U.S. patent application number 12/260892 was filed with the patent office on 2010-10-14 for vimentin directed diagnostics and therapeutics for multidrug resistant neoplastic disease.
Invention is credited to Anne-Marie Bonneau, Frederic Dallaire, Elias Georges, Lucile Serfass.
Application Number | 20100260667 12/260892 |
Document ID | / |
Family ID | 35599927 |
Filed Date | 2010-10-14 |
United States Patent
Application |
20100260667 |
Kind Code |
A1 |
Georges; Elias ; et
al. |
October 14, 2010 |
Vimentin Directed Diagnostics And Therapeutics For Multidrug
Resistant Neoplastic Disease
Abstract
Disclosed are methods for detecting multidrug resistance in
neoplastic or damaged cells or multidrug resistant (MDR) neoplastic
or damaged cells by detecting an increase in the cell surface
expression of vimentin protein in such cells as compared to the
level of cell surface expression of vimentin protein in a normal
cell or a non-MDR neoplastic cell.
Inventors: |
Georges; Elias; (Laval,
CA) ; Serfass; Lucile; (Montreal, CA) ;
Bonneau; Anne-Marie; (Laval, CA) ; Dallaire;
Frederic; (Montreal, CA) |
Correspondence
Address: |
WILMERHALE/BOSTON
60 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
35599927 |
Appl. No.: |
12/260892 |
Filed: |
October 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10736889 |
Dec 15, 2003 |
7550256 |
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12260892 |
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60433480 |
Dec 13, 2002 |
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Current U.S.
Class: |
424/1.49 ;
424/174.1; 424/178.1; 424/277.1; 424/278.1; 424/450; 435/6.14;
435/7.23; 536/24.31 |
Current CPC
Class: |
A61K 51/1045 20130101;
A61K 47/6823 20170801; A61K 47/6851 20170801; A61P 35/00 20180101;
A61K 45/06 20130101; G01N 2800/44 20130101 |
Class at
Publication: |
424/1.49 ;
435/7.23; 435/6; 536/24.31; 424/174.1; 424/450; 424/277.1;
424/278.1; 424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/574 20060101 G01N033/574; C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; A61K 9/127 20060101
A61K009/127; A61K 51/10 20060101 A61K051/10; A61K 39/00 20060101
A61K039/00; A61P 35/00 20060101 A61P035/00 |
Claims
1.-9. (canceled)
10. A method for detecting a multidrug resistant cell in a patient
comprising: (a) administering to the patient, a vimentin binding
agent operably linked to a detectable label; and (b) detecting the
label operably linked to the vimentin binding agent, wherein the
vimentin binding agent specifically binds to cell surface-expressed
vimentin present on a multidrug resistant cell in the patient.
11. The method of claim 10, wherein the vimentin binding agent is
an antibody or fragment thereof.
12. The method of claim 10, wherein the vimentin binding agent is
selected from the group consisting of modified LDL, NLK1 protein,
vimentin, desmin, glial fibrillary acidic protein, and peripherin,
fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A.
13. The method of claim 10, wherein the vimentin binding agent is
selected from the group consisting of natural ligands, synthetic
small molecules, chemicals, nucleic acids, peptides, proteins, and
antibodies.
14. The method of claim 10, wherein the detectable label is
selected from the group consisting of fluorophores, chemical dyes,
radioactive compounds, chemoluminescent compounds, magnetic
compounds, paramagnetic compounds, promagnetic compounds, enzymes
that yield a colored product, enzymes that yield a chemoluminescent
product, and enzymes that yield a magnetic product.
15. The method of claim 14, wherein the multidrug resistant cell is
a neoplastic cell.
16. The method of claim 15, wherein the neoplastic cell is selected
from the group consisting of a breast cancer cell, an ovarian
cancer cell, a myeloma cancer cell, a lymphoma cancer cell, a
melanoma cancer cell, a sarcoma cancer cell, a leukemia cancer
cell, a retinoblastoma cancer cell, a hepatoma cancer cell, a
glioma cancer cell, a mesothelioma cancer cell, and a carcinoma
cancer cell.
17. The method of claim 15, wherein the neoplastic cell is selected
from the group consisting of a promyleocytic leukemia cell, a T
lymphoblastoid cell, a breast epithelial cell, and an ovarian
cell.
18. The method of claim 10, wherein the patient is a human.
19. The method of claim 18, wherein the patient is suffering from a
disease or disorder caused by the presence of the multidrug
resistant cell.
20. A kit for diagnosing or detecting multidrug resistance in a
test neoplastic cell comprising: a) a first probe for the detection
of vimentin; and b) a second probe for the detection of a multidrug
resistance marker selected from the group consisting of
nucleophosmin and HSC70.
21. A kit for diagnosing or detecting multidrug resistance in a
test neoplastic cell comprising: a) a first probe for the detection
of vimentin; and b) a second probe for the detection of a marker
selected from the group consisting of MDR1, MDR3, MRP1, MRP5, and
LRP.
22. The kit of claim 21, wherein the probe for detecting vimentin
is an anti-vimentin antibody.
23. The kit of claim 21, wherein the probe for detecting vimentin
is a vimentin ligand selected from the group consisting of LDL,
NLK1 protein, vimentin, desmin, glial fibrillary acidic protein,
and peripherin, fimbrin, RhoA-binding kinase alpha, and protein
phosphatase 2A.
24. The kit of claim 20, wherein the second probe is selected from
the group consisting of a nucleophosmin antibody and an HSC70
antibody.
25. The kit of claim 20, wherein the second probe is selected from
the group consisting of a nucleophosmin ligand and an HSC70
ligand.
26. The kit of claim 21, wherein the first probe detects vimentin
present on the surface of the test neoplastic cell.
27. The kit of claim 21, wherein the second probe detects a marker
present of the surface of the test neoplastic cell.
28. The kit of claim 21, wherein the second probe is selected from
the group consisting of: an MDR1 antibody, an MDR3 antibody, an
MRP1 antibody, an MRP3 antibody, and an LRP antibody.
29. A cell surface vimentin in situ detection probe for the
detection of cell surface vimentin in a patient, comprising a
vimentin binding component and a detectable label for detection in
situ.
30. The cell surface vimentin in situ detection probe of claim 29,
wherein the vimentin binding component is an antibody.
31. The cell surface vimentin in situ detection probe of claim 29,
wherein the detectable label is Technetium.
32. A cell surface vimentin-targeted agent for treating or
preventing a multi-drug resistant neoplasm, comprising a vimentin
binding component and a therapeutic component, wherein the vimentin
binding component targets the therapeutic component to the
multi-drug resistant neoplasm and thereby treats the multi-drug
resistant neoplasm.
33. The agent of claim 32, wherein the vimentin binding component
is an anti-vimentin antibody.
34. The agent of claim 32, wherein the vimentin binding component
is selected from the group consisting of LDL, NLK1 protein,
vimentin, desmin, glial fibrillary acidic protein, peripherin,
fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A.
35. The agent of claim 32, wherein said vimentin binding component
is selected from the group consisting of natural ligands, synthetic
small molecules, chemicals, nucleic acids, peptides, proteins,
antibodies, and vimentin binding fragments thereof.
36. The agent of claim 32, wherein the therapeutic component is
selected from the group consisting of Actinomycin, Adriamycin,
Altretamine, Asparaginase, Bleomycin, Busulfan, Capecitabine,
Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,
Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,
Daunorubicin, Docetaxel, Doxorubicin, Epoetin, Etoposide,
Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin,
Ifosfamide, Imatinib, Irinotecan, Lomustine, Mechlorethamine,
Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane,
Mitoxantrone, Paclitaxel, Pentostatin, Procarbazine, Taxol,
Teniposide, Topotecan, Vinblastine, Vincristine, and
Vinorelbine.
37. The agent of claim 32, wherein the therapeutic component is in
a liposome formulation.
38. The agent of claim 32, wherein the therapeutic component is a
radioisotope.
39. The agent of claim 38, wherein the radioisotope is selected
from the group consisting of .sup.90Y, .sup.125I, .sup.131I,
.sup.211At, and .sup.213Bi.
40. The agent of claim 32, wherein the therapeutic component is a
toxin capable of killing or inducing the killing of the targeted
multi-drug resistant neoplastic cell.
41. The agent of claim 40, wherein the toxin is selected from the
group consisting of a Pseudomonas exotoxin, a diphtheria toxin, a
plant ricin toxin, a plant abrin toxin, a plant saporin toxin, a
plant gelonin toxin, and pokeweed antiviral protein.
42. The agent of claim 32, wherein the vimentin binding component
binds to the surface of the target cell and the therapeutic element
is internalized and arrests growth of the cell, compromises
viability of the cell or kills the cell.
43. A vaccine for treating or preventing a multi-drug resistant
neoplasm, comprising a vimentin polypeptide, or vimentin
polypeptide subsequence thereof, and at least one pharmaceutically
acceptable vaccine component.
44. The vaccine of claim 43, wherein the vimentin polypeptide or
polypeptide subsequence is a human vimentin polypeptide sequence of
SEQ ID NO.: 1.
45. The vaccine of claim 43, wherein the vimentin polypeptide
subsequence is at least eight amino acids long.
46. The vaccine of claim 45, wherein the vimentin polypeptide
subsequence comprises a hapten.
47. The vaccine of claim 43, wherein the pharmaceutically
acceptable vaccine component is an adjuvant.
48. The vaccine of claim 47, wherein the adjuvant is selected from
the group consisting of aluminum hydroxide, aluminum phosphate,
calcium phosphate, oil emulsion, a bacterial product, whole
inactivated bacteria, an endotoxins, cholesterol, a fatty acid, an
aliphatic amine, a paraffinic compound, a vegetable oil,
monophosphoryl lipid A, a saponin, and squalene.
49. A method of treating or preventing a multidrug resistant
neoplasm in a subject comprising administering a cell surface
vimentin-targeted therapeutic agent of claim 32.
50. The method of claim 49, wherein the neoplasm is selected from
the group consisting of a breast cancer, an ovarian cancer, a
myeloma, a lymphoma, a melanoma, a sarcoma, a leukemia, a
retinoblastoma, a hepatoma, a glioma, a mesothelioma, and a
carcinoma.
51. The method of claim 49, wherein the subject is a human
patient.
52. The method of claim 51, wherein the human patient is suffering
from a disease or disorder caused by the presence of the multi-drug
resistant cell.
53. The method of claim 49, wherein the neoplasm is from a tissue
selected from the group consisting of blood, bone marrow, spleen,
lymph node, liver, thymus, kidney, brain, skin, gastrointestinal
tract, eye, breast, prostate, and ovary.
54. A method of treating or preventing a multidrug resistant
neoplasm in a subject comprising administering a vimentin vaccine
of claim 43.
55. The method of claim 54, wherein the neoplasm is selected from
the group consisting of a breast cancer, an ovarian cancer, a
myeloma, a lymphoma, a melanoma, a sarcoma, a leukemia, a
retinoblastoma, a hepatoma, a glioma, a mesothelioma, and a
carcinoma.
56. The method of claim 54, wherein the subject is a human
patient.
57. The method of claim 56, wherein the human patient is suffering
from a disease or disorder caused by the presence of the multi-drug
resistant cell.
58. The method of claim 54, wherein the neoplasm is from a tissue
selected from the group consisting of blood, bone marrow, spleen,
lymph node, liver, thymus, kidney, brain, skin, gastrointestinal
tract, eye, breast, prostate, and ovary.
59-65. (canceled)
66. A method for detecting a neoplastic cell in a patient
comprising: (a) administering to the patient, a vimentin binding
agent operably linked to a detectable label; and (b) detecting the
label operably linked to the vimentin binding agent, wherein the
vimentin binding agent specifically binds to cell surface-expressed
vimentin present on a neoplastic cell in the patient.
67. The method of claim 66, wherein the vimentin binding agent is
an antibody or fragment thereof.
68. The method of claim 66, wherein the vimentin binding agent is
selected from the group consisting of modified LDL, NLK1 protein,
vimentin, desmin, glial fibrillary acidic protein, and peripherin,
fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A.
69. The method of claim 66, wherein the vimentin binding agent is
selected from the group consisting of natural ligands, synthetic
small molecules, chemicals, nucleic acids, peptides, proteins,
antibodies, and fragments thereof.
70. The method of claim 66, wherein the detectable label is
selected from the group consisting of fluorophores, chemical dyes,
radioactive compounds, chemoluminescent compounds, magnetic
compounds, paramagnetic compounds, promagnetic compounds, enzymes
that yield a colored product, enzymes that yield a chemoluminescent
product, and enzymes that yield a magnetic product.
71. The method of claim 66, wherein the neoplastic cell is selected
from the group consisting of a breast cancer cell, an ovarian
cancer cell, a myeloma cancer cell, a lymphoma cancer cell, a
melanoma cancer cell, a sarcoma cancer cell, a leukemia cancer
cell, a retinoblastoma cancer cell, a hepatoma cancer cell, a
glioma cancer cell, a mesothelioma cancer cell, and a carcinoma
cancer cell.
72. The method of claim 66, wherein the neoplastic cell is selected
from the group consisting of a promyleocytic leukemia cell, a T
lymphoblastoid cell, a breast epithelial cell, and an ovarian
cell.
73. The method of claim 66, wherein the patient is a human.
74. The method of claim 73, wherein the patient is suffering from a
disease or disorder caused by the presence of the neoplastic
cell.
75. A kit for diagnosing or detecting neoplasia, comprising: a) a
first probe for the detection of vimentin; and b) a second probe
for the detection of a neoplasia marker selected from the group
consisting of nucleophosmin and HSC70.
76. The kit of claim 75, wherein the probe for detecting vimentin
is an anti-vimentin antibody or binding fragment thereof.
77. The kit of claim 75, wherein the probe for detecting vimentin
is a vimentin ligand selected from the group consisting of LDL,
NLK1 protein, vimentin, desmin, glial fibrillary acidic protein,
and peripherin, fimbrin, RhoA-binding kinase alpha, and protein
phosphatase 2A.
78. The kit of claim 75, wherein the second probe is selected from
the group consisting of a nucleophosmin antibody and an HSC70
antibody.
79. The kit of claim 75, wherein the second probe is selected from
the group consisting of a nucleophosmin ligand and an HSC70
ligand.
80. The kit of claim 75, wherein the first probe detects vimentin
present on the surface of the test cell if it is neoplastic.
81. The kit of claim 75, wherein the second probe detects a marker
present of the surface of the test cell if it is neoplastic.
82. A cell surface vimentin-targeted agent for treating a cancerous
neoplastic cell growth comprising a vimentin binding component and
a therapeutic component, wherein the vimentin binding component
targets the therapeutic component to the neoplastic cell growth and
thereby treats the cancer.
83. The agent of claim 82, wherein the vimentin binding component
is an anti-vimentin antibody.
84. The agent of claim 82, wherein the vimentin binding component
is selected from the group consisting of LDL, NLK1 protein,
vimentin, desmin, glial fibrillary acidic protein, and peripherin,
fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A.
85. The agent of claim 82, wherein said vimentin binding component
is selected from the group consisting of natural ligands, synthetic
small molecules, chemicals, nucleic acids, peptides, proteins,
antibodies, and vimentin binding fragments thereof.
86. The agent of claim 82, wherein the therapeutic component is
selected from the group consisting of Actinomycin, Adriamycin,
Altretamine, Asparaginase, Bleomycin, Busulfan, Capecitabine,
Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,
Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,
Daunorubicin, Docetaxel, Doxorubicin, Epoetin, Etoposide,
Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin,
Ifosfamide, Imatinib, Irinotecan, Lomustine, Mechlorethamine,
Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane,
Mitoxantrone, Paclitaxel, Pentostatin, Procarbazine, Taxol,
Teniposide, Topotecan, Vinblastine, Vincristine, and Vinorelbine
and combinations thereof.
87. The agent of claim 82, wherein the therapeutic component is in
a liposome formulation.
88. The agent of claim 82, wherein the therapeutic component is a
radioisotope.
89. The agent of claim 88, wherein the radioisotope is selected
from the group consisting of .sup.90Y, .sup.111In, .sup.125I,
.sup.131I, .sup.211At, and .sup.213Bi.
90. The agent of claim 82, wherein the therapeutic component is a
toxin capable of killing or inducing the killing of the targeted
neoplastic cell.
91. The agent of claim 90, wherein the toxin is selected from the
group consisting of a Pseudomonas exotoxin, a diphtheria toxin, a
plant ricin toxin, a plant abrin toxin, a plant saporin toxin, a
plant gelonin toxin, and pokeweed antiviral protein.
92. The agent of claim 82, wherein the vimentin binding component
binds to the surface of the target cell and the therapeutic element
is internalized and arrests growth of the cell, compromises
viability of the cell, or kills the cell.
93. A vaccine for treating or preventing a neoplasm comprising a
vimentin polypeptide, or vimentin polypeptide subsequence thereof,
and at least one pharmaceutically acceptable vaccine component.
94. The vaccine of claim 93, wherein the vimentin polypeptide or
polypeptide subsequence is a human vimentin polypeptide sequence
set forth in SEQ ID NO.: 1.
95. The vaccine of claim 93, wherein the vimentin polypeptide
subsequence is at least eight amino acids long.
96. The vaccine of claim 95, wherein the vimentin polypeptide
subsequence comprises a hapten.
97. The vaccine of claim 93, wherein the pharmaceutically
acceptable vaccine component is an adjuvant.
98. The vaccine of claim 97, wherein the adjuvant is selected from
the group consisting of aluminum hydroxide, aluminum phosphate,
calcium phosphate, oil emulsion, a bacterial product, whole
inactivated bacteria, an endotoxins, cholesterol, a fatty acid, an
aliphatic amine, a paraffinic compound, a vegetable oil,
monophosphoryl lipid A, a saponin, and squalene.
99. A method of treating or preventing a neoplasm in a subject
comprising administering a cell surface vimentin-targeted
therapeutic agent of claim 82.
100. The method of claim 99, wherein the neoplasm is selected from
the group consisting of a breast cancer, an ovarian cancer, a
myeloma, a lymphoma, a melanoma, a sarcoma, a leukemia, a
retinoblastoma, a hepatoma, a glioma, a mesothelioma, and a
carcinoma.
101. The method of claim 99, wherein the subject is a human
patient.
102. The method of claim 101, wherein said human patient is
suffering from a disease or disorder caused by the presence of the
multi-drug resistant cell.
103. The method of claim 99, wherein the neoplasm is from a tissue
selected from the group consisting of blood, bone marrow, spleen,
lymph node, liver, thymus, kidney, brain, skin, gastrointestinal
tract, eye, breast, prostate and ovary.
104. A method of treating or preventing a neoplasm in a subject
comprising administering a vimentin vaccine of claim 93.
105. The method of claim 104, wherein the neoplasm is selected from
the group consisting of a breast cancer, an ovarian cancer, a
myeloma, a lymphoma, a melanoma, a sarcoma, a leukemia, a
retinoblastoma, a hepatoma, a glioma, a mesothelioma, and a
carcinoma.
106. The method of claim 104, wherein said subject is a human
patient.
107. The method of claim 106, wherein said human patient is
suffering from a disease or disorder caused by the presence of the
neoplastic cell.
108. The method of claim 104, wherein the neoplasm is from a tissue
selected from the group consisting of blood, bone marrow, spleen,
lymph node, liver, thymus, kidney, brain, skin, gastrointestinal
tract, eye, breast, prostate, and ovary.
109. The method of claim 10, wherein the vimentin binding agent is
an anti-vimentin antibody.
110. The method of claim 109, wherein the anti-vimentin antibody is
operably linked to a radiolabel.
Description
[0001] This Application claims the benefit of priority to U.S.
Provisional Application No. 60/433,480, filed Dec. 13, 2002, the
specification of which is incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the field of diagnostics and
therapeutics. In particular, this invention relates to the
detection and treatment of neoplastic and/or damaged cells and, in
addition, to the detection and treatment of multidrug resistant
neoplastic and/or damaged cells.
1. BACKGROUND OF THE INVENTION
[0003] Diseases such as cancer or those caused by
pathogen-infection are often treated with drugs (e.g.,
chemotherapeutics and antibiotics). In order to kill the cancer or
diseased cells, the drug(s) must enter the cells and reach an
effective dose so as to interfere with essential biochemical
pathways. However, some cells evade being killed by the drug by
developing resistance to it (termed "drug resistance"). Moreover,
in some cases, cancer cells (also called tumor cells or neoplastic
cells) and damaged cells (e.g., pathogen-infected cells), or the
pathogens themselves, develop resistance to a broad spectrum of
drugs, including drugs that were not originally used for treatment.
This phenomenon is termed "multidrug resistance" (MDR). There are
different types of MDR, each associated with a different biological
mechanism, and there are specific biological "markers" for
different types of MDR that are clinically useful for detecting and
diagnosing each type of MDR.
[0004] MDR can involve cancer cells or infectious pathogens such as
viruses, bacteria, parasites and other microorganisms that may be
present in cells or body fluids. The emergence of the MDR phenotype
is the major cause of failure in the treatment of infectious
diseases (see Davies J., Science 264: 375-382, 1994; Poole, K.,
Cur. Opin. Microbiol. 4: 500-5008, 2001). Patient cross-resistance
to different anti-microbial and anti-cancer agents, which are
structurally and functionally distinct, can cause serious problems
for pathogen-infected patients. Similarly, the development of
multidrug resistant cancer cells is the principal reason for
treatment failure in cancer patients (see Gottesman, M. M., Ann.
Rev. Med. 53: 615-627, 2000).
[0005] Multidrug resistance is multifactoral. The classic MDR
mechanism involves alterations in the gene for the highly
evolutionarily conserved plasma membrane protein (P-glycoprotein or
MDR 1) that actively transports or pumps drugs out of the cell or
microorganism (Volm M. et al., Cancer 71: 3981-3987, 1993); Bradley
and Ling, Cancer Metastasis Rev. 13: 223-233, 1994). Both human
cancer cells and infectious bacterial pathogens may develop classic
MDR via mechanisms involving the overexpression of P-glycoprotein
due to amplification of the gene encoding P-glycoprotein. The
overexpression of P-glycoprotein mRNA or protein in MDR cancer
cells or pathogen-infected cells is a biological marker for MDR.
Diagnostic tests and therapeutic methods have been developed that
make use of the overexpression of P-glycoprotein marker to diagnose
and to treat MDR cancer and pathogen infections (Szakacs G. et al.,
Pathol. Oncol. Res. 4: 251-257, 1998). However, because various
normal tissues express different amounts of P-glycoprotein, there
are significant problems with side effects as any therapy that
targets P-glycoprotein on the cell surface of MDR cancer cells
would also affect those normal tissues that also have a relatively
high level of P-glycoprotein expression, such as liver, kidney,
stem cells, and blood-brain barrier epithelium.
[0006] "Atypical MDR" is a term used to describe MDR cancer cells
or pathogens wherein the mechanism of multidrug resistance is
unknown, novel, or different from the classic mechanism involving
P-glycoprotein. For example, human lung tumors are multidrug
resistant but do not have alterations in P-glycoprotein (see Cole
S. P. et al., Science 258: 1650-1654, 1992). Rather, they express
another drug transporter (the multidrug resistance associated
protein or "MRP1"). A new mechanism of MDR was recently described
that involves lung resistance related protein, which is a marker
for this type of atypical MDR (Rome L. H. et al., PCT Pub. No.
WO9962547). Other examples of atypical markers for MDR include
MRP5, which is a novel mammalian efflux pump for nucleoside analog
drugs (see Fridland and Schuetz, PCT Pub. No. WO0058471) and
certain sphingoglycolipids (see U.S. Pat. No. 6,090,565). It should
be noted that MDR cells may express more than one MDR marker (both
classical P-glycoprotein and atypical markers) simultaneously on
the same cell, and that the markers are expressed independently
(Grandjean F, et al. Anticancer Drugs, 12:247-258, 2001). It is
therefore possible to combine treatments directed against more than
one cell surface MDR marker and potentially against more than one
mechanism of MDR.
[0007] Intermediate filaments are a major component of the
cytoskeleton of higher eukaryotic cells. Intermediate filaments are
composed of a number of different structurally related proteins and
different intermediate filament protein genes are expressed in
different tissues. Studies involving spontaneous and experimentally
produced mutations in intermediate filament genes have demonstrated
that intermediate filaments function to enhance the mechanical
stability of epidermal and muscle cells (Evans R. M., BioEssays,
20: 79-86, 1998). Vimentin (gi/4507895) is a homodimeric
intracellular protein found in class III intermediate filaments in
mesenchymal and other nonepithelial cells and in the Z disk of
skeletal and cardiac muscle cells. (See, e.g., Evans, R. M. (1998)
Bioassays 20:79-86; Herrmann, H., Aebi, U. (2000) Curr. Opin. Cell
Biol. 12:79-90, ibid. (1998) Subcell Biochem 31:319-62.). Vimentin
is a phosphoprotein whose phosphorylation is enhanced during cell
division. Both the human and murine vimentin genes have been
characterized (see, e.g., Kryszke et al. (1998) Pathol. Biol.
46:39-45; Paulin, D. (1989) Pathol. Biol. 37:277-82). Purified
eukaryotic vimentin has a molecular mass of 54 kDa, is soluble in
its native form and insoluble when denatured. Vimentin gene
regulation appears to participate in several steps of viral
infections (reviewed in Kryszke et al. (1998) Pathol. Biol.
46:39-45).
[0008] Various cytoskeleton modifications are associated with
malignant cell transformation and have been used as prognostic
factors. In contrast to the in vivo situation where vimentin
expression is characteristic of cells of mesenchymal origin,
vimentin synthesis is characteristic of all proliferating cells in
vitro regardless of their embryonal origin, and is switched off
upon differentiation of certain precursor cells. Vimentin gene
expression is upregulated in some metastatic tumor cells, and is
thus a marker for oncogenic progression (see, Kryszke et al. (1998)
Pathol. Biol. 46:39-45; Osborn et al. (1989) Curr. Comm. in Mol.
Biol., Cold Spring Harbor Press).
[0009] Vimentin and other IF proteins have been used in the
histological classification of human tumors (for reviews see
Ramaekers et al. (1982) Cold Spring Harb. Symp. Quant. Biol. 46:
331-339; Thomas et al. (1999) Clin. Cancer Res. 5:2698-2703).
Vimentin in particular has been used as a marker for
de-differentiation in several types of tumors.
[0010] Tumor markers that co-localize with vimentin and other
intermediate filaments have been described. For example, De
Bernard, Marina (WO0127269) describes a novel marker for
neuroblastomas called VIP54, a protein that is closely associated
with vimentin and desmin filaments, and the use of this internal
marker for the detection and cellular imaging of intermediate
filaments (particularly class-III vimentin or desmin filaments) as
a markers for tumor development and/or progression.
[0011] Finally, there are several examples that show changes in the
expressed level and intracellular distribution of vimentin in
different types of human solid tumors and solid tumor cell lines,
often in association with the development of multidrug resistance
(Conforti G, et al., Br. J. Cancer, 3:505-511, 1995; Moran E. et
al., Eur J. Cancer, 33:652-660, 1997).
[0012] There remains a need in both humans and animals for
detecting, treating, preventing, and/or reversing the development
of both classical and atypical MDR phenotypes in cancer cells and
non-cancerous damaged cells, regardless of how the MDR arises
(e.g., naturally occurring or drug-induced). In addition, the
ability to identify and to make use of reagents that identify
multiple drug resistant cells has clinical potential for
improvements in the treatment, monitoring, diagnosis, and medical
imaging of multidrug resistant cancer and multidrug resistant
damaged cells.
2. SUMMARY OF THE INVENTION
[0013] The invention is based, in part, upon the discovery that
full-length vimentin, a normally intracellular protein, is
expressed in full length on the cell surface of neoplastic cells
and damaged cells, and is expressed more abundantly on the cell
surfaces of multidrug resistant (MDR) neoplastic cells and MDR
damaged cells. Although lower levels of vimentin are expressed on
the cell surface of drug-sensitive neoplastic cells, in contrast to
other cell surface MDR markers (such as P-glycoprotein), vimentin
is expressed in only negligible amounts on the cell surface of
normal cells of the body. By "negligible amounts" is meant fewer
than 100 molecules of vimentin on the cell surface. Thus, the
invention allows the use of binding agents, to which are bound
toxins or other therapeutic or diagnostic agents, that specifically
bind to vimentin without detrimental side effects, since the only
non-vimentin cells that are being killed are drug-sensitive
neoplastic cells or damaged cells; normal cells remain
unharmed.
[0014] In one aspect, the invention provides a method for detecting
multidrug resistance or multidrug resistance potential in a test
neoplastic cell by measuring a level of cell surface-expressed
vimentin protein in the test neoplastic cell of a given origin or
cell type, and comparing it to the level of cell surface-expressed
vimentin in a nonresistant neoplastic cell of the same origin or
cell type. If the level of cell surface-expressed vimentin in the
test neoplastic cell is greater than the level of cell
surface-expressed vimentin in the nonresistant neoplastic cell of
the same given origin or cell type, then the test neoplastic cell
is multidrug resistant or has multidrug resistance potential. In
certain embodiments, the level of cell surface-expressed vimentin
in the test neoplastic cell is measured by isolating a cytoplasmic
membrane fraction from the cell and measuring the level of vimentin
in the cytoplasmic membrane fraction. In other embodiments, the
level of cell surface-expressed vimentin in the test neoplastic
cell is measured by contacting the cell with an anti-vimentin
antibody and measuring the level of antibody bound to cell surface
vimentin. For example, the level of antibody bound to cell surface
vimentin may be measured by immunofluorescence emission or
radiolabel.
[0015] In certain embodiments of this aspect of the invention, the
test neoplastic cell is a promyleocytic leukemia cell, a T
lymphoblastoid cell, a breast epithelial cell, or an ovarian cell.
In other embodiments the test neoplastic cell is a lymphoma cell, a
melanoma cell, a sarcoma cell, a leukemia cell, a retinoblastoma
cell, a hepatoma cell, a myeloma cell, a glioma cell, a
mesothelioma cell, or a carcinoma cell. In still other embodiments
of the invention, the test neoplastic cell is from a tissue such as
blood, bone marrow, spleen, lymph node, liver, thymus, kidney,
brain, skin, gastrointestinal tract, eye, breast, prostate, or
ovary. In further embodiments, the nonresistant neoplastic
(control) cell is a drug-sensistive neoplastic cell line such as
HL60, NB4, CEM, HSB2 Molt4, MCF-7, MDA, SKOV-3, or 2008.
[0016] In another aspect, the invention provides a method for
detecting a multidrug resistant cell or cells in a patient by
administering to the patient, a vimentin binding agent operably
linked to a detectable label. The label is operably linked to the
vimentin binding agent, which specifically binds to cell
surface-expressed vimentin present on the multidrug resistant
cell(s) in the patient, and is then detected, thereby locating the
presence of the multidrug resistant cell(s) (if any) in the
patient. In certain embodiments, the vimentin binding agent used is
an antibody or fragment thereof. In other embodiments, the vimentin
binding agent is a vimentin ligand such as modified LDL, NLK1
protein, vimentin, desmin, glial fibrillary acidic protein, or
peripherin, fimbrin, RhoA-binding kinase alpha, or protein
phosphatase 2A. In particular embodiments, the vimentin binding
agent is a natural ligand, a synthetic small molecule, a chemical,
a nucleic acid, a peptide, a protein or an antibody. In other
embodiments, the detectable label is a fluorophore, a chemical dye,
a radioactive compound, a chemoluminescent compound, a magnetic
compound, a paramagnetic compound, a promagnetic compound, an
enzyme that yields a colored product, an enzyme that yields a
chemoluminescent product, or an enzymes that yields a magnetic
product.
[0017] In certain embodiments of this aspect, the multidrug
resistant cell is a neoplastic cell. In particular embodiments, the
neoplastic cell is a breast cancer cell, an ovarian cancer cell, a
myeloma cancer cell, a lymphoma cancer cell, a melanoma cancer
cell, a sarcoma cancer cell, a leukemia cancer cell, a
retinoblastoma cancer cell, a hepatoma cancer cell, a glioma cancer
cell, a mesothelioma cancer cell, or a carcinoma cancer cell. In
some embodiments, the neoplastic cell is a promyleocytic leukemia
cell, a T lymphoblastoid cell, a breast epithelial cell, or an
ovarian cell. In particular embodiments, the patient is a human,
such as a human patient that is suffering from a disease or
disorder caused by the presence of the multidrug resistant
cell.
[0018] In another aspect, the invention provides kits for
diagnosing or detecting multidrug resistance in a test neoplastic
cell. The kits include one probe for the detection of vimentin and
a second probe for the detection of another multidrug resistance
marker such as nucleophosmin or HSC70. In certain embodiment, these
kits of the invention include a first probe for the detection of
vimentin and a second probe for the detection of another multidrug
resistance marker such as MDR1, MDR3, MRP1, MRP5, or LRP. In
particular embodiments, the kits include anti-vimentin antibody as
the probe for detecting vimentin. In other embodiments, the kits
include a vimentin ligand such as LDL, NLK1 protein, vimentin,
desmin, glial fibrillary acidic protein, and peripherin, fimbrin,
RhoA-binding kinase alpha, or protein phosphatase 2A. In further
embodiments, the kits include a nucleophosmin antibody or an HSC70
antibody as probes for detecting the second, non-vimentin multidrug
resistance marker. In other embodiments, the second probe may be a
nucleophosmin ligand (such as protein kinase R (PKR), RNA,
retinoblastoma protein, IRF-1, or a nuclear localization signals
(NLS) such as the N-terminal nuclear localization signal of Rex
protein) or an HSC70 ligand (such as Alzheimer's tau protein,
BAG-1, small glutamine-rich tetratricopeptide repeat-containing
protein (SGT), (aa 642-658) of rotavirus VP5 protein, auxilin, or
the immunosuppressant 5-deoxyspergualin (DSG)).
[0019] In another embodiment of this aspect of the invention, the
kit includes a probe that detects vimentin present on the surface
of the test neoplastic cell. In other embodiments, the kit includes
a second probe that detects another (non-vimentin) marker present
of the surface of the test MDR neoplastic cell. In certain
embodiment, the kit includes a second probe that is an MDR1
antibody, an MDR3 antibody, an MRP1 antibody, an MRP3 antibody, or
an LRP antibody.
[0020] In another aspect, the invention provides a cell surface
vimentin in situ detection probe for the detection of cell surface
vimentin in a patient. This cell surface vimentin probe has a
vimentin binding component and a detectable label for detection in
situ (e.g., a Technetium label). In some embodiments, the vimentin
binding component is an antibody.
[0021] In yet another aspect, the invention provides a cell surface
vimentin-targeted agent for treating or preventing a multi-drug
resistant neoplasm. This vimentin-targeted agent includes both a
vimentin binding component and a therapeutic component which act
together such that the vimentin binding component targets the
therapeutic component to the multi-drug resistant neoplasm and
thereby treats the multi-drug resistant neoplasm. In certain
embodiments, the vimentin binding component is an anti-vimentin
antibody. In other embodiments, the vimentin binding component is a
vimentin ligand such as LDL, NLK1 protein, vimentin, desmin, glial
fibrillary acidic protein, peripherin, fimbrin, RhoA-binding kinase
alpha, or protein phosphatase 2A. In particular embodiments, the
vimentin binding component is a natural ligands, synthetic small
molecules, chemicals, nucleic acids, peptides or protein.
[0022] In certain useful embodiments of this aspect of the
invention, the therapeutic component is a chemotherapeutic agent
such as Actinomycin, Adriamycin, Altretamine, Asparaginase,
Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine,
Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine,
Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin,
Epoetin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine,
Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Irinotecan,
Lomustine, Mechlorethamine, Melphalan, Mercaptopurine,
Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Paclitaxel,
Pentostatin, Procarbazine, Taxol, Teniposide, Topotecan,
Vinblastine, Vincristine, or Vinorelbine. In particular
embodiments, the therapeutic component is in a liposome
formulation.
[0023] In other embodiments, the therapeutic component is a
radioisotope such as .sup.90Y, .sup.125I, .sup.131I, .sup.211At or
.sup.213Bi.
[0024] In still other embodiments, the therapeutic component is a
toxin capable of killing or inducing the killing of the targeted
multi-drug resistant neoplastic cell. Such toxins for use in the
invention include Pseudomonas exotoxin, diphtheria toxin, plant
ricin toxin, plant abrin toxin, plant saporin toxin, plant gelonin
toxin and pokeweed antiviral protein.
[0025] In particularly useful embodiments, the vimentin binding
component of the cell surface vimentin-targeted therapeutic agent
binds to the surface of the target cell, and the therapeutic
element is internalized and arrests growth of the cell, compromises
viability of the cell or kills the cell.
[0026] In another aspect, the invention provides a method of
treating or preventing a multidrug resistant neoplasm in a subject
by administering any of the cell surface vimentin-targeted
therapeutic agents described above. In certain embodiments of this
aspect, the neoplasm is a breast cancer, an ovarian cancer, a
myeloma, a lymphoma, a melanoma, a sarcoma, a leukemia, a
retinoblastoma, a hepatoma, a glioma, a mesothelioma, or a
carcinoma. In further embodiments, the neoplasm is from a tissue
such as blood, bone marrow, spleen, lymph node, liver, thymus,
kidney, brain, skin, gastrointestinal tract, eye, breast, prostate,
or ovary. In particular embodiments, the subject is a human
patient, such as a human patient suffering from a disease or
disorder caused by the presence of the multi-drug resistant
cell.
[0027] In yet another aspect, the invention provides vaccines for
treating or preventing multi-drug resistant neoplasms, comprising a
vimentin polypeptide, or vimentin polypeptide subsequence thereof,
and at least one pharmaceutically acceptable vaccine component. In
certain embodiments, the vimentin polypeptide or polypeptide
subsequence is a human vimentin polypeptide sequence having an
amino acid sequence of SEQ ID NO.: 1. In particular embodiments,
the vimentin polypeptide subsequence is at least eight amino acids
long, and, in certain embodiments, functions as a hapten.
[0028] In certain embodiments, the vaccine formulation includes an
adjuvant or other pharmaceutically acceptable vaccine component. In
particular embodiments, the adjuvant is aluminum hydroxide,
aluminum phosphate, calcium phosphate, oil emulsion, a bacterial
product, whole inactivated bacteria, an endotoxins, cholesterol, a
fatty acid, an aliphatic amine, a paraffinic compound, a vegetable
oil, monophosphoryl lipid A, a saponin, or squalene.
