RNA polymerase, or by a synthetic
reaction. A polynucleotide
may comprise modified nucleotides, such as methylated nucleotides and their
analogs. If present,
modification to the nucleotide structure may be imparted before or after
assembly of the polymer. The
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sequence of nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be
further modified after synthesis, such as by conjugation with a label. Other
types of modifications
include, for example, "caps", substitution of one or more of the naturally
occurring nucleotides with an
analog, internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with
charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), those containing pendant
moieties, such as, for example,
proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,
etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron,
oxidative metals, etc.), those containing alkylators, those with modified
linkages (e.g., alpha anomeric
nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s).
Further, any of the hydroxyl
groups ordinarily present in the sugars may be replaced, for example, by
phosphonate groups, phosphate
groups, protected by standard protecting groups, or activated to prepare
additional linkages to additional
nucleotides, or may be conjugated to solid or semi-solid supports. The 5 and
3' terminal OH can be
phosphorylated or substituted with amines or organic capping group moieties of
from 1 to 20 carbon
atoms. Other hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also
contain analogous forms of ribose or deoxyribose sugars that are generally
known in the art, including,
for example, 2.-0-methyl-, 2.-0-allyl, T-fluoro- or T-azido-ribose,
carbocyclic sugar analogs, a-
anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose
sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as
methyl riboside. One or
more phosphodiester linkages may be replaced by alternative linking groups.
These alternative linking
groups include, but are not limited to, embodiments wherein phosphate is
replaced by P(0)S("thioate"),
P(S)S ("dithioate"), "(0)NR2 ("amidate"), P(0)R, P(0)0R., CO or CH2
("formacetal"), in which each R
or R. is independently H or substituted or unsubstituted alkyl (1-20 C)
optionally containing an ether (-
0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all
linkages in a polynucleotide need
be identical. The preceding description applies to all polynucleotides
referred to herein, including RNA
and DNA.
[0039] The term "small molecule" refers to any molecule with a molecular
weight of about 2000
daltons or less, preferably of about 500 daltons or less.
[0040] An "isolated" antibody is one which has been separated from a component
of its natural
environment. In some embodiments, an antibody is purified to greater than 95%
or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric
focusing (IEF), capillary
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electrophoresis) or chromatographic (e.g., ion exchange or reverse phase
HPLC). For review of methods
for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B
848:79-87 (2007).
[0041] The term "antibody" herein is used in the broadest sense and
encompasses various antibody
structures, including but not limited to monoclonal antibodies, polyclonal
antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so long as
they exhibit the desired
antigen-binding activity.
[0042] The terms anti-polypeptide of interest antibody and "an antibody that
binds to" a polypeptide of
interest refer to an antibody that is capable of binding a polypeptide of
interest with sufficient affinity
such that the antibody is useful as a diagnostic and/or therapeutic agent in
targeting a polypeptide of
interest. In one embodiment, the extent of binding of an anti-polypeptide of
interest antibody to an
unrelated, non- polypeptide of interest protein is less than about 10% of the
binding of the antibody to a
polypeptide of interest as measured, e.g., by a radioimmunoassay (RIA). In
certain embodiments, an
antibody that binds to a polypeptide of interest has a dissociation constant
(Kd) of < 1pM, < 100 nM, <
10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10-8 M or less, e.g.,
from 10-8 M to 10-13 M,
e.g., from 10-9 M to 10-13 M). In certain embodiments, an anti- polypeptide of
interest antibody binds to
an epitope of a polypeptide of interest that is conserved among polypeptides
of interest from different
species.
[0043] A "blocking antibody" or an "antagonist antibody" is one which inhibits
or reduces biological
activity of the antigen it binds. Preferred blocking antibodies or antagonist
antibodies substantially or
completely inhibit the biological activity of the antigen.
[0044] "Affinity" refers to the strength of the sum total of noncovalent
interactions between a single
binding site of a molecule (e.g., an antibody) and its binding partner (e.g.,
an antigen). Unless indicated
otherwise, as used herein, "binding affinity" refers to intrinsic binding
affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and antigen).
The affinity of a molecule X
for its partner Y can generally be represented by the dissociation constant
(Kd). Affinity can be
measured by common methods known in the art, including those described herein.
Specific illustrative
and exemplary embodiments for measuring binding affinity are described in the
following.
[0045] An "antibody fragment" refers to a molecule other than an intact
antibody that comprises a
portion of an intact antibody that binds the antigen to which the intact
antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH,
F(ab.)2; diabodies; linear
antibodies; single-chain antibody molecules (e.g., scFv); and multispecific
antibodies formed from
antibody fragments.
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[0046] An "antibody that binds to the same epitope" as a reference antibody
refers to an antibody that
blocks binding of the reference antibody to its antigen in a competition assay
by 50% or more, and
conversely, the reference antibody blocks binding of the antibody to its
antigen in a competition assay by
50% or more.
[0047] The term "chimeric" antibody refers to an antibody in which a portion
of the heavy and/or light
chain is derived from a particular source or species, while the remainder of
the heavy and/or light chain
is derived from a different source or species.
[0048] The terms "full length antibody," "intact antibody," and "whole
antibody" are used herein
interchangeably to refer to an antibody having a structure substantially
similar to a native antibody
structure or having heavy chains that contain an Fc region.
[0049] The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population
of substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population are
identical and/or bind the same epitope, except for possible variant
antibodies, e.g., containing naturally
occurring mutations or arising during production of a monoclonal antibody
preparation, such variants
generally being present in minor amounts. In contrast to polyclonal antibody
preparations, which
typically include different antibodies directed against different determinants
(epitopes), each monoclonal
antibody of a monoclonal antibody preparation is directed against a single
determinant on an antigen.
Thus, the modifier "monoclonal" indicates the character of the antibody as
being obtained from a
substantially homogeneous population of antibodies, and is not to be construed
as requiring production
of the antibody by any particular method. For example, the monoclonal
antibodies to be used in
accordance with the present invention may be made by a variety of techniques,
including but not limited
to the hybridoma method, recombinant DNA methods, phage-display methods, and
methods utilizing
transgenic animals containing all or part of the human immunoglobulin loci,
such methods and other
exemplary methods for making monoclonal antibodies.
[0050] A "human antibody" is one which possesses an amino acid sequence which
corresponds to that
of an antibody produced by a human or a human cell or derived from a non-human
source that utilizes
human antibody repertoires or other human antibody-encoding sequences. This
definition of a human
antibody specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0051] A "humanized" antibody refers to a chimeric antibody comprising amino
acid residues from
non-human HVRs and amino acid residues from human FRs. In certain embodiments,
a humanized
antibody will comprise substantially all of at least one, and typically two,
variable domains, in which all
or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-
human antibody, and all or
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substantially all of the FRs correspond to those of a human antibody. A
humanized antibody optionally
may comprise at least a portion of an antibody constant region derived from a
human antibody. A
"humanized form" of an antibody, e.g., a non-human antibody, refers to an
antibody that has undergone
humanization.
[0052] An "immunoconjugate" is an antibody conjugated to one or more
heterologous molecule(s),
including but not limited to a cytotoxic agent.
[0053] "Individual response" or "response" can be assessed using any endpoint
indicating a benefit to
the individual, including, without limitation, (1) inhibition, to some extent,
of disease progression (e.g.,
cancer progression), including slowing down and complete arrest; (2) a
reduction in tumor size; (3)
inhibition (i.e., reduction, slowing down or complete stopping) of cancer cell
infiltration into adjacent
peripheral organs and/or tissues; (4) inhibition (i.e. reduction, slowing down
or complete stopping) of
metasisis; (5) relief, to some extent, of one or more symptoms associated with
the disease or disorder
(e.g., cancer); (6) increase in the length of progression free survival;
and/or (9) decreased mortality at a
given point of time following treatment.
[0054] The term "substantially the same," as used herein, denotes a
sufficiently high degree of
similarity between two numeric values, such that one of skill in the art would
consider the difference
between the two values to be of little or no biological and/or statistical
significance within the context of
the biological characteristic measured by said values (e.g., Kd values or
expression). The difference
between said two values is, for example, less than about 50%, less than about
40%, less than about 30%,
less than about 20%, and/or less than about 10% as a function of the
reference/comparator value.
[0055] The phrase "substantially different," as used herein, denotes a
sufficiently high degree of
difference between two numeric values such that one of skill in the art would
consider the difference
between the two values to be of statistical significance within the context of
the biological characteristic
measured by said values (e.g., Kd values). The difference between said two
values is, for example,
greater than about 10%, greater than about 20%, greater than about 30%,
greater than about 40%, and/or
greater than about 50% as a function of the value for the reference/comparator
molecule.
[0056] An "effective amount" of a substance/molecule, e.g., pharmaceutical
composition, refers to an
amount effective, at dosages and for periods of time necessary, to achieve the
desired therapeutic or
prophylactic result.
[0057] A "therapeutically effective amount" of a substance/molecule may vary
according to factors
such as the disease state, age, sex, and weight of the individual, and the
ability of the substance/molecule
to elicit a desired response in the individual. A therapeutically effective
amount is also one in which any
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toxic or detrimental effects of the substance/molecule are outweighed by the
therapeutically beneficial
effects. A "prophylactically effective amount" refers to an amount effective,
at dosages and for periods
of time necessary, to achieve the desired prophylactic result. Typically but
not necessarily, since a
prophylactic dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically
effective amount will be less than the therapeutically effective amount.
[0058] The term "pharmaceutical formulation" refers to a preparation which is
in such form as to permit
the biological activity of an active ingredient contained therein to be
effective, and which contains no
additional components which are unacceptably toxic to a subject to which the
formulation would be
administered.
[0059] A "pharmaceutically acceptable carrier" refers to an ingredient in a
pharmaceutical formulation,
other than an active ingredient, which is nontoxic to a subject., A
pharmaceutically acceptable carrier
includes, but is not limited to, a buffer, excipient, stabilizer, or
preservative.
[0060] The phrase "pharmaceutically acceptable salt" as used herein, refers to
pharmaceutically
acceptable organic or inorganic salts of a compound.
[0061] As used herein, "treatment" (and grammatical variations thereof such as
"treat" or "treating")
refers to clinical intervention in an attempt to alter the natural course of
the individual being treated, and
can be performed either for prophylaxis or during the course of clinical
pathology. Desirable effects of
treatment include, but are not limited to, preventing occurrence or recurrence
of disease, alleviation of
symptoms, diminishment of any direct or indirect pathological consequences of
the disease, preventing
metastasis, decreasing the rate of disease progression, amelioration or
palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of the
invention are used to delay
development of a disease or to slow the progression of a disease.
[0062] The term "anti-cancer therapy" refers to a therapy useful in treating
cancer. Examples of anti-
cancer therapeutic agents include, but are limited to, e.g., chemotherapeutic
agents, growth inhibitory
agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis
agents, apoptotic agents,
anti-tubulin agents, and other agents to treat cancer, anti-CD20 antibodies,
platelet derived growth
factor inhibitors (e.g., GleevecTM (Imatinib Mesylate)), a COX-2 inhibitor
(e.g., celecoxib), interferons,
cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or
more of the following targets
PDGFR-beta, BlyS, APRIL, BCMA receptor(s), TRAIL/Apo2, and other bioactive and
organic chemical
agents, etc. Combinations thereof are also included in the invention.
[0063] The term "cytotoxic agent" as used herein refers to a substance that
inhibits or prevents a cellular
function and/or causes cell death or destruction. The term is intended to
include radioactive isotopes
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(e.g., At211, /131, /125, y90 , Re 186, Re 188, sm153, B/212, P32, pb212,
and radioactive isotopes of Lu),
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca
alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,
daunorubicin or other
intercalating agents), growth inhibitory agents, enzymes and fragments thereof
such as nucleolytic
enzymes, antibiotics, and toxins such as small molecule toxins or
enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments and/or variants
thereof, and the various
antitumor or anticancer agents disclosed below. Other cytotoxic agents are
described below. A
tumoricidal agent causes destruction of tumor cells.
[0064] A "chemotherapeutic agent" refers to a chemical compound useful in the
treatment of cancer.
Examples of chemotherapeutic agents include alkylating agents such as thiotepa
and cyclosphosphamide
(CYTOXANC)); alkyl sulfonates such as busulfan, improsulfan and piposulfan;
aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including
altretamine, triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and bullatacinone);
delta-9-
tetrahydrocannabinol (dronabinol, MARINOLC)); beta-lapachone; lapachol;
colchicines; betulinic acid;
a camptothecin (including the synthetic analogue topotecan (HYCAMTINC), CPT-11
(irinotecan,
CAMPTOSARC), acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin;
CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic
analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and
cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-
TM1); eleutherobin;
pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as
chlorambucil, chlornaphazine,
chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine
oxide hydrochloride,
melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosoureas such
as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and
ranimnustine; antibiotics such as
the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin
gammalI and calicheamicin
omegaIl (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186
(1994)); CDP323, an oral
alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin;
as well as
neocarzinostatin chromophore and related chromoprotein enediyne antibiotic
chromophores),
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin,
carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-
L-norleucine, doxorubicin (including ADRIAMYCIN , morpholino-doxorubicin,
cyanomorpholino-
doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection
(DOXILC), liposomal
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doxorubicin TLC D-99 (MYOCETO), peglylated liposomal doxorubicin (CAELYXO),
and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,
mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin,
puromycin, quelamycin,
rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin,
zorubicin; anti-metabolites
such as methotrexate, gemcitabine (GEMZARO), tegafur (UFTORALO), capecitabine
(XELODAO), an
epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as
denopterin, methotrexate,
pteropterin, trimetrexate; purine analogs such as fludarabine, 6-
mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine,
dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as
aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid; aceglatone;
aldophosphamide glycoside;
aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene;
edatraxate; defofamine;
demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone;
etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone;
mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; 2-
ethylhydrazide; procarbazine; PSKO polysaccharide complex (JHS Natural
Products, Eugene, OR);
razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2'-
trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A,
roridin A and anguidine);
urethan; vindesine (ELDISINEO, FILDESINO); dacarbazine; mannomustine;
mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid, e.g.,
paclitaxel (TAXOLO), albumin-
engineered nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel
(TAXOTEREO);
chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents
such as cisplatin,
oxaliplatin (e.g., ELOXATINO), and carboplatin; vincas, which prevent tubulin
polymerization from
forming microtubules, including vinblastine (VELBANO), vincristine (ONCOVINO),
vindesine
(ELDISINEO, FILDESINO), and vinorelbine (NAVELBINEO); etoposide (VP-16);
ifosfamide;
mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin;
ibandronate;
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMF0); retinoids
such as retinoic acid,
including bexarotene (TARGRETINO); bisphosphonates such as clodronate (for
example, BONEFOSO
or OSTACO), etidronate (DIDROCALO), NE-58095, zoledronic acid/zoledronate
(ZOMETAO),
alendronate (FOSAMAXO), pamidronate (AREDIAO), tiludronate (SKELIDO), or
risedronate
(ACTONELO); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides,
particularly those that inhibit expression of genes in signaling pathways
implicated in aberrant cell
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proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal
growth factor receptor (EGF-
R); vaccines such as THERATOPEO vaccine and gene therapy vaccines, for
example, ALLOVECTINO
vaccine, LEUVECTINO vaccine, and VAXIDO vaccine; topoisomerase 1 inhibitor
(e.g.,
LURTOTECANO); rmRH (e.g., ABARELIXO); BAY439006 (sorafenib; Bayer); SU-11248
(sunitinib,
SUTENTO, Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib),
proteosome inhibitor
(e.g., PS341); bortezomib (VELCADEO); CCI-779; tipifarnib (R11577); orafenib,
ABT510; Bc1-2
inhibitor such as oblimersen sodium (GENASENSEO); pixantrone; EGFR inhibitors
(see definition
below); tyrosine kinase inhibitors (see definition below); serine-threonine
kinase inhibitors such as
rapamycin (sirolimus, RAPAMUNEO); farnesyltransferase inhibitors such as
lonafarnib (SCH 6636,
SARASARTm); and pharmaceutically acceptable salts, acids or derivatives of any
of the above; as well
as combinations of two or more of the above such as CHOP, an abbreviation for
a combined therapy of
cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an
abbreviation for a
treatment regimen with oxaliplatin (ELOXATINTm) combined with 5-FU and
leucovorin.
