U.S. patent application number 09/737255 was filed with the patent office on 2002-06-13 for compositions and methods for detecting proteolytic activity.
Invention is credited to Rehemtulla, Alnawaz, Ross, Brian D..
Application Number | 20020073441 09/737255 |
Document ID | / |
Family ID | 24963189 |
Filed Date | 2002-06-13 |
United States Patent
Application |
20020073441 |
Kind Code |
A1 |
Ross, Brian D. ; et
al. |
June 13, 2002 |
Compositions and methods for detecting proteolytic activity
Abstract
The invention provides compositions and methods for non-invasive
imaging of enzyme (e.g., protease) activity in cells, tissues and
organs and entire bodies in vitro, in vivo and in situ. The
invention provides a chimeric polypeptide having a bioluminescent
or chemiluminescent polypeptide, or a heterologous kinase, and at
least one silencing moiety, and a protease cleavage motif
positioned between the first and second domains. The imaging can be
by computer assisted tomography (CAT), magnetic resonance
spectroscopy (MRS), magnetic resonance imaging (MRI), positron
emission tomography (PET), single-photon emission computed
tomography (SPECT), or bioluminescence imaging (BLI).
Inventors: |
Ross, Brian D.; (Ann Arbor,
MI) ; Rehemtulla, Alnawaz; (Plymouth, MI) |
Correspondence
Address: |
GREGORY P. EINHORN
Fish & Richardson P.C.
Suite 500
4350 La Jolla Village Drive
San Diego
CA
92122
US
|
Family ID: |
24963189 |
Appl. No.: |
09/737255 |
Filed: |
December 13, 2000 |
Current U.S.
Class: |
800/18 ; 435/189;
435/354; 435/4; 530/350; 536/23.2 |
Current CPC
Class: |
G01N 2333/96466
20130101; C12Q 1/37 20130101; G01N 2500/10 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
800/18 ; 435/4;
435/189; 536/23.2; 530/350; 435/354 |
International
Class: |
A01K 067/027; C12Q
001/00; C07H 021/04; C12N 009/02; C12N 005/06 |
Claims
What is claimed is:
1. A chimeric polypeptide comprising a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains.
2. The chimeric polypeptide of claim 1, wherein the
chemiluminescent polypeptide comprises luciferase.
3. The chimeric polypeptide of claim 1, wherein the bioluminescent
or chemiluminescent polypeptide comprises an aequorin, an obelin, a
ninemiopsin or a berovin.
4. The chimeric polypeptide of claim 1, wherein the bioluminescent
or chemiluminescent polypeptide comprises a green fluorescent
protein, an alpha-galactosidase or a chloramphenicol
acetyltransferase.
5. The chimeric polypeptide of claim 1, wherein the heterologous
kinase comprises a herpes simplex virus-1 thymidine kinase (HSV-1
TK).
6. The chimeric polypeptide of claim 1, wherein the silencing
moiety comprises a ligand binding domain.
7. The chimeric polypeptide of claim 6, wherein the ligand binding
domain comprises a steroid hormone receptor ligand binding
domain.
8. The chimeric polypeptide of claim 6, wherein the steroid hormone
receptor ligand binding domain comprises an estrogen receptor
ligand binding domain.
9. The chimeric polypeptide of claim 6, wherein the hormone
receptor is selected from the group consisting of glucocorticoid
receptor, progesterone receptor, androgen receptor,
mineralcorticoid receptor, thyroid hormone receptor, retinoic acid
receptor and RXR receptor.
10. The chimeric polypeptide of claim 1, wherein the silencing
moiety comprises a sequence of a transcription factor.
11. The chimeric polypeptide of claim 10, wherein the transcription
factor comprises an estrogen receptor ligand binding domain
polypeptide as set forth in SEQ ID NO:1.
12. The chimeric polypeptide of claim 1, wherein the endogenous
protease cleavage motif is specifically cleaved by an endogenous
cellular protease.
13. The chimeric polypeptide of claim 1, wherein the activity of
the endogenous cellular protease is increased or decreased during
apoptosis.
14. The chimeric polypeptide of claim 1, wherein the endogenous
cellular protease cleavage motif comprises a caspase recognition
motif.
15. The chimeric polypeptide of claim 14, wherein the caspase
recognition motif comprises an amino acid sequence selected from
group consisting of DEVD (SEQ ID NO:1), IETD (SEQ ID NO:2) and LEHD
(SEQ ID NO:3).
16. The chimeric polypeptide of claim 12, wherein the endogenous
cellular protease comprises caspase 3, caspase 6, caspase 7,
procaspase 8, caspase 8, caspase 9, caspase 10, matrix
metalloproteinase (MMP) or gamma-secretase.
17. The chimeric polypeptide of claim 1, wherein the endogenous
protease cleavage recognition motif comprises a PACE/furin cleavage
recognition motif.
18. The chimeric polypeptide of claim 1, wherein the endogenous
protease cleavage motif comprises a metalloprotease cleavage
recognition motif, a serine protease cleavage recognition motif, or
a gamma-secretase cleavage recognition motif.
19. The chimeric polypeptide of claim 1, wherein the endogenous
protease recognition motif further comprises at least one glycine
residue flanking the carboxy or amino terminal amino acid of the
motif.
20. The chimeric polypeptide of claim 1, further comprising a
cellular transport sequence.
21. The chimeric polypeptide of claim 20, wherein the cellular
transport sequence comprises a herpes simplex virus (HSV) VP-22
sequence.
22. The chimeric polypeptide of claim 20, wherein the cellular
transport sequence is located between the endogenous cleavage
protease motif and the first domain or between the endogenous
protease cleavage motif and the second domain.
23. The chimeric polypeptide of claim 1, wherein the polypeptide
comprises at least about 30 amino acids, at least about 50 amino
acids, at least about 100 amino acids or at least about 200 amino
acids.
24. The chimeric polypeptide of claim 1, wherein the polypeptide is
a recombinant fusion protein.
25. The chimeric polypeptide of claim 1, wherein the bioluminescent
or cbemiluminescent polypeptide, or the heterologous kinase, can
directly or by enzymatic reaction with a reagent generate a
molecule that can be imaged by computer assisted tomography (CAT),
magnetic resonance spectroscopy (MRS), magnetic resonance imaging
(MRI), positron emission tomography (PET), single-photon emission
computed tomography (SPECT), bioluminescence image (BLI) or
equivalent.
26. A chimeric polypeptide comprising a bioluminescent or
chemiluminescent polypeptide domain flanked on both sides by a
silencing moiety and an endogenous protease cleavage motif
positioned between the bioluminescent or chemiluminescent
polypeptide domain and each silencing moiety.
27. The chimeric polypeptide of claim 26, wherein the
chemiluminescent polypeptide is luciferase and the silencing domain
is an estrogen receptor ligand binding domain.
28. A chimeric polypeptide comprising a luciferase flanked on both
sides by an estrogen receptor ligand binding domain and an
endogenous protease cleavage motif positioned between the
luciferase and the estrogen receptor ligand binding domain.
29. A nucleic acid encoding a chimeric polypeptide comprising a
first domain comprising a bioluminescent or chemiluminescent
polypeptide, or a heterologous kinase, and a second domain
comprising at least one silencing moiety, and an endogenous
protease cleavage motif positioned between the first and second
domains.
30. An expression cassette comprising a nucleic acid encoding a
chimeric polypeptide comprising a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains.
31. A transformed host cell comprising a nucleic acid encoding a
chimeric polypeptide comprising a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains.
32. A non-human transgenic animal that expresses a chimeric
polypeptide comprising a first domain comprising a bioluminescent
or chemiluminescent polypeptide, or a heterologous kinase, and a
second domain comprising at least one silencing moiety, and an
endogenous protease cleavage motif positioned between the first and
second domains.
33. A non-human transgenic animal comprising a nucleic acid
encoding a chimeric polypeptide comprising a first domain
comprising a bioluminescent or chemiluminescent polypeptide, or a
heterologous kinase, and a second domain comprising at least one
silencing moiety, and an endogenous protease cleavage motif
positioned between the first and second domains.
34. The non-human transgenic animal of claim 33, wherein the animal
is a mouse or a rat.
35. A kit comprising a chimeric polypeptide or a nucleic acid and
instructions for use wherein the chimeric polypeptide comprises a
first domain comprising a bioluminescent or chemiluminescent
polypeptide, or a heterologous kinase, and a second domain
comprising at least one silencing moiety, and an endogenous
protease cleavage motif positioned between the first and second
domains wherein the nucleic acid encodes a chimeric polypeptide
comprising a first domain comprising a bioluminescent or
chemiluminescent polypeptide, or a heterologous kinase, and a
second domain comprising at least one silencing moiety, and an
endogenous protease cleavage motif positioned between the first and
second domains.
36. The kit of claim 35, further comprising a substrate for the
bioluminescent or chemiluminescent polypeptide or the heterologous
kinase.
37. The kit of claim 35, wherein the instructions are for measuring
apoptosis in a living subject.
38. A pharmaceutical formulation comprising a chimeric polypeptide
and a pharmaceutically acceptable excipient suitable, wherein the
chimeric polypeptide comprises a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains.
39. The pharmaceutical composition of claim 38, further comprising
a substrate for the bioluminescent or the chemiluminescent
polypeptide or the heterologous kinase.
40. The pharmaceutical composition of claim 39, wherein the
chemiluminescent polypeptide is luciferase and the substrate is
luciferin.
41. The pharmaceutical composition of claim 39, wherein the
heterologous kinase is 8-[18F] fluoroganciclovir (FGCV) and the
substrate is a herpes simplex virus-1 thymidine kinase (HSV-1
TK).
42. The pharmaceutical composition of claim 38, further comprising
a liposome.
43. A method for detecting a protease activity in a sample
comprising (a) contacting a polypeptide as set forth in claim 1
with a sample containing or suspected of containing a protease
under conditions allowing cleavage of the endogenous protease
cleavage motif, wherein the protease is capable of cleaving the
endogenous protease cleavage motif; and (b) detecting the amount of
bioluminescent or a chemiluminescent signal in the sample, thereby
detecting a protease activity in the sample.
44. The method of claim 43, wherein the contacting is in solution,
in solid phase or is in a cell in vitro, in situ, or in vivo.
45. The method of claim 43, wherein the endogenous protease
specifically cleaves the endogenous protease cleavage motif.
46. The method of claim 45, wherein the endogenous cellular
protease comprises a caspase.
47. The method of claim 43, wherein the endogenous cellular
protease comprises a caspase 3, a caspase 6, a caspase 7, a
procaspase 8, a caspase 8, a caspase 9, a caspase 10, a matrix
metalloproteinase (MMP) or a gamma-secretase.
48. The method of claim 43, further comprising providing a
substrate for the bioluminescent or chemiluminescent polypeptide,
or the heterologous kinase, and administering the substrate before,
with or after administration of the bioluminescent or
chemiluminescent polypeptide or the heterologous kinase.
49. The method of claim 48, wherein the chemiluminescent
polypeptide is luciferase and the substrate is luciferin.
50. The method of claim 48, wherein the heterologous kinase is
8-[18F] fluoroganciclovir (FGCV) and the substrate is a herpes
simplex virus-1 thymidine kinase (HSV-1 TK).
51. A method for identifying the presence of a caspase in a sample
comprising (a) contacting a polypeptide with a sample under
conditions allowing cleavage of the endogenous protease cleavage
motif, wherein the polypeptide comprises a chimeric polypeptide
comprising a first domain comprising a bioluminescent or
chemiluminescent polypeptide, or a heterologous kinase, and a
second domain comprising at least one silencing moiety, and an
endogenous protease cleavage motif positioned between the first and
second domains, and, the endogenous protease cleavage motif is a
cleavage motif specific for the caspase; and (b) detecting the
amount of bioluminescent or a chemiluminescent signal in the
sample, thereby identifying the presence of the caspase.
52. A method for detecting apoptosis in a cell comprising (a)
contacting a polypeptide with the cell under conditions allowing
cleavage of the endogenous protease cleavage motif by a cellular
enzyme, wherein the polypeptide comprises a chimeric polypeptide
comprising a first domain comprising a bioluminescent or
chemiluminescent polypeptide, or a heterologous kinase, and a
second domain comprising at least one silencing moiety, and an
endogenous protease cleavage motif positioned between the first and
second domains, and, the endogenous protease cleavage motif is a
cleavage motif specific for an enzyme associated with apoptosis;
and (b) detecting the amount of bioluminescent or chemiluminescent
signal in the cell, thereby identifying the presence and activity
of the apoptosis-associated enzyme and detecting apoptosis.
53. The method of claim 52, wherein the contacting is in a cell, a
tissue, an organ or an entire body in vitro, in situ, or in
vivo.
