U.S. patent application number 14/979055 was filed with the patent office on 2016-07-07 for anaplastic lymphoma kinase (alk) as an oncogene capable of transforming normal human cells.
The applicant listed for this patent is The Trustees of the University of Pennsylvania. Invention is credited to James L. Riley, Mariusz Wasik, Fang Wei, Qian Zhang.
Application Number | 20160194616 14/979055 |
Document ID | / |
Family ID | 52391040 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160194616 |
Kind Code |
A1 |
Wasik; Mariusz ; et
al. |
July 7, 2016 |
Anaplastic Lymphoma Kinase (ALK) As An Oncogene Capable Of
Transforming Normal Human Cells
Abstract
The present invention provides compositions and methods for
transforming primary mammalian cells using an oncogenic form of ALK
wherein the transformed cells display features of that of a
corresponding tumor cell isolated from a cancer subject. The
invention also provides a method for immortalizing normal CD4+ T
lymphocytes with a lymphoma-characteristic form of ALK such as
NPM-ALK.
Inventors: |
Wasik; Mariusz; (Ardmore,
PA) ; Riley; James L.; (Downingtown, PA) ;
Zhang; Qian; (Philadelphia, PA) ; Wei; Fang;
(Drexel Hill, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of the University of Pennsylvania |
Philadelphia |
PA |
US |
|
|
Family ID: |
52391040 |
Appl. No.: |
14/979055 |
Filed: |
December 22, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14197829 |
Mar 5, 2014 |
9255279 |
|
|
14979055 |
|
|
|
|
61774770 |
Mar 8, 2013 |
|
|
|
Current U.S.
Class: |
435/366 ;
435/325 |
Current CPC
Class: |
A01K 2267/0331 20130101;
C12N 9/12 20130101; C12N 2310/14 20130101; C12N 2501/727 20130101;
C12N 15/85 20130101; A01K 2207/12 20130101; G01N 2333/91205
20130101; C12N 2510/00 20130101; C12N 9/1205 20130101; G01N 33/5011
20130101; C12N 15/1137 20130101; C12N 5/0694 20130101; C12N 2503/02
20130101; C12Y 207/10001 20130101 |
International
Class: |
C12N 9/12 20060101
C12N009/12; C12N 15/85 20060101 C12N015/85 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
R01-CA89194, R01-CA96856, P01-A1080192, and R01-CA147795 awarded by
the National Institutes of Health (NIH). The government has certain
rights in the invention.
Claims
1. An isolated transformed mammalian cell, wherein the transformed
cell is genetically modified to express an oncogenic anaplastic
lymphoma kinase (ALK), and wherein the transformed cell exhibits
one or more features of that of a corresponding tumor cell isolated
from a cancer subject.
2. The transformed cell of claim 1, wherein the oncogenic ALK is
selected from the group consisting of NPM-ALK, EML4-ALK, RANBP-ALK,
TPM3-ALK, TFG-ALK, KIF5B-ALK, ATIC-ALK, and CLTC-ALK.
3. The transformed cell of claim 1, wherein the oncogenic ALK is a
mutant carrying a mutation selected from the group consisting of
R1275Q and F117L.
4. The transformed cell of claim 1, wherein the cell is a T
cell.
5. The transformed cell of claim 1, wherein the cell exhibits one
or more features of that of a corresponding tumor cell isolated
from a cancer subject having anaplastic large-cell lymphoma
(ALCL).
6. The transformed cell of claim 1, wherein the cell exhibits one
or more of perpetual cell growth, characteristic cell morphology,
activation of the key signal transduction pathways and expression
of CD30, IL-10 and PD-L1/CD274.
7. The composition of claim 1, wherein the mammalian cell is a
human cell and the subject is a human.
8.-13. (canceled)
14. An isolated immortalized cell produced by the method of claim
8.
15. A cell culture comprising the isolated cell of claim 14.
16.-18. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of, and claims
priority to U.S. patent application Ser. No. 14/197,829, filed Mar.
5, 2014, now allowed, which claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 61/774,770, filed
Mar. 8, 2013, all of which applications are hereby incorporated
herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] Anaplastic large-cell lymphomas (ALCL) carrying anaplastic
lymphoma kinase (ALK) comprise a distinct clinical-pathological
entity (Li et al, 2007, Med Res Rev 28(3):372-412; Wasik et al.,
2009, Semin Oncol 36(2 Suppl 1):527-35; Tabbo et al, 2012, Front
Oncol 2:41). ALK+ALCL are derived from CD4+ T lymphocytes,
typically occur in children and young adults, and involve soft
tissues and other extranodal sites. As the name implies, they are
comprised of large highly atypical cells with prominent nuclei and
abundant cytoplasm and, hence, bear little resemblance to their
normal CD4+ T-cell counterparts, either resting or activated. They
also display a unique phenotype with the variable loss of CD3 and
other T-cell markers and strong expression of CD30; a cell surface
receptor from the TNF-R family.
[0004] While ALK is physiologically expressed only in a subset of
immature neuronal cells (Li et al, 2007, Med Res Rev
28(3):372-412), its aberrant expression has been identified in a
subset of ALCL (Morris et al., 1994, Science 263(5151):1281-1284;
Shiota et al., 1994, Oncogene 9(6):1567-1574) and, subsequently, in
a spectrum of histologically diverse malignancies including subsets
of a large B-cell lymphoma, inflammatory myofibroblastic tumor,
non-small cell lung carcinoma (Li et al, 2007, Med Res Rev
28(3):372-412; Wasik et al., 2009, Semin Oncol 36(2 Suppl
1):S27-35; Tabbo et al, 2012, Front Oncol 2:41) and several other
types of cancer. The aberrant expression of ALK typically results
in these malignancies from chromosomal translocations involving the
ALK gene and various partner genes with the nucleophosmin (NPM)
gene being by far the most frequent partner in ALK+ALCL (Li et al,
2007, Med Res Rev 28(3):372-412) and EML4 in lung carcinoma (Soda
et al., 2007, Nature 448:561-566). The NPM-ALK, EML4-ALK and other
chimeric proteins are constitutively activated through
autophosphorylation (Morris et al., 1994, Science
263(5151):1281-1284; Shiota et al., 1994, Oncogene 9(6):1567-1574)
and highly oncogenic as documented mainly by using patient-derived
cell lines and transgenic mouse models (Fujimoto et al., 1996, Proc
Natl Acad Sci USA 93(9):4181-4186; Kuefer et al., 1997, Blood
90(8):2901-2910; Chiarle et al, 2003, Blood 101(5):1919-1927;
Turner et al., 2006, Anticancer Res 26(5A):3275-3279; Giuriato et
al., 2010, Blood 115(20):4061-4070). NPM-ALK acts by activating a
number of signal transduction pathways such as STAT3 (Zhang et al.,
2002, J Immunol 168(1):466-474; Zamo et al., 2002, Oncogene
21(7):1038-1047) and mTORC1 including its down-stream target S6RP
(Marzec et al., 2007, Oncogene 26(38):5606-5614). The chronic
activity of these signaling pathways leads to the persistent
modulation of a number of genes and results in sustained cell
proliferation, resistance to cell death, and other oncogenic
properties. NPM-ALK is capable of fostering evasion of the
anti-tumor immune response by inducing expression of potent
immunosuppressive proteins: the cytokine IL-10 and the cell
membrane bound ligand PD-L1/CD274 (Marzec et al., 2008, Proc Natl
Acad Sci USA 105(52):20852-20857; Kasprzycka et al., 2006, Natl
Acad Sci USA 103(26):9964-9969).
[0005] Cellular transformation by NPM-ALK has been demonstrated in
immortalized rodent fibroblasts (Bai, R Y. et al. (1998) Mol Cell
Biol. 18:6951-6961), and confirmed in studies which have shown that
ALK protects Ba/F3 and PC12 cells from interleukin-3 or growth
factor withdrawal (Stoica, G E., et al. (2001) J Biol Chem.
276:16772-16779 and (Bai R Y., et al. (1998) Mol Cell Biol.
18:6951-6961). Transfer of NPM-ALK transduced bone-marrow cells
into irradiated host recipient mice resulted in the generation in
vivo of large cell B-cell lymphomas (Kuefer, M U. et al. (1997)
Blood. 90:2901-2910). The later, even more refined studies using
T-cell specific promoters resulted in the development of T-cell
malignancies in the host mice (Chiarle et al. (2003) Blood
101:1919-1827). However, these tumors comprise of immature rather
than mature T lymphocytes and lack key morphologic, phenotypic and
other characteristics of human, patient-derived ALCL.
[0006] In the past few years, the molecular mechanisms of
ALK-mediated cellular transformation have also been partially
elucidated (Duyster, J. et al. (2001) Oncogene. 20:5623-5637). It
has been shown that the ALK portion of the fusion protein,
corresponding to the cytoplasmic tail of the ALK receptor and
containing the catalytic domain, is absolutely required for
transformation (Bai, R Y. et al. (1998) Mol Cell Biol.
18:6951-6961), whereas all the N-terminal regions of the ALK
chimeras function as dimerization domains (Bischof, D. et al.
(1997) Mol Cell Biol. 17:2312-2325) and (Duyster, J. et al. (2001)
Oncogene. 20:5623-5637). As a result of spontaneous dimerization,
ALK undergoes autophosphorylation and becomes catalytically active.
Constitutively active ALK fusion proteins can bind multiple adaptor
proteins and activate a series of pathways involved in cell
proliferation, transformation and survival. These include the
PLC-Shc PI3-K/Akt and the Jak3-Stat3 pathways (Bai, R Y. et al.
(1998) Mol Cell Biol. 18:6951-6961; Bai R Y., et al. (2000) Blood.
96:4319-4327 and Zamo, A. et al. (2002) Oncogene.
21:1038-1047).
[0007] Given limitations of the existing mouse models and cell
lines derived from patient tumors, there is a clear need to develop
new models of ALK-driven malignancies. In addition, there are no
good models to transform normal human cells including T lymphocytes
and bronchial epithelial cells, ALCL and lung carcinoma are derived
from, respectively. Thus, there is a need in the art for a model of
transformation by ALK chimeras in primary normal cells. The present
invention satisfies this need in the art.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides compositions and methods for
transforming primary mammalian cells, particularly human cells,
using an oncogenic form of anaplastic lymphoma kinase (ALK) wherein
the transformed cells display features of that of a corresponding
tumor cell isolated from a cancer patient. The invention also
provides a method for immortalizing normal CD4+ T lymphocytes with
a lymphoma-characteristic form of ALK such as NPM-ALK.
[0009] The invention includes a composition comprising an isolated
transformed mammalian cell, wherein the transformed cell is
genetically modified to express an oncogenic ALK, and wherein the
transformed cell exhibits one or more features of that of a
corresponding tumor cell isolated from a cancer subject.
[0010] The invention further includes a method of immortalizing a
primary normal mammalian cell. The method comprises genetically
modifying the primary normal mammalian cell with an oncogenic ALK,
wherein the immortalized cell exhibits a one or more features of
that of a corresponding tumor cell isolated from a cancer
subject.
[0011] The invention also includes a method for screening a test
compound for antitumor activity. The method comprises contacting a
transformed mammalian cell with a test compound, wherein the cell
is genetically modified to express an oncogenic ALK, further
wherein the cell exhibits a morphology of that of a corresponding
cell isolated from a cancer subject. The method further comprises
monitoring the antitumor activity of the test compound, wherein
modulation of the morphology of the transformed cell after contact
with the test compound is indicative of a compound having antitumor
activity in comparison to the morphology of the transformed cell
prior to contact with the test compound.
[0012] In certain embodiments, the composition comprises a
transformed cell wherein the oncogenic ALK is selected from the
group consisting of NPM-ALK, EML4-ALK, RANBP-ALK, TPM3-ALK,
TFG-ALK, KIF5B-ALK, ATIC-ALK, CLTC-ALK and other ALK translocation
variants as well as mutants such as R1275Q and F117L, and the like.
In other embodiments, the composition comprises a transformed cell,
wherein the cell is a T cell. In yet other embodiments, the
composition further comprises a transformed cell wherein the cell
exhibits one or more features of that of a corresponding tumor cell
isolated from a cancer subject having anaplastic large-cell
lymphoma. In yet other embodiments, the transformed cell exhibits
one or more of perpetual cell growth, characteristic cell
morphology, activation of the key signal transduction pathways and
expression of CD30, IL-10 and PD-L1/CD274. In yet other
embodiments, the mammalian cell is a human cell and the subject is
a human.
[0013] In certain embodiments, prior to genetically modifying the
primary normal mammalian cell, the cell is cultured in the presence
of a composition comprising a first agent that is capable of
providing a primary activation signal to a T cell and a second
agent that is capable of activating a co-stimulatory molecule on a
T cell. In other embodiments, the first agent is an anti-CD3
antibody and the second agent is an anti-CD28 antibody. In yet
other embodiments, the cell is genetically modified using a
lentivirus expressing the oncogenic ALK. In yet other embodiments,
the cell exhibits one or more features of that of a corresponding
tumor cell isolated from a cancer subject having anaplastic
large-cell lymphoma morphology. In yet other embodiments, the
immortalized cell is isolated. In yet other embodiments, the
isolated immortalized cell can be grown in cell culture.