[0065] Chemotherapeutic agents as defined herein include "anti-hormonal
agents" or "endocrine
therapeutics" which act to regulate, reduce, block, or inhibit the effects of
hormones that can promote
the growth of cancer. They may be hormones themselves, including, but not
limited to: anti-estrogens
with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEXO), 4-
hydroxytamoxifen,
toremifene (FARESTONO), idoxifene, droloxifene, raloxifene (EVISTAO),
trioxifene, keoxifene, and
selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-
estrogens without agonist
properties, such as fulvestrant (FASLODEXO), and EM800 (such agents may block
estrogen receptor
(ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress
ER levels); aromatase
inhibitors, including steroidal aromatase inhibitors such as formestane and
exemestane (AROMASINO),
and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEXO),
letrozole (FEMARAO) and
aminoglutethimide, and other aromatase inhibitors include vorozole (RIVISORO),
megestrol acetate
(MEGASEO), fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing
hormone agonists,
including leuprolide (LUPRONO and ELIGARDO), goserelin, buserelin, and
tripterelin; sex steroids,
including progestines such as megestrol acetate and medroxyprogesterone
acetate, estrogens such as
diethylstilbestrol and premarin, and androgens/retinoids such as
fluoxymesterone, all transretionic acid
and fenretinide; onapristone; anti-progesterones; estrogen receptor down-
regulators (ERDs); anti-
androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically
acceptable salts, acids
or derivatives of any of the above; as well as combinations of two or more of
the above.
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[0066] The term "prodrug" as used in this application refers to a precursor or
derivative form of a
pharmaceutically active substance that is less cytotoxic to tumor cells
compared to the parent drug and is
capable of being enzymatically activated or converted into the more active
parent form. See, e.g.,
Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions,
14, pp. 375-382, 615th
Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to
Targeted Drug Delivery,"
Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this
invention include, but are not limited to, phosphate-containing prodrugs,
thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified prodrugs,
glycosylated prodrugs, [3-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-
containing prodrugs or optionally substituted phenylacetamide-containing
prodrugs, 5-fluorocytosine
and other 5-fluorouridine prodrugs which can be converted into the more active
cytotoxic free drug.
Examples of cytotoxic drugs that can be derivatized into a prodrug form for
use in this invention
include, but are not limited to, those chemotherapeutic agents described
above.
[0067] A "growth inhibitory agent" when used herein refers to a compound or
composition which
inhibits growth of a cell (e.g., a cell whose growth is dependent upon the
activity of the polypeptide of
interest either in vitro or in vivo). Examples of growth inhibitory agents
include agents that block cell
cycle progression (at a place other than S phase), such as agents that induce
G1 arrest and M-phase
arrest. Classical M-phase blockers include the vincas (vincristine and
vinblastine), taxanes, and
topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest, for example,
DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and
ara-C. Further information can be found in THE MOLECULAR BASIS OF CANCER,
Mendelsohn and
Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by
Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The
taxanes (paclitaxel and
docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel
(TAXOTERE , Rhone-
Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL ,
Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of
microtubules from tubulin
dimers and stabilize microtubules by preventing depolymerization, which
results in the inhibition of
mitosis in cells.
[0068] By "radiation therapy" is meant the use of directed gamma rays or beta
rays to induce sufficient
damage to a cell so as to limit its ability to function normally or to destroy
the cell altogether. It will be
appreciated that there will be many ways known in the art to determine the
dosage and duration of
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treatment. Typical treatments are given as a one-time administration and
typical dosages range from 10
to 200 units (Grays) per day.
[0069] An "individual" or "subject" is a mammal. Mammals include, but are not
limited to,
domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates
(e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In
certain embodiments, the
individual or subject is a human.
[0070] The term "concomitantly" is used herein to refer to administration of
two or more therapeutic
agents, give in close enough temporal proximity where their individual
therapeutic effects overlap in
time. Accordingly, concurrent administration includes a dosing regimen when
the administration of one
or more agent(s) continues after discontinuing the administration of one or
more other agent(s). In some
embodiments, the concomitantly administration is concurrently, sequentially,
and/or simultaneously.
[0071] By "reduce or inhibit" is meant the ability to cause an overall
decrease of 20%, 30%, 40%, 50%,
60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to
the symptoms of the
disorder being treated, the presence or size of metastases, or the size of the
primary tumor.
[0072] The term "package insert" is used to refer to instructions customarily
included in commercial
packages of therapeutic products, that contain information about the
indications, usage, dosage,
administration, combination therapy, contraindications and/or warnings
concerning the use of such
therapeutic products.
[0073] An "article of manufacture" is any manufacture (e.g., a package or
container) or kit comprising
at least one reagent, e.g., a medicament for treatment of a disease or
disorder (e.g., cancer), or a probe
for specifically detecting a biomarker described herein. In certain
embodiments, the manufacture or kit
is promoted, distributed, or sold as a unit for performing the methods
described herein.
[0074] As is understood by one skilled in the art, reference to "about" a
value or parameter herein
includes (and describes) embodiments that are directed to that value or
parameter per se. For example,
description referring to "about X" includes description of "X".
[0075] It is understood that aspect and embodiments of the invention described
herein include
"consisting" and/or "consisting essentially of' aspects and embodiments. As
used herein, the singular
form "a", "an", and "the" includes plural references unless indicated
otherwise.
II. Methods and Uses
[0076] Provided herein are methods utilizing an ALDH inhibitor (e.g.,
disulfiram and/or derivatives
thereof) and a targeted therapeutic (e.g., TKI) for treating cancer.
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[0077] In particular, provided herein are methods of treating cancer in an
individual comprising
concomitantly administering to the individual an ALDH inhibitor (e.g.,
disulfiram and/or derivatives
thereof) and a targeted therapeutic (e.g., TKI). In some embodiments, the
respective amounts of the
ALDH inhibitor (e.g., disulfiram and/or derivatives thereof) and the targeted
therapeutic (e.g., TKI) are
effective to increase the period of cancer sensitivity and/or delay the
development of cell resistance to
the targeted therapeutic (e.g., TKI). In some embodiments, the respective
amounts of the ALDH
inhibitor (e.g., disulfiram and/or derivatives thereof) and the targeted
therapeutic (e.g., TKI) are
effective to increase efficacy of a cancer treatment comprising a targeted
therapeutic (e.g., TM). For
example, in some embodiments, the respective amounts of the ALDH inhibitor
(e.g., disulfiram and/or
derivatives thereof) and the targeted therapeutic (e.g., TKI) are effective to
increased efficacy compared
to a standard treatment comprising administering an effective amount of the
targeted therapeutic (e.g.,
TKI) without (in the absence of) the ALDH inhibitor (e.g., disulfiram and/or
derivatives thereof). In
some embodiments, the respective amounts of the ALDH inhibitor (e.g.,
disulfiram and/or derivatives
thereof) and the targeted therapeutic (e.g., TM) are effective to increased
response (e.g., complete
response) compared to a standard treatment comprising administering an
effective amount of the
targeted therapeutic (e.g., TKI) without (in the absence of) the ALDH
inhibitor (e.g., disulfiram and/or
derivatives thereof). In some embodiments, the targeted therapeutic is a
tyrosine kinase inhibitor (TKI).
In some embodiments, the TM is an EGFR inhibitor, HER2 inhibitor, MET/HGF
inhibitor, ALK
inhibitor, BRAF inhibitor, ROS1 inhibitor, and/or MEK inhibitor. In some
embodiments, the TM is a
receptor tyrosine kinase inhibitor (RTKI). In some embodiments, the RTKI is an
EGFR inhibitor, HER2
inhibitor, MET inhibitor, and/or ALK inhibitor.
[0078] Further provided herein are methods of increasing efficacy of a cancer
treatment comprising a
targeted therapeutic (e.g., TKI) in an individual comprises concomitantly
administering to the individual
an effective amount of the targeted therapeutic (e.g., TKI) and an effective
amount of an ALDH
inhibitor (e.g., disulfiram and/or derivatives thereof). Provided herein are
also methods of treating
cancer in an individual wherein cancer treatment comprising concomitantly
administering to the
individual an effective amount of targeted therapeutic (e.g., TKI) and an
effective amount of an ALDH
inhibitor (e.g., disulfiram and/or derivatives thereof), wherein the cancer
treatment has increased
efficacy compared to a standard treatment comprising administering an
effective amount of the targeted
therapeutic (e.g., TKI) without (in the absence of) the ALDH inhibitor (e.g.,
disulfiram and/or
derivatives thereof). In some embodiments, the targeted therapeutic is a TKI.
In some embodiments, the
TKI is an EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor, ALK inhibitor,
BRAF inhibitor, ROS1
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inhibitor, and/or MEK inhibitor. In some embodiments, the TM is a RTKI. In
some embodiments, the
RTKI is an EGFR inhibitor, HER2 inhibitor, MET/HGF inhibitor, and/or ALK
inhibitor.
[0079] In addition, provided herein are methods of delaying and/or preventing
development of cancer
resistance to a targeted therapeutic (e.g., TKI) in an individual, comprising
concomitantly administering
to the individual an effective amount of an ALDH inhibitor (e.g., disulfiram
and/or derivatives thereof)
and an effective amount of the targeted therapeutic (e.g., TM). Provided
herein are also methods of
increasing sensitivity to a targeted therapeutic (e.g., TKI) in an individual
comprising concomitantly
administering to the individual an effective amount of an ALDH inhibitor
(e.g., disulfiram and/or
derivatives thereof) and an effective amount of the targeted therapeutic
(e.g., TKI). In some
embodiments, the targeted therapeutic is a TKI. In some embodiments, the TM is
an EGFR inhibitor,
HER2 inhibitor, MET/HGFinhibitor, ALK inhibitor, BRAF inhibitor, ROS1
inhibitor, and/or MEK
inhibitor. In some embodiments, the TM is a RTKI. In some embodiments, the
RTKI is an EGFR
inhibitor, HER2 inhibitor, MET/HGFinhibitor, and/or ALK inhibitor.
[0080] Further, provided herein are methods of extending the period of a
targeted therapeutic (e.g., TKI)
sensitivity in an individual with cancer comprising concomitantly
administering to the individual an
effective amount of an ALDH inhibitor (e.g., disulfiram and/or derivatives
thereof) and an effective
amount of the targeted therapeutic (e.g., TKI). Provided herein are also
methods of extending the
duration of response to a targeted therapeutic (e.g., TKI) in an individual
with cancer comprising
concomitantly administering to the individual an effective amount of an ALDH
inhibitor (e.g.,
disulfiram and/or derivatives thereof) and an effective amount of the targeted
therapeutic (e.g., TM). In
some embodiments, the targeted therapeutic is a TM. In some embodiments, the
TKI is an EGFR
inhibitor, HER2 inhibitor, MET/HGFinhibitor, ALK inhibitor, BRAF inhibitor,
ROS1 inhibitor, and/or
MEK inhibitor. In some embodiments, the TKI is a RTKI. In some embodiments,
the RTKI is an EGFR
inhibitor, HER2 inhibitor, MET/HGFinhibitor, and/or ALK inhibitor.
[0081] In some embodiments of any of the methods, the ALDH inhibitor and/or
targeted therapeutic
(e.g., TM) is an antibody, binding polypeptide, binding small molecule, or
polynucleotide such as those
described herein. In some embodiments, the ALDH inhibitor is disulfiram and/or
derivatives thereof. In
some embodiments, the ALDH inhibitor is disulfiram. In some embodiments of any
of the methods, the
ALDH inhibitor is gossypol and/or or an ALDH-inhibiting derivative or
metabolite thereof. In some
embodiments, the ALDH inhibitor is gossypol. In some embodiments, the ALDH
inhibitor is 2,2'-bis-
(Formy1-1,6,7-trihydroxy-5-isopropy1-3-methylnaphthalene) or pharmaceutically
acceptable salt thereof.
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In some embodiments, the ALDH inhibitor is 2,2'-bis-(Formy1-1,6,7-trihydroxy-5-
isopropy1-3-
methylnaphthalene).
[0082] Cancer having resistance to a therapy as used herein includes a cancer
which is not responsive
and/or reduced ability of producing a significant response (e.g., partial
response and/or complete
response) to the therapy. Resistance may be acquired resistance which arises
in the course of a treatment
method. In some embodiments, the acquired drug resistance is transcient and/or
reversible drug
tolerance. Transcient and/or reversible drug resistance to a therapy includes
wherein the drug resistance
is capable of regaining sensitivity to the therapy after a break in the
treatment method. In some
embodiments, the acquired resistance is permanent resistance. Permanent
resistance to a therapy
includes a genetic change conferring drug resistance. Permanent resistance can
occur as a result of
treatment with general chemotherapies-cyclophosphomide, platinum agent, and/or
taxol.
[0083] Cancer having sensitivity to a therapy as used herein includes cancer
which is responsive and/or
capable of producing a significant response (e.g., partial response and/or
complete response).
[0084] Methods of determining of assessing acquisition of resistance and/or
maintenance of sensitivity
to a therapy are known in the art and described in the Examples. Changes in
acquisition of resistance
and/or maintenance of sensitivity such as drug tolerance may be assessed by
assaying the growth of drug
tolerant persisters as described in the Examples and Sharma et al. Changes in
acquisition of resistance
and/or maintenance of sensitivity such as permanent resistance and/or expanded
resisters may be
assessed by assaying the growth of drug tolerant expanded persisters as
described in the Examples and
Sharma et al. In some embodiments, resistance may be indicated by a change in
IC50, EC50 or decrease
in tumor growth in drug tolerant persisters and/or drug tolerant expanded
persisters. In some
embodiments, the change is greater than about any of 50%, 100%, and/or 200%.
In addition, changes in
acquisition of resistance and/or maintenance of sensitivity may be assessed in
vivo for examples by
assessing response, duration of response, and/or time to progression to a
therapy, e.g., partial response
and complete response. Changes in acquisition of resistance and/or maintenance
of sensitivity may be
based on changes in response, duration of response, and/or time to progression
to a therapy in a
population of individuals, e.g., number of partial responses and complete
responses.