54. The method of claim 53, wherein the cell, tissue or organ is
undergoing aberrant proliferation or aberrant degeneration.
55. The method of claim 53, wherein the aberrant proliferation
comprises hyperproliferation.
56. The method of claim 53, wherein the cell comprises a benign or
a metastatic tumor.
57. The method of claim 53, wherein the cell comprises a solid
tumor.
58. The method of claim 53, wherein the aberrant proliferation
comprises deficient proliferation.
59. The method of claim 53, wherein the aberrant degeneration
comprises decreased cell death or increased cell death.
60. The method of claim 53, wherein the amount of bioluminescent or
chemiluminescent signal in the cell can be imaged by computer
assisted tomography (CAT), magnetic resonance spectroscopy (MRS),
magnetic resonance imaging (MRI), positron emission tomography
(PET), single-photon emission computed tomography (SPECT),
bioluminescence image (BLI) or equivalent.
61. A method for detecting changes in apoptosis in a cell
comprising: a) contacting a polypeptide with the cell under
conditions allowing cleavage of the endogenous protease cleavage
motif by a cellular enzyme, wherein the polypeptide comprises a
chimeric polypeptide comprising a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains, and, the endogenous protease
cleavage motif is a cleavage motif specific for an enzyme
associated with apoptosis; and b) detecting any change in an amount
of bioluminescent or chemiluminescent signal in the cell, thereby
identifying the presence and activity of the apoptosis-associated
enzyme and detecting a change in cell apoptosis.
62. The method of claim 61, wherein the apoptosis-associated enzyme
comprises a caspase.
63. The method of claim 61, wherein the contacting is in a cell, a
tissue, an organ or an entire body in vitro, in situ, or in
vivo.
64. The method of claim 63, wherein the tissue comprises breast,
brain, head or neck, eye, nasopharynx, lung, liver, pancreas,
kidney, esophagus, stomach, small or large intestine, bladder,
rectum, prostate, testicle, ovary, uterus, bone, muscle, skin or
blood.
65. The method of claim 63, wherein the cell comprises a subject
undergoing a therapy that increases or decreases cell proliferation
or cell death.
66. The method of claim 65, wherein the subject has or is at risk
of having a cell proliferative disorder or a cell degenerative
disorder.
67. The method of claim 66, wherein the cell proliferative disorder
comprises cell hyperplasia.
68. The method of claim 67, wherein the cell hyperplasia comprises
a cancer.
69. The method of claim 68, wherein the cancer is a lymphoma, a
myeloma or a leukemia.
70. The method of claim 68, wherein the cancer is a solid
tumor.
71. The method of claim 68, wherein the cancer is a metastatic
tumor.
72. The method of claim 68, wherein the cancer comprises a sarcoma
or fibrosarcoma.
73. The method of claim 68, wherein the cancer is a glioma or
neuroblastoma.
74. The method of claim 66, wherein the cell degenerative disorder
is produced by a stroke, a heart attack or a partial or complete
arterial obstruction.
75. The method of claim 66, wherein the cell degenerative disorder
afflicts neural tissue, muscular tissue, cardiac tissue or bone
marrow cells.
76. The method of claim 66, wherein the cell degenerative disorder
is dementia, Alzheimer's disease, Parkinson's disease, ALS,
Huntington's disease, MachadoJoseph disease, spino-cerebellar
ataxias, Kennedy's disease, muscular dystrophy, multiple sclerosis,
beta-thalasemia, sickle cell anemia, aplastic anemia,
ischemia/reperfusion injury, rheumatoid arthritis or graft versus
host disease.
77. A method for monitoring the effectiveness of a therapy that
modulates cell proliferation or cell survival in a subject
comprising: a) contacting a cell, a tissue or an organ in the
subject, before therapy, with a polypeptide, under conditions
allowing cleavage of the polypeptide by an endogenous cell protease
associated with cell proliferation or cell survival, wherein the
polypeptide comprises a chimeric polypeptide comprising a first
domain comprising a bioluminescent or chemiluminescent polypeptide,
or a heterologous kinase, and a second domain comprising at least
one silencing moiety, and an endogenous protease cleavage motif
positioned between the first and second domains, and, the
endogenous protease cleavage motif is a cleavage motif specific for
an enzyme associated with cell proliferation or cell survival; b)
detecting the amount of bioluminescent or chemiluminescent signal
in the cell, tissue or organ, thereby identifying the presence and
activity of the cell proliferation- or cell survival-associated
enzyme; c) administering a therapy to the subject; and d) detecting
any change in the amount of bioluminescent or chemiluminescent
signal in the cell, tissue or organ after the therapy, wherein a
change in the amount of bioluminescent or chemiluminescent signal
after the therapy indicates the amount of cell proliferation or
cell survival, thereby indicating the effectiveness of the
therapy.
78. The method of claim 77, further comprising re-contacting the
cell, tissue or organ with the polypeptide as set forth in claim 1
after administering the therapy to the subject.
79. The method of claim 77, wherein effectiveness of the therapy is
indicated by an increase in the bioluminescent or chemiluminescent
signal after the therapy.
80. The method of claim 77, wherein effectiveness of the therapy is
indicated by a decrease in the bioluminescent or chemiluminescent
signal after the therapy.
81. The method of claim 77, wherein the therapy inhibits cell
proliferation.
82. The method of claim 77, wherein the therapy stimulates cell
death.
83. The method of claim 77, wherein the therapy stimulates cell
proliferation.
84. The method of claim 77, wherein the therapy inhibits cell
death.
85. The method of claim 77, wherein the therapy comprises
anti-cancer therapy.
86. The method of claim 77, wherein the therapy comprises
chemotherapy or radiation therapy.
87. The method of claim 77, wherein the therapy comprises a
treatment for a cell degenerative disorder.
88. The method of claim 87, wherein the cell degenerative disorder
afflicts neural tissue, muscle tissue, cardiac tissue or bone
marrow cells.
89. The method of claim 77, wherein the therapy comprises a
treatment for ischemia.
90. The method of claim 77, wherein the therapy comprises a
treatment for stroke, a heart attack or a partial or complete
arterial obstruction.
91. The method of claim 77, wherein the therapy comprises a
treatment for dementia, Alzheimer's disease, Parkinson's disease,
ALS, Huntington's disease, Machado-Joseph disease, spino-cerebellar
ataxias, Kennedy's disease, muscular dystrophy, multiple sclerosis,
beta-thalasemia, sickle cell anemia, aplastic anemia,
ischemia/reperfusion injury, rheumatoid arthritis and graft versus
host disease.
92. A computer-implemented method for monitoring relative
effectiveness of a therapy that modulates cell proliferation or
cell survival in a subject comprising: (a) providing an imaging
device in operable association with a computer, wherein the imaging
device is a computer assisted tomography (CAT) device, a magnetic
resonance spectroscopy (MRS) device, a magnetic resonance imaging
(MRI) device, a positron emission tomography (PET) device, a
single-photon emission computed tomography (SPECT) device, a
bioluminescence imaging (BLI) device or equivalent; (b) taking an
image of a defined area of the subject before or during therapy in
which the area has been contacted with a polypeptide to image the
amount of bioluminescent or chemiluminescent signal, wherein the
endogenous protease cleavage motif is an endogenous cleavage motif
specific for an enzyme associated with cell proliferation or cell
survival, and the polypeptide comprises a chimeric polypeptide
comprising a first domain comprising a bioluminescent or
chemiluminescent polypeptide, or a heterologous kinase, and a
second domain comprising at least one silencing moiety, and an
endogenous protease cleavage motif positioned between the first and
second domains; (c) outputting the image data to the computer; (d)
administering a therapy to the subject and again imaging the
defined area; (e) comparing the image data obtained in step (b)
with the image data obtained in step (d) with the computer to
generate a differential histogram; and (f) analyzing the
differential histogram to quantitate any change in bioluminescent
or chemiluminescent signal after therapy, wherein a change in the
amount or activity of the bioluminescent or cbemiluminescent signal
after the therapy indicates a relative effectiveness of the therapy
in modulating cell proliferation or cell survival.
93. A method for identifying an agent that modulates an enzyme
activity comprising: (a) contacting a sample comprising the enzyme
with a polypeptide in the presence and absence of a test agent,
wherein the endogenous cleavage motif is an endogenous cleavage
motif specific for the enzyme, wherein the polypeptide comprises a
chimeric polypeptide comprising a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains, and; (b) measuring the amount
of bioluminescent or chemiluminescent signal in the sample after
adding the polypeptide and in the presence and absence of the test
agent, wherein an increase or decrease in the amount or activity of
the bioluminescent or chemiluminescent signal in the presence of
the test agent identifies the test agent as a modulator of the
enzyme's activity.
94. The method of claim 93, wherein the contacting occurs in
solution or in solid phase.
95. The method of claim 93, wherein the contacting occurs in a
cell.
96. The method of claim 93, wherein the cell comprises a tissue, an
organ or an entire body.
97. The method of claim 95, wherein the cell comprises a non-human
transgenic animal.
98. The method of claim 95, wherein the non-human transgenic animal
is a mouse or a rat.
Description
TECHNICAL FIELD
[0001] This invention generally pertains to the fields of medicine
and non-invasive imaging. The invention provides compositions and
methods for non-invasive imaging of enzyme (e.g., protease)
activity in cells, tissues, organs and entire bodies in vitro, in
vivo and in situ. The imaging can be by computer assisted
tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic
resonance imaging (MRI), positron emission tomography (PET),
single-photon emission computed tomography (SPECT), or
bioluminescence imaging (BLI).
BACKGROUND
[0002] The revolution in molecular biology along with the nearly
completed mapping of the human genome provides an unprecedented
opportunity to transform our understanding and treatment of human
diseases. Paralleling these discoveries, the imaging sciences have
made remarkable advances and have reached a stage in which anatomic
and functional imaging can be achieved in vivo at submillimeter
resolution in both animals and humans. These developments have
created a historic opportunity to non-invasively probe cellular and
molecular events associated with the ever-expanding myriad of newly
identified pathology-related genes and proteins in vivo in both
animals and humans. The revolution in molecular biology has
expanded our understanding of the genetics and biochemistry of
transformed cells. These tremendous advances have been made largely
through studies of cultured cells or ex vivo studies on tumor
specimens. However, it is clear that extrapolations between in
vitro and in vivo situations do not always hold true. Their exists
a significant opportunity to bridge this great divide between in
vitro and in vivo studies in research, e.g., cancer therapies,
through the development of novel molecular imaging approaches.
[0003] For example, research would be aided by in vitro and in vivo
imaging of apoptosis, or programmed cell death. Apoptosis is a
physiologic process important in the normal development and
homeostasis of multicellular organisms. The molecular components
comprising the cell death machinery have been identified. Apoptosis
can be physiologically activated by the activation of death
receptors (Fas, TNFR, DR4, DR5 etc) or when a cell undergoes
stress. Growth factor withdrawal, environmental conditions that
damage mitochondrial function or homeostasis, DNA damaging events,
hypoxia, heat, cold and chemical injury, result in activation of
apoptosis.
[0004] Lack of balance between apoptosis and proliferation has been
implicated in a wide variety of pathologic conditions including
stroke, dementia, bone marrow diseases and cancer. In stroke, death
of white and gray matter by apoptosis plays a central role in
hypoxic-ischemic injury in adults. Because the actual loss of cells
in these patients is gradual there may be a therapeutic window
wherein pharmacological inhibition of apoptosis may prevent the
long term debilitating effects of a stroke. In dementia, neuronal
and glial cell apoptosis occurs in AIDS, encephalitis, and in
neurodegenerative disorders such as Alzheimer's disease,
Parkinson's disease and ALS. The gradual loss of white and gray
matter in these disorders is primarily due to apoptosis. In bone
marrow diseases, beta-thalasemia, sickle cell disease and aplastic
anemia are diseases in which excessive apoptosis within the bone
marrow is the central cause of the pathology. The ability to image
apoptosis within the marrow would not only facilitate diagnosis of
these disorders but would also provide a direct measure of
therapeutic efficacy of experimental drugs. Cancer is as much a
disease of cell death as one of cell proliferation (see, e.g.,
Korsmeyer (1990) Curr. Top.
[0005] Microbiol. Immunol. 166:203). For example, constitutive
activation of the anti-apoptotic gene bcl-2 leads to B-cell
lymphoma (see, e.g., Holgren (1995) Nature Med. 1:149). It is
believed that mutations that attenuate apoptotic responses
facilitate neoplastic transformation. The mutations may be allowing
the accumulation of other growth-promoting mutations that would
otherwise commit a cell to suicide in the absence of external
growth cues. Tumor progression may also exert a selective pressure
for cells resistant to apoptosis. Evolution toward a "survivor"
phenotype may be a product of the hypoxia, nutrient starvation, and
falling pH that may be produced as tumor cells outgrow their blood
supply. The selective pressure may be especially strong just prior
to the "angiogenic switch" when dormant tumor growth is thought to
be the result of balanced cellular proliferation and apoptosis
(see, e.g., Holgren (1995) supra). The resulting defects in the
apoptotic response of tumor cells arising from early events in
carcinogenesis or as a result of selective pressures are also
thought to contribute to the resistance of tumor cells to cytotoxic
therapies.