[0014] In certain embodiments, the method for screening a test
compound for antitumor activity enables the identification of such
a compound of interest.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0016] FIGS. 1A-1F are series of images demonstrating
NPM-ALK-induced malignant transformation of normal human CD4+ T
lymphocytes. FIG. 1A depicts cell growth curves of
NPM-ALK-expressing CD4+ T cells. Purified CD4+ T cells were
stimulated with bead-immobilized CD3 and CD28 antibodies (ab) and
either transduced with wild-type NPM-ALK (NA1) or enzymatically
inactive NPM-ALK mutant (KD) or left untransfected. The cells were
counted at the depicted days using a cell counter. FIG. 1B depicts
cell growth curves of NPM-ALK-transfected CD4+ T cells (NA1, NA2,
NA3) from three separate, consecutive experiments. FIG. 1C depicts
activation of ALK, STAT3, and mTORC1 pathway as determined by
phosphorylation status of the listed proteins. Expression of total
NPM-ALK and .beta.-actin (ACTB) served as controls.
ALK+ALCL-derived cell line SUDHL-1, ALK-ALCL cell line 2B, and
normal unstimulated CD4+ T cells were used as additional positive
and negative controls. FIG. 1D depicts cell volume of
NPM-ALK-transfected and untransfected CD4+ T cells as determined by
cell counter counter analysis (Beckman Coulter, Brea, Calif.).
ALK+ALCL-derived cell line SUDHL-1 served as a positive control.
FIG. 1E depicts migration of NPM-ALK-transfected cells determined
using a Transwell culture system. Cells transfected with the
enzymatically inactive NPM-ALK (KD) and untransfected cells (Ctrl)
served as controls. P=0.01 for NA1 and P=0.04 for NA3 versus
combined KD and Ctrl. FIG. 1F depicts colony formation by the
NPM-ALK-transfected NA1, NA 2, NA3 and control ALK+ALCL-derived
SUDHL-1 cells.
[0017] FIGS. 2A-2E are series of images depicting morphologic and
immunophenotypic features of NPM-ALK-transformed CD4+ T cells. FIG.
2A shows H&E stain and immunohistochemical analysis for the
depicted proteins of NA1 cells. Main image: 200.times.
magnification, inset: 400.times.. FIG. 2B shows a multiparameter
flow cytometry analysis of NA1 cells for expression of T-cell
markers CD2 and CD3 (left panel) and CD4 and CD25 (right panel).
FIG. 2C shows a flow cytometry analysis of the NA1 and NA2 for CD30
expression. ALK+ALCL-derived SUDHL-1 and mantle cell lymphoma
derived Jeko cell line served as a positive and negative control,
respectively. FIG. 2D shows the expression of IL-10 mRNA determined
by RT-PCR in NA1 and NA2 cells, with CD3 and CD28-stimulated,
NPM-ALK-untransfecte CD4+ T cells (Ctrl) serving as negative
control. P=0.01 for the experimental versus control cells. FIG. 2E
shows the expression of the immunosuppressive PDL-1/CD274 protein
by NA1, and NA2, and control SUDHL-1 cells. Original magnification:
.times.200; .times.400 (insets).
[0018] FIGS. 3A-3I are series of images demonstrating
NPM-ALK-dependence of the transformed CD4+ T cells. Suppressive
effect of ALK inhibitor (CEP-28122, 100 nM) on (FIG. 3A)
phosphorylation of the depicted cell signaling proteins in NA1, NA2
and control SUDHL-1 cells, (FIG. 3B) expression of CD30 (with
CD30+ALK- T-cell line 2A serving as a negative control, (FIG. 3C)
synthesis of IL-10, and (FIG. 3D) expression of PDL-1. FIG. 3E
depicts dose-dependent inhibition of cell growth by CEP-28122 ALK
inhibitor. ALK- T-cell lines MyLa2059 and MyLa3675 served as
negative controls. Depletion of NPM-ALK mediated by ALK siRNA (FIG.
3F; left panel) and its effect on phosphorylation of the depicted
signaling proteins (FIG. 3F; right panel), expression of IL-10
(FIG. 3G) and PDL-1 (FIG. 3H), and cell growth (FIG. 3I).
Non-specific (NS) siRNA was used as a negative control (FIGS.
3F-3I).
[0019] FIGS. 4A-4C are series of images showing growth of the
transformed CD4+ T cells in vivo. FIG. 4A shows the ability of
NPM-ALK-transfected CD4+ T lymphocytes to form tumors in
immunodeficient mice. NA1 and control SUDHL-1 cells were
transfected with a vector containing luciferase gene, injected
intraperitoneally at 3.times.10.sup.6 cells/mouse. The mice were
examined for the presence of bioluminescence 5 weeks later. FIG. 4B
shows representative 400.times. images of an H&E and
immunohistochemical stains for expression of the depicted proteins
by the tumor tissues. FIG. 4C shows flow cytometry-detected
expression of T-cell markers CD5 and CD3 (left panel) and CD4 and
CD3 (right panel) by the isolated tumor cells.
[0020] FIG. 5 is an image showing the transfection efficiency of
the NPM-ALK WT and KD constructs. Normal CD4+ T cells prestimulated
with CD3 and CD28 antibody-coated beads (ab/beads) were transfected
with the NPM-ALK and stained with an anti-ALK ab (marked by "1") or
IgG (marked by symbol "2") five days later. The data are
representative of three independent experiments in which expression
of NPM-ALK or GFP (for the NPM-ALK-GFP constructs) was
measured.
[0021] FIG. 6 is an image showing the growth time of CD4+ T
lymphocytes transfected with NPM-ALK. Normal CD4+ T lymphocytes
were transfected with NPM-ALK and remain in on-going culture for
the indicated periods of time. The CD4+ T cells either
non-transfected or transfected with the enzymatically inactive
NPM-ALK-KD mutant served as controls.
[0022] FIG. 7 is a chart showing the immunophenotype of cell lines
derived from the NPM-ALKtransfected CD4+ T cells.
[0023] FIG. 8 is an image showing cytogenetic analysis of NA1 and
NA2 cell lines derived from the NPM-ALK-transfected CD4+ T cells.
Representative normal karyotypes of the NA1 (upper) and NA2 (lower)
lines are shown.
[0024] FIG. 9 is a chart showing T-cell receptor (TCR) gene
rearrangement in NPM-ALK-transfected CD4+ T cells.
[0025] FIG. 10 is an image showing the effect of ALK inhibitor (ALK
inh) CEP-28122 on activation of ALK and its targets. The cells were
exposed to the inhibitor at the indicated doses and examined for
the expression of the depicted phospho-proteins with the total
proteins serving as controls.
[0026] FIG. 11 is an image showing the results of flow cytometry
analysis of tumor cells derived from the NPM-ALK-transformed CD4+ T
cells (line NA1).
[0027] FIGS. 12A-12B are series of images depicting
NPM-ALK-dependent survival of transduced CD4+ T cells. FIG. 12A
shows the effect of 100 nmol/L ALK inhibitor CEP-28122 on
cell-surface annexin V expression and propidium iodide (PI)
incorporation in cell lines NA1, NA2, SUDHL-1 (positive control),
and MyLA3675 (negative control), FIG. 12B shows the effect NPM-ALK
and nonspecific (NS) control siRNA on NA1 cell staining for annexin
V and PI; the western blots show the degree of NPM-ALK depletion,
with .beta.-actin (ACTB) serving as control.
DETAILED DESCRIPTION OF THE INVENTION
[0028] It has been discovered that a primary normal human cell can
be immortalized by introducing an oncogenic ALK into the cell,
wherein the immortalized cell is morphologically and
immunophenotypically indistinguishable from a corresponding
malignant cell isolated from an ALK+ cancer patient. Accordingly,
the invention provides an in vitro model of mechanisms of malignant
transformation of normal human cells.
[0029] The invention is based partly on the successful in vitro
transduction of normal human CD4+ T lymphocytes with the chimeric
ALK gene that resulted in their malignant transformation. The
transformed cells exhibit morphology and immunophenotype of that of
a "naturally occurring" human malignancy derived from patients.
[0030] The invention allows for the generation of a malignant cell
that recapitulates malignant cells found in a cancer patient. The
cells of the invention are valuable in studying the early stages of
oncogenesis and mechanisms of progression thereof. The cells of the
invention offer an in vitro model for evaluating cancer features
and its development as well as for screening of anti-cancer
agents.
[0031] In one embodiment, the invention provides compositions and
methods for transforming normal cells, such as CD4+ T lymphocytes,
using a single oncogene (e.g., oncogenic ALK) wherein the
transformed cells are morphologically and immunophenotypically
indistinguishable from a malignant cell isolated from a cancer
patient. In one embodiment, because the cells of the invention are
immortalized due only to the expression of an oncogenic kinase
(e.g., oncogenic ALK), the activities of the oncogenic kinase can
be evaluated separately from other confounding mechanisms of
oncogenesis that are present when not starting from a primary
normal cell.
[0032] The present invention relates to a normal primary cell
transduced with an oncogenic ALK and methods for their production
and use. In certain forms of the invention, a method of screening
for, or otherwise identifying, drugs or agents that modulate
oncogene-mediated neoplastic or hyperplastic transformation, or
increase the sensitivity of the cells of the invention to the toxic
effects of radiation or chemotherapy, are provided. Additionally,
the cells of the invention may be used as a model to study
conserved pathways that lead to oncogene-mediated cancer
progression.
DEFINITIONS
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein. In describing and claiming the present invention, the
following terminology will be used.
[0034] It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
[0035] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0036] The term "about" will be understood by persons of ordinary
skill in the art and will vary to some extent on the context in
which it is used.
[0037] "Allogeneic" refers to a graft derived from a different
animal of the same species.
[0038] "Alloantigen" is an antigen that differs from an antigen
expressed by the recipient.
[0039] The term "ALK" includes the human ALK protein encoded by the
ALK (Anaplastic Lymphoma Kinase) gene which in its native form is a
membrane-spanning protein tyrosine kinase (PTK)/receptor.
[0040] The term "antibody" as used herein, refers to an
immunoglobulin molecule, which is able to specifically bind to a
specific epitope on an antigen. Antibodies can be intact
immunoglobulins derived from natural sources or from recombinant
sources and can be immunoactive portions of intact immunoglobulins.
Antibodies are typically tetramers of immunoglobulin molecules. The
antibodies included in the present invention may exist in a variety
of forms including, for example, polyclonal antibodies, monoclonal
antibodies, Fv, Fab and F(ab).sub.2, as well as single chain
antibodies and humanized antibodies (Harlow et al., 1999, Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual,
Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl.
Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science
242:423-426).
[0041] The term "antigen" or "Ag" as used herein is defined as a
molecule that provokes an immune response. This immune response may
involve either antibody production, or the activation of specific
immunologically-competent cells, or both. The skilled artisan will
understand that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen. Furthermore, antigens can be
derived from recombinant or genomic DNA. A skilled artisan will
understand that any DNA, which comprises a nucleotide sequences or
a partial nucleotide sequence encoding a protein that elicits an
immune response therefore encodes an "antigen" as that term is used
herein. Furthermore, one skilled in the art will understand that an
antigen need not be encoded soley by a full length nucleotide
sequence of a gene. It is readily apparent that the present
invention includes, but is not limited to, the use of partial
nucleotide sequences of more than one gene and that these
nucleotide sequences are arranged in various combinations to elicit
the desired immune response. Moreover, a skilled artisan will
understand that an antigen need not be encoded by a "gene" at all.
It is readily apparent that an antigen can be generated synthesized
or can be derived from a biological sample. Such a biological
sample can include, but is not limited to a tissue sample, a tumor
sample, a cell or a biological fluid.
[0042] As used herein, the term "autologous" is meant to refer to
any material derived from the same individual to which it is later
to be re-introduced into the individual.
[0043] The term "cancer" as used herein is defined as disease
characterized by the rapid and uncontrolled growth of cells. Cancer
cells can spread locally or through the bloodstream and lymphatic
system to other parts of the body. Examples of various cancers
include but are not limited to, breast cancer, prostate cancer,
ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,
colorectal cancer, renal cancer, liver cancer, brain cancer,
lymphoma, leukemia, lung cancer and the like.
[0044] The term "DNA" as used herein is defined as deoxyribonucleic
acid.
[0045] As used herein, an "effector cell" refers to a cell which
mediates an immune response against an antigen. An example of an
effector cell includes, but is not limited to a T cell and a B
cell.
[0046] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0047] As used herein "endogenous" refers to any material from or
produced inside an organism, cell, tissue or system.
[0048] As used herein, the term "exogenous" refers to any material
introduced from or produced outside an organism, cell, tissue or
system.
[0049] The term "epitope" as used herein is defined as a part of an
antigen that is recognized by the immune system and that can elicit
an immune response, inducing B and/or T cell responses. An antigen
can have one or more epitopes. Most antigens have many epitopes;
i.e., they are multivalent. In general, an epitope is roughly five
amino acids and/or sugars in size. One skilled in the art
understands that generally the overall three-dimensional structure,
rather than the specific linear sequence of the molecule, is the
main criterion of antigenic specificity and therefore distinguishes
one epitope from another.
[0050] The term "expression" as used herein is defined as the
transcription and/or translation of a particular nucleotide
sequence driven by its promoter.