[0029] In another aspect, the invention provides a method of
treating or preventing a multidrug resistant neoplasm in a subject
by administering any of the vimentin vaccines described above. In
certain embodiments of this aspect, the neoplasm to be treated is a
breast cancer, an ovarian cancer, a myeloma, a lymphoma, a
melanoma, a sarcoma, a leukemia, a retinoblastoma, a hepatoma, a
glioma, a mesothelioma, or a carcinoma. In further embodiments, the
neoplasm is from a tissue such as blood, bone marrow, spleen, lymph
node, liver, thymus, kidney, brain, skin, gastrointestinal tract,
eye, breast, prostate, or ovary. In particular embodiments, the
subject is a human patient, such as a human patient is suffering
from a disease or disorder caused by the presence of the multi-drug
resistant cell.
[0030] In yet another aspect, the invention provides a method for
detecting whether a test cell is neoplastic by measuring the level
of cell surface-expressed vimentin protein in the test cell of a
given origin or cell type, and comparing it to the level of cell
surface-expressed vimentin in a normeoplastic cell of the same
origin or cell type. If the level of cell surface-expressed
vimentin in the test cell is greater than the level of cell
surface-expressed vimentin in the normeoplastic cell of the same
given origin or cell type, then the test cell is neoplastic.
[0031] In certain embodiments, the level of cell surface-expressed
vimentin in the test cell is measured by isolating a cytoplasmic
membrane fraction from the cell and measuring the level of vimentin
in the cytoplasmic membrane fraction. In other embodiments, the
level of cell surface-expressed vimentin in the test cell is
measured by contacting the cell with an anti-vimentin antibody and
measuring the level of antibody bound to cell surface vimentin. For
example, the level of antibody bound to cell surface vimentin may
be measured by immunofluorescence emission or radiolabel.
[0032] In certain embodiments of this aspect of the invention, the
test cell is a promyleocytic leukemia cell, a T lymphoblastoid
cell, a breast epithelial cell, or an ovarian cell. In other
embodiments the test cell is a lymphoma cell, a melanoma cell, a
sarcoma cell, a leukemia cell, a retinoblastoma cell, a hepatoma
cell, a myeloma cell, a glioma cell, a mesothelioma cell, or a
carcinoma cell. In still other embodiments of the invention, the
test cell is from a tissue such as blood, bone marrow, spleen,
lymph node, liver, thymus, kidney, brain, skin, gastrointestinal
tract, eye, breast, prostate, or ovary.
[0033] In another aspect, the invention provides a method for
detecting a neoplastic cell or cells in a patient by administering
to the patient, a vimentin binding agent operably linked to a
detectable label. The label is operably linked to the vimentin
binding agent, which specifically binds to cell surface-expressed
vimentin present on the neoplastic cell(s) in the patient, and is
then detected, thereby locating the presence of the neoplastic
cell(s) (if any) in the patient. In certain embodiments, the
vimentin binding agent used is an antibody or fragment thereof. In
other embodiments, the vimentin binding agent is a vimentin ligand
such as modified LDL, NLK1 protein, vimentin, desmin, glial
fibrillary acidic protein, or peripherin, fimbrin, RhoA-binding
kinase alpha, or protein phosphatase 2A. In particular embodiments,
the vimentin binding agent is a natural ligand, a synthetic small
molecule, a chemical, a nucleic acid, a peptide, a protein or an
antibody. In other embodiments, the detectable label is a
fluorophore, a chemical dye, a radioactive compound, a
chemoluminescent compound, a magnetic compound, a paramagnetic
compound, a promagnetic compound, an enzyme that yields a colored
product, an enzyme that yields a chemoluminescent product, or an
enzymes that yields a magnetic product.
[0034] In certain embodiments of this aspect, the neoplastic cell
is a breast cancer cell, an ovarian cancer cell, a myeloma cancer
cell, a lymphoma cancer cell, a melanoma cancer cell, a sarcoma
cancer cell, a leukemia cancer cell, a retinoblastoma cancer cell,
a hepatoma cancer cell, a glioma cancer cell, a mesothelioma cancer
cell, or a carcinoma cancer cell. In certain embodiments, the
neoplastic cell is a promyleocytic leukemia cell, a T
lymphoblastoid cell, a breast epithelial cell, or an ovarian cell.
In particular embodiments, the patient is a human, such as a human
patient that is suffering from a disease or disorder caused by the
presence of the neoplastic cell(s).
[0035] In another aspect, the invention provides kits for
diagnosing or detecting a neoplasm, which include at least one
probe for detecting vimentin, and at least one other probe for
detecting another neoplastic marker such as nucleophosmin or HSC70.
In one embodiment, the probe for detecting vimentin is an
anti-vimentin antibody or a binding fragment thereof. In other
embodiments, the probe for detecting vimentin is a vimentin ligand
such as modified LDL, NLK1 protein, vimentin, desmin, glial
fibrillary acidic protein, and peripherin, fimbrin, RhoA-binding
kinase alpha, or protein phosphatase 2A.
[0036] In certain embodiments, the second probe for detecting a
non-vimentin neoplastic marker is a nucleophosmin antibody or an
HSC70 antibody. In other embodiments, the second probe for
detecting a non-vimentin neoplastic marker is a nucleophosmin
ligand, such as protein kinase R (PKR), RNA, retinoblastoma
protein, IRF-1, or a nuclear localization signals (NLS) such as the
N-terminal nuclear localization signal of Rex protein as and an
HSC70 ligand. In still other embodiments, the second probe for
detecting a non-vimentin neoplastic marker is an HSC70 ligand, such
as Alzheimer's tau protein, BAG-1, small glutamine-rich
tetratricopeptide repeat-containing protein (SGT), (aa 642-658) of
rotavirus VP5 protein, auxilin, or the immunosuppressant
5-deoxyspergualin (DSG).
[0037] In particularly useful embodiments, the kit includes a first
probe which detects vimentin present on the surface of the test
cell if it is neoplastic, and a second probe which detects another
(non-vimentin) marker present of the surface of the test cell if it
is neoplastic.
[0038] In yet another aspect, the invention provides a cell surface
vimentin-targeted agent for treating a cancerous neoplastic cell
growth. The cell surface vimentin-targeted agent generally includes
a vimentin binding component and a therapeutic component. The
vimentin binding component targets the therapeutic component to the
neoplastic cell growth and thereby treats the cancer. The vimentin
binding component and the therapeutic component, therefore, act
together such that the vimentin binding component targets the
therapeutic component to the neoplasm to treat the neoplasm.
[0039] In certain embodiments, the vimentin binding component is an
anti-vimentin antibody. In other embodiments, the vimentin binding
component is a vimentin ligand such as LDL, NLK1 protein, vimentin,
desmin, glial fibrillary acidic protein, peripherin, fimbrin,
RhoA-binding kinase alpha, or protein phosphatase 2A. In particular
embodiments, the vimentin binding component is a natural ligands,
synthetic small molecules, chemicals, nucleic acids, peptides or
protein.
[0040] In certain useful embodiments of this aspect of the
invention, the therapeutic component is a chemotherapeutic agent
such as Actinomycin, Adriamycin, Altretamine, Asparaginase,
Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine,
Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide, Cytarabine,
Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin,
Epoetin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine,
Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Irinotecan,
Lomustine, Mechlorethamine, Melphalan, Mercaptopurine,
Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Paclitaxel,
Pentostatin, Procarbazine, Taxol, Teniposide, Topotecan,
Vinblastine, Vincristine, or Vinorelbine. In particular
embodiments, the therapeutic component is in a liposome
formulation.
[0041] In other embodiments, the therapeutic component is a
radioisotope such as .sup.90Y, .sup.125I, .sup.131I, .sup.211At or
.sup.213Bi.
[0042] In still other embodiments, the therapeutic component is a
toxin capable of killing or inducing the killing of the targeted
multi-drug resistant neoplastic cell. Such toxins for use in the
invention include Pseudomonas exotoxin, diphtheria toxin, plant
ricin toxin, plant abrin toxin, plant saporin toxin, plant gelonin
toxin and pokeweed antiviral protein.
[0043] In particularly useful embodiments, the vimentin binding
component of the cell surface vimentin-targeted therapeutic agent
binds to the surface of the target cell, and the therapeutic
element is internalized and arrests growth of the cell, compromises
viability of the cell or kills the cell.
[0044] In another aspect, the invention provides a method of
treating a neoplasm in a subject by administering any of the cell
surface vimentin-targeted therapeutic agents described above. In
certain embodiments of this aspect, the neoplasm is a breast
cancer, an ovarian cancer, a myeloma, a lymphoma, a melanoma, a
sarcoma, a leukemia, a retinoblastoma, a hepatoma, a glioma, a
mesothelioma, or a carcinoma. In further embodiments, the neoplasm
is from a tissue such as blood, bone marrow, spleen, lymph node,
liver, thymus, kidney, brain, skin, gastrointestinal tract, eye,
breast, prostate, or ovary. In particular embodiments, the subject
is a human patient, such as a human patient suffering from a
disease or disorder caused by the presence of the neoplasm.
[0045] In yet another aspect, the invention provides vaccines for
treating or preventing a neoplasm. These vaccines of the invention
include a vimentin polypeptide, or vimentin polypeptide subsequence
thereof, and at least one pharmaceutically acceptable vaccine
component. In certain embodiments, the vimentin polypeptide or
polypeptide subsequence is a human vimentin polypeptide sequence
having an amino acid sequence of SEQ ID NO: 1. In particular
embodiments, the vimentin polypeptide subsequence is at least eight
amino acids long, and, in certain embodiments, functions as a
hapten.
[0046] In certain embodiments, the vaccine formulation includes an
adjuvant or other pharmaceutically acceptable vaccine component. In
particular embodiments, the adjuvant is aluminum hydroxide,
aluminum phosphate, calcium phosphate, oil emulsion, a bacterial
product, whole inactivated bacteria, an endotoxins, cholesterol, a
fatty acid, an aliphatic amine, a paraffinic compound, a vegetable
oil, monophosphoryl lipid A, a saponin, or squalene.
[0047] In another aspect, the invention provides a method of
treating or preventing a neoplasm in a subject by administering any
of the vimentin vaccines described above. In certain embodiments of
this aspect, the neoplasm to be treated is a breast cancer, an
ovarian cancer, a myeloma, a lymphoma, a melanoma, a sarcoma, a
leukemia, a retinoblastoma, a hepatoma, a glioma, a mesothelioma,
or a carcinoma. In further embodiments, the neoplasm is from a
tissue such as blood, bone marrow, spleen, lymph node, liver,
thymus, kidney, brain, skin, gastrointestinal tract, eye, breast,
prostate, or ovary. In particular embodiments, the subject is a
human patient, such as a human patient is suffering from a disease
or disorder caused by the presence of the multi-drug resistant
cell.
[0048] In still another aspect, the invention provides a method for
detecting damage (e.g., pathogen infection) in a test cell by
measuring a level of cell surface-expressed vimentin protein in the
test cell of a given origin or cell type, and comparing it to the
level of cell surface-expressed vimentin in a nondamaged cell of
the same origin or cell type. If the level of cell
surface-expressed vimentin in the test cell is greater than the
level of cell surface-expressed vimentin in the nondamaged cell of
the same given origin or cell type, then the test cell is damaged
(e.g., infected).
[0049] In certain embodiments, the damaged cell is infected with a
pathogen. In particular embodiments, the level of cell
surface-expressed vimentin in the test cell is measured by
isolating a cytoplasmic membrane fraction from the cell and
measuring the level of vimentin in the cytoplasmic membrane
fraction. In certain embodiments, the level of cell
surface-expressed vimentin in the test cell is measured with an
anti-vimentin antibody. In particular embodiments, the
anti-vimentin antibody measures the level of cell surface vimentin
present on the intact test cell. For example, the level of antibody
bound to cell surface vimentin may be measured by
immunofluorescence emission or radiolabel.
[0050] In particular embodiments, damaged cell is infected with a
pathogen that is a virus, a bacterium or a parasite. In certain
embodiments, the pathogen is a virus such as HIV, West Nile virus
or Dengue virus. In other embodiments, the pathogen is a bacterium
such as a Mycobacteria, Rickettsia, or Chlamydia. In still other
embodiments, the pathogen is a parasite such as a Plasmodium,
Leishmania, or Taxoplasma.
[0051] In certain other embodiments, the test cell is from a tissue
such as blood, bone marrow, spleen, lymph node, liver, thymus,
kidney, brain, skin, gastrointestinal tract, eye, breast, prostate,
or ovary. In particular embodiments, the test cell is from a human.
In particular embodiments, the human patient is suffering from a
disease or disorder caused by the presence of the pathogen infected
cell.
[0052] In another aspect, the invention provides a method for
detecting a damaged (e.g., pathogen-infected) cell or cells in a
patient by administering to the patient, a vimentin binding agent
operably linked to a detectable label. The label is operably linked
to the vimentin binding agent, which specifically binds to cell
surface-expressed vimentin present on the damaged (e.g.,
pathogen-infected) cell(s) in the patient, and is then detected,
thereby locating the presence of the damaged (e.g.,
pathogen-infected) (if any) in the patient. In certain embodiments,
the vimentin binding agent used is an antibody or fragment thereof.
In other embodiments, the vimentin binding agent is a vimentin
ligand such as modified LDL, NLK1 protein, vimentin, desmin, glial
fibrillary acidic protein, or peripherin, fimbrin, RhoA-binding
kinase alpha, or protein phosphatase 2A. In particular embodiments,
the vimentin binding agent is a natural ligand, a synthetic small
molecule, a chemical, a nucleic acid, a peptide, a protein or an
antibody. In other embodiments, the detectable label is a
fluorophore, a chemical dye, a radioactive compound, a
chemoluminescent compound, a magnetic compound, a paramagnetic
compound, a promagnetic compound, an enzyme that yields a colored
product, an enzyme that yields a chemoluminescent product, or an
enzyme that yields a magnetic product.
[0053] In another aspect, the invention provides kits for
diagnosing or detecting pathogen infection in a test cell. The kits
include one probe for the detection of vimentin and a second probe
for the detection of another marker of damage (e.g., pathogen
infection) such as nucleophosmin or HSC70. In particular
embodiments, the kits include anti-vimentin antibody as the probe
for detecting vimentin. In other embodiments, the kits include a
vimentin ligand such as LDL, NLK1 protein, vimentin, desmin, glial
fibrillary acidic protein, and peripherin, fimbrin, RhoA-binding
kinase alpha, or protein phosphatase 2A. In further embodiments,
the kits include a nucleophosmin antibody or an HSC70 antibody as
probes for detecting the second, non-vimentin damage (e.g.,
pathogen infection) marker. In other embodiments, the second probe
may be a nucleophosmin ligand (such as protein kinase R (PKR), RNA,
retinoblastoma protein, IRF-1, or a nuclear localization signals
(NLS) such as the N-terminal nuclear localization signal of Rex
protein), or an HSC70 ligand (such as Alzheimer's tau protein,
BAG-1, small glutamine-rich tetratricopeptide repeat-containing
protein (SGT), (aa 642-658) of rotavirus VP5 protein, auxilin, or
the immunosuppressant 5-deoxyspergualin (DSG)). In certain
embodiments, the vimentin binding component is a natural ligand, a
synthetic small molecule, a chemical, a nucleic acid, a peptide, a
protein, or an antibody or fragments thereof.
[0054] In yet another aspect, the invention provides cell surface
vimentin-targeted agents for treating infection by a pathogen. The
vimentin-targeted agent includes a vimentin binding component and a
therapeutic component. The vimentin binding component targets the
therapeutic component to the pathogen infected cell and thereby
treats the infection. In certain embodiments, the vimentin binding
agent is an anti-vimentin antibody. In other embodiments the
vimentin binding component is a vimentin ligand such as modified
LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic
protein, and peripherin, fimbrin, RhoA-binding kinase alpha, or
protein phosphatase 2A. In certain embodiments, the vimentin
binding component is a natural ligand, a synthetic small molecule,
a chemical, a nucleic acid, a peptide, a protein, or an antibody or
fragments thereof.
[0055] In particular embodiments, the therapeutic component is an
antibacterial, antiviral or antiparasitic agent. In certain
embodiments, the vimentin binding component binds to the surface of
the target cell and the therapeutic element is internalized and
arrests growth of the pathogen, compromises viability of the
pathogen or kills the pathogen-infected cell.
[0056] In yet another aspect, the invention provides vaccines for
treating or preventing infection by a pathogen. These vaccines
include a vimentin polypeptide or polypeptide subsequence at least
one pharmaceutically acceptable vaccine component. In certain
embodiments, the vimentin polypeptide is a human vimentin
polypeptide sequence having an amino acid sequence of SEQ ID NO: 1.
In particular embodiments, the vimentin polypeptide subsequence is
at least eight amino acids long, and, in certain embodiments,
functions as a hapten.
[0057] In certain embodiments, the vaccine formulation includes an
adjuvant or other pharmaceutically acceptable vaccine component. In
particular embodiments, the adjuvant is aluminum hydroxide,
aluminum phosphate, calcium phosphate, oil emulsion, a bacterial
product, whole inactivated bacteria, an endotoxins, cholesterol, a
fatty acid, an aliphatic amine, a paraffinic compound, a vegetable
oil, monophosphoryl lipid A, a saponin, or squalene.
[0058] In another aspect, the invention provides a method of
treating or preventing an infection in a subject by administering
any of the vimentin vaccines described above. In certain
embodiments of this aspect, the subject is a human patient. In
particular embodiments the human patient is suffering from a
disease or disorder caused by the presence of infection. In certain
embodiments, the infection is in a tissue such as blood, bone
marrow, spleen, lymph node, liver, thymus, kidney, brain, skin,
gastrointestinal tract, eye, breast, prostate or ovary.
3. BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1A is a photographic representation of a silver stained
2-D gel resolving total cell extracts from MCF-7 cells showing the
presence of vimentin as a 53 kDa spot.
[0060] FIG. 1B is a photographic representation of a silver stained
2-D gel resolving total cell extracts from multidrug resistant
MCF-7/AR cells showing the presence of vimentin as a 53 kDa
spot.
[0061] FIG. 2A is a representation of an anti-vimentin immunoblot
of total cell extracts from MCF-7 cells resolved by 2-D gels and
transferred onto nitrocellulose.
[0062] FIG. 2B is a representation of an anti-vimentin immunoblot
of total cell extracts from multidrug resistant MCF-7AR cells
resolved by 2-D gels and transferred onto nitrocellulose.
[0063] FIG. 3A is a representation of a streptavidin-HRP blot of
surface biotinylated total cell extracts from CEM cells resolved by
2-D gels and transferred onto nitrocellulose.
[0064] FIG. 3B is a representation of a streptavidin-HRP blot of
surface biotinylated total cell extracts from multidrug resistant
CEM/VLB cells resolved by 2-D gels and transferred onto
nitrocellulose.
[0065] FIG. 4A is a photographic representation of a Western blot
analysis of 10% SDS-polyacrylamide gel-resolved biotinylated total
cell extracts prepared from HL60 and HL60/AR cells using an
anti-vimentin antibody.
[0066] FIG. 4B is a photographic representation of a Western blot
analysis of 10% SDS-polyacrylamide gel resolved-biotinylated total
cell extracts prepared from streptavidin purified extracts prepared
from surface biotinylated total cell extracts and using an
anti-vimentin antibody.
[0067] FIG. 4C is a photographic representation of a Western blot
analyses of 10% SDS-polyacrylamide gel resolved-biotinylated total
cell extracts prepared from anti-vimentin immunoprecipitates of
surface biotinylated total cell extracts and using an anti-vimentin
antibody.
[0068] FIG. 4D is a photographic representation of a Western blot
analysis of 10% SDS-polyacrylamide gel resolved-biotinylated total
cell extracts prepared from anti-vimentin immunoprecipitates of
surface biotinylated total cell extracts using a streptavidin-HRP
probe.
[0069] FIG. 5A is a photographic representation of a Western blot
analysis of 10% SDS-polyacrylamide gel resolved biotinylated total
cell extracts prepared from MCF-7 and MCF-7/AR cells using an
anti-vimentin antibody.
[0070] FIG. 5B is a photographic representation of a Western blot
analysis of 10% SDS-polyacrylamide gel resolved biotinylated total
cell extracts prepared from streptavidin purified extracts prepared
from surface biotinylated total cell extracts and using an
anti-vimentin antibody.
[0071] FIG. 5C is a photographic representation of Western blot
analyses of 10% SDS-polyacrylamide gel resolved biotinylated total
cell extracts prepared from anti-vimentin immunoprecipitates of
surface biotinylated total cell extracts and using an anti-vimentin
antibody.
[0072] FIG. 5D is a photographic representation of Western blot
analyses of 10% SDS-polyacrylamide gel resolved biotinylated total
cell extracts prepared from anti-vimentin immunoprecipitates of
surface biotinylated total cell extracts and using an
HRP-streptavidin probe.
[0073] FIG. 6 is a graphic representation of the results of FACS
analysis for the surface expression of vimentin using polyclonal
anti-vimentin antibody on HL60 (white bar) and HL60/AR (gray bar)
cell lines.
[0074] FIG. 7 is a graphic representation of the result of FACS
analysis for the surface expression of vimentin at saturating
amounts of monoclonal anti-vimentin antibody on HL60 (white bar)
and HL60/AR (gray bar) cell lines.
[0075] FIG. 8 is a graphic representation of the results of FACS
analysis for the surface expression of vimentin using monoclonal
anti-vimentin antibody on Molt4 (white bars) and Molt4/DOX (gray
bars) cell lines.
[0076] FIG. 9 is a graphic representation of the results of FACS
analysis for the surface expression of vimentin using monoclonal
anti-vimentin antibody on breast cancer sensitive and
drug-resistant model cell lines.
[0077] FIG. 10 is a graphic representation of the results of FACS
analysis for the surface expression of vimentin using monoclonal
anti-vimentin antibody on MDA drug-sensitive and MDA/MITO
drug-resistant cell lines.
[0078] FIG. 11 is a graphic representation of the results of
quantitative FACS analysis of the surface expression of vimentin on
MCF-7 and MCF-7/AR cell lines.
[0079] FIG. 12A is a schematic representation of the polypeptide
sequence of a human vimentin corresponding to GenBank Accession No.
P08670 (SEQ ID NO. 1).
[0080] FIG. 12B is a schematic representation of the nucleotide
sequence of a human vimentin-encoding nucleic acid sequence
corresponding to GenBank Accession No. X56134 (SEQ ID NO. 2). The
initiation and termination codons of the vimentin protein open
reading frame are underlined.
[0081] FIG. 13A is a flow chart showing a procedure for
immunohistochemical staining of non-permeabilized adherent tumor
cells.
[0082] FIG. 13B is a flow chart showing a procedure for
immunohistochemical staining of non-permeabilized adherent tumor
cells.
[0083] FIG. 14 is a photographic representation of permeabilized
and non-permeabilized MCF-7 and MCF-7/AR cells immunostained with
anti-vimentin antibody (clone V9, NeoMarker MS-129-P) using the
procedure described in FIGS. 13A&B. Mouse IgG1 was used as
negative isotype matching control and didn't show any staining (not
shown). Only MCF-7/AR shows surface exposed Vimentin.
[0084] FIG. 15 is a graphic representation of the results of the
intact cell radioimmunoassay for the surface exposure of vimentin
in HL60 drug sensitive and HL60/AR drug-resistant cell lines. CD33
was used as positive control for surface exposure
[0085] FIG. 16 is a graphic representation of the results of the
cytotoxicity of radiolabeled anti-vimentin on HL60 and HL60/AR
cells. CD33 was used as positive control for surface exposure
[0086] FIG. 17A is a table showing the results of 5 independent
Scatchard analyses of the surface exposure of vimentin in MDA/mito
cells. R.sup.2 values represent the quality of the non-linear curve
fit used to determine K.sub.d and Bmax. The analysis was performed
with Prizm GraphPad.
[0087] FIG. 17B is a representation of the Scatchard analysis
performed in the first experiment reported in table 17A.
[0088] FIG. 17C is a table reporting the number of molecules of
vimentin present on the surface of various breast and ovarian cell
lines. R/S represents the folds overexpression of surface vimentin
when comparing sensitive and resistant counterpart cells.
[0089] FIG. 18 is a graphic representation of the results of the
intact cell radioimmunoassay for the induction of surface
expression of vimentin with doxorubicin, taxol, and
mitoxanthrone.
[0090] FIG. 19A is a graphic representation of the results of the
assessment of the internalization of vimentin for MCF-7/AR cells
kept in suspension for the duration of the experiment.
[0091] FIG. 19B is a graphic representation of the results of the
assessment of the internalization of vimentin for adherent
MCF-7/AR, MDA, MDA/AR and MDA/mito cells.
4. DETAILED DESCRIPTION
[0092] The patent and scientific literature referred to herein
establishes knowledge that is available to those of skill in the
art. The issued U.S. patents, allowed applications, published
foreign applications, and references, including GenBank database
sequences, that are cited herein are hereby incorporated by
reference to the same extent as if each was specifically and
individually indicated to be incorporated by reference.
[0093] In particular, this application incorporates the following
patent applications by reference in their entirety: U.S. Ser. No.
60/433,480, filed Dec. 13, 2002 and entitled "Vimentin
Detection-Based Methods for Diagnosing and Treating Damaged Cells,
Neoplastic Cells and Multidrug Resistance;" U.S. Ser. No.
60/433,351, filed Dec. 13, 2002 and entitled "Nucleophosmin
Detection-Based Methods for Diagnosing and Treating Damaged Cells,
Neoplastic Cells and Multidrug Resistance", as well as U.S. Ser.
No. ______, filed Dec. 15, 2003 and entitled "Nucleophosmin
Directed Diagnostics and Therapeutics for Multidrug Resistant
Neoplastic Disease;" and U.S. Ser. No. 60/438,012, filed Jan. 1,
2003 and entitled "HSC70 Detection-Based Methods for Diagnosing and
Treating Damaged Cells, Neoplastic Cells and Multidrug Resistance,"
as well as U.S. Ser. No. ______, filed Dec. 15, 2003 and entitled
"HSC70 Directed Therapeutics and Diagnostics for Multidrug
Resistant Neoplastic Disease."
4.1 General
[0094] The invention provides methods and reagents for diagnosing,
preventing and/or treating cancer, and for diagnosing, preventing
and/or treating the development of both naturally occurring and
drug-induced MDR phenotypes of both non-cancerous and cancerous
cells. The invention allows for improvement of the clinical
management of multidrug resistant tumors and pathogen infections.
Moreover, the invention provides a reagent that allows the
identification of patients having neoplastic or damaged cells,
including MDR cells, thus allowing improvements in the treatment,
monitoring, diagnosis, and medical imaging of multidrug resistant
cancer and pathogen infections.
[0095] Accordingly, an embodiment of the invention provides a
method for detecting multidrug resistance in a test damaged cell.
The method includes measuring the level of cell surface expression
of vimentin protein damaged cell of a specific cell type; measuring
the level of cell surface-expressed vimentin protein on a drug
susceptible damaged cell of the same cell type; and determining
that the test damaged cell is multidrug resistant if an increased
level of cell surface-expressed vimentin is present compared to the
level of cell surface-expressed vimentin present on the
drug-susceptible damaged cell. In particular embodiments, the level
of cell surface-expressed vimentin is measured by separating the
cellular components of the test damaged cell and the drug
susceptible damaged cell into fractions, and measuring the level of
vimentin in the cellular component fraction containing the
cytoplasmic or plasma membrane of the cells.
[0096] In some embodiments, the test damaged cell is infected with
a pathogen. In particular embodiments, the pathogen is a virus, a
bacterium, or a parasite. Exemplary viruses include, but are not
limited to, HIV, West Nile virus and Dengue virus. Exemplary
bacteria include, but are not limited to, Mycobacteria, Rickettsia,
and Chlamydia. Exemplary parasites include, but are not limited to
Plasmodium, Leishmania, and Taxoplasma. In some embodiments, the
test damaged cell is from a tissue selected from the group
consisting of blood, bone marrow, spleen, lymph node, liver,
thymus, kidney, brain, skin, gastrointestinal tract, eye, breast,
prostate and ovary. In certain embodiments, the test damaged cell
is from a human.
[0097] The invention also provides a method for detecting multidrug
resistance in a test neoplastic cell. The method includes measuring
the level of cell surface-expressed vimentin protein on the test
neoplastic cell of a specific cell type; measuring the level of
cell surface-expressed vimentin protein on a drug susceptible
neoplastic cell of the same type; and determining that the test
neoplastic cell is multidrug resistant if an increased level of
cell surface expression of vimentin is present compared to the
level of cell surface expression present on the drug susceptible
neoplastic cell. In particular embodiments, the level of cell
surface expression of vimentin is measured by separating the
cellular components of the test neoplastic cell and the drug
susceptible neoplastic cell into fractions, and measuring the level
of vimentin in the cellular component fraction containing the
cytoplasmic or plasma membrane of the cells.
[0098] Exemplary neoplastic cells include, but are not limited to,
lymphoma cells, melanoma cells, sarcoma cells, leukemia cells,
retinoblastoma cells, hepatoma cells, myeloma cells, glioma cells,
mesothelioma cells and carcinoma cells. In certain embodiments, the
test neoplastic cell is from a tissue selected from the group
consisting of blood, bone marrow, spleen, lymph node, liver,
thymus, kidney, brain, skin, gastrointestinal tract, eye, breast,
prostate and ovary. In certain embodiments, the test neoplastic
cell is from a human.
[0099] The invention further provides a method for detecting a
multidrug resistant cell in a patient. The method includes
administering a binding agent that specifically binds to vimentin
protein operably linked to a detectable label, and detecting
increased binding of the binding agent specifically bound to
vimentin protein on the surface of a multidrug resistant cell in
the patient compared to the amount of binding agent bound to
vimentin protein on the surface of a drug susceptible cell. In some
embodiments of the invention, a medical imaging device or system
detects the binding agent specifically bound to the cell surface of
a multidrug resistant cell in the patient. Exemplary binding agents
include, but are not limited to, natural ligands, synthetic small
molecules, chemicals, nucleic acids, peptides, proteins, antibodies
and fragments thereof. In certain embodiments, the binding agent is
an antibody.
[0100] Exemplary detectable labels include, but are not limited to,
fluorophores, chemical dyes, radioactive compounds,
chemoluminescent compounds, magnetic compounds, paramagnetic
compounds, promagnetic compounds, enzymes that yield a colored
product, enzymes that yield a chemoluminescent product and enzymes
that yield a magnetic product. In certain embodiments, the patient
is a human. In some embodiments, the multidrug resistant cell is a
damaged cell or a neoplastic cell. In certain embodiments, the
damaged cell is infected with a pathogen. Exemplary pathogens
include, but are not limited to, viruses, bacteria and parasites.
Exemplary viruses include, but are not limited to, HIV, West Nile
virus and Dengue virus. Exemplary bacteria include, but are not
limited to, Mycobacteria, Rickettsia, and Chlamydia. Exemplary
parasites include, but are not limited to, Plasmodium, Leishmania,
and Taxoplasma. In particular embodiments, the neoplastic cell is
selected from the group consisting of a breast cancer cell, an
ovarian cancer cell, a myeloma cancer cell, a lymphoma cancer cell,
a melanoma cancer cell, a sarcoma cancer cell, a leukemia cancer
cell, a retinoblastoma cancer cell, a hepatoma cancer cell, a
glioma cancer cell, a mesothelioma cancer cell, or a carcinoma
cancer cell. In particular embodiments, the patient is a human. In
some embodiments, the patient is suffering from a disease or
disorder caused by the presence of the multidrug resistant
cell.
[0101] The invention also provides a method for detecting a
neoplastic cell. The method includes measuring the cell
surface-expressed vimentin protein on the suspected neoplastic cell
and determining that the cell is neoplastic if an increased level
of cell surface-expressed vimentin protein is present compared to
the level of cell surface-expressed vimentin present on a normal
cell of the same type. In certain embodiments, cell
surface-expressed vimentin is measured by separating the cellular
components of the suspected neoplastic cell and the normal cell
into fractions, and measuring the level of vimentin in the cellular
component fraction containing the cytoplasmic or plasma membrane of
said cells. In some embodiments, the cellular components of the
test and normal cells are contacted with a detectable binding
agent, followed by detection of the binding agent to determine the
level of cell surface-expressed vimentin protein on each cell. In
other embodiments, cell surface-expressed vimentin is measured by
contacting an intact, suspected neoplastic cell of a specific cell
type and an intact normal cell of the same type cell with a
detectable binding agent that specifically binds to vimentin
protein, and detecting the binding of the agent to determine the
level of cell surface-expressed vimentin protein on the intact
cells.
[0102] In certain embodiments, the test cell is from a tissue
selected from the group consisting of blood, bone marrow, spleen,
lymph node, liver, thymus, kidney, brain, skin, gastrointestinal
tract, eye, breast, prostate and ovary. In a particular embodiment,
the suspected neoplastic cell is from a human. In one embodiment,
the human is suffering from a cancer caused by the presence of the
neoplastic cell.
[0103] As used herein, a "neoplastic cell" is a cell that shows
aberrant cell growth, such as increased cell growth. A neoplastic
cell may be a hyperplastic cell, a cell that shows a lack of
contact inhibition of growth in vitro, a tumor cell that is
incapable of metastasis in vivo, or a cancer cell that is capable
of metastasis in vivo. Non-limiting examples of neoplastic cells
include lymphoma cells, melanoma cells, breast cancer cells,
ovarian cancer cells, prostate cancer cells, sarcoma cells,
leukemic cells, retinoblastoma cells, hepatoma cells, myeloma
cells, glioma cells, mesothelioma cells, and carcinoma cells.
[0104] As used in accordance with the invention, a "damaged cell"
means a cell that is non-neoplastic, but that has been otherwise
injured. For example, the non-neoplastic damaged cell may be a cell
infected with a pathogen, such as a virus, a bacterium, or a
parasite. In one non-limiting example, the cells may be damaged by
infection with a multi-cellular parasite, or damaged by the effects
of infection by a parasite. Such non-limiting parasites include
schistosomes, plasmodiums, trypanosomes, Leishmania, and
Taxoplasma.
[0105] As used herein, the terms, "multidrug resistant" and
"multidrug resistance," are used to refer to the development, in a
neoplastic cell or damaged cell, of resistance to a number of
different drugs, including drugs to which the neoplastic cell or
damaged cell was never exposed. For example, if a patient suffering
from leukemia being treated with vincristine develops leukemia
cells resistant to vincristine as well as other chemotherapeutics
that the patient had never received (e.g., methotrexate or
mercaptopurine), that patient's leukemic cells are multidrug
resistant. Similarly, if a patient suffering from tuberculosis
being treated with penicillin develops tuberculosis-infected cells
resistant to penicillin as well as other drugs that the patient had
never received (e.g., erythromycin), that patient's
tuberculosis-infected cells are multidrug resistant. Notably,
multidrug resistance (MDR) may include acquired simultaneous
resistance to a wide spectrum of drugs, including drugs with little
structural or even functional similarity to the original drug(s),
and results in reduced efficacy of all the drugs concerned.
[0106] Note that the terms, "multidrug resistant" and "multidrug
resistance," are used to describe a neoplastic cell or a damaged
cell that is multidrug resistant due to either the classical
mechanism (i.e., involving P-glycoprotein or another MDR protein)
or an atypical mechanism (non-classical mechanism) that does not
involve P-glycoprotein (e.g., an atypical mechanism that involves
the MRP1 multidrug resistance marker).
[0107] As used herein, the term "MDR protein" includes any of
several integral transmembrane glycoproteins of the ABC type that
are involved in (multiple) drug resistance. These include MDR 1
(P-glycoprotein or P-glycoprotein 1), an energy-dependent efflux
pump responsible for decreased drug accumulation in multidrug
resistant cells. Examples of MDR 1 include human MDR 1 (see, e.g.,
database code MDR1_HUMAN, GenBank Accession No. P08183, 1280 amino
acids (141.34 kDa)). Other MDR proteins include MDR 3 (or
P-glycoprotein 3), which is an energy-dependent efflux pump that
causes decreased drug accumulation but is not capable of conferring
drug resistance by itself. Examples of MDR 3 include human MDR 3
(see, e.g., database code MDR3_HUMAN, GenBank Accession No. P21439,
1279 amino acids (140.52 kDa). Other MDR-associated proteins
participate in the active transport of drugs into subcellular
organelles. Examples from human include MRP 1, Multidrug
Resistance-associated Protein 1, database code MRP HUMAN, GenBank
Accession No. P33527, 1531 amino acids (171.47 kDa).
[0108] In accordance with the invention, a cell (e.g., a neoplastic
or damaged cell) that develops multidrug resistance can develop
such MDR status either by being exposed to a drug (e.g., a
chemotherapeutic drug or an antibiotic drug), or by naturally
developing such MDR (i.e., without having been exposed to the drug
to which the cell has developed resistance). In this respect, the
invention allows the detection of the potentially multidrug
resistant character of a neoplasm even before the neoplasm has been
treated. Similarly, the invention allows for the effective
treatment of, for example, potentially multidrug resistant
neoplasms even before the neoplasm has been treated and shown to be
drug resistant.
[0109] The invention also allows the early identification of
patients having such MDR neoplastic or damaged cells. For example,
where the patient identified as having such cells is an
asymptomatic patient who is being treated for an infectious
disease, or had received treatment for an infectious disease (e.g.,
hepatitis B), the invention allows identification of these patients
prior to resurgence of symptoms, as well as allows the monitoring
of these patients during treatment with a drug, such that the
treatment regimen can be altered if such MDR cells are detected.
Similarly, where the patient identified as having such cells is a
patient in remission of cancer or is being treated for cancer
(e.g., a patient suffering from breast cancer, ovarian cancer,
prostate cancer, leukemia, etc.), the invention allows
identification of these patients prior to resurgence and/or
progression of their cancer, as well as allows the monitoring of
these patients during treatment with a drug, such that the
treatment regimen can be altered.
[0110] The invention stems from the discovery that cell surface
expressed vimentin is expressed on the surface of multidrug
resistant cells at measurably higher levels than that found on
other cells and, thus, is useful in part as a marker for multidrug
resistance of a cell. An important advantage of the vimentin
protein cell surface marker is that vimentin is present only in
negligible levels on the surface of normal cells of the body. This
expression is in contrast to the situation with other known MDR
markers such as P-glycoprotein and the multidrug resistance protein
(MRP), which are present at variable levels on the surface of cells
of different normal tissues, including high levels on the surface
of liver, kidney, stem cells, and blood-brain barrier epithelial
cells (Cordon-Cardo C. et al., J. Histochem. Cytochem. 38:
1277-1287, 1990; Nakamara T. et al., Drug Metabatabolism &
Disposition, 30: 4-6, 2002). As a consequence, use of agents
directed against MDR cancer cell markers such as P-glycoprotein and
MRP, have been limited by side effects caused by the killing of
normal cells that also express high levels of cell surface
P-glycoprotein and MRP (see, e.g., FitzGerald, D. J. et al., Proc.