[0006] Currently available techniques for studying apoptosis in
vivo, such as in solid tumors, make it difficult to study these
problems. Scoring apoptotic indices by morphological criteria is
time consuming and requires skilled observers. Specific staining of
apoptotic cells, such as the TUNEL method for marking the 3'
termini of cleaved DNA, is also time consuming and may have a
significant false-positive rate.
SUMMARY
[0007] The invention provides compositions and methods for
non-invasive imaging of enzyme (e.g., protease) activity in cells,
tissues and organs and entire bodies in vitro, in vivo and in situ.
Because many enzymes and proteases are specifically associated with
certain normal and abnormal conditions and diseases, such as
apoptosis and cancer, in vitro, in vivo and in situ imaging of
protease activity is useful for identification, targeting,
diagnosis and the like. The imaging can be by computer assisted
tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic
resonance imaging (MRI), positron emission tomography (PET),
single-photon emission computed tomography (SPECT), or
bioluminescence imaging (BLI).
[0008] The invention provides a chimeric polypeptide comprising a
first domain comprising a bioluminescent or chemiluminescent
polypeptide, or a heterologous kinase, and a second domain
comprising at least one silencing moiety, and an endogenous
protease cleavage motif positioned between the first and second
domains. The ability of the bioluminescent or chemiluminescent
polypeptide, or heterologous kinase, to directly or indirectly
(e.g., by action on a substrate) generate a signal readable by CAT,
MRS, PET, SPECT or BLI or equivalent is completely or substantially
suppressed by the "silencing" or "quenching" moiety. Upon cleavage
of the endogenous protease cleavage motif, the "silencing" or
"quenching" domain no longer suppresses the ability of the
bioluminescent or chemiluminescent polypeptide to directly or
indirectly generate a readable signal (e.g., by the physical
separation of the first and second domains). Accordingly, if an
endogenous protease cleavage motif is specific for a particular
enzyme (e.g., an endogenous cellular protease), or class of
enzymes, then after administration of the chimeric polypeptide of
the invention, generation of a readable image indicates the
presence of that enzyme in active form.
[0009] The invention provides a chimeric polypeptide comprising a
first domain comprising a bioluminescent or chemiluminescent
polypeptide, or a heterologous kinase, and a second domain
comprising at least one silencing moiety, and an endogenous
protease cleavage motif positioned between the first and second
domains. In various aspects of the chimeric polypeptide of the
invention, the chemiluminescent polypeptide comprises luciferase,
aequorin, obelin, mnemiopsin or berovin, or equivalents thereof.
The bioluminescent or chemiluminescent polypeptide can comprise a
green fluorescent protein, an alpha-galactosidase or a
chloramphenicol acetyltransferase. The heterologous kinase can
comprise a herpes simplex virus-1 thymidine kinase (HSV-1 TK), or
equivalents thereof.
[0010] In one aspect of the chimeric polypeptide of the invention,
the silencing moiety comprises a ligand binding domain, such as a
steroid hormone receptor ligand binding domain (e.g., a
transcription factor), e.g., an estrogen receptor ligand binding
domain, such as one derived from a mouse, e.g., a polypeptide
comprising a sequence as set forth in SEQ ID NO:4. The hormone
receptor can also be a glucocorticoid receptor, a progesterone
receptor, an androgen receptor, a mineralcorticoid receptor, a
thyroid hormone receptor, a retinoic acid receptor or a retinoid X
receptor ("RXR receptor").
[0011] In one aspect of the chimeric polypeptide of the invention,
the endogenous protease cleavage motif is specifically cleaved by
an endogenous cellular protease. In one aspect, the activity of the
endogenous cellular protease is increased or decreased during
apoptosis (thus, the rate of digestion, or cleavage, of the
endogenous protease cleavage motif by the protease is increased or
decreased during apoptosis). The endogenous cellular protease
cleavage motif can comprise a caspase recognition motif, including
any of the known caspases, e.g., caspases 1 through 10. For
example, in one aspect the endogenous cellular protease can
comprise caspase 3, caspase 6, caspase 7, procaspase 8, caspase 8,
caspase 9, caspase 10. The caspase recognition motif can comprise
an amino acid sequence selected from group consisting of DEVD (SEQ
ID NO:1), IETD (SEQ ID NO:2) and LEHD (SEQ ID NO:3). The endogenous
cellular protease can comprise matrix metalloproteinase (MMP) or
gamma-secretase. The endogenous protease cleavage recognition motif
can comprise a PACE/furin cleavage recognition motif. The
endogenous protease cleavage motif can comprise a metalloprotease
cleavage recognition motif, a serine protease cleavage recognition
motif, or a gamma-secretase cleavage recognition motif. The
endogenous protease recognition motif can further comprise at least
one glycine residue flanking the carboxy or amino terminal amino
acid of the motif, or both.
[0012] In one aspect, the chimeric polypeptide of the invention
further comprises a cellular transport sequence. The cellular
transport sequence can comprise a herpes simplex virus (HSV) VP-22
sequence (see, e.g., U.S. Pat. No. 6,017,735), or equivalent. The
cellular transport sequence can be located between the endogenous
protease cleavage motif and the first domain or between the
endogenous protease cleavage motif and the second domain.
[0013] In one aspect, the chimeric polypeptide of the invention
comprises at least about 30 amino acids, at least about 50 amino
acids, at least about 100 amino acids or at least about 200 amino
acids. The chimeric polypeptide of the invention can be a
recombinant fusion protein.
[0014] In one aspect, the bioluminescent or chemiluminescent, or
the heterologous kinase, domain of the chimeric polypeptide can
directly, or by enzymatic reaction with a reagent, generate a
molecule that can be imaged, for example, by computer assisted
tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic
resonance imaging (MRI), positron emission tomography (PET),
single-photon emission computed tomography (SPECT), bioluminescence
image (BLI) or equivalent.
[0015] In one aspect, the bioluminescent or chemiluminescent
polypeptide domain is flanked on both sides (i.e., carboxy and
amino terminal sides) by a silencing moiety and an endogenous
protease cleavage motif is positioned between the bioluminescent or
chemiluminescent polypeptide domain and each silencing moiety.
[0016] In one aspect, the chemiluminescent polypeptide is
luciferase and the silencing domain is an estrogen receptor ligand
binding domain. In one aspect, the chimeric polypeptide comprises a
luciferase flanked on both sides by an estrogen receptor ligand
binding domain and an endogenous protease cleavage motif positioned
between the luciferase and the estrogen receptor ligand binding
domain.
[0017] The invention provides a nucleic acid encoding a chimeric
polypeptide of the invention. The invention provides a vector
comprising a nucleic acid encoding a polypeptide of the invention.
The invention provides a transformed or infected host cell
comprising a nucleic acid (or expression cassette, e.g., a
vector).
[0018] The invention provides a non-human transgenic animal that
expresses a chimeric polypeptide comprising a first domain
comprising a bioluminescent or chemiluminescent polypeptide, or a
heterologous kinase, and a second domain comprising at least one
silencing moiety, and an endogenous protease cleavage motif
positioned between the first and second domains.
[0019] The invention provides a kit comprising the chimeric
polypeptide of the invention or a nucleic acid of the invention
(including, e.g., a vector) and instructions for use. The kit can
further comprise a substrate for the bioluminescent or
chemiluminescent polypeptide or the heterologous kinase. The kit
can further comprise instructions for measuring apoptosis in a
living subject.
[0020] The invention provides a pharmaceutical formulation
comprising a chimeric polypeptide and a pharmaceutically acceptable
excipient suitable, wherein the chimeric polypeptide comprises a
first domain comprising a bioluminescent or chemiluminescent
polypeptide, or a heterologous kinase, and a second domain
comprising at least one silencing moiety, and an endogenous
protease cleavage motif positioned between the first and second
domains. The pharmaceutical composition can further comprise a
substrate for the bioluminescent or the chemiluminescent
polypeptide or the heterologous kinase. In one aspect of the
pharmaceutical composition, the chemiluminescent polypeptide is
luciferase and the substrate is luciferin. The heterologous kinase
can be 8-[18F] fluoroganciclovir (FGCV) and the substrate a herpes
simplex virus-I thymidine kinase (HSV-1 TK).
[0021] The pharmaceutical composition of the invention can further
comprise a liposome or other detergent or lipid. A substrate for a
bioluminescent or a chemiluminescent enzyme can be administered in
the same lipid vehicle (e.g., liposome) as the bioluminescent or
the chemiluminescent polypeptide, or, it can be administered
separately.
[0022] The invention provides a method for detecting a protease
activity in a sample comprising: (a) contacting a polypeptide with
a sample containing or suspected of containing a protease under
conditions allowing cleavage of the endogenous protease cleavage
motif, wherein the protease is capable of cleaving the endogenous
protease cleavage motif, wherein the polypeptide comprises a
chimeric polypeptide comprising a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains; and, (b) detecting the amount
of bioluminescent or a chemiluminescent signal in the sample,
thereby detecting a protease activity in the sample. The contacting
can be in solution, in solid phase or is in a cell in vitro, in
situ, or in vivo. For this and other methods of the invention, the
endogenous cellular protease can specifically cleave the endogenous
protease cleavage motif. The endogenous cellular protease can
comprise a caspase. In one aspect, the endogenous cellular protease
comprises a caspase 3, a caspase 6, a caspase 7, a procaspase 8, a
caspase 8, a caspase 9, or a caspase 10. In one aspect, the
endogenous cellular protease comprises a matrix metalloproteinase
(MMP) or a gamma-secretase.
[0023] The invention provides a method further comprising providing
a substrate for the bioluminescent or chemiluminescent polypeptide,
or the heterologous kinase, and administering the substrate before,
with or after administration of the bioluminescent or
chemiluminescent polypeptide or the heterologous kinase. In one
aspect, the chemiluminescent polypeptide is luciferase and the
substrate is luciferin. The heterologous kinase can be 8-[18F]
fluoroganciclovir (FGCV) and the substrate can be a herpes simplex
virus-1 thymidine kinase (HSV-1 TK).
[0024] The invention provides a method for identifying the presence
of a caspase in a sample comprising (a) contacting a polypeptide
with a sample under conditions allowing cleavage of the endogenous
protease cleavage motif, wherein the polypeptide comprises a
chimeric polypeptide comprising a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains, and, the endogenous protease
cleavage motif is a cleavage motif specific for the caspase; and,
(b) detecting the amount of bioluminescent or a chemiluminescent
signal in the sample, thereby identifying the presence of the
caspase.
[0025] The invention provides a method for detecting apoptosis in a
cell comprising (a) contacting a polypeptide with the cell under
conditions allowing cleavage of the endogenous protease cleavage
motif by a cellular enzyme, wherein the polypeptide comprises a
chimeric polypeptide comprising a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains, and the endogenous protease
cleavage motif is a cleavage motif specific for an enzyme
associated with apoptosis; and, (b) detecting the amount of
bioluminescent or chemiluminescent signal in the cell, thereby
identifying the presence and activity of the apoptosis-associated
enzyme and detecting apoptosis. In this, as in all methods of the
invention, the contacting can be in a cell, a tissue, an organ or
an entire body in vitro, in situ, or in vivo. In alternative
aspects, the cell, tissue or organ is undergoing aberrant
proliferation or aberrant degeneration. The aberrant proliferation
can comprise hyperproliferation. In one aspect, the cell comprises
a benign or a metastatic tumor, or a solid tumor.
[0026] In one aspect, the aberrant proliferation can comprise
deficient proliferation. The aberrant degeneration can comprise
decreased cell death or increased cell death.
[0027] In the methods of the invention, the amount of
bioluminescent or chemiluminescent signal in a cell or tissue or
organ or entire body can be imaged by computer assisted tomography
(CAT), magnetic resonance spectroscopy (MRS), magnetic resonance
imaging (MRI), positron emission tomography (PET), single-photon
emission computed tomography (SPECT), bioluminescence image (BLI)
or equivalent.
[0028] The invention provides a method for detecting apoptosis, or,
changes in apoptosis, in a cell (or tissue) comprising: (a)
contacting a polypeptide with the cell under conditions allowing
cleavage of the endogenous protease cleavage motif by a cellular
enzyme, wherein the polypeptide comprises a chimeric polypeptide
comprising a first domain comprising a bioluminescent or
chemiluminescent polypeptide, or a heterologous kinase, and a
second domain comprising at least one silencing moiety, and an
endogenous protease cleavage motif positioned between the first and
second domains, and the endogenous protease cleavage motif is a
cleavage motif specific for an enzyme associated with apoptosis;
and, (b) detecting any change in an amount of bioluminescent or
chemiluminescent signal in the cell, thereby identifying the
presence and activity of the apoptosis-associated enzyme and
detecting a change in cell apoptosis. In one aspect, the
apoptosis-associated enzyme comprises a caspase, e.g., caspase 3 or
caspase 9.