[0085] In some embodiments of any of the methods, the cancer is a solid tumor
cancer. In some
embodiments, the cancer is gastric cancer. In some embodiments, the cancer is
lung cancer (e.g., non-
small cell lung cancer (NSCL)). In some embodiments, the cancer is breast
cancer. In some
embodiments, the cancer is colorectal cancer (e.g., colon cancer and/or rectal
cancer). In some
embodiments, the cancer is basel cell carcinoma. In some embodiments of any of
the cancers, the cancer
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is adenocarcinoma. The cancer in any of the combination therapies methods
described herein when
starting the method of treatment comprising the ALDH inhibitor (e.g.,
disulfiram and/or derivatives
thereof) and the targeted therapeutic (e.g., TM) may be sensitive (examples of
sensitive include, but are
not limited to, responsive and/or capable of producing a significant response
(e.g., partial response
and/or complete response)) to a method of treatment comprising the targeted
therapeutic alone. The
cancer in any of the combination therapies methods described herein when
starting the method of
treatment comprising the ALDH inhibitor (e.g., disulfiram and/or derivatives
thereof) and the targeted
therapeutic (e.g., TKI) may not be resistant (examples of resistance include,
but are not limited to, not
responsive and/or reduced ability and/or incapable of producing a significant
response (e.g., partial
response and/or complete response)) to a method of treatment comprising the
targeted therapeutic alone.
[0086] In some embodiments of any of the methods, the individual according to
any of the above
embodiments may be a human.
[0087] In some embodiments of any of the methods, the combination therapies
noted above encompass
combined administration (where two or more therapeutic agents are included in
the same or separate
formulations), and separate administration, in which case, administration of
the antagonist of the
invention can occur prior to, simultaneously, sequentially, concurrently
and/or following, administration
of the additional therapeutic agent and/or adjuvant. In some embodiments, the
combination therapy
further comprises radiation therapy and/or additional therapeutic agents.
[0088] An ALDH inhibitor (e.g., disulfiram and/or derivatives thereof) and/or
targeted therapeutic (e.g.,
TKI) described herein can be administered by any suitable means, including
oral, parenteral,
intrapulmonary, and intranasal, and, if desired for local treatment,
intralesional administration.
Parenteral infusions include intramuscular, intravenous, intraarterial,
intraperitoneal, or subcutaneous
administration. Dosing can be by any suitable route, e.g., by injections, such
as intravenous or
subcutaneous injections, depending in part on whether the administration is
brief or chronic. Various
dosing schedules including but not limited to single or multiple
administrations over various time-
points, bolus administration, and pulse infusion are contemplated herein.
[0089] An ALDH inhibitor (e.g., disulfiram and/or derivatives thereof) and/or
targeted therapeutic (e.g.,
TKI) described herein may be formulated, dosed, and administered in a fashion
consistent with good
medical practice. Factors for consideration in this context include the
particular disorder being treated,
the particular mammal being treated, the clinical condition of the individual
patient, the cause of the
disorder, the site of delivery of the agent, the method of administration, the
scheduling of administration,
and other factors known to medical practitioners. The ALDH inhibitor (e.g.,
disulfiram and/or
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derivatives thereof) and/or targeted therapeutic (e.g., TM) need not be, but
is optionally formulated with
one or more agents currently used to prevent or treat the disorder in
question. The effective amount of
such other agents depends on the amount of the ALDH inhibitor (e.g.,
disulfiram and/or derivatives
thereof) and/or targeted therapeutic (e.g., TKI) present in the formulation,
the type of disorder or
treatment, and other factors discussed above. These are generally used in the
same dosages and with
administration routes as described herein, or about from 1 to 99% of the
dosages described herein, or in
any dosage and by any route that is empirically/clinically determined to be
appropriate.
[0090] For the prevention or treatment of disease, the appropriate dosage of
an ALDH inhibitor (e.g.,
disulfiram and/or derivatives thereof) and/or targeted therapeutic (e.g., TKI)
described herein (when
used alone or in combination with one or more other additional therapeutic
agents) will depend on the
type of disease to be treated, the severity and course of the disease, whether
the ALDH inhibitor (e.g.,
disulfiram and/or derivatives thereof) and/or targeted therapeutic (e.g., TKI)
is administered for
preventive or therapeutic purposes, previous therapy, the patient's clinical
history and response to the
ALDH inhibitor (e.g., disulfiram and/or derivatives thereof), and the
discretion of the attending
physician. The ALDH inhibitor (e.g., disulfiram and/or derivatives thereof) is
suitably administered to
the patient at one time or over a series of treatments. For repeated
administrations over several days or
longer, depending on the condition, the treatment would generally be sustained
until a desired
suppression of disease symptoms occurs. Such doses may be administered
intermittently, e.g., every
week or every three weeks (e.g., such that the patient receives from about two
to about twenty, or e.g.,
about six doses of the ALDH inhibitor (e.g., disulfiram and/or derivatives
thereof)) and/or targeted
therapeutic (e.g., TKI). An initial higher loading dose, followed by one or
more lower doses may be
administered. An exemplary dosing regimen comprises administering. However,
other dosage regimens
may be useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
[0091] It is understood that any of the above formulations or therapeutic
methods may be carried out
using an immunoconjugate as the ALDH inhibitor and/or targeted therapeutic
(e.g., TM).
III. Therapeutic Compositions
[0092] Provided herein are combinations comprising an ALDH inhibitor (e.g.,
disulfiram and/or
derivatives thereof) and a targeted therapeutic (e.g., TM). In one aspect,
there is provided a
pharmaceutical product comprising a) as a first component an effective amount
of an ALDH inhibitor,
and b) as a second component an effective amount of a targeting agent
(targeted therapeutic) for the
concomitant or sequential use for the treatment of cancer. In certain
embodiments, the combination
increases the efficacy of the targeted therapeutic administered alone. In
certain embodiments, the
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combination delays and/or prevents development of cancer resistance to the
targeted therapeutic. In
certain embodiments, the combination extends the period of the targeted
therapeutic sensitivity in an
individual with cancer.
[0093] Also provided herein are ALDH inhibitors and/or targeted therapeutics
useful in the combination
therapy methods described herein. In some embodiments, the ALDH inhibitors
and/or targeted
therapeutics are an antibody, binding polypeptide, binding small molecule,
and/or polynucleotide.
[0094] In some embodiments of any of the combination therapy methods described
herein, the ALDH
inhibitor inhibits one or more of ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1,
ALDH1L2, ALDH2, ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2, ALDH4A1, ALDH5A1,
ALDH6A1, ALDH7A1, ALDH8A1, ALDH9A1, ALDH16A1, and/or ALDH18A1. In some
embodiments, ALDH inhibitors according to the invention are compounds that are
capable of inhibiting
the activity of one or more of the several isozymes of ALDH. In some
embodiments, the ALDH
inhibitor is a pan-ALDH inhibitor. ALDH inhibitors include, but are not
limited to, disulfiram, coprine,
cyanamide, 1-aminocyclopropanol (ACP), daidzin (i.e., the 7-glucoside of 4',7-
dihydroxyisoflavone),
cephalosporins, antidiabetic sulfonyl ureas, metronidazole,
diethyldithiocarbamate, phenethyl
isothiocyanate (PEITC), prunetin (4',5-dihydroxy-7-methoxyisoflavone), 5-
hydroxydaidzin (genistin),
and any of their metabolites or analogs exhibiting ALDH-inhibiting activity.
In another embodiment, the
ALDH inhibitor is disulfiram or an ALDH-inhibiting metabolite thereof. Such
metabolites include, e.g.,
S-methyl N,N-diethyldithiocarbamae, S-methyl N,N-diethyldithiocarbamate
sulfoxide, and S-methyl
N,N-diethylthiocarbamate sulfoxide. In some embodiments, the ALDH inhibitor is
disulfiram. In some
embodiments of any of the methods, the ALDH inhibitor is gossypol and/or or an
ALDH-inhibiting
derivative or metabolite thereof. In some embodiments, the ALDH inhibitor is
gossypol. In some
embodiments, the ALDH inhibitor is 2,2'-bis-(Formy1-1,6,7-trihydroxy-5-
isopropy1-3-
methylnaphthalene) or pharmaceutically acceptable salt thereof. In some
embodiments, the ALDH
inhibitor is 2,2'-bis-(Formy1-1,6,7-trihydroxy-5-isopropy1-3-
methylnaphthalene).
[0095] ALDH inhibitors also include compounds of the formula:
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R2
R10 10 0 R3
410
R7 R4
R6 0
R5
Formula I
wherein:
R1 is selected from the group consisting of hydrogen, carboxy, halo, branched
or
unbranched (C1-C6)haloalkyl, (C3-C6)cyclo alkoxy,
(C1-C6)haloalkoxy, (C3-
C6)cyclohaloalkoxy, (C3-C6)cycloalkoxyalkyl, (C1-C6)alkoxy(C3-C6)cycloalkyl,
(C3-
C6)cycloalkylcarbonyl, substituted or unsubstituted phenyl, phenyl(C1-
C6)alkyl, heterocyclyl,
and heterocyclyloxy, heterocyclylcarbonyl, wherein substituents are from one
to four and are
selected from the group consisting of halo, aminocarbonyl, aminothiocarbonyl,
carboxy,
formyl, hydroxy, amino, carbamoyl, (C1-C3)alkyl, (C1-C3)haloalkyl, (C1-
C3)alkoxy, (C1-
C3)haloalkoxy, (C1-C3)alkylamino, di(Ci-C3)alkylamino, (C1-C2)alkoxy(C1-
C2)alicYl, (C1-
C2)alkylamino(C1-C2)alkyl, di(C1-C2)alkylamino(C1-C2)alkyl, (C1-
C3)alkylcarbonyl, (C1-
C3)allcoxycarbonyl, (C1C3)alkylaminocarbonyl, and di (C1-
C3)alkylaminocarbonyl;
R2 is selected from the group consisting of hydrogen and alkoxy;
R3 is selected from the group consisting of hydrogen Ci-C6 alkoxycarbonyl,
carboxy
and sugar;
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R4 is selected from the group consisting of hydrogen and hydroxide;
R5 is selected from the group consisting of hydrogen, carboxy, hydroxy, halo,
branched or unbranched (C1-C6)alkyl, (C1-C6)haloalkyl, (C2-C6)alkenyl, (C3-
C6)alkadienyl,
(C1-C6)alkoxy, (C3-C6)cycloalkoxy, (C1-C6)haloalkoxy, (C3-C6)cyclohaloalkoxy,
(C2-
C6)alkynyloxy, (C1-C6)alkoxy(Ci-C6)alkyl, (C3-C6)cycloalkoxyalkyl, (C1-
C6)alkoxy(C3-
C6)cycloalkyl, (C1-C6jalkylcarbonyl, (C3C6)cyc1oa1ky1carbonyl, (C1-
C6)alkoxycarbonyl, (C4-
C6)alkoxycarbonylalkyl, (C1C6)hydroxyalkyl, substituted or tinsubstituted
phenyl, phenyl(Ci-
C6)alkyl, heterocyclyl, heterocyclyloxy, heterocyclylcarbonyl, wherein
substituents are from
one to four and are selected from the group consisting of halo, aminocarbonyl,
arninothiocalbonyl, carboxy, formyl, hydroxy, amino, earbamoyl, (C1-C3)alkyl,
(C1-
C3)haloalkyl, (C1-C3)alkoxy,
F-C3)haloalkoxy, (C1-C3)alkylamino, di(C -COalkylainino,
(C1-C2) alkox y(C -C2)alicyl, (C1 -C2)alkyl amino (C -COancyl,
di(CI-C2)alkylamino(Ci-
C2)allcyl, (C1-C3)alkylcarbonyl, (C1-C3)alkoxycarbonyl,
(C1C3)alkylaminocarbonyl, and di
(Ci-C3)alkylaminocarbonyl;
R6 is selected from the group consisting of hydrogen and hydroxide; and
R7 is selected from the group consisting of hydrogen, halogen, and C1-C6
alkoxy.
[0096] ALDH inhibitors also include compounds with CAS Registry numbers:
1069117-57-2, 1069117-
56-1, 1069117-55-0, 1055417-23-6, 1055417-22-5, 1055417-21-4, 1055417-20-3,
1055417-19-0,
1055417-18-9, 1055417-17-8, 1055417-16-7, 1055417-15-6 and 1055417-13-4, and
salts thereof.
[0097] ALDH inhibitors also include compounds of the formula:
NH2
R1
0
SR3
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wherein R1, R2 and R3, independently represent a saturated or unsaturated
linear or branched Ci-C6 alkyl
radical, or a salt thereof.
[0098] ALDH inhibitors also include 4-amino-4-methyl-2-pentynethioic acid (S)-
methyl ester, and salts
thereof.
[0099] In some embodiments of any of the combination therapy methods described
herein, the targeted
therapeutic is a TM. In some embodiments, the TM is an EGFR inhibitor, HER2
inhibitor, MET/HGF
inhibitor, ALK inhibitor, BRAF inhibitor, ROS1 inhibitor, and/or MEK
inhibitor. In some embodiments,
the TM is a RTKI. In some embodiments, the RTKI is an EGFR inhibitor, HER2
inhibitor, MET/HGF
inhibitor, and/or ALK inhibitor.
[0100] In some embodiments of any of the combination therapy methods described
herein, the targeted
therapeutic is an EGFR inhibitor. Exemplary EGFR inhibitors (anti-EGFR
antibodies) include antibodies
such as humanized monoclonal antibody known as nimotuzumab (YM Biosciences),
fully human ABX-EGF
(panitumumab, Abgenix Inc.) as well as fully human antibodies known as E1.1,
E2.4, E2.5, E6.2, E6.4,
E2.11, E6. 3 and E7.6. 3 and described in US 6,235,883; MDX-447 (Medarex Inc).
Pertuzumab (2C4) is a
humanized antibody that binds directly to HER2 but interferes with HER2-EGFR
dimerization thereby
inhibiting EGFR signaling. Other examples of antibodies which bind to EGFR
include GA201 (RG7160;
Roche Glycart AG), MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb
225 (ATCC
CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No. 4,943, 533, Mendelsohn
et al.) and variants
thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIXO) and reshaped
human 225 (H225) (see,
WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted
antibody (Imclone);
antibodies that bind type II mutant EGFR (US Patent No. 5,212,290); humanized
and chimeric antibodies
that bind EGFR as described in US Patent No. 5,891,996; and human antibodies
that bind EGFR, such as
ABX-EGF (see W098/50433, Abgenix); EMD 55900 (Stragliotto et al. Eur. J.
Cancer 32A:636-640
(1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR
that competes with
both EGF and TGF-alpha for EGFR binding; and mAb 806 or humanized mAb 806
(Johns et al., J. Biol.
Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be conjugated
with a cytotoxic agent,
thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent
GmbH). In some embodiments,
the anti-EGFR antibody is cetuximab. In some embodiments, the anti-EGFR
antibody is panitumumab. In
some embodiments, the anti-EGFR antibobdy is zalutumumab, nimotuzumab, and/or
matuzumab.
[0101] Anti-EGFR antibodies that are useful in the methods include any
antibody that binds with sufficient
affinity and specificity to EGFR and can reduce or inhibit EGFR activity. The
antibody selected will
normally have a sufficiently strong binding affinity for EGFR, for example,
the antibody may bind human c-
met with a Kd value of between 100 nM-1 pM. Antibody affinities may be
determined by a surface plasmon
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resonance based assay (such as the BIAcore assay as described in PCT
Application Publication No.