[0051] The term "expression vector" as used herein refers to a
vector containing a nucleic acid sequence coding for at least part
of a gene product capable of being transcribed. In some cases, RNA
molecules are then translated into a protein, polypeptide, or
peptide. In other cases, these sequences are not translated, for
example, in the production of antisense molecules, siRNA,
ribozymes, and the like. Expression vectors can contain a variety
of control sequences, which refer to nucleic acid sequences
necessary for the transcription and possibly translation of an
operatively linked coding sequence in a particular host organism.
In addition to control sequences that govern transcription and
translation, vectors and expression vectors may contain nucleic
acid sequences that serve other functions as well.
[0052] The term "heterologous" as used herein is defined as DNA or
RNA sequences or proteins that are derived from the different
species.
[0053] "Homologous" as used herein, refers to the subunit sequence
similarity between two polymeric molecules, e.g., between two
nucleic acid molecules, e.g., two DNA molecules or two RNA
molecules, or between two polypeptide molecules. When a subunit
position in both of the two molecules is occupied by the same
monomeric subunit, e.g., if a position in each of two DNA molecules
is occupied by adenine, then they are homologous at that position.
The homology between two sequences is a direct function of the
number of matching or homologous positions, e.g., if half (e.g.,
five positions in a polymer ten subunits in length) of the
positions in two compound sequences are homologous then the two
sequences are 50% homologous, if 90% of the positions, e.g., 9 of
10, are matched or homologous, the two sequences share 90%
homology. By way of example, the DNA sequences 5'-ATTGCC-3' and
5'-TATGGC-3' share 50% homology.
[0054] As used herein, "homology" is used synonymously with
"identity."
[0055] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, i.e., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, i.e., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, i.e., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (i.e., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0056] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0057] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The phrase nucleotide sequence that encodes a
protein or an RNA may also include introns to the extent that the
nucleotide sequence encoding the protein may in some version
contain an intron(s).
[0058] The term "polynucleotide" as used herein is defined as a
chain of nucleotides. Furthermore, nucleic acids are polymers of
nucleotides. Thus, nucleic acids and polynucleotides as used herein
are interchangeable. One skilled in the art has the general
knowledge that nucleic acids are polynucleotides, which can be
hydrolyzed into the monomeric "nucleotides." The monomeric
nucleotides can be hydrolyzed into nucleosides. As used herein
polynucleotides include, but are not limited to, all nucleic acid
sequences which are obtained by any means available in the art,
including, without limitation, recombinant means, i.e., the cloning
of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR.TM., and the
like, and by synthetic means.
[0059] The term "polypeptide" as used herein is defined as a chain
of amino acid residues, usually having a defined sequence. As used
herein the term polypeptide is mutually inclusive of the terms
"peptide" and "protein".
[0060] "Proliferation" is used herein to refer to the reproduction
or multiplication of similar forms of entities, for example
proliferation of a cell. That is, proliferation encompasses
production of a greater number of cells, and can be measured by,
among other things, simply counting the numbers of cells, measuring
incorporation of .sup.3H-thymidine into the cell, and the like.
[0061] The term "promoter" as used herein is defined as a DNA
sequence recognized by the synthetic machinery of the cell, or
introduced synthetic machinery, required to initiate the specific
transcription of a polynucleotide sequence.
[0062] As used herein, the term "promoter/regulatory sequence"
means a nucleic acid sequence which is required for expression of a
gene product operably linked to the promoter/regulatory sequence.
In some instances, this sequence may be the core promoter sequence
and in other instances, this sequence may also include an enhancer
sequence and other regulatory elements which are required for
expression of the gene product. The promoter/regulatory sequence
may, for example, be one which expresses the gene product in a
tissue specific manner.
[0063] A "constitutive" promoter is a nucleotide sequence which,
when operably linked with a polynucleotide which encodes or
specifies a gene product, causes the gene product to be produced in
a cell under most or all physiological conditions of the cell.
[0064] An "inducible" promoter is a nucleotide sequence which, when
operably linked with a polynucleotide which encodes or specifies a
gene product, causes the gene product to be produced in a cell
substantially only when an inducer which corresponds to the
promoter is present in the cell.
[0065] A "tissue-specific" promoter is a nucleotide sequence which,
when operably linked with a polynucleotide which encodes or
specifies a gene product, causes the gene product to be produced in
a cell substantially only if the cell is a cell of the tissue type
corresponding to the promoter.
[0066] The term "RNA" as used herein is defined as ribonucleic
acid.
[0067] The term "recombinant DNA" as used herein is defined as DNA
produced by joining pieces of DNA from different sources.
[0068] The term "recombinant polypeptide" as used herein is defined
as a polypeptide produced by using recombinant DNA methods.
[0069] As used herein, a "substantially purified" cell is a cell
that is essentially free of other cell types. A substantially
purified cell also refers to a cell which has been separated from
other cell types with which it is normally associated in its
naturally occurring state. In some instances, a population of
substantially purified cells refers to a homogenous population of
cells. In other instances, this term refers simply to cell that
have been separated from the cells with which they are naturally
associated in their natural state. In some embodiments, the cells
are culture in vitro. In other embodiments, the cells are not
cultured in vitro.
[0070] "Therapeutically effective amount" is an amount of a
compound of the invention, that when administered to a patient,
ameliorates a symptom of the disease. The amount of a compound of
the invention which constitutes a "therapeutically effective
amount" will vary depending on the compound, the disease state and
its severity, the age of the patient to be treated, and the like.
The therapeutically effective amount can be determined routinely by
one of ordinary skill in the art in light of this disclosure and
their knowledge of the art.
[0071] "Patient" for the purposes of the present invention includes
humans and other animals, particularly mammals, and other
organisms. Thus the methods are applicable to both human therapy
and veterinary applications. In a preferred embodiment the patient
is a mammal, and in a most preferred embodiment the patient is
human.
[0072] The terms "treat," "treating," and "treatment," refer to
therapeutic or preventative measures described herein. The methods
of "treatment" employ administration to a subject, in need of such
treatment, a composition of the present invention, for example, a
subject having a disorder mediated by ALK or other oncoprotein or a
subject who ultimately may acquire such a disorder, in order to
prevent, cure, delay, reduce the severity of, or ameliorate one or
more symptoms of the disorder or recurring disorder, or in order to
prolong the survival of a subject beyond that expected in the
absence of such treatment.
[0073] The term "ALK-mediated disorder" refers to disease states
and/or symptoms associated with ALK-mediated cancers or tumors. In
general, the term "ALK-mediated disorder" refers to any disorder,
the onset, progression or the persistence of the symptoms of which
requires the participation of ALK. Exemplary ALK-mediated disorders
include, but are not limited to, cancer.
[0074] As used herein, an "oncogenic protein" refers to a protein
that causes cancer. In some instances, activation of an oncogenic
protein increases the chance that a normal cell will develop into a
tumor cell. Non-limiting examples of an oncogenic protein is the
NPM/ALK tyrosine kinase or other forms of oncogenic ALK, other
chimeric tyrosine kinases, other oncogenic kinase, and the
like.
[0075] The term "transfected" or "transformed" or "transduced" as
used herein refers to a process by which exogenous nucleic acid is
transferred or introduced into the host cell. A "transfected" or
"transformed" or "transduced" cell is one which has been
transfected, transformed or transduced with exogenous nucleic acid.
The cell includes the primary subject cell and its progeny.
[0076] The phrase "under transcriptional control" or "operatively
linked" as used herein means that the promoter is in the correct
location and orientation in relation to a polynucleotide to control
the initiation of transcription by RNA polymerase and expression of
the polynucleotide.
[0077] A "vector" is a composition of matter which comprises an
isolated nucleic acid and which can be used to deliver the isolated
nucleic acid to the interior of a cell. Numerous vectors are known
in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds,
plasmids, and viruses. Thus, the term "vector" includes an
autonomously replicating plasmid or a virus. The term should also
be construed to include non-plasmid and non-viral compounds which
facilitate transfer of nucleic acid into cells, such as, for
example, polylysine compounds, liposomes, and the like. Examples of
viral vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, and the
like.
[0078] "Xenogeneic" refers to a graft derived from an animal of a
different species.
[0079] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
Description
[0080] The present invention provides compositions and methods for
transforming a primary normal human cell into an immortalized cell
that exhibits a morphology and immunophenotype of a corresponding
cell isolated from a cancer patient. In one embodiment, the primary
normal human cell is genetically modified with an oncogenic kinase,
such as ALK, to transform the primary cell into an immortalized
cell. Non-limiting examples of an oncogenic ALK includes but are
not limited to NPM-ALK, EML4-ALK, RANBP-ALK, TPM3-ALK, TFG-ALK,
KIF5B-ALK, ATIC-ALK, CLTC-ALK and other ALK translocation variants
as well as mutants such as R1275Q and F117L, and the like.
[0081] In one embodiment, transforming a primary normal human cell
into an immortalized cell that exhibits a morphology and
immunophenotype of a corresponding cell isolated from a cancer
patient can be accomplished by genetically modifying the normal
cell with an oncogenic kinase, such as ALK as well as genes, RNA,
proteins and the like that are regulated by ALK.
[0082] In one embodiment, the invention provides compositions and
methods for transforming normal T cells with a lentiviral vector
expressing an oncogenic kinase, such as ALK. Preferably, the T cell
is CD4+ and the oncogenic ALK is NPM-ALK, wherein the transformed
cell becomes immortalized and exhibits a morphology and
immunophenotype of that of a lymphoma. In one embodiment, the
immortalized cell exhibits a morphology and immunophenotype of that
of a patient-derived anaplastic large-cell lymphoma (ALCL).
Compositions
[0083] Anaplastic Large Cell Lymphomas (ALCLs) carry translocations
in which the anaplastic lymphoma kinase (ALK) gene is juxtaposed to
various genes, the most common of which is the NPM/B23 gene. ALK
fusion proteins result in the constitutive activation of ALK
tyrosine kinase, thereby enhancing proliferation and increasing
cell survival. The present invention is based on the discovery that
normal cells can be immortalized by introducing an oncogenic ALK
into a normal cell. In some embodiments, the normal cell is a T
cell. In some embodiments, the T cell is a CD4+ T cell.
[0084] In one embodiment, the present invention provides an in
vitro cell model of carcinogenesis in which human normal cells are
transduced with an oncogenic ALK (e.g., NPM-ALK). Accordingly, the
present invention provides compositions and methods for expressing
oncogenic ALK in a normal cell to generate a cell that truly
recapitulates features of a malignancy associated with expression
of oncogenic ALK. In one embodiment, the malignancy associated with
expression of oncogenic ALK is ALK+ALCL, a malignancy of mature
CD4+ T lymphocytes with highly distinct morphology and
phenotype.
[0085] In one embodiment, the invention provides an in vitro model
of malignancy in which normal cells are genetically modified to
express oncogenic ALK, such as NPM-ALK. Accordingly, the in vitro
model of the invention can be generated by transducing normal
cells, such as CD4+ T lymphocytes, with a lentiviral vector
expressing the oncogenic ALK.
[0086] The cells or cell lines of the invention that are
genetically modified with an oncogenic ALK may be used for
screening new therapeutic agents for inhibition of cellular
proliferation in vitro. In addition, the growth of these cells can
be assessed in vivo following transplant into animal models. Novel
therapeutic agents can then be tested in animals bearing these
tumor cells containing the oncogenic ALK or variants and
translocations thereof.
[0087] Accordingly, the cells of the invention provide a means for
testing new therapeutic regimens and for screening and identifying
novel compounds for use in treating cancers, including but not
limited to ALK+ALCL, ALK+ non-small cell carcinoma, ALK+
inflammatory myofibroblastic tumor and other malignancies
expressing oncogenic form of ALK.
[0088] The cells of the invention also provide a research tool for
studying the effects of early therapeutic intervention and the
mechanisms of malignant cell transformation and tumor progression.
This is because the cells of the invention become immortalized and
exhibit a morphology and immunophenotype to that of a corresponding
cancer cell isolated from a cancer patient. In addition, because
these cells are immortalized due to the expression of an oncogenic
kinase (e.g., oncogenic ALK), the activities of the oncogenic
kinase can be evaluated separately from other confounding
mechanisms of oncogenesis that are present in tumors derived from
cancer patients, such as established cell lines. Therefore the
cells of the invention provide insight in the early stages and
other key aspects of human oncogenesis.
[0089] The cells of the invention provide proof that malignant
transformation of normal human cells recapitulating the "natural"
carcinogenesis can be reproducibly achieved experimentally. Thus,
the cells of the invention permit, among other things, the study of
early stages of carcinogenesis, in particular the initial
oncogene-host cell interactions. In one embodiment, the cells of
the invention allow for testing new therapeutic regimens and for
screening and identifying novel compounds for use in treating
cancers.
Sources of T Lymphocytes and Other Cells
[0090] The present invention provides compositions and methods for
transforming primary normal human cells. The invention is partly
based on the discovery that the oncogenic tyrosine kinase NPM-ALK
is capable of transforming in vitro normal human CD4+ T lymphocytes
and conferring upon these cells morphologic and immunophenotypic
features characteristic of the patient-derived ALK+ALCL cells and
tissues. However, the invention should not be limited to only CD4+
T cells. Rather, the invention encompasses any normal cells capable
of becoming transformed by an oncogenic form of ALK.