[0029] As in all methods of the invention, the contacting can be in
a cell, a tissue, an organ or an entire body in vitro, in situ, or
in vivo. In one aspect, the tissue can comprise breast, brain, head
or neck, eye, nasopharynx, lung, liver, pancreas, kidney,
esophagus, stomach, small or large intestine, bladder, rectum,
prostate, testicle, ovary, uterus, bone, muscle, skin or blood. The
cell can comprise a subject undergoing a therapy that increases or
decreases cell proliferation or cell death. In one aspect, the
subject has or is at risk of having a cell proliferative disorder
or a cell degenerative disorder. The cell proliferative disorder
can comprise a cell hyperplasia, such as a cancer, e.g., a
lymphoma, a myeloma or a leukemia, a solid tumor, a metastatic
tumor, a sarcoma or fibrosarcoma, a glioma or a neuroblastoma.
[0030] In one aspect, the cell degenerative disorder is produced by
a stroke, a heart attack or a partial or complete arterial
obstruction. The cell degenerative disorder can afflicts neural
tissue, muscular tissue, cardiac tissue or bone marrow cells. The
cell degenerative disorder can be dementia, Alzheimer's disease,
Parkinson's disease, ALS, Huntington's disease, Machado-Joseph
disease, spino-cerebellar ataxias, Kennedy's disease, muscular
dystrophy, multiple sclerosis, beta-thalasemia, sickle cell anemia,
aplastic anemia, ischemia/reperfusion injury, rheumatoid arthritis
or graft versus host disease.
[0031] The invention provides a method for monitoring the
effectiveness of a therapy that modulates cell proliferation or
cell survival in a subject comprising: (a) contacting a cell, a
tissue or an organ in the subject, before therapy, with a
polypeptide, under conditions allowing cleavage of the polypeptide
by an endogenous cellular protease associated with cell
proliferation or cell survival, wherein the polypeptide comprises a
chimeric polypeptide comprising a first domain comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a second domain comprising at least one silencing
moiety, and an endogenous protease cleavage motif positioned
between the first and second domains, and the endogenous protease
cleavage motif is a cleavage motif specific for an enzyme
associated with cell proliferation or cell survival; (b) detecting
the amount of bioluminescent or chemiluminescent signal in the
cell, tissue or organ, thereby identifying the presence and
activity of the cell proliferation- or cell survival -associated
enzyme; (c) administering a therapy to the subject; and, (d)
detecting any change in the amount of bioluminescent or
chemiluminescent signal in the cell, tissue or organ after the
therapy, wherein a change in the amount of bioluminescent or
chemiluminescent signal after the therapy indicates the amount of
cell proliferation or cell survival, thereby indicating the
effectiveness of the therapy.
[0032] In one aspect, the methods of the invention further comprise
re-contacting the cell, tissue or organ with a polypeptide of the
invention after administering the therapy to the subject. The
effectiveness of the therapy is indicated by an increase, a
decrease, in the bioluminescent or chemiluminescent signal after
the therapy; thus allowing detection of apoptosis, or, changes in
rates of apoptosis or locations of apoptotic events, in cells or
tissues. In the methods of the invention, the therapy can inhibit
cell proliferation, or the therapy can stimulate cell death, or the
therapy can stimulate cell proliferation, or the therapy can
inhibit cell death. In the methods of the invention, the therapy
can comprise anti-cancer therapy. The therapy can comprise
chemotherapy or radiation therapy. The therapy can comprise
treatment (i.e., amelioration) of a cell degenerative disorder,
such as a disorder afflicting neural tissue, muscle tissue, cardiac
tissue or bone marrow cells. The therapy can comprise treatment
(i.e., amelioration) of ischemia, stroke, a heart attack or a
partial or complete arterial obstruction, or dementia, Alzheimer's
disease, Parkinson's disease, ALS, Huntington's disease,
Machado-Joseph disease, spino-cerebellar ataxias, Kennedy's
disease, muscular dystrophy, multiple sclerosis, beta-thalasemia,
sickle cell anemia, aplastic anemia, ischemia/reperfusion injury,
rheumatoid arthritis and graft versus host disease.
[0033] The invention provides a computer-implemented method for
monitoring relative effectiveness of a therapy that modulates cell
proliferation or cell survival in a subject comprising: (a)
providing an imaging device in operable association with a
computer, (b) taking an image of a defined area of the subject
before or during therapy in which the area has been contacted with
a polypeptide of the invention to image the amount of
bioluminescent or chemiluminescent signal, wherein the endogenous
protease cleavage motif of the polypeptide is an endogenous
cleavage motif specific for an enzyme associated with cell
proliferation or cell survival; (c) outputting the image data to
the computer; (d) administering a therapy to the subject and taking
another image of the defined area; (e) comparing the image data
obtained in step (b) with the image data obtained in step (d) with
the computer to generate a differential histogram; and (g)
analyzing the differential histogram to quantitate any change in
bioluminescent or chemiluminescent signal after therapy, wherein a
change in the amount or activity of the bioluminescent or
chemiluminescent signal after the therapy indicates a relative
effectiveness (or ineffectiveness) of the therapy in modulating
cell proliferation or cell survival. The imaging device can be a
computer assisted tomography (CAT) device, a magnetic resonance
spectroscopy (MRS) device, a magnetic resonance imaging (MRI)
device, a positron emission tomography (PET) device, a
single-photon emission computed tomography (SPECT) device, a
bioluminescence imaging (BLI) device or equivalent. The polypeptide
of the invention (and, if appropriate, its substrate) can be
administered two or more (several) times, e.g., it can be
re-administered several times before a first imaging or after a
first imaging.
[0034] The invention provides a method for identifying an agent
that modulates an enzyme activity comprising: (a) contacting a
sample comprising the enzyme with a polypeptide of the invention in
the presence and absence of a test agent, wherein the endogenous
cleavage motif is an endogenous cleavage motif specific for the
enzyme; and, (b) measuring the amount of bioluminescent or
chemiluminescent signal in the sample after adding the polypeptide
and in the presence and absence of the test agent, wherein an
increase or decrease in the amount or activity of the
bioluminescent or chemiluminescent signal in the presence of the
test agent identifies the test agent as a modulator of the enzyme's
activity. In alternative aspects, the contacting between the test
agent and the sample can occur in solution or in solid phase, or,
it can occur in a cell. The cell can further comprise a tissue, an
organ or an entire body. The cell can further comprise a non-human
transgenic animal, such as a mouse or a rat, or other animal, as
described below.
[0035] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
[0036] All publications, patents, patent applications, GenBank
sequences and ATCC deposits, cited herein are hereby expressly
incorporated by reference for all purposes.
DESCRIPTION OF DRAWINGS
[0037] FIG. 1 is a schematic showing apoptosis-associated enzymes,
as described in detail in Example 1, below.
[0038] FIG. 2 shows a western blot of extracts from 293T cells
transfected with constructs encoding chimeric polypeptides of the
invention comprising the caspase 3 protease cleavage recognition
domain, with and without glycine "spacers," blotted with a
luciferase specific antibody, as described in detail in Example 1,
below.
[0039] FIG. 3 shows a bioluminescence imaging station reading of
293 cells transfected with various constructs, including two
encoding chimeric polypeptides of the invention, as described in
detail in Example 1, below.
[0040] FIG. 4 shows a magnetic resonance image revealing the size
and location of the tumor within the brain parenchyma using
bioluminescence imaging of cells transfected nucleic acids encoding
chimeric polypeptides of the invention, as described in detail in
Example 1, below.
[0041] FIG. 5 shows kinetics of intracranial glioma growth in vivo
by implanted 9L-Luc (luciferase expressing) cells with MRI (top
panel) and BLI (lower panel), as described in detail in Example 1,
below.
[0042] FIG. 6 shows the correlation of tumor volume as measured
using BLI with in vivo photon emission as imaged by MRI, as
discussed in detail in Example 1, below.
[0043] FIG. 7 summarizes data demonstrating that bioluminescence
imaging (BLI) can measure events dynamic changes in tumor volume,
as discussed in detail in Example 1, below.
[0044] FIG. 8 shows a western blot comparing extracts from 293T
cells transfected with constructs encoding chimeric polypeptides of
the invention comprising one and two "silencing" domains as
described in detail in Example 1, below.
[0045] FIG. 9 shows a western blot of extracts from 293T cells
transfected with constructs encoding chimeric polypeptides of the
invention comprising the caspase 8 and caspase 9 protease cleavage
recognition domains, blotted with a luciferase specific antibody,
as described in detail in Example 1, below.
[0046] FIG. 10 shows an exemplary "silencing domain" of the
invention, a mouse estrogen receptor regulatory domain (ER) (SEQ ID
NO:4).
[0047] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0048] The invention provides chimeric polypeptides and methods for
using them to non-invasively image enzyme (e.g., protease) activity
in vitro, in vivo and in situ. The noninvasive imaging can be in
cells, tissues and organs and entire bodies. Because many enzymes
and proteases are specifically associated with certain normal and
abnormal conditions and diseases, such as cell proliferation, cell
death (e.g., apoptosis), cancer, and other diseases, infections and
conditions, in vitro, in vivo and in situ imaging of enzyme (e.g.,
protease) activity is useful for identification, targeting,
diagnosis and the like. The imaging can be by computer assisted
tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic
resonance imaging (MRI), positron emission tomography (PET),
single-photon emission computed tomography (SPECT), or
bioluminescence imaging (BLI).
[0049] Definitions
[0050] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. As used herein,
the following terms have the meanings ascribed to them unless
specified otherwise.
[0051] "Caspase-3" or "CPP32" is a mammalian homolog of the C.
elegens death effector Ced-3. Caspase-3 cleaves a number of enzymes
and structural proteins containing the DEVD (SEQ ID NO:1) consensus
cleavage site. Cleavage of these "death substrates" commits cells
to suicide, and thus caspase-3 is thought to be an end-effector in
the caspase cascade induced by apoptotic stimuli.
[0052] The term "non-endogenous kinase" or "heterologous kinase"
means a kinase not normally associated with a cell or tissue, as
described in further detail, below. In some aspects, an exemplary
kinase used in the compositions and methods of the invention
includes herpes simplex virus-1 thymidine kinase (HSV-1 TK). For
example, in the methods of the invention a chimeric polypeptide
comprising a domain comprising a non-mammalian kinase is
administered to a mammal, e.g., a human.
[0053] As used herein, "matrix metalloproteinase (MMP)" includes
interstitial collagenases, stromelysins, gelatinases and
membrane-type metalloproteinases and MMPs secreted by cancer cells.
See, e.g., Yip (1999) Invest. New Drugs 17:387-399.
[0054] As used herein, the term "bioluminescence imaging" or "BLI"
includes all bioluminescence, fluorescence or chemiluminescence or
other photon detection systems and devices capable of detecting
bioluminescence, fluorescence or chemiluminescence or other photon
detection systems. Since light can be transmitted through mammalian
tissues at a low level, bioluminescent and fluorescent proteins can
be detected externally using sensitive photon detection systems;
see, e.g., Contag (2000) Neoplasia 2:41-52; Zhang (1994) Clin. Exp.
Metastasis 12: 87-92. The methods of the invention can be practiced
using any such photon detection device, or variation or equivalent
thereof, or in conjunction with any known photon detection
methodology, including visual imaging. An exemplary photodetector
device is an intensified charge-coupled device (ICCD) camera
coupled to an image processor. See, e.g., U.S. Pat. No. 5,650,135.
Photon detection devices are manufactured by, e.g., Xenogen
(Alameda, Calif.) (the Xenogen IVIS.TM. imaging system); or,
Hamamatsu Corp., Bridgewater, N.J.
[0055] As used herein, a "computer assisted tomography (CAT)" or a
"computerized axial tomography (CAT)" incorporates all
computer-assisted tomography imaging systems or equivalents and
devices capable of computer assisted tomography imaging. The
methods of the invention can be practiced using any such device, or
variation of a CAT device or equivalent, or in conjunction with any
known CAT methodology. See, e.g., U.S. Pat. Nos. 6,151,377;
5,946,371; 5,446,799; 5,406,479; 5,208,581; 5,109,397. Animal
imaging modalities are also included, such as MicroCAT.TM. (ImTek,
Inc., Knoxville, Tenn.).