W02005/012359); enzyme-linked immunoabsorbent assay (ELISA); and competition
assays (e.g., RIA's),
for example. Preferably, the anti-EGFR antibody of the invention can be used
as a therapeutic agent in
targeting and interfering with diseases or conditions wherein EGFR/EGFR ligand
activity is involved. Also,
the antibody may be subjected to other biological activity assays, e.g., in
order to evaluate its effectiveness as
a therapeutic. Such assays are known in the art and depend on the target
antigen and intended use for the
antibody. In some embodiments, a EGFR arm may be combined with an arm which
binds to a triggering
molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3),
or Fc receptors for IgG
(Fc7R), such as Fc7RI (CD64), Fc7RII (CD32) and Fc7RIII (CD16) so as to focus
cellular defense
mechanisms to the EGFR-expressing cell. Bispecific antibodies may also be used
to localize cytotoxic
agents to cells which express EGFR. These antibodies possess an EGFR-binding
arm and an arm which
binds the cytotoxic agent (e.g. saporin, anti-interferon-a, vinca alkaloid,
ricin A chain, methotrexate or
radioactive isotope hapten). Bispecific antibodies can be prepared as full
length antibodies or antibody
fragments (e.g., F(ab')2bispecific antibodies).
[0102] Exemplary EGFR inhibitors also include small molecules such as
compounds described in
U55616582, U55457105, U55475001, U55654307, U55679683, U56084095, U56265410,
U56455534,
U56521620, U56596726, U56713484, U55770599, U56140332, U55866572, U56399602,
U56344459,
U56602863, US6391874, W09814451, W09850038, W09909016, W09924037, W09935146,
W00132651, U56344455, U55760041, U56002008, and/or U55747498. Particular small
molecule EGFR
antagonists include OSI-774 (CP-358774, erlotinib, OSI Pharmaceuticals); PD
183805 (CI 1033, 2-
propenamide, N44-[(3-chloro-4-fluorophenyl)amino]-743-(4-morpholinyl)propoxy]-
6-quinazoliny1]-,
dihydrochloride, Pfizer Inc.); Iressa (ZD1839, gefitinib, AstraZeneca); ZM
105180 ((6-amino-4-(3-
methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-
pheny1)-N2-(1-methyl-
piperidin-4-y1)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim);
PKI-166 ((R)-4-[4-[(1-
phenylethyflamino]-1H-pyrrolo[2,3-c]pyrimidin-6-yfl-phenol); (R)-6-(4-
hydroxypheny1)-4-[(1-
phenylethyflamino]-7H-pyrrolo[2,3-c]pyrimidine); CL-387785 (N-[4-[(3-
bromophenyl)amino]-6-
quinazoliny1]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-
cyano-7-ethoxy-6-
quinoliny1]-4-(dimethylamino)-2-butenamide); lapatinib (Tykerb,
GlaxoSmithKline); ZD6474 (Zactima,
AstraZeneca); CUDC-101 (Curis); canertinib (CI-1033); AEE788 (644-R4-ethyl-I-
piperazinyl)methyflphenyfl-N-R1R)-1-phenylethyfl-7H-pyrrolo[2,3-d]pyrimidin-4-
amine, W02003013541,
Novartis) and PKI166 444-[[(1R)-1-phenylethyflamino]-7H-pyrrolo[2,3-
c]pyrimidin-6-yfl-phenol,
W09702266 Novartis). In some embodiments, the EGFR antagonist is N-(3-
ethynylpheny1)-6,7-bis(2-
methoxyethoxy)-4-quinazolinamine and/or a pharmaceutical acceptable salt
thereof (e.g., N-(3-
ethynylpheny1)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine-HC1). In some
embodiments, the EGFR
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antagonist is gefitinib and/or a pharmaceutical acceptable salt thereof. In
some embodiments, the EGFR
antagonist is lapatinib and/or a pharmaceutical acceptable salt thereof. In
some embodiments, the EGFR
antagonist is gefitinib and/or erlobtinib.
[0103] In some embodiments of any of the combination therapy methods described
herein, the targeted
therapeutic is an HGF/MET inhibitor. Exemplary HGF/MET inhibitors (anti-HGF
and/or anti-MET
antibodies) include antibodies such as anti-MET antibodies disclosed in
W005/016382 (including but not
limited to antibodies 13.3.2, 9.1.2, 8.70.2, 8.90.3); an anti- met antibodies
produced by the hybridoma cell
line deposited with ICLC number PD 03001 at the CBA in Genoa, or that
recognizes an epitope on the
extracellular domain of the 13 chain of the HGF receptor, and said epitope is
the same as that recognized by
the monoclonal antibody); anti-met antibodies disclosed in W02007/126799
(including but not limited to
04536, 05087, 05088, 05091, 05092, 04687, 05097, 05098, 05100, 05101, 04541,
05093, 05094, 04537,
05102, 05105, 04696, 04682); anti met antibodies disclosed in W02009/007427
(including but not limited to
an antibody deposited at CNCM, Institut Pasteur, Paris, France, on March 14,
2007 under the number 1-3731,
on March 14, 2007 under the number 1-3732, on July 6, 2007 under the number 1-
3786, on March 14, 2007
under the number 1-3724; an anti-met antibody disclosed in 20110129481; an
anti-met antibody disclosed in
US20110104176; an anti-met antibody disclosed in W02009/134776; an anti-met
antibody disclosed in
W02010/059654; an anti-met antibody disclosed in W02011020925 (including but
not limited to an
antibody secreted from a hybridoma deposited at the CNCM, Institut Pasteur,
Paris, France, on Mar. 12,
2008 under the number 1-3949 and the hybridoma deposited on January 14, 2010
under the number 1-4273);
and/or MetMAb (onartuzumab) or a biosimilar version thereof( W02006/015371;
Jin et al, Cancer Res
(2008) 68:4360). In some embodiments, the MET/HGF inhibitor is onartuzumab.
[0104] In some embodiments of any of the combination therapy methods described
herein, the
MET/HGFinhibitor is an anti-hepatocyte growth factor (HGF) antibody, for
example, humanized anti-HGF
antibody TAK701, rilotumumab, Ficlatuzumab, and/or humanized antibody 2B8
described in
W02007/143090. In some embodiments, the anti-HGF antibody is the anti-HGF
antibody described in
US7718174B2.
[0105] In certain embodiments of any of the combination therapy methods
described herein, the
MET/HGFinhibitor is any one of: SGX-523, Crizotinib (PF-02341066; 3-[(1R)-1-
(2,6-dichloro-3-
fluorophenyl)ethoxy1-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine; CAS no.
877399-52-5); JNJ-
38877605 (CAS no. 943540-75-8), BMS-698769, PHA-665752 (Pfizer), SU5416, INC-
280 (Incyte;
SU11274 (Sugen; [(3Z)-N-(3-chloropheny1)-3-({3,5-dimethyl-4-[(4-
methylpiperazin-l-yl)carbony1]-1H-
pyrrol-2-yllmethylene)-N-methyl-2-oxoindoline-5-sulfonamide; CAS no. 658084-23-
21), Foretinib
(GSK1363089), XL880 (CAS no. 849217-64-7; XL880 is a inhibitor of MET/HGFand
VEGFR2 and KDR);
MGCD-265 (MethylGene; MGCD-265 targets the met, VEGFR1, VEGFR2, VEGFR3, Ron
and Tie-2
31
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receptors; CAS no. 875337-44-3), Tivantinib (ARQ 197; (-)-(3R,4R)-3-(5,6-
dihydro-4H-pyrrolo[3,2,1-
ij]quinolin-1-y1)-4-(1H-indo1-3-yl)pyrrolidine-2,5-dione; see Munchi et al,
Mol Cancer Ther June 2010 9;
1544; CAS no. 905854-02-6), LY-2801653 (Lilly), LY2875358 (Lilly), MP-470,
Rilotumumab (AMG 102,
anti-HGF monoclonal antibody), antibody 223C4 or humanized antibody 223C4
(W02009/007427),
humanized L2G7 (humanized TAK701; humanized anti-HGF monoclonal antibody); EMD
1214063 (Merck
Sorono), EMD 1204831 (Merck Serono), NK4, Cabozantinib (XL-184, CAS no. 849217-
68-1; carbozantinib
is a dual inhibitor of M ET/HGFand VEGFR2), MP-470 (SuperGen; is a novel
inhibitor of c-KIT, MET,
PDGFR, F1t3, and AXL), Comp-1, Ficlatuzumab (AV-299; anti-HGF monoclonal
antibody), E7050 (Cas no.
1196681-49-8; E7050 is a dual MET/HGF and VEGFR2 inhibitor (Esai); MK-2461
(Merck; N-((2R)-1,4-
Dioxan-2-ylmethyl)-N-methyl-N'-[3-(1-methy1-1H-pyrazol-4-y1)-5-oxo-5H-
benzo[4,5]cyclohepta[1,2-
b]pyridin-7-yl]sulfamide; CAS no. 917879-39-1); MK8066 (Merck), PF4217903
(Pfizer), AMG208
(Amgen), SGX-126, RP1040, LY2801653, AMG458, EMD637830, BAY-853474, DP-3590.
In certain
embodiments, the met inhibitor is any one or more of crizotinib, tivantinib,
carbozantinib, MGCD-265,
ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224G11, DN-30, GDC-
0712, MK-2461,
E7050, MK-8033, PF-4217903, AMG208, JNJ-38877605, EMD1204831, INC-280, LY-
2801653, SGX-126,
RP1040, LY2801653, BAY-853474, and/or LA480. In certain embodiments, the met
inhibitor is any one or
more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab,
humanized TAK-701, rilotumumab,
and/or foretinib. In some emboidments, the met inhibitor is crizotnib.
[0106] In some embodiments of any of the combination therapy methods described
herein, the targeted
therapeutic is a BRAF inhibitor. Exemplary BRAF inhibitors are known in the
art and include, for
example, sorafenib, PLX4720, PLX-3603, dabrafenib (GSK2118436), GDC-0879,
RAF265 (Novartis),
XL281, ARQ736, BAY73-4506, vemurafenib and those described in W02007/002325,
W02007/002433, W02009111278, W02009111279, W02009111277, W02009111280 and U.S.
Pat.
No. 7,491,829. In some embodiments, the BRAF inhibitor is a selective BRAF
inhibitor. In some
embodiments, the BRAF inhibitor is a selective inhibitor of BRAF V600. In some
embodiments, BRAF
V600 is BRAF V600E, BRAF V600K, and/or V600D. In some embodiments, BRAF V600
is BRAF
V600R. In some embodiments, the BRAF inhibitor is vemurafenib. In some
embodiments, the BRAF
inhibitor is vemurafenib.
[0107] Vemurafenib (RG7204, PLX-4032, CAS Reg. No. 1029872-55-5) has been
shown to cause
programmed cell death in various cancer call lines, for example melanoma cell
lines. Vemurafenib
interrupts the BRAF/MEK step on the BRAF/MEK/ERK pathway - if the BRAF has the
common
V600E mutation. Vemurafenib works in patients, for example in melanoma
patients as approved by the
FDA, whose cancer has a V600E BRAF mutation (that is, at amino acid position
number 600 on the
32
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BRAF protein, the normal valine is replaced by glutamic acid). About 60% of
melanomas have the
V600E BRAF mutation. The V600E mutation is present in a variety of other
cancers, including
lymphoma, colon cancer, melanoma, thyroid cancer and lung cancer. Vemurafenib
has the following
structure:
0, "õ-..,......,
:S,
HN v
F 0
CI 0 0 *
I ,
\ F
N IN
H .
[0108] ZELBORAF (vemurafenib) (Genentech, Inc.) is a drug product approved in
the U.S. and
indicated for treatment of patients with unresectable or metastatic melanoma
with BRAF V600E
mutation as detected by an FDA-approved test. ZELBORAF (vemurafenib) is not
recommended for
use in melanoma patients who lack the BRAF V600E mutation (wild-type BRAF
melanoma).
[0109] In some embodiments of any of the combination therapy methods described
herein, the targeted
therapeutic is an ALK inhibitor. In some embodiments, the ALK inhibitor is
crizotinib. Crizotinib (also
known as PF-02341066 or 1066), is a Met and ALK (anaplastic lymphoma kinase)
inhibitor of the
aminopyridine chemical series that is being developed by Pfizer Incorporated
(see Zou et al., Cancer
Research 67: 4408-4417, 2007 and supplemental data). Other exemplary ALK
inhibitors include, for
example, TAE-684 (from Novartis; see Galkin, et al., Proc. National Acad. Sci.
104(1) 270-275, 2007),
AP26113 (Ariad Pharmaceuticals, Inc.), and CEP-14083, CEP-14513, and CEP-11988
(Cephalon; see
Wan et al., Blood 107: 1617-1623, 2006); and WHI-P131 and WHI-P154 (EMD
Biosciences), 5-chloro-
N4-112-(isopropylsulfonyl)phenyll-N2-12-methoxy-4-114-(4- -methylpiperazin-l-
yl)piperidin-l-
yllphenyllpyrimidine-2,4-diamine and 21(5-bromo-2-1 [2-methoxy-4-(4-
methylpiperazin-1-
yl)phenyllaminolpyrimidi- -n-4-yllaminol-N-methylbenzenesulfonamide (see Mosse
et al., Clin Cancer
Res. 2009 Sep. 15; 15(18):5609-14, 2009; J. of Med. Chem. 49: 1006-1015, 2006;
Cancer Research,
(US), 2004, 64: 8919-8923, 2004; Proc. Natl. Acad. Sci. 101:13306-13311, 2004;
Annual Review of
Medicine, (US) 54: 73, 2003; Science 278: 1309-1312, 1997; Oncogene 14 (4):
439-449, 1997;
Oncogene 9: 1567-1574, 1994; Am J Pathol 160: 1487-1494, 2002; Am J Pathol
157: 377-384, 2000;
Blood 90: 2901-2910, 1997; Am J. Pathol. 156 (3): 781-9, 2000; J Comb Chem. 8:
401-409, 2006 and
U.S. Pub. Nos. 20100152182; 20100099658; 20100048576; 20090286778;
20090221555;
33
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20090186801; 20090118216; 20090099193; 20080176881; 20080090776; 2008/0300273;
WO
2005/097765; WO 2005/009389; WO 2005/016894; WO 2004/080980; and
W02004079326).
[0110] In some embodiments of any of the combination therapy methods described
herein, the targeted
therapeutic is a MEK inhibitor. In some embodiments, the MEK inhibitor is a
MEK1 inhibitor, MEK2
inhibitor, and/or MEK1/2 inhibitor. Exemplary MEK inhibitors include, but are
not limited to,
trametinib (GSK 1120212), MEK162, selumetinib (AZD 6244, ARRY-142886),
pimasertib
(MSC1936369B, AS-703026, AS703026), GDC-0973, GDC-0623, PD-325901, GDC-0973,
CI-1040,
PD035901. In some embodiments, the Mek inhibitor is selumetinib, pimasertib,
GDC-0973, GDC-0623
or trametinib. In certain embodiments, the Mek inhibitor is GDC-0973.