[0091] Prior to expansion and genetic modification, a source of T
cells is obtained from a subject. The term "subject" is intended to
include living organisms in which an immune response can be
elicited (e.g., mammals). Examples of subjects include humans,
dogs, cats, mice, rats, and transgenic species thereof. T cells can
be obtained from a number of sources, including peripheral blood
mononuclear cells, bone marrow, lymph node tissue, cord blood,
thymus tissue, tissue from a site of infection, ascites, pleural
effusion, spleen tissue, and tumors. In certain embodiments of the
present invention, any number of T cell lines available in the art,
may be used. In certain embodiments of the present invention, T
cells can be obtained from a unit of blood collected from a subject
using any number of techniques known to the skilled artisan, such
as Ficoll.TM. separation. In one embodiment, cells from the
circulating blood of an individual are obtained by apheresis. The
apheresis product typically contains lymphocytes, including T
cells, monocytes, granulocytes, B cells, other nucleated white
blood cells, red blood cells, and platelets. In one embodiment, the
cells collected by apheresis may be washed to remove the plasma
fraction and to place the cells in an appropriate buffer or media
for subsequent processing steps. In one embodiment of the
invention, the cells are washed with phosphate buffered saline
(PBS). In an alternative embodiment, the wash solution lacks
calcium and may lack magnesium or may lack many if not all divalent
cations. Again, surprisingly, initial activation steps in the
absence of calcium lead to magnified activation. As those of
ordinary skill in the art would readily appreciate a washing step
may be accomplished by methods known to those in the art, such as
by using a semi-automated "flow-through" centrifuge (for example,
the Cobe 2991 cell processor, the Baxter CytoMate, or the
Haemonetics Cell Saver 5) according to the manufacturer's
instructions. After washing, the cells may be resuspended in a
variety of biocompatible buffers, such as, for example, Ca-free,
Mg-free PBS, PlasmaLyte A, or other saline solution with or without
buffer. Alternatively, the undesirable components of the apheresis
sample may be removed and the cells directly resuspended in culture
media.
[0092] In another embodiment, T cells are isolated from peripheral
blood lymphocytes by lysing the red blood cells and depleting the
monocytes, for example, by centrifugation through a PERCOLL.TM.
gradient or by counterflow centrifugal elutriation. A specific
subpopulation of T cells, such as CD3.sup.+, CD28.sup.+, CD4.sup.+,
CD8.sup.+, CD45RA.sup.+, and CD45RO.sup.+ T cells, can be further
isolated by positive or negative selection techniques. For example,
in one embodiment, T cells are isolated by incubation with
anti-CD3/anti-CD28 (i.e., 3.times.28)-conjugated beads, such as
DYNABEADS.RTM. M-450 CD3 and CD28 T, for a time period sufficient
for positive selection of the desired T cells. In one embodiment,
the time period is about 30 minutes. In a further embodiment, the
time period ranges from 30 minutes to 36 hours or longer and all
integer values there between. In a further embodiment, the time
period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another
preferred embodiment, the time period is 10 to 24 hours. In one
preferred embodiment, the incubation time period is 24 hours. For
isolation of T cells from patients with leukemia, use of longer
incubation times, such as 24 hours, can increase cell yield. Longer
incubation times may be used to isolate T cells in any situation
where there are few T cells as compared to other cell types, such
in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue
or from immunocompromised individuals. Further, use of longer
incubation times can increase the efficiency of capture of CD8+ T
cells. Thus, by simply shortening or lengthening the time T cells
are allowed to bind to the CD3 and CD28 beads and/or by increasing
or decreasing the ratio of beads to T cells (as described further
herein), subpopulations of T cells can be preferentially selected
for or against at culture initiation or at other time points during
the process. Additionally, by increasing or decreasing the ratio of
anti-CD3 and/or anti-CD28 antibodies on the beads or other surface,
subpopulations of T cells can be preferentially selected for or
against at culture initiation or at other desired time points. The
skilled artisan would recognize that multiple rounds of selection
can also be used in the context of this invention. In certain
embodiments, it may be desirable to perform the selection procedure
and use the "unselected" cells in the activation and expansion
process. "Unselected" cells can also be subjected to further rounds
of selection.
[0093] Enrichment of a T cell population by negative selection can
be accomplished with a combination of antibodies directed to
surface markers unique to the negatively selected cells. One method
is cell sorting and/or selection via negative magnetic
immunoadherence or flow cytometry that uses a cocktail of
monoclonal antibodies directed to cell surface markers present on
the cells negatively selected. For example, to enrich for CD4.sup.+
cells by negative selection, a monoclonal antibody cocktail
typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR,
and CD8. In certain embodiments, it may be desirable to enrich for
or positively select for regulatory T cells which typically express
CD4.sup.+, CD25.sup.+, CD62L.sup.hi, GITR.sup.+, and FoxP3.sup.+.
Alternatively, in certain embodiments, T regulatory cells are
depleted by anti-C25 conjugated beads or other similar method of
selection.
[0094] For isolation of a desired population of cells by positive
or negative selection, the concentration of cells and surface
(e.g., particles such as beads) can be varied. In certain
embodiments, it may be desirable to significantly decrease the
volume in which beads and cells are mixed together (i.e., increase
the concentration of cells), to ensure maximum contact of cells and
beads. For example, in one embodiment, a concentration of 2 billion
cells/ml is used. In one embodiment, a concentration of 1 billion
cells/ml is used. In a further embodiment, greater than 100 million
cells/ml is used. In a further embodiment, a concentration of cells
of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
In yet another embodiment, a concentration of cells from 75, 80,
85, 90, 95, or 100 million cells/ml is used. In further
embodiments, concentrations of 125 or 150 million cells/ml can be
used. Using high concentrations can result in increased cell yield,
cell activation, and cell expansion. Further, use of high cell
concentrations allows more efficient capture of cells that may
weakly express target antigens of interest, such as CD28-negative T
cells, or from samples where there are many tumor cells present
(i.e., leukemic blood, tumor tissue, etc.). Such populations of
cells may have therapeutic value and would be desirable to obtain.
For example, using high concentration of cells allows more
efficient selection of CD8.sup.+ T cells that normally have weaker
CD28 expression.
[0095] In a related embodiment, it may be desirable to use lower
concentrations of cells. By significantly diluting the mixture of T
cells and surface (e.g., particles such as beads), interactions
between the particles and cells is minimized. This selects for
cells that express high amounts of desired antigens to be bound to
the particles. For example, CD4.sup.+ T cells express higher levels
of CD28 and are more efficiently captured than CD8.sup.+ T cells in
dilute concentrations. In one embodiment, the concentration of
cells used is 5.times.10.sup.6/ml. In other embodiments, the
concentration used can be from about 1.times.10.sup.5/ml to
1.times.10.sup.6/ml, and any integer value in between.
[0096] In other embodiments, the cells may be incubated on a
rotator for varying lengths of time at varying speeds at either
2-10.degree. C. or at room temperature.
[0097] T cells for stimulation can also be frozen after a washing
step. Wishing not to be bound by theory, the freeze and subsequent
thaw step provides a more uniform product by removing granulocytes
and to some extent monocytes in the cell population. After the
washing step that removes plasma and platelets, the cells may be
suspended in a freezing solution. While many freezing solutions and
parameters are known in the art and will be useful in this context,
one method involves using PBS containing 20% DMSO and 8% human
serum albumin, or culture media containing 10% Dextran 40 and 5%
Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25%
Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5%
Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable
cell freezing media containing for example, Hespan and PlasmaLyte
A, the cells then are frozen to -80.degree. C. at a rate of
1.degree. per minute and stored in the vapor phase of a liquid
nitrogen storage tank. Other methods of controlled freezing may be
used as well as uncontrolled freezing immediately at -20.degree. C.
or in liquid nitrogen.
[0098] In certain embodiments, cryopreserved cells are thawed and
washed as described herein and allowed to rest for one hour at room
temperature prior to activation using the methods of the present
invention.
[0099] Also contemplated in the context of the invention is the
collection of blood samples or apheresis product from a subject at
a time period prior to when the expanded cells as described herein
might be needed. As such, the source of the cells to be expanded
can be collected at any time point necessary, and desired cells,
such as T cells, isolated and frozen for later use in T cell
therapy for any number of diseases or conditions that would benefit
from T cell therapy, such as those described herein. In one
embodiment a blood sample or an apheresis is taken from a generally
healthy subject. In certain embodiments, a blood sample or an
apheresis is taken from a generally healthy subject who is at risk
of developing a disease, but who has not yet developed a disease,
and the cells of interest are isolated and frozen for later use. In
certain embodiments, the T cells may be expanded, frozen, and used
at a later time.
[0100] Following the isolation of the desired cells from a subject,
the cells can be cultured as described elsewhere herein.
Activation and Expansion of Cells
[0101] In one embodiment, the cells of the invention are generated
by transducing normal cells, preferably purified normal CD4+ T
lymphocytes, with a lentiviral vector expressing oncogenic ALK
after pre-activating the cells with anti-CD3 and CD28 antibodies to
foster an effective transduction.
[0102] In one embodiment, prior to genetically modifying the
primary normal human cell with an oncogenic ALK or an equivalent
thereof, the cell is cultured in the presence of a composition
comprising a first agent that is capable of providing a primary
activation signal to a T cell and a second agent that is capable of
activating a co-stimulatory molecule on a T cell or their
functional equivalents in epithelial and other cell types.
[0103] T cells are activated and expanded generally using methods
as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;
6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;
7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;
6,797,514; 6,867,041; and U.S. Patent Application Publication No.
20060121005.
[0104] Generally, the T cells of the invention are expanded by
contact with a surface having attached thereto an agent that
stimulates a CD3/TCR complex associated signal and a ligand that
stimulates a co-stimulatory molecule on the surface of the T cells.
In particular, T cell populations may be stimulated as described
herein, such as by contact with an anti-CD3 antibody, or
antigen-binding fragment thereof, or an anti-CD2 antibody
immobilized on a surface, or by contact with a protein kinase C
activator (e.g., bryostatin) in conjunction with a calcium
ionophore. For co-stimulation of an accessory molecule on the
surface of the T cells, a ligand that binds the accessory molecule
is used. For example, a population of T cells can be contacted with
an anti-CD3 antibody and an anti-CD28 antibody, under conditions
appropriate for stimulating proliferation of the T cells. To
stimulate proliferation of either CD4.sup.+ T cells or CD8.sup.+ T
cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of
an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone,
Besancon, France) can be used as can other methods commonly known
in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998;
Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al.,
J. Immunol Meth. 227(1-2):53-63, 1999).
[0105] In certain embodiments, the primary stimulatory signal and
the co-stimulatory signal for the T cell may be provided by
different protocols. For example, the agents providing each signal
may be in solution or coupled to a surface. When coupled to a
surface, the agents may be coupled to the same surface (i.e., in
"cis" formation) or to separate surfaces (i.e., in "trans"
formation). Alternatively, one agent may be coupled to a surface
and the other agent in solution. In one embodiment, the agent
providing the co-stimulatory signal is bound to a cell surface and
the agent providing the primary activation signal is in solution or
coupled to a surface. In certain embodiments, both agents can be in
solution. In another embodiment, the agents may be in soluble form,
and then cross-linked to a surface, such as a cell expressing Fc
receptors or an antibody or other binding agent which will bind to
the agents. In this regard, see for example, U.S. Patent
Application Publication Nos. 20040101519 and 20060034810 for
artificial antigen presenting cells (aAPCs) that are contemplated
for use in activating and expanding T cells in the present
invention.
[0106] In one embodiment, the two agents are immobilized on beads,
either on the same bead, i.e., "cis," or to separate beads, i.e.,
"trans." By way of example, the agent providing the primary
activation signal is an anti-CD3 antibody or an antigen-binding
fragment thereof and the agent providing the co-stimulatory signal
is an anti-CD28 antibody or antigen-binding fragment thereof; and
both agents are co-immobilized to the same bead in equivalent
molecular amounts. In one embodiment, a 1:1 ratio of each antibody
bound to the beads for CD4.sup.+ T cell expansion and T cell growth
is used. In certain aspects of the present invention, a ratio of
anti CD3:CD28 antibodies bound to the beads is used such that an
increase in T cell expansion is observed as compared to the
expansion observed using a ratio of 1:1. In one particular
embodiment an increase of from about 1 to about 3 fold is observed
as compared to the expansion observed using a ratio of 1:1. In one
embodiment, the ratio of CD3:CD28 antibody bound to the beads
ranges from 100:1 to 1:100 and all integer values there between. In
one aspect of the present invention, more anti-CD28 antibody is
bound to the particles than anti-CD3 antibody, i.e., the ratio of
CD3:CD28 is less than one. In certain embodiments of the invention,
the ratio of anti CD28 antibody to anti CD3 antibody bound to the
beads is greater than 2:1. In one particular embodiment, a 1:100
CD3:CD28 ratio of antibody bound to beads is used. In another
embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is
used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody
bound to beads is used. In another embodiment, a 1:30 CD3:CD28
ratio of antibody bound to beads is used. In one preferred
embodiment, a 1:10 CD3:CD28 ratio of antibody bound to beads is
used. In another embodiment, a 1:3 CD3:CD28 ratio of antibody bound
to the beads is used. In yet another embodiment, a 3:1 CD3:CD28
ratio of antibody bound to the beads is used.