Natl. Acad. Sci. 84: 4288-4292, 1987). The present invention
overcomes this detrimental side effect because the vimentin MDR
marker is expressed on the cell surface of drug-sensitive cancer
cells at moderate levels (as compared to the very low to negligible
levels on drug-sensitive non-cancerous normal cells), and at higher
levels on MDR cancer cells and MDR non-cancerous damaged cells
(e.g., cells infected with a virus). Thus, the invention provides
agents directed toward cell surface-expressed vimentin which kills
MDR-damaged or MDR-neoplastic cells as well as drug-sensitive
neoplastic cells, while leaving normal cells unscathed.
[0111] It should be noted that the same MDR neoplastic or damaged
cell may express more than one MDR marker (e.g., may express both
P-glycoprotein and vimentin) simultaneously, or may express these
MDR markers independently. This joint expression on the same MDR
cell offers the possibility of combining binding agents directed
against more than one cell surface MDR marker. For example, a
sub-lethal dosage of a binding agent that specifically binds to
vimentin can be combined with a sub-lethal dosage of a binding
agent that specifically binds to P-glycoprotein. Since normal cells
do not express vimentin on their cell surface, they will not be
harmed by the binding agent that specifically binds to vimentin.
Rather, only MDR cells that express both P-glycoprotein and
vimentin on their cell surface will be killed by this combination
therapy.
[0112] Thus, cell surface-expressed vimentin is a superior marker
for use in therapies (such as immunotoxin therapy) that kill MDR
neoplastic cells and MDR damaged cells bearing the vimentin marker
on their cell surface, since normal cells would be spared from cell
killing, thus reducing or eliminating harmful side effects of
treatment. Similarly, diagnosis and imaging of MDR neoplastic or
damaged cells using the cell surface vimentin marker are more
sensitive and accurate, and provide fewer false positives as
compared to diagnosis and imaging of MDR neoplastic and damaged
cells using the other MDR markers such as P-glycoprotein or MRP
that are also expressed on normal tissues. Moreover, cell surface
vimentin is useful as an anti-MDR cancer vaccine antigen or an
anti-MDR damaged cell vaccine antigen for vaccination of patients
against their cancers or damaged cell tissue expressing vimentin on
their cell surface.
[0113] The invention also allows the identification of those
patients whose damaged or neoplastic cells have acquired multidrug
resistance. In some situations, the patient is identified when
he/she no longer responds to the drug being used in his/her
treatment. For example, a breast cancer patient in remission being
treated with a chemotherapeutic agent (e.g., vincristine) may
suddenly come out of remission, despite being constantly treated
with the chemotherapeutic agent. Unfortunately, such a patient is
often found also to be unresponsive to other chemotherapeutic
agents, including some to which the patient has never been exposed.
Of course, after these patients become multidrug resistant,
treating these patients to control their now-resurgent cancer or
disease caused by a damaged cell is difficult and may require more
drastic therapies, such as radiotherapy or surgery (e.g., bone
marrow transplantation or amputation of necrotic tissue).
[0114] The invention allows an early diagnosis of multidrug
resistance by detecting increased amounts of cell surface
expression of vimentin on the neoplastic or damaged cells of the
patient. Such an early diagnosis allows patients who are initially
drug responders and sensitive to drug treatment to be distinguished
from those who are initially drug non-responders. Further,
diagnostic procedures using vimentin cell surface expression may
also be used to follow the development and emergence of MDR damaged
or neoplastic cells that are resistant to the treatment drug and
that arise during the course of drug treatment. For example, such
procedures are useful for treating AIDS patients that have been
treated with AZT and that have been reported to subsequently
develop multidrug resistance to a wide spectrum of anti-viral,
antibacterial, and anticancer drugs (see Gollapudi et al., Biochem.
Biophys. Res. Commun. 171: 1002-1007, 1990; Antonelli et al., AIDS
Research and Human Retroviruses 8: 1839-1844, 1992).
[0115] In addition, diagnostic assays for vimentin cell surface
expression are useful for selecting patients in clinical studies
involving therapy for treatment of damaged or neoplastic cells.
Hence, the presence of vimentin on the cell surface of a patient's
cells either qualifies or disqualifies that patient from being
included in a given clinical study.
[0116] Accordingly, in one aspect, the invention provides a method
for detecting multidrug resistance in a test damaged cell suspected
of being multidrug resistant. The method includes measuring the
level of cell surface-expressed vimentin protein on a test damaged
cell of a specific cell type; measuring the level of cell
surface-expressed vimentin protein on a drug susceptible damaged
cell of the same type; and determining that the test damaged cell
is multidrug resistant if an increased level of cell
surface-expressed vimentin protein is present compared to the level
of cell surface-expressed the vimentin protein present on the
non-MDR damaged cell (e.g., a damaged cell that is susceptible to a
drug).
[0117] In another aspect, the invention provides a method for
detecting multidrug resistance in a test neoplastic cell suspected
of being multidrug resistant. The method includes measuring the
level of cell surface-expressed vimentin protein on a test
neoplastic cell of a specific cell type; measuring the level of
cell surface-expressed vimentin on a drug susceptible neoplastic
cell of the same type; and determining that the test cell is
multidrug resistant if an increased level of cell surface-expressed
vimentin protein is present compared to the level of cell
surface-expressed vimentin present on the drug susceptible
neoplastic cell. In some embodiments, the test neoplastic cell or
test damaged cell expresses an amount of vimentin on its cell
surface that is at least two-fold higher than the level of cell
surface expression of the vimentin protein on a normal cell or on a
non-MDR neoplastic cell or non-MDR damaged cell, respectively. Such
a determination of level of cell surface-expressed vimentin can be
made by any number of known methods including, without limitation,
those methods described below.
[0118] As described below, although all normal cells express the
vimentin protein intracellularly, it was unexpectedly discovered
that neoplastic cells and damaged cells express the full length
vimentin protein on their cell surface. Moreover, as these
neoplastic cells or damaged cells become multidrug resistant, they
express increased amounts of the full length vimentin protein on
their cell surface. Drug-sensitive normal cells contain vimentin
protein mainly in their nucleus, nucleolus, and cytoplasm. A
moderate amount of vimentin protein is also expressed on the
surface of drug-sensitive neoplastic cells. Drug-sensitive cells
that do not express vimentin on their cell surface (or that express
a small amount of vimentin at their cell surface) are killed by
treatment with a drug. In contrast, neoplastic or damaged cells
that acquire multidrug resistance, whether naturally or after
treatment with a drug, are recognizable because they express
elevated levels of vimentin protein on their cell surface. Thus,
antibodies or other binding agents that specifically bind to
cell-surface vimentin protein are useful for developing methods for
diagnosis, treatment, screening, and imaging of MDR damaged cells
and MDR neoplastic cells that express vimentin on their cell
surface.
[0119] In certain embodiments, the test damaged cell is from a
tissue, for example, from a biopsy of damaged tissue (e.g.,
necrotic tissue), or from a type of cell that is infected by the
pathogen. For example, the hepatitis B virus typically infects only
liver cells; thus, a damaged cell (i.e., a liver cell infected by
hepatitis B virus) is from a tissue (i.e., liver). Similarly, the
Human Immunodeficiency Virus (HIV) typically infects only CD4+ T
cells and macrophages; thus a damaged cell (e.g., a CD4+ T cell
infected with HIV) is from a tissue (i.e., blood or bone
marrow).
[0120] Note that in some limited situations, infection by a virus
may cause a cell to become neoplastic. For example, some B cells,
when infected with the Epstein Barr Virus (EBV), become neoplastic.
Such a neoplastic B cell, although damaged by virtue of its
infection with a virus, is included herein as a "neoplastic cell",
not a damaged cell.
[0121] In certain embodiments, the test neoplastic cell is from a
tissue, for example, from a biopsy of a hyperplastic tissue (e.g.,
a lump in the breast). Non-limiting examples of tissues from which
a test neoplastic cell can be from include blood, bone marrow,
spleen, lymph node, liver, thymus, kidney, brain, skin,
gastro-intestinal tract, eye, breast, prostate, and ovary.
[0122] In accordance with the invention, the neoplastic cell may be
from a patient, such as a human, who is suffering from a disease or
disorder where the disease or disorder is caused by the presence of
the neoplastic cell. For example, where the neoplastic cell is a
neoplastic melanoma cell, the disease is a cancer of the melanoma
cell (i.e., the cancer is melanoma which is caused by aberrant cell
growth and metastasis of the neoplastic cell).
[0123] In accordance with the invention, the damaged cell may be
from a patient, such as a human, who is suffering from a disease or
disorder where the disease or disorder is caused by the presence of
the damaged cell. For example, where the damaged cell is infected
with a pathogen, the disease is an infection caused by the presence
of those damaged cells infected by the pathogen or lack thereof
(e.g., AIDS caused by the lack of CD4.sup.+ T cells which were
infected by the HIV virus).
[0124] As used herein, a "patient suffering from a disease or
disorder" means a patient who has the clinical manifestations
and/or symptoms of a disease or disorder. In certain situations, a
patient with a disease or disorder may be asymptomatic, and yet
still have clinical manifestations of the disease or disorder. For
example, a patient suffering from leukemia, may not be symptomatic
(e.g., may not be sick or weak), but shows the clinical
manifestation in that the patient has a larger number of white
blood cells as compared to a healthy individual of the same age and
weight. In another non-limiting example, a patient suffering from
infection with a virus (e.g., HIV), may not be symptomatic (e.g.,
may not show a diminished CD4+ T cell count), but shows the
clinical manifestation in that the patient has anti-HIV
antibodies.
[0125] According to the invention, those damaged cells or
neoplastic cells that have become multidrug resistant are
distinguishable from those cells that are not multidrug resistant
by the increased expression of the full length vimentin protein on
the cell surface of multidrug resistant cells. Representative
nucleotide and amino acid sequences of vimentin are set forth in
FIG. 12. Thus, when the cellular components are separated into
fractions, those cells that are multidrug resistant contain
vimentin in their cytoplasmic membrane fraction. Cell surface
expression of vimentin protein may also be routinely detected by
non-limiting methods such as FACS analysis, cell surface
biotinylation followed by 2-D gels, immunoprecipitation (see
Examples), or immunofluorescent analysis of fixed clinical
specimens, and other types of routine methods performed by those
skilled in the art.
[0126] In some embodiments, measuring the level of expression of a
vimentin protein on the surface of the test damaged cell or test
neoplastic cell comprises separating the cellular components of the
test cell into fractions and then examining the fraction containing
the cytoplasmic or plasma membrane for vimentin.
[0127] Alternatively, the measuring step comprises separating the
products of enzymatic digestion of cell surface expressed vimentin
protein from the test cell. The resulting peptides from the
digested surface exposed proteins are isolated by quickly spinning
the cells and leaving the digested peptides in the supernatant.
These digested peptides are then analyzed by various methods (e.g.,
immunological methods or mass spectroscopy) to determine if they
are vimentin peptides.
[0128] Separation of cellular components may be performed by any
standard separation procedure including, without limitation, thin
layer chromatography, gas chromatography, high performance liquid
chromatography, paper chromatography, affinity chromatography,
supercritical flow chromatography, gel electrophoresis, and the
procedures described below in the Examples section. Separation
procedures are generally known (see, e.g., Scopes and Scopes,
Protein Purification: Principles and Practice, Springer Verlag
1994).
[0129] In some embodiments, measuring the level of expression of a
vimentin protein on the surface of the test damaged cell includes
contacting the intact test damaged cell or test neoplastic cell
with a detectable binding agent that specifically binds to a
vimentin protein. In some embodiments, the cell is then fractioned
and then amount of labeled vimentin in the fraction containing the
cytoplasmic or plasma membrane is then measured.
[0130] The binding agents useful in the present invention are any
which specifically bind to a vimentin protein or fragment thereof.
The binding agent can specifically bind to any portion of the
vimentin protein since the entire protein is expressed on the cell
surface of multidrug resistant damaged or neoplastic cells. The
binding agent of the invention specifically binds to the surface of
a neoplastic or damaged cell expressing vimentin on its cell
surface at high levels compared to that amount expressed in the
surface of a drug-susceptible neoplastic or drug-susceptible
damaged cell, thereby identifying that cell as being multidrug
resistant.
[0131] Of course, since vimentin is also expressed inside of normal
cells, drug-sensitive neoplastic cells, and drug-sensitive damaged
cells, if such normal cells and drug-sensitive neoplastic or
damaged cells are first lysed or if their membranes are
permeabilized prior to addition of the binding agent, the binding
agent will also bind to intracellular vimentin in normal and
drug-sensitive neoplastic or damaged cells.
[0132] In some embodiments, MDR neoplastic cells or MDR damaged
cells that express vimentin on their cell surface are
distinguishable from other types of cells, in that the MDR
neoplastic or damaged cells express the vimentin protein on their
cell surface at levels that are at least two-fold higher than the
normal cells from the tissue of origin of the neoplastic or damaged
cells, or at levels that are at least two-fold higher than
drug-sensitive neoplastic or damaged cells from the tissue of
origin. For example, a leukemic T cell expresses more vimentin on
its cell surface than a normal T cell. Moreover, as described
below, a MDR leukemic T cell expresses at least twice as much
vimentin on its cell surface as a leukemic T cell that is not
multidrug resistant. Similarly, as described in the examples below,
an MDR breast cancer cell expresses at least twice as much vimentin
on its cell surface as its drug-sensitive counterpart (i.e., the
drug-sensitive counterpart is not multidrug resistant). Thus, when
the cellular components are separated (e.g., into cytoplasmic
membrane fraction and cytosolic fraction), those MDR neoplastic or
damaged cells that express vimentin on their cell surface contain
vimentin in their cytoplasmic membrane fractions at two fold or
higher levels than do other cells from the same tissue that are not
multidrug resistant, regardless whether or not the non-multidrug
resistant cell is normal, neoplastic, or damaged.
[0133] In another aspect, the invention provides a binding agent
that specifically binds to a vimentin protein or fragment thereof.
As used herein, "specifically binds" means that a binding agent
recognizes and binds to a vimentin protein or fragment thereof, but
does not substantially recognize and bind to other molecules in a
sample. Thus, a binding agent of the invention specifically binds
to the surface of a MDR cell that expresses vimentin on its cell
surface. A useful binding agent forms an association with the
vimentin protein with an affinity of at least 10.sup.6M.sup.-1, or
at least 10.sup.7M.sup.-1, or at least 10.sup.8M.sup.-1, or at
least 10.sup.9M.sup.-1 either in water, under physiological
conditions, or under conditions which approximate physiological
conditions with respect to ionic strength, e.g., 140 mM NaCl, 5 mM
MgCl.sub.2.
[0134] As used herein, a "binding agent" is a molecule that
specifically binds or attaches to a vimentin protein or fragment
thereof. A binding agent need not be any particular size or have
any particular structure so long as it specifically binds to the
vimentin protein or fragment thereof. Thus, a "binding agent" is a
molecule that specifically binds to or attaches to any region
(e.g., three dimensional structure, amino acid sequence, or
particular small chemical groups) so long as it specifically binds
to the vimentin protein or fragment thereof. Non-limiting examples
of binding agents include natural ligands (such as hormones or
GTP), as well as synthetic small molecules, chemicals, nucleic
acids, peptides, and proteins such as hormones, antibodies, and
portions thereof. Typically, the binding agent's ability to
specifically bind an epitope is based on highly complementary
structures. That is, the shape of the binding agent contains
structures that are the complement of the portion on the antigen to
which the binding agent specifically binds. The portion of the
antigen to which an antibody binds is called an "epitope."
[0135] In certain embodiments, the binding agent is an antibody.
Where the binding agent that specifically binds a vimentin protein
is an antibody, the antibody may be, without limitation, a
polyclonal antibody, a monoclonal antibody, a chimeric antibody, a
humanized antibody, a genetically engineered antibody, a bispecific
antibody (where one of the specificities of the bispecific antibody
specifically binds to the vimentin protein), antibody fragments
(including but not limited to "Fv," "F(ab').sub.2," "F(ab)," and
"Dab"); and single chains representing the reactive portion of an
antibody ("SC-MAb"). Methods for making antibodies and other
binding agents are well known (see, e.g., Coligan et al., Current
Protocols in Immunology, John Wiley and Sons, New York City, N.Y.,
1991; Jones et al., Nature 321: 522-525, 1986; Marx, Science 229:
455-456, 1985; Rodwell, Nature 342: 99-100, 1989; Clackson, Br. J.
Rheumatol. 3052: 36-39, 1991; Reichman et al., Nature 332: 323-327,
1988; Verhoeyen, et al., Science 239: 1534-1536, 1988).
[0136] As used herein, "detectably labeled" means that a binding
agent of the invention is operably linked to a moiety that is
detectable. "Operably linked" means that the moiety is attached to
the binding agent by either a covalent or non-covalent (e.g.,
ionic) bond. Methods for creating covalent bonds are known (see
general protocols in, e.g., Wong, S. S., Chemistry of Protein
Conjugation and Cross-Linking, CRC Press 1991; Burkhart et al., The
Chemistry and Application of Amino Crosslinking Agents or
Aminoplasts, John Wiley & Sons Inc., New York City, N.Y.
1999).
[0137] In accordance with the invention, a detectably labeled
binding agent of the invention includes a binding agent that is
conjugated to a detectable moiety. Another detectably labeled
binding agent of the invention is a fusion protein, where one
partner is the binding agent and the other partner is a detectable
label. Yet a further non-limiting example of a detectably labeled
binding agent is a first fusion protein comprising a binding agent
and a first moiety with high affinity to a second moiety, and a
second fusion protein comprising a second moiety and a detectable
label. For example, a binding agent that specifically binds to a
vimentin protein may be operably linked to a streptavidin moiety. A
second fusion protein comprising a biotin moiety operably linked to
a fluorescein moiety may be added to the binding agent-streptavidin
fusion protein, where the combination of the second fusion protein
to the binding agent-streptavidin fusion protein results in a
detectably labeled binding agent (i.e., a binding agent operably
linked to a detectable label).
[0138] According to the invention, a detectable label is a moiety
that can be tracked, and includes, without limitation, fluorophores
(e.g., fluorescein (FITC), phycoerythrin, rhodamine), chemical
dyes, or compounds that are radioactive, chemoluminescent,
magnetic, paramagnetic, promagnetic, or enzymes that yield a
product that may be colored, chemoluminescent, or magnetic. In
particular embodiments, the detectable label is detectable to a
medical imaging device or system. For example, where the medical
imaging system is an X-ray machine, the detectable label that can
be detected by the X-ray machine is a radioactive label (e.g.,
.sup.32P). Note that a binding agent need not be directly
conjugated to the detectable moiety. For example, a binding agent
(e.g., a mouse anti-human vimentin antibody) that is itself
specifically bound by a secondary detectable binding agent (e.g., a
FITC labeled goat anti-mouse secondary antibody) is operably linked
to a detectable moiety (i.e., the FITC moiety).
[0139] In some embodiments, measuring the level of expression of a
vimentin protein on the surface of the test damaged cell includes
contacting the intact test damaged cell with a detectable binding
agent that specifically binds to a vimentin protein. For example,
where the detectable binding agent is detectably labeled by being
operably linked to a fluorophore, cells staining with the
fluorophore (i.e., those that are specifically bound by the binding
agent) can be identified by fluorescent activated cell sorter
analysis (see Examples), or by routine fluorescent microscopy of
clinical specimens prepared on slides.
[0140] In addition to detectable moieties, other non-limiting
moieties that may be operably linked to a binding agent of the
invention include, without limitation, a toxin (e.g., a radioactive
isotope), an enzyme, an antibody (or a portion thereof), a
cytotoxic drug, or a conjugate of these. Where a toxin is operably
linked to a binding agent of the invention, non-limiting examples
of a toxin which can be operably linked to a binding agent of the
invention include a radioactive isotope, Diptheria toxin, a
nuclease (e.g., DNAse or RNAse), a protease, a degradative enzyme,
Pseudomonas exotoxin (PE), ricin A or B chains, and ribonuclease A
(Fizgerald D., Semin. Cancer Biol. 7: 87-95, 1996).
[0141] In some embodiments, the binding agent is an immunotoxin
(e.g., an antibody-toxin conjugate or antibody-drug conjugate).
Non-limiting examples of immunotoxins include
antibody-anthracycline conjugates (Braslawsky G. R. et al.,
European Patent No. EP0398305), antibody-cytokine conjugates
(Gilles S. D., PCT Pub. No. WO9953958), and monoclonal antibody-PE
conjugates (Roffler S. R. et al., Cancer Res. 51: 4001-4007,
1991).
[0142] In a further aspect, the invention provides a therapeutic
composition comprising a cytotoxic drug, a binding agent that
specifically binds to a vimentin protein or fragment thereof, and a
pharmaceutically-acceptable carrier. Non-limiting examples of such
pharmaceutically-acceptable carriers are described in more detail
in Remington: The Science and Practice of Pharmacy, Gennaro et al.
(eds), 20.sup.th Edition, Lippincott Williams & Wilkins,
Philadelphia, Pa., 2001 (ISBN 0-683-306472), a standard reference
text. In certain embodiments, binding of the binding agent is toxic
to damaged cells, regardless of whether such cells are
drug-sensitive or multidrug resistant. In some embodiments, binding
of the binding agent is toxic to neoplastic cells, regardless of
whether such cells are drug-sensitive or multidrug resistant. In
certain embodiments, the binding agent of the composition is
operably linked to a toxin.
[0143] Actual methods for preparing therapeutic compositions are
known or apparent to those skilled in the art, and are described in
detail in Remington: The Science and Practice of Pharmacy, 2001
(supra); and in Pharmaceutical Dosage Forms and Drug Delivery
Systems, 6th ed., Williams & Wilkins (1995). The therapeutic
compositions of the invention may be in any form suitable for
administration including, without limitation, in the form of a
tablet, a capsule, a powder, a solution, or an elixir.
[0144] Note that a cytotoxic drug of the therapeutic composition of
the invention need not be cytotoxic to all cells. In some
embodiments, where the therapeutic composition is being
administered to a patient suffering from a disease caused by the
presence of a damaged cell, the cytotoxic drug of the therapeutic
composition is an antipathogenic or anti-microbial drug. In some
embodiments, where the damaged cells are infected with a pathogen
(e.g., a virus, a bacterium, or a multi-cellular parasite) and the
disease is caused by the infection, the cytotoxic drug of the
therapeutic composition is an antipathogenic drug. Where the
damaged cells are infected by a pathogen, non-limiting examples of
the drug which differs depending upon the infecting pathogen
include, but are not limited to, Acyclovir, amphotericin,
ampicillin, anthracyclin, b-lactam antibiotics, cephalothin,
chloramphenicol, chloroquine (CQ), cidofovir (CDV), ciprofloxacin,
erythromycin, fluconazole, 5 flucytosine, fluoroquinolone,
foscarnet, gancyclovir, halofantrine, Itraconazole, lamivudine,
macrolides, mefloquine, methicillin, metronidazole, miconazole,
nelfinavir, ofloxacin, penicillin, primaquine, quinoline,
Streptomycin, Sulfonamides, teicoplanin, terbinafine, tetracycline,
vancomycin, voriconazole. Therapeutically effective amounts of such
drugs are known to routinely skilled physicians and pharmacists. In
addition, such information can be obtained from the manufacturer of
the drug, or from the Physician's Desk Reference, Medical Economics
Co. (published yearly).
[0145] In some embodiments, where the therapeutic composition is
being administered to a patient suffering from a cancer caused by
the presence of a neoplastic cell, the cytotoxic drug of the
therapeutic composition is an anti-cancer drug. Such anti-cancer
drugs include, without limitation, chemotherapeutic drugs and
radiotherapeutic drugs. Non-limiting examples of such anti-cancer
drugs include Actinomycin, Adriamycin (AR), Altretamine,
Asparaginase, Bleomycin, Busulfan, Capecitabine, Carboplatin,
Carmustine, Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide,
Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel,
Doxorubicin (DOX), Epoetin, Etoposide, Fludarabine, Fluorouracil,
Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib,
Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine,
Methotrexate, Mitomycin (MITO), Mitotane, Mitoxantrone, Paclitaxel,
Pentostatin, Procarbazine, Taxol, Teniposide, Topotecan,
Vinblastine (VLB), Vincristine, and Vinorelbine. Therapeutically
effective amounts of such drugs are known to routinely skilled
physicians and pharmacists. In addition, such information can be
obtained from the manufacturer of the drug, or from the Physician's
Desk Reference, Medical Economics Co. (published yearly).
[0146] In another aspect, the invention provides a method for
treating a patient suffering from a disease caused by the presence
of damaged cells. This method includes administering to the patient
a therapeutically effective amount of a drug and a therapeutically
effective amount of a binding agent that specifically binds to a
vimentin protein or fragment thereof. In some embodiments, the
binding agent kills damaged cells that are multidrug resistant
while the drug kills damaged cells that are drug-sensitive. Of
course, since the vimentin protein is expressed at moderate levels
on drug-sensitive damaged cells, the binding agent, which, in some
embodiments is different than the drug, will also kill
drug-sensitive damaged cells. According to this method, the patient
shows an improved prognosis for the disease as compared to an
untreated patient. In some embodiments, the untreated patient
receives no binding agent, but does receive the drug. An "untreated
patient" or "control patient" is one that receives no binding
agent, but does receive the drug; or one that receives no treatment
at all (i.e., receives neither the binding agent nor the drug). The
drug and the binding agent can be separately administered at
different times in any order or can be administered together. In
some embodiments, the patient is a human.
[0147] In certain embodiments, the damaged cells of the patient are
infected with a pathogen. In particular embodiments, the pathogen
is a virus, a bacterium, or a parasite (HIV, West Nile virus and
Dengue virus; Mycobacteria, Rickettsia, and Chlamydia; Plasmodium,
Leishmania, and Taxoplasma)
[0148] In another aspect, the invention provides a method for
treating a patient suffering from a disease (e.g., cancer) caused
by the presence of neoplastic cells. This method includes
administering to the patient a therapeutically effective amount of
a drug and a therapeutically effective amount of a binding agent
that specifically binds to a vimentin protein or fragment thereof.
In some embodiments, the binding agent kills neoplastic cells that
are multidrug resistant while the drug kills neoplastic cells that
are drug-sensitive. Since the vimentin protein is expressed at
moderate levels on drug-sensitive neoplastic cells, in some
embodiments, the binding agent, which, in some embodiments is
different than the drug, will also kill drug-sensitive neoplastic
cells. According to this method, the patient shows an improved
prognosis for the disease as compared to an untreated patient. In
some embodiments, the untreated patient receives no binding agent,
but does receive the drug. In some embodiments, the untreated
patient receives no treatment at all (i.e., receives neither the
binding agent nor the drug). The drug and the binding agent (e.g.,
an antibody) can be separately administered in any order at
different times or can be administered together. In some
embodiments, the patient is a human.
[0149] In certain embodiments, the neoplastic cells of the patient
are breast cancer cells, ovarian cancer cells, myeloma cancer
cells, lymphoma cancer cells, melanoma cancer cells, sarcoma cancer
cells, leukemia cancer cells, retinoblastoma cancer cells, hepatoma
cancer cells, glioma cancer cells, mesothelioma cancer cells, or
carcinoma cancer cells.
[0150] In some embodiments, the binding agent is operably linked to
a toxin. Non-limiting examples of such toxins are described
above.
[0151] As used herein, the term "therapeutically effective amount"
is used to denote known treatments of a drug at dosages and for
periods of time effective to kill a damaged cell. Administration
may be by any route including, without limitation, intravenous,
parenteral, oral, sublingual, transdermal, topical, intranasal,
intraocular, intravaginal, intrarectal, intraarterial,
intramuscular, subcutaneous, and intraperitoneal.
[0152] The dose and dosage regimen of a binding agent, drug, and/or
therapeutic composition in accordance with the invention, will
depend mainly on the degree of symptoms of the disease or cancer,
the type of drug used (e.g., chemotherapeutic agent,
radiotherapeutic agent, or antibiotic), the patient (e.g., the
patient's gender, age, and/or weight), the patient's history, and
the patient's response to treatment. The doses of binding agent,
drug, and/or therapeutic composition may be single doses or
multiple doses. If multiple doses are employed, the frequency of
administration (schedule) will depend, for example, on the patient,
type of response, and type of drug used. Administration once a week
may be effective for some patients; whereas for others, daily
administration or administration every other day or every third day
may be effective. The practitioner will be able to ascertain upon
routine experimentation, which route of administration and
frequency of administration are most effective in any particular
case.
[0153] In yet another aspect, the invention features a method for
detecting a multidrug resistant cell in a patient. The method
includes administering a binding agent that specifically binds to a
vimentin protein or fragment thereof to a patient suspected of
having a multidrug resistant cell, wherein the binding agent is
operably linked to a label that is detectable by a medical imaging
device or system and examining the patient with the medical imaging
device or system. According to this method, the medical imaging
device or system detects the binding agent (e.g., an antibody)
specifically bound to the cell surface of a multidrug resistant
cell in the patient.
[0154] Medical imaging devices and systems are known, as are labels
that are detectable by such systems. As discussed above, one
non-limiting example of such a system and label is an X-ray machine
which can detect radiolabeled binding agents. Other non-limiting
examples of medical imaging systems include (a) X-ray based
Computer Tomography (CT), positron emission tomography (PET), and
new combinations and improvedments on these technologies [(PET+CT,
spiral CT, single photon emission CT (SPECT), high resolution PET
(microPET), and immunoscintigraphy (using radiolabeled antibodies
(Czernin, J. and M. E. Phelps, Ann. Rev. Med. 53:89-112, 2002;
Goldenberg, D. M., Cancer 80 (12):2431-2435, 1997; Langer, S. G. et
al. (2001) World J. Surg. 25:1428-1437; Middleton M L, Shell E G.,
Postgrad Med. 111(5):89-90, 93-6, 2002); (b) magnetic resonance
imaging (MRI) (Helbich, T. H., J. Radiol. 34:208-219, 2000; Langer,
S. G. et al. World Journal of Surgery 25:1428-1437, 2001; Nabi, H.
A. and Zubeldia, J. M., Oncology J. Nuclear Med. Technol. 30
(1):3-9, 2002); ultrasonic imaging (US) (Harvey, C. J., et al.
Advances in Ultrasound Clin. Radiol. 57:157-177, 2002; Langer, S.
G. et al. W. J. Surg. 25:1428-1437, 2001); (c) fiber optic
endoscope (Shelhase D E, Curr. Opin. Pediatr. 14:327-33, 2002); (d)
gamma scintillation detectors (detect gamma emitters, e.g. 192-Ir),
and beta scintillation detectors (detect beta emitters, e.g.
90-Sr/Y) (Hanefeld C, Amirie, S. et al., Circulation 105:2493-6,
2002).
[0155] In certain embodiments, the patient is a mammal such as a
human. The patient may be, for example, a human suffering from a
disease caused by the presence of the multidrug resistant cell. For
example, the patient may be suffering from cancer caused by a
multidrug resistant neoplastic cell. Such a multidrug resistant
neoplastic cell includes, without limitation, an ovarian cancer
cell, and myeloma cancer cell, a lymphoma cancer cell, a melanoma
cancer cell, a sarcoma cancer cell, a leukemia cancer cell, a
retinoblastoma cancer cell a hepatoma cancer cell, a glioma cancer
cell, a mesothelioma cancer cell, or a carcinoma cancer cell.
[0156] In some embodiments, the multidrug resistant cell is a
damaged cell, and the patient is suffering from a disease caused by
the presence of such a multidrug resistant damaged cell.
Non-limiting ways in which a cell may be damaged include infection
by a pathogen (e.g., virus, bacteria or parasite), or a cell may
suffer damage by necrosis. In particular embodiments, the damaged
cell is infected with a pathogen (e.g., a virus, parasite, or
bacterium). For example, the patient may be suffering from
tuberculosis caused by a multidrug resistant strain of
Mycobacterium tuberculosis.
[0157] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
4.2 Vimentin Antibodies
[0158] The invention provides antibodies directed against vimentin
for use in detection, imaging and treatment of cancers and damaged
(e.g., pathogen-infected) cells. Anti-vimentin antibodies for use
in the invention are available from several commercial vendors. For
example, CHEMICON (Temecula, Calif.) and ABR-Affinity BioReagents
(Golden, Colo.) both produce such anti-human vimentin mouse
monoclonal and/or rabbit polyclonal antibodies.
[0159] The term "antibody" is used in the broadest sense and
specifically covers single anti-vimentin monoclonal and polyclonal
antibodies, as well as anti-vimentin antibody fragments (e.g., Fab,
F(ab)2, and Fv) and anti-vimentin antibody compositions with
polyepitopic specificity (including binding and non-binding
antibodies). The term "monoclonal antibody" as used herein refers
to an antibody obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible naturally
occurring mutations that may be present in minor-amounts.
Monoclonal antibodies are highly specific, being directed against a
single antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. Novel monoclonal antibodies or fragments thereof mean in
principle all immunoglobulin classes such as IgM, IgG, IgD, IgE,
IgA or their subclasses such as the IgG subclasses or mixtures
thereof. IgG and its subclasses are included, such as IgG1, IgG2,
IgG2a, IgG2b, IgG3 or IgGM. The IgG subtypes IgG1/kappa and IgG
2b/kapp are also included as embodiments.
[0160] The monoclonal antibodies herein include hybrid and
recombinant antibodies produced by splicing a variable (including
hypervariable) domain of an anti-vimentin antibody with a constant
domain (e.g., "humanized" antibodies), or a light chain with a
heavy chain, or a chain from one species with a chain from another
species, or fusions with heterologous proteins, regardless of
species of origin or immunoglobulin class or subclass designation,
as well as antibody fragments (e.g., Fab, F(ab)2, and Fv), so long
as they exhibit the desired biological activity. (See, e.g., U.S.
Pat. No. 4,816,567 and Mage & Lamoyi, in Monoclonal Antibody
Production Techniques and Applications, pp. 79-97 (Marcel Dekker,
Inc.), New York (1987)). Thus, the modified "monoclonal" indicates
the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method. For example, the monoclonal antibodies to be
used in accordance with the present invention may be made by the
hybridoma method first described by Kohler and Milstein, Nature
256:495 (1975), or may be made by recombinant DNA methods (U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage libraries generated using the techniques
described in McCafferty et al., Nature 348:552-554 (1990), for
example.
[0161] "Humanized" forms of non-human (e.g., murine) antibodies are
specific chimeric immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab)2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from the complementary determining regions (CDRs)
of the recipient antibody are replaced by residues from the CDRs of
a non-human species (donor antibody) such as mouse, rat or rabbit
having the desired specificity, affinity and capacity. In some
instances, Fv framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human FR residues.
Furthermore, the humanized antibody may comprise residues that are
found neither in the recipient antibody nor in the imported CDR or
FR sequences. These modifications are made to further refine and
optimize antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR residues are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin.
[0162] Vimentin or anti-vimentin monoclonal antibodies or fragments
thereof mean in principle all immunoglobulin classes such as IgM,
IgG, IgD, IgE, IgA or their subclasses such as the IgG subclasses
or mixtures thereof. IgG and its subclasses are, such as IgG1,
IgG2, IgG2a, IgG2b, IgG3 or IgGM. The IgG subtypes IgG1/kappa and
IgG 2b/kapp are included as embodiments. Fragments which may be
mentioned are all truncated or modified antibody fragments with one
or two antigen-complementary binding sites which show high binding
and binding activity toward mammalian vimentin, such as parts of
antibodies having a binding site which corresponds to the antibody
and is formed by light and heavy chains, such as Fv, Fab or F(ab')2
fragments, or single-stranded fragments. Truncated double-stranded
fragments such as Fv, Fab or F(ab')2 are. These fragments can be
obtained, for example, by enzymatic means by eliminating the Fc
part of the antibody with enzymes such as papain or pepsin, by
chemical oxidation or by genetic manipulation of the antibody
genes. It is also possible and advantageous to use genetically
manipulated, non-truncated fragments. The anti-vimentin antibodies
or fragments thereof can be used alone or in mixtures.
[0163] The novel antibodies, antibody fragments, mixtures or
derivatives thereof advantageously have a binding affinity for
vimentin with a dissociation constant value within a log-range of
from about 1.times.10.sup.-11 M (0.01 nM) to about
1.times.10.sup.-8 M (10 nM), or about 1.times.10.sup.-10 M (0.1 nM)
to about 3.times.10.sup.99 M (3 nM).
[0164] The antibody genes for the genetic manipulations can be
isolated, for example from hybridoma cells, in a manner known to
the skilled worker. For this purpose, antibody-producing cells are
cultured and, when the optical density of the cells is sufficient,
the mRNA is isolated from the cells in a known manner by lysing the
cells with guanidinium thiocyanate, acidifying with sodium acetate,
extracting with phenol, chloroform/isoamyl alcohol, precipitating
with isopropanol and washing with ethanol. cDNA is then synthesized
from the mRNA using reverse transcriptase. The synthesized cDNA can
be inserted, directly or after genetic manipulation, for example by
site-directed mutagenesis, introduction of insertions, inversions,
deletions or base exchanges, into suitable animal, fungal,
bacterial or viral vectors and be expressed in appropriate host
organisms. Preference is given to bacterial or yeast vectors such
as pBR322, pUC18/19, pACYC184, lambda or yeast mu vectors for the
cloning of the genes and expression in bacteria such as E. coli or
in yeasts such as Saccharomyces cerevisiae.
[0165] The invention furthermore relates to cells that synthesize
vimentin antibodies. These include animal, fungal, bacterial cells
or yeast cells after transformation as mentioned above. They are
advantageously hybridoma cells or trioma cells, preferably
hybridoma cells. These hybridoma cells can be produced, for
example, in a known manner from animals immunized with vimentin and
isolation of their antibody-producing B cells, selecting these
cells for vimentin-binding antibodies and subsequently fusing these
cells to, for example, human or animal, for example, mouse mylemoa
cells, human lymphoblastoid cells or heterohybridoma cells (see,
e.g., Koehler et al., (1975) Nature 256: 496) or by infecting these
cells with appropriate viruses to produce immortalized cell lines.