[0056] As used herein, "positron emission tomography imaging (PET)"
incorporates all positron emission tomography imaging systems or
equivalents and all devices capable of positron emission tomography
imaging. The methods of the invention can be practiced using any
such device, or variation of a PET device or equivalent, or in
conjunction with any known PET methodology. See, e.g., U.S. Pat.
Nos. 6,151,377; 6,072,177; 5,900,636; 5,608,221; 5,532,489;
5,272,343; 5,103,098. Animal imaging modalities are included, e.g.
micro-PETs (Corcorde Microsystems, Inc.).
[0057] As used herein, "single-photon emission computed tomography
(SPECT) device" incorporates all single-photon emission computed
tomography imaging systems or equivalents and all devices capable
of single-photon emission computed tomography imaging. The methods
of the invention can be practiced using any such device, or
variation of a SPECT device or equivalent, or in conjunction with
any known SPECT methodology. See, e.g., U.S. Pat. Nos. 6,115,446;
6,072,177; 5,608,221; 5,600,145; 5,210,421; 5,103,098. Animal
imaging modalities are also included, such as micro-SPECTs.
[0058] As used herein, "magnetic resonance imaging (MRI) device"
incorporates all magnetic resonance imaging systems or equivalents
and all devices capable of magnetic resonance imaging. The methods
of the invention can be practiced using any such device, or
variation of an MRI device or equivalent, or in conjunction with
any known MRI methodology. In magnetic resonance methods and
apparatus a static magnetic field is applied to a tissue or a body
under investigation in order to define an equilibrium axis of
magnetic alignment in a region of interest. A radio frequency field
is then applied to that region in a direction orthogonal to the
static magnetic field direction in order to excite magnetic
resonance in the region. The resulting radio frequency signals are
detected and processed. The exciting radio frequency field is
applied. The resulting signals are detected by radio-frequency
coils placed adjacent the tissue or area of the body of interest.
See, e.g., U.S. Pat. Nos. 6,151,377; 6,144,202; 6,128,522;
6,127,825; 6,121,775; 6,119,032; 6,115,446; 6,111,410; 602,891;
5,555,251; 5,455,512; 5,450,010; 5,378,987; 5,214,382; 5,031,624;
5,207,222; 4,985,678; 4,906,931; 4,558,279. MRI and supporting
devices are manufactured by, e.g., Bruker Medical GMBH; Caprius;
Esaote Biomedica (Indianapolis, Ind.); Fonar; GE Medical Systems
(GEMS); Hitachi Medical Systems America; Intermagnetics General
Corporation; Lunar Corp.; MagneVu; Marconi Medicals; Philips
Medical Systems; Shimadzu; Siemens; Toshiba America Medical
Systems; including imaging systems, by, e.g., Silicon Graphics.
Animal imaging modalities are also included, such as
micro-MRIs.
[0059] As used herein, the terms "computer" and "processor" are
used in their broadest general contexts and incorporate all such
devices. The methods of the invention can be practiced using any
computer/processor and in conjunction with any known software or
methodology. For example, a computer/processor can be a
conventional general-purpose digital computer, e.g., a personal
"workstation" computer, including conventional elements such as
microprocessor and data transfer bus. The computer/processor can
further include any form of memory elements, such as dynamic random
access memory, flash memory or the like, or mass storage such as
magnetic disc optional storage.
[0060] As used herein, "bioluminescent" and "chemiluminescent"
polypeptides include all known polypeptides known to be
bioluminescent or chemiluminescent, or, acting as enzymes on a
specific substrate (reagent), can generate (by their enzymatic
action) a bioluminescent or chemiluminescent molecule. They
include, e.g., isolated and recombinant luciferases, aequorin,
obelin, mnemiopsin, berovin and variations thereof and combinations
thereof, as discussed in detail, below. In some aspects, the
bioluminescent or chemiluminescent are enzymes that act on a
substrate that reacts with the reagent in situ to generate a
molecule that can be imaged. The substrate can be administered
before, at the same time (e.g., in the same formulation), or after
administration of the chimeric polypeptide (including the
enzyme).
[0061] The term "pharmaceutical composition" refers to a
composition suitable for pharmaceutical use in a subject (including
human or veterinary). The pharmaceutical compositions of this
invention are formulations that comprise a pharmacologically
effective amount of a composition comprising, e.g., a chimeric
composition, a recombinant polypeptide, a nucleic acid encoding a
chimeric polypeptide of the invention, a vector comprising a
nucleic acid of the invention, or a cell of the invention, and a
pharmaceutically acceptable carrier. The pharmaceutical formulation
of the invention can further comprise a substrate for the
bioluminescent or chemiluminescent polypeptide. For example, the
chemiluminescent polypeptide can be luciferase and the reagent
luciferin. Alternatively, the substrate reagent can be
co-administered or administered before or after the chimeric
polypeptide (enzyme) formulation.
[0062] As used herein, "recombinant" refers to a polynucleotide
synthesized or otherwise manipulated in vitro (e.g., "recombinant
polynucleotide"), to methods of using recombinant polynucleotides
to produce gene products in cells or other biological systems, or
to a polypeptide ("recombinant protein") encoded by a recombinant
polynucleotide.
[0063] The term "nucleic acid" or "nucleic acid sequence" refers to
a deoxyribonucleotide or ribonucleotide oligonucleotide, including
single- or double-stranded, or coding or non-coding (e.g.,
"antisense") forms. The term encompasses nucleic acids, i.e.,
oligonucleotides, containing known analogues of natural
nucleotides. The term also encompasses nucleic-acid-like structures
with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl.
Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry
36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev
6:153-156.
[0064] The term "expression cassette" refers to any recombinant
expression system for the purpose of expressing a nucleic acid
sequence of the invention in vitro or in vivo, constitutively or
inducibly, in any cell, including, in addition to mammalian cells,
insect cells, plant cells, prokaryotic, yeast, fungal or mammalian
cells. The term includes linear or circular expression systems. The
term includes all vectors. The cassettes can remain episomal or
integrate into the host cell genome. The expression cassettes can
have the ability to self-replicate or not, i.e., drive only
transient expression in a cell. The term includes recombinant
expression cassettes that contain only the minimum elements needed
for transcription of the recombinant nucleic acid.
[0065] As used herein the terms "polypeptide," "protein," and
"peptide" are used interchangeably and include compositions of the
invention that also include "analogs," or "conservative variants"
and "mimetics" (e.g., "peptidomimetics") with structures and
activity that substantially correspond to the polypeptides of the
invention, including the chimeric polypeptide comprising a
bioluminescent or chemiluminescent polypeptide, or a heterologous
kinase, and a silencing moiety, and an endogenous protease cleavage
motif positioned between the first and second domains. Thus, the
terms "conservative variant" or "analog" or "mimetic" also refer to
a polypeptide or peptide which has a modified amino acid sequence,
such that the change(s) do not substantially alter the
polypeptide's (the conservative variant's) structure and/or
activity (e.g., binding specificity), as defined herein. These
include conservatively modified variations of an amino acid
sequence, i.e., amino acid substitutions, additions or deletions of
those residues that are not critical for protein activity, or
substitution of amino acids with residues having similar properties
(e.g., acidic, basic, positively or negatively charged, polar or
non-polar, etc.) such that the substitutions of even critical amino
acids does not substantially alter structure and/or activity.
Conservative substitution tables providing functionally similar
amino acids are well known in the art. For example, one exemplary
guideline to select conservative substitutions includes (original
residue followed by exemplary substitution): ala/gly or ser; arg/
lys; asn/ gin or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala
or pro; his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or
gin or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr;
thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. An alternative
exemplary guideline uses the following six groups, each containing
amino acids that are conservative substitutions for one another: 1)
Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
(see also, e.g., Creighton (1984) Proteins, W.H. Freeman and
Company; Schulz and Schimer (1979) Principles of Protein Structure,
Springer-Verlag). One of skill in the art will appreciate that the
above-identified substitutions are not the only possible
conservative substitutions. For example, for some purposes, one may
regard all charged amino acids as conservative substitutions for
each other whether they are positive or negative. In addition,
individual substitutions, deletions or additions that alter, add or
delete a single amino acid or a small percentage of amino acids in
an encoded sequence can also be considered "conservatively modified
variations."
[0066] The terms "mimetic" and "peptidomimetic" refer to a
synthetic chemical compound that has substantially the same
structural and/or functional characteristics of the polypeptides of
the invention (e.g., ability to be specifically recognized and
cleaved by enzymes, including proteases). The mimetic can be either
entirely composed of synthetic, non-natural analogues of amino
acids, or, is a chimeric molecule of partly natural peptide amino
acids and partly non-natural analogs of amino acids. The mimetic
can also incorporate any amount of natural amino acid conservative
substitutions as long as such substitutions also do not
substantially alter the mimetics' structure and/or activity. As
with polypeptides of the invention which are conservative variants,
routine experimentation will determine whether a mimetic is within
the scope of the invention, i.e., that its structure and/or
function is not substantially altered. Polypeptide mimetic
compositions can contain any combination of non-natural structural
components, which are typically from three structural groups: a)
residue linkage groups other than the natural amide bond ("peptide
bond") linkages; b) non-natural residues in place of naturally
occurring amino acid residues; or c) residues which induce
secondary structural mimicry, i.e., to induce or stabilize a
secondary structure, e.g., a beta turn, gamma turn, beta sheet,
alpha helix conformation, and the like. A polypeptide can be
characterized as a mimetic when all or some of its residues are
joined by chemical means other than natural peptide bonds.
Individual peptidomimetic residues can be joined by peptide bonds,
other chemical bonds or coupling means, such as, e.g.,
glutaraldehyde, N-hydroxysuccinimide esters, bifunctional
maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or
N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be an
alternative to the traditional amide bond ("peptide bond") linkages
include, e.g., ketomethylene (e.g., --C(.dbd.O)--CH.sub.2-- for
--C(.dbd.O)--NH--), aminomethylene (CH.sub.2--NH), ethylene, olefin
(CH.dbd.CH), ether (CH.sub.2--O), thioether (CH.sub.2--S),
tetrazole (CN.sub.4--), thiazole, retroamide, thioamide, or ester
(see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino
Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone
Modifications," Marcell Dekker, N.Y.). A polypeptide can also be
characterized as a mimetic by containing all or some non-natural
residues in place of naturally occurring amino acid residues;
non-natural residues are well described in the scientific and
patent literature.
[0067] Bioluminescent or Chemiluminescent Polypeptides
[0068] The invention provides a chimeric polypeptide, e.g., a
recombinant polypeptide, and a pharmaceutical composition,
comprising a bioluminescent or chemiluminescent polypeptide. As
defined above, these polypeptides include enzymes that act on a
specific reagent to generate a molecule that can be imaged (e.g.,
luciferase reacting with luciferin in situ). Once cleaved, the
bioluminescent or chemiluminescent domain is "liberated" from its
"silencer" to be used as a reporter in quantitative assays to
noninvasively image the endogenous enzyme (e.g., protease) activity
(the protease specific for the cleavage motif). The kinase activity
can be imaged in living animals using MRI, PET, SPECT and the
like.
[0069] In alternative aspects, these polypeptides include, e.g.,
luciferase, aequorin, halistaurin, phialidin, obelin, mnemiopsin or
berovin, or, equivalent photoproteins, and combinations thereof.
The compositions and methods of the invention also include
recombinant forms of these polypeptides as recombinant chimeric or
"fusion" proteins, including chimeric nucleic acids and constructs
encoding them. Methods of making recombinant forms of these
polypeptides are well known in the art, e.g., luciferase reporter
plasmids are described, e.g., by Everett (1999) J. Steroid Biochem.
Mol. Biol. 70:197-201. Sala-Newby (1998) Immunology 93:601-609,
described used of a recombinant cytosolic fusion protein of firefly
luciferase and aequorin (luciferase-aequorin). The
Ca.sup.2+-activated photoprotein obelin is described by, e.g.,
Dormer (1978) Biochim. Biophys. Acta 538:87105; and, recombinant
obelin is described by, e.g., Illarionov (2000) Methods Enzymol.
305:223-249. The photoprotein mnemiopsin is described by, e.g.,
Anctil (1984) Biochem J. 221:269-272. The monomeric Ca2+-binding
protein aequorin is described by, e.g., Kurose (1989) Proc. Natl.
Acad. Sci. USA 86:80-84; Shimomura (1995) Biochem. Biophys. Res.
Commun. 211:359-363. The aequorin-type photoproteins halistaurin
and phialidin are described by, e.g., Shimomura (1985) Biochem J.