[0107] Ratios of particles to cells from 1:500 to 500:1 and any
integer values in between may be used to stimulate T cells or other
target cells. As those of ordinary skill in the art can readily
appreciate, the ratio of particles to cells may depend on particle
size relative to the target cell. For example, small sized beads
could only bind a few cells, while larger beads could bind many. In
certain embodiments the ratio of cells to particles ranges from
1:100 to 100:1 and any integer values in-between and in further
embodiments the ratio comprises 1:9 to 9:1 and any integer values
in between, can also be used to stimulate T cells. The ratio of
anti-CD3- and anti-CD28-coupled particles to T cells that result in
T cell stimulation can vary as noted above, however certain
preferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9,
1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,
7:1, 8:1, 9:1, 10:1, and 15:1 with one preferred ratio being at
least 1:1 particles per T cell. In one embodiment, a ratio of
particles to cells of 1:1 or less is used. In one particular
embodiment, a preferred particle:cell ratio is 1:5. In further
embodiments, the ratio of particles to cells can be varied
depending on the day of stimulation. For example, in one
embodiment, the ratio of particles to cells is from 1:1 to 10:1 on
the first day and additional particles are added to the cells every
day or every other day thereafter for up to 10 days, at final
ratios of from 1:1 to 1:10 (based on cell counts on the day of
addition). In one particular embodiment, the ratio of particles to
cells is 1:1 on the first day of stimulation and adjusted to 1:5 on
the third and fifth days of stimulation. In another embodiment,
particles are added on a daily or every other day basis to a final
ratio of 1:1 on the first day, and 1:5 on the third and fifth days
of stimulation. In another embodiment, the ratio of particles to
cells is 2:1 on the first day of stimulation and adjusted to 1:10
on the third and fifth days of stimulation. In another embodiment,
particles are added on a daily or every other day basis to a final
ratio of 1:1 on the first day, and 1:10 on the third and fifth days
of stimulation. One of skill in the art will appreciate that a
variety of other ratios may be suitable for use in the present
invention. In particular, ratios will vary depending on particle
size and on cell size and type.
[0108] In further embodiments of the present invention, the cells,
such as T cells, are combined with agent-coated beads, the beads
and the cells are subsequently separated, and then the cells are
cultured. In an alternative embodiment, prior to culture, the
agent-coated beads and cells are not separated but are cultured
together. In a further embodiment, the beads and cells are first
concentrated by application of a force, such as a magnetic force,
resulting in increased ligation of cell surface markers, thereby
inducing cell stimulation.
[0109] By way of example, cell surface proteins may be ligated by
allowing paramagnetic beads to which anti-CD3 and anti-CD28 are
attached (3.times.28 beads) to contact the T cells. In one
embodiment the cells (for example, 10.sup.4 to 10.sup.9 T cells)
and beads (for example, DYNABEADS.RTM. M-450 CD3/CD28 T
paramagnetic beads at a ratio of 1:1) are combined in a buffer,
preferably PBS (without divalent cations such as, calcium and
magnesium). Again, those of ordinary skill in the art can readily
appreciate any cell concentration may be used. For example, the
target cell may be very rare in the sample and comprise only 0.01%
of the sample or the entire sample (i.e., 100%) may comprise the
target cell of interest. Accordingly, any cell number is within the
context of the present invention. In certain embodiments, it may be
desirable to significantly decrease the volume in which particles
and cells are mixed together (i.e., increase the concentration of
cells), to ensure maximum contact of cells and particles. For
example, in one embodiment, a concentration of about 2 billion
cells/ml is used. In another embodiment, greater than 100 million
cells/ml is used. In a further embodiment, a concentration of cells
of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
In yet another embodiment, a concentration of cells from 75, 80,
85, 90, 95, or 100 million cells/ml is used. In further
embodiments, concentrations of 125 or 150 million cells/ml can be
used. Using high concentrations can result in increased cell yield,
cell activation, and cell expansion. Further, use of high cell
concentrations allows more efficient capture of cells that may
weakly express target antigens of interest, such as CD28-negative T
cells. Such populations of cells may have therapeutic value and
would be desirable to obtain in certain embodiments. For example,
using high concentration of cells allows more efficient selection
of CD8+ T cells that normally have weaker CD28 expression.
[0110] In one embodiment of the present invention, the mixture may
be cultured for several hours (about 3 hours) to about 14 days or
any hourly integer value in between. In another embodiment, the
mixture may be cultured for 21 days. In one embodiment of the
invention the beads and the T cells are cultured together for about
eight days. In another embodiment, the beads and T cells are
cultured together for 2-3 days. Several cycles of stimulation may
also be desired such that culture time of T cells can be 60 days or
more. Conditions appropriate for T cell culture include an
appropriate media (e.g., Minimal Essential Media or RPMI Media 1640
or, X-vivo 15, (Lonza)) that may contain factors necessary for
proliferation and viability, including serum (e.g., fetal bovine or
human serum), interleukin-2 (IL-2), insulin, IFN-.gamma., IL-4,
IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF.beta., and TNF-.alpha. or
any other additives for the growth of cells known to the skilled
artisan. Other additives for the growth of cells include, but are
not limited to, surfactant, plasmanate, and reducing agents such as
N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI
1640, AIM-V, DMEM, MEM, .alpha.-MEM, F-12, X-Vivo 15, and X-Vivo
20, Optimizer, with added amino acids, sodium pyruvate, and
vitamins, either serum-free or supplemented with an appropriate
amount of serum (or plasma) or a defined set of hormones, and/or an
amount of cytokine(s) sufficient for the growth and expansion of T
cells. Antibiotics, e.g., penicillin and streptomycin, are included
only in experimental cultures, not in cultures of cells that are to
be infused into a subject. The target cells are maintained under
conditions necessary to support growth, for example, an appropriate
temperature (e.g., 37.degree. C.) and atmosphere (e.g., air plus 5%
CO.sub.2).
[0111] T cells that have been exposed to varied stimulation times
may exhibit different characteristics. For example, typical blood
or apheresed peripheral blood mononuclear cell products have a
helper T cell population (T.sub.H, CD4.sup.+) that is greater than
the cytotoxic or suppressor T cell population (T.sub.C, CD8.sup.+).
Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors
produces a population of T cells that prior to about days 8-9
consists predominately of T.sub.H cells, while after about days
8-9, the population of T cells comprises an increasingly greater
population of T.sub.C cells. Accordingly, depending on the purpose
of treatment, infusing a subject with a T cell population
comprising predominately of T.sub.H cells may be advantageous.
Similarly, if an antigen-specific subset of T.sub.C cells has been
isolated it may be beneficial to expand this subset to a greater
degree.
[0112] Further, in addition to CD4 and CD8 markers, other
phenotypic markers vary significantly, but in large part,
reproducibly during the course of the cell expansion process. Thus,
such reproducibility enables the ability to tailor an activated T
cell product for specific purposes.
Vectors
[0113] Following activation by stimulating CD3 and CD28 receptors
on the T cell, the activated cell is genetically modified to
express an oncogenic kinase, such as ALK. Accordingly, the
invention includes vectors useful for transducing an oncogenic
kinase, such as ALK, into a T cell.
[0114] The present invention also provides vectors in which a DNA
of the present invention is inserted. Vectors derived from
retroviruses such as the lentivirus are suitable tools to achieve
long-term gene transfer since they allow long-term, stable
integration of a transgene and its propagation in daughter cells.
Lentiviral vectors have the added advantage over vectors derived
from onco-retroviruses such as murine leukemia viruses in that they
can transduce non-proliferating cells, such as hepatocytes. They
also have the added advantage of low immunogenicity.
[0115] In brief summary, the expression of natural or synthetic
nucleic acids encoding oncogenic ALK is typically achieved by
operably linking a nucleic acid encoding the oncogenic ALK
polypeptide or portions thereof to a promoter, and incorporating
the construct into an expression vector. The vectors can be
suitable for replication and integration eukaryotes. Typical
cloning vectors contain transcription and translation terminators,
initiation sequences, and promoters useful for regulation of the
expression of the desired nucleic acid sequence.
[0116] The expression constructs of the present invention may also
be used for nucleic acid immunization and gene therapy, using
standard gene delivery protocols. Methods for gene delivery are
known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859,
5,589,466, incorporated by reference herein in their entireties. In
another embodiment, the invention provides a gene therapy
vector.
[0117] The nucleic acid can be cloned into a number of types of
vectors. For example, the nucleic acid can be cloned into a vector
including, but not limited to a plasmid, a phagemid, a phage
derivative, an animal virus, and a cosmid. Vectors of particular
interest include expression vectors, replication vectors, probe
generation vectors, and sequencing vectors.
[0118] Further, the expression vector may be provided to a cell in
the form of a viral vector. Viral vector technology is well known
in the art and is described, for example, in Sambrook et al. (2001,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York), and in other virology and molecular biology
manuals. Viruses, which are useful as vectors include, but are not
limited to, retroviruses, adenoviruses, adeno-associated viruses,
herpes viruses, and lentiviruses. In general, a suitable vector
contains an origin of replication functional in at least one
organism, a promoter sequence, convenient restriction endonuclease
sites, and one or more selectable markers, (e.g., WO 01/96584; WO
01/29058; and U.S. Pat. No. 6,326,193).
[0119] A number of viral based systems have been developed for gene
transfer into mammalian cells. For example, retroviruses provide a
convenient platform for gene delivery systems. A selected gene can
be inserted into a vector and packaged in retroviral particles
using techniques known in the art. The recombinant virus can then
be isolated and delivered to cells of the subject either in vivo or
ex vivo. A number of retroviral systems are known in the art. In
some embodiments, adenovirus vectors are used. A number of
adenovirus vectors are known in the art. In one embodiment,
lentivirus vectors are used.
[0120] Additional promoter elements, e.g., enhancers, regulate the
frequency of transcriptional initiation. Typically, these are
located in the region 30-110 bp upstream of the start site,
although a number of promoters have recently been shown to contain
functional elements downstream of the start site as well. The
spacing between promoter elements frequently is flexible, so that
promoter function is preserved when elements are inverted or moved
relative to one another. In the thymidine kinase (tk) promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either cooperatively
or independently to activate transcription.
[0121] One example of a suitable promoter is the immediate early
cytomegalovirus (CMV) promoter sequence. This promoter sequence is
a strong constitutive promoter sequence capable of driving high
levels of expression of any polynucleotide sequence operatively
linked thereto. Another example of a suitable promoter is
Elongation Growth Factor-1.alpha. (EF-1.alpha.). However, other
constitutive promoter sequences may also be used, including, but
not limited to the simian virus 40 (SV40) early promoter, mouse
mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long
terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia
virus promoter, an Epstein-Barr virus immediate early promoter, a
Rous sarcoma virus promoter, as well as human gene promoters such
as, but not limited to, the actin promoter, the myosin promoter,
the hemoglobin promoter, and the creatine kinase promoter. Further,
the invention should not be limited to the use of constitutive
promoters. Inducible promoters are also contemplated as part of the
invention. The use of an inducible promoter provides a molecular
switch capable of turning on expression of the polynucleotide
sequence which it is operatively linked when such expression is
desired, or turning off the expression when expression is not
desired. Examples of inducible promoters include, but are not
limited to a metallothionine promoter, a glucocorticoid promoter, a
progesterone promoter, and a tetracycline promoter.
[0122] In order to assess the expression of a CAR polypeptide or
portions thereof, the expression vector to be introduced into a
cell can also contain either a selectable marker gene or a reporter
gene or both to facilitate identification and selection of
expressing cells from the population of cells sought to be
transfected or infected through viral vectors. In other aspects,
the selectable marker may be carried on a separate piece of DNA and
used in a co-transfection procedure. Both selectable markers and
reporter genes may be flanked with appropriate regulatory sequences
to enable expression in the host cells. Useful selectable markers
include, for example, antibiotic-resistance genes, such as neo and
the like.
[0123] Reporter genes are used for identifying potentially
transfected cells and for evaluating the functionality of
regulatory sequences. In general, a reporter gene is a gene that is
not present in or expressed by the recipient organism or tissue and
that encodes a polypeptide whose expression is manifested by some
easily detectable property, e.g., enzymatic activity. Expression of
the reporter gene is assayed at a suitable time after the DNA has
been introduced into the recipient cells. Suitable reporter genes
may include genes encoding luciferase, beta-galactosidase,
chloramphenicol acetyl transferase, secreted alkaline phosphatase,
or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000
FEBS Letters 479: 79-82). Suitable expression systems are well
known and may be prepared using known techniques or obtained
commercially. In general, the construct with the minimal 5'
flanking region showing the highest level of expression of reporter
gene is identified as the promoter. Such promoter regions may be
linked to a reporter gene and used to evaluate agents for the
ability to modulate promoter-driven transcription.
[0124] Methods of introducing and expressing genes into a cell are
known in the art. In the context of an expression vector, the
vector can be readily introduced into a host cell, e.g., mammalian,
bacterial, yeast, or insect cell by any method in the art. For
example, the expression vector can be transferred into a host cell
by physical, chemical, or biological means.
[0125] Physical methods for introducing a polynucleotide into a
host cell include calcium phosphate precipitation, lipofection,
particle bombardment, microinjection, electroporation, and the
like. Methods for producing cells comprising vectors and/or
exogenous nucleic acids are well-known in the art. See, for
example, Sambrook et al. (2012, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York). A preferred
method for the introduction of a polynucleotide into a host cell is
calcium phosphate transfection.