Hybridoma cell lines produced by fusion are particularly useful,
mouse hybridoma cell lines are very useful. The hybridoma cell
lines of the invention secrete antibodies of the IgG type. The
binding of the mAb antibodies of the invention bind with high
affinity to vimentin.
[0166] The invention further includes derivates of these
anti-vimentin, which preferably retain their vimentin-binding
activity while altering one or more other properties related to
their use as a pharmaceutical agent, e.g., serum stability or
efficiency of production. Examples of such antivimentin antibody
derivatives include peptides, peptidomimetics derived from the
antigen-binding regions of the antibodies, and antibodies,
fragments or peptides bound to solid or liquid carriers such as
polyethylene glycol, glass, synthetic polymers such as
polyacrylamide, polystyrene, polypropylene, polyethylene or natural
polymers such as cellulose, Sepharose or agarose, or conjugates
with enzymes, toxins or radioactive or nonradioactive markers such
as .sup.3H, .sup.123I, .sup.125I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sup.36Cl, .sup.57Co, .sup.55Fe, .sup.59Fe, .sup.90Y,
.sup.99mTc (metastable isomer of Technetium 99), .sup.75Se, or
antibodies, fragments or peptides covalently bonded to
fluorescent/chemiluminescent labels such as rhodamine, fluorescein,
isothiocyanate, phycoerythrin, phycocyanin, fluorescamine, metal
chelates, avidin, streptavidin or biotin.
[0167] The novel antibodies, antibody fragments, mixtures and
derivatives thereof can be used directly, after drying, for example
freeze drying, after attachment to the abovementioned carriers or
after formulation with other pharmaceutical active and ancillary
substances for producing pharmaceutical preparations. Examples of
active and ancillary substances which may be mentioned are other
antibodies, antimicrobial active substances with a microbiocidal or
microbiostatic action such as antibiotics in general or
sulfonamides, antitumor agents, water, buffers, salines, alcohols,
fats, waxes, inert vehicles or other substances customary for
parenteral products, such as amino acids, thickeners or sugars.
These pharmaceutical preparations are used to control diseases,
preferably to control arthritic disturbances, advantageously
disturbances of joint cartilage.
[0168] The anti-vimentin antibodies of the invention can be
administered orally or parenterally subcutaneously,
intramuscularly, intravenously or interperitoneally.
[0169] The antibodies, antibody fragments, mixtures or derivatives
thereof can be used in therapy or diagnosis directly or after
coupling to solid or liquid carriers, enzymes, toxins, radioactive
or nonradioactive labels or to fluorescent/chemiluminescent labels
as described above. Vimentin can be detected on a wide variety of
cell types--particularly neoplastic cells. The human vimentin
monoclonal antibody of the present invention may be obtained as
follows. Those of skill in the art will recognize that other
equivalent procedures for obtaining vimentin antibodies are also
available and are included in the invention.
[0170] First, a mammal is immunized with human vimentin. Purified
human vimentin is commercially available from Sigma (St. Louis,
Mo., catalog A6152), as well as other commercial vendors. Human
vimentin may be readily purified from human placental tissue.
Furthermore, methods of immunoaffinity purification for obtaining
highly purified vimentin immunogen are known (see, e.g., Vladutiu
et al., (1975) 5: 147-59 Prep. Biochem.). The mammal used for
raising anti-human vimentin antibody is not restricted and may be a
primate, a rodent such as mouse, rat or rabbit, bovine, sheep, goat
or dog.
[0171] Next, antibody-producing cells such as spleen cells are
removed from the immunized animal and are fused with myeloma cells.
The myeloma cells are well-known in the art (e.g., p3x63-Ag8-653,
NS-0, NS-1 or P3U1 cells may be used). The cell fusion operation
may be carried out by a well-known conventional method.
[0172] The cells, after being subjected to the cell fusion
operation, are then cultured in HAT selection medium so as to
select hybridomas. Hybridomas, which produce antihuman monoclonal
antibodies, are then screened. This screening may be carried out
by, for example, sandwich ELISA (enzyme-linked immunosorbent assay)
or the like in which the produced monoclonal antibodies are bound
to the wells to which human vimentin is immobilized. In this case,
as the secondary antibody, an antibody specific to the
immunoglobulin of the immunized animal, which is labeled with an
enzyme such as peroxidase, alkaline phosphatase, glucose oxidase,
beta-D-galactosidase or the like, may be employed. The label may be
detected by reacting the labeling enzyme with its substrate and
measuring the generated color. As the substrate,
3,3-diaminobenzidine, 2,2-diaminobis-o-dianisidine,
4-chloronaphthol, 4-aminoantipyrine, o-phenylenediamine or the like
may be produced.
[0173] By the above-described operation, hybridomas, which produce
anti-human vimentin antibodies, can be selected. The selected
hybridomas are then cloned by the conventional limiting dilution
method or soft agar method. If desired, the cloned hybridomas may
be cultured on a large scale using a serum-containing or a serum
free medium, or may be inoculated into the abdominal cavity of mice
and recovered from ascites, thereby a large number of the cloned
hybridomas may be obtained.
[0174] From among the selected anti-human vimentin monoclonal
antibodies, those that have an ability to bind cell surface
vimentin are then chosen for further analysis and manipulation.
[0175] The monoclonal antibodies herein further include hybrid and
recombinant antibodies produced by splicing a variable (including
hypervariable) domain of an anti-vimentin antibody with a constant
domain (e.g., "humanized" antibodies), or a light chain with a
heavy chain, or a chain from one species with a chain from another
species, or fusions with heterologous proteins, regardless of
species of origin or immunoglobulin class or subclass designation,
as well as antibody fragments (e.g., Fab, F(ab)2, and Fv), so long
as they exhibit the desired biological activity. (See, e.g., U.S.
Pat. No. 4,816,567 and Mage & Lamoyi, in Monoclonal Antibody
Production Techniques and Applications, pp. 79-97 (Marcel Dekker,
Inc.), New York (1987)).
[0176] Thus, the term "monoclonal" indicates that the character of
the antibody obtained is from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler and Milstein, Nature 256:495 (1975), or may be made by
recombinant DNA methods (U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" may also be isolated from phage libraries generated
using the techniques described in McCafferty et al., Nature
348:552-554 (1990), for example.
[0177] "Humanized" forms of non-human (e.g., murine) antibodies are
specific chimeric immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab)2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from the complementary determining regions (CDRs)
of the recipient antibody are replaced by residues from the CDRs of
a non-human species (donor antibody) such as mouse, rat or rabbit
having the desired specificity, affinity and capacity. In some
instances, Fv framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human FR residues.
Furthermore, the humanized antibody may comprise residues that are
found neither in the recipient antibody nor in the imported CDR or
FR sequences. These modifications are made to further refine and
optimize antibody performance. In general, the humanized antibody
comprises substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR residues are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also comprises at least a portion of an immununoglobulin constant
region (Fc), typically that of a human immunoglobulin.
[0178] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source, which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al., (1986)
Nature 321: 522-525; Riechmann et al., (1988) Nature, 332: 323-327;
and Verhoeyen et al., (1988) Science 239: 1534-1536), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies, wherein substantially less than
an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0179] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., (1993) J. Immunol., 151:2296; and
Chothia and Lesk (1987) J. Mol. Biol., 196:901). Another method
uses a particular framework derived from the consensus sequence of
all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., (1992) Proc. Natl. Acad. Sci.
(USA), 89: 4285; and Presta et al., (1993) J. Immunol.,
151:2623).
[0180] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a method,
humanized antibodies are prepared by a process of analysis of the
parental sequences and various conceptual humanized products using
three-dimensional models of the parental and humanized sequences.
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the consensus and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the CDR residues are directly
and most substantially involved in influencing antigen binding.
[0181] Human antibodies directed against vimentin are also included
in the invention. Such antibodies can be made, for example, by the
hybridoma method. Human myeloma and mouse-human heteromyeloma cell
lines for the production of human monoclonal antibodies have been
described, for example, by Kozbor (1984) J. Immunol., 133, 3001;
Brodeur, et al., Monoclonal Antibody Production Techniques and
Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and
Boerner et al., (1991) J. Immunol., 147:86-95. Specific methods for
the generation of such human antibodies using, for example, phage
display, transgenic mouse technologies and/or in vitro display
technologies, such as ribosome display or covalent display, have
been described (see Osbourn et al. (2003) Drug Discov. Today 8:
845-51; Maynard and Georgiou (2000) Ann. Rev. Biomed. Eng. 2:
339-76; and U.S. Pat. Nos. 4,833,077; 5,811,524; 5,958,765;
6,413,771; and 6,537,809.
[0182] It is now possible to produce transgenic animals (e.g.,
mice) that are capable, upon immunization, of producing a full
repertoire of human antibodies in the absence of endogenous
immunoglobulin production. For example, it has been described that
the homozygous deletion of the antibody heavy-chain joining region
(JH) gene in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array in such gem-line mutant mice
will result in the production of human antibodies upon antigen
challenge (see, e.g., Jakobovits et al., (1993) Proc. Natl. Acad.
Sci. (USA), 90: 2551; Jakobovits et al., (1993) Nature,
362:255-258; and Bruggermann et al., (1993) Year in Immuno.,
7:33).
[0183] Alternatively, phage display technology (McCafferty et al.,
(1990) Nature, 348: 552-553) can be used to produce human
antibodies and antibody fragments in vitro, from immunoglobulin
variable (V) domain gene repertoires from unimmunized donors.
According to this technique, antibody V domain genes are cloned
in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, such as M13 or fd, and displayed as
functional antibody fragments on the surface of the phage particle.
Because the filamentous particle contains a single-stranded DNA
copy of the phage genome, selections based on the functional
properties of the antibody also result in selection of the gene
encoding the antibody exhibiting those properties. Thus, the phage
mimics some of the properties of the B-cell. Phage display can be
performed in a variety of formats (for review see, e.g., Johnson et
al., (1993) Curr. Opin. in Struct. Bio., 3:564-571). Several
sources of V-gene segments can be used for phage display. For
example, Clackson et al., ((1991) Nature, 352: 624-628) isolated a
diverse array of anti-oxazolone antibodies from a small random
combinatorial library of V genes derived from the spleens of
immunized mice. A repertoire of V genes from unimmunized human
donors can be constructed and antibodies to a diverse array of
antigens (including self-antigens) can be isolated essentially
following the techniques described by Marks et al., ((1991) J. Mol.
Biol., 222:581-597, or Griffith et al., (1993) EMBO J.,
12:725-734).
[0184] In a natural immune response, antibody genes accumulate
mutations at a high rate (somatic hypermutation). Some of the
changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process can be mimicked by employing the technique
known as "chain shuffling" (see Marks et al., (1992) Bio/Technol.,
10:779-783). In this method, the affinity of "primary" human
antibodies obtained by phage display can be improved by
sequentially replacing the heavy and light chain V region genes
with repertoires of naturally occurring variants (repertoires) of V
domain genes obtained from unimmunized donors. This technique
allows the production of antibodies and antibody fragments with
affinities in the nM range. A strategy for making very large phage
antibody repertoires has been described by Waterhouse et al.,
((1993) Nucl. Acids Res., 21:2265-2266).
[0185] Gene shuffling can also be used to derive human antibodies
from rodent antibodies, where the human antibody has similar
affinities and specificities to the starting rodent antibody.
According to this method, which is also referred to as "epitope
imprinting", the heavy or light chain V domain gene of rodent
antibodies obtained by phage display technique is replaced with a
repertoire of human V domain genes, creating rodent-human chimeras.
Selection on antigen results in isolation of human variable capable
of restoring a functional antigen-binding site, i.e., the epitope
governs (imprints) the choice of partner. When the process is
repeated in order to replace the remaining rodent V domain, a human
antibody is obtained (see PCT WO 93/06213, published 1 Apr. 1993).
Unlike traditional humanization of rodent antibodies by CDR
grafting, this technique provides completely human antibodies,
which have no framework or CDR residues of rodent origin.
[0186] By using the above-described monoclonal antibody of the
present invention, human vimentin in a sample can be detected or
quantified. The detection or quantification of the human vimentin
in a sample can be carried out by an immunoassay utilizing the
specific binding reaction between the monoclonal antibody of the
present invention and human vimentin. Various immunoassays are
well-known in the art and any of them can be employed. Examples of
the immunoassays include sandwich method employing the monoclonal
antibody and another monoclonal antibody as primary and secondary
antibodies, respectively, sandwich methods employing the monoclonal
antibody and a polyclonal antibody as primary and secondary
antibodies, staining methods employing gold colloid, agglutination
method, latex method and chemical luminescence. Among these,
especially is sandwich ELISA. As is well-known, in this method, a
primary antibody is immobilized on, for example, the inner wall of
a well and then a sample is reacted with the immobilized primary
antibody. After washing, a secondary antibody is reacted with the
antigen-antibody complex immobilized in the well. After washing,
the immobilized secondary antibody is quantified. As the primary
antibody, an antibody specifically reacts with human vimentin is
preferably employed.
[0187] The quantification of the secondary antibody may be carried
out by reacting a labeled antibody (e.g., enzyme-labeled antibody)
specific to the immunoglobulin of the animal from which the
secondary antibody was obtained with the secondary antibody, and
then measuring the label. Alternatively, a labeled (e.g.,
enzyme-labeled) antibody is used as the secondary antibody and the
quantification of the secondary antibody may be carried out by
measuring the label on the secondary antibody.
4.3 Vimentin Binding Agents
[0188] In another aspect, the invention provides a binding agent
that specifically binds to a vimentin protein or fragment thereof.
As used herein, "specifically binds" means that a binding agent
recognizes and binds to a vimentin protein or fragment thereof, but
does not substantially recognize and bind to other molecules in a
sample. Thus, a binding agent of the invention specifically binds
to the surface of a MDR cell that expresses vimentin on its cell
surface. A useful binding agent forms an association with the
vimentin protein with an affinity of at least at least about
10.sup.6M-1, or at least about 10.sup.7 M-1, or at least about
10.sup.8 M-1, or at least about 10.sup.9 M-1 either in water, under
physiological conditions, or under conditions which approximate
physiological conditions with respect to ionic strength, e.g., 140
mM NaCl, 5 mM MgCl.sub.2. As used herein, a "binding agent" is a
molecule that specifically binds or attaches to a vimentin protein
or fragment thereof.
[0189] A binding agent need not be any particular size or have any
particular structure so long as it specifically binds to the
vimentin protein or fragment thereof. Thus, a "binding agent" is a
molecule that specifically binds to or attaches to any region
(e.g., three dimensional structure, amino acid sequence, or
particular small chemical groups) so long as it specifically binds
to the vimentin protein or fragment thereof. Non-limiting examples
of binding agents include natural ligands (such as hormones or
GTP), as well as synthetic small molecules, chemicals, nucleic
acids, peptides, and proteins such as hormones, antibodies, and
portions thereof.
[0190] There are a number of examples of non-antibody vimentin
binding agents known in the art. For example, modified LDL has been
shown to bind specifically to vimentin at an affinity of
approximately Kd of 1.7.times.10.sup.-7 M (corresponding to a Ka of
about 5.9.times.10.sup.6 M-1) (see Heidenthal, et al. (2000)
Biochem. Biophys. Res. Comm. 267: 49-53). The modified LDL is,
optimally, oxidized and acetylated LDL. Methods of producing such
modified LDL are known in the art. For example, oxidized LDL may be
prepared by incubating LDL with 5 uM CuSO.sub.4 in EDTA-free,
O.sub.2-saturated PBS (see Hrboticky et al. (1999) Arterioscler.
Throm. Basc. Biol. 19: 1267-75). Acetylation, and iodination, of
the oxidized LDL may be performed using methods known in the art
(see Basu et al. (1976) Proc. Natl. Acad. Sci. USA 73: 3178-82
(acetylation) and Bilheimer et al. (Biochim. Biophys. Acta 260:
212-21 (iodination)). Other examples of non-antibody vimentin
binding agents include the Nlk1 protein (U.S. Pat. No. 6,476,193)
and SNAP23 protein (see Faigle et al. (2000) Mol. Biol. Cell. 11:
3485-3494), as well as desmin, glial fibrillary acidic protein, and
peripherin, fimbrin, RhoA-binding kinase alpha and protein
phosphatase 2A. Vimentin itself is known to multimerize through
self-association, and so, is also a non-antibody vimentin binding
agent.
4.4 Vimentin-Targeted Diagnostics
[0191] The invention further allows the early identification of
patients having such MDR neoplastic or damaged cells. For example,
where the patient identified as having such cells is a patient in
remission of cancer or is being treated for cancer (e.g., a patient
suffering from breast cancer, ovarian cancer, prostate cancer,
leukemia, etc.), the invention allows identification of these
patients prior to resurgence and/or progression of their cancer, as
well as allows the monitoring of these patients during treatment
with a drug, such that the treatment regimen can be altered.
Similarly, where the patient identified as having such cells is an
asymptomatic patient who is being treated for an infectious
disease, or had received treatment for an infectious disease (e.g.,
hepatitis B), the invention allows identification of these patients
prior to resurgence of symptoms, as well as allows the monitoring
of these patients during treatment with a drug, such that the
treatment regimen can be altered if such MDR cells are detected.
Furthermore, diagnostic applications of the invention allow early
diagnosis and imaging of neoplastic, MDR neoplastic or damaged
(e.g., pathogen-infected) cells using the cell surface vimentin
marker
[0192] The diagnostic applications of the invention include probes
and other detectable agents that are joined to a vimentin binding
agent, such as an anti-vimentin antibody. As used herein, the term
"detectably labeled" means that a binding agent of the invention is
operably linked to a moiety that is detectable. "Operably linked"
means that the moiety is attached to the binding agent by either a
covalent or non-covalent (e.g., ionic) bond. Methods for creating
covalent bonds are known (see general protocols in, e.g., Wong, S.
S., Chemistry of Protein Conjugation and Cross-Linking, CRC Press
1991; Burkhart et al., The Chemistry and Application of Amino
Crosslinking Agents or Aminoplasts, John Wiley & Sons Inc., New
York City, N.Y. 1999).
[0193] In accordance with the invention, a detectably labeled
binding agent of the invention includes a binding agent that is
conjugated to a detectable moiety. Another detectably labeled
binding agent of the invention is a fusion protein, where one
partner is the binding agent and the other partner is a detectable
label. Yet a further non-limiting example of a detectably labeled
binding agent is a first fusion protein comprising a binding agent
and a first moiety with high affinity to a second moiety, and a
second fusion protein comprising a second moiety and a detectable
label. For example, a binding agent that specifically binds to a
vimentin protein may be operably linked to a streptavidin moiety. A
second fusion protein comprising a biotin moiety operably linked to
a fluorescein moiety may be added to the binding agent-streptavidin
fusion protein, where the combination of the second fusion protein
to the binding agent-streptavidin fusion protein results in a
detectably labeled binding agent (i.e., a binding agent operably
linked to a detectable label).
[0194] The detectable label of the invention is a moiety that can
be tracked, and includes, without limitation, fluorophores (e.g.,
fluorescein (FITC), phycoerythrin, rhodamine), chemical dyes, or
compounds that are radioactive, chemoluminescent, magnetic,
paramagnetic, promagnetic, or enzymes that yield a product that may
be colored, chemoluminescent, or magnetic. In particular
embodiments, the detectable label is detectable to a medical
imaging device or system. For example, where the medical imaging
system is an X-ray machine, the detectable label that can be
detected by the X-ray machine is a radioactive label (e.g.,
.sup.32P). Note that a binding agent need not be directly
conjugated to the detectable moiety. For example, a binding agent
(e.g., a mouse anti-human vimentin antibody) that is itself
specifically bound by a secondary detectable binding agent (e.g., a
FITC labeled goat anti-mouse secondary antibody) is operably linked
to a detectable moiety (i.e., the FITC moiety).
[0195] In some embodiments, measuring the level of expression of a
vimentin protein on the surface of the test damaged cell includes
contacting the intact test damaged cell with a detectable binding
agent that specifically binds to a vimentin protein. For example,
where the detectable binding agent is detectably labeled by being
operably linked to a fluorophore, cells staining with the
fluorophore (i.e., those that are specifically bound by the binding
agent) can be identified by fluorescent activated cell sorter
analysis (see Examples), or by routine fluorescent microscopy of
clinical specimens prepared on slides.
[0196] Medical imaging devices and systems are known, as are labels
that are detectable by such systems. As discussed above, one
non-limiting example of such a system and label is an X-ray machine
which can detect radiolabeled binding agents. Other non-limiting
examples of medical imaging systems include (a) X-ray based
Computer Tomography (CT), positron emission tomography (PET), and
new combinations and improvedments on these technologies [(PET+CT,
spiral CT, single photon emission CT (SPECT), high resolution PET
(microPET), and immunoscintigraphy (using radiolabeled antibodies
(Czemin, J. and M. E. Phelps (2002) Annual Reviews of Medicine
53:89-112; Goldenberg, D. M. (1997) Cancer 80 (12):2431-2435;
Langer, S. G. et al. (2001) World Journal of Surgery 25:1428-1437;
Middleton M L and Shell E G (2002) Postgrad Med 111(5):89-90, 93-6;
(b) magnetic resonance imaging (MRI) (Helbich, T. H, (2002) Journal
of Radiology 34:208-219; Langer, S. G. et al. (2001) World Journal
of Surgery 25:1428-1437; Nabi, H. A. and Zubeldia, J. M. (2002)
Oncology Journal of Nuclear Medicine Technology 30 (1):3-9);
ultrasonic imaging (US) (Harvey, C. J, et al. (2002) Advances in
Ultrasound Clinical Radiology 57:157-177; Langer, S. G. et al.
(2001) World Journal of Surgery 25:1428-1437); (c) fiber optic
endoscope (Shelhase D E (2002) Curr Opin Pediatr 14:327-33); (d)
gamma scintillation detectors (detect gamma emitters, e.g. 192-Ir),
and beta scintillation detectors (detect beta emitters, e.g.
90-Sr/Y) (Hanefeld C, Amirie, S. et al. (2002) Circulation
105:2493-6, 2002).
[0197] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to vimentin polypeptide can be used for
diagnostic purposes to detect, diagnose, or monitor diseases and/or
disorders associated with the aberrant expression of a cell surface
vimentin. The invention provides for the detection of aberrant
expression of cell surface vimentin (a) assaying the expression of
the polypeptide of interest in cells or cell surface membrane
fractions of an individual using one or more antibodies specific to
vimentin and (b) comparing the level of gene expression with a
standard gene expression level, whereby an increase or decrease in
the assayed cell surface vimentin expression level compared to the
standard expression level is indicative of aberrant expression. For
example, where multidrug resistance in a neoplastic cell is to be
detected, the "standard expression level" to which comparison
should be made is a nonmultidrug resistant neoplastic cell of the
same or similar origin or cell type. Similarly, where neoplasia in
a test cell is to be detected, the "standard expression level" to
which comparison should be made is a non-neoplastic cell of the
same or similar origin or cell type. Furthermore, where "damage" in
a test cell (e.g., pathogen infection) is to be detected, the
"standard expression level" to which comparison should be made is a
non-damage cell (e.g., uninfected cell) of the same or similar
origin or cell type.
[0198] With respect to cancer, the presence of a relatively high
amount of cell surface vimentin in biopsied tissue or test cell
from an individual may indicate a predisposition for the
development of the disease, or may provide a means for detecting
the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0199] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, M.,
et al., (1985) J. Cell. Biol. 101:976-985); Jalkanen, M., et al.
(1987) J. Cell. Biol. 105:3087-3096). Other antibody-based methods
useful for detecting vimentin protein expression include
immunoassays, such as the enzyme linked immunosorbent assay (ELISA)
and the radioimmunoassay (RIA). Suitable antibody assay labels are
known in the art and include enzyme labels, such as, glucose
oxidase; radioisotopes, such as iodine (.sup.125I, .sup.121I),
carbon (.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In) and technetium (.sup.99Tc); luminescent labels, such
as luminol; and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0200] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of
cell surface vimentin in an animal, such as a mammal, e.g., a
human. In one embodiment, diagnosis comprises: a) administering
(for example, parenterally, subcutaneously, or intraperitoneally)
to a subject an effective amount of a labeled anti-vimentin
antibody, or other vimentin binding agent, which specifically binds
to cell surface vimentin in the animal; b) waiting for a time
interval following administration for permitting the labeled
molecule to preferentially concentrate at sites in the subject
where the polypeptide is expressed (and for unbound labeled
molecule to be cleared to background level); c) determining
background level; and d) detecting the labeled molecule in the
subject, such that detection of labeled molecule above the
background level indicates that the subject has a particular
disease or disorder associated with aberrant expression of the
polypeptide of interest. Background level can be determined by
various methods including, comparing the amount of labeled molecule
detected to a standard value previously determined for a particular
system.
[0201] A vimentin-specific antibody or antibody portion which has
been labeled with an appropriate detectable imaging moiety, such as
a radioisotope (for example, .sup.131I, .sup.111In, .sup.99mTc), a
radio-opaque substance, or a material detectable by nuclear
magnetic resonance, is introduced (for example, parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined
for a disorder. Generally, suitable radioisotopes for imaging and
detection include radioisotopes that emit alpha, beta, or gamma
radiation. Gamma radiation may be particularly easy to image using
current technology. Examples are radioisotopes derived from
Gallium, Indium, Technetium, Yttrium, Ytterbium, Rhenium, Platinum,
Thallium, and Astatine, e.g., .sup.67Ga, .sup.111In, .sup.99mTc,
.sup.90Y, .sup.86Y, .sup.169Yb, .sup.188Re, .sup.195mPt,
.sup.201Ti, and .sup.211At. It is understood in the art that the
size of the subject and the imaging system used will determine the
quantity of imaging moiety needed to produce diagnostic images. In
the case of a radioisotope moiety, for a human subject, the
quantity of radioactivity injected will normally range from about 5
to 20 millicuries of .sup.99mTc. The labeled antibody or antibody
fragment will then preferentially accumulate at the location of
cells which express cell surface vimentin protein. Reagents and
methods for tumor imaging in vivo (i.e., in situ) are known in the
art and described in, for example, S. W. Burchiel et al. (1982)
"Immunopharmacokinetics of Radiolabeled Antibodies and Their
Fragments." (Chapter 13 in Tumor Imaging. The Radiochemical
Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson
Publishing Inc.). For example, antibody labels or markers for in
vivo imaging of endokine alpha protein include those detectable by
X-radiography, NMR or ESR. For X-radiography, suitable labels
include radioisotopes such as barium or cesium, which emit
detectable radiation but are not overtly harmful to the subject.
Suitable markers for NMR and ESR include those with a detectable
characteristic spin, such as deuterium, which may be incorporated
into the antibody by labeling of nutrients for the relevant
hybridoma.
[0202] Depending on several variables which can be optimized using
routine practice, including the type of label used and the mode of
administration, the time interval following the administration for
permitting the labeled molecule to preferentially concentrate at
sites in the subject and for unbound labeled molecule to be cleared
to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12
hours. In another embodiment the time interval following
administration is 5 to 20 days or 5 to 10 days.
[0203] In certain embodiment, monitoring of the disease or disorder
is carried out by repeating the method for diagnosing the disease
or disease, for example, one month after initial diagnosis, six
months after initial diagnosis, one year after initial diagnosis,
etc.
[0204] Significantly, the presence of the labeled anti-vimentin
antibody or other vimentin binding can be detected in the patient
using methods known in the art for in vivo scanning. These methods
depend upon the type of label used. Skilled artisans will be able
to determine the appropriate method for detecting a particular
label. Methods and devices that may be used in the diagnostic
methods of the invention include, but are not limited to, computed
tomography (CT), whole body scan such as position emission
tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0205] For example, in a specific embodiment, the anti-vimentin
antibody is labeled with a radioisotope and is detected in the
patient using a radiation responsive surgical instrument (Thurston
et al., U.S. Pat. No. 5,441,050). In another embodiment, the
anti-vimentin antibody is labeled with a fluorescent compound and
is detected in the patient using a fluorescence responsive scanning
instrument. In another embodiment, the anti-vimentin antibody is
labeled with a positron emitting metal and is detected in the
patent using positron emission-tomography. In yet another
embodiment, the anti-vimentin antibody is labeled with a
paramagnetic label and is detected in a patient using magnetic
resonance imaging (MRI).
4.5 Vimentin-Targeted Therapeutics
[0206] The invention takes advantage of the fact that vimentin
protein cell surface marker is present only in negligible levels on
the surface of normal cells of the body, but occurs on the cell
surface of neoplastic and, expecially, in multidrug resistant
neoplastic cells. In contrast, other markers, and particularly the
MDR markers such as P-glycoprotein and the multidrug resistance
protein (MRP), are present at variable levels on the surface of
many different normal cell and tissues, including high levels on
the surface of liver, kidney, stem cells, and blood-brain barrier
epithelial cells. Accordingly, the invention provides a highly
specific way of targeting therapeutics to neoplastic and,
particularly, multidrug resistant neoplastic, cells using a binding
agent that binds to cell surface vimentin.
[0207] Therapeutic agents to be targeted to vimentin by the methods
of the invention include, but are not limited to, antimetabolites
(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II)
(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0208] Anti-Vimentin Antibodies
[0209] In one approach, anti-vimentin antibodies specific to cell
surface expressed vimentin expressed on damaged (e.g.,
pathogen-infected), neoplastic, and MDR neoplastic cells are
administered systemically to a patient with cancer. Adhesion of
antibody to tumor cells results in tumor cell death by activation
of the complement system (complement-mediated cytotoxicity) or by
activation of T cells (antibody-dependent cell-mediated
cytotoxicity). Other antibody-induced antitumor effects include
induction of apoptosis, enhancement of the cytotoxic effects of a
second agent (e.g., an anti-cancer chemotherapeutic drug), and
induction of anti-idiotype network response. In certain
embodiments, humanized anti-vimentin antibodies may be utilized.
Humanized antibodies avoid the potential problem of causing human
patients to develop anti-animal (e.g., anti-mouse or anti-rat)
antibodies. Humanized antibodies consist of human antibody containt
the compelementarity-determining region from a nonhuman source.
[0210] Antibody based therapeutics have been used successfully in a
number of cases. For example, Rituximab is a genetically engineered
monoclonal anti-CD20 antibody used to treat non-Hodgkins lymphoma
(NHL), a relatively common malignancy affecting both young and old
populations. The CD20 antigen typically present on these B-cell
lymphomas serves as an ideal targeting antigen because it is not
present on plasma cells, B-cell precursors, stem cells, or
dendritic (antigen-presenting) cells. The Rituximab antibody (or
Rituxan) is neither shed nor internalized by NHL cells and it does
not undergo modification following antigen binding. Rituximab was
approved by the FDA in 1997 for the treatment of relapsed or
refractory CD20-positive B-cell NHL and for low-grade or follicular
type lymphoma (see Abou-Jawde et al. (2003) Clin. Therap. 25:
2121-37; and Kim (2003) Am. J. Surg. 186: 264-68). It functions by
mediating antibody-dependent cytotoxicity, inhibiting cell growth,
sensitizing chemoresistant cells to toxins and chemotherapy, and
inducing apoptosis in a dose-dependent manner (White et al. (2001)
Annu. Rev. Med. 52: 125-45; Press (1999) Semin. Oncol. 26 (Suppl
14): 58-65).
[0211] Another example of an anti-tumor antigen antibody
therapeutic is Alemtuzumab. Alemtuzumab is a humanized anti-CD52
monoclonal antibody approved by the FDA in 2001 for the treatment
of patients with B-cell chronic lymphocytic leukemia (CLL), a
prevalent form of adult malignancy (see Abou-Jawde et al. (2003)
Clin. Therap. 25: 2121-37). Even though the function of CD52 is not
well identified, Alemtuzumab has been shown to elicit tumor
response even in the presence of bulky disease (Ferrajoli et al.
(2001) Expert Opin. Biol. Ther. 1: 1059-1065).
[0212] Still another example of an anti-tumor antigen antibody
therapeutic is Trastuzumab (also Herceptin), a recombinant
humanized anti-HER 2 monoclonal antibody approved by the FDA in
1998 for the treatment of metastatic breast cancer (see Abou-Jawde
et al. (2003) Clin. Therap. 25: 2121-37; and Kim (2003) Am. J.
Surg. 186: 264-68). HER 2 is an epidermal growth factor receptor
(EGFR) family member expressed by many breast cancers tumors and,
accordingly, this antibody therapeutic is effective against solid
tumors. Several randomized, controlled studies were conducted and
showed efficacy and improved quality of life in HER2-positive
breast cancer patients treated with Trastuzumab (Vogel et al.
(2002) J. Clin. Oncol. 20: 719-26).
[0213] Finally, Cetuximab is a chimeric anti-HER1 monoclonal
antibody that is effective in treating several HER1/erb-B1
expressing solid tumors, including colorectal, pancreatic,
non-small cell lung cancer (NSCLC), and head and neck cancers (see,
e.g., O'dwyer et al. (2002) Semin. Oncol. 29 (Suppl. 14): 10-17).
Cetuximab competes for the binding of the EGFR and removes receptor
from the cell membrane by stimulating internalization and thereby
dirsuptin the cellular process responsible for proliferation growth
and metastasis (see Abou-Jawde et al. (2003) Clin. Therap. 25:
2121-37).
[0214] Vimentin-Targeted Antibody and Ligand Conjugates
[0215] In addition to the `naked` antibody approach described
above, antibodies can be conjugated, or otherwise "operably linked"
to biological or chemical toxins or radioisotopes. An anti-vimentin
antibody or antibody fragment thereof may be conjugated or
otherwise operably linked to a therapeutic moiety such as a
cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic
agent or a radioactive metal ion. "Operably linked" means that the
therapeutic moiety is attached to the binding agent by either a
covalent or non-covalent (e.g., hydrophobic or ionic) bond. Methods
for creating covalent bonds are known (see general protocols in,
e.g., Wong, S. S., Chemistry of Protein Conjugation and
Cross-Linking, CRC Press 1991; Burkhart et al., The Chemistry and
Application of Amino Crosslinking Agents or Aminoplasts, John Wiley
& Sons Inc., New York City, N.Y. 1999). Following systemic
administration, the therapy is targeted to the cancer cell (or MDR
cancer cell or damaged (e.g., pathogen-infected) cell) by the
antibody.
[0216] The invention further includes vimentin-targeted agents made
up of a vimentin targeting element and a toxic agent, such as a
biological toxin, a chemical toxin or a radioisotope. A cytotoxin
or cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Biological toxins have been conjugated, or genetically
fused in frame, to antibodies, and other tumor marker-localizing
agents. These biological toxins include ricin, diphtheria toxin and
Pseudomonas exotoxin. Following binding to cell surface vimentin,
the toxins generally cross the cell membrane, and may then be
processed, before killing the targeted cell. The toxic effect is
typically due to inhibition of protein-synthesis by the active
biological toxin.
[0217] For example, in one embodiment, anti-vimentin antibodies are
conjugated to cobra venom factor. In accordance with the invention,
vimentin specific antibodies conjugated to cobra venom factor are
used to treat cancer, including especially multidrug resistant
cancer in a human. Methods of conjugating antibodies to cobra venom
factor are taught in U.S. Pat. No. 5,773,243. In some embodiments,
the binding agent is an immunotoxin (e.g., an antibody-toxin
conjugate or antibody-drug conjugate). Non-limiting examples of
immunotoxins include antibody-anthracycline conjugates (Braslawsky
G. R. et al., European Patent No. EP0398305), antibody-cytokine
conjugates (Gilles S. D., PCT Publication No. WO9953958), and
monoclonal antibody-PE conjugates (Roffler S. R. et al., Cancer
Res. 51: 4001-4007, 1991).
[0218] Techniques for conjugating other therapeutic moieties to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982); each of which is incorporated
herein by reference. Alternatively, an antibody can be conjugated
to a second antibody to form an antibody heteroconjugate as
described by Segal in U.S. Pat. No. 4,676,980, which is
incorporated herein by reference.
[0219] Drug Attachment
[0220] A number of approaches to drug and therapeutics attachment
and release have been described in the literature, and the
strategies employed in soluble polymer-drug conjugates have been
recently reviewed (Soyez, et al., (1966) Adv. Drug Del. Rev.
21:81-106). The chemistry of many of these conjugation methods is
described in textbooks (e.g., Ref. Wong (1991) CRC Press, Boca
Raton, Fla.), site of the attachment to the antibody.
[0221] The most used site is that of the e-amino groups of the
lysine residues, as these are chemically convenient to use, either
by amide bond forming reagents such as carbodiimides or by
heterobifunctional agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson, et
al., (1978) Biochem J. 173:723-737) which can introduce reactive
thiol groups. Antibodies have a variable number of lysine residues,
which are spread over the whole of the antibody, and there is no
evidence for any subset of more reactive residues. Consequently,
lysine residues at the active site are as likely to be modified as
any other residues leading to loss of antibody activity, and the
greater the number of lysine residues modified, the greater the
likelihood of loss of binding. Linking to lysine .epsilon.-amino
groups can also have other effects. Firstly by neutralizing a
positive charge on the protein could have structural effects, or
affect the solubility of the antibody (Hudecz, et al., (1990)
Bioconjug. Chem. 1:197-204) although this can be overcome by using
a reagent such as 2-iminothiolane which provides a thiol
functionality without altering the charge (Jue, et al., (1978)
Biochemistry 17:5399-5406). Finally, the number of lysine residues
modified is statistical, so a range of modified residues is
provided within a population of antibody molecules, leading to a
variable amount of drug attached and loss of binding activity
within a single preparation (Firestone, et al., (1996) BR96-Dox, J.
Control 39:251-259).
[0222] A second site for modification is the sugar residues
attached to the hinge region of the antibody. As this is a site of
unique chemical reactivity situated away from the antibody binding
site this is a useful area for attachment. This has been exploited
by several groups by periodate oxidation of the sugars to provide
aldehyde groups (O'Shanessy, et al., (1984) Immunol. Lett.
8:273-277; O'Shanessy, et al., (1987) J. Immunol. Methods
99:153-161; and Rodwell, et al., (1986) Proc. Natl. Acad. Sci. USA
83:2632-2636) which can be used in a number of coupling procedures.
Aldehyde groups can also be generated on immunoglobulins by an
enzymic reaction involving neuraminidase and glucose oxidase
(Rodwell, et al., (1986) Proc. Natl. Acad. Sci. USA 83:2632-2636
and Stan, et al., (1999) Cancer Res. 59:115-121). Antibodies have
also been modified by genetic engineering to produce new
oligosaccharides sites which are reported to be more favourably
located for attachment of carrier-drug molecules (Qu, et al.,
(1998) J. Immunol. Methods 213:131-144).