[0111] GDC-0973 (XL518) is a selective inhibitor of MEK, also known as mitogen
activated protein
kinase kinase (MAPKK), which is a key component of the RAS/RAF/MEK/ERK pathway
that is
frequently activated in human tumors. Inappropriate activation of the MEK/ERK
pathway promotes cell
growth in the absence of exogenous growth factors. Clinical trials evaluating
GDC-0973 for solid
tumors is ongoing. GDC-0973 can be prepared as described in International
Patent Application
Publication Number W02007044515(A1). GDC-0973 has the name: (S)-(3,4-difluoro-
2-(2-fluoro-4-
iodophenylamino)phenyl)(3-hydroxy-3-(piperidin-2-ypazetidin-1-yl)methanone,
and the following
structure:
HO
r\O0 F
N
EN-I 0
NH
I
0 F
F
[0112] Trametinib (GSK 1120212, CAS Registry No. 871700-17-3) has the name N-
(3-13-Cyclopropyl-
5-R2-fluoro-4-iodophenyllaminol-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-
tetrahydropyridol4,3-dlpyrimidin-
1(2H)-yllphenypacetamide, and the following structure:
H
NI(
lel 0
ON 0
\
N N
0 NH 0
I F
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[0113] In some embodiments of any of the TKI and/or RTKI, the inhibitor may be
a specific inhibitor
for the polypeptide of interest, e.g., an inhibitor specific for EGFR, HER2,
MET/HGF, ALK, BRAF,
ROS1, and/or MEK. In some embodiments of any of the TM and/or RTKI, the
inhibitor may be a dual
inhibitor or pan inhibitor wherein the TM and/or RTKI inhibits one or more
polypeptides of interest,
e.g., an inhibitor specific for EGFR, HER2, MET/HGF, ALK, BRAF, ROS1, and/or
MEK, and one or
more other target polypeptides.
A. Antibodies
[0114] Provided herein isolated antibodies that bind to a polypeptide of
interest, such as ALDH and/or
tyrosine kinase (e.g., receptor tyrosine kinase), for use in the methods
described herein. In any of the
above embodiments, an antibody is humanized. Further, the antibody according
to any of the above
embodiments is a monoclonal antibody, including a chimeric, humanized or human
antibody. In one
embodiment, the antibody is an antibody fragment, e.g., a Fv, Fab, Fab', scFv,
diabody, or F(ab')2
fragment. In another embodiment, the antibody is a full length antibody, e.g.,
an "intact IgG 1" antibody
or other antibody class or isotype as defined herein.
[0115] In a further aspect, an antibody according to any of the above
embodiments may incorporate any
of the features, singly or in combination, as described in Sections below:
1. Antibody Affinity
[0116] In certain embodiments, an antibody provided herein has a dissociation
constant (Kd) of < 1pM,
< 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10-8 M or
less, e.g., from 10-8
M to 10-13 M, e.g., from 10-9 M to 10-13 M). In one embodiment, Kd is measured
by a radiolabeled
antigen binding assay (RIA). In one embodiment, the RIA is performed with the
Fab version of an
antibody of interest and its antigen. For example, solution binding affinity
of Fabs for antigen is
measured by equilibrating Fab with a minimal concentration of (125I)-labeled
antigen in the presence of a
titration series of unlabeled antigen, then capturing bound antigen with an
anti-Fab antibody-coated
plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish
conditions for the assay,
MICROTITER multi-well plates (Thermo Scientific) are coated overnight with 5
pg/ml of a capturing
anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and
subsequently blocked with
2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature
(approximately
23 C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM
1112511-antigen are mixed with serial
dilutions of a Fab of interest (e.g., consistent with assessment of the anti-
VEGF antibody, Fab-12, in
Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then
incubated overnight;
however, the incubation may continue for a longer period (e.g., about 65
hours) to ensure that
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equilibrium is reached. Thereafter, the mixtures are transferred to the
capture plate for incubation at
room temperature (e.g., for one hour). The solution is then removed and the
plate washed eight times
with 0.1% polysorbate 20 (TWEEN-20 ) in PBS. When the plates have dried, 150
pl/well of scintillant
(MICROSCINT-20 TM ; Packard) is added, and the plates are counted on a
TOPCOUNT TM gamma
counter (Packard) for ten minutes. Concentrations of each Fab that give less
than or equal to 20% of
maximal binding are chosen for use in competitive binding assays.
[0117] According to another embodiment, Kd is measured using a BIACORE
surface plasmon
resonance assay. For example, an assay using a BIACORE -2000 or a BIACORE (1)-
3000 (BIAeore, Inc.,
Piscataway, NJ) is performed at 25 C with immobilized antigen CM5 chips at ¨10
response units (RU).
In one embodiment, earboxymethylated dextran biosensor chips (CM5, BIACORE,
Inc.) are activated
with N-ethyl-N'- (3-dimethylaminopropy1)-earbodiimide hydrochloride (EDC) and
N-
hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is
diluted with 10 mM
sodium acetate, pH 4.8, to 5 g/m1 (-0.2 pM) before injection at a flow rate
of 5 pl/minute to achieve
approximately 10 response units (RU) of coupled protein. Following the
injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics measurements,
two-fold serial dilutions
of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20
(TWEEN-20) surfactant
(PBST) at 25 C at a flow rate of approximately 25 pl/min. Association rates
(kon) and dissociation rates
(kat.) are calculated using a simple one-to-one Langmuir binding model
(BIACORE Evaluation
Software version 3.2) by simultaneously fitting the association and
dissociation sensorgrams. The
equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon.
See, e.g., Chen et al., J. Mol.
Biol. 293:865-881 (1999). If the on-rate exceeds 106 M-1 5-1 by the surface
plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent quenching
technique that measures the
increase or decrease in fluorescence emission intensity (excitation = 295 nm;
emission = 340 nm, 16 nm
band-pass) at 25 C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2,
in the presence of
increasing concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped
spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO TM
spectrophotometer
(ThermoSpectronic) with a stirred euvette.
2. Antibody Fragments
[0118] In certain embodiments, an antibody provided herein is an antibody
fragment. Antibody
fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv,
and seFv fragments, and other
fragments described below. For a review of certain antibody fragments, see
Hudson et al. Nat. Med.
9:129-134 (2003). For a review of seFv fragments, see, e.g., Pluekthiin, in
The Pharmacology of
36
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Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag,
New York), pp. 269-
315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and
5,587,458. For discussion of
Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues
and having increased in
vivo half-life, see U.S. Patent No. 5,869,046.
[0119] Diabodies are antibody fragments with two antigen-binding sites that
may be bivalent or
bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat.
Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).
Triabodies and tetrabodies are
also described in Hudson et al., Nat. Med. 9:129-134 (2003).
[0120] Single-domain antibodies are antibody fragments comprising all or a
portion of the heavy chain
variable domain or all or a portion of the light chain variable domain of an
antibody. In certain
embodiments, a single-domain antibody is a human single-domain antibody
(Domantis, Inc., Waltham,
MA; see, e.g., U.S. Patent No. 6,248,516 B1).
[0121] Antibody fragments can be made by various techniques, including but not
limited to proteolytic
digestion of an intact antibody as well as production by recombinant host
cells (e.g., E. coli or phage), as
described herein.
3. Chimeric and Humanized Antibodies
[0122] In certain embodiments, an antibody provided herein is a chimeric
antibody. Certain chimeric
antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et
al., Proc. Natl. Acad. Sci.
USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-
human variable
region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or
non-human primate, such as
a monkey) and a human constant region. In a further example, a chimeric
antibody is a "class switched"
antibody in which the class or subclass has been changed from that of the
parent antibody. Chimeric
antibodies include antigen-binding fragments thereof.
[0123] In certain embodiments, a chimeric antibody is a humanized antibody.
Typically, a non-human
antibody is humanized to reduce immunogenicity to humans, while retaining the
specificity and affinity
of the parental non-human antibody. Generally, a humanized antibody comprises
one or more variable
domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a
non-human antibody, and
FRs (or portions thereof) are derived from human antibody sequences. A
humanized antibody optionally
will also comprise at least a portion of a human constant region. In some
embodiments, some FR
residues in a humanized antibody are substituted with corresponding residues
from a non-human
antibody (e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve
antibody specificity or affinity.
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[0124] Humanized antibodies and methods of making them are reviewed, e.g., in
Almagro and
Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g.,
in Riechmann et al.,
Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-
10033 (1989); US
Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al.,
Methods 36:25-34 (2005)
(describing specificity-determining region (SDR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991)
(describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005)
(describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,
83:252-260 (2000)
(describing the "guided selection" approach to FR shuffling).
[0125] Human framework regions that may be used for humanization include but
are not limited to:
framework regions selected using the "best-fit" method (see, e.g., Sims et al.
J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of human
antibodies of a particular
subgroup of light or heavy chain variable regions (see, e.g., Carter et al.
Proc. Natl. Acad. Sci. USA,
89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature
(somatically mutated)
framework regions or human germline framework regions (see, e.g., Almagro and
Fransson, Front.
Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR
libraries (see, e.g.,
Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol.
Chem. 271:22611-22618
(1996)).
4. Human Antibodies
[0126] In certain embodiments, an antibody provided herein is a human
antibody. Human antibodies
can be produced using various techniques known in the art. Human antibodies
are described generally in
van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and
Lonberg, Curr. Opin.
Immunol. 20:450-459 (2008).
[0127] Human antibodies may be prepared by administering an immunogen to a
transgenic animal that
has been modified to produce intact human antibodies or intact antibodies with
human variable regions
in response to antigenic challenge. Such animals typically contain all or a
portion of the human
immunoglobulin loci, which replace the endogenous immunoglobulin loci, or
which are present
extrachromosomally or integrated randomly into the animal's chromosomes. In
such transgenic mice,
the endogenous immunoglobulin loci have generally been inactivated. For review
of methods for
obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech.
23:1117-1125 (2005).
See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing
XENOMOUSETm technology; U.S.
Patent No. 5,770,429 describing HuMab technology; U.S. Patent No. 7,041,870
describing K-M
MOUSE technology, and U.S. Patent Application Publication No. US
2007/0061900, describing
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VelociMouse technology). Human variable regions from intact antibodies
generated by such animals
may be further modified, e.g., by combining with a different human constant
region.
[0128] Human antibodies can also be made by hybridoma-based methods. Human
myeloma and mouse-
human heteromyeloma cell lines for the production of human monoclonal
antibodies have been
described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987); and
Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via
human B-cell hybridoma
technology are also described in Li et al., Proc. Natl. Acad. Sci. USA,
103:3557-3562 (2006). Additional
methods include those described, for example, in U.S. Patent No. 7,189,826
(describing production of
monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai
Mianyixue, 26(4):265-
268 (2006) (describing human-human hybridomas). Human hybridoma technology
(Trioma technology)
is also described in Vollmers and Brandlein, Hist. & Histopath., 20(3):927-937
(2005) and Vollmers and
Brandlein, Methods Find Exp. Clin. Pharmacol., 27(3):185-91 (2005).
[0129] Human antibodies may also be generated by isolating Fv clone variable
domain sequences
selected from human-derived phage display libraries. Such variable domain
sequences may then be
combined with a desired human constant domain. Techniques for selecting human
antibodies from
antibody libraries are described below.
5. Library-Derived Antibodies
[0130] Antibodies may be isolated by screening combinatorial libraries for
antibodies with the desired
activity or activities. For example, a variety of methods are known in the art
for generating phage
display libraries and screening such libraries for antibodies possessing the
desired binding
characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. Methods
Mol. Biol. 178:1-37
(O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described,
e.g., in the McCafferty et
al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et
al., J. Mol. Biol. 222:
581-597 (1992); Marks and Bradbury, Methods Mol. Biol. 248:161-175 (Lo, ed.,
Human Press, Totowa,
NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J.
Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and
Lee et al., J. Immunol.
Methods 284(1-2): 119-132(2004).
[0131] In certain phage display methods, repertoires of VH and VL genes are
separately cloned by
polymerase chain reaction (PCR) and recombined randomly in phage libraries,
which can then be
screened for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455
(1994). Phage typically display antibody fragments, either as single-chain Fv
(scFv) fragments or as Fab
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fragments. Libraries from immunized sources provide high-affinity antibodies
to the immunogen
without the requirement of constructing hybridomas. Alternatively, the naive
repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a wide range of
non-self and also self
antigens without any immunization as described by Griffiths et al., EMBO J,
12: 725-734 (1993).
Finally, naive libraries can also be made synthetically by cloning
unrearranged V-gene segments from
stem cells, and using PCR primers containing random sequence to encode the
highly variable CDR3
regions and to accomplish rearrangement in vitro, as described by Hoogenboom
and Winter, J. Mol.
Biol., 227: 381-388 (1992). Patent publications describing human antibody
phage libraries include, for
example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574,
2005/0119455,
2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0132] Antibodies or antibody fragments isolated from human antibody libraries
are considered human
antibodies or human antibody fragments herein.
6. Multispecific Antibodies
[0133] In certain embodiments, an antibody provided herein is a multispecific
antibody, e.g., a
bispecific antibody. Multispecific antibodies are monoclonal antibodies that
have binding specificities
for at least two different sites. In certain embodiments, one of the binding
specificities is a polypeptide
of interest, such as ALDH and/or tyrosine kinase (e.g., receptor tyrosine
kinase), and the other is for any
other antigen. In certain embodiments, bispecific antibodies may bind to two
different epitopes of a
polypeptide of interest, such as ALDH and/or tyrosine kinase (e.g., receptor
tyrosine kinase). Bispecific
antibodies may also be used to localize cytotoxic agents to cells which
express a polypeptide of interest,
such as ALDH and/or tyrosine kinase (e.g., receptor tyrosine kinase).
Bispecific antibodies can be
prepared as full length antibodies or antibody fragments.
[0134] Techniques for making multispecific antibodies include, but are not
limited to, recombinant co-
expression of two immunoglobulin heavy chain-light chain pairs having
different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et
al., EMBO J. 10: 3655
(1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No.
5,731,168). Multi-specific antibodies
may also be made by engineering electrostatic steering effects for making
antibody Fc-heterodimeric
molecules (WO 2009/089004A1); cross-linking two or more antibodies or
fragments (see, e.g., US
Patent No. 4,676,980, and Brennan et al., Science, 229: 81(1985)); using
leucine zippers to produce bi-
specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553
(1992)); using "diabody"
technology for making bispecific antibody fragments (see, e.g., Hollinger et
al., Proc. Natl. Acad. Sci.
USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see,e.g.,
Gruber et al., J. Immunol.,
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152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in
Tutt et al. J. Immunol. 147:
60 (1991).
[0135] Engineered antibodies with three or more functional antigen binding
sites, including "Octopus
antibodies," are also included herein (see, e.g., US 2006/0025576A1).
[0136] The antibody or fragment herein also includes a "Dual Acting FAb" or
"DAF" comprising an
antigen binding site that binds to a polypeptide of interest, such as ALDH
and/or tyrosine kinase (e.g.,
receptor tyrosine kinase), as well as another, different antigen (see, US
2008/0069820, for example).
7. Antibody Variants
a) Glycosylation variants
[0137] In certain embodiments, an antibody provided herein is altered to
increase or decrease the extent
to which the antibody is glycosylated. Addition or deletion of glycosylation
sites to an antibody may be
conveniently accomplished by altering the amino acid sequence such that one or
more glycosylation
sites is created or removed.
[0138] Where the antibody comprises an Fc region, the carbohydrate attached
thereto may be altered.
Native antibodies produced by mammalian cells typically comprise a branched,
biantennary
oligosaccharide that is generally attached by an N-linkage to Asn297 of the
CH2 domain of the Fc
region. See, e.g., Wright et al. TIBlECH 15:26-32 (1997). The oligosaccharide
may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (G1cNAc), galactose, and
sialic acid, as well as a
fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide
structure. In some
embodiments, modifications of the oligosaccharide in an antibody of the
invention may be made in order
to create antibody variants with certain improved properties.