228:745-749. Ward (1975) Proc. Natl. Acad. Sci USA 72:2530-2534,
describes the purification of mnemiopsin, aequorin and berovin. The
recombinant bioluminescent or chemiluminescent chimeric
polypeptides of the invention can be made by any method, see, e.g.,
U.S. Pat. No. 6,087,476, that describes making recombinant,
chimeric luminescent proteins. U.S. Pat. Nos. 6,143,50; 6,074,859;
6,074,859, 5,229,285, describe making recombinant luminescent
proteins. The bioluminescent or chemiluminescent activity of the
chimeric recombinant polypeptides of the invention can be assayed,
e.g., using assays described in, e.g., U.S. Pat. Nos. 6,132,983;
6,087,476; 6,060,261; 5,866,348; 5,094,939; 5,744,320. Various
photoproteins that can be used in compositions of the invention are
described in, e.g., U.S. Pat. Nos. 5,648,218; 5,360,728;
5,098,828.
[0070] Silencing or Quenching Moieties
[0071] The invention provides chimeric polypeptides comprising at
least one "silencing" or "quenching" moiety. As noted above, in the
intact chimeric polypeptide, the "silencing" or "quenching" domain
suppresses the activity of the bioluminescent or chemiluminescent
domain sufficiently to dampen the signal to unreadable or
relatively low levels (the quenching also can be complete). After
cleavage of the endogenous protease cleavage motif, the "silencing"
or "quenching" is sufficiently lost to increase the bioluminescent
or chemiluminescent signal; the loss can be complete, or, just
enough to generate a significantly different signal, i.e., a
readably different signal, after endogenous protease activity
(cleavage of the chimeric polypeptide of the invention). Any
"silencing" or "quenching" moiety can be used. For example, a
steroid hormone receptor ligand binding domain (e.g., a
transcription factor), or, a ligand for a kinase, can serve as a
"quenching" moiety. In one aspect, the steroid hormone receptor
ligand binding domain is an estrogen receptor regulatory domain
(ER), such as one derived from a mouse (SEQ ID NO:4) (see FIG. 10),
which can be the "silencing domain" for a variety of bioluminescent
or chemiluminescent polypeptides, including, e.g., for the
quenching of luciferase or thymidine kinase activity.
[0072] In other aspects, the hormone receptor (e.g., a
transcription factor) acting as a "silencing" or a "quenching"
moiety can also be a glucocorticoid receptor, a progesterone
receptor (see, e.g., Bain (2000) J. Biol. Chem. 275:7313-7320), an
androgen receptor, a mineralcorticoid receptor, a thyroid hormone
receptor (see, e.g., Ikeda (1996) J. Biol. Chem. 271:23096-23104),
a retinoic acid receptor, pregnane X receptor ("PXR," see, e.g.,
Moore (2000) Toxicology 153:1-10), liver X receptor ("LXR," see,
e.g., Repa (2000) Genes Dev. 14:2819-2830), vitamin D(3) receptor
("VDR," see, e.g., Toell (2000) Biochem J. 352:301-309), or a
retinoid X receptor ("RXR," see, e.g., Ikeda (1996) supra).
[0073] One exemplary chimeric polypeptide of the invention
comprises two ER domains, one amino-and another carboxy- terminal
to a luciferase, to maximize the silencing efficiency of the ER on
the bioluminescent or chemiluminescent polypeptide, e.g., a
luciferase. An endogenous protease cleavage recognition site (from,
e.g., a caspase) is inserted on either side of the bioluminescent
or chemiluminescent polypeptide (e.g., luciferase) such that during
induction of apoptosis both the ER sequences would separate from
the polypeptide. Glycine spacers can also be included between one,
several or all the various domains of the chimeric polypeptide
(e.g., ER-DEVD-G.sub.3-Luc-G.sub.3-DEVD-ER).
[0074] Identification of a "silencing" or "quenching" moiety and
the capacity of a silencing moiety to "quench" or "silence" the
bioluminescence or chemiluminescence of the (uncleaved) chimeric
recombinant polypeptides of the invention also can be assayed using
assays described in, e.g., U.S. Pat. Nos. 6,132,983; 6,087,476;
6,060,261; 5,866,348; 5,094,939; 5,744,320.
[0075] Endogenous Protease Cleavage Motifs
[0076] The invention provides chimeric polypeptides comprising at
least one endogenous protease cleavage motif. The endogenous
protease cleavage motif can be specifically cleaved by an enzyme
endogenous to the system studied (e.g., a cell proliferation or
cell death-associated protease) or exogenous to the studied cells
(e.g., a transfected enzyme). Any of the many known endogenous
protease cleavage motifs can be used in the chimeric polypeptide of
the invention. Alternatively, entirely synthetic protease cleavage
motifs can be devised and incorporated. For example, in one aspect,
the endogenous protease cleavage motif is aspartic acid-glutamic
acid-valine-aspartic acid (DEVD) (SEQ ID NO:1), which is
specifically recognized by the apoptosis-associated enzyme caspase
(see, e.g., U.S. Pat. No. 5,976,822). Protease cleavage motifs
specific for apoptosis-associated, endogenous cellular proteases
include, e.g., caspase 3, caspase 6, caspase 7, procaspase 8,
caspase 8, caspase 9, caspase 10, scan be used. For example, the
protease cleavage recognition domain for caspase 8, IETD, or
isoleucine-glutamic acid-threonine-aspartic acid, (SEQ ID NO:2) and
caspase 9, LEHD, or leucine- glutamic acid-histidine-aspartic acid,
(SEQ ID NO:3) can be used. For other enzymes associated with
apoptosis, see, e.g., U.S. Pat. Nos. 6,143,522; 6,107,088;
6,072,031; 6,010,853; 5,985,829; 5,955,429; 5,935,931; 5,858,715;
5,846,768.
[0077] Endogenous protease cleavage recognition domains can also be
derived from matrix metalloproteinase (MMP) enzymes (see, e.g.,
U.S. Pat. Nos. 6,140,099; 6,114,568; 6,093,398; 5,595,885);
secretins; gamma-secretase associated with Alzheimer's disease
(see, e.g., Zhang (2000) Nat. Cell Biol. 2:463-465); calpain
proteases (also associated with Alzheimer's disease, see e.g., Nath
(2000) Biochem. Biophys. Res. Commun. 274:16-21; Wang (2000) Trends
Neurosci. 23:20-26). Other examples include cleavage site
recognized by thrombin, H64A subtilisin, and enterokinase described
by Forsberg (1992) J. Protein Chem. 11:201-211. Humphreys (2000)
Protein Eng. 13:201-206, described an improved efficiency of the
site-specific copper (II) ion-catalyzed protein cleavage peptide
sequence (N)DKTH(C) effected by mutagenesis of cleavage site.
Various virus-specified protease cleavage recognition sites are
described in U.S. Pat. No. 4,952,493.
[0078] The endogenous protease cleavage motif positioned between
the first and second domains of the chimeric polypeptide. In one
aspect, the protease cleavage motif is flanked by a "spacer" on one
or both sides (i.e., a spacer is between the cleavage motif and
either or both the silencing domain and the bioluminescent or
chemiluminescent polypeptide domain. The spacer can be, e.g., a
poly-glycine moiety. Other "spacers" are known in the art; for
example, to improve site-specific cleavage of a methionyl porcine
growth hormone [[Met1]-pGH(1-46)-IGF-II] fision protein by the
enzyme H64A subtilisin, Polyak (1997) Protein Eng. 10:615-619,
introduced a series of flexible, unstructured spacer peptides
N-terminal to the cleavage site.
[0079] Non-endogenous Kinases
[0080] The invention provides a chimeric polypeptide comprising a
non-endogenous kinase, such as a herpes simplex virus-1 thymidine
kinase (HSV-1 TK). Once cleaved, the kinase domain is liberated"
from its "silencer" to be used as a reporter in quantitative assays
to non-invasively image enzyme (e.g., protease) activity (the
endogenous protease specific for the cleavage motif). The kinase
activity can be imaged in living animals using MRI, PET, SPECT, BLI
and the like.
[0081] In one aspect, after administration of the chimeric
polypeptide of the invention, a kinase substrate (a "reporter
probe") is administered, e.g., the positron-emitting 8-[18F]
fluoroganciclovir (FGCV). In one aspect, the herpes simplex virus 1
thymidine kinase enzyme (HSV1-TK) is the kinase.
Adenovirus-directed hepatic expression of the HSV-1 TK gene in
living mice has been shown to be detectable by PET. See, e.g.,
Tjuvajev (1995) Cancer Res. 55:6126-6132; Gambhir (1999) Proc.
Natl. Acad. Sci. USA 96:2333-2338; Gambhir (2000) Proc. Natl. Acad.
Sci. USA 97:2785-2790; Gambhir (2000) Neoplasia 2:118-138; MacLaren
(2000) Biol. Psychiatry 48:337-348; Schwimmer (2000) Q. J. Nucl.
Med. 44:153-167; Yu (2000) Nat. Med. 6:933-937.
[0082] In Vivo Bioluminescent Imaging
[0083] The invention provides compositions and methods to enhance
the imaging of cells and tissues by, e.g., bioluminescence imaging
(BLI). In vivo Bioluminescent Imaging (BLI) is a relatively new
imaging modality; see discussion above and, e.g., Contag (2000)
Neoplasia 2:41-52. This modality consists of the detection of a
photoprotein (i.e., an optical reporter), such as luciferase from
the firefly, using a sensitive photon detection system. The number
of photons emitted from cells expressing the photoprotein (e.g.,
luciferase) can be quantitatively detected and overlayed
(projected) onto a visual picture of the animal (including humans).
This imaging approach provides a two-dimensional image data set and
thus provides some spatial information as to the origin of the
signal within the animal. An exciting aspect of BLI is its
excellent sensitivity along with its ability to report on
"molecular events" using specifically designed luciferase reporter
constructs.
[0084] Nanoparticles and Imaging of Brain Tumors
[0085] The invention provides pharmaceutical formulations
comprising the chimeric polypeptides of the invention that can
further comprise imaging contrast agents (see, e.g., U.S. Pat. No.
4,731,239). The pharmaceutical formulations and/or the contrast
agents can be administered by nanoencapsulation, e.g., by hydrogel
nanoparticles (and liposomes, which are discussed below).
Nanoencapsulation can be used to manipulate the environment
surrounding the pharmaceutical formulation and/or the contrast
agent. Although the contrast between healthy and abnormal tissues
is strong, there exists considerable overlap of magnetic resonance
imaging (MRI) T.sub.1 and T.sub.2 signals in all tissues. This
physical property of biological tissues renders necessary the use
of contrast agents for adequate resolution of many lesions: in
particular, the diffuse margins of some lesions. Contrast agents
for magnetic resonance imaging typically affect the protons on
adjacent water molecules shortening either the T.sub.1 or T.sub.2
signals generated in the magnetic field. The most important factor
in enhancement of relaxation is the difference between T.sub.1 and
T.sub.2. There must be direct contact between protons and the
magnetic parts of the contrast agent in order to shorten the
T.sub.1 component significantly. This effect can be clearly
observed when gadolinium chelates are encapsulated in liposomes
with resulting weakening of the T.sub.1 signal. Weakening of the
T.sub.1 signal is thought to be due to the reduced access of water
to the cavity of the liposome.
[0086] Enhancement of T.sub.2 effects, however, requires clustering
of the contrast agent and proximity to each other. This clustering
of magnetic contrast agent exerts a greater influence over a much
larger localized field. Thus incorporation into liposomes increases
the proximity of T.sub.2 contrast agents and enhances their
effectiveness. Incorporation of contrast agents into the body of
hydrogel nanoparticles has with it the potential advantages of both
immobilizing and clustering the contrast agent and providing a
material through which water can freely diffuse.
[0087] The pharmaceutical compositions of the invention can further
comprise monocrystalline iron oxide nanoparticles (MION), which
have been successfully used in a variety of biological and clinical
applications. MION has an average diameter of approximately 18 to
24 nm and thus are able to penetrate endothelial fenestrations
throughout the body and are cleared through the reticuloendothelial
system and are disposed of by hepatic metabolism of iron. MION has
excellent contrast characteristics in vivo and out-performs the
most effective dendrimer-conjugated contrast agents.
[0088] Polypeptides and Peptides
[0089] The invention provides a chimeric polypeptide comprising a
bioluminescent or chemiluminescent domain or a heterologous kinase,
and a second domain comprising at least one silencing moiety, and
an endogenous protease cleavage motif positioned between the first
and second domains. As noted above, the term polypeptide includes
peptides and peptidomimetics, etc. Polypeptides and peptides of the
invention can be isolated from natural sources, be synthetic, or be
recombinantly generated polypeptides. Peptides and proteins can be
recombinantly expressed in vitro or in vivo. The peptides and
polypeptides of the invention can be made and isolated using any
method known in the art.
[0090] Polypeptide and peptides of the invention can also be
synthesized, whole or in part, using chemical methods well known in
the art. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser.