[0223] The third major possibility for attachment is through
internal disulphide bonds within the antibody. Disulphide linkages
play an important role in the structure of antibodies, providing
both interchain and intrachain linkages. The intrachain linkages
which stabilize the antibody domain structure can be selectively
cleaved by dithiothreitol without affecting the interchain
disulphide holding the antibody chains together. This procedure has
been exploited by (Willner et al., (1993) Bioconjug. Chem.
4:521-527) to produce more soluble conjugates with better binding
activity and with a defined number of drug molecules per antibody
molecule. Surprisingly, this procedure had no detectable effect on
the stability and immunoreactivity of the antibody. There are a
maximum number of intrachain disulphide sites which can be
exploited depending on the antibody subclass. A branched chain
hydrazone linker has not also been described to exploit this
binding site further, by doubling the number of drug molecules
which can be chemically attached (King, et al., (1999) Bioconjug.
Chem. 10:279-288).
[0224] Antibody fragments have also been used in a number of drug
targeting studies. From a conjugation viewpoint the Fab' fragment
is particularly convenient, bearing a single free sulphydryl group
which can be readily used for attachment to drugs or other
macromolecules (Hashida, et al., (1984) J. Appl. Biochem.
6:56-63).
[0225] One of the simplest methods of attachment is the use of
peptide bond forming reagents such as carbodiimides or active
esters which have been used to attach carboxyl-bearing drug such as
methotrexate (MTX). Early work with polylysine conjugates, has
shown that biodegradable MTX-poly(-.sub.L-lysine) shows some
cytotoxicity, but that MTX-poly(-.sub.D-Lysine) is non-toxic,
suggesting that free drug is released through cleavage of the
polymer (Ryser, et al., (1980) Cancer 45:1207-1211). In that case
we would also expect biological molecules such as albumin and
immunoglobulins to be cleaved to release free drug. Both
MTX-immunoglobulin (Kanellos, et al., (1985) J. Natl. Cancer Inst.
75:319-332) and MTX-HSA-Immunoglobulin (Garnett, et al., (1983)
Int. J. Cancer 31:661-670) conjugates have been reported using this
principal. These reactions do not cleanly produce a single product,
a mixture of labile ester and stable amide bonds being formed
(Endo, et al., (1988) Cancer Res. 48:3330-3335 and Hudecz, et al.,
(1992) Biomed. Chromatogr. 6:128-132). The ester-linked material
can form up to 24% of the conjugated drug, and is gradually
released from the conjugate by hydrolysis in storage at 4.degree.
C. over 20 days (Hudecz, et al., (1992) Biomed. Chromatogr.
6:128-132). The inhibition of conjugate cytotoxicity by ammonium
chloride, and the lysosomal protease inhibitors leupeptin and E64
(Garnett, et al., (1985) Anti-Cancer Drug Design 1:3-12) suggested
that the free drug was being released through degradation in a
lysosomal compartment. However, detailed studies on the release of
MTX from HSA-MTX conjugates (Fitzpatrick et al., (1995) Anti-Cancer
Drug Design 10:11-24) have shown that the amount of low-molecular
weight drug released by rat liver tritosomes is very low (5.6% in
55 h). Further, only about 10% of the material released was free
MTX, the rest being amino acids are not readily cleaved from the
drug enzymically and are significantly less cytotoxic than
unmodified methotrexate (Rosowsky, et al., (1984) J. Med. Chem.
27:888-893). Conjugates designed to release these amino acid
derivatives of MTX were also less cytotoxic than conjugates
releasing free MTX. Release of MTX and MTX derivatives from
antibody-MTX conjugates was much lower, estimated to be <0.05%
over the same period, which was attributed to both the low
substitution ratio for methotrexate (Rosowsky, et al., (1984) J.
Med. Chem. 27:888-893) and the poor degradation of the Fab region
of antibodies by tritosomes (Schneider, et al., (1981) J. Cell.
Biol. 88:380-387). A linker which specifically releases free drug
from conjugate is therefore a vital component of targeted drug
conjugates. Various types of linkage have been reported.
[0226] Aldehyde/Schiff base linkages may also be used to link
therapeutic agents to antibodies or other localizing agents. Sugar
residues with vicinal hydroxyl groups can be converted into
aldehyde groups by oxidation with periodate (O'Shanessy, et al.,
(1984) Immunol. Lett. 8:273-277). This enables sugar residues in
polysaccharides such as dextran, or sugar moieties in drugs, e.g.,
nucleoside sugar groups in fluorouridine, or the amino sugar in
daunomycin to have a more useful aldehyde group inserted. Aldehydes
will readily react with hydrazides to form a hydrazone, or with
amines to form a Schiff base. The Schiff base itself is relatively
unstable (Cordes, et al., (1963) J. Am. Chem. Soc. 85:2843-2848),
and can reform its starting materials so is usually reduced with a
reagent such as sodium borohydride or sodium cyanoborohydride to
stabilize the linkage. Dialdehydes such as glutaraldehyde can also
be used to crosslink between drug and macromolecule, or between
macromolecules, but in this case the linkage of the products is
usually irreversible (Wong, (1991) CRC Press Boca Raton, Fla.
101-102). The use of glutaraldehyde as a cross-linking agent also
has the disadvantage that it is a homobifunctional reagent, so
unwanted cross-links and aggregates can be readily generated.
[0227] Sulphydryl linkages may also be used to link therapeutic
agents to antibodies or other localizing agents. Disulphide
linkages are found connecting the chains of plant toxins, and are
essential for their activity (Masuho, et al., (1982) J. Biochem.
91:1583-1591) so that the enzymic A chain can dissociate from the
binding chain and enter the cytoplasm. The necessity of this
linkage suggests that this is a possible way of releasing drugs
from antibody or polymer molecules in intracellular compartments.
Conjugates of methotrexate to poly(.sub.D-lysine) through a
disulphide linkage were shown to be cytotoxic (Shen, et al., (1985)
J. Biol. Chem. 260:10905-10908). Cleavage of these conjugates was
shown to occur initially at the cell surface, but did not take
place within the endosomal or lysosomal compartments (Feener, et
al., (1990) J. Biol. Chem. 265:18780-18785). It was suggested that
the Golgi apparatus was the most likely site of cleavage. Although
cleavable intracellularly, this linkage was shown to be unstable in
the circulation, and hindered disulphide bonds have therefore been
developed to reduce this problem (Thorpe, et al., (1987) Cancer
Res. 47:5924-5931 and Worrell, et al., (1986) Anti-Cancer Drug
Design 1:179-188). For conjugates where a stable, chemically
convenient coupling via a sulphydryl group is required a thioether
bond is more stable and can be produced through the use of
maleimide (Hashida, et al., (1984) J. Appl. Biochem. 6:56-63 and
Lau, et al., (1995) Bioorg. Med. Chem. 3:1299-1304) or iodacetate
(Rector, et al., (1978) J. Immunol. Methods 24:321-336) coupling
reagents.
[0228] Acid-labile linkages may also be used to link therapeutic
agents to antibodies or other localizing agents. Chemically labile
linkages could be used to release drug in the presence of more acid
conditions. These conditions can occur either in the tumour
environment which is reported to be 0.5-1 pH unit more acidic than
health tissue and blood (Lavie, et al., (1991) Cancer Immunol.
Immunther. 33:223-230 and Ashby (1966) Lancet ii:312-315), or
during passage through the endosomal/lysosomal compartment, where
pH of 6-6.8 and 4.5-5.5, respectively, can be found. The major
drawback to the use of an acid-labile linkage is that this is a
rate-dependent phenomenon, where the rate of cleavage is
proportional to pH: a 10-fold difference in rate can be expected
for each pH unit decreased. This means that the hydrolysis will
always be a compromise between a fast rate at low pH in the
intracellular compartment, and a slow rate for serum stability.
[0229] The first acid sensitive linker described was the
cis-aconityl linkage described by Shen and Ryser (Shen, et al.,
(1981) Biochem. Biophys. Res. Commun 102:1048-1054. Daunomycin was
first reacted with cis-aconitic anhydride, which was then
subsequently coupled to poly-lysine using a water-soluble
carbodiimide. This linkage was reported to have a half-life of less
than 3 h at pH 4 and greater then 96 h at pH 6. However, only about
50% of the drug was released from an affi-gel matrix. This may be
due to inappropriate binding of the gel matrix to the remaining
cis-carboxyl group responsible for the acid-sensitive release from
this linker. The optimum conditions for the use of this reagent
have been described in detail by (Hudecz, et al., (1990) Bioconjug.
Chem. 1:197-204). An improved version of this release mechanism has
also been described by (Blattler, et al., (1985) Biochemistry
24:1517-1524), as a heterobifunctional agent for coupling of toxin
molecules. In this method, the conjugation through the third
carboxyl of the cis-aconitate has been replaced with a specific
maleimido group which will eliminate the possibility of
inactivation of the acid release properties of the cis-carbonyl
group.
[0230] A range of acid-sensitive homo- and heterobifunctional
agents originally prepared to give acid-sensitive release for toxin
immunoconjugates have been described by Srinivasachar, based on
ortho esters, acetals and ketals (Srinivasachar, et al., (1989)
Biochemistry 28:2501-2509). These could also potentially be of use
for constructing chemoimmunoconjugates. These reagents vary in
their rate of hydrolysis at the pH found in intracellular
compartments. Hydrazone linkages may also be used to link
therapeutic agents to antibodies or other localizing agents.
[0231] Hydrazide derivatives are also acid labile and have been
used to produce both vindesine and adriamycin conjugates (Laguzza,
et al., (1989) J. Med. Chem. 32:548-555 and Greenfield, et al.,
(1990) Cancer Res. 50:6600-6607). In the former, (Laguzza, et al.,
(1989) J. Med. Chem. 32:548-555) vindesine was first reacted with
hydrazine, and the hydrazide derivative then reacted with the
oxidised sugar residue of antibody. In the adriamycin conjugate
(Greenfield, et al., (1990) Cancer Res. 50:6600-6607), an SPDP
hydrazine derivative, was prepared which was reacted with a
thiolated antibody. In both of these conjugates low-molecular
weight drug was released under acid conditions. In the former case,
up to about 30% vinca hydrazide was released at pH 5.3 over 7 days
at 37.degree. C., in the latter case, unmodified adriamycin was
released rapidly from the conjugate at pH 4.0-5.5. A study of
different hydrazone derivatives of adriamycin has been reported
(Kaneko, et al., (1991) Bioconjug. Chem. 2:133-141), which show
that the acid instability of the various linkers is
acylhydrazide>semicarbazide>carbonic acid
dihydrazide>thiosemicarbazide>hydrazine
carboxylate=arylhydrazide, all releasing adriamycin as the only
product. With the exception of the arylhydrazide, all of these
compounds were stable at pH 7.4. The acyl hydrazine released 85% of
the theoretical amount of drug at pH 5.0, 37.degree. C. in 3 h, and
when conjugated to an anti-transferrin receptor antibody, was
nearly as cytotoxic as free adriamycin. A maleimidocaproylhydrazone
derivative has also been synthesized to provide a thioether-linked
conjugate which is more stable in serum, and which can be readily
coupled to reduced intrachain disulphide groups in antibodies
(Firestone, et al., (1996) BR96-Dox, J. Control 39:251-259).
Further long-chain arylhydrazide linkers for conjugation of
anthracyclines have been described by (Lau, et al., (1995) Bioorg.
Med. Chem. 3:1299-1304).
[0232] Enzymically degradable linkers may also be used to link
therapeutic agents to antibodies or other localizing agents. The
gold standard for attaching and releasing drugs from macromolecules
is a linker which is stable in serum but can be cleaved
intracellularly by specific enzymes. Linkers of this type have been
described containing a variety of amino acids. Some of these
linkers have been used in targeted drug conjugates with antibodies,
but others only in polymer-drug conjugates. Cleavable amino acid
pro-drugs of daunomycin (Dau) were first produced by Levin and Sela
(Levin, et al., (1979) FEBS Lett. 98:119-122), although these were
designated as low-molecular weight pro-drugs. The first systematic
studies investigating amino acid sequences and lengths for
lysosomal digestion were reported by (Masquelier et al. (1980) J.
Med. Chem. 23:1166-1170). These studies identified an Ala-Leu-Dau
derivative which could be converted back to the free drug by
lysosomal hydrolases in 2 h. The activity was ascribed to a
lysosomal dipeptidyl aminopeptidase. While these dipeptide
derivatives were much less potent than Dau in vitro, they showed
greater potency in vivo (Baurain, et al., (1980) J. Med. Chem.
23:1171-1174). Further work reported by this group (Trouet, et al.,
(1982) Proc. Natl. Acad. Sci. USA 79:626-629) resulted in
conjugates in which daunorubicin was linked to succinylated serum
albumin by a spacer arm of one to four amino acids. A minimum tri
or peptide spacer was found to be essential for good release of
drug. A release of 75% of free drug was achieved in 8 h with an
albumin conjugate with an Ala-Leu-Ala-Leu-Dau linkage, which was
stable in the presence of serum (only 2.5% drug released in 24 h).
No drug was released by lysosomal enzymes from Dau conjugated to
succinylated serum albumin without a peptide spacer.
[0233] Another tetrapeptide spacer was derived from a long
collaboration between Duncan and Kopecek, in which the release of
p-nitroaniline as a model drug from
poly[N-(2-hydroxypropyl)methacrylamide] co-polymers was
investigated (described in Duncan [(Duncan, (1986) CRC Crit. Rev.
Biocompat. 2:127-145)]). These studies resulted in a greater
understanding of lysosomal enzyme specificity and the development
of a Gly-Phe-Leu-Gly-Dau linker which released 80% of bound
p-nitroaniline over a 50-h incubation period. Daunomycin was
subsequently coupled to the polymer delivery systems (Duncan, et
al., (1987) Br. J. Cancer 55:165-174) and as antibody carrier drug
conjugates.
[0234] A tetrapeptide spacer has been incorporated into monoclonal
antibody-methotrexate conjugates by (Umemoto, et al., (1989) Int.
J. Cancer 43:677-684). This is a MTX-Leu-Ala-Leu-Ala-hydrazide
linker based on the tetrapeptide described by Trouet. However, in
Trouet's study the Dau was attached to the C-terminal of the
peptide, and in this conjugate MTX was attached to the N terminal
of the peptide. In addition there is also a hydrazide incorporated
into the linkage which may give some acid-sensitive release of the
drug-linker part of the conjugate. No studies were reported on the
effect of lysosomal enzymes on this linker, and what products were
released, nor the rate of release of products. However, these
linkers gave a substantial increase in efficiency of the conjugate
compared to directly linked MTX, and release was shown by
inhibitors such as leupeptin to be lysosomally mediated.
[0235] The development of a further tetrapeptide spacer for an HAS
carrier molecule has been described by (Fitzpatrick, et al., (1995)
Anti-Cancer Drug Design 10:1-9). An appropriate spacer was
developed using a lysosomal enzyme degradation system, where
attachment of the terminal residue of the peptide chain to an
.epsilon.-amino lysine residue was used as a model for conjugation
to protein. Using this system it was shown that a variety of amino
acids coupled to the carboxyl groups of MTX could release free
drug, and the rate of release of free drug was dependent on the
length of the spacer, a tetrapeptide spacer giving about 90%
release of free drug. Conjugation of the MTX-tetrapeptide to HAS
further reduced the rate of release of free drug to about 30% over
48 h. These experiments show that the tetrapeptide spacer is not
just to overcome steric constraints of a polymer molecules, but
also relate to the efficiency of binding of the cleavage site to
the enzyme active site.
[0236] An efficient and general method for linking anthracyclines
to peptides by an oxime linkage has been described by
(Ingallinella, et al., (2001) Bioorg. Med. Chem. Lett.
11:1343-1346; however, no immunoconjugates were reported using this
linkage.
[0237] Generally the simplest way of producing an immunoconjugate
is to couple the drug directly to the antibody. This may involve a
direct linkage between the functional group of the drug, and one of
the functional groups on the antibody, or alternatively may involve
the interposition of a linker or spacer group between these two
parts of the conjugate. A linker group may be used merely to make
the chemistry of the coupling possible, but may have the second
function of allowing a specific type of release of the drug. If the
release is mediated by an enzyme, located either intra- or
extracellularly, the group may be termed a spacer group, its
purpose being to allow sufficient space, or reduce steric
constraints so that the enzyme can access the relevant bond
adequately.
[0238] Methotrexate was one of the first cytotoxic drugs to be
linked to antibodies. In these early studies using immune sera
(Marthe, et al., (1958) C.R. Acad. Sci. 246:1626-1628 and Burstein,
et al., (1977) J. Med. Chem. 20:950-952) with coupling either
through diazotization or mixed anhydride procedures. Both of these
procedures resulted in therapeutically active conjugates in mouse
models.
[0239] The conjugates that have been produced have been documented
in many reviews (e.g., Magerstadt (1991) CRC Press Boca Raton, Fla.
77-215; Dubowchik, et al., (1999) Pharmacol. Ther. 83:67-123; and
Pietersz, et al., (1994) Adv. Immunol. 56:301-387). Early work with
vinblastine-antibody conjugates used a variety of methods for
conjugating drug to antibody, with some reports showing increased
cytotoxicity of conjugate compared to free drug (Johnson, et al.,
(1981) Br. J. Cancer 44:372). Clinical studies on vinblastine
conjugates have been reported. The first of these studies involved
a conjugate with the murine monoclonal antibody KS1/4, using a
hemisuccinate derivative of DAVLB rather then the optimized DAVLBHY
(Schneck, et al., (1990) Clin. Pharmacol. Exp. 47:36-41).
[0240] The two main anthracyclines used in antibody conjugates are
daunomycin (synonymous with daunorubicin) and adriamycin
(synonymous with doxorubicin), differing in only the terminal C14
of the side chain, which is a methyl group in the former and a less
hydrophobic methoxy group in the latter. Daunomycin (Dau) is
reported to be more cytotoxic than doxorubicin (Dox). Idarubicin
and epirubicin are slightly more cytotoxic derivatives, with an
improved toxicity profile compared to Dau or Dox (Arcamone, (1985)
Cain Memorial Aware Lecture, Cancer Res. 45:5995-5999).
Morpholinodoxorubicin and cyanomorpholino doxorubicin were reported
to be highly cytotoxic derivatives (Newman, et al., (1985) Science
228:1544-1546).
[0241] Immunoconjugate preparations of anthracyclines (Dau and Dox)
to immunoglobulin assessed (Hurwitz, et al., (1975) Cancer Res.
35:1175-1181): (1) periodate oxidation of the sugar moiety of the
drug, conjugation to the lysyl groups of antibody and subsequent
reduction of the Schiff base reduction, (2) glutaraldehyde coupling
between the sugar amino group and lysyl groups of antibody. Drug
activity was best preserved with glutaraldehyde activity, but both
periodate-oxidised and glutaraldehyde-linked conjugates showed good
activity against target cells. The periodate-oxidised conjugates
were assessed in more detail (Levy, et al., (1975) Cancer Res.
35:1182-1186) and shown to retain about 50% of the activity of free
drug and have specificity in a cytotoxicity assay involving a brief
exposure to immunoconjugate. The antitumour effects of the
conjugate on PC5 B-cell leukemia were better than the free
drug.
[0242] Idarubicin (Ida) immunoconjugates were prepared from
14-bromo-idarubicin with anti-ly2.1 antibodies with an MR of 1-5
(Pietersz, et al., (1988) Cancer Res. 48:926-931).
[0243] Conjugates showed selective cytotoxicity that was less
active than free drug on E3 target cells (IC.sub.50=430 and 120 nM,
respectively). Anti-tumour activity was shown on E3 thymoma
xenografts by reduction of tumour growth rate which was greater
than that produced by Ida alone. Further studies using IDA
immunoconjugates prepared from anti-CD19 antibody gave activities
of 240 nM for immunoconjugate compared to 12 nM for free Ida
(Rowland, et al., (1993) Cancer Immunol. Immunother.
37:195-202).
[0244] The anthracyclines have been a popular choice of drug for
targeted delivery. The morpholino group makes the sugar amino group
commonly used for conjugation unavailable so linkers via the C13 on
the side chain were used.
[0245] Immunoconjugates with 5-fluoro-2'-deoxyuridine (FUDR) were
constructed by reacting an active ester derivative of succinylated
FUDR with anti-ly1.2 monoclonal antibody giving a conjugate with
drug to antibody MR of 7-9 (Krauer, et al., (1992) Cancer Res.
52:132-137). On the antigen-positive E3 cell line succinylated
FUDR, and immunoconjugate both gave similar cytotoxicities
(IC.sub.50=5 and 3 nM, respectively) which were about 10-fold lower
than free drug (0.4 nM). In vivo a greater inhibition of E3 thymoma
tumour growth was seen with the immunoconjugate than with an
equivalent amount of free drug.
[0246] A taxol immunoconjugate has been reported by Guillemard and
Saragovi (Guillemard, et al., (2001) Cancer Res. 61:694-699). Taxol
was first modified with glutaric anhydride, to give a cleavable
ester linkage to the drug, and then conjugated directly to antibody
using carbodiimide. Immunoconjugate to anti-mouse and anti-rat IgG
were made with an MR of drug to antibody of 1. Cytotoxicity tests
appeared to show that conjugates were more cytotoxic than free
drug. In vivo, immunoconjugate showed a small but significant
reduction of tumour growth on neuroblastoma xenografts.
[0247] Antibody concentration is an important determinant of the
rate of drug uptake; therefore, if more drug molecules can be
conjugated per antibody molecule, cytotoxicity should increase.
However, as the loss of antibody binding activity is the
rate-limiting factor in the number of drug molecule, the use of
carrier molecules for targeted drug conjugates offers a solution to
this problem. A number of types of conjugate have been explored,
mostly using dextran, human serum albumin or
hydroxypropylmethacrylamide (HPMA) as the carrier molecules, and
using the drugs doxorubicin or methotrexate. The earliest carrier
conjugate was of phenylene diamine mustard to antibody via a
polyglutamic acid carrier, showing a 45:1 molar substitution ratio
(Rowland, et al., (1975) Nature 255:487-488).
[0248] Another solution to the difficulties of delivering
sufficient drug molecules to kill cancer cells is to use more
potent drugs, which require fewer molecules of drug to kill a cell.
A number of these molecules have been discovered and investigated
as potential anti-tumour agents. These include: CC-1065-like
alkylating agents such as Duocarmycin; Enediynes, including the
dynemicins, the calicheamicins/esperamicins, and the chromoproteins
(Borders, et al., (1994) Marcel Dekker New York) (e.g.,
Neocarzinostatin, and Calicheamicin), and Macrolide antibiotics
such as Geldanamycin and maytansine.
[0249] Application of tumor-targeted cytotoxic therapeutics has
been used successfully with many tumor antigens. For example,
Gemtuzumab ozogamicin, used to treat acute myelogenous leukemias
(AMLs), is composed of an anti-CD33 antibody attached to
calicheamicin, an antitumor chemotherapeutic agent. The CD33
antigen is present on myeloid precursors, but not on hematopoietic
stem cells, so the CD33-targeted therapeutic is selective for AML
cells while sparing critical normal cell types. Binding of this
agent to the CD33 antigen present of AML cells results in the
formation of a complex that is internalized into the cell. After
internalization, the antitumor moiety of this agent is released
into the myeloid cells and causes cell death (Naito et al. (2000)
Leukemia 14: 14636-43). This drug has been approved by the FDA for
treatment of relapsed or refractory AML in patients over 60 years
old (Abou-Jawde et al. (2003) Clin. Therap. 25: 2121-37).
Furthermore, gemtuzumab ozogamicin has shown benefit in treating
recurrent AMS and, as such, may help many patients.
[0250] Immuntoxins
[0251] The therapeutic agent or drug moiety is not to be construed
as limited to classical chemical or radiological therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, in addition to toxins such as abrin,
ricin A, pseudomonas exotoxin, or diphtheria toxin, other proteins
with biological activity such as tumor necrosis factor,
alpha-interferon, beta-interferon, nerve growth factor, platelet
derived growth factor, tissue plasminogen activator, a thrombotic
agent or an anti-angiogenic agent, e.g., angiostatin or endostatin;
or, biological response modifiers such as, for example,
lymphokines, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, 11-8, 11-9, 11-10,
IL-12, IL-15, granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0252] Immunotoxins contain a ligand such as a growth factor,
monoclonal antibody, or fragment of an antibody which is connected
to a protein toxin. After the ligand subunit binds to the surface
of the target cell, the molecule internalizes and the toxin kills
the cell. Bacterial toxins which have been targeted to cancer cells
include Pseudomonas exotoxin and diphtheria toxin, which are well
suited to forming recombinant single-chain or double-chain fusion
toxins. Plant toxins include ricin, abrin, pokeweed antiviral
protein, saporin and gelonin, and have generally been connected to
ligands by disulfide-bond chemistry. Immunotoxins have been
produced to target hematologic malignancies and solid tumors via
wide variety of growth factor receptors and antigens.
[0253] The goal of immunotoxin therapy is to target a cytotoxic
agent to cell surface molecules which will internalize the
cytotoxic agent and result in cell death. Since immunotoxins differ
greatly from chemotherapy in their mode of action and toxicity
profile, immunotoxins provide improved systemic treatment of
tumors.
[0254] Immunotoxins can be simply defined as proteins containing a
toxin and an antibody. Toxins reviewed here include catalytic
proteins produced by plants or bacteria which kill target cells.
While the term `immunotoxin` generally refers to a toxin targeted
by either an intact IgG, an Fab fragment or an Fv fragment, toxins
targeted by growth factors or other ligands are also referred to as
`chimeric toxins`. In some immunotoxins or chimeric toxins, the
linkage between the ligand and the toxin is made chemically, and
the proteins may be referred to as `chemical conjugates`.
Otherwise, when the linkage is a peptide bond produced by genetic
engineering, the proteins are referred to as `recombinant toxins`
or `fusion toxins`. Finally, a select group of immunotoxins contain
an Fv sequence fused to the toxin, and these proteins, being both
immunotoxins and recombinant toxins, are often referred to as
`recombinant immunotoxins`.
[0255] Protein toxins are well suited because of their extreme
potency. It has been shown that one or a few molecules of protein
toxins can kill a cell when injected into the cytoplasm (see
Yamaizumi, et al., (1978) Cell 15:245-250 and Eiklid, et al.,
(1980) Exp. Cell Res. 126:321-326).
[0256] Plant toxins exist in nature as holotoxins and hemitoxins.
Holotoxins (also referred to as class II ribosome in activating
proteins) include ricin, abrin, misdetoe lectin and modeccin, which
contain a binding domain disulfide-bonded to an enzymatic domain.
Hemitoxins, such as pokeweed antiviral protein (PAP), saporin and
gelonin contain an enzymatic but no binding domain.
[0257] To make immunotoxins, plant toxins are generally conjugated
chemically to ligands (see e.g., Kreitman, et al., (1998) Adv. Drug
Del. Rev., 31:53-88).
[0258] Two bacterial toxins generally used to make immunotoxins
include Pseudomonas exotoxin (PE), made by Pseudomonas aeruginosa,
and diphtheria toxin (DT), made by Corynebacterium diphtherae. Both
PE and DT catalytically ADP ribosylate EF-2 in the cytosol (see
Carroll et al., (1987) J. Biol. Chem. 262:8707-8711; Uchida et al.,
(1972) Science 175:901-903; and Uchida et al., (1973) J. Biol.
Chem. 248:3838-3844).
[0259] Mutated and truncated forms of DT and PE may also be used
(see Kreitman, et al., (1998) Adv. Drug Del. Rev., 31:53-88). For
example, mutant toxins will be designed in which the binding domain
of the toxin was deleted or made non-functional by mutation. In the
case of DT this could be done chemically by treating the toxin with
trypsin and purifying the A-chain (see Masuho, et al., (1979)
Biochem. Biophys. Res. Commun. 90:320-326). Recombinant toxins
which are full-length and contain mutated binding domains include
PE.sup.4E, containing glutamate replacing basic residues at
positions 57, 246, 247 and 249 of PE, and CRM107, containing
phenylalanines replacing a leucine at position 390 and serine at
position 525 of DT (see Greenfield, et al., (1987) Science
238:536-539 and Chaudhary, et al., (1990) J. Biol. Chem.
265:16306-16310).
[0260] A wide variety of trace immunotoxins and recombinant toxins
have been made and tested against malignant target cells (see
Kreitman, et al., (1998) Adv. Drug Del. Rev., 31:53-88).
[0261] The next generation of immunotoxin include recombinant
toxins, immunotoxins containing an antibody or antibody fragment
like an Fab' chemically conjugated to a toxin have several
disadvantages. Firstly, their large size (100-200 kDa), often
results in reduces tumor penetration. Secondly, for conjugation to
antibodies, toxins such as PE40 and PE38 must be derivatized with
reagents which modify the lysine residues, many of which are near
the carboxyl terminus. Similarly, the antibody may require
derivatization of lysine residues within the antigen binding
domains. The resulting immunotoxins are therefore a heterogeneous
mixture with respect to sites of attachment of the antibody and
toxin, as well as the number of toxin and antibody components per
immunotoxin molecule. Finally, chemical conjugates are difficult to
produce, because the toxin and antibody must be purified
separately, conjugated, and then the product repurified.
[0262] Toxins can be targeted to cells without chemically
conjugating the ligand and the toxin if both are connected as one
polypeptide unit.
[0263] The bacterial toxins PE and DT are optimal for making these
fusion toxins because each toxin contains a proteolytic processing
site within a disulfide loop which allows the catalytic domain to
separate from the rest of the toxin after internalization and
translocate efficiently to the cytosol (see Chiron, et al., 1994)
J. Biol. Chem. 269:18167-18176); Fryling, et al., (1992) Infect.
Immun. 60:497-502; Ogata, et al., (1992) J. Biol. Chem.
267:25396-25401; and Williams, et al., (1990) J. Biol. Chem.
265:20673-20677).
[0264] In 1981 it is reported that the Mab B3/25 was conjugated to
truncated DT or RTA and used to inhibit the growth of human
melanoma in nude mice (see Trowbridge, et al., (1981) Nature
294:171-173). These and similar immunotoxins have displayed
antitumor activity against a variety of solid tumors, including
gastrointestinal adenocarcinomas, mesothelioma, cervical cancer and
glioblastoma (see Griffin, et al., (1998) J. Biol. Response Mod.
7:559-567; Griffin, et al., (1987) Cancer Res. 47:4266-4270; and
Martell, et al., (1993) Cancer Res. 53:1348-1353). The Mab HB21 was
conjugated to full-length PE and delivered intraperitoneally to
increase the survival of mice harboring human ovarian carcinoma
(see FitzGerald, et al., (1986) Proc. Natl. Acad. Sci. USA
83:6627-6630). HB21 as well as its Fab', (Fab').sub.2 and Fv
fragments have also been conjugated or fused to truncated PE or DT
and shown to cause antitumor activity in a variety of models (see
Batra, et al., (1989) Proc. Natl. Acad. Sci. USA 86:8545-8549;
Debinski, et al., (1991) Cancer Res. 52:5379-5385; and Batra, et
al., (1991) Mol. Cell. Biol. 11:2200-2205).
[0265] Many of the cell lines that are targets for immunotoxins
targeting a variety of antigens are relatively resistant to
chemotherapy Immunotoxins have also been made to specifically
target cells resistant to multiple chemotherapeutic agents by
targeting the p-glycoprotein molecule which is responsible for
increased export of chemotherapeutic agents from cells. The Mab
MRK16 conjugated to PE was very cytotoxic toward those cell lines
that were most resistant to chemotherapy due to expression of
p-glycoprotein (see FitzGerald, et al., (1987) Proc. Natl. Acad.
Sci. USA 84:4288-4292). This immunotoxin also killed
multidrug-resistant carcinoma cells in MDR-transgenic mice (see
Mickisch, et al., (1993) J. Urol. 149:174-178). This antibody has
also been conjugated to saporin to form an immunotoxin able to
purge multidrug resistant cells from bone marrow (see Dinota, et
al., (1990) Cancer Res. 50:291-4294.
[0266] Targeted Radiotherapy
[0267] Radioisotopes may also be used as cytotoxic agents for
vimentin-targeted therapeutics. Anti-vimentin antibodies of the
present invention may be coupled to one or more therapeutic agents.
Suitable agents in this regard include radionuclides. Suitable
radionuclides include .sup.90Y, .sup.123I, .sup.125I, .sup.131I,
.sup.186Re, .sup.211At or .sup.212Bi. Carriers specific for
radionuclide agents, to facilitate attachment to the vimentin
targeting agent, include radiohalogenated small molecules and
chelating compounds. For example, U.S. Pat. No. 4,735,792 discloses
representative radiohalogenated small molecules and their
synthesis. A radionuclide chelate may be formed from chelating
compounds that include those containing nitrogen and sulfur atoms
as the donor atoms for binding the metal, or metal oxide,
radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et
al. discloses representative chelating compounds and their
synthesis.
[0268] An ideal radioligand therapy agent would accumulate
selectively in target cells. The effectiveness of radiotherapy is
due to the destruction of dividing cells resulting from
radiation-induced damage to cellular DNA (see, e.g., W. D. Bloomer
et al., (1977) "Therapeutic Application of Iodine-125 Labeled
Iododeoxyuridine in an Early Ascites Tumour Model," Current Topics
in Radiation Research Quarterly 12:513-25). In both therapeutic and
imaging applications, any unbound, circulating radioligand is
rapidly cleared by excretory systems, which helps protect normal
organs and tissues. The radioligand may also be degraded by body
processes which will increase the clearance of the free
radioisotope (see G. A. Wiseman et al. (1995) "Therapy of
Neuroendocrine Tumors with Radiolabelled MIBG and Somatostatin
Analogues," Seminars in Nuclear Medicine, vol. XXV, No. 3, pp.
272-278).
[0269] Radioisotopes most suitable for therapeutic treatment
include Auger-electron-emitting radioisotopes, e.g. .sup.125I,
.sup.123I, .sup.124I, .sup.129I, .sup.131I, .sup.111In, .sup.77Br,
and other radiolabeled halogens. The choice of a suitable
radioisotope can be optimized based on a variety of factors
including the type of radiation emitted, the emission energies, the
distance over which energy is deposited, and the physical half-life
of the radioisotope. In certain instances, the radioisotopes used
are those having a radioactive half-life corresponding to, or
longer than, the biological half-life of the vimentin-targeted
therapeutic. For example, in certain examples the radioisotope has
a half-life between about 1 hour and 60 days, preferably between 5
hours and 60 days, more preferably between 12 hours and 60 days.
.sup.125I has an advantage over other emitters that produce
high-energy gamma rays (i.e., .sup.111In and .sup.131I) which
require inpatient hospitalization and isolation .sup.125I will
allow the development of outpatient-based treatments due to the
limited amounts of radiation that escapes the body.
[0270] Radiolabeled therapeutics have typically been administered
by intravenous, bolus injection (see, e.g., H. P. Kalofonos et al.,
(1989)"Antibody Guided Diagnosis and Therapy of Brain Gliomas using
Radiolabeled Monoclonal Antibodies Against Epidermal Growth Factor
Receptor and Placental Alkaline Phosphatase" The Journal of Nuclear
Medicine vol. 30, pp. 163-645; I. Virgolini et al., (1994)
"Vasoactive Intestinal Peptide-Receptor Imaging for the
Localization of Intestinal Adenocarcinomas and Endocrine Tumors"
The New England Journal of Medicine, vol. 331, pp., 1116-21; G. A.
Wiseman et al., (1995) "Therapy of Neuroendocrine Tumors with
Radiolabelled MIBG and Somatostatin Analogues" Seminars in Nuclear
Medicine, vol. XXV, no. 3, pp. 272-78; S. W. J. Lamberts et al.,
(1990) "Somatostatin-Receptor Imaging in the Localization of
Endocrine Tumors" The New England Journal of Medicine vol. 323, pp.
126-49; E. P. Krenning et al. (1992) "Somatostatin Receptor
Scintigraphy with Indium-111-DTPA-D-Phe-1-Octreotide in Man:
Metabolism, Dosimetry and Comparison with
Iodine-123-Tyr-3-Octreotide" The Journal of Nuclear Medicine vol.
33, pp. 652-58; E. P. Krenning et al. (1989) "Localisation of
Endocrine-Related Tumours with Radioiodinated Analogue of
Somatostatin," The Lancet vol. 1989, no. 1, pp. 242-244.
[0271] Targeted Gene Therapy
[0272] Gene vectors may also be used as cytotoxic agents for
vimentin-targeted therapeutics. For example, a gene vector encoding
an antibody gene (or fragment thereof) inside the tumor cell. The
transgene expression product binds intracellular proteins, e.g.,
those derived from oncogenes, and thereby down-regulates oncogenic
protein expression. Targeted gene therapy may be facilitated by the
use of bifunctional crosslinkers to target adenoviral and
retroviral vectors, by inserting short targeting peptides and
larger polypeptide-binding domains into the coat protein of a
number of different viral vectors, and by the use of
replication-competent vectors (see Wand and Liu (2003) Acta
Biochimica et Biophysica Sinica 35(4): 311-6). Other non-viral
therapeutic agents, including DNA complexes and bacterial vehicles,
have also been developed. Gene therapy methods for
vimentin-targeted compositions and methods of the invention may be
adapted from gene therapy methods known in the art or adapted from
U.S. Pat. Nos. 5,871,726, 5,885,806, 5,888,767, 5,981,274,
6,207,426, 6,210,708, 6,232,120, 6,498,033, 6,537,805, 6,555,107,
and 6,569,426.
[0273] In one approach, targeted replicative or non-replicative
viral vectors may be used to deliver the gene therapeutic. For
example, andoviral gene therapy vectors have been adapted for the
targeting of neoplastic cells (see Rots, et al. (2003) Journal of
Controlled Release 87: 159-165). Selective targeting of adenovirus
vectors limits the inflammatory and immune response against the
viral vector and decreases the toxicity of the treatment because
lower doses of virus can be used. Adenoviral infection is normally
initiated by the binding of target cells by the C-terminal part of
the adenovirus fiber protein, termed know, and the primary cellular
receptor, coxsackie B virus and adenovirus receptor (CAR). After
this step, entry of the virus into the cell occurs via interaction
of the RGD (arg-gly-asp) sequence of viral penton base protein and
cellular integrins. Selective targeting of adenovirus vectors can
be achieved. Linking (e.g., conjugation) of a vimentin-specific
antibody to the adenoviral vector will target the resulting
construct to vimentin-expressing neoplastic, MDR neoplastic and
damaged (e.g. pathogen infected) cells. For example, this strategy
has been successfully adapted to target adenovirus to the EGP-2
antigen present on tumor cells (Heiderman et al. (2001) Cancer Gene
Ther. 8: 342-51) by conjugating a neutralizing anti-fiber protein
antibody to an antibody against the Epithelial Cell Adhesion
Molecule (EGP-2). The resulting EGP-2 adenovirus was targeted to
cancer cells expressing EGP-2, and infection was shown to be
independent of CAR. Another strategy is to use bispecific
antibodies to bridge cell surface vimentin to the therapeutic gene
delivery vector (e.g., adenoviral vector) (see, e.g., Haisma et al.