[0139] In one embodiment, antibody variants are provided having a carbohydrate
structure that lacks
fucose attached (directly or indirectly) to an Fc region. For example, the
amount of fucose in such
antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to
40%. The amount
of fucose is determined by calculating the average amount of fucose within the
sugar chain at Asn297,
relative to the sum of all glycostructures attached to Asn 297 (e. g. complex,
hybrid and high mannose
structures) as measured by MALDI-TOF mass spectrometry, as described in WO
2008/077546, for
example. Asn297 refers to the asparagine residue located at about position 297
in the Fc region (Eu
numbering of Fc region residues); however, Asn297 may also be located about
3 amino acids
upstream or downstream of position 297, i.e., between positions 294 and 300,
due to minor sequence
variations in antibodies. Such fucosylation variants may have improved ADCC
function. See, e.g., US
Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa
Hakko Kogyo Co.,
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Ltd). Examples of publications related to "defucosylated" or "fucose-
deficient" antibody variants
include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US
2002/0164328;
US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US
2004/0109865; WO
2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742;
W02002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-
Ohnuki et al., Biotech.
Bioeng. 87: 614 (2004). Examples of cell lines capable of producing
defucosylated antibodies include
Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem.
Biophys. 249:533-545
(1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al,
Adams et al.,
especially at Example 11), and knockout cell lines, such as alpha-1,6-
fucosyltransferase gene, FUT8,
knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614
(2004); Kanda, Y. et al.,
Biotechnol. Bioeng., 94(4):680-688 (2006); and W02003/085107).
[0140] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a
biantennary oligosaccharide attached to the Fc region of the antibody is
bisected by GlcNAc. Such
antibody variants may have reduced fucosylation and/or improved ADCC function.
Examples of such
antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.);
US Patent No. 6,602,684
(Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at
least one galactose
residue in the oligosaccharide attached to the Fc region are also provided.
Such antibody variants may
have improved CDC function. Such antibody variants are described, e.g., in WO
1997/30087 (Patel et
al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
b) Fc region variants
[0141] In certain embodiments, one or more amino acid modifications may be
introduced into the Fc
region of an antibody provided herein, thereby generating an Fc region
variant. The Fc region variant
may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or
IgG4 Fc region)
comprising an amino acid modification (e.g., a substitution) at one or more
amino acid positions.
[0142] In certain embodiments, the invention contemplates an antibody variant
that possesses some but
not all effector functions, which make it a desirable candidate for
applications in which the half life of
the antibody in vivo is important yet certain effector functions (such as
complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be
conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor
(FcR) binding assays can
be conducted to ensure that the antibody lacks FcyR binding (hence likely
lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC, NK cells,
express Fc(RIII only,
whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression on
hematopoietic cells is
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summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.
9:457-492 (1991). Non-
limiting examples of in vitro assays to assess ADCC activity of a molecule of
interest is described in
U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad.
Sci. USA 83:7059-7063
(1986)) and Hellstrom, let al., Proc. Nat'l Acad. Sci. USA 82:1499-1502
(1985); 5,821,337 (see
Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-
radioactive assays
methods may be employed (see, for example, ACTITm non-radioactive cytotoxicity
assay for flow
cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 non-
radioactive cytotoxicity
assay (Promega, Madison, WI). Useful effector cells for such assays include
peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity
of the molecule of interest may be assessed in vivo, e.g., in an animal model
such as that disclosed in
Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays
may also be carried out
to confirm that the antibody is unable to bind Clq and hence lacks CDC
activity. See, e.g., Clq and C3c
binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement
activation, a CDC
assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol.
Methods 202:163
(1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and
M.J. Glennie, Blood
103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life
determinations can also be
performed using methods known in the art (see, e.g., Petkova, S.B. et al.,
Int'l. Immunol. 18(12):1759-
1769 (2006)).
[0143] Antibodies with reduced effector function include those with
substitution of one or more of Fc
region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No.
6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid positions
265, 269, 270, 297 and
327, including the so-called "DANA" Fc mutant with substitution of residues
265 and 297 to alanine
(US Patent No. 7,332,581).
[0144] Certain antibody variants with improved or diminished binding to FcRs
are described. (See, e.g.,
U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem.
9(2): 6591-6604
(2001).) In certain embodiments, an antibody variant comprises an Fc region
with one or more amino
acid substitutions which improve ADCC, e.g., substitutions at positions 298,
333, and/or 334 of the Fc
region (EU numbering of residues). In some embodiments, alterations are made
in the Fc region that
result in altered (i.e., either improved or diminished) Clq binding and/or
Complement Dependent
Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO
99/51642, and Idusogie et al. J.
Immunol. 164: 4178-4184 (2000).
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[0145] Antibodies with increased half-lives and improved binding to the
neonatal Fc receptor (FcRn),
which is responsible for the transfer of maternal IgGs to the fetus (Guyer et
al., J. Immunol. 117:587
(1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in
US2005/0014934A1 (Hinton et al.).
Those antibodies comprise an Fc region with one or more substitutions therein
which improve binding
of the Fc region to FcRn. Such Fc variants include those with substitutions at
one or more of Fc region
residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356,
360, 362, 376, 378, 380, 382,
413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No.
7,371,826). See also Duncan
& Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No.
5,624,821; and WO
94/29351 concerning other examples of Fc region variants.
c) Cysteine engineered antibody variants
[0146] In certain embodiments, it may be desirable to create cysteine
engineered antibodies, e.g.,
"thioMAbs," in which one or more residues of an antibody are substituted with
cysteine residues. In
particular embodiments, the substituted residues occur at accessible sites of
the antibody. By substituting
those residues with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the
antibody and may be used to conjugate the antibody to other moieties, such as
drug moieties or linker-
drug moieties, to create an immunoconjugate, as described further herein. In
certain embodiments, any
one or more of the following residues may be substituted with cysteine: V205
(Kabat numbering) of the
light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering)
of the heavy chain Fc
region. Cysteine engineered antibodies may be generated as described, e.g., in
U.S. Patent No.
7,521,541.
B. Immunoconjugates
[0147] Further provided herein are immunoconjugates comprising antibody which
binds a polypeptide
of interest, such as ALDH and/or tyrosine kinase (e.g., receptor tyrosine
kinase), or immunoconjugates
comprising an antibody which binds a polypeptide of interest, such as ALDH
and/or tyrosine kinase
(e.g., receptor tyrosine kinase), conjugated to one or more cytotoxic agents,
such as chemotherapeutic
agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins,
enzymatically active toxins of
bacterial, fungal, plant, or animal origin, or fragments thereof), or
radioactive isotopes for use in the
methods described herein.
[0148] In one embodiment, an immunoconjugate is an antibody-drug conjugate
(ADC) in which an
antibody is conjugated to one or more drugs, including but not limited to a
maytansinoid (see U.S.
Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as
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monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent
Nos. 5,635,483
and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative
thereof (see U.S. Patent Nos.
5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,
and 5,877,296; Hinman et
al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-
2928 (1998)); an
anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current
Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006);
Torgov et al., Bioconj.
Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834
(2000); Dubowchik et
al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.
Chem. 45:4336-4343
(2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane such
as docetaxel, paclitaxel,
larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.
[0149] In another embodiment, an immunoconjugate comprises an antibody as
described herein
conjugated to an enzymatically active toxin or fragment thereof, including but
not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain
(from Pseudomonas
aeluginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia
inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin,
enomycin, and the tricothecenes.
[0150] In another embodiment, an immunoconjugate comprises an antibody as
described herein
conjugated to a radioactive atom to form a radioconjugate. A variety of
radioactive isotopes are
available for the production of radioconjugates. Examples include At211, 1131,
1125, y90, Re186, Re188,
sm153, Bi212, P32, Pb 212
and radioactive isotopes of Lu. When the radioconjugate is used for detection,
it
may comprise a radioactive atom for scintigraphic studies, for example Tc99m
or 1123, or a spin label for
nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance
imaging, mri), such as
iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium,
manganese or iron.
[0151] Conjugates of an antibody and cytotoxic agent may be made using a
variety of bifunctional
protein coupling agents such as N-succinimidy1-3-(2-pyridyldithio) propionate
(SPDP), succinimidy1-4-
(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT),
bifunctional derivatives
of imidoesters (such as dimethyl adipimidate HC1), active esters (such as
disuccinimidyl suberate),
aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-
azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as bis-(p-diazoniumbenzoy1)-ethylenediamine),
diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-
difluoro-2,4-dinitrobenzene).
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For example, a ricin immunotoxin can be prepared as described in Vitetta et
al., Science 238:1098
(1987). Carbon-14-labeled 1-isothiocyanatobenzy1-3-methyldiethylene
triaminepentaacetic acid (MX-
DTPA) is an exemplary chelating agent for conjugation of radionucleotide to
the antibody. See
W094/11026. The linker may be a "cleavable linker" facilitating release of a
cytotoxic drug in the cell.
For example, an acid-labile linker, peptidase-sensitive linker, photolabile
linker, dimethyl linker or
disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S.
Patent No. 5,208,020)
may be used.
[0152] The immunuoconjugates or ADCs herein expressly contemplate, but are not
limited to such
conjugates prepared with cross-linker reagents including, but not limited to,
BMPS, EMCS, GMBS,
HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-
GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidy1-(4-vinylsulfone)benzoate) which are commercially available
(e.g., from Pierce
Biotechnology, Inc., Rockford, IL., U.S.A).
C. Binding Polypeptides
[0153] Binding polypeptides are polypeptides that bind, preferably
specifically, to a polypeptide of
interest, such as ALDH and/or tyrosine kinase (e.g., receptor tyrosine
kinase),are also provided for use in
the methods described herein as described herein. In some embodiments, the
binding polypeptides are
antagonists of a polypeptide of interest, such as ALDH and/or tyrosine kinase
(e.g., receptor tyrosine
kinase).
[0154] Binding polypeptides may be chemically synthesized using known
polypeptide synthesis
methodology or may be prepared and purified using recombinant technology.
Binding polypeptides are
usually at least about 5 amino acids in length, alternatively at least about
6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or 100 amino acids in length or more, wherein such binding polypeptides
that are capable of binding,
preferably specifically, to a polypeptide of interest, such as ALDH and/or
tyrosine kinase (e.g., receptor
tyrosine kinase).
[0155] Binding polypeptides may be identified without undue experimentation
using well known
techniques. In this regard, it is noted that techniques for screening
polypeptide libraries for binding
polypeptides that are capable of specifically binding to a polypeptide target
are well known in the art
(see, e.g., U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092,
5,223,409, 5,403,484,
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5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and W084/03564; Geysen
et al., Proc. Natl.
Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci.
U.S.A., 82:178-182 (1985);
Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et
al., J. Immunol. Meth,,
102:259-274 (1987); Schoofs et al., J. Immunot, 140:611-616 (1988), Cwirla, S.
E. et al. (1990) Proc.
Natl. Acad. Sci. USA, 87:6378; Lowman, H.B. et al. (1991) Biochemistry,
30:10832; Clackson, T. et al.
(1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581;
Kang, A.S. et al. (1991)
Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin.
Biotechnol., 2:668).
Methods of generating peptide libraries and screening these libraries are also
disclosed in U.S. Patent
Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434,
5,734,018, 5,698,426,
5,763,192, and 5,723,323.
D. Binding Small Molecules
[0156] Provided herein are binding small molecules for use as a small molecule
ALDH inhibitor (e.g.,
disulfiram and/or derivatives thereof) and/or small molecule targeted
therapeutic (e.g., small molecule
TKI (e.g., small molecule RTKI) for use in the methods described above.
[0157] Binding small molecules are preferably organic molecules other than
binding polypeptides or
antibodies as defined herein that bind, preferably specifically, to a
polypeptide of interest, such as
ALDH and/or tyrosine kinase (e.g., receptor tyrosine kinase). Binding organic
small molecules may be
identified and chemically synthesized using known methodology (see, e.g., PCT
Publication Nos.
W000/00823 and W000/39585). Binding organic small molecules are usually less
than about 2000
daltons in size, alternatively less than about 1500, 750, 500, 250 or 200
daltons in size, wherein such
organic small molecules that are capable of binding, preferably specifically,
to a polypeptide as
described herein may be identified without undue experimentation using well
known techniques. In this
regard, it is noted that techniques for screening organic small molecule
libraries for molecules that are
capable of binding to a polypeptide of interest are well known in the art
(see, e.g., PCT Publication Nos.
W000/00823 and W000/39585). Binding organic small molecules may be, for
example, aldehydes,
ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines,
secondary amines, tertiary
amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,
thioethers, disulfides, carboxylic
acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals,
acetals, thioacetals, aryl halides,
aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds,
heterocyclic compounds, anilines,
alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines,
thiazolidines, thiazolines, enamines,
sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo
compounds, acid chlorides, or
the like.
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E. Antagonist Polynucleotides
[0158] Provided herein are also polynucleotide antagonists for use in the
methods described herein. The
polynucleotide may be an antisense nucleic acid and/or a ribozyme. The
antisense nucleic acids
comprise a sequence complementary to at least a portion of an RNA transcript
of a gene of interest, such
as ALDH and/or tyrosine kinase (e.g., receptor tyrosine kinase). However,
absolute complementarity,
although preferred, is not required.
[0159] A sequence "complementary to at least a portion of an RNA," referred to
herein, means a
sequence having sufficient complementarity to be able to hybridize with the
RNA, forming a stable
duplex; in the case of double stranded antisense nucleic acids, a single
strand of the duplex DNA may
thus be tested, or triplex formation may be assayed. The ability to hybridize
will depend on both the
degree of complementarity and the length of the antisense nucleic acid.
Generally, the larger the
hybridizing nucleic acid, the more base mismatches with a RNA it may contain
and still form a stable
duplex (or triplex as the case may be). One skilled in the art can ascertain a
tolerable degree of mismatch
by use of standard procedures to determine the melting point of the hybridized
complex.
[0160] Polynucleotides that are complementary to the 5 end of the message,
e.g., the 5' untranslated
sequence up to and including the AUG initiation codon, should work most
efficiently at inhibiting
translation. However, sequences complementary to the 3' untranslated sequences
of mRNAs have been
shown to be effective at inhibiting translation of mRNAs as well. See
generally, Wagner, R., 1994,
Nature 372:333-335. Thus, oligonucleotides complementary to either the 5'- or
3'-non-translated, non-
coding regions of the gene, could be used in an antisense approach to inhibit
translation of endogenous
mRNA. Polynucleotides complementary to the 5' untranslated region of the mRNA
should include the
complement of the AUG start codon. Antisense polynucleotides complementary to
mRNA coding
regions are less efficient inhibitors of translation but could be used in
accordance with the invention.
Whether designed to hybridize to the 5'-, 3'- or coding region of an mRNA,
antisense nucleic acids
should be at least six nucleotides in length, and are preferably
oligonucleotides ranging from 6 to about
50 nucleotides in length. In specific aspects the oligonucleotide is at least
10 nucleotides, at least 17
nucleotides, at least 25 nucleotides or at least 50 nucleotides.