215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga,
A. K., Therapeutic Peptides and Proteins, Formulation, Processing
and Delivery Systems (1995) Technomic Publishing Co., Lancaster,
Pa. For example, peptide synthesis can be performed using various
solid-phase techniques (see e.g., Roberge (1995) Science 269:202;
Merrifield (1997) Methods Enzymol. 289:3-13) and automated
synthesis may be achieved, e.g., using the ABI 431A Peptide
Synthesizer (Perkin Elmer). The skilled artisan will recognize that
individual synthetic residues and polypeptides incorporating
mimetics can be synthesized using a variety of procedures and
methodologies, which are well described in the scientific and
patent literature, e.g., Organic Syntheses Collective Volumes,
Gilman, et al. (Eds) John Wiley & Sons, Inc., NY. Polypeptides
incorporating mimetics can also be made using solid phase synthetic
procedures, as described, e.g., by Di Marchi, et al., U.S. Pat. No.
5,422,426. Peptides and peptide mimetics of the invention can also
be synthesized using combinatorial methodologies. Various
techniques for generation of peptide and peptidomimetic libraries
are well known, and include, e.g., multipin, tea bag, and
split-couple-mix techniques; see, e.g., al-Obeidi (1998) Mol.
Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol.
1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996)
Methods Enzymol. 267:220-234. Modified peptides of the invention
can be further produced by chemical modification methods, see,
e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel
(1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994)
Biochemistry 33:7886-7896.
[0091] Peptides and polypeptides of the invention can also be
synthesized and expressed as chimeric or "fusion" proteins with one
or more additional domains linked thereto for, e.g., to more
readily isolate a recombinantly synthesized peptide, and the like.
Detection and purification facilitating domains include, e.g.,
metal chelating peptides such as polyhistidine tracts and
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle WA).
The inclusion of a cleavable linker sequences such as Factor Xa or
enterokinase (Invitrogen, San Diego Calif.) between the
purification domain and GCA-associated peptide or polypeptide can
be useful to facilitate purification. For example, an expression
vector can include an epitope-encoding nucleic acid sequence linked
to six histidine residues followed by a thioredoxin and an
enterokinase cleavage site (see e.g., Williams (1995) Biochemistry
34:1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-14). The
histidine residues facilitate detection and purification while the
enterokinase cleavage site provides a means for purifying the
epitope from the remainder of the fusion protein.
[0092] Nucleic Acids and Expression Vectors
[0093] This invention provides nucleic acids encoding the chimeric
polypeptides of the invention. As the genes and expression
cassettes (e.g., vectors) of the invention can be made and
expressed in vitro or in vivo, the invention provides for a variety
of means of making and expressing these genes and vectors. One of
skill will recognize that desired phenotypes can be obtained by
modulating the expression or activity of the genes and nucleic
acids (e.g., promoters) within the expression cassettes of the
invention. Any of the known methods described for increasing or
decreasing expression or activity can be used for this invention.
The invention can be practiced in conjunction with any method or
protocol known in the art, which are well described in the
scientific and patent literature.
[0094] The nucleic acid sequences of the invention and other
nucleic acids used to practice this invention, whether RNA, cDNA,
genomic DNA, expression cassettes, vectors, viruses or hybrids
thereof, may be isolated from a variety of sources, genetically
engineered, amplified, and/or expressed recombinantly. Any
recombinant expression system can be used, including, in addition
to bacterial cells, e.g., mammalian, yeast, insect or plant cell
expression systems.
[0095] Alternatively, these nucleic acids can be synthesized in
vitro by well-known chemical synthesis techniques, as described in,
e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel
(1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994)
Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;
Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.
22:1859; U.S. Pat. No. 4,458,066.
[0096] Techniques for the manipulation of nucleic acids, such as,
e.g., generating mutations in sequences, subcloning, labeling
probes, sequencing, hybridization and the like are well described
in the scientific and patent literature, see, e.g., Sambrook, ed.,
Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold
Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997);
Laboratory Techniques In Biochemistry And Molecular Biology:
Hybridization With Nucleic Acid Probes, Part 1. Theory and Nucleic
Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
[0097] Transgenic Non-Human Animals
[0098] The invention provides transgenic non-human animals, e.g.,
goats, rats and mice, comprising the chimeric nucleic acids of the
invention. These animals can be used, e.g., as in vivo models to
study apoptosis, or, as models to screen for enzyme activity in
vivo. For example, an increase in the activity of an enzyme capable
of cleaving the endogenous protease cleavage domain on the in vivo
produced chimeric polypeptide can be read by BLI, PET, MRI, etc. As
demonstrated in Example 1, below, such transgenic non-human animals
are excellent models for imaging apoptosis in vivo by determining
the activity of apoptosis-associated enzymes. The coding sequences
for the chimeric polypeptides can be designed to be constitutive,
or, under the control of tissue-specific, developmental-specific or
inducible transcriptional regulatory factors.
[0099] Transgenic non-human animals can be designed and generated
using any method known in the art; see, e.g., U.S. Pat. Nos.
6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;
5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933, describing
making and using transgenic mice, rats, rabbits, sheep, pigs and
cows. See also, e.g., Pollock (1999) J. Immunol. Methods
231:147-157, describing the production of recombinant proteins in
the milk of transgenic dairy animals; Baguisi (1999) Nat.
Biotechnol. 17:456-461, demonstrating the production of transgenic
goats.
[0100] Formulation and Administration Pharmaceuticals
[0101] The invention provides pharmaceutical formulations
comprising the chimeric molecules of the invention and a
pharmaceutically acceptable excipient suitable for administration
to image endogenous enzyme (e.g., protease) activity, and methods
for making and using these compositions. These pharmaceuticals can
be administered by any means in any appropriate formulation.
Routine means to determine drug regimens and formulations to
practice the methods of the invention are well described in the
patent and scientific literature. For example, details on
techniques for formulation, dosages, administration and the like
are described in, e.g., the latest edition of Remington's
Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.
[0102] The formulations of the invention can include
pharmaceutically acceptable carriers that can contain a
physiologically acceptable compound that acts, e.g., to stabilize
the composition or to increase or decrease the absorption of the
pharmaceutical composition. Physiologically acceptable compounds
can include, for example, carbohydrates, such as glucose, sucrose,
or dextrans, antioxidants, such as ascorbic acid or glutathione,
chelating agents, low molecular weight proteins, compositions that
reduce the clearance or hydrolysis of any co-administered agents,
or excipients or other stabilizers and/or buffers. Detergents can
also used to stabilize the composition or to increase or decrease
the absorption of the pharmaceutical composition. Other
physiologically acceptable compounds include wetting agents,
emulsifying agents, dispersing agents or preservatives that are
particularly useful for preventing the growth or action of
microorganisms. Various preservatives are well known, e.g.,
ascorbic acid. One skilled in the art would appreciate that the
choice of a pharmaceutically acceptable carrier, including a
physiologically acceptable compound depends, e.g., on the route of
administration and on the particular physio-chemical
characteristics of any co-administered agent.
[0103] In one aspect, the composition for administration comprises
a chimeric polypeptide of the invention in a pharmaceutically
acceptable carrier, e.g., an aqueous carrier. A variety of carriers
can be used, e.g., buffered saline and the like. These solutions
are sterile and generally free of undesirable matter. These
compositions may be sterilized by conventional, well-known
sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
active agent in these formulations can vary widely, and will be
selected primarily based on fluid volumes, viscosities, body weight
and the like in accordance with the particular mode of
administration and imaging modality selected.
[0104] The pharmaceutical formulations of the invention can be
administered in a variety of unit dosage forms, depending upon the
particular enzyme-expressing cell or tissue or cancer to be imaged,
the general medical condition of each patient, the method of
administration, and the like. Details on dosages are well described
in the scientific and patent literature, see, e.g., the latest
edition of Remington's Pharmaceutical Sciences. The exact amount
and concentration of chimeric polypeptide or pharmaceutical of the
invention and the amount of formulation in a given dose, or the
"effective dose" can be routinely determined by, e.g., the
clinician. The "dosing regimen," will depend upon a variety of
factors, e.g., whether the enzyme-expressing cell or tissue or
tumor to be imaged is disseminated or local, the general state of
the patient's health, age and the like. Using guidelines describing
alternative dosaging regimens, e.g., from the use of other imaging
contrast agents, the skilled artisan can determine by routine
trials optimal effective concentrations of pharmaceutical
compositions of the invention. The invention is not limited by any
particular dosage range.
[0105] The pharmaceutical compositions of the invention (e.g.,
chimeric polypeptides) can be delivered by any means known in the
art systemically (e.g., intravenously), regionally, or locally
(e.g., intra- or peri-tumoral or intracystic injection, e.g., to
image bladder cancer) by, e.g., intraarterial, intratumoral,
intravenous (IV), parenteral, intra-pleural cavity, topical, oral,
or local administration, as subcutaneous, intra-tracheal (e.g., by
aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine,
rectal, nasal mucosa), intra-tumoral (e.g., transdermal application
or local injection). For example, intraarterial injections can be
used to have a "regional effect," e.g., to focus on a specific
organ (e.g., brain, liver, spleen, lungs). For example,
intra-hepatic artery injection or intra-carotid artery injection.
If it is desired to deliver the preparation to the brain, it can be
injected into a carotid artery or an artery of the carotid system
of arteries (e.g., occipital artery, auricular artery, temporal
artery, cerebral artery, maxillary artery, etc.).
[0106] The pharmaceutical formulations of the invention can be
presented in unitdose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets.
Therapeutic compositions can also be administered in a lipid
formulation, e.g., complexed with liposomes or in lipid/nucleic
acid complexes or encapsulated in liposomes, as in immunoliposomes
directed to specific cells. These lipid formulations can be
administered topically, systemically, or delivered via aerosol.
See, e.g., U.S. Pat. Nos. 6,149,937; 6,146,659; 6,143,716;
6,133,243; 6,110,490; 6,083,530; 6,063,400; 6,013,278; 5,958,378;
5,552,157.
[0107] Kits
[0108] The invention provides kits comprising the compositions,
e.g., the pharmaceutical compositions, nucleic acids, expression
cassettes, vectors, cells of the invention, to image the activity
of endogenous enzymes. The kits also can contain instructional
material teaching methodologies, e.g., how and when to administer
the pharmaceutical compositions, how to apply the compositions and
methods of the invention to imaging systems, e.g., computer
assisted tomography (CAT), magnetic resonance spectroscopy (MRS),
magnetic resonance imaging (MRI), positron emission tomography
(PET), single-photon emission computed tomography (SPECT) or
bioluminescence imaging (BLI). Kits containing pharmaceutical
preparations (e.g., chimeric polypeptides, expression cassettes,
vectors, nucleic acids) can include directions as to indications,
dosages, routes and methods of administration, and the like.
EXAMPLES
[0109] The following example is offered to illustrate, but not to
limit the claimed invention.
Example 1
In Vivo Imaging of Protease Activity
[0110] The following example demonstrates use of the compositions
and methods of the invention to in vivo image the activity of
endogenous enzymes associated with apoptosis, e.g., caspase-3,
caspase-8, caspase-9. Specifically, these experiments demonstrate
use of the compositions and methods of the invention to measure the
induction of apoptosis by quantitative and non-invasive imaging of
endogenous enzyme activation.
[0111] Caspase-3, an end-effector in the caspase cascade induced by
apoptotic stimuli, was imaged in vivo using a chimeric polypeptide
of the invention. Its function in the cell's "cell death," or
"apoptosis" pathway is shown in FIG. 1. This schematic also
illustrates other apoptosis-associated enzymes that can be included
in the invention's strategy for the imaging of the activity of
these enzymes, and, thus, apoptosis. The chimeric polypeptide of
the invention is designed to include the desired cleavage domain;
in this example, the protease cleavage domain specific for
caspase-3 was used. However, in other exemplary chimeric
polypeptides, the protease cleavage domain is specifically cleaved
by, e.g., caspase 3, caspase 6, caspase 7, procaspase 8, caspase 8,
caspase 9, caspase 10, matrix metalloproteinase (MMP) or
gamma-secretase.
[0112] For "normal" cells (i.e., cells not undergoing apoptosis),
the chimeric polypeptide is not cleaved and the "reporter" domain
(in this example, a luciferase enzyme) is not liberated from the
"silencing" domain (in this example, an estrogen receptor
regulatory domain (ER).
[0113] Other "reporters" can be used e.g., as described above, such
as HSV TK, a silencing domain, which also can be an estrogen
receptor regulatory domain (ER), and PET imaging. While any
"quenching" moiety can be used, in this exemplary fusion protein,
the estrogen receptor regulatory domain (ER) "silenced" luciferase.
Some luciferase activity may be present; however, this amount is
sufficiently low such that upon cleavage a detectable change in
bioluminescent signal is imaged by BLI.