(2000) Cancer Gene 7: 901-4; Grill et al. (2001) Clin. Cancer Res.
7: 641-50; Krasnykh et al. (1998) J. Virol. 72: 1844-52; and van
Beusechem et al. (2000) Gene Ther. 7: 1940-46).
[0274] In general, the terms "viral vectors" and "viruses" are used
interchangeably herein to refer to any of the obligate
intracellular parasites having no protein-synthesizing or
energy-generating mechanism. The viral genome may be RNA or DNA
contained with a coated structure of protein of a lipid membrane.
The terms virus(es) and viral vector(s) are used interchangeably
herein. The viruses useful in the practice of the present invention
include recombinantly modified enveloped or non-enveloped DNA and
RNA viruses, preferably selected from baculoviridiae,
parvoviridiae, picornoviridiae, herpesviridiae, poxyiridae, or
adenoviridiae. The viruses may be naturally occurring viruses or
their viral genomes may be modified by recombinant DNA techniques
to include expression of exogenous transgenes and may be engineered
to be replication deficient, conditionally replicating or
replication competent. Chimeric viral vectors which exploit
advantageous elements of each of the parent vector properties (See
e.g., Feng, et al., (1997) Nature Biotechnology 15:866-870) may
also be useful in the practice of the present invention. Minimal
vector systems in which the viral backbone contains only the
sequences need for packaging of the viral vector and may optionally
include a transgene expression cassette may also be produced
according to the practice of the present invention. Although it is
generally favored to employ a virus from the species to be treated,
in some instances it may be advantageous to use vectors derived
from different species that possess favorable pathogenic features.
For example, equine herpes virus vectors for human gene therapy are
described in WO98/27216 published Aug. 5, 1998. The vectors are
described as useful for the treatment of humans as the equine virus
is not pathogenic to humans. Similarly, ovine adenoviral vectors
may be used in human gene therapy as they are claimed to avoid the
antibodies against the human adenoviral vectors. Such vectors are
described in WO 97/06826 published Apr. 10, 1997.
[0275] The term "replication deficient" refers to vectors which are
incapable of replication in a wild type mammalian cell. In order to
produce such vectors in quantity, the producer cell line must be
cotransfected with a helper virus or modified to complement the
missing functions. For example, 293 cells have been engineered to
complement adenoviral E1 deletions allowing propagation of the E1
deleted replication deficient adenoviral vectors in 293 cells. The
term "replication competent viral vectors" refers to a viral vector
which is capable of infection, DNA replication, packaging and lysis
of an infected cell. The term "conditionally replicating viral
vectors" is used herein to refer to replication competent vectors
which are designed to achieve selective expression in particular
cell types while avoiding untoward broad spectrum infection. Such
conditional replication may be achieved by operably linking tissue
specific, tumor specific or cell type specific or other selectively
induced regulatory control sequences to early genes (e.g. the E1
gene of adenoviral vectors).
[0276] In addition to targeting, cell type specificity with viral
vectors may be improved through the use of a pathway responsive
promoters driving a repressor of viral replication. The term
"pathway-responsive promoter" refers to DNA sequences that bind a
certain protein and cause nearby genes to respond transcriptionally
to the binding of the protein in normal cells. Such promoters may
be generated by incorporating response elements which are sequences
to which transcription factors bind. Such responses are generally
inductive, though there are several cases where increasing protein
levels decrease transcription. Pathway-responsive promoters may be
naturally occurring or synthetic. Pathway-responsive promoters are
typically constructed in reference to the pathway or a functional
protein that is targeted. For example, a naturally occurring p53
pathway-responsive promoter would include transcriptional control
elements activated by the presence of functional p53 such as the
p21 or bax promoter. Alternatively, synthetic promoters containing
p53 binding sites upstream of a minimal promoter (e.g. the SV40
TATA box region) may be employed to create a synthetic
pathway-responsive promoter. Synthetic pathway-responsive promoters
are generally constructed from one or more copies of a sequence
that matches a consensus binding motif. Such consensus DNA binding
motifs can readily be determined. Such consensus sequences are
generally arranged as a direct or head-to-tail repeat separated by
a few base pairs.
[0277] Examples of pathway-responsive promoters useful in the
practice of the present invention include synthetic insulin
pathway-responsive promoters containing the consensus insulin
binding sequence (Jacob, et al. (1995) J. Biol. Chem.
270:27773-27779), the cytokine pathway-responsive promoter, the
glucocorticoid pathway-responsive promoter (Lange, et al. (1992) J.
Biol. Chem. 267:15673-80), IL1 and IL6 pathway-responsive promoters
(Won K.-A and Baumann H. (1990) Mol. Cell. Biol. 10: 3965-3978), T3
pathway-responsive promoters, thyroid hormone pathway-responsive
promoters containing the consensus motif, the TPA
pathway-responsive promoters (TREs), TGF-beta pathway-responsive
promoters (as described in Grotendorst, et al. (1996) Cell Growth
and Differentiation 7: 469-480). Additionally, natural or synthetic
E2F pathway responsive promoters may be used. An example of an E2F
pathway responsive promoter is described in Parr, et al. (1997)
Nature Medicine 3:1145-1149) which describes an E2F-1 promoter
containing 4 E2F binding sites and is reportedly active in tumor
cells with rapid cycling. Examples of other pathway-responsive
promoters are well known in the art and can be identified in the
Database of Transcription Regulatory Regions on Eukaryotic Genomes
accessible through the Internet at
http://www.eimb.rssi.ru/TRRD.
[0278] In the certain applications of the invention, the viral
vector is an adenovirus. The term "adenovirus" is synonomous with
the term "adenoviral vector" and refers to viruses of the genus
adenoviridiae. The term adenoviridiae refers collectively to animal
adenoviruses of the genus mastadenovirus including but no limited
to human, bovine, ovine, equine, canine, porcine, murine and simian
adenovirus subgenera. In particular, human adenoviruses includes
the A-F sugenera as well as the individual serotypes thereof the
individual serotypes and A-F subgenera including but not limited to
human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Ad11A
and Ad 11P), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. The term bovine
adenoviruses includes but is not limited to bovine adenovirus types
1, 2, 3, 4, 7, and 10. The term canine adenoviruses includes but is
not limited to canine types 1 (strains CLL, Glaxo, RI261, Utrect,
Toronto 26-61) and 2. The term equine adenoviruses includes but is
not limited to equine types 1 and 2. The term porcine adenoviruses
includes but is not limited to porcine types 3 and 4. The term
viral vector includes replication deficient, replication competent
and conditionally replicating viral vectors.
[0279] Particularly useful are vectors derived from human
adenovirus types 2 and 5. These vectors may incorporate particular
modifications to enhance their therapeutic potential. For example
they may include deletions of E1a and E1b genes. Certain other
regions may be enhanced or deleted to provide specific features.
For example upregulation of the E3 region is described to reduce
the immunogenicity associated with human adenoviral vectors
administered to human subjects. The E4 region has been implicated
as important to expression of transgenes from the CMV promoter,
however the E4orf 6 protein has been described as leading to the
degradation of p53 in target cells in the presence of E1b large
protein (Steegenga, et al. (1998) Oncogene 16:345-347).
[0280] The therapeutic gene to be delivered is generally a
cytotoxic gene, a tumor suppressor gene, a toxin gene, a
pro-apoptotic gene, a pro-drug activating gene, or a cytokine gene.
The term "cytotoxic transgene" refers to a nucleotide sequence the
expression of which in the target cell induces lysis or apoptosis
of the cell. The term cytotoxic transgene includes but is not
limited to tumor suppressor genes, toxin genes, cytostatic genes,
pro-drug activating genes, or apoptotic genes. The vectors of the
present invention may be used to produce one or more therapeutic
transgenes, either in tandem through the use of IRES elements or
through independently regulated promoters.
[0281] The term "tumor suppressor gene" refers to a nucleotide
sequence, the expression of which in the target cell is capable of
supressing the neoplastic phenotype and/or inducing apoptosis.
Examples of tumor suppressor genes useful in the practice of the
present invention include the p53 gene, the APC gene, the DPC-4
gene, the BRCA-1 gene, the BRCA-2 gene, the WT-1 gene, the
retinoblastoma gene (Lee, et al. (1987) Nature 329:642), the MMAC-1
gene, the adenomatous polyposis coli protein (U.S. Pat. No.
5,783,666), the deleted in colon carcinoma (DCC) gene, the MMSC-2
gene, the NF-1 gene, nasopharyngeal carcinoma tumor suppressor gene
that maps at chromosome 3p21.3. (Cheng, et al. (1998) Proc. Nat.
Acad. Sci. 95:3042-3047), the MTS1 gene, the CDK4 gene, the NF-1
gene, the NF2 gene, and the VHL gene.
[0282] The term "toxin gene" refers to nucleotide sequence, the
expression of which in a cell produces a toxic effect. Examples of
such toxin genes include nucleotide sequences encoding pseudomonas
exotoxin, ricin toxin, diptheria toxin, and the like.
[0283] The term "pro-apoptotic gene" refers to a nucleotide
sequence, the expression thereof results in the programmed cell
death of the cell. Examples of pro-apoptotic genes include p53,
adenovirus E3-11.6K, the adenovirus E4orf4 gene, p53 pathway genes,
and genes encoding the caspases.
[0284] The term "pro-drug activating genes" refers to nucleotide
sequences, the expression of which, results in the production of
protein capable of converting a non-therapeutic compound into a
therapeutic compound, which renders the cell susceptible to killing
by external factors or causes a toxic condition in the cell. An
example of a prodrug activating gene is the cytosine deaminase
gene. Cytosine deaminase converts 5-fluorocytosine to
5-fluorouracil, a potent antitumor agent). The lysis of the tumor
cell provides a localized burst of cytosine deaminase capable of
converting 5FC to 5FU at the localized point of the tumor resulting
in the killing of many surrounding tumor cells. This results in the
killing of a large number of tumor cells without the necessity of
infecting these cells with an adenovirus (the so-called bystander
effect"). Additionally, the thymidine kinase (TK) gene (see U.S.
Pat. No. 5,631,236 and U.S. Pat. No. 5,601,818) in which the cells
expressing the TK gene product are susceptible to selective killing
by the administration of gancyclovir may be employed. The term
"cytokine gene" refers to a nucleotide sequence, the expression of
which in a cell produces a cytokine. Examples of such cytokines
include GM-CSF, the interleukins, especially IL-1, IL-2, IL-4,
IL-12, IL-10, IL-19, IL-20, interferons of the alpha, beta and
gamma subtypes especially interferon alpha-2b and fusions such as
interferon alpha-2-alpha-1.
[0285] Modifications and/or deletions to the above referenced genes
so as to encode functional subfragments of the wild type protein
may be readily adapted for use in the practice of the present
invention. For example, the reference to the p53 gene includes not
only the wild type protein but also modified p53 proteins. Examples
of such modified p53 proteins include modifications to p53 to
increase nuclear retention, deletions such as the delta13-19 amino
acids to eliminate the calpain consensus cleavage site,
modifications to the oligomerization domains (as described in
Bracco, et al. PCT published application WO97/0492 or U.S. Pat. No.
5,573,925).
[0286] The invention further includes use of gene-targeted
non-viral vectors. "Non-viral vector" for use in this aspect of the
invention include autonomously replicating, extrachromosomal
circular DNA molecules, distinct from the normal genome and
nonessential for cell survival under non-selective conditions
capable of effecting the expression of a DNA sequence in the target
cell. Plasmids autonomously replicate in bacteria to facilitate
bacterial production. Additional genes, such as those encoding drug
resistance, can be included to allow selection or screening for the
presence of the recombinant vector. Such additional genes can
include, for example, genes encoding neomycin resistance,
multi-drug resistance, thymidine kinase, beta-galactosidase,
dihydrofolate reductase (DHFR), and chloramphenicol acetyl
transferase.
[0287] In order to target the therapeutic gene to neoplastic, MDR
neoplastic and damaged (e.g., pathogen-infected) cells, it is
advantageous, in certain instances, to incorporate additional
elements into non-viral gene delivery systems which facilitate
cellular targeting. For example, a lipid encapsulated expression
plasmid may incorporate vimentin antibodies or ligands to
facilitate targeting. Although a simple liposome formulation may be
administered, the liposomes either filled or decorated with a
desired composition of the invention of the invention can delivered
systemically, or can be directed to a tissue of interest, where the
liposomes then deliver the selected therapeutic/immunogenic peptide
compositions. Vimentin antibodies and ligand for use in this
application include antibodies, monoclonal antibodies, humanized
antibodies, single chain antibodies, chimeric antibodies or
functional fragments (Fv, Fab, Fab') thereof. Alternatively,
non-viral vectors can be linked through a polylysine moiety to a
targeting moiety as described in Wu, et al. U.S. Pat. No. 5,166,320
and U.S. Pat. No. 5,635,383.
[0288] Liposomal Formulations
[0289] Another strategy that may be employed for vimentin-targeted
delivery of therapeutic agents is the use of immunoliposomes.
Immunoliposomes incorporate antibodies against tumor-associated
antigens into liposomes, which carry the therapeutic agent or an
enzyme that activates an otherwise inactive prodrug (see, e.g.,
Lasic et al. (1995) Science 267: 1275-76). A number of pre-clinical
reports have reported successful targeting and enhanced anti-cancer
efficacy with immunoliposomal drugs (Maruyama et al. (1990) J.
Pharm. Sci. 74: 978-84); Maruyama et al. (1995) Biochim. Biophys.
Acta 1234: 74-80; Otsubo et al. (1998) Antimicrob. Agents
Chemother. 42: 40-44; Lopes de Menezes et al. (1998) Cancer Res.
58: 3320-30).
[0290] Alternatively, non-antibody vimentin binding agents such as
modified LDL may be used as tumor-specific ligands in targeting
liposoomal formulations of therapeutics. For example,
folate-coupled liposomes can be used to target therapeutics to
tumors which overexpress the folate receptor. Folate-coupled
liposomes have been successfully delivered to folate
receptor-overexpressing cancer cells in vitro as well as in vivo
(Lee and Low (1994) J. Biol. Chem. 269: 3198-204; Lee and Low
(1995) Biochim. Biophys. Acta 1233: 134-44; Rui et al. (1998) J.
Am. Chem. Soc. 120: 11213-18; and Gabizon et al. (1999) Bioconj.
Chem. 10: 289-98). Indeed, several pre-clinical reports have
described the successful targeting of liposomal drugs coupled to
such ligands (Ichinose et al. (1998) Anticancer Res. 18: 401-4;
Yamamoto et al. (2000) Oncol. Rep. 7: 107-11; Rui et al. (1998) J.
Am. Chem. Soc. 120: 11213-18; and Gabizon et al. (1999) Bioconj.
Chem. 10: 289-98). Transferrin has been employed as a targeting
ligand to direct liposomal drugs to various types of cancer cell in
vivo (Ishida and Maruyama (1998) Nippon Rinsho 56: 657-62; Kirpotin
et al. (1997) Biochem. 36: 66-75). PEG-immunoliposomes with
anti-transferrin antibodies coupled to the distal ends of the PEG
preferentially associate with C6 glima cells in vitro and
significantly increased gliomal doxorubicin uptake after treatment
with the tumor-specific long-circulating liposomes containing
doxorubicin (Eavarone et al. (2000) J. Biomed. Mater. Res. 51:
10-14).
[0291] Methods of forming liposomal micelle/drug formulations are
known in the art. For example, therapeutic drug micelles can be
formed by combining a therapeutic drug and a phosphatidyl glycerol
lipid derivative (PGL derivative). Briefly, the therapeutic drug
and PGL derivative are mixed in a range of 1:1 to 1:2.1 to form a
therapeutic drug mixture. Alternatively, the range of therapeutic
drug to PGL derivative is in the ranges 1:1.2; or 1:1.4; or 1:1.5;
or 1:1.6; or 1:1.8 or 1:1.9 or 1:2.0 or 1:2.1. The mixture is then
combined with an effective amount of at least a 20% organic solvent
such as an ethanol solution to form micelles containing the
therapeutic drug. Methods for inclusion of an antibody or tumor
targeting ligand into the micelle formulation to produce
immunoliposomes are known in the art and described further below.
For example, methods for preparation and use of immunoliposomes are
described in U.S. Pat. Nos. 4,957,735, 5,248,590, 5,464,630,
5,527,528, 5,620,689, 5,618,916, 5,977,861, 6,004,534, 6,027,726,
6,056,973, 6,060,082, 6,316,024, 6,379,699, 6,387,397, 6,511,676
and 6,593,308.
[0292] As used herein, the term "phosphatidyl glycerol lipid
derivative (PGL derivative)" is any lipid derivative having the
ability to form micelles and have a net negatively charged head
group. This includes but is not limited to dipalmitoyl phosphatidyl
glycerol (DPPG), dimyristoyl phosphatidyl glycerol, and dicapryl
phosphatidyl glycerol. In one aspect, phosphatidyl derivatives with
a carbon chain of 10 to 28 carbons and having unsaturated side
aliphatic side chain are within the scope of this invention. The
complexing of a therapeutic drug with negatively-charged
phosphatidyl glycerol lipids having variations in the molar ratio
giving the particles a net positive (1:1) neutral (1:2) or slightly
negative (1:2.1) charge will allow targeting of different tissues
in the body after administration. However, complexing of a
therapeutic drug with negatively charged PGL has been shown to
enhance the solubility of the therapeutic drug in many instances,
thus reducing the volume of the drug required for effective
antineoplastic therapy. In addition, the complexing of a
therapeutic drug and negatively charged PGL proceeds to very high
encapsulation efficiency, thereby minimizing drug loss during the
manufacturing process. These complexes are stable, do not form
precipitates and retain therapeutic efficacy after storage at
4.degree. C. for at least 4 months. In order to achieve maximum
therapeutic efficacy by avoiding rapid clearance from the blood
circulation by the reticuloendothelial system (RES),
immunoliposomal drug formulations may incorporate components such
as polyethylene glycol (PEG) (see Klibanov et al. (1990) FEBS Lett.
268: 235-7; Mayuryama et al. (1992) Biochim. Biophys. Acta 1128:
44-49; Allen et al. (1991) Biochim. Biophys. Acta 1066: 29-36). PEG
conjugation to immunoliposomes has been shown to prolong liposome
circulation in blood, as well as to enhance the therapeutic
efficacy of liposomal drugs (Daemen et al. (1997) J. Control Rel.
44: 1-9; Storm et al. (1998) Clin. Cancer Res. 4: 111-115; Vaage et
al. (1997) Br. J. Cancer 75: 482-6; Gabizon et al. (1994) Cancer
Res. 54: 987-92). Long-circulating immunoliposomes can be
classified into two types: those with antibodies coupled to a lipid
head growth (Maruyama et al. (1990) J. Pharm. Sci. 74: 978-84); and
those with antibodies coupled to the distal end of PEG (Maruyama et
al. (1997) Adv. Drug Del. Rev. 24: 235-42). In certain instances,
it way be advantageous to place the tumor-specific antibodies at
the distal end of the PEG polymer to obtain efficient target
binding by avoiding steric hindrance from the PEG chains. This type
of immunoliposome formulation has been used successfully for in
vivo targeting to the lungs (Maruyama et al. (1995) Biochim.
Biophys. Acta 1234: 74-80; brain (Huwyler et al. (1996) Proc. Natl.
Acad. Sci. USA 93: 14164-69); and tumors (Allen et al. (1995)
Biochem. Soc. Transact. 23: 1073-79).
[0293] Effective delivery of drugs by immunoliposome formulations
is generally enhanced by active uptake of the bound immunoliposome
through endocytosis. Human scFv antibodies can be selected for
optimized internalization into tumor cells from a phage display
library to ensure optimal targeting and delivery of the
immunoliposomes into which they are incorporated (see Poul et al.
(2000) J. Mol. Biol. 301: 1149-61; Schier et al., (1996) J. Mol.
Biol. 263: 551-67).
[0294] Another strategy related to antibody-mediated tumor
targeting is antibody-directed enzyme pro-drug (ADEPT), which is a
two step therapeutic approach designed to generate a high
concentration of anticancer drugs in proximity to tumor cell
membranes (Springer et al. (1996) Adv. Drug Deliv. Rev. 22:
351-64). Using this strategy, an enzyme-antibody conjugate that
preferentially binds to a given tumor-associated antigen is
administered first, followed by injection of a nontoxic prod-drug,
which becomes activated by the action of the targeted enzyme. An
improved ADEPT using immunoliposomes as a targeted carrier for the
pro-drug-activating enzymes instead of an enzyme-antibody conjugate
has been developed and tested (Storm et al. (1997) Adv. Deliv. Rev.
24: 225-31; Vingerhoeds et al. (1993) FEBS Lett. 336: 485-90).
[0295] Therapies
[0296] The invention provides for treatment or prevention of
cancer, including, but not limited to, neoplasms, tumors,
metastases, or any disease or disorder characterized by
uncontrolled cell growth, and particularly multidrug resistant
forms thereof by the administration of therapeutically or
prophylactically effective amounts of anti-vimentin antibodies or
nucleic acid molecules encoding said antibodies. Examples of types
of cancer and proliferative disorders to be treated with the
vimentin-targeted therapeutics of the invention include, but are
not limited to, leukemia (e.g., myeloblastic, promyelocytic,
myelomonocytic, monocytic, erythroleukemia, chronic myelocytic
(granulocytic) leukemia, and chronic lymphocytic leukemia),
lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease),
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, colon
carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, renal cell carcinoma, hepatoma, Wilms' tumor,
cervical cancer, uterine cancer, testicular tumor, lung carcinoma,
small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma,
retinoblastoma, dysplasia and hyperplasia. In a particular
embodiment, therapeutic compounds of the invention are administered
to men with prostate cancer (e.g., prostatitis, benign prostatic
hypertrophy, benign prostatic hyperplasia (BPH), prostatic
paraganglioma, prostate adenocarcinoma, prostatic intraepithelial
neoplasia, prostato-rectal fistulas, and atypical prostatic stromal
lesions). The treatment and/or prevention of cancer includes, but
is not limited to, alleviating symptoms associated with cancer, the
inhibition of the progression of cancer, the promotion of the
regression of cancer, and the promotion of the immune response. In
one embodiment, commercially available or naturally occurring
anti-vimentin antibodies, functionally active fragments or
derivatives thereof are used in the present invention.
[0297] The vimentin therapeutics may be administered alone or in
combination with other types of cancer treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Examples of anti-tumor agents include, but are
not limited to, cisplatin, ifosfamide, paclitaxel, taxanes,
topoisomerase I inhibitors (e.g., CPT-11, topotecan, 9-AC, and
GG-211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil
(5-FU), leucovorin, vinorelbine, temodal, and taxol. In one
embodiment, one or more anti-vimentin antibodies are administered
to an animal, preferably a mammal and most preferably a human,
after surgical resection of cancer. In another embodiment, one or
more anti-vimentin antibodies are administered to an animal,
preferably a mammal and most preferably a human, in conjugation
with chemotherapy or radiotherapy. In another embodiment, one or
more anti-vimentin antibodies are administered to an animal,
preferably a mammal and most preferably a human, for the prevention
or treatment of cancer prior to (e.g., 1 minute, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12
hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1
minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week
after), or concomitantly with the administration of plasma to the
animal.
[0298] The anti-vimentin antibodies, and other vimentin-targeted
therapeutics described herein, may be administered to an animal,
preferably a mammal and most preferably a human, for the prevention
or treatment of cancer prior to (e.g., 1 minute, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12
hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1
minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week
after), or concomitantly with the administration of IgG antibodies,
IgM antibodies and/or one or more complement components to the
animal. In another preferred embodiment, one or more anti-vimentin
antibodies are administered to an animal, preferably a mammal and
most preferably a human, prior to (e.g., 1 minute, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12
hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1
minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week
after), or concomitantly with the administration of antibodies
immunospecific for one or more cancer cell antigens. In yet another
preferred embodiment, one or more anti-vimentin antibodies are
administered to an animal, preferably a mammal and most preferably
a human, for the prevention or treatment of cancer prior to (e.g.,
1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week
before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45
minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24
hours, 2 days, or 1 week after), or concomitantly with the
administration of antibodies currently used for the treatment of
cancer. Examples of such antibodies include, but are not limited
to, Herceptin, Retuxan, OvaRex, Panorex, BEC2, IMC-C225, Vitaxin,
Campath I/H, Smart MI95, LymphoCide, Smart I D10, and Oncolym.
[0299] The invention further provides methods for the treatment or
prevention of viral and other pathogen infections in an animal,
preferably a mammal and most preferably a human, said methods
comprising the administration of a therapeutically or
prophylactically effective amount of anti-vimentin antibodies or
nucleic acid molecules encoding said antibodies or other
vimentin-targeted therapeutics described herein. Examples of viral
infections which can be treated or prevented in accordance with
this invention include, but are limited to, viral infections caused
by retroviruses (e.g., human T-cell lymphotrophic virus (HTLV)
types I and II and human immunodeficiency virus (HIV)), herpes
viruses (e.g., herpes simplex virus (HSV) types I and II,
Epstein-Barr virus and cytomegalovirus), arenaviruses (e.g., lassa
fever virus), paramyxoviruses (e.g., morbillivirus virus, human
respiratory syncytial virus, and pneumovirus), adenoviruses,
bunyaviruses (e.g., hantavirus), cornaviruses, filoviruses (e.g.,
Ebola virus), flaviviruses (e.g., hepatitis C virus (HCV), yellow
fever virus, and Japanese encephalitis virus), hepadnaviruses
(e.g., hepatitis B viruses (HBV)), orthomyoviruses (e.g., Sendai
virus and influenza viruses A, B and C), papovaviruses (e.g.,
papillomavirues), picornaviruses (e.g., rhinoviruses, enteroviruses
and hepatitis A viruses), poxviruses, reoviruses (e.g.,
rotavirues), togaviruses (e.g., rubella virus), and rhabdoviruses
(e.g., rabies virus). The treatment and/or prevention of a viral
infection includes, but is not limited to, alleviating symptoms
associated with said infection, the inhibition or suppression of
viral replication, and the enhancement of the immune response.
[0300] The vimentin-targeted therapeutics described herein may be
administered alone or in combination with other types of anti-viral
or other anti-pathogen agents. Examples of anti-viral agents
include, but are not limited to: cytokines (e.g., IFN-.alpha.,
IFN-.beta., and IFN-.gamma); inhibitors of reverse transcriptase
(e.g., AZT, 3TC, D4T, ddC, ddI, d4T, 3TC, adefovir, efavirenz,
delavirdine, nevirapine, abacavir, and other dideoxynucleosides or
dideoxyfluoronucleosides); inhibitors of viral mRNA capping, such
as ribavirin; inhibitors of proteases such HIV protease inhibitors
(e.g., amprenavir, indinavir, nelfinavir, ritonavir, and
saquinavir); amphotericin B; castanospermine as an inhibitor of
glycoprotein processing; inhibitors of neuraminidase such as
influenza virus neuraminidase inhibitors (e.g., zanamivir and
oseltamivir); topoisomerase I inhibitors (e.g., camptothecins and
analogs thereof); amantadine and rimantadine. For example, one or
more anti-vimentin antibodies-drug conjugates are administered to
an animal, preferably a mammal and most preferably a human, for the
prevention or treatment of a viral infection prior to (e.g., 1
minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week
before subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45
minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24
hours, 2 days, or 1 week after), or concomitantly with the
administration of plasma to the animal.
[0301] In other examples, one or more vimentin-targeted
therapeutics are administered to an animal, preferably a mammal and
most preferably a human, for the prevention or treatment of a viral
infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45
minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24
hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,
8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or
concomitantly with the administration of IgG antibodies, IgM
antibodies and/or one or more complement components to the animal.
In another preferred embodiment, anti-vimentin antibodies are
administered to an animal, preferably a mammal and most preferably
a human, for the prevention or treatment of a viral infection prior
to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1
week before), subsequent to (e.g., 1 minute, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12
hours, 24 hours, 2 days, or 1 week after), or concomitantly with
the administration of antibodies immunospecific for one or more
viral antigens. Example of antibodies immunospecific for viral
antigens include, but are not limited to, Synagis.RTM., PRO542,
Ostavir, and Protovir.
[0302] The invention further provides methods for the treatment or
prevention of microbial infections in an animal, preferably a
mammal and most preferably a human, said methods comprising the
administration of a therapeutically or prophylactically effective
amount of anti-vimentin-targeted therapeutics. Examples of
microbial infections which can be treated or prevented in
accordance with this invention include, but are not limited to,
yeast infections, fungal infections, protozoan infections and
bacterial infections. Bacteria which cause microbial infections
include, but are not limited to, Streptococcus pyogenes,
Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria
meningitidis, Corynebacterium diphtheriae, Clostridium botulinum,
Clostridium perfringens, Clostridium tetani, Haemophilus
influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella
rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae,
Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio)
fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus,
Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis,
Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella
flexneri, Shigella sonnei, Salmonella typhimurium, Treponema
pallidum, Treponema pertenue, Treponema carateneum, Borrelia
vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,
Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis
carinii, Francisella tularensis, Brucella abortus, Brucella suis,
Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki,
Rickettsia tsutsugumushi, Chlamydia spp., and Helicobacter pylori.
The treatment and/or prevention of a microbial infection includes,
but is not limited to, alleviating symptoms associated with said
infection, the inhibition or suppression of replication, and the
enhancement of the immune response.
[0303] Vimentin-targeted therapeutics may be administered alone or
in combination with other types of anti-microbial agents. Examples
of anti-microbial agents include, but are not limited to:
antibiotics such as penicillin, amoxicillin, ampicillin,
carbenicillin, ticarcillin, piperacillin, cepalospolin, vancomycin,
tetracycline, erythromycin, amphotericin B, nystatin, metroidazole,
ketoconazole, and pentamidine. In one embodiment, a
vimentin-targeted therapeutic is administered to an animal,
preferably a mammal and most preferably a human, for the prevention
or treatment of a microbial infection prior to (e.g., 1 minute, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,
8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent
to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1
week after) or concomitantly with the administration of plasma to
the animal.
[0304] In certain instances, one or more vimentin-targeted
therapeutics are administered to an animal, preferably a mammal and
most preferably a human, for the prevention or treatment of a
microbial infection prior to (e.g., 1 minute, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12
hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1
minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week
after), or concomitantly with the administration of IgG antibodies,
IgM antibodies and/or one or more complement components to the
animal. In other instances, one or more vimentin-targeted
therapeutics are administered to an animal, preferably a mammal and
most preferably a human, for the prevention or treatment of a
microbial infection prior to (e.g., 1 minute, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12
hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1
minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week
after), or concomitantly with the administration of antibodies
immunospecific for one or more microbial antigens. Example of
antibodies immunospecific for microbial antigens include, but are
not limited to, antibodies immunospecific for LPS and capsular
polysaccharide 5/8. In certain embodiments, animals with increased
risk of a viral or bacterial infection are administered a
composition of the invention. Examples of such animals include, but
are not limited to, human burn patients, infants, immunocompromised
or immunodeficient humans, and the elderly.
4.6 Kits
[0305] The invention further provides kits for use in diagnostics
or prognostic, as well as therapeutic, methods for neoplasias and
multidrug resistant neoplasias. The diagnostic kits are useful, for
example, for detecting cell surface vimentin-expressing neoplasias
and for monitoring the occurrence of multidrug resistant cells in a
patient sample or in situ in a patient. For example, during the
course of patient chemotherapeutic treatment, monitoring of cell
surface vimentin, and other MDR-associated markers described
herein, provides valuable information regarding the efficacy of the
treatment and for avoiding the development of multidrug resistance.
For example, the kit can comprise a labeled compound or agent
capable of detecting cell surface vimentin protein in a biological
sample; as well as means for determining the amount of cell surface
vimentin in the sample; and means for comparing the amount of
vimentin in the sample with a standard (e.g., normal non-neoplastic
cells or non-MDR neoplastic cells). The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect cell surface vimentin
protein, as well as other MDR-associated markers. Such a kit can
comprise, e.g., one or more antibodies capable of binding
specifically to at least a portion of a cell surface vimentin
protein.
4.7 Vimentin Vaccines
[0306] Immunological compositions, including vaccines, and other
pharmaceutical compositions containing the vimentin protein, or
portions thereof, are included within the scope of the present
invention. One or more of the vimentin proteins, or active or
antigenic fragments thereof, or fusion proteins thereof can be
formulated and packaged, alone or in combination with other
antigens, using methods and materials known to those skilled in the
art for vaccines. The immunological response may be used
therapeutically or prophylactically and may provide antibody
immunity or cellular immunity, such as that produced by T
lymphocytes.
[0307] To enhance immunogenicity, the proteins may be conjugated to
a carrier molecule. Suitable immunogenic carriers include proteins,
polypeptides or peptides such as albumin, hemocyanin, thyroglobulin
and derivatives thereof, particularly bovine serum albumin (BSA)
and keyhole limpet hemocyanin (KLH), polysaccharides,
carbohydrates, polymers, and solid phases. Other protein derived or
non-protein derived substances are known to those skilled in the
art. An immunogenic carrier typically has a molecular mass of at
least 1,000 Daltons, preferably greater than 10,000 Daltons.
Carrier molecules often contain a reactive group to facilitate
covalent conjugation to the hapten. The carboxylic acid group or
amine group of amino acids or the sugar groups of glycoproteins are
often used in this manner. Carriers lacking such groups can often
be reacted with an appropriate chemical to produce them.
Preferably, an immune response is produced when the immunogen is
injected into animals such as mice, rabbits, rats, goats, sheep,
guinea pigs, chickens, and other animals, most preferably mice and
rabbits. Alternatively, a multiple antigenic peptide comprising
multiple copies of the protein or polypeptide, or an antigenically
or immunologically equivalent polypeptide may be sufficiently
antigenic to improve immunogenicity without the use of a
carrier.
[0308] The vimentin protein or portions thereof, such as consensus
or variable sequence amino acid motifs, or combination of proteins
may be administered with an adjuvant in an amount effective to
enhance the immunogenic response against the conjugate. One
adjuvant widely used in humans has been alum (aluminum phosphate or
aluminum hydroxide). Saponin and its purified component Quil A,
Freund's complete adjuvant and other adjuvants used in research and
veterinary applications are also available. Chemically defined
preparations such as muramyl dipeptide, monophosphoryl lipid A,
phospholipid conjugates such as those described by Goodman-Snitkoff
et al. (1991) J. Immunol. 147:410-415 and incorporated by reference
herein, encapsulation of the conjugate within a proteoliposome as
described by Miller et al. (1992) J. Exp. Med. 176:1739-1744 and
incorporated by reference herein, and encapsulation of the protein
in lipid vesicles such as Novasome.TM. lipid vesicles (Micro
Vescular Systems, Inc., Nashua, N.H.) may also be useful.
[0309] The invention includes the vimentin polypeptide fragments,
or subsequences of the intact vimentin polypeptide shown in FIG.
12A (SEQ ID NO. 1). Such vimentin polypeptide subsequences, or a
corresponding nucleic acid sequence that encodes them in the case
of DNA vaccines, are preferably selected so as to be highly
immunogenic. The principles of antigenicity for the purpose of
producing anti-vimentin vaccines apply also to the use of vimentin
polypeptide sequences for use as immunogens for generating
anti-vimentin polyclonal and monoclonal antibodies for use in the
vimentin-based diagnostics and therapeutics described herein.
[0310] Computer assisted algorithms for predicting polypeptide
subsequence antigenicity are widely available. For example
"Antigenic" looks for potential antigenic regions using the method
of Kolaskar (see Kolaskar and Tongaonkar (1990) FEBS Letters
276:172-174 "A semi-empirical method for prediction of antigenic
determinants on protein antigens"). In their initial study,
Kolaskar and Tongaonkar experimentally tested 169 antigenic. The
156 which have less than 20 amino acids per determinant were
selected (total 2066 residues). f(Ag) was calculated as the
frequency of occurrence of each residue in antigenic determinants
[f(Ag)=Epitope_occurrence/2066]. The Hydrophilicity, Accessibility
and Flexibility values are from Parker, et al. (see Parker, et al.
(1986) Biochemistry 25:5425-5432). In a given protein, the average
for each 7-mer is calculated, and values are assigned to the
central residue of the 7-mer. A residue is considered to be on the
surface if any of the 7-mer values was above the average for the
protein. These results were used to obtain f(s) as the frequency of
occurrence of amino acids at the surface. The prediction algorithm
includes the following steps: calculate the average propensity for
each overlapping 7-mer and assign the result to the central residue
(i+3) of the 7-mer; calculate the average for the whole protein; if
the average for the whole protein is above 1.0 then all residues
having above 1.0 are potentially antigenic; if the average for the
whole protein is below 1.0 then all residues having above the
average for the whole protein (note: the original paper has a
mangled formula here) are potentially antigenic; find 6-mers where
all residues are selected by step 3.
[0311] Another method for determining antigenicity of a polypeptide
subsequence is the algorithm of Hopp and Woods ((1981) Proc. Natl.
Acad. Sci. 86: 152-6). There are publicly available web sites for
Hopp and Woods algorithm analysis of a user-input polypeptide
sequence and convenient graphical output of the resulting analysis
(see, e.g.,
http://hometown.aol.com/_ht_a/lucatoldo/myhomepage/JaMBW/3/1/7/).
Using this algorithm to analyze the full-length human vimentin
sequence shown in FIG. 12A, several suitable sequence having a high
Hopp and Woods antigenic index of an adequate length for
immunogenicity were revealed. These include vimentin amino acid
residues: 45-60 (i.e., RPSTSRSLYASSPGGV); 295-315 (i.e.,
FADLSEAANRNNDALRQAKQE) and 330-345 (i.e., VDALKGTNESLERQMR).