F. Antibody and Binding Polypeptide Variants
[0161] In certain embodiments, amino acid sequence variants of the antibodies
and/or the binding
polypeptides provided herein are contemplated. For example, it may be
desirable to improve the binding
affinity and/or other biological properties of the antibody and/or binding
polypeptide. Amino acid
sequence variants of an antibody and/or binding polypeptides may be prepared
by introducing
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appropriate modifications into the nucleotide sequence encoding the antibody
and/or binding
polypeptide, or by peptide synthesis. Such modifications include, for example,
deletions from, and/or
insertions into and/or substitutions of residues within the amino acid
sequences of the antibody and/or
binding polypeptide. Any combination of deletion, insertion, and substitution
can be made to arrive at
the final construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-
binding.
[0162] In certain embodiments, antibody variants and/or binding polypeptide
variants having one or
more amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the
HVRs and FRs. Conservative substitutions are shown in Table 1 under the
heading of "preferred
substitutions." More substantial changes are provided in Table 1 under the
heading of "exemplary
substitutions," and as further described below in reference to amino acid side
chain classes. Amino acid
substitutions may be introduced into an antibody and/or binding polypeptide of
interest and the products
screened for a desired activity, e.g., retained/improved antigen binding,
decreased immunogenicity, or
improved ADCC or CDC.
TABLE 1
Original Residue Exemplary Substitutions Preferred
Substitutions
Ala (A) Val; Leu; Ile Val
Arg (R) Lys; Gln; Asn Lys
Asn (N) Gln; His; Asp, Lys; Arg Gln
Asp (D) Glu; Asn Glu
Cys (C) Ser; Ala Ser
Gln (Q) Asn; Glu Asn
Glu (E) Asp; Gln Asp
Gly (G) Ala Ala
His (H) Asn; Gln; Lys; Arg Arg
Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu
Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile
Lys (K) Arg; Gln; Asn Arg
Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr
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Original Residue Exemplary Substitutions Preferred
Substitutions
Pro (P) Ala Ala
Ser (S) Thr Thr
Thr (T) Val; Ser Ser
Trp (W) Tyr; Phe Tyr
Tyr (Y) Trp; Phe; Thr; Ser Phe
Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0163] Amino acids may be grouped according to common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
[0164] Non-conservative substitutions will entail exchanging a member of one
of these classes for
another class.
G. Antibody and Binding Polypeptide Derivatives
[0165] In certain embodiments, an antibody and/or binding polypeptide provided
herein may be further
modified to contain additional nonproteinaceous moieties that are known in the
art and readily available.
The moieties suitable for derivatization of the antibody and/or binding
polypeptide include but are not
limited to water soluble polymers. Non-limiting examples of water soluble
polymers include, but are not
limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene
glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone,
poly-1, 3-dioxolane, poly-
1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random
copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol
homopolymers, prolypropylene oxide/ethylene oxide co-polymers,
polyoxyethylated polyols (e.g.,
glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have
advantages in manufacturing due to its stability in water. The polymer may be
of any molecular weight,
and may be branched or unbranched. The number of polymers attached to the
antibody and/or binding
polypeptide may vary, and if more than one polymer are attached, they can be
the same or different
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molecules. In general, the number and/or type of polymers used for
derivatization can be determined
based on considerations including, but not limited to, the particular
properties or functions of the
antibody and/or binding polypeptide to be improved, whether the antibody
derivative and/or binding
polypeptide derivative will be used in a therapy under defined conditions,
etc.
[0166] In another embodiment, conjugates of an antibody and/or binding
polypeptide to
nonproteinaceous moiety that may be selectively heated by exposure to
radiation are provided. In one
embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al.,
Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength, and
includes, but is not limited to,
wavelengths that do not harm ordinary cells, but which heat the
nonproteinaceous moiety to a
temperature at which cells proximal to the antibody and/or binding polypeptide-
nonproteinaceous
moiety are killed.
IV. Methods of Screening and/or Identifying ALDH inhibitor and/or Targeted
TherapeuticsWith
Desired Function
[0167] Additional antagonists of a polypeptide of interest, such as ALDH
and/or tyrosine kinase (e.g.,
receptor tyrosine kinase) for use in the methods described herein, including
antibodies, binding
polypeptides, and/or binding small molecules provided herein may be
identified, screened for, or
characterized for their physical/chemical properties and/or biological
activities by various assays known
in the art.
[0168] Amino acid sequences of various human ALDH family members (e.g.,
"isozymes") are known in
the art and are publicly available. See, e.g., GenBank Accession No. NP<sub>--</sub>
000680 (ALDH 1,
member Al); GenBank Accession No. NP_000684 (ALDH 1, member A3); GenBank
Accession Nos.
AAH02967 and NP<sub>--000681</sub> (ALDH 2); GenBank Accession No. NP<sub>--</sub>
001026976 (ALDH 3,
member A2, isoform 1); GenBank Accession No. CA139494 (ALDH 4, member Al);
GenBank
Accession No. CAA20248 (ALDH 5, member Al); GenBank Accession No. EAW81160
(ALDH 6,
member Al, isoform CRA_b); GenBank Accession No. AAH02515 (ALDH 7, member Al);
GenBank
Accession No. NP<sub>--072090</sub> (ALDH 8, member Al, isoform 1); GenBank
Accession No. NP<sub>--</sub>
000687 (ALDH 9, member Al); GenBank Accession No. AAG42417 (ALDH 12); GenBank
Accession
No. AAG42417 (ALDH 12); GenBank Accession No. NP<sub>--699160</sub> (ALDH 16); and
GenBank
Accession No. CAI16766 (ALDH 18, member Al).
[0169] The crystal structures of wild-type ALDH2 and a C3025 mutant of ALDH2
are known in the art
(U.S. Pat. No. 8,124,389), and can be used in the design and preparation of
ALDH inhibitors for use in
the methods and compositions described herein.
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[0170] In certain embodiments, a computer system comprising a memory
comprising atomic
coordinates of an ALDH polypeptide are useful as models for rationally
identifying compounds that a
ligand binding site of an ALDH polypeptide. Such compounds may be designed
either de novo, or by
modification of a known compound, for example. In other cases, binding
compounds may be identified
by testing known compounds to determine if the "dock" with a molecular model
of an ALDH
polypeptide. Such docking methods are generally well known in the art.
[0171] ALDH crystal structure data can be used in conjunction with computer-
modeling techniques to
develop models of binding of various ALDH-binding compounds by analysis of the
crystal structure
data. The site models characterize the three-dimensional topography of site
surface, as well as factors
including van der Waals contacts, electrostatic interactions, and hydrogen-
bonding opportunities.
Computer simulation techniques are then used to map interaction positions for
functional groups
including but not limited to protons, hydroxyl groups, amine groups, divalent
cations, aromatic and
aliphatic functional groups, amide groups, alcohol groups, etc. that are
designed to interact with the
model site. These groups may be designed into a pharmacophore or candidate
compound with the
expectation that the candidate compound will specifically bind to the site.
Pharmacophore design thus
involves a consideration of the ability of the candidate compounds falling
within the pharmacophore to
interact with a site through any or all of the available types of chemical
interactions, including hydrogen
bonding, van der Waals, electrostatic, and covalent interactions, although in
general, pharmacophores
interact with a site through non-covalent mechanisms.
[0172] The ability of a pharmacophore or candidate compound to bind to an ALDH
polypeptide can be
analyzed in addition to actual synthesis using computer modeling techniques.
Only those candidates that
are indicated by computer modeling to bind the target (e.g., an ALDH
polypeptide binding site) with
sufficient binding energy (in one example, binding energy corresponding to a
dissociation constant with
the target on the order of 10-2 M or tighter) may be synthesized and tested
for their ability to bind to an
ALDH polypeptide and to inhibit ALDH enzymatic function using enzyme assays
known to those of
skill in the art and/or as described herein. The computational evaluation step
thus avoids the unnecessary
synthesis of compounds that are unlikely to bind an ALDH polypeptide with
adequate affinity.
[0173] An ALDH pharmacophore or candidate compound may be computationally
evaluated and
designed by means of a series of steps in which chemical entities or fragments
are screened and selected
for their ability to associate with individual binding target sites on an ALDH
polypeptide. One skilled in
the art may use one of several methods to screen chemical entities or
fragments for their ability to
associate with an ALDH polypeptide, and more particularly with target sites on
an ALDH polypeptide.
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The process may begin by visual inspection of, for example a target site on a
computer screen, based on
the ALDH polypeptide coordinates, or a subset of those coordinates known in
the art.
[0174] To select for an ALDH inhibitor which enhances induction cancer cell
death, loss of membrane
integrity as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD
uptake in combination with a
targeted therapeutic (e.g., TKI) may be assessed relative to a reference. A PI
uptake assay can be
performed in the absence of complement and immune effector cells. Tumor cells
are incubated with
medium alone or medium containing the appropriate combination of an ALDH
and/or targeted
therapeutic (TKI). The cells are incubated for a 3-day time period. Following
each treatment, cells are
washed and aliquoted into 35 mm strainer-capped 12 x 75 tubes (1 ml per tube,
3 tubes per treatment
group) for removal of cell clumps. Tubes then receive P1(10 pg/ml). Samples
may be analyzed using a
FACSCAN flow cytometer and FACSCONVERT CellQuest software (Becton
Dickinson). Those
ALDH inhibitor in combination with a targeted therapeutic (e.g., TM) that
induce statistically
significant levels of cell death compared to media alone and/or targeted
therapeutic (e.g., TM) alone as
determined by PI uptake may be selected as cell death-inducing antibodies,
binding polypeptides or
binding small molecules.
[0175] In some embodiments of any of the methods of screening and/or
identifying, the candidate
ALDH inhibitor is an antibody, binding polypeptide, binding small molecule, or
polynucleotide. In some
embodiments, the ALDH inhibitor (e.g., disulfiram and/or derivatives thereof)
is a small molecule.
V. Pharmaceutical Formulations
[0176] Pharmaceutical formulations of an antagonist of an ALDH inhibitor
(e.g., disulfiram and/or
derivatives thereof) and/or a targeted therapeutic (e.g., TKI) as described
herein are prepared by mixing
such antibody having the desired degree of purity with one or more optional
pharmaceutically
acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol,
A. Ed. (1980)), in the
form of lyophilized formulations or aqueous solutions. In some embodiments,
the antagonist of an
ALDH inhibitor (e.g., disulfiram and/or derivatives thereof) and/or a targeted
therapeutic (e.g., TKI) is a
binding small molecule, an antibody, binding polypeptide, and/or
polynucleotide. Pharmaceutically
acceptable carriers are generally nontoxic to recipients at the dosages and
concentrations employed, and
include, but are not limited to: buffers such as phosphate, citrate, and other
organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride; benzethonium
chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol;
resorcinol; cyclohexanol; 3-
pentanol; and m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such
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as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine, arginine, or
lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose, or
dextrins; chelating agents such as
EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming
counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic
surfactants such as
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers
herein further include
insterstitial drug dispersion agents such as soluble neutral-active
hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such
as rHuPH20
(HYLENEX , Baxter International, Inc.). Certain exemplary sHASEGPs and methods
of use, including
rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and
2006/0104968. In one aspect,
a sHASEGP is combined with one or more additional glycosaminoglycanases such
as chondroitinases.
[0177] Exemplary lyophilized formulations are described in US Patent No.
6,267,958. Aqueous
antibody formulations include those described in US Patent No. 6,171,586 and
W02006/044908, the
latter formulations including a histidine-acetate buffer.
[0178] The formulation herein may also contain more than one active
ingredients as necessary for the
particular indication being treated, preferably those with complementary
activities that do not adversely
affect each other. Such active ingredients are suitably present in combination
in amounts that are
effective for the purpose intended.
[0179] Active ingredients may be entrapped in microcapsules prepared, for
example, by coacervation
techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-
microcapsules and poly-(methylmethacylate) microcapsules, respectively, in
colloidal drug delivery
systems (for example, liposomes, albumin microspheres, microemulsions, nano-
particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed in
Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980).
[0180] Sustained-release preparations may be prepared. Suitable examples of
sustained-release
preparations include semipermeable matrices of solid hydrophobic polymers
containing the antagonist
of an ALDH inhibitor (e.g., disulfiram and/or derivatives thereof) and/or a
targeted therapeutic (e.g.,
TKI), which matrices are in the form of shaped articles, e.g., films, or
microcapsules.
[0181] The formulations to be used for in vivo administration are generally
sterile. Sterility may be
readily accomplished, e.g., by filtration through sterile filtration
membranes.
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VI. Articles of Manufacture
[0182] In another aspect of the invention, an article of manufacture
containing materials useful for the
treatment, prevention and/or diagnosis of the disorders described above is
provided. The article of
manufacture comprises a container and a label or package insert on or
associated with the container.
Suitable containers include, for example, bottles, vials, syringes, IV
solution bags, etc. The containers
may be formed from a variety of materials such as glass or plastic. The
container holds a composition
which is by itself or combined with another composition effective for
treating, preventing and/or
diagnosing the condition and may have a sterile access port (for example the
container may be an
intravenous solution bag or a vial having a stopper pierceable by a hypodermic
injection needle). At
least one active agent in the composition is an antagonist of an ALDH
inhibitor (e.g., disulfiram and/or
derivatives thereof) and/or a targeted therapeutic (e.g., TKI) described
herein. The label or package
insert indicates that the composition is used for treating the condition of
choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition contained
therein, wherein the
composition comprises an antagonist of an ALDH inhibitor (e.g., disulfiram
and/or derivatives thereof)
and/or a targeted therapeutic (e.g., TM); and (b) a second container with a
composition contained
therein, wherein the composition comprises a further cytotoxic or otherwise
therapeutic agent.
[0183] In some embodiments, the article of manufacture comprises a container,
a label on said
container, and a composition contained within said container; wherein the
composition includes one or
more reagents (e.g., primary antibodies that bind to one or more biomarkers or
probes and/or primers to
one or more of the biomarkers described herein), the label on the container
indicating that the
composition can be used to evaluate the presence of one or more biomarkers in
a sample, and
instructions for using the reagents for evaluating the presence of one or more
biomarkers in a sample.
The article of manufacture can further comprise a set of instructions and
materials for preparing the
sample and utilizing the reagents. In some embodiments, the article of
manufacture may include
reagents such as both a primary and secondary antibody, wherein the secondary
antibody is conjugated
to a label, e.g., an enzymatic label. In some embodiments, the article of
manufacture one or more probes
and/or primers to one or more of the biomarkers described herein.
[0184] In some embodiments of any of the article of manufacture, the
antagonist of an ALDH inhibitor
(e.g., disulfiram and/or derivatives thereof) and/or a targeted therapeutic
(e.g., TKI) is an antibody,
binding polypeptide, binding small molecule, or polynucleotide. In some
embodiments, the antagonist of
an ALDH inhibitor (e.g., disulfiram and/or derivatives thereof) and/or a
targeted therapeutic (e.g., TM)
is a small molecule. In some embodiments, the antagonist of an ALDH inhibitor
(e.g., disulfiram and/or
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derivatives thereof) and/or a targeted therapeutic (e.g., TKI) is an antibody.
In some embodiments, the
antibody is a monoclonal antibody. In some embodiments, the antibody is a
human, humanized, or
chimeric antibody. In some embodiments, the antibody is an antibody fragment
and the antibody
fragment binds an ALDH inhibitor (e.g., disulfiram and/or derivatives thereof)
and/or a targeted
therapeutic (e.g., TKI).