[0126] Biological methods for introducing a polynucleotide of
interest into a host cell include the use of DNA and RNA vectors.
Viral vectors, and especially retroviral vectors, have become the
most widely used method for inserting genes into mammalian, e.g.,
human cells. Other viral vectors can be derived from lentivirus,
poxviruses, herpes simplex virus I, adenoviruses and
adeno-associated viruses, and the like. See, for example, U.S. Pat.
Nos. 5,350,674 and 5,585,362.
[0127] Chemical means for introducing a polynucleotide into a host
cell include colloidal dispersion systems, such as macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes. An exemplary colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (e.g., an artificial
membrane vesicle).
[0128] In the case where a non-viral delivery system is utilized,
an exemplary delivery vehicle is a liposome. The use of lipid
formulations is contemplated for the introduction of the nucleic
acids into a host cell (in vitro, ex vivo or in vivo). In another
aspect, the nucleic acid may be associated with a lipid. The
nucleic acid associated with a lipid may be encapsulated in the
aqueous interior of a liposome, interspersed within the lipid
bilayer of a liposome, attached to a liposome via a linking
molecule that is associated with both the liposome and the
oligonucleotide, entrapped in a liposome, complexed with a
liposome, dispersed in a solution containing a lipid, mixed with a
lipid, combined with a lipid, contained as a suspension in a lipid,
contained or complexed with a micelle, or otherwise associated with
a lipid. Lipid, lipid/DNA or lipid/expression vector associated
compositions are not limited to any particular structure in
solution. For example, they may be present in a bilayer structure,
as micelles, or with a "collapsed" structure. They may also simply
be interspersed in a solution, possibly forming aggregates that are
not uniform in size or shape. Lipids are fatty substances which may
be naturally occurring or synthetic lipids. For example, lipids
include the fatty droplets that naturally occur in the cytoplasm as
well as the class of compounds which contain long-chain aliphatic
hydrocarbons and their derivatives, such as fatty acids, alcohols,
amines, amino alcohols, and aldehydes.
[0129] Lipids suitable for use can be obtained from commercial
sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP")
can be obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Choi") can be obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform or chloroform/methanol can be
stored at about -20.degree. C. Chloroform is used as the only
solvent since it is more readily evaporated than methanol.
"Liposome" is a generic term encompassing a variety of single and
multilamellar lipid vehicles formed by the generation of enclosed
lipid bilayers or aggregates. Liposomes can be characterized as
having vesicular structures with a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved
solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology
5: 505-10). However, compositions that have different structures in
solution than the normal vesicular structure are also encompassed.
For example, the lipids may assume a micellar structure or merely
exist as nonuniform aggregates of lipid molecules. Also
contemplated are lipofectamine-nucleic acid complexes.
[0130] Regardless of the method used to introduce exogenous nucleic
acids into a host cell or otherwise expose a cell to the inhibitor
of the present invention, in order to confirm the presence of the
recombinant DNA sequence in the host cell, a variety of assays may
be performed. Such assays include, for example, "molecular
biological" assays well known to those of skill in the art, such as
Southern and Northern blotting, RT-PCR and PCR; "biochemical"
assays, such as detecting the presence or absence of a particular
peptide, e.g., by immunological means (ELISAs and Western blots) or
by assays described herein to identify agents falling within the
scope of the invention.
Screening Agents
[0131] The invention includes a cell-based screening method
comprises: bringing test substances into contact with a cell
comprising an oncogenic kinase, such as ALK, of the invention by
mixing (i.e., addition) (contact step); analyzing whether the cell
expressing an oncogenic ALK of the present invention exhibits
morphological or phenotypical changes or not by the test
substance(s), by comparison with the morphological or phenotypical
of the cell present invention not brought into contact with the
test substances (analysis step); and selecting a substance altering
the morphological or phenotypical of the cell of the present
invention (i.e., a therapeutic agent for cancer, particularly, a
therapeutic agent for lymphoma or lung cancer).
[0132] The invention includes a cell-based screening method
comprises: bringing test substances into contact with a cell
comprising an oncogenic ALK of the invention by mixing (i.e.,
addition) (contact step); analyzing whether the activity of the
oncogenic ALK of the present invention is inhibited or not by the
test substance(s), by comparison with the activity of the oncogenic
ALK of the present invention not brought into contact with the test
substances (analysis step); and selecting a substance inhibiting
the activity of the oncogenic ALK of the present invention (i.e., a
therapeutic agent for cancer, particularly, a therapeutic agent for
lymphoma or lung cancer).
[0133] The invention also includes an expression inhibition-based
screening method comprises: bringing test substances into contact
with a cell expressing an oncogenic ALK by mixing (i.e., addition)
(contact step); analyzing whether the expression of the oncogenic
ALK is inhibited or not by the test substance(s), by comparison
with the expression of the oncogenic ALK not brought into contact
with the test substances (analysis step); and selecting a substance
inhibiting the expression of oncogenic ALK (i.e., a therapeutic
agent for cancer, particularly, a therapeutic agent for lymphoma or
lung cancer, that is shown to be positive for the polynucleotide of
the present invention).
[0134] In one embodiment, the screening method of the present
invention further comprises, in addition to analyzing whether the
oncogenic ALK is inhibited or not and selecting a substance
inhibiting the polypeptide of the present invention, the step of
confirming that the selected test substance has a therapeutic
activity against cancer (particularly, lymphoma or lung cancer).
Examples of the step of confirming that the selected substance has
a therapeutic activity against cancer include a step of practicing
an evaluation method known in the art or a modified method
thereof.
[0135] A growth-inhibiting effect or cell death-inducing effect on
the cells of the invention by the test substance can be confirmed
by adding the test substance selected by the screening method of
the present invention to a culture medium of the cells of the
invention and measuring a cell count or cell death rate after
culture by a standard method. If the selected test substance
exhibits the growth-inhibiting effect and/or cell death-inducing
effect on the cells, this selected test substance is confirmed to
have a therapeutic activity against cancer. The test substance may
be added to the medium under conditions in which the test substance
is added at the start of culture or during culture once or any
number of times without limitations. A culture period in the
presence of the test substance can be set appropriately and is 5
minutes to 2 weeks, preferably 1 hour to 72 hours. Any of a variety
of cell measurement methods may be used, such as trypan blue
staining, Sulforhodamine, MTT, intracellular ATP measurement, and
thymidine uptake methods, and any of a variety of cell death
measurement methods may be used, such as LDH release measurement,
annexin V staining, and caspase activity measurement methods.
[0136] The inhibitory effect of the test substance on the growth of
the transformed cells of the invention can be examined with
anchorage-independent growth, one feature of cancer cells, as an
index to thereby determine a therapeutic activity against cancer.
The anchorage-independent growth refers to, in contrast to adherent
normal cells that must adhere to the extracellular matrix
(anchorage) for their survival and growth, the general essential
property of cancer cells capable of growing even without such an
anchorage. One of most reliable methods for examining the
carcinogenesis of cells is to confirm that the cells can grow
without an anchorage. Whether cells transformed from normal cells
by gene expression exhibit an anchorage-independent growth ability
can be examined to determine whether the gene is an oncogene. The
transformed cells of the invention also acquire an
anchorage-independent growth ability. Therefore, the therapeutic
activity of the test substance against cancer that is shown to be
positive can be examined with this property as an index. The
anchorage-independent growth of the transformed cells of the
invention can be achieved by a method for cell culture in a soft
agar medium or a method for cell culture in a plate capable of
cell-culturing spheroids (cell aggregates).
[0137] In one embodiment, the invention provides methods of
screening for drugs or agents that modulate (e.g., enhance or
suppress) oncogene-mediated neoplastic or hyperplastic
transformation. In one embodiment, a method includes (a) contacting
or otherwise exposing a cell of the invention (e.g., a normal cell
genetically modified with an oncogenic ALK) to a test drug or
agent; (b) determining if the test drug or agent modulates (e.g.,
enhances or suppresses) oncogene-mediated neoplastic or
hyperplastic transformation; and (c) classifying the test drug or
agent as an drug or agent that modulates oncogene-mediated
neoplastic or hyperplastic transformation if the test drug or agent
suppresses or enhances oncogene-mediated neoplastic or hyperplastic
transformation.
[0138] In another embodiment, the invention provides methods of
screening for drugs or agents that modulate the sensitivity of the
cells of the invention to treatment with radiation or chemotherapy.
In one embodiment, the method comprises (a) contacting or otherwise
exposing a cell of the invention to a test drug or agent; (b)
determining if the test drug or agent modulates (e.g., suppresses
or enhances) sensitivity to radiation- or chemotherapy-induced
programmed cell death; and (c) classifying the test drug or agent
as an drug or agent that modulates sensitivity to radiation- or
chemotherapy-induced programmed cell death if the test drug or
agent suppresses or enhances sensitivity to radiation- or
chemotherapy-induced programmed cell death.
[0139] In one embodiment, the test drug or agent may suppress, or
otherwise alter, or enhance expression of oncogene RNA and/or the
oncogenic protein product, or RNA or protein expression of other
genes involved in the oncogenic transformation process.
Additionally, the test drug or agent may inhibit or stimulate the
activity of other molecules involved, directly or indirectly, in
the neoplastic/hyperplastic transformation process, or in the
sensitivity of transgenic cells to treatments with radiation or
chemotherapy. A wide variety of drugs or agents may be tested in
the screening methods of the present invention. For example, small
molecule compounds similar to those identified in Peterson, R. T.,
et al., Proc. Natl. Acad. Sci. U.S.A, 97: 12965-12969, (2000) and
Peterson, R. T., et al. Curr. Biol., 11: 1481-1491, (2001) or a
panel of FDA approved chemicals may be assayed. Small molecule
compounds are identified by screening large chemical libraries for
the effects of compound addition to the water of developing fish.
Additionally, proteins such as oligo- and polypeptides, may also
act as test drugs or agents.
[0140] Further examples of such test drugs or agents include
oligonucleotides or polynucleotides, such as, for example,
antisense deoxyribonucleic acid (DNA), antisense ribonucleic acid
(RNA), and small interfering RNAs. The antisense nucleotide
sequences typically include a nucleotide sequence that is
complementary to, or is otherwise able to hybridize with, a portion
of the target nucleotide sequence, such as the target nucleotide
sequences described herein and others described herein. The
antisense nucleotide sequence may have a length of at least about
10 nucleotides, but may range in length from about 10 to about 1000
nucleotides, or may be the entire length of the gene target. The
skilled artisan can select an appropriate target and an appropriate
length of antisense nucleic acid in order to have the desired
therapeutic effect by standard procedures known to the art, and as
described, for example, in Methods in Enzymology, Antisense
Technology, Parts A and B (Volumes 313 and 314) (M. Phillips, ed.,
Academic Press, 1999).
[0141] Examples of the test substances used in the screening method
of the present invention can also include, but not particularly
limited to, commercially available compounds (including peptides),
a variety of compounds (including peptides) known in the art and
registered in chemical files, compound groups obtained by a
combinatorial chemistry technique (N. Terrett et al., Drug Discov.
Today, 4 (1): 41, 1999), microorganism culture supernatants, plant-
or marine organism-derived natural components, animal tissue
extracts, double-stranded nucleic acids, antibodies or antibody
fragments, and compounds (including peptides) chemically or
biologically modified from compounds (including peptides) selected
by the screening method of the present invention.
[0142] In various embodiments, in vitro assays can be carried out
with cells that harbor the NPM-ALK gene and that are representative
of the tumor cell type involved in a subject's disease, to
determine if a compound has a desired effect upon such tumor cell
types. In one embodiment, the cells are T or B cell lymphomas,
anaplastic large cell lymphomas, or multiple myelomas.
EXPERIMENTAL EXAMPLES
[0143] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0144] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out the
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
Example 1
A Potent Oncogene NPM-ALK Mediates Malignant Transformation of
Normal Human CD4+ T Lymphocytes
[0145] The results presented herein show that in vitro transduction
of normal human CD4+ T lymphocytes with NPM-ALK results in their
malignant transformation. The transformed cells become immortalized
and display morphology and immunophenotype of patient-derived
anaplastic large-cell lymphomas (ALCL). These unique features
including the perpetual cell growth, activation of the key signal
transduction pathways and expression of CD30, IL-10 and PD-L1/CD274
are strictly dependent on NPM-ALK activity and expression.
Implantation of the NPM-ALK-transformed CD4+ T lymphocytes into
immunodeficient mice results in formation of tumors
indistinguishable from patient ALCL. This study demonstrates that
the early stages and other key aspects of human oncogenesis can be
faithfully reproduced in vitro when potent oncogenic stimulus is
used to transform the "natural" target cells. The study provides
proof-of-principle evidence that malignant transformation of normal
human cells recapitulating the "natural" carcinogenesis can be
reproducibly achieved experimentally. This finding stresses the key
role in carcinogenesis of potent oncogenes when they become
expressed in the relevant target cells. The transformed cells of
this kind permit the study of early stages of carcinogenesis, in
particular the initial oncogene-host cell interactions. This
experimental approach also fosters studies into the effects of
early therapeutic intervention and the mechanisms of malignant
progression.
[0146] The materials and methods employed in the experiments
disclosed herein are now described.