[0312] In addition, the present invention provides a composition
comprising the vimentin protein or polypeptide fragment of the
invention in combination with a suitable adjuvant. Such a
composition can be in a pharmaceutically acceptable carrier, as
described herein. As used herein, "adjuvant" or "suitable adjuvant"
describes a substance capable of being combined with the vimentin
protein or polypeptide to enhance an immune response in a subject
without deleterious effect on the subject. A suitable adjuvant can
be, but is not limited to, for example, an immunostimulatory
cytokine, SYNTEX adjuvant formulation 1 (SAF-1) composed of 5
percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent
Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2
percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline.
Other suitable adjuvants are well known in the art and include
QS-21, Freund's adjuvant (complete and incomplete), alum, aluminum
phosphate, aluminum hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to
as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dip-
almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A,
referred to as MTP-PE) and RIBI, which contains three components
extracted from bacteria, monophosphoryl lipid A, trealose
dimycolate and cell wall skeleton (MPL+TDM+CWS) in 2%
squalene/Tween 80 emulsion. The adjuvant, such as an
immunostimulatory cytokine can be administered before the
administration of the vimentin protein or vimentin-encoding nucleic
acid, concurrent with the administration of the vimentin protein or
vimentin-encoding nucleic acid or up to five days after the
administration of the vimentin protein or vimentin-encoding nucleic
acid to a subject. QS-21, similarly to alum, complete Freund's
adjuvant, SAF, etc., can be administered within hours of
administration of the fusion protein.
[0313] The invention may also utilize combinations of adjuvants,
such as immunostimulatory cytokines co-administered to the subject
before, after or concurrent with the administration of the vimentin
protein or vimentin-encoding nucleic acid. For example,
combinations of adjuvants, such as immunostimulatory cytokines, can
consist of two or more of immunostimulatory cytokines of this
invention, such as GM/CSF, interleukin-2, interleukin-12,
interferon-gamma, interleukin-4, tumor necrosis factor-alpha,
interleukin-1, hematopoietic factor flt3L, CD40L, B7.1
co-stimulatory molecules and B7.2 co-stimulatory molecules. The
effectiveness of an adjuvant or combination of adjuvants may be
determined by measuring the immune response directed against the
vimentin polypeptide with and without the adjuvant or combination
of adjuvants, using standard procedures, as described herein.
[0314] Furthermore, the present invention provides a composition
comprising the vimentin protein or vimentin-encoding nucleic acid
and an adjuvant, such as an immunostimulatory cytokine or a nucleic
acid encoding an adjuvant, such as an immunostimulatory cytokine.
Such a composition can be in a pharmaceutically acceptable carrier,
as described herein. The immunostimulatory cytokine used in this
invention can be, but is not limited to, GM/CSF, interleukin-2,
interleukin-12, interferon-gamma, interleukin-4, tumor necrosis
factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L,
B7.1 con-stimulatory molecules and B7.2 co-stimulatory
molecules.
[0315] The term "vaccine" as used herein includes DNA vaccines in
which the nucleic acid molecule encoding vimentin or antigenic
portions thereof, such as any consensus or variable sequence amino
acid motif, in a pharmaceutical composition is administered to a
patient. For genetic immunization, suitable delivery methods known
to those skilled in the art include direct injection of plasmid DNA
into muscles (Wolff et al. (1992) Hum. Mol. Genet. 1:363), delivery
of DNA complexed with specific protein carriers (Wu et al. (1989)
J. Biol. Chem. 264:16985, coprecipitation of DNA with calcium
phosphate (Benvenisty and Reshef (1986) Proc. Natl. Acad. Sci.
83:9551), encapsulation of DNA in liposomes (Kaneda et al. (1989)
Science 243:375), particle bombardment (Tang et al., (1992) Nature
356:152, and Eisenbraun et al. (1993) DNA Cell Biol. 12:791), and
in vivo infection using cloned retroviral vectors (Seeger et al.
(1984) Proc. Natl. Acad. Sci. 81:5849).
[0316] In another embodiment, the invention is a polynucleotide
which comprises contiguous nucleic acid sequences capable of being
expressed to produce a vimentin or immunostimulant gene product
upon introduction of said polynucleotide into eukaryotic tissues in
vivo. The encoded gene product preferably either acts as an
immunostimulant or as an antigen capable of generating an immune
response. Thus, the nucleic acid sequences in this embodiment
encode an immunogenic epitope, and optionally a cytokine or a
T-cell costimulatory element, such as a member of the B7 family of
proteins.
[0317] Advantages to immunization with a gene rather than its gene
product include the following. First, is the relative simplicity
with which native or nearly native antigen can be presented to the
immune system. Mammalian proteins expressed recombinantly in
bacteria, yeast, or even mammalian cells often require extensive
treatment to ensure appropriate antigenicity. A second advantage of
DNA immunization is the potential for the immunogen to enter the
MHC class I pathway and evoke a cytotoxic T cell response
Immunization of mice with DNA encoding the influenza A
nucleoprotein (NP) elicited a CD8.sup.+ response to NP that
protected mice against challenge with heterologous strains of flu.
(Montgomery, D. L. et al. (1997) Cell Mol Biol 43(3):285-92; and
Ulmer, J. et al. (1997) Vaccine 15(8):792-794). Cell-mediated
immunity is important in controlling infection. Since DNA
immunization can evoke both humoral and cell-mediated immune
responses, its greatest advantage may be that it provides a
relatively simple method to survey a large number of vimentin genes
and gene fragments for their vaccine potential.
[0318] The invention also includes known methods of preparing and
using tumor antigen vaccines for use in treating or preventing
cancers. For example, U.S. Pat. No. 6,562,347 which teaches the use
of a fusion polypeptide including a chemokine and a tumor antigen
which is administered as either a protein or nucleic acid vaccine
to elicit an immune response effective in treating or preventing
cancer. Chemokines are a group of usually small secreted proteins
(7-15 kDa) induced by inflammatory stimuli and are involved in
orchestrating the selective migration, diapedesis and activation of
blood-born leukocytes that mediate the inflammatory response (see
Wallack (1993) Annals New York Academy of Sciences 178). Chemokines
mediate their function through interaction with specific cell
surface receptor proteins (23). At least four chemokine subfamilies
have been identified as defined by a cysteine signature motif,
termed CC, CXC, C and CX.sub.3C, where C is a cysteine and X is any
amino acid residue. Structural studies have revealed that at least
both CXC and CC chemokines share very similar tertiary structure
(monomer), but different quaternary structure (dimer). For the most
part, conformational differences are localized to sections of loop
or the N-terminus. In the instant invention, for example, a human
vimentin polypeptide sequence (such as that shown in FIG. 12A), or
polypeptide fragment thereof, and a chemokine sequence are fused
together and used in an immunizing vaccine. The chemokine portion
of the fusion can be a human monocyte chemotactic protein-3, a
human macrophage-derived chemokine or a human SDF-1 chemokine. The
vimentin portion of the fusion is, preferably, a portion shown in
routine screening to have a strong antigenic potential.
4.8 Pharmaceutical Formulations and Methods of Treatment
[0319] The present invention provides for both prophylactic and
therapeutic methods of treating a subject having a neoplastic
disease. Subjects at risk for such a disease can be identified by a
diagnostic or prognostic assay, e.g., as described herein.
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the neoplasm, such that
development of the neoplasm is prevented or, alternatively, delayed
in its progression. In general, the prophylactic or therapeutic
methods comprise administering to the subject an effective amount
of a compound which comprises a vimentin targeting component that
is capable of binding to cell surface vimentin present on
neoplastic, and particularly multidrug resistant neoplastic, cells
and which compound is linked to a therapeutic component.
[0320] Examples of vimentin targeting components include monoclonal
anti-vimentin antibodies and fragments thereof. Examples of
suitable therapeutic components include traditional
chemotherapeutic agents such as Actinomycin, Adriamycin,
Altretamine, Asparaginase, Bleomycin, Busulfan, Capecitabine,
Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,
Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,
Daunorubicin, Docetaxel, Doxorubicin, Epoetin, Etoposide,
Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin,
Ifosfamide, Imatinib, Irinotecan, Lomustine, Mechlorethamine,
Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane,
Mitoxantrone, Paclitaxel, Pentostatin, Procarbazine, Taxol,
Teniposide, Topotecan, Vinblastine, Vincristine, and Vinorelbine.
Other examples of suitable therapeutic components include
immunotoxins such as Pseudomonas exotoxin, a diphtheria toxin, a
plant ricin toxin, a plant abrin toxin, a plant saporin toxin, a
plant gelonin toxin, and pokeweed antiviral protein. Such
immunotoxins are targeted to the vimentin expressing neoplastic, or
multidrug resistant neoplastic, cell by the vimentin targeting
component of the therapeutic compound and, upon binding of cell
surface vimentin and uptake into the cell, function to kill or
block the growth of the neoplastic cell.
4.8.1 Effective Dose
[0321] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (The
Dose Lethal To 50% Of The Population) And The ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
that exhibit large therapeutic induces are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0322] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
4.8.2 Formulation and Use
[0323] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and
solvates may be formulated for administration by, for example,
injection, inhalation or insulation (either through the mouth or
the nose) or oral, buccal, parenteral or rectal administration.
[0324] For such therapy, the compounds of the invention can be
formulated for a variety of loads of administration, including
systemic and topical or localized administration. Techniques and
formulations generally may be found in Remrnington's Pharmaceutical
Sciences, Meade Publishing Co., Easton, Pa. For systemic
administration, injection is preferred, including intramuscular,
intravenous, intraperitoneal, and subcutaneous. For injection, the
compounds of the invention can be formulated in liquid solutions,
preferably in physiologically compatible buffers such as Hank's
solution or Ringer's solution. In addition, the compounds may be
formulated in solid form and redissolved or suspended immediately
prior to use. Lyophilized forms are also included.
[0325] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., ationd oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0326] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner. For administration
by inhalation, the compounds for use according to the present
invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebuliser, with the
use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g., gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch.
[0327] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0328] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0329] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt. Other suitable delivery systems include microspheres which
offer the possibility of local noninvasive delivery of drugs over
an extended period of time. This technology utilizes microspheres
of precapillary size which can be injected via a coronary catheter
into any selected part of the e.g. heart or other organs without
causing inflammation or ischemia. The administered therapeutic is
slowly released from these microspheres and taken up by surrounding
tissue cells (e.g. endothelial cells).
[0330] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration bile
salts and fusidic acid derivatives. in addition, detergents may be
used to facilitate permeation. Transmucosal administration may be
through nasal sprays or using suppositories. For topical
administration, the oligomers of the invention are formulated into
ointments, salves, gels, or creams as generally known in the art. A
wash solution can be used locally to treat an injury or
inflammation to accelerate healing.
[0331] In clinical settings, a therapeutic and gene delivery system
for the vimentin-targeted therapeutic can be introduced into a
patient by any of a number of methods, each of which is familiar in
the art. For instance, a pharmaceutical preparation of the
vimentin-targeted therapeutic can be introduced systemically, e.g.,
by intravenous injection.
[0332] The pharmaceutical preparation of the vimentin-targeted
therapeutic compound of the invention can consist essentially of
the compound in an acceptable diluent, or can comprise a slow
release matrix in which the gene delivery vehicle or compound is
imbedded.
[0333] The compositions may, if desired, be presented in a pack or
dispenser device that may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
5. EXAMPLES
[0334] This invention is further illustrated by the following
examples, which should not be construed as limiting. The contents
of all references, patents and published patent applications cited
throughout this application are hereby incorporated by reference.
Nucleotide and amino acid sequences deposited in public databases
as referred to herein are also hereby incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain,
using no more than routine experimentation, numerous equivalents to
the specific substances and procedures described herein. Such
equivalents are intended to be encompassed in the scope of the
claims that follow the examples below.
Example 1
5.1 Overexpression of a 53 kDa Protein in MDR Solid Tumor/Cancer
Cells
[0335] Studies were performed to determine what proteins, if any,
were differentially expressed in multidrug resistant tumor cell
lines as compared to their drug-sensitive counterparts. The seven
different cell lines used in the Examples are described in Table I
below.
TABLE-US-00001 TABLE I Multidrug resistant cell line derived from a
clone Cancer cell tissue Drug-sensitive of the "parent" drug- type
"parent" cell line sensitive cell line* Source of cells
Promyelocytic HL60 HL60/AR American Tissue leukemia Culture
Collection (ATCC, Manassas, VA) & Aurelium BioPharma
Promyelocytic NB4 NB4/VLB Deutsche Sammlung leukemia NB4/DOX von
Midroorganismen und Zellkulturen GmbH (DSMZ, Germany) &
Aurelium BioPharma T lymphoblastoid CEM CEM/VLB ATCC, Dr. William
CEM/DOX Beck and Aurelium BioPharma T lymphoblastoid HSB2 HSB2/VLB
ATCC and Aurelium HSB2/DOX BioPharma T lymphoblastoid Molt4
Molt4/DOX ATCC and Aurelium Molt4/VLB BioPharma Breast epithelial
MCF-7 MCF-7/AR ATCC and Aurelium MDA/MITO BioPharma Breast
epithelial MDA MDA/AR ATCC and Aurelium MDA/MITO BioPharma Ovarian
SKOV-3 SKOV-3/T320 ATCC and Aurelium 2008 2008/T320 BioPharma *MDR
cells lines are named systematically using the parent cell line
followed by a forward slash and abbreviation for the name of the
drug used in selecting resistance in the parent cell line. Drug
abbreviations in this table include: AR (adriamycin); VLB
(vinblastine); DOX (doxorubicin); MITO (mitomycin); and T320
(taxol).
[0336] Suspension cells were grown in RPMI or .alpha.-MEM medium,
containing 10% to 15% fetal calf serum (commercially available from
Hyclone Inc., Logan, Utah). The cells were grown in the absence of
antibiotics at 37.degree. C. in humid atmosphere of 5% CO.sub.2 and
95% air, and passaged when cultures were 1.times.10.sup.6 cells/ml.
Multidrug resistant cells (HL60/AR, NB4/VLB, NB4/DOX, CEM/VLB,
CEM/DOX, HSB2/VLB, HSB2/DOX, Molt4/VLB Molt4/DOX) (Aurelium
Biopharma Inc., Montreal (Quebec), Canada) were grown continuously
with appropriate concentrations of cytotoxic drugs. Similarly,
adherent cells were grown in .alpha.-MEM medium (MCF-7) or DMEM
(MDA), containing 10% fetal calf serum. Multidrug resistant cells
(MCF-7/AR, MDA/AR and MDA/MITO) were grown continuously with
appropriate concentrations of cytotoxic drugs. All cell lines were
examined for and determined to be free of mycoplasma contamination
using a PCR-based mycoplasma detection kit according to
manufacturer's instructions commercially available (e.g., from
Stratagene Inc., San Diego, Calif.). All multidrug resistant cell
lines were routinely tested for multidrug resistance using a panel
of different drugs representing different classes of drugs. The MDR
cells also expressed other MDR markers on their cell surface.
[0337] Different types of extracts were prepared from each cell
type. Cells were concentrated and lysed according to standard
procedures to obtain total cell extracts from the cells (e.g.,
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons Inc., New York City, N.Y. 1993). Alternatively, cells
were first surface biotinylated and then lysed to obtain
biotinylated total cell extracts (as shown in Examples below). To
do these studies, intact drug sensitive (CEM, HL60 and MCF-7) and
multidrug resistant cells (CEM/VLB, HL60/AR and MCF-7/AR) were
biotinylated with a membrane impermeable biotinylating agent,
Sulfo-NHS-LC-LC-Biotin (Pierce #21338). To do this, cells were
biotinylated by washing 3.times. with 50 ml PBS, pH 8. Next,
Sulfo-NHS-LC-LC-Biotin, which is a membrane impermeable reagent,
was prepared at 0.1-0.5 mg/ml and added to cells. The incubation
with Sulfo-NHS-LC-LC-Biotin was allowed to continue for 1 hour at
4.degree. C. with rotation. The reaction was stopped by washing
cells one time with 50 ml PBS pH 8, containing 10 mM glycine and
several times with 50 ml PBS, without glycine. Cells were then
lysed in 200 .mu.l of buffer A (1% SDS and 0.05 M Tris/HCl, pH
7.4), containing proteases inhibitors (Proteases inhibitors: 1
.mu.g/ml pepstatin, 1 .mu.g/ml leupeptin; 1 .mu.g/ml benzamidine;
0.2 mM PMSF) and incubated 5 minutes on ice. The cell lysate was
then sonicated with a Vibracell sonicator amplitude 40 setting #25
for 3.times.10 seconds with 1 minute on ice between shots. The
sonicated cell lysate was mixed with 800 .mu.l of buffer B (1.25%
Triton-X100, 0.05 M Tris/HCl, pH 7.4, 190 mM NaCl), containing
proteases inhibitors and incubated 5 minutes on ice. The cell
lysate was next centrifuged at 14,000 rpm in an Eppendorf microfuge
for 5 minutes. The supernatant was removed, and its protein
concentration was determined using the DC protein assay kit from
BIORAD according to manufacturer's instructions (BioRad
Laboratories, Hercules, Calif.) (see also Lowry et al., J. Biol.
Chem. 193: 265-275, 1951).
[0338] The use of the sulfo-LC-LC-biotinylating agent ensured the
modification of the c amino group on the lysine side chain in
proteins exposed on the cell surface; conversely, intracellular
proteins were not expected to be biotinylated since this
sulfo-biotin cannot cross the cell membrane of intact cells.
[0339] In addition, plasma membrane preparations were prepared from
surface biotinylated or nonbiotinylated cells of each type. To do
this, 3.times.10.sup.9 cells (of each cell type) were suspended in
12.5 ml of hypotonic buffer 1 (10 mM NaCl, 1.5 mM MgCl.sub.2, 10 mM
Tris-HCl pH 7.4) and incubated for 10 min on ice. The cells were
then homogenized in a Dounce glass homogenizer type B (15 ml). The
degree of cell lysis was determined by examining cells under the
microscope. Approximately 40 strokes were required to break about
85% of the cells. Immediately after homogenization, half volume
(6.25 ml) of 2.5.times. buffer II (Buffer 1.times.: 210 mM
mannitol, 70 mM sucrose, 5 mM Tris-HCl pH 7.5, 1 mM EDTA pH 7.5)
was added to the cell homogenate and mixed. The homogenate was spun
at 1300.times.g (3300 rpm) for 5 min in a Sorvall centrifuge using
SS34 rotor (brake off). The pellet containing the nuclei fraction
was separated from the supernatant containing cell membranes and
organelles. The post-nuclei supernatant was spun or centrifuged
again at 17000.times.g (11900 rpm) 15 min in a Sorvall centrifuge
using the SS34 rotor (brake off). The mitochondrial-enriched pellet
was separated from the membrane enriched supernatant fraction
(post-mitochondrial fraction). The latter supernatant was
centrifuged for 2 hours at 100,000.times.g in the Sorvall
Ultracentrifuge using the AH-629 rotor and PA UltraClear tubes from
Beckman #cat 344058 at 4.degree. C. The cytosolic enriched
supernatant was carefully removed and the membrane enriched
membrane pellet was resuspended in 300 .mu.l of buffer 1 above and
mixed well using a 27 gauge needle.
[0340] The cell membranes were further enriched by resolving the
last membrane pellet on a discontinuous sucrose gradient (16%, 31%,
45%, 60% w/v sucrose/buffer 1). Briefly, equal volume (about 350
.mu.l) of 32% w/v of sucrose in buffer 1 (16% w/v final) was added
to the resuspended pellet of enriched membrane material following
100,000.times.g centrifugation step. The sucrose gradient was
prepared with 6.9 ml 60% at the bottom of the tube followed by 9.9
ml of 45% sucrose, 13.9 ml 31% sucrose and 6.9 ml of 16% sucrose. A
16% sucrose containing crude plasma membranes was slowly poured on
the top of the gradient and the sample spun for 18 hours at
100,000.times.g at 4.degree. C. in the Sorvall ultracentrifuge
using the AH-629 rotor and PA UltraClear tubes from Beckman #cat
344058. The interphase between the 16% and 31% sucrose, containing
a highly enriched cell membrane was collected and washed once by
centrifugation with buffer 1. Following a 100,000.times.g
centrifugation in the ultracentrifuge the highly enriched cell
membrane pellet was resuspended in an appropriate volume (about 50
.mu.l) of buffer 1 and stored at -80.degree. C.
[0341] Equivalent amounts of protein from surface biotinylated
total cell extracts (containing biotinylated and non-biotinylated
proteins) and plasma membrane preparations from each of the cell
types (HL60, HL60/AR, NB4, NB4/DOX, NB4/VLB, CEM, CEM/VLB, CEM/DOX,
Molt4, Molt4/AR, Molt4/VLB, HSB2, HSB2/VLB, HSB2/DOX, MCF-7,
MCF-7/AR, MDA, MDA/AR, MDA/MITO) were analyzed by two dimensional
(2-D) gel electrophoresis and visualized by either silver staining
or immunoblotting with anti-VIM antibody or streptavidin-HPR
conjugate (streptavidin binds biotin). This allowed resolution of
protein samples according to differences in their isoelectric
points in the first dimension and molecular masses in the second
dimension. For the first dimension, isoelectric focusing was
achieved using 13 cm immobilized pH gradient strips (Amersham
Pharmacia Biotech, Piscataway, N.J.). Briefly, 13 cm strips were
rehydrated in a ceramic strip holder in 250 .mu.l rehydration
buffer containing the protein samples (0.5 mg-2 mg proteins) for 15
hours at 30 volts. Electrode pads were then placed over each
electrode and the proteins separated on an IPGphor unit using the
following program:
13 cm strips (pH 4-7): [0342] -500V for 500 Vh [0343] -1000V for
1000 Vh [0344] -8000V for 16000 Vh Strips were then slightly rinsed
with water and equilibrated in 1% DTT in equilibration buffer for
15 min, followed by 4% iodoacetamide in equilibration buffer for 15
minutes.
[0345] For the second dimension, the above isoelectric strips were
subject to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) using an 8% or 10% gel, according to the
method of Laemmli (Laemmli U.K., Nature 227:680-685, 1970).
Molecular weight markers were loaded onto a 2.times.3 mm filter
paper and placed at one end of the strip. The strip and molecular
weight marker filter were then sealed onto the polyacrylamide gel
with a 0.5% agarose solution in running buffer. Proteins were
slowly transferred from the strip to the gel at 30 V at room
temperature for an hour and the separation was carried out at
4-8.degree. C. for 17-18 hours at 70 V or 75 V for 8% or 10% gels
respectively.
[0346] The gels were next stained with silver stain, and
photographed. (Note, that at this point, the gel-resolved proteins
can also be transferred onto Hybond C nitrocellulose membrane and
then immunoblotted with antibody or streptavidin-HRP, as described
below in the Examples).
[0347] An about 53 kDa protein (with a pI of about 5) was
identified in silver stained 2D-gels of total cell extracts of
MCF-7 and MCF-7/AR cells. The 53 kDa protein was highly
overexpressed in the total cell extracts of multidrug resistant
MCF-7/AR cells compared to drug sensitive MCF-7 cells (FIGS. 1(A)
and (B)).
Example 2
5.2 Identification of the 53 kDa Protein of MCF-7/AR Breast Cancer
Cells as Vimentin
[0348] To discover the identity of the 53 kDa protein of breast
MCF-7 and MCF-7/AR cells, 2-D gels were loaded with MCF-7 or
MCF-7/AR total cells extract (2.times.750 .mu.g, pI 4 to 7, 10%
gel), silver-stained, and the 53 kDa spots were excised. The spots
were next processed using optimized procedures for
staining/destaining of gels, trypsin digestion, peptide extraction
and peptide purification. Briefly, gels were stained with
SilverQuest silver stain (Invitrogen, Carlsbad, Calif.) using the
recipe given by the manufacturer. The protein spots of interest
were excised with an acid washed razor blade and cut into small
pieces on a clean (acid washed) glass plate and transferred to a
200 .mu.l PCR tube (MeOH treated). The gel pieces were mixed with
50 .mu.l destainer A and 50 .mu.l destainer B (provided with
SilverQuest kit) (or 100 .mu.l of the destainers premix prepared
fresh), and incubated for 15 min at room temperature without
agitation. The wash was removed using a capillary tip. Water was
added to the gel pieces, mixed and incubated 10 min at room
temperature. The latter step was repeated three times. The gel
pieces were then dehydrated in 100 .mu.l 100% methanol for 5
minutes at room temperature, followed by re-hydration in 30%
methanol/water for 5 min. Gel pieces were then washed twice in
water for ten minutes and twice in 25 mM Ambic (ammonium
bicarbonate)/30% acetonitrile (for 5 minutes) followed by a gel
drying step. Tryptic digestion of the destained and washed gel
pieces was performed by adding about 1 volume of trypsin solution
(130 ng of enzyme in 25 mM ammonium bicarbonate, 5 mM CaCl.sub.2)
to 1 volume of gel pieces and the samples were left on ice for 45
minutes. The digestion was allowed to proceed for 15-16 hr at
37.degree. C. Digested peptides were extracted with acetonitrile
for 15 minutes at room temperature with shaking. The gel
pieces/solvent were sonicated 5 minutes and re-extracted with 25 mM
Ambic:50% acetonitrile without sonication. Digested peptides were
further extracted with 5% formic acid:50% acetonitrile:45% water
freshly prepared at room temperature with shaking for 15 minutes.
The mix was completed with one volume of acetonitrile and the gel
pieces/solvent were sonicated 5 minutes and re-extracted the same
way without sonication. The collected material was combined and
dried. The extracted peptides were re-suspended in 5% methanol with
0.2% trifluoroacetic acid (or 0.5% formic acid) then loaded on an
equilibrated C18 bed (Ziptip from Millipore). The loaded Ziptip was
washed with 5% acetonitrile containing 0.2% TFA (or 0.5% formic
acid) and then eluted with 10 .mu.l of 60% acetonitrile. The eluted
peptide solution was dried and analyzed using MALDI mass
spectroscopy (Mann M, et al. Ann. Rev. Biochem. 70:437-473,
2001).
[0349] The mass spectrogram of the 53 kDa spot (following
purification and protease digestion) consisted of over 20 tryptic
peptides, of which 16 peptides were from the 53 kDa protein while
the remaining peptides were trypsin autodigestion products (data
not shown). The 53 kDa peptides were further analyzed using a
sequence database search shareware software program called
ProFound.TM. (http://www.proteomics.com/prowl-cgi/Profound.exe).
PROFOUND was used to search public databases for protein sequences
(e.g., non-redundant collection of sequences at the US National
Center for Biotechnology Information (NCBI)). The NCBI database
contains translated protein sequences from the entire collection of
DNA sequences kept at GenBank, and also the protein sequences in
the PDB, SWISS-PROT and PIR databases.
[0350] Using the ProFound.TM. program, the 53 kDa protein was
identified as vimentin (Chan et al., Biochemistry 28: 1033-1039,
1989; Hale and Mansfield, Nucleic Acids Res. 17(23): 10112, 1989)
with a probability Z-score of 2.43, which is in the 99.9.sup.th
percentile, based on the analysis of the 16 tryptic peptide
sequences that covered 43% of vimentin's complete amino acid
sequence (FIG. 12). Two values were taken into account when
evaluating the PROFOUND analysis result: the Z probability score
and the percent coverage (the percent of the peptides' amino acid
sequences relative to the identified protein's complete amino acid
sequence). Z=1.65-2.43 is an acceptable range of scores. Z=1.65
means that the result is in the 95th percentile and Z=2.43 means
that the result is in the 99.9th percentile. Thus the Z score of
2.43 indicated that the 53 kDa spot peptide sequences corresponded
to those of vimentin with the highest degree of probability.
[0351] The sequences of the 16 peptides were spread throughout the
vimentin molecule and together corresponded to 43% of the complete
vimentin protein amino acid sequence, hence the protein identified
was the full length protein and not a fragment or fusion
protein.
[0352] To confirm that vimentin is overexpressed in MCF-7/AR cells
total extracts were resolved by 2D-PAGE, transferred onto
nitrocellulose and blotted with anti-vimentin (MS-129-P,
NeoMarker).
[0353] As shown in FIGS. 2(A) and (B), Vimentin was overexpressed
in the total cell extracts of multidrug resistant MCF-7/AR cells by
at least a factor of 11 as compared to the total cell extracts of
drug-sensitive MCF-7 cells.
Example 3
5.3 Cell Surface Expression of Vimentin in MDR Lymphoblastic
Leukemia Cancer Cells
[0354] To determine whether vimentin was present on the inside or
outside of the cell membrane, intact lymphoblastic leukemia tumor
cells (CEM and CEM/VLB) were treated the same way as in Example 2,
with a membrane impermeable biotinylating reagent that reacts with
lysines, and total cell extracts from both drug sensitive (CEM) and
multidrug resistant (CEM/VLB) were prepared. Two equivalent sets of
2-D gels of CEM and CEM/VLB were prepared and silver stained or
transferred onto nitrocellulose and blotted with
streptavidin-HRP.
[0355] FIGS. 3A and B represent the 2-D streptavidin-HRP blot of
CEM and CEM/VLB total surface biotinylated cell extracts,
respectively the area represented corresponds to the presumed
location of vimentin on a 2-D gel (MW=53 kDa, pI=5.1). The 50 kDa
protein was overexpressed on the surface of multidrug resistant
CEM/VLB cells by at least a factor of two as compared to the
drug-sensitive CEM cells (compare FIGS. 3A and B).
[0356] To confirm the identity of this protein as vimentin, the 50
kDa spot was localized on the silver stained gel using the
coordinates obtained from the corresponding blot, was excized and
processed to prepare a sample for MALDI analysis as described in
example II. The mass spectrogram of the 50 kDa spot (following
tryptic digestion and peptide purification) consisted of over 20
tryptic peptides, of which 15 peptides were from the 50 kDa protein
while the remaining peptides were trypsin autodigestion products.
The 50 kDa peptides were further analyzed using the sequence
database search shareware software program called ProFound.TM.
(http://www.proteomics.com/prowl-cgi/Profound.exe).
[0357] The 50 kDa protein was identified as vimentin (Chan et al.,
Biochem. 28: 1033-1039, 1989; Hale and Mansfield, Nucleic Acids
Res. 17(23): 10112, 1989) with a probability Z-score of 2.43, which
is in the 99.9.sup.th percentile, based on the analysis of 14
tryptic peptide sequences that covered 52% of its complete amino
acid sequence. The Z score of 2.43 indicated that the about 50 kDa
spot peptide sequence corresponded to those of vimentin with the
highest degree of probability.
[0358] Full length vimentin protein was expressed on the cell
surface of CEM/VLB cells and was biotinylated at one site: at a
lysine residue located relatively close to the C-terminus of the
molecule. The sequences of the 15 peptides were spread throughout
the vimentin molecule and together corresponded to 52% of the
complete vimentin protein amino acid sequence, hence the protein
identified was, again, the full length protein and not a fragment
or fusion protein. The biotinylated region was detected in two
ways. First, the bound biotin moiety changed the mass of the
peptide containing it relative to the unbiotinylated peptide, and
secondly, biotinylated peptides bound to streptavidin beads
(immobilized streptavidin beads commercially available from Pierce
#20347, Dallas, Tex.), whereas non-biotinylated peptides did
not.
Example 4
5.4 Cell Surface Expression of Vimentin in MDR Promyelocytic
Leukemia Cancer Cells
[0359] To determine whether vimentin was present on the inside or
outside of the cell membrane, intact promyelocytic leukemia tumor
cells (HL60 and HL60/AR) were reacted with a membrane impermeable
biotinylating reagent that reacts with the amino acid lysine, and
total cell extracts from both drug sensitive (HL60) and multidrug
resistant (HL60/AR) were prepared.
[0360] To do this, cells were biotinylated by washing 3.times. with
50 ml PBS, pH 8. Next, Sulfo-NHS-LC-LC-Biotin (Pierce Chemicals,
Dallas, Tex.), which is a membrane impermeable reagent, was
prepared at 0.1-0.5 mg/ml and added to cells. The incubation with
Sulfo-NHS-LC-LC-Biotin was allowed to continue for 1 hour at
4.degree. C. with rotation. The reaction was stopped by washing
cells one time with 50 ml PBS pH 8, containing 10 mM glycine and
several times with 50 ml PBS, without glycine. Cells were then
lysed in 200 .mu.l of buffer A (1% SDS and 0.05 M Tris/HCl, pH
7.4), containing proteases inhibitors (Proteases inhibitors: 1
.mu.g/ml pepstatin, 1 .mu.g/ml Leupeptin; 1 .mu.g/ml benzamidine;
0.2 mM PMSF) and incubated 5 minutes on ice. The cell lysate was
then sonicated with a Vibracell sonicator amplitude 40 setting #25
for 3.times.10 seconds with 1 minute on ice between shots. The
sonicated cell lysate was mixed with 800 .mu.l of buffer B (1.25%
Triton-X100, 0.05 M Tris/HCl, pH 7.4, 190 mM NaCl), containing
proteases inhibitors and incubated 5 minutes on ice. The cell
lysate was next centrifuged at 14,000 rpm in an Eppendorf microfuge
for 5 minutes. The supernatant was removed, and its protein
concentration was determined using the DC protein assay kit from
BIORAD according to manufacturer's instructions (BioRad
Laboratories, Hercules, Calif.) (see also Lowry et al., J. Biol.
Chem. 193: 265-275, 1951).
[0361] In addition, streptavidin purified biotinylated proteins
were prepared from the surface-biotinylated total cell extracts of
HL60 and HL60/AR cells using immobilized streptavidin (commercially
available from Pierce, catalog no #20347 or Amersham Pharmacia
Biotech RPN1231 (Piscataway, N.J.).) To do this, 50 .mu.l samples
containing 500 .mu.g to 2 mg protein were diluted to 450 .mu.l
final with buffer C (1:4 v/v of buffers A and B above), containing
proteases inhibitors. Samples were then centrifuged at 14,000 rpm
in an eppendorf microfuge for 1 minute. The supernatant was
transferred to a new Eppendorf tube and mixed with 100 .mu.l of
Streptavidin-linked sepharose beads. The protein lysate together
with the linked sepharose beads were incubated with rotation
overnight at 4.degree. C. The mix was centrifuged at 14,000 rpm for
30 seconds in an eppendorf microfuge. The supernatant was removed
and the protein loaded beads were washed 3 times with buffer C,
then with 500 mM NaCl in buffer C and buffer C again. Proteins were
eluted from the Streptavidin-linked beads with SDS sample buffer
following 10 minutes boiling. Elution was repeated and volumes
pooled.
[0362] Equivalent amounts of protein from HL60 and HL60/AR
(promyelocytic leukemia) cell surface biotinylated total cell
extracts and streptavidin purified cell surface biotinylated
extracts were resolved by SDS-PAGE according to the method of
Laemmli (supra) and subjected to Western blotting analysis and
probed with either anti-vimentin antibody (mouse monoclonal
MS-129-P, NeoMarker) or with horseradish peroxidase (HRP)-linked
streptavidin (which specifically binds to biotinylated proteins,
Amersham RPN 1231). To do this, gels containing separated proteins
were transferred onto Hybond C nitrocellulose membrane (Amersham,
Piscataway, N.J.) according to the method of Towbin (Towbin, H. T.,
Proc. Natl. Acad. Sci. USA 76:4350-4354, 1979). The nitrocellulose
membranes were then probed with antibody or HPR-streptavidin.
Binding of the antibody was detected with HRP conjugated goat
anti-mouse secondary antibody (BioRad). Both secondary antibody and
HRP-linked streptavidin were detected using the ECL
chemiluminescent detection kit commercially available from Pierce.
Relative protein levels were detected by exposure in the dark to
XAR films (Kodak, Rochester, N.Y.).
[0363] Anti-vimentin antibody bound to vimentin protein that was
expressed at significantly higher levels in surface biotinylated
total cell extracts of multidrug resistant HL60/AR cells compared
to HL60 cells (see FIG. 4A). Vimentin was also overexpressed in
streptavidin purified cell surface biotinylated proteins of HL60/AR
cells compared to HL60 cells (see FIG. 4B).
[0364] To confirm that vimentin present on the cell surface was
indeed biotinylated, surface biotinylated total cell extracts from
HL60 and HL/60AR cells were immunoprecipated with anti-vimentin
antibody and the immunoprecipates were resolved on SDS-PAGE and
Western blotted. To do this, samples were prepared as described
above using Protein A Sepharose beads instead of streptavidin
beads. To elute the proteins from the Protein A Sepharose beads,
the loaded beads were washed five times with Buffer D (0.03% SDS,
0.05 M Tris-HCl, pH 7.4, 0.1% Triton X-100, 5 mg/ml BSA fraction V,
150 mM NaCl) and one time with Buffer E (150 mM NaCl, 0.05 M
Tris-HCl, pH 7.4), containing protease inhibitors as above.
Proteins were eluted from the beads with SDS sample buffer
following 10 minutes incubation at room temperature with vortex
every 1 minute. Protein elution from the beads was repeated one
more time and the volumes pooled. Proteins were resolved by
SDS-PAGE and Western blotting as before with either anti-VIM
monoclonal antibody or HRP-labeled streptavidin.
[0365] The blots were probed with anti-vimentin antibody and with
streptavidin-HRP. As expected, vimentin was detected in the
immunoprecipitates from both cell types, however, significantly
more cell surface biotinylated vimentin was present in the
anti-vimentin immunoprecipitates from HL60/AR cells compared with
those from HL60 cells (see FIGS. 4C and 4D). Vimentin has a highly
sensitive protease reactive site at D90 which may account for a
lower molecular weight band observed when vimentin was
immunoprecipitated with anti-vimentin.
[0366] These results, taken together, suggest that translocation of
vimentin across the plasma membrane to the cell surface, as well as
additional de novo synthesis of vimentin, was associated with
multidrug resistance in HL60/AR cells.
Example 5
5.5 Cell Surface Expression of Vimentin in MDR Breast Cancer
[0367] To determine whether vimentin was present on the inside or
outside of the cell membrane, intact breast tumor cells (MCF-7 and
MCF-7/AR) were treated with a membrane impermeable biotinylating
reagent that reacts with the amino acid lysine, and total cell
extracts from both drug sensitive (MCF-7) and multidrug resistant
(MCF-7/AR) were prepared. In addition, streptavidin purified
biotinylated proteins as well as anti-vimentin immunoprecipates
were prepared from the surface biotinylated total cell extracts of
MCF-7 and MCF-7/AR cells, resolved on SDS-PAGE and transferred onto
nitrocellulose the same way as in Example 4.