[0185] The article of manufacture in this embodiment of the invention may
further comprise a package
insert indicating that the compositions can be used to treat a particular
condition. Alternatively, or
additionally, the article of manufacture may further comprise a second (or
third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water for injection
(BWFI), phosphate-
buffered saline, Ringer's solution and dextrose solution. It may further
include other materials desirable
from a commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0186] Other optional components in the article of manufacture include one or
more buffers (e.g., block
buffer, wash buffer, substrate buffer, etc), other reagents such as substrate
(e.g., chromogen) which is
chemically altered by an enzymatic label, epitope retrieval solution, control
samples (positive and/or
negative controls), control slide(s) etc.
[0187] It is understood that any of the above articles of manufacture may
include an immunoconjugate
described herein in place of or in addition to an antagonist of an ALDH
inhibitor (e.g., disulfiram and/or
derivatives thereof) and/or a targeted therapeutic (e.g., TKI).
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EXAMPLES
[0188] The following are examples of methods and compositions of the
invention. It is understood that
various other embodiments may be practiced, given the general description
provided above.
Example 1
Materials and Methods
Human Cancer Cell Lines and Reagents
[0189] Human cancer cell lines were grown in RPMI media supplemented with
sodium pyruvate, 10%
Fetal bovine serum and antibiotics penicillin and streptomycin at 37 C in the
presence of 5% CO2.
ALDH Activity Assay
[0190] A bodipy labeled ALDH substrate (Aldefluor Kit, Stem Cell Technology),
reconstituted
according to the vendor's protocol, was used to detect ALDH activity. The
substrate was diluted in
RPMI media (Sul substrate/ml media) and added to the adherent cells. After 30
minutes of incubation at
37 C in the CO2 incubator cells were washed twice with RPMI media and the
images were taken using
IncuCyte HD System (Essen BioScience) and a 10x objective.
Flow Cytometry and RNA Extraction
[0191] Aldefluor assay was used to detect ALDH activity in Kato II parental
cells. ALDHingh and
ALDH10w cells representing ¨5% of parental cells with highest and lowest ALDH
activity, respectively,
were sorted using flow cytometry. Kato II cells incubated with the bodipy
labeled substrate in the
presence of DEAB, a cold competitive substrate, were used as negative control.
Total RNA extracted
using RNAEasy column (Qiagen) was used for microarray based gene expression
analysis.
Cell Viability Assay
[0192] The cells were fixed with 4% paraformaldehyde at the end of the assay
period and the viability
was determined using nucleic acid stain Syto60 (Life Technologies) diluted
1:5000 in water. The
fluorescence intensity was measured using SpectraMax M5 (excitation 635nm and
emission 695nm;
Molecular Device). The viability was expressed as % of no treatment control.
Generation of Drug Tolerant Cells
[0193] Kato II and GTL-16 DTPs were generated by treating parental cells with
luM crizotinib for 30
days. PC9 parental cells were treated with 2uM erlotinib for 9 days for DTP
generation. In all cases
media was changed every three days.
Immunobloting
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[0194] Proteins were extracted from cell pellet using NP-40 lysis buffer
containing protease and
phosphatase inhibitors. Proteins were separated using SDS-PAGE gels (BioRad)
and immunodetection
was performed using standard protocols. The antibodies to ALDH1A1 was
purchased from R&D
Systems, GAPDH, cleaved PARP and phospho-ATM/ATR substrate antibodies were
purchased from
Cell Signaling Technology and phospho-yH2A.x antibody was purchased from
Millipore.
ROS Assay
[0195] ROS assay was performed using carboxy derivative of fluorescein, CM-
H2DCFDA (Molecular
Probes). Reconstituted ROS indicator was added to the growth media in the
plates containing DTPs and
incubated for 30 minutes. DTPs were detached from the plate using trypsini-
EDTA and ROS level was
detected using flow-cytometry using untreated parental cells as controls.
Xenograft Tumor Studies
[0196] PC-9, PC-9-GFP, EBC-1, and GTL-16 cells were cultured in growth media
(RPMI 1640, 10%
heat-inactivated fetal calf serum, 2 mM L-glutamine) to 80% confluency and
then trypsinized, washed
once with PBS, and resuspended in either Hank's Balanced Salt Solution (HBSS)
or a 1:1 mixture of
HBSS with matrigel [growth factor reduced; catalog #356231 (BD Biosciences,
West Grove, PA)] to a
final concentration of 5 x 107 cells/ml. Each xenograft tumor model was
established using 5 x106 cells
(100 pL) inoculated subcutaneously (s.c.) in the rear right flank of
immunocompromised mice. GTL-16
cells were implanted in HBSS without matrigel in nude (nu/nu) mice (Charles
River Laboratories,
Hollister, CA). PC-9 and PC-9-GFP cells were implanted in HBSS with matrigel
in nude (nu/nu) mice
(Charles River Laboratories, Hollister, CA). EBC-1 cells were implanted in
HBSS without matrigel in
nude (nu/nu) mice (Charles River Laboratories, Hollister, CA). When tumor
volumes reach
approximately 100-200 mm3, mice were separated into groups of 10-15 animals
with similarly sized
tumors, and treatment was initiated the day after grouping. Mice were dosed
via daily (QD) oral gavage
(PO) with GDC-0712 (Genentech, Inc. ¨ a MET small molecule inhibitor, at 100
mg/kg formulated in
water), erlotinib (50 mg/kg in 7.5% Captisol) and/or disulfiram (Sigma -
Tetraethylthiuram, Catalog #
86720, dosed at 200 mg/kg formulated in safflower oil 95%, benzyl alcohol 5%),
or with corresponding
vehicle only. Tumor volumes were determined using digital calipers (Fred V.
Fowler Company, Inc.)
using the formula (L x W x W)/2. Tumor growth inhibition (%TGI) was calculated
as the percentage of
the area under the fitted curve (AUC) for the respective dose group per day in
relation to the vehicle,
such that %TGI = 100 x 1 ¨(AUC treatment/day)/(AUC vehicle/day). Curve fitting
was applied to Log2
transformed individual tumor volume data using a linear mixed-effects (LME)
model using the R
package nlme, version 3.1-97 in R v2.12Ø
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Seahorse Assay
[0197] Approximately 5,000 parental cells and 15,000 DTPs cells were plated
per well in XF 96-well
cell culture microplates (SeahorseBioscience) and incubated for 24 h at 37 C
in a5% CO2 incubator.
Disulfiram and NAC treatment was performed for 48h in the presence of TKI. The
oxygen consumption
rate (OCR) and extracellular acidification rate (ECAR) measurements were
performed in bicarbonate-
free, serum-free, 37oC pre-warmed media. After completion of analysis the
cells were fixed with 4%
paraformaldehyde, stained with Hoechst and 4 quadrants/well was imaged using a
Molecular Devices
ImageXpress HCS and average nuclei number per quadrant was counted. The bar
graphs represented the
mean +/- SEM of normalized (cell number) OCR and ECAR measurements from six
wells.
Results
Cancer stem cell marker gene ALDH1A1 is differentially expressed in drug
tolerant cells
[0198] Microarray based gene expression analysis was performed to identify
genes differentially
expressed in crizotinib tolerant Kato II gastric carcinoma cells. Among many
up-regulated genes
ALDH1A1, a cancer stem cell marker gene, was identified in drug tolerant
persister (DTP) population.
ALDH activity in live Kato II cells using Aldefluor assay (Stem Cell
Technology) was measured and
high ALDH activity was detected in a small (-5%) population of Kato II
parental cells (Fig. 1A) and in
almost all Kato II DTPs after a month of crizotinib treatment. A microarray
based gene expression
analysis performed on RNAs isolated ALDHhigh cells identified ALDH1A1 as the
only member of the
ALDH family of genes up-regulated ¨8 fold compared to ALDH1' cells (Fig. lb).
Consistent with the
RNA level ALDH1A1 protein level in Kato II was detected in ALDHhigh cells and
in DTPs derived from
Kato II and GTL-16 cells (gastric carcinoma) (Fig.1c). Crizotinib treatment
increased ALDH1A1
protein level in Kato II parental cells within 24h (Fig. 1c), before any drug
induced apoptosis was
detected as measured by the appearance of cleaved PARP protein product (data
not shown). Knockdown
of ALDH1A1 expression in Kato II and GTL-16 cells had no significant effect on
drug sensitivity or
DTP formation.
Disulfiram, an ALDH inhibitor, Kills Drug Tolerant Cells
[0199] To understand the role of ALDH in drug tolerance, Kato II and GTL-16
parental cells were
treated with an irreversible ALDH inhibitor called Disulfiram (DS). DS and its
metabolites inhibit
enzymatic activity of multiple ALDH family members (Koppaka et al., 2012).
Disulfiram alone had no
significant effect on the growth of these cancer cells, but in combination
with crizotinib, DS eliminated
drug tolerant Kato II and GTL-16 cells (Fig.2A,B). Similar effect of DS was
observed on non-small cell
lung carcinoma PC9 cells that do not express ALDH1A1 but express other ALDH
family members (Fig.
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2C). Approximately 20% PC9 DTPs starts dividing like parental PC9 cells while
maintaining their drug
tolerance property when maintained over 10 days in erlotinib. This growing
population of erlotinib
tolerant PC9 DTPs are called drug tolerant expanded persister or DTEPs (Sharma
et al., 2010), which
unlike DTPs are much less sensitive to DS. The bar graphs in Fig.2 represent
data from triplicate wells
and illustrate the combined effect of DS and TM on the viability of DTPs from
all three cell lines
mentioned above. Pre-treatment of PC9 and GTL-16 cells with DS alone for 3-6
days prior to TM
exposure did not eliminate DTPs, indicating continuous suppression of ALDH
activity is critical for the
killing DTPs.
[0200] The effectiveness of various TKI-DS combinations were tested on eight
other cancer cell lines of
breast, colon and lung cancer origins and addicted to various oncogenes. While
DS alone had no
significant effect on the viability, all TKI-DS combinations were highly
effective in either eliminating or
significantly reducing the number of corresponding DTPs (Fig.3). These results
further emphasize the
dependence of drug tolerant cells, in general, on ALDH activity for their
survival and implicate potential
beneficial effect of the use of DS in combination of TMs in
eliminating/delaying relapse of various type
of cancer.
Drug Tolerant Cells Have High ROS Level
[0201] Cancer cells, compared to their normal counterpart, have higher ROS
level, which is believed to
promote cell proliferation (Szatrowski et al., 1991; Boonstra et al., 2004).
Exposure to chemotherapy
and radiation therapies increase ROS level even higher in cancer cells, which
can cause generation of
various aldehyde products through peroxidation of membrane lipid. Some of
these aldehyde products
like malonaldehyde and 4-hydroxy-nonenal (4-HNE) have longer half-life and can
cause DNA damage
and subsequent cell death (Chiu et al., 2012; Casares et al., 2012, Li et al.,
2009). A prompt activation
of DNA repair pathway in CD133+ glioblastoma stem cells in response to
increase in ROS level
provided the basis for their resistance to radiation (Bao et al. 2006). To
determine whether similar
mechanisms involving ROS play role in drug tolerance, the bioenergetics, ROS
level, extent of DNA
damage and activity of DNA repair pathway was measured in DTPs. An over six-
fold increase in ROS
level was observed in both PC9 and GTL-16-derived DTPs compared to their
parental cells (Fig.4A).
DS treatment for 48h caused further increase in ROS level in DTPs, which can
be reversed by adding
NAC to the media. Increased ROS level led to increased oxygen consumption rate
(OCR) (Fig.4B) and
increased double-stranded DNA breaks in drug tolerant cells and activated DNA
repair mechanism.
(Fig.4C). These results suggest that ALDH family members play a ROS scavenger
role, which is critical
for the survival of DTPs.
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N-acetyl cycteine rescue the lethal effect of disulfiram on DTPs
[0202] Next we asked whether NAC treatment is sufficient to prevent the
killing of DTPs by TKI+DS
treatment. PC9 and GTL-16 cells were treated with erlotinib and crizotinib
respectively either alone or
in combination with DS and NAC. As expected DS and TKI combination killed all
PC9 DTPs within
14days, which was almost completely rescued by NAC when added along with DS
and TKI (Fig.5A).
Similar results were obtained with GTL-16 DTPs where inclusion of NAC during
the treatment with
TKI+DS (Fig.5B) rescued ¨80% of GTL-16 DTPs from DS induced death.
[0203] To understand the mechanism of DS action, GTL-16-derived DTPs were
treated with DS and
NAC for 48h and performed immunoblot experiments with the extracted proteins.
DS treatment caused
decrease in ALDH1A1 and NFKB levels and resulted in several fold increase in
yH2A.x, which
suggested extensive DNA damage in DS treated DTPs and subsequent activation of
apoptotic pathway
as revealed by significant increase in cleaved PARP level. Presence of NAC, a
ROS scavenger, restored
ALDH1A1 and NFKB levels and prevented increase in yH2A.x and apoptosis of
DTPs.
Disulfiram Delays Tumor Relapse in Xenograft Mouse Models
[0204] Xenograft mouse models were used to investigate the efficacy of DS in
eliminating/delaying
tumor relapse in vivo. The treatment regimen for PC9 in vivo study was first
tested in an in vitro
experiment where PC9 cells were treated with either erlotinib alone or in
combination with DS for six
days. PC9 DTPs in the erlotinib, DS and one of the erlotinib+DS group were
allowed to grow without
any drug while the other erlotinib+DS group continued to receive DS. As shown
in Fig.6A, a significant
delay in the growth of PC9 DTPs was observed from erlotinib+DS group where
both drugs were
withdrawn as compared to erlotinib group. As expected, no PC9 DTPs survived
from erlotinib+DS sub-
group which continued to receive DS. The bar graph represents data from
triplicate wells illustrating the
effect of DS.
[0205] For the PC9 xenograph study the mice were inoculated with PC9 cells and
the tumors were
allowed to grow to 100-200 mm3 in size, which were then divided into four
treatment groups, namely,
vehicle control, DS control, TM alone and TKI+DS groups. The treatments were
stopped after eleven
days except for the animals in the TKI+DS group, which continued to receive DS
untill the end of the
study. As shown in Fig. 6B, near complete regression of PC9 tumors was oserved
upon erlotinib
treatment. The time of tumor progression (TTP) measured as 5xTTP was 60 days
for the erlotinib
treatment group with 10 PR and 1CR whereas for the erlotinib+DS group the
average tumor size with
9PR and 6CR was below the initial volume of the tumor (P=0.0007). In agreement
with the cell line data
xenograft data showed that TKI and DS combination can significantly delay
tumor relapse.
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References
Koppaka, V et al. (2012). Pharmacol Rev 64, 520-539.
Szatrowski, T.P. and Nathan, C. F. (1991) Cancer Research, 51, 794-798.
Boonstra, J. and Post, J. A. (2004) Gene, 337, 1-13.
Chiu, W. H. et al. (2012). Biochemical Pharmacology, 83, 1159-1171.
Li, Y. et al. (2009). Anti-Cancer Drugs, 20, 770-778.
Bao, S. et al. (2006) Nature 444:756-760.
[0206] Although the foregoing invention has been described in some detail by
way of illustration and
example for purposes of clarity of understanding, the descriptions and
examples should not be construed
as limiting the scope of the invention. The disclosures of all patent and
scientific literature cited herein
are expressly incorporated in their entirety by reference.
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