Materials and Methods
[0147] Lentiviral Transduction of CD4+ T Lymphocytes
[0148] De-identified purified human CD4+ T cells were obtained from
the Human Immunology Core of the University of Pennsylvania under
an IRB approved protocol. CD4+ T cells were activated by co-culture
with anti-CD3/28 Ab-coated beads at the cell:bead ratio of 1:3. The
cells were transduced 24 hr later by being exposed to lentiviral
vectors containing NPM-ALK (wild type or kinase-deficient K210R
mutant), either alone or together with GFP as part of T2A fusion
construct (REF 18768965). Fresh medium was added to the cells on
day 3 and twice weekly thereafter. On day 5, the magnetic beads
were removed. The transduction efficiency was determined by flow
cytometry by examining expression of GFP or NPM-ALK, the latter
accomplished using anti-ALK ab (J606, BD Biosciences, San Jose,
Calif.) and a Cytofix/Cytoperm.TM. fixation and permeablization Kit
(BD Biosciences, San Jose, Calif.).
[0149] Cell Lines
[0150] The standard cell lines used in this study have been
described previously (Marzec et al., 2007, Oncogene
26(38):5606-5614; Marzec et al., 2008, Proc Natl Acad Sci USA
105(52):20852-20857; Kasprzycka et al., 2006, Natl Acad Sci USA
103(26):9964-9969). In brief, SUDHL-1 and JB6 cell lines were
developed from ALK+ALCL and 2A (Mac-2A) cell line from the primary
cutaneous ALK-ALCL. MyLa2059 and MyLa3675 cell lines were derived
from cutaneous T-cell lymphomas. Jeko cell line was established
from a mantle B-cell lymphoma. HEK 293 cells have been derived from
human embryonic kidney (purchased from ATCC, Manassas, Va.).
[0151] Western Blotting
[0152] These experiments were performed using antibodies against
phosphorylated (p)-ALK, p-STAT3, p-S6RP, total S6RP (Cell Signaling
Technology), total NPM-ALK (Pharmigen, San Diego, Calif.), and
total STAT3 and actin (ACTB), both from Santa Cruz Biotechnology
(Santa Cruz, Calif.), according to the standard protocols.
[0153] Cell Migration
[0154] The cells were incubated for 20 hr in the FBS-free RPMI
medium, washed, re-suspended in quenching medium (5% BSA-RPMI).
They were applied at concentration of 2.times.10.sup.6/ml in 250 ul
to the top chamber of the Transwell system (Chemicon International)
and 400 ul of RPMI with FBS media were added to the lower chamber.
The plates were covered and incubated for 24 hr at 37.degree. C. in
an atmosphere containing 5% CO.sub.2. Cells that passed through the
membrane were collected from the lower chamber and added to a
96-well plate. Lysis Buffer/Dye Solution containing the CyQUANT
green dye was added to all samples for 15' at room temperature and
the plate was examined with a fluorescence plate reader (Molecular
Devices) using the 480/520 nm filter set.
[0155] Colony Formation
[0156] The cells were plated for 21 days in the semi-solid agar
prepared according to the standard protocol. The number of growing
colonies was counted using an inverted microscope.
[0157] Flow Cytometry
[0158] The cells were analyzed using FACSCalibur (BD Biosciences)
and, for data analysis, CellQuest Pro software version 6.03 (BD
Biosciences). For the standard cell-surface staining,
0.5-1.0.times.10.sup.6 cells were incubated for 20 minutes at
4.degree. C. with 10-20 ul of fluorescein isothiocyanate-conjugated
(FITC), phycoerythrin-conjugated (PE), or
allophycocyanin-conjugated (APC) standard anti-T- and B-cell
antibodies, PD-L1 or isotype control antibody (Bio-Legend, San
Diego, Calif.). The intracellular staining was performed by using
commercially available fixation and permeabilization reagents (BD
Biosciences or Life Technologies, Carlsbad, Calif.). In brief,
0.5-1.0.times.10.sup.6 washed membrane-stained or unstained cells
were fixed for 15 minutes at room temperature with 100 ul of
Fix/Per-solution or Fixation medium. After washing the cells were
re-suspended in 100 ul of PBS or Permeabilization medium, and
incubated for 15 minutes at room temperature with 10-20 ul of
PE-labeled ALK or isotype control antibody (BD Biosciences). After
additional washing, cells were analyzed by flow cytometry
(FACSCalibur; BD Biosciences). Data acquisition and analysis were
performed using CellQuest Pro software version 6.03 (BD
Biosciences).
[0159] Immunohistochemistry
[0160] The immunohistochemical stainings were performed on
formalin-fixed, paraffin-embedded cell blocks or xenotransplant
tumor tissues using standard methods. In brief, the slides were
heat-treated for antigen retrieval in 10-mM citrate buffer and
sections were incubated with the diluted primary antibodies to ALK,
CD30, CD2, MUM1, Ki67 (all from Dako, Carpinteria, Calif.) and CD3
(Novocastra, Leica Microsystems, Wetzlar, Germany). For
interpretation, the immunostained slides were evaluated by light
microscopy.
[0161] Cytogenetics
[0162] A metaphase arresting agent, Colcemid solution was added to
cell cultures for 2 hours. Cells were exposed to hypotonic solution
for 40 minutes at 37.degree. C., followed with three changes of 1:3
glacial acetic acid and methanol solution. Cell suspension was
dropped onto water wet microscope slides. Dried slides were aged at
600.degree. C. oven for 14 hours and stained with Wright's stain
for G-banding. Metaphase spreads were analyzed under 100.times.
magnification of the bright field microscope, images captured and
karyotypes were prepared using Gene Vision (Applied Imaging
Computer Karyotyping System, Santa Clara, Calif.).
[0163] T-Cell Receptor Gene Rearrangement
[0164] DNA was extracted from cultured cells using conventional
column based methods (Qiagen, Valencia, Calif.). Two separate
multiplex PCR amplifications were performed using primers to
relatively well-conserved regions in the V and J gene segments of
the T cell receptor gamma locus (TRGg). PCR products were separated
by capillary electrophoresis using an ABI 3130xl system (Life
Technologies). Peak size and height were determined using
GeneMapper v3.7 software (Life Technologies). PCR product sizes are
expected to range between 200 bp and 250 bp for the TRGg V gene
segments 1 to 8 primer mix, and 150 bp and 200 bp for the TRGg V
gene segments 9 to 11 primer mix.
[0165] siRNA Assay
[0166] A mixture of four siRNAs specific for ALK or control siRNAs
(all from Dharmacon; Thermo Fisher Scientific, Waltham, Mass.) was
introduced into cells using Lipofectamine 2000 as described
previously (Marzec et al., 2008, Proc Natl Acad Sci USA
105(52):20852-20857) for SUDHL-1 cells and Nucleofector T-solution
(Amaxa; Lonza, Walkersville, Md.) for CD4+ T-cell derived NA1
cells.
[0167] IL-10 Assay
[0168] IL-10 expression was using a Human Cytokine 10-Plex Antibody
Bead Kit (Life Technologies) as per manufacturer protocol. Sample
acquisition employed a Luminex FlexMAP-3D (Life Technologies), and
analyses performed using xPONENT software (version 4.0). A
nine-point standard curve at threefold dilutions was employed with
the range defined by 80%-120% of expected/observed values. Samples
were tested in duplicate and CV was <10%. The results are
presented as the decrease in the ALK inhibitor (CEP-28122)-treated
cells relative to untreated cells.
[0169] RT-qPCR
[0170] Total RNA was extracted (RNeasy kit; Qiagen) and reverse
transcribed using High Capacity RNA-to-cDNA kit (ABI). Expression
levels of NPM-ALK mRNA were quantified by using an ABI/PRISM 7700
sequence detection system with TaqMan Gene Expression Assay kits
(NPM-ALK, Hs03024829; .beta.-actin, Hs9999903) (Life Technologies)
and SYBR Green assay (Life Technologies) using primers for IL-10
(5'-AAGGCGCATGTGAACTCC-3'; SEQ ID NO: 1 and
5'-AAGGCATTCTTCACCTGCTC-3'; SEQ ID NO: 2) and for GAPDH
(5'-TCTCCAGAACATCATCCCTGCCTC-3; SEQ ID NO: 3 and
5'-TGGGCCATGAGGTCCACCACCCTG-3; SEQ ID NO: 4. All assays were
performed in duplicate. The fold difference in RNA levels was
calculated on the basis of the difference between C.sub.T values
obtained for control and individual mRNA (.DELTA.C.sub.T).
[0171] MTT Enzymatic Conversion Assay
[0172] The cells suspended at 2.times.10.sup.4/well were incubated
at 37.degree. C. in microtiter plates for up to two days, then
incubated with MTT (Promega Madison, N.J.) for 4 hours. Well
contents were solubilized overnight in the medium containing 10%
SDS and 0.01M HCL. Absorbance at 570 nm in each well was measured
using a Titertek Multiskan reader (Thermo Fisher Scientific).
[0173] Bromodeoxyuridine Incorporation Assay
[0174] The assay was performed using a cell-proliferation enzyme
linked immunosorbent assay (ELISA; Roche Diagnostics, Indianapolis,
Ind.) according to the manufacturer's protocol. In brief, cells
were cultured at a concentration of 2.times.10.sup.4 cells per well
for 44 hours and labeled with bromodeoxyuridine for 4 hours. After
centrifugation, supernatant removal, and plate drying, the cells
were fixed and the DNA was denatured using FixDenat reagent (Roche
Diagnostics).
The amount of incorporated bromodeoxyuridine was determined by
incubation with a specific antibody conjugated with horseradish
peroxidase, followed by colorimetric conversion of the substrate
and absorbance evaluation in the ELISA plate reader.
[0175] TUNEL Assay for DNA Fragmentation
[0176] TUNEL assay was performed using an ApoAlert DNA
fragmentation assay kit (BD Biosciences) according to the
manufacturer's protocol. In brief, cells were cultured at
0.5.times.10.sup.4 cells per well for 48 hours and then were
collected, washed, fixed, permeabilized with 70% ethanol, washed
again, and incubated in terminal deoxynucleotidyl transferase
incubation buffer for 1 hour at 37.degree. C. The reaction was
stopped by adding 20 mmol/L EDTA. The cells were washed twice,
resuspended in 0.5 mL of propidium iodide-RNase-PBS, collected, and
analyzed by flow cytometry using a FACSCalibur system (BD
Biosciences); data acquisition and analysis were performed using
Cell-Quest Pro software (BD Biosciences).
[0177] Annexin V Expression Assay
[0178] For annexin V expression assay, cells were treated with the
ALK inhibitor CEP-28122 (100 nmol/L) for 48 hours or with ALK siRNA
(100 pmol/L) for 72 hours. After treatment, cells were washed with
PBS and stained with anti-annexin V antibody and propidium iodide
for 10 minutes, according to the manufacturer's instructions (Roche
Diagnostics). The stained cells were analyzed by flow cytometry
using a FACSCalibur system (BD Biosciences); data acquisition and
analysis were performed using Cell-Quest Pro software (BD
Biosciences).
[0179] Mouse Xenograft Tumor Formation
[0180] For tumor growth studies, NOD/SCID/IL-2R.gamma.c.sup.null
(NSG, JAX stock #005557, Jackson Laboratory, Bar Harbor, Me.) mice
were generated by the Stem Cell and Xenograft Core (University of
Pennsylvania School of Medicine) using stock breeders obtained from
the Jackson Laboratory. Mice were housed in sterile conditions
using HEPA-filtered microisolators and fed with irradiated food and
acidified water. All experiments were conducted using mice aged 8
weeks in accordance with a protocol reviewed and approved by the
Institutional Animal Care and Use Committee. The NPM-ALK
transformed CD4+ T-cell lines NA1 and ALK+ALCL-derived SUDHL-1 line
were transduced to express luciferase. On day 0, individual mice
were implanted with 3.times.10.sup.6 cells in 100 ul PBS by
intraperitoneal administration. Mice were monitored weekly for
tumor growth by visual examination and, starting with the week 3,
bioluminescence imaging, which was conducted on anesthetized mice
using a Xenogen Spectrum system and Living Image v3.2 software. For
imaging, 10 mg/kg D-luciferin (Caliper Life Sciences) re-suspended
in sterile PBS at a concentration of 15 mg/ml was administered
intraperitoneally. Mice were imaged 12 minutes post-luciferin
injection and serial images were collected at various exposures.
Data were analyzed with Living Image v3.2 software using images
taken with identical settings for mice in each group at each time
point. Imaging data were converted to
photons/second/cm.sup.2/steradian to normalize each image for
exposure time, f-stop, binning and mouse size.
[0181] The results of the experiments presented in this Example are
now described.
NPM-ALK Induces Malignant Transformation of Normal CD4+ T
Lymphocytes
[0182] Given the highly oncogenic phenotype of NPM-ALK and the CD4+
T-cell derivation of ALK+ALCL (Li et al, 2007, Med Res Rev
28(3):372-412; Wasik et al., 2009, Semin Oncol 36(2 Suppl
1):527-35; Tabbo et al, 2012, Front Oncol 2:41), experiments were
designed to transduce purified normal CD4+ T lymphocytes with
lentiviral vector expressing the kinase after pre-activating the
cells with anti-CD3 and CD28 antibodies to foster an effective
transduction. Separate pools of the pre-activated CD4+ T cells were
transduced with either a NPM-ALK mutant devoid of enzymatic
activity (NPM-ALK-KD) or left untransduced. As shown in FIG. 1A,
transfection with the native NPM-ALK led to sustained growth of the
target cells. Although they displayed somewhat higher transfection
efficiency (FIG. 5), the cells expressing inactive NPM-ALK reached
the growth plateau by the second week and began to decline shortly
afterwards, similar to untransfected cells. The same pattern of
cell growth was seen in three independent consecutive experiments
where the CD4+ T cell transfection with wild-type NPM-ALK resulted
in the establishment of cell lines designated NA1, NA2, and NA3.