[0368] Anti-vimentin antibody bound to vimentin protein which was
expressed at significantly higher levels in surface biotinylated
total cell extracts of multidrug resistant MCF-7/AR cells compared
to MCF-7 cells (see FIG. 5A). Vimentin was also overexpressed in
streptavidin purified cell surface biotinylated proteins of
MCF-7/AR cells compared to MCF-7 cells (see FIG. 5B). In addition,
the immunoprecipitate blots were probed with anti-vimentin antibody
and with streptavidin-HRP. As expected, significantly more cell
surface biotinylated vimentin was present in the anti-vimentin
immunoprecipitates from MCF-7/AR cells compared with those from
MCF-7 cells (see FIGS. 5C and 5D).
[0369] These results, taken together, suggest that translocation of
vimentin across the plasma membrane to the cell surface, as well as
additional de novo synthesis of vimentin, was associated with
multidrug resistance in MCF-7/AR cells.
Example 6
5.6 Cell Surface Expression of Vimentin in MDR Promyelocytic
Leukemia Cancer Cells
[0370] HL60 and HL60/AR cells were analyzed by cell surface
staining and fluorescence-activated cell sorter (FACS) analysis to
determine the difference in cell surface expression of vimentin on
the two cell lines. To do this, indirect immunofluorescence
analysis was performed using 1 .mu.g of rabbit polyclonal
anti-vimentin as primary antibody (CBL46, Cymbus Biotech,
Hampshire, UK), followed by goat anti-rabbit IgG FITC-conjugated
secondary antibody (Cat # F9887, Sigma).
[0371] The following indirect staining procedure was used to
prepare the cells for FACS analysis:
[0372] Cells were washed three times in 50 ml PBS, pH 7.4 and 0.1%
NaN.sub.3 and counted.
[0373] 1.times.10.sup.6 cells per sample were placed in 100 .mu.l
PBS and 0.1% NaN.sub.3 in 12.times.75 mm tubes or deep 96 well
plate. The first antibody (rabbit polyclonal anti-vimentin
antibody) was added and incubated for 20 minutes at 37.degree. C. 3
ml (or 1.25 ml in plate) PBS pH 7.4 and 0.1% NaN.sub.3 was added
the mixture was spun for 5 min (1000 rpm/200.times.g). The
supernatant was discarded and the pellet was resuspended in 100
.mu.l PBS pH 7.4 and 0.1% NaN.sub.3.
[0374] The second Ab (FITC-conjugated) was added. The mixture was
prepared by diluting 1/2 in PBS, pH 7.4 and 0.1% NaN.sub.3 and spun
at maximum speed for 30 min. at 4.degree. C. Separate from pellet
and use a 1/10 for staining. Incubate for 20 min at 37.degree. C. 3
ml (or 1.25 ml in plate) PBS pH 7.4 and 0.1% NaN.sub.3 was added
and the mixture spun 5 min (1000 rpm/200.times.g). The supernatant
was discarded and the addition of PBS and NaN.sub.3 was repeated.
The mixture was spun again for 5 min (1000 rpm/200.times.g).
[0375] 1 .mu.g-2 .mu.g/.mu.l of EMA was added. The mixture was
incubated in white light on ice for 10 minutes. 3 ml (or 1.25 ml in
plate) PBS pH 7.4 and 0.1% NaN.sub.3 was added and the mixture spun
5 min (1000 rpm/200.times.g). The supernatant was discarded and the
addition of PBS and NaN.sub.3 was repeated. The mixture was spun
again for 5 min (1000 rpm/200.times.g). Resuspend in 500 .mu.l PBS
pH 7.2/2% paraformaldehyde and store in the dark at 4.degree.
C.
[0376] For each sample, 10,000 cells were analyzed using a
fluorescence-activated cell sorter (Beckman Coulter XL MCL). The
fluorescence emission corresponding to specifically stained cells
was calculated by subtracting the emission measured for cells at
530 nm stained with precleared rabbit IgG negative control. In some
cases, rabbit IgG controls were precleared with HL60 or HL60/AR
cells in order to reduce the non-specific staining on each cell
line. This was done by incubating 1 .mu.g of rabbit IgG with
1-2.times.10.sup.6 cells in 50 .mu.l-100 .mu.l PBS for 20 min at
37.degree. C. The mix was then spun and the supernatant used as
negative control or carried through one or two additional
incubations. The supernatant was used as rabbit IgG control for
HL60 and HL60/AR when incubated with HL60 and HL60/AR
respectively.
[0377] The results showed that vimentin was overexpressed on the
surface of HL60/AR cells at a level five fold higher than the level
of vimentin expressed on the surface of HL60 cells (see FIG.
6).
[0378] As expected, the difference between the expression level of
vimentin on the surface of HL60/AR cells and the expression level
of vimentin on the surface of HL60 cells was better observed when
precleared rabbit IgG controls were used. Additionally, the
difference between the expression level of vimentin on the surface
of HL60/AR cells and the expression level of vimentin on the
surface of HL60 cells was also better observed when low levels of
primary antibody were used. Thus, when indirect immunofluorescence
analysis was performed using 0.1 .mu.g rather than 1 .mu.g of
polyclonal anti-vimentin as primary antibody (CBL46, Cymbus
Biotech, Hampshire, UK) and 0.1 .mu.g rather than 1 .mu.g of
precleared or non-precleared rabbit IgG as negative control, the
fold difference in expression levels of vimentin on HL60/AR versus
HL60 cells was 26 (precleared) and 9 (non-precleared)
respectively.
[0379] In addition, indirect immunofluorescence analysis was
performed using increasing amounts 10 .mu.g-20 .mu.g-40 .mu.g of
monoclonal anti-VIM as primary antibody (commercially available
from NeoMarker, catalog no. MS-129-P), followed by anti-mouse
IgG-FITC conjugated secondary antibody at 1:10 dilution
(commercially available from Chemicon, catalog #AP181F) The
fluorescence emission corresponding to specifically stained cells
was calculated by subtracting the emission measured for cells at
530 nm stained with mouse IgG.sub.1 isotype control. 40 .mu.g of
anti-VIM was saturating for HL60/AR.
[0380] Similarly, experiments were performed with amounts of
antibody ranging from 2.5 .mu.g to 40 .mu.g to determine the
concentration of anti-vimentin that would be saturating HL60 cells.
The values of fluorescence intensities obtained didn't increase in
a concentration dependent manner and ranged between 0.2 and 0.9 RFI
indicating that the cells were already saturated at 2.5 .mu.g. 40
.mu.g of anti-vimentin were therefore used to quantify the number
of molecules of vimentin present at the surface of HL60 and HL60/AR
cells.
[0381] At saturating concentration (40 .mu.g of monoclonal
anti-vimentin), vimentin was expressed on the surface of HL60/AR
cells at a level 37-fold higher than the level of vimentin
expressed on the surface of HL60 cells. The number of molecules of
vimentin expressed on the surface of HL60/HL60/AR cells has been
determined using a quantum simply cellular flow cytometry
quantification kit (Serotec #FCSC814: 498-44298 molecules, Raleigh,
N.C.). The QSC system consists of microbeads of approximately the
size of lymphocytes that are coupled to goat anti-mouse antibodies.
A set of five populations of microbeads bearing different known
amounts of goat anti-mouse antibodies are provided in the kit. The
microbeads are incubated with saturating amounts of the mouse
monoclonal antibody of interest. The fluorescence intensities
obtained with the microbeads are plotted to create a standard curve
of the fluorescence intensity to the number of antibody molecules
bound on the beads. The signal obtained with the cells (at
saturating amounts) is then, using the standard curve, correlated
to the number of antibody molecules bound to the cells, which
corresponds to the number of antigens present on the surface of the
cell. At saturating conditions of antibody for the beads and the
cell lines, HL60 and HL60/AR have been shown to bear approximately
186 and 5052 molecules of vimentin respectively when using the
mouse monoclonal anti-vimentin purchased from NeoMarker (see FIG.
7). This corresponds to a greater than 27 fold increase in cell
surface vimentin expression in the multidrug resistant HL60/AR cell
line as compared to the corresponding non-MDR HL60 cell line.
Example 7
5.7 Characterization of Cell Surface Vimentin Expression in MDR
Lymphoblastic Leukemia Cancer Cells
[0382] Molt4 and Molt4/DOX cells were analyzed by cell surface
staining and fluorescence-activated cell sorter (FACS) analysis to
determine the difference in cell surface expression of vimentin on
the two cell lines. To do this, indirect immunofluorescence
analysis was performed using 20 .mu.g of mouse monoclonal
anti-vimentin as primary antibody (MS-129-P, NeoMarker), followed
by goat anti-mouse IgG FITC-conjugated secondary antibody
(Chemicon, catalog #AP181F).
[0383] The cells were prepared according to the procedure set forth
in Example 6 above. For each sample, 10,000 cells were analyzed
using a fluorescence-activated cell sorter (Beckman Coulter XL MCL,
Fullerton, Calif.). The fluorescence emission corresponding to
specifically stained cells was calculated by subtracting the
emission measured for cells at 530 nm stained with 20 .mu.g of
mouse IgG.sub.1 isotype control.
[0384] The results of the FACS analysis demonstrate that vimentin
was expressed on the surface of Molt4/AR cells at a level 1.5 fold
higher than the level of vimentin expressed on the surface of Molt4
cells (see FIG. 8).
Example 8
5.8 Characterization of Cell Surface Vimentin Expression in MDR
Breast Cancer Cells
[0385] HS574M, MCF-7, MCF-7/AR, MDA, MDA/AR and MDA/MITO breast
cancer cells were analyzed by cell surface staining and
fluorescence-activated cell sorter (FACS) analysis to determine the
difference in cell surface expression of vimentin on the two cell
lines. To do this, indirect immunofluorescence analysis was
performed using 5 and/or 20 .mu.g of mouse monoclonal anti-vimentin
as primary antibody (MS-129-P, NeoMarker), followed by goat
anti-mouse IgG FITC-conjugated secondary antibody (Chemicon,
catalog #AP181F, Temecula, Calif.).
[0386] The cells were prepared according to the procedure set forth
in Example VI above. For each sample, 10,000 cells were analyzed
using a fluorescence-activated cell sorter (Beckman Coulter XL
MCL). The fluorescence emission corresponding to specifically
stained cells was calculated by subtracting the emission measured
for cells at 530 nm stained with 5 .mu.g or 20 .mu.g of mouse
IgG.sub.1 isotype control.
[0387] The results of FACS analysis demonstrate that while vimentin
was not expressed on the surface of HS574M cells, it was expressed
on the surface of MCF-7/AR cells at a level 1.5 fold higher than
the level of vimentin expressed on the surface of MCF-7 cells (see
FIG. 9). Similarly, vimentin was expressed on the surface of MDA/AR
cells at a level 3 to 5 fold higher than the level of vimentin
expressed on the surface of MDA cells (see FIG. 9). Vimentin was
expressed on the surface of MDA/MITO cells at a level 3 folds
higher than the level of vimentin expressed on the surface of MDA
cells (see FIG. 10).
[0388] The number of molecules of vimentin expressed on the surface
of MCF-7 and MCF-7/AR cells was determined using a quantum simply
cellular flow cytometry quantification kit (Serotec #FCSC814:
498-44298 molecules) the same way as described in example 5.6. At
saturating conditions of antibody for the beads and the cell lines
(50 .mu.g), MCF-7 and MCF-7/AR was shown to bear approximately
14,556 and 22,010 molecules of vimentin respectively when using the
mouse monoclonal anti-vimentin purchased from NeoMarker, which
corresponds to a 1.5 fold difference (see FIG. 11).
Example 9
5.9 Comparison of Vimentin Expression in Normal, Neoplastic and MDR
Neoplastic Cell
[0389] Normal cells were next compared to hematological cancer
cells and MDR hematological cancer cells to determine the
difference in levels of cell surface expressed vimentin. To do
this, human blood samples (collected in heparin tubes) were
obtained from donors and were processed within an hour. Briefly,
erythrocytes were separated from leukocytes and plasma on a Ficoll
hypaque gradient (Histopaque Sigma 1077-1, St. Louis, Mo.).
[0390] Specifically, 15 ml of blood was diluted one-half in
phosphate buffered saline (PBS), pH 7.4 and put over equal volume
of Ficoll gradient. Cells were separated by centrifugation at
400.times.g for 30 min. at room temperature. The upper phase
(plasma) was removed until 0.5 cm from the plasma/Ficoll interface.
Then, mononuclear cells (at the interface) were collected in a 50
ml Falcon tubes. The Ficoll was removed and red blood cells (at the
bottom of the tube) were collected. All cells types were washed
with PBS two times by spinning for 10 min. at 250.times.g at
4.degree. C. and counted. Cells were then re-suspended at
1.times.10.sup.7 cells/ml in PBS, and 100 .mu.l (1.times.10.sup.6
cells) aliquots were used for flow cytometry (FACS) analysis.
[0391] The cells were stained with primary and secondary antibody
for FACS analysis as described in Example 6 above. For each sample,
10,000 cells were analyzed using a fluorescence-activated cell
sorter (Beckman Coulter XL MCL). The fluorescence emission
corresponding to specifically stained cells was calculated by
subtracting the emission measured for cells at 530 nm stained with
an isotype matching control.
[0392] Each FACS experiment was carried out with several controls,
including cells alone to determine autofluorescence; cells plus EMA
to identify dead cells during analysis; cells plus secondary
antibody (Ab) alone to identify non-specific interactions due to
secondary antibody; cells plus isotype matching antibodies (Abs) or
appropriate host primary Abs control; cells (mononuclear cells)
plus CD45-PC5 mouse monoclonal, phycoerythrin-Cyanine 5 conjugate
(Immunotech PN IM2653) to establish and gate the different
leukocyte populations; and erythrocytes plus Glycophorin A-FITC
mouse monoclonal, Cymbus CBL 409F as positive control for
erythrocytes.
[0393] When red blood cells or white blood cells from normal,
healthy donors were stained with 0.25 .mu.g-0.5 .mu.g or 1 .mu.g
(3, 6 and 21 donors, respectively) of polyclonal anti-vimentin
antibody (CBL46, Cymbus Biotech), in contrast to HL60 and HL60/AR
stained with 1 .mu.g of polyclonal anti-vimentin antibody, the
normal RBC and WBC were found not to express any vimentin on their
cell surface (results not shown).
[0394] Therefore, as well as showing an increase in MDR neoplastic
versus non-MDR neoplastic cells of the same type, cell surface
vimentin expression is increased in neoplastic versus
non-neoplastic cells of the same or similar origin. Accordingly,
levels of cell surface vimentin may be used to detect and or
diagnose neoplasms as wells as MDR neoplasms.
Example 10
5.10 Vimentin-Based Vaccine Protection Against MDR Hematological
Cancer Cells and MDR Mammary Adenocarcinoma Cells
[0395] To determine whether or not the full length vimentin protein
expressed on the cell surface of MDR hematological cancer cells is
useful as an antigen for a vaccine to immune animals against MDR
hematological cancer cells, purified vimentin protein is combined
with an adjuvant (e.g., Freund's), and administered to groups of
mice having a hematological cancer caused by the presence of a
hematological cancer cell. One such non-limiting hematological
cancer is Acute lymphocytic leukemia.
[0396] To do this, the mice are injected with hematological tumor
cells that are compatible with the mice's MHC type (or are injected
into SCID mice). Some of the mice receive the injected tumor cells
prior to being immunized with purified full length murine vimentin
protein; some receive the hematological tumor cell injection after
being immunized with purified full length murine vimentin protein.
Note that the purified vimentin protein may be administered with an
adjuvant. Proper controls are performed for each group of mice
(i.e., one control group receives only the purified murine vimentin
protein; another receives only the hematological tumor cell
injection).
[0397] The tumors that form in the mice are weighed or measured
(e.g., tumor cell number counted, tumor excised and weighed, or
tumor measured by calipers). The mice that are vaccinated with
vimentin prior to injection of the tumor cells are found to have
tumors that are smaller after treatment than those that were not
vaccinated with vimentin prior to injection of the tumor cells.
[0398] In further studies, the efficacy of vimentin as an antigen
against MDR mammary adenocarcinoma cells (MCF/AR) is assessed.
Briefly, six week-old female mice are injected with 30-250 ug of
whole vimentin or control antigen administered with or without
adjuvant S.C. and I.P or into rear footpads on days 1, 7, 14, 21,
28 and 35. After various intervals, blood samples are collected
from the retro-orbital venous plexus for anti-vimentin antibody
assay. Mice are challenged on day 59 with 1.5.times.10.sup.4 viable
MDR mammary adenocarcinoma cells (MCF/AR) administered S.C. into
the right flauk. Mice are examined twice a week and tumor incidence
is determined from the number of mice bearing tumors. Tumor size
was measured with a Vernier caliper. Survival rates are measured up
to 80 days post challenge with adenocarcinoma.
Example 11
5.11 Vimentin-Targeted Therapy Against MDR Hematological Cancer
Cells
[0399] In order to determine whether targeting a therapeutic to
cell surface vimentin would be useful in treating a preexisting
cancerous condition, hematological tumor cells are administered to
MHC-matched mice, and tumors are allowed to form. Next, the mice
are administered vincristine (or another chemotherapeutic drug) at
a dosage predicted to kill most, but not all of the tumor cells in
the mice. Those mice that are identified as having developed
multidrug resistant tumor cells are administered a composition
comprising vincristine and a binding agent that specifically binds
to murine vimentin protein, where the binding agent is operably
linked to ricin toxin.
[0400] The mice that receive the composition show a better
prognosis (i.e., smaller tumor or fewer tumor cells) as compared to
mice that receive only the binding agent or only the
vincristine.
[0401] In further studies, the efficacy of a vimentin-targeted
therapeutic in treating an MDR mammary adenocarcinoma cells
(MCF/AR) is assessed. Briefly, Athymic nude mice are used for the
MCF-7/ADR xenografts. Male mice 5-7 weeks old, weighing 18-22 g,
are used. Mice receive a subcutaneous (s.c.) injection of the cells
using 0.5 million cells/inoculation under the shoulder. After s.c.
implantation of the cells, when the s.c. tumour is approximately
5.5 mm in size, mice are randomized into treatment groups of four
including controls and groups receiving vincristine or doxorubicin
alone (4 mg/kg), intraperitoneally (i.p.) every 2 days,
anti-vimentin alone (100 ug-1 mg/kg) or both vincristine or
doxorubicin and anti-vimentin mAb (100 ug-1 mg/Kg), i.p. The
animal's weight is measured every 4 days. Each animal is tagged in
the ear and followed individually throughout the experiments. Tumor
growth starting on the first day of treatment is measured and the
volume of the xenograft is monitored every 4 days. The mice are
anaesthetized and killed when the mean tumor weights is over 1 g in
the control group. Tumor tissue is excised from the mice and its
weight is measured.
Example 12
5.12 Qualitative Analysis of Cell Surface Vimentin Expression in
MDR Breast Cancer Cells
[0402] MCF-7 and MCF-7/AR cells were analyzed by immunostaining to
determine the difference in cell surface expression of vimentin on
the two cell lines. To do this, immunofluorescence analysis was
performed using 0.5 .mu.g of anti-vimentin as primary antibody
(clone V9, NeoMarker MS-129-P) on permeabilized and
non-permeabilized cells. Ethidium monoazide (EMA) was used to stain
the nucleus of permeabilized or damaged cells.
[0403] FIGS. 13A and 13B provides flow charts depicting the steps
taken to stain cells. The control cells were MCF-7 and MCF-7/AR
stained with 0.5 .mu.g mouse IgG1 and no staining was observed in
either the permeabilized or non-permabilized cells (data not
shown). As shown in FIG. 14, vimentin was clearly expressed on the
surface of intact multidrug resistant MCF-7/AR cells while MCF-7
didn't show any staining. When cells were permabilized, MCF-7
showed minimal staining whereas MCF-7/AR showed a very abundant
network of vimentin filaments (FIG. 14).
Example 13
5.13 Quantitative Analysis of Cell Surface Vimentin Expression in
MDR Promyelocytic Leukemia Cancer Cells
[0404] HL60 and HL60/AR cells were analyzed for surface exposure of
vimentin by direct binding of 125-iodine labeled anti-vimentin to
the surface of the cells. To do this, anti-vimentin (NeoMarker,
MS-129-PABX), mouse IgG1 (isotype matching negative control, Sigma,
M-9035) and anti-CD33 (positive control, Serotec, #MCA1271) were
iodinated using IODO-GEN.RTM. precoated iodination tubes (Pierce,
#28601) following the procedure given by the manufacturer (average
activity obtained: 7.5 .mu.Ci/.mu.g).
[0405] Cells were washed twice with RPMI 1640 and resuspended at
10.sup.6 cells/100 .mu.L in the same media. Viability was assessed
by trypan blue staining and was less than 5%. Cells
(1.times.10.sup.6) were aliquoted into borosilicate tubes and
incubated for 1 hour on ice in the presence of 1 .mu.g of
radiolabeled anti-vimentin, IgG1 or anti-CD33. After incubation,
cells were washed 3 times with 1 ml RPMI 1640 and the cell pellet
was counted in a gamma-counter.
[0406] The results of the intact cell radioimmunoassay are
represented in a bar graph in FIG. 15. The numbers are expressed in
counts per minute obtained for the cell pellet from which the IgG1
background has been subtracted. Vimentin was expressed on the
surface of the resistant HL60/AR cells at a level 3 folds higher
than the level of vimentin expressed on the surface of
drug-sensitive HL60 cells.
Example 14
5.14 Cytotoxic Effect of Radioiodinated Anti-Vimentin on HL60 and
HL60/AR Cells
[0407] Cells were washed twice with RPMI 1640 and resuspended at
10.sup.6 cells/100 .mu.l in the same media. Viability was assessed
by trypan blue staining and was less than 5%. Cells
(1.times.10.sup.6) were aliquoted into borosilicate tubes and
incubated for 4 hour at 37.degree. C. (0.5% CO.sub.2) in the
presence of 5 .mu.g of sterile filtered radiolabeled anti-vimentin,
IgG1 or anti-CD33 (iodination procedure was described in example
5.13). After incubation, cells were washed once with 1 ml RPMI
1640, 10% FBS, 1 mM HEPES, resuspended in 1 ml of the same media
and seeded at 5000 cells per well into a flat bottom tissue culture
96 well plate. The cytotoxicity of the radiolabeled antibodies was
assessed after 72 hours in an MTT based assay. Anti-CD33 was used a
positive control for surface binding of the antibody and 120 nM
doxorubicin (Doxo) was used as positive control for
cytotoxicity.
[0408] The results of the cytotoxicity assay are represented in a
bar graph in FIG. 16. Values are expressed as percent viability.
Values for percent survival with anti-vimentin and anti-CD33
antibody-.sup.125I conjugates were normalized against the values
obtained for the radiolabeled IgG1 (non-significant background
binding, 100% viability). Values obtained with the anticancer drug
doxorubicin were normalized against the values obtained for cells
non-treated with drug.
[0409] The results obtained demonstrate that radiolabeled
anti-vimentin has a cytotoxic effect on HL60 and HL60/AR which both
express vimentin on their surface. The slight increase in
cytotoxicity observed for HL60/AR (10%) might be due to the fact
that HL60/AR drug-resistant cells express more vimentin on their
surface than HL60 drug-sensitive cells.
Example 15
5.15 Molecular Quantitation of Cell Surface Vimentin in Drug
Sensitive and Drug Resistant Breast and Ovarian Cancer Cell
Lines
[0410] Breast MCF-7, MCF-7/AR, MDA, MDA/mito, and ovarian SKOV3,
SKOV/T320 (resistant to taxol) 2008 and 2008/T320 (resistant to
taxol) cells were analyzed for surface exposure of vimentin by
direct binding of 125-iodine labeled anti-vimentin to the surface
of the cells. To do this, anti-vimentin (NeoMarker, #MS-129-PABX)
and mouse IgG1 (isotype matching negative control Sigma, M-9035)
were iodinated using IODO-GEN.RTM. precoated iodination tubes
(Pierce, #28601) following the procedure given by the manufacturer
(average activity obtained: 7.5 .mu.Ci/.mu.g).
[0411] Cells were seeded at 20,000 cells per well into a 96
Stripwell.TM. plate (Costar, #9102). After an overnight growth in
complete media (37.degree. C., 0.5% CO.sub.2), wells were gently
washed with 100 .mu.l media containing 1% FBS and incubated for 1
hour at 37.degree. C. (0.5% CO.sub.2) in 100 .mu.L media containing
1% FBS and 0.1% sodium azide as well as 100 ng of radioiodinated
anti-vimentin (or IgG1). Mortality was checked prior to incubation
by trypan blue staining and was typically less than 1%. After
incubation, media was discarded, wells washed twice with 350 .mu.L
media containing 1% FBS, and individually counted in a
gamma-counter.
[0412] Binding studies were performed on MDA/mito cells using
increasing amounts of radiolabeled anti-vimentin in an intact cell
radioimmunoassay. Scatchard analysis was used to calculate the
apparent dissociation constant (K.sub.d) and the number of
molecules of antibody bound per cell. The average number of
molecules of anti-vimentin bound per cell was obtained from the
average of 5 independent Scatchard determinations and is reported
in the table in FIG. 17A. FIG. 17B shows a Scatchard plot for the
first experiment described in the table in FIG. 17A.
[0413] The average number of vimentin epitopes present on the
surface of MDA/mito cells was 2.49.times.10.sup.6 per cell and the
average K.sub.d was 5.7 nM. The K.sub.d was determined for MDA
cells in a separate experiment and was 9.3 nM (see Table in FIG.
17C), which is equivalent to the K.sub.d measured for MDA/mito
cells.
[0414] The number of vimentin epitopes present on MCF-7, MCF-7/AR,
MDA, SKOV3, SKOV/T320, 2008 and 2008/T320 was calculated from the
fold difference in surface exposure between these cell lines and
MDA/mito. This fold was determined by testing the same number of
MDA/mito cells and cells listed above (about 20,000 cells) using
the above intact cell radioimmunoassay with 100 ng of radiolabeled
anti-vimentin. The latter numbers of surface vimentin epitopes
represent the average from multiple independent experiments (n=2 to
5). These numbers are reported in the table in FIG. 17C and show
that MCF-7/AR, MDA, MDA/mito, SKOV3 and SKOV/T320 cells express
large amounts of vimentin on their surface (up to
2.5.times.10.sup.6 for MDA/mito), whereas MCF-7, 2008 and 2008/T320
numbers are relatively low (note that "2.5.E+06 represents base 10
Exponential, i.e. 2.5.times.10.sup.6). The folds difference in
surface exposure of vimentin between sensitive and resistant
counterpart cells are given as R/S and were 41.2, 4, 1.6, and 2 for
MCF-7/AR, MDA/mito, SKOV/T320 and 2008/T320 respectively.
Example 16
5.16 Induction of Cell Surface Exposure of Vimentin in Breast and
Ovarian Cancer Cells
[0415] Given the findings reported in example 5.15 above, which
shows that various breast and ovarian cancer cells selected with
doxorubicin, mitoxanthrone or taxol demonstrate increased surface
exposure of vimentin when compared to their sensitive counterpart,
it was of interest to determine whether these drugs were capable of
inducing surface exposure of vimentin in MDA and SKOV3 cell lines.
To do this, 20,000 cells were seeded into a 96 Stripwell.TM. plate
(Costar, #9102). After 5 h, the culture media was removed and
replaced with culture media containing various drugs (as indicated
in FIG. 18) and incubated for 12 to 16 hours at 37.degree. C. (0.5%
CO.sub.2). After incubation, wells were gently washed with 100
.mu.L media containing 1% FBS and incubated for 1 hour at
37.degree. C. (0.5% CO.sub.2) in 100 .mu.L media containing 1% FBS
and 0.1% sodium azide as well as 100 ng of radioiodinated
anti-vimentin (or IgG1) (labeling procedure is given in example
5.13). Mortality was checked prior to incubation by trypan blue
staining and was typically less than 1%. After incubation, media
was discarded, wells washed twice with 350 .mu.L media containing
1% FBS, and individually counted in a gamma-counter. FIG. 18
represents the surface exposure of vimentin when MDA cells were
incubated with 1 or 10 .mu.M taxol, 1 or 10 .mu.M doxorubicin, or
0.1 or 1 .mu.M mitoxanthrone. Values were corrected for
non-specific binding with IgG1. A slight induction of surface
exposure of vimentin was observed with all drugs, mitoxanthrone
having the most significant effect. A similar but less intense
effect was observed when SKOV3 cells were treated with 1 .mu.M
taxol. These finding are of importance in the context of
immunotherapy combined with drugs.
Example 17
5.17 Internalization of .sup.125I-labeled Anti-Vimentin by
MCF-7/AR, MDA, MDA/AR and MDA/Mito Cells
[0416] Internalization of cell surface vimentin was measured on
breast cancer cells kept in suspension (see FIG. 19A) or cells
adhered in a 96 well plate (FIG. 19B). FIG. 19A, shows 10.sup.6
cells were subcultured with a dissociation buffer (Gibco,
#13150-016), and then transferred to a borosilicate tube, washed in
PBS and resuspended in 200 .mu.l alpha-MEM, 3% BSA containing 1
.mu.g of radioiodinated anti-vimentin, or 1 .mu.g of anti-Mucin-1
as positive control for internalization or 1 .mu.g of IgG1 as
background control. Mucin-1 (CD227) is a highly glycosylated
protein ubiquitously present in many human tissues that, in tumor
cells, is often produced at elevated levels and with an abnormal
glycosylation pattern. Mouse antibodies have been used in clinical
trials for the purpose of treating such cancers and are
commercially available (e.g., from Fitzgerald Industries, Inc.,
Concord, Mass.). After 1 hour incubation at 4.degree. C., cells
were washed twice and further incubated additional 4 hours at
4.degree. C. or 37.degree. C. Cell viability was 100% according to
trypan blue staining of cells prior to incubation with radiolabeled
IgGs. After incubation, cells were washed and the radiolabel
determined by counting samples in a gamma-counter. Cells were then
stripped for 10 min at RT with 50 mM L-Gly, pH 2.8, 150 mM NaCl,
washed and counted for residual activity in a gamma counter. The
graph shown in FIG. 19A represents the percent of surface
associated radiolabel (stripped with L-Gly) and internalized
(remaining after stripping) for MCF-7/AR cells at 4.degree. C. and
37.degree. C. The % internalization was obtained from the
difference in internalization measured at 4 and 37.degree. C. and
was 38.2% for vimentin and 12.5% for Mucin-1.
[0417] The results show that anti-vimentin antibodies attached to a
radionuclide therapeutic agent (or diagnostic probe) bind to cell
surface vimentin present on the surface of multidrug resistant
breast epithelial neoplastic cells, and are actively internalized
in a temperature-dependent manner. The temperature dependence
suggests that the antibody-.sup.125I conjugate is actively taken up
by endocytosis. Regardless of the mechanism of uptake, the results
indicate that anti-vimentin antibodies are capable of recognizing
and transporting therapeutic agents (or diagnostic agents) into MDR
neoplastic cells for the treatment (or diagnostic detection) of
such cells.
[0418] For FIG. 19B, cells were prepared the same way as for an
intact cell radioimmunoassay (20,000 cells per well). Briefly,
after an overnight growth, wells were washed and preincubated in
100 .mu.l media+1% FBS containing 100 ng of radioiodinated
anti-vimentin or IgG1 as background control, for 1 hour at
4.degree. C. Mortality was checked prior to preincubation by trypan
blue staining and was less than 1%. After the preincubation, media
was discarded, wells washed twice with 350 .mu.L media containing
1% FBS, and further incubated for 4 hours at 4.degree. C. or
37.degree. C. After incubation, wells were washed the same way as
earlier and cells were stripped for 10 min at RT with 50 mM L-Gly,
pH 2.8, 150 mM NaCl, and washed. Stripped material and wells were
then counted for activity in a gamma counter. The graph in FIG. 19B
represents the percent of surface associated cpm (stripped with
L-Gly) and internalized (remaining after stripping) for MCF-7/AR,
MDA, MDA/AR, MDA/mito cells at 4 and 37.degree. C. The percent
internalization of vimentin was obtained from the difference in
internalization measured at 4 and 37.degree. C. and was 11.3, 25.3,
24.8 and 8.9% for MCF-7/AR, MDA, MDA/AR and MDA/mito
respectively.
[0419] These results further support the usefulness of
anti-vimentin antibodies for the targeting and uptake of linked
therapeutic and diagnostic agents for both neoplastic, and MDR
neoplastic cells.
EQUIVALENTS
[0420] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific embodiments described specifically
herein. Such equivalents are intended to be encompassed in the
scope of the following claims.
Sequence CWU 1
1
51466PRTHomo sapiens 1Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr
Arg Arg Met Phe Gly 1 5 10 15Gly Pro Gly Thr Ala Ser Arg Pro Ser
Ser Ser Arg Ser Tyr Val Thr 20 25 30Thr Ser Thr Arg Thr Tyr Ser Leu
Gly Ser Ala Leu Arg Pro Ser Thr 35 40 45Ser Arg Ser Leu Tyr Ala Ser
Ser Pro Gly Gly Val Tyr Ala Thr Arg 50 55 60Ser Ser Ala Val Arg Leu
Arg Ser Ser Val Pro Gly Val Arg Leu Leu 65 70 75 80Gln Asp Ser Val
Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe 85 90 95Lys Asn Thr
Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp 100 105 110Arg
Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn 115 120
125Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser
130 135 140Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg
Arg Gln145 150 155 160Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val
Glu Val Glu Arg Asp 165 170 175Asn Leu Ala Glu Asp Ile Met Arg Leu
Arg Glu Lys Leu Gln Glu Glu 180 185 190Met Leu Gln Arg Glu Glu Ala
Glu Asn Thr Leu Gln Ser Phe Arg Gln 195 200 205Asp Val Asp Asn Ala
Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val 210 215 220Glu Ser Leu
Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu225 230 235
240Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile
245 250 255Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg
Asp Val 260 265 270Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu
Gln Glu Ala Glu 275 280 285Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu
Ser Glu Ala Ala Asn Arg 290 295 300Asn Asn Asp Ala Leu Arg Gln Ala
Lys Gln Glu Ser Thr Glu Tyr Arg305 310 315 320Arg Gln Val Gln Ser
Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr 325 330 335Asn Glu Ser
Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala 340 345 350Val
Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu 355 360
365Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln
370 375 380Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala
Thr Tyr385 390 395 400Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile
Ser Leu Pro Leu Pro 405 410 415Asn Phe Ser Ser Leu Asn Leu Arg Glu
Thr Asn Leu Asp Ser Leu Pro 420 425 430Leu Val Asp Thr His Ser Lys
Arg Thr Phe Leu Ile Lys Thr Val Glu 435 440 445Thr Arg Asp Gly Gln
Val Ile Asn Glu Thr Ser Gln His His Asp Asp 450 455 460Leu
Glu46521766DNAHomo sapiens 2cgcgccaccg ccgccgccca ggccatcgcc
accctccgca gccatgtcca ccaggtccgt 60gtcctcgtcc tcctaccgca ggatgttcgg
cggcccgggc accgcgagcc ggccgagctc 120cagccggagc tacgtgacta
cgtccacccg cacctacagc ctgggcagcg cgctgcgccc 180cagcaccagc
cgcagcctct acgcctcgtc cccgggcggc gtgtatgcca cgcgctcctc
240tgccgtgcgc ctgcggagca gcgtgcccgg ggtgcggctc ctgcaggact
cggtggactt 300ctcgctggcc gacgccatca acaccgagtt caagaacacc
cgcaccaacg agaaggtgga 360gctgcaggag ctgaatgacc gcttcgccaa
ctacatcgac aaggtgcgct tcctggagca 420gcagaataag atcctgctgg
ccgagctcga gcagctcaag ggccaaggca agtcgcgcct 480gggggacctc
tacgaggagg agatgcggga gctgcgccgg caggtggacc agctaaccaa
540cgacaaagcc cgcgtcgagg tggagcgcga caacctggcc gaggacatca
tgcgcctccg 600ggagaaattg caggaggaga tgcttcagag agaggaagcc
gaaaacaccc tgcaatcttt 660cagacaggat gttgacaatg cgtctctggc
acgtcttgac cttgaacgca aagtggaatc 720tttgcaagaa gagattgcct
ttttgaagaa actccacgaa gaggaaatcc aggagctgca 780ggctcagatt
caggaacagc atgtccaaat cgatgtggat gtttccaagc ctgacctcac
840ggctgccctg cgtgacgtac gtcagcaata tgaaagtgtg gctgccaaga
acctgcagga 900ggcagaagaa tggtacaaat ccaagtttgc tgacctctct
gaggctgcca accggaacaa 960tgacgccctg cgccaggcaa agcaggagtc
cactgagtac cggagacagg tgcagtccct 1020cacctgtgaa gtggatgccc
ttaaaggaac caatgagtcc ctggaacgcc agatgcgtga 1080aatggaagag
aactttgccg ttgaagctgc taactaccaa gacactattg gccgcctgca
1140ggatgagatt cagaatatga aggaggaaat ggctcgtcac cttcgtgaat
accaagacct 1200gctcaatgtt aagatggccc ttgacattga gattgccacc
tacaggaagc tgctggaagg 1260cgaggagagc aggatttctc tgcctcttcc
aaacttttcc tccctgaacc tgagggaaac 1320taatctggat tcactccctc
tggttgatac ccactcaaaa aggacacttc tgattaagac 1380ggttgaaact
agagatggac aggttatcaa cgaaacttct cagcatcacg atgaccttga
1440ataaaaattg cacacactca gtgcagcaat atattaccag caagaataaa
aaagaaatcc 1500atatcttaaa gaaacagctt tcaagtgcct ttctgcagtt
tttcaggagc gcaagataga 1560tttggaatag gaataagctc tagttcttaa
caaccgacac tcctacaaga tttagaaaaa 1620agtttacaac ataatctagt
ttacagaaaa atcttgtgct agaatacttt ttaaaaggta 1680ttttgaatac
cattaaaact gctttttttt ttccagcaag tatccaacca acttggttct
1740gcttcaataa atctttggaa aaacta 176634PRTArtificial
SequenceDescription of Artificial Sequence Synthetic linker peptide
3Ala Leu Ala Leu 144PRTArtificial SequenceDescription of Artificial
Sequence Synthetic linker peptide 4Gly Phe Leu Gly 154PRTArtificial
SequenceDescription of Artificial Sequence Synthetic linker peptide
5Leu Ala Leu Ala 1
* * * * *
References