[0114] For cells undergoing apoptosis, caspase-3 enzyme activity
increases significantly. This activation of caspase-3 during
apoptosis results in cleavage of the chimeric polypeptide of the
invention at the protease cleavage domain, in this example, the
(SEQ ID NO:1) sequence. This releases luciferase from the silencing
effects of the ER. The "unsilenced" luciferase can react with an
appropriate substrate, e.g., luciferin, to generate a signal
readable by BLI. In one exemplary chimeric polypeptide, glycine
residue spacers were used on both sides of the DEVD (SEQ ID NO:1)
cleavage sequence.
[0115] In this example, BLI was used; it is a very sensitive
imaging technique. BLI has an additional advantage of being more
cost effective than PET. It does not require a cyclotron run along
with radiochemical synthesis. However, PET is useful for obtaining
high resolution imaging of in vivo apoptosis.
[0116] Using PCR mutagenesis, three versions of the ER-luciferase
chimeric molecule was constructed. The first, ER-Luc), is a simple
fusion of the coding sequence for the mouse ER (SEQ ID NO:4) (see
FIG. 10) to the firefly luciferase complete coding sequence. A
second version, ER-DEVD-Luc, contains the DEVD (SEQ ID NO:1)
sequence, as described above. This protease cleavage recognition
domain was inserted between the ER and the luciferase sequence. A
third version, ER-G.sub.3-DEVD-G.sub.3-- Luc, is identical to the
second except that it contains three glycine residues amino- and
carboxy- terminal to the DEVD (SEQ ID NO:1) sequence. The ER-Luc
construct was a negative control; in the absence of a caspase-3
cleavage site there shouldn't be any cleavage of the polypeptide
during apoptosis, even in the presence of caspase-3. The
ER-DEVD-Luc chimeric polypeptide should be cleaved during
apoptosis. The ER-G3-DEVD-G3-Luc molecule, with the glycine
residues "spacers," may be less conformationally restrained (e.g.,
more "floppy") than the ER-DEVD-Luc polypeptide; thus, it would
make the DEVD (SEQ ID NO:1) sequence more accessible to
caspase-3.
[0117] Construction of these fusion constructs involved the use of
PCR primers. The ER and luciferase coding sequence were PCR
amplified such that the 3' end of the ER coding sequence contained
the caspase cleavage site (with or without the glycine spacers) and
the 5' end of the luciferase coding sequence. Since the 3' of the
ER and the 5' of the luciferase are complimentary, they can be
joined in a PCR reaction using the ER 5' primer and the luciferase
3' primer. Three independent isolates of each construct were
characterized to ensure that PCR errors were not been introduced.
In addition, a hi-fidelity thermostable polymerase was used. All
the constructs were inserted in the bicistronic vector pZ (Genetics
Institute, Mass.). Each the constructs was transfected into 293T
cells to ensure that the correct polypeptide was being made (i.e.
60 kDa of Luc+30 kDa of ER=90 kDa) and that it reacted with the
appropriate antibodies (luciferase and mouse estrogen receptor
specific), indicating that the amino acid sequence generated by the
constructs were appropriate.
[0118] As shown by the western blot illustrated in FIG. 2, after
transfection into cells, ER-DEVD-Luc was cleaved in an apoptosis
dependent manner. The three constructs described above (two
independent clones of each) were transfected into 293T cells. At 48
hr after transfection the cells were either left untreated (lanes
labeled C in the figure) or treated with staurosporine to induce
apoptosis (labeled T). In an adjacent lane cells transfected with
the luciferase (pLuc) expression plasmid were used as control (lane
labeled Luc). As shown in the blot, the ER-Luc construct remains as
a 90 kDa (60 kDa luc+30 kDa ER) polypeptide under control
conditions, as well as under conditions were the cells are
undergoing apoptosis.
[0119] In contrast, while the ER-DEVD-Luc construct is
predominantly 90 kDa under control conditions, it is cleaved to
yield a 60 kDa polypeptide (luciferase only) when apoptosis is
induced. About 50% of the ER-DEVD-Luc protein was cleaved in cells
undergoing apoptosis. In contrast, the ER-G3-DEVD-G3-Luc was very
efficiently cleaved when apoptosis was induced.
[0120] These constructs were next transfected into 293 cells tissue
culture cells. FIG. 3 shows a bioluminescence imaging station
reading of the transfected cells. Forty-eight hours after
transfection cells were left untreated or treated with
staurosporine to induce apoptosis. Three hours after treatment the
levels of luciferase activity was measured by adding luciferin
(0.15 mg/ml) to the tissue culture dish. The cells were then imaged
on a bioluminescence imaging station, specifically, the
cryogenically-cooled Xenogen IVIS.TM. (Alameda, Calif.) system
coupled to a data acquisition PC running IGOR.TM. under Windows
98.TM.. This system provides outstanding signal-to-noise images of
luciferase signals emerging from within living cells and animals.
Cell temperature is regulated using a digitally thermostated stage
located in the system. A greyscale image is collected in the
chamber followed by acquisition and overlay of a pseudocolor image
representing the spatial distribution of photon counts emerging
from the active luciferase, e.g., from a multi-well plate or an
animal model. Digital image processing software provided by
manufacturer, or equivalent, is used to quantitate the photon
counts from each digital image data set. The intensity of
bioluminescence was represented in the image by pseudo-colors with
blue being the least intense, followed by green, yellow and red
(although FIG. 3 is in black and white). The release of luciferase
from ER correlated with an increase in bioluminescent activity. The
data presented here demonstrate that induction of apoptosis results
in the activation of luciferase activity in the ER-DEVD-Luc and the
ER-G3-DEVD-G3-Luc polypeptide, but not in the molecule that lacks a
caspase-3 cleavage site.
[0121] A rat brain tumor cell line that stably expresses luciferase
was used to assess the sensitivity of bioluminescence imaging in
vivo, as shown in FIG. 4. The right-hand panel shows an MRI image
revealing the size and location of the tumor within the brain
parenchyma. The image on the left-hand panel reveals the greyscale
image of the rat head on which an overlay of the photon counts
emitted from the tumor is shown. This data was acquired using a
Hamamatsu (Bridgewater, N.J.) image system. A Xenogen system (see
above), estimated to have about 100 to 1,000-fold more sensitivity,
can also be used. This data reveals for the first time that
bioluminescent images (BLI) can be acquired from cells located
within the inner regions of a cranium, in this example, a rat
skull, thus making the caspase-3 activity imaging method of the
invention applicable for the imaging of brain tumors. Tumors as
small as 1.5 mm growing in the brain of a rat can be non-invasively
imaged in 5 minutes using this approach.
[0122] Next, it was determined that the luciferase-generated signal
imaged by the BLI was proportional to the number of cells emitting
the signal. A rat with an intracerebral 9L/luciferase-expressing
tumor was imaged at three to four day intervals. As shown in FIG.
5, BLI images show the kinetics of intracranial glioma growth in a
representative animal. The "9L-Luc" cells were implanted
intra-cerebrally at 16 days prior to sham treatment with ethanol
vehicle. Tumor progression was monitored with MRI (top panel) and
BLI (lower panel). The days, post sham treatment, on which the
images were obtained are indicated at the top ("-2" is two days
before, "0" is day zero, and 4 days and 10 days after treatment).
The MRI images are T2-weighted and are of a representative slice
from the multi-slice dataset. The scale to the right of the BLI
images describes the color map for the pixel photon counts.
[0123] The ability of BLI to determine tumor size was comparable to
tumor volume as measured by MRI. The relationship between the two
measurements (MRI and BLI) was defined by regression analysis. FIG.
6 shows the correlation of the BLI determined tumor volume with the
in vivo photon emission as detected by MRI. The number of cells
expressing luciferase was represented by the number of photon
counts emerging from the tumor mass. The photon counts from the
intracerebral luciferase-expressing tumors were plotted against the
MRI-determined tumor volume (units of microliters) from five rats
in which data was obtained over time during untreated growth. The
amount and size of the bioluminescent signal increased with time as
the tumor expanded. There was an excellent correlation of the
number of cells expressing luciferase and tumor mass. As indicated
by the good correlation coefficient, there was excellent
correlation between the BLI and MRI imaging modalities.
[0124] This data also demonstrates that, using the compositions and
methods of the invention, bioluminescent imaging (BLI) can be used
to obtain quantitative data relating to the number of cells
expressing caspase 3, i.e., the number of cells undergoing
apoptosis. Thus, this approach can provide quantitative data as to
the numbers of cells undergoing caspase 3 activation (and
apoptosis) within a subject, e.g., a non-human transgenic host
animal (e.g., mouse, rat) for study. This is a breakthrough
approach for the noninvasive assessment of apoptosis and for
quantifying the numbers of cell within a specific organ that have
received an apoptotic signaling event. Thus, these methods also
provide for the in vivo detection of apoptosis and the screening
and testing of novel anti-apoptotic therapeutic strategies in cell
and animal models, e.g., transgenic animal.
[0125] In order to demonstrate that therapeutic interventions that
affect cell numbers relate proportionally to photons emitted,
experiments were conducted in which rats harboring intracerebral 9L
luciferase-expressing tumors were treated with a chemotherapeutic
agent. As the tumor cells begin to die, the amount of light
proportionally decreases in response to cell death. In this study,
both MRI images and bioluminescent images were acquired in
succession over time.
[0126] To demonstrate that the compositions (including transgenic
non-human animals, such as transgenic mice and rats, expressing the
nucleic acids of the invention) and methods of the invention can be
applied as in vivo models for screening for novel antiapoptotic
therapeutic agents, experiments were conducted in rats harboring
intracerebral 9L luciferase-expressing tumors treated with a known
chemotherapeutic agent, the nitrosourea bischloroethylnitrosourea
(BCNU, see, e.g., U.S. Pat. Nos. 6,147,060; 5,736,129). As the
tumor cells die in response to the BCNU treatment, the amount of
light proportionally decreases. Both MRI and BLI images were
acquired over various times.
[0127] FIG. 7 summarizes the data demonstrating that
bioluminescence imaging (BLI) can measure events dynamic changes in
tumor volume. Three time points were acquired prior to chemotherapy
using BCNU, which was administered at time zero. Tumor volume and
photon counts continued to increase for three time points after
BCNU treatment followed by a decline in both parameters over time.
These changes both occurred in a similar time frame and degree of
change. Tumor volume and light output should not exactly correlate
as the tumor volume becomes non-proportional to living cells
following treatment, as it takes time for re-absorption of cellular
debris to occur following cell killing. This study clearly
demonstrates that the methods of the invention can be used to
noninvasively monitor therapeutic intervention of cells with
luciferase activity (i.e., cells expressing exogenous luciferase).
These data indicate that the invention's novel molecular imaging
constructs coupled to a "reporter" (e.g., a luciferase) can be
quantitatively and noninvasively imaged to provide dynamic
information relating to apoptotic cell death in intact animals,
e.g., subjects and non-human transgenic animals.
[0128] In one aspect of the invention, a chimeric polypeptide
comprises two "silencing" domains. The second "silencing domain"
maximizes the quenching effect on the bioluminescent or
chemiluminescent polypeptide. Experiments to confirm that the
constructs are cleaved in an apoptosis-dependent manner were
performed by transfection of tissue culture cells with the chimeric
polypeptides: ER-DEVD-Luc-DEVD-ER, at 120 kD, and Luc-DEVD-ER, at
90 kD (Luc alone is 60 kD). Tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) was used to induce apoptosis in
one set of transformed cells; it is a potent inducer of apoptosis
of transformed and cancer cells but not of most normal cells
(Ozoren (2000) Cancer Res. 60:6259-6265). FIG. 8 shows a western
blot comparing extracts from 293T cells transfected with these
constructs. The "control" lane cells were normal, the "+TRAIL" lane
cells were induced to undergo apoptosis. The western was blotted
with a luciferase specific. As demonstrated by the western blots,
endogenous enzymes whose activity is induced by apoptosis can
cleave the "double" silencer-containing chimeric polypeptide.
[0129] In one aspect of the invention, a chimeric polypeptide
comprises protease cleavage recognition domains from a caspase,
including, e.g., caspase 3, caspase 6, caspase 7, procaspase 8,
caspase 8, caspase 9, caspase 10. To confirm that the cleavage
recognition domains for the exemplary caspase 8 and caspase 9,
which are IETD (SEQ ID NO:2) and LEHD (SEQ ID NO:3), respectively,
are cleaved in an apoptosis-dependent manner, experiments were
performed by transfection of tissue culture cells with chimeric
polypeptides containing these cleavage domains separating Luc from
ER. FIG. 9 shows westerns blotted with a luciferase specific
antibody of extracts from 293T cells transfected these constructs.
As above, TRAIL was used to induce apoptosis. As demonstrated by
the western blots, endogenous enzymes whose activity is induced by
apoptosis cleave the caspase 8 and caspase 9 cleavage recognition
domains.
* * * * *