These cell lines display a steady growth rate (FIG. 1B) and remain
in continuous culture for at least eight months while the control
cell populations ceased to grow by the 3-4 week of culture (FIG.
6). The established cell lines exhibited sustained expression of
NPM-ALK as well as phosphorylation of the kinase at the
concentration similar (NA1) or visibly lower (NA2) then the
control, ALK+ALCL-derived SUDHL-1 cells (FIG. 1C). They also
exhibited phosphorylation of direct target of NPM-ALK STAT3 and of
its indirect, mTORC1-dependent target S6RP. These cells were also
observed to be very large matching the size of the SUDHL-1 cells
and markedly exceeding the size of the control CD3 and
CD28-stimulated CD4+ T cells (FIG. 1D). They migrated (FIG. 1E) and
formed colonies (FIG. 1F) and, hence, exhibited additional features
of transformed cells.
Characteristics of the NPM-ALK Transformed Cells
[0183] Experiments were designed to examine the morphology and
immunophenotype of the NPM-ALK-transformed CD4+ T cells. As shown
in FIG. 2A, the cells displayed predominantly large nuclei with
prominent nucleoli and moderate to abundant amount of the
eosinophilic cytoplasm. In addition to expressing NPM-ALK, their
subset weakly expressed T-cell related CD3 antigen and strongly
expressed proliferation-related Ki67 antigen as determined by
immunohistochemistry. Of note, the cells universally and strongly
expressed CD30 and IRF4/MUM1 antigens. The marked loss of CD3
expression was confirmed by flow cytometry where diminished
expression of CD5 and strong expression of CD4 and CD25 were also
observed (FIG. 2B). The strong CD30 expression was also confirmed
by this method (FIG. 2C). As summarized in FIG. 7, the cells
further mimicked ALK+ALCL cells by variably expressing T-cell
markers: CD2 and CD7, among other features. Because ALK+ALCL cells
universally express the immunosuppressive molecules IL-10.sup.14
and PD-L1 (Marzec et al., 2008, Proc Natl Acad Sci USA
105(52):20852-20857), experiments were designed to examine the
NPM-ALK-transformed CD4+ cells for the expression of these two
immunosuppressive molecules. Indeed, both were expressed by the
transformed cells (FIGS. 2D and 2E, respectively).
[0184] To further characterize the NPM-ALK-transformed CD4+ T
cells, their karyotype and clonality were evaluated. Both NA1 and
NA2 analyzed displayed essentially normal cytogenetics with only
occasional random changes identified (FIG. 8). However, these two
cell lines as well as NA3 showed two to four distinct peaks in the
T-cell receptor gamma chain rearrangement PCR study that used two
separate primer pairs (FIG. 9), indicating mono-to oligoclonal
nature of the transformed CD4+ T lymphocytes.
Transformed Cells are Strictly NPM-ALK-Dependent
[0185] In the next series of experiments, the effects of
suppression of NPM-ALK on the cells were evaluated. A highly
selective ALK inhibitor was used (Cheng et al., 2012, Mol Cancer
Ther 11(3):670-679) followed by ALK-targeting siRNA. Cell signaling
is ALK-dependent in the transformed cells as documented by the ALK
inhibitor-induced suppression of phosphorylation of the key
proteins: ALK itself, STAT3, and S6RP (FIG. 3A). A similar result
was obtained in the NPM-ALK-transfected epithelial HEK cells,
although higher dose of the inhibitor had to be used to suppress
ALK, STAT3, and S6RP phosphorylation due to high concentration of
NPM-ALK expressed by these easily transfectable cells (FIG. 10).
Furthermore, HEK cells transfected with the enzymatically inactive
NPM-ALK-KD failed to phosphorylate ALK, STAT3, and S6RP, further
supporting the key role of NPM-ALK in their activation. Because
expression of CD30, the hallmark of ALK+ALCL, has been reported as
being NPM-ALK-dependent (Hsu et al., 2006, Cancer Res
15(18):9002-9008), experiments were designed to examine whether
this is the case in the NPM-ALK-transformed CD4+ T cells. Indeed,
ALK inhibition diminished CD30 expression not only in the
ALK+ALCL-derived cells but also in the transformed CD4+ T cells,
while having no effect on CD30 expression in the cells from a
different type T-cell lymphoma that is ALK negative (FIG. 3B).
Similarly to CD30, expression of the immunosuppressive proteins
IL-10 and PD-L1 is ALK-dependent in the NPM-ALK-transformed CD4+ T
cells (FIGS. 3C and 3D, respectively). ALK is also critical for the
transformed CD4+ T cells on the functional level, since ALK
inhibition suppressed their growth (FIG. 3E and FIG. 12A).
Depletion of NPM-ALK by siRNA yielded results similar to its
inhibition including loss of ALK, STAT3, and S6RP phosphorylation
(FIG. 3F), IL-10 (FIG. 3G) and PD-L1 expression (FIG. 3H), and
impairment of cell growth (FIG. 3I and FIG. 12B).
In Vivo Tumor Formation
[0186] As shown in FIGS. 4A-4C, the NPM-ALK-transformed cells are
capable of forming tumors in vivo as xenotransplants in the
immunodeficient NSG mice. Within five weeks from injection, three
out of five mice developed large tumors as compared to four out of
five mice injected with ALK+ALCL cells (FIG. 4A). The tumors are
indistinguishable from native ALK+ALCL as determined by anaplastic
large cell morphology and the immunophenotype including expression
of NPM-ALK, CD30, variable loss of T-cell antigens, and high
proliferative rate (FIGS. 4B and 4C and FIG. 11). All the above
findings indicate that NPM-ALK induces malignant transformation of
normal human CD4+ T lymphocytes.
NPM-ALK Induces Malignant Transformation of Normal Human CD4+ T
Lymphocytes
[0187] The understanding of carcinogenesis has been facilitated by
development of various experimental models including tumor-derived
cell lines and oncogene-expressing transgenic mice. However, these
models have significant limitations. Only a handful of cell lines
exist for any given malignancy and they originate almost
exclusively from aggressive, clinically advanced tumors precluding
studies of the early stages of carcinogenesis and the mechanisms of
progression. Essentially all transgenic mouse models recapitulate
only some features of the human malignancies; this certainly is the
case in regard to the NPM-ALK transgenic mice. Efforts of several
research groups using different gene promoters resulted in
development of NPM-ALK-driven lymphomas but all these lymphomas are
of either diverse B-cell or immature T-cell origin (Kuefer et al.,
1997, Blood 90(8):2901-2910; Chiarle et al, 2003, Blood
101(5):1919-1927). None of the transgenic mouse models truly
recapitulates features of ALK+ALCL, a malignancy of mature CD4+ T
lymphocytes with highly distinct morphology and phenotype.
[0188] In vitro malignant transformation of normal human cells has
been a goal of cancer research for quite some time. The efforts to
recreate carcinogenesis in this manner were met with some success,
most notably by immortalizing B lymphocytes using an oncogenic
virus and epithelial cells using a combination of oncogenes. While
normal B lymphocytes can routinely be immortalized by the
Epstein-Barr virus (EBV) (Klein et al., 2010, Biochem. Biophys.
Res. Commun 396(1):67-73), the transformed cells resemble most
closely lymphoproliferative disorders seen in transplant patients
and other immunodeficient individuals rather than bona fide
lymphomas occurring in the population at large. Furthermore, EBV
genome contains almost 100 genes with expression of at least nine
members from the EBNA and LMP gene families being seemingly
critical to achieve the immortalization. Other investigations
successfully transformed normal human fibroblasts and primary
breast epithelial cells into neoplasms (Ince et al., 2007, Cancer
Cell 12(2):160-170; Elenbaas et al., 2001, Genes Dev 15(1):50-65;
Hahn et al., 1999, Nature 400(6743):464-468) using simultaneously
three separate retroviral vectors containing and genes encoding
hTERT unit of telomerase, H-ras oncogene, and simian virus SV40
early response region coding for large and small viral T antigens.
Hence, three, if not four, distinct genes were required to
accomplish the transformation. Perhaps more importantly, these gene
combinations and specifically the SV40 T antigen(s) have not been
unequivocally implicated in pathogenesis of the naturally occurring
human malignancies.
[0189] In this context, it is remarkable that the results presented
herein demonstrate the successful transformation of normal CD4+ T
lymphocytes using a single oncogene NPM-ALK and that the
transformed cells are morphologically and immunophenotypically
virtually indistinguishable from the patient-derived ALK+ALCL.
Previous studies have demonstrated that NPM-ALK tyrosine kinase is
a very powerful oncogene capable of activating several key cell
signaling pathways including STAT3 and mTORC1. (Li et al, 2007, Med
Res Rev 28(3):372-412; Wasik et al., 2009, Semin Oncol 36(2 Suppl
1):S27-35; Tabbo et al, 2012, Front Oncol 2:41; Zhang et al., 2002,
J Immunol 168(1):466-474; Marzec et al., 2007, Oncogene
26(38):5606-5614; Zamo et al., 2002, Oncogene 21(7):1038-1047).
Activation of these multiple pathways modulates expression of a
myriad of diverse genes which regulate the key oncogenic cell
functions including the sustained cell growth and evasion of immune
response. This pluripotency of ALK, a cell-surface receptor in its
native form, is the most likely cause of its ability to transform
the normal CD4+ T cells. In principle, other cell surface tyrosine
kinase receptors such as members of the EGF-R family or even the
IL-3R-mimicking cytoplasmic BCR-ABL, a very powerful oncogene in
its own right sharing a number of characteristics with NPM-ALK
(Shah et al., 2012, Blood 119(15):3374-3376), not to mention the
other oncogenic forms of ALK including ELM4-ALK (Li et al, 2007,
Med Res Rev 28(3):372-412; Wasik et al., 2009, Semin Oncol 36(2
Suppl 1):527-35; Tabbo et al, 2012, Front Oncol 2:41), should also
be able to effectively transform in vitro the normal cells they
target in vivo.
[0190] It is interesting that the NPM-ALK immortalized cells are
clonal, given the high transduction rate of CD4+ T cells (FIG. 5).
Similar phenomenon has also been noted by others in the multigene,
SV40 T antigen-based cell transformation system. (Ince et al.,
2007, Cancer Cell 12(2):160-170). At least two scenarios may be
considered to explain this phenomenon. First is that additional
genetic changes are required to achieve the transformation. On one
hand, the high success rate of immortalization achieved in several
separate experiments, normal karyotype of the NPM-ALK transformed
clones, and the young age of the ALK+ALCL patients argue to some
degree against this option. The recent observation that highly
malignant rhabdoid cancers in children display very few genetic
changes with a loss of a single gene SMARCB1 being the sole common
genomic lesion (Lee et al., 2012, J Clin Invest 122(8):2983-2988)
supports this conclusion. In contrast, the ability of NPM-ALK to
impair DNA repair resulting in increased mutation rate (Young et
al., 2011, Am J Pathol 179(1):411-421), suggest that the secondary
genetic changes may be required and can occur. The second scenario
is that only a small subset of the CD4+ T lymphocytes undergoes the
effective NPM-ALK-induced malignant transformation. CD4+ T cells
are phenotypically and functionally quite diverse so it is possible
that only their minor subset becomes transformed by NPM-ALK.
Accordingly, a recent study indicates a resemblance of ALK+ALCL
cells to the Th17 subset of the CD4+ T lymphocytes (Matsuyama et
al., 2011, Blood 118(26):6881-6892). Furthermore, ALK+ALCL may
contain a very minor stem cell population critical for their
development, as postulated recently for MALT B-cell lymphoma
(Vicente-Duenas et al., 2012, Proc Natl Acad Sci USA
109(26):10534-10539).
[0191] In summary, the results presented herein demonstrate that
the oncogenic tyrosine kinase NPM-ALK is capable of transforming in
vitro normal CD4+ T cells and conferring upon these cells
morphologic and immunophenotypic features characteristic of the
patient-derived ALK+ALCL cells and tissues. This study documents
effective malignant transformation of normal human cells by a
potent oncogene with the product cells faithfully recapitulating
malignant cells encountered in ALK+ALCL patients. The cells
generated by this manner should prove invaluable in studying the
early stages of oncogenesis and, possibly, the mechanisms of
progression. They should also be useful in evaluating anti-cancer
agents including ALK inhibitors that have already shown substantial
efficacy in ALK-driven malignancies (Kwak et al., 2010, N Engl J
Med 363:1693-1703; Gambacorti-Passerini et al., 2011, N Engl J Med
364(8):775-776).
[0192] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0193] While the invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
4118DNAArtificial SequencePrimer 1aaggcgcatg tgaactcc
18220DNAArtificial SequencePrimer 2aaggcattct tcacctgctc
20324DNAArtificial SequencePrimer 3tctccagaac atcatccctg cctc
24424DNAArtificial SequencePrimer 4tgggccatga ggtccaccac cctg
24
* * * * *