U.S. patent application number 11/801415 was filed with the patent office on 2009-01-01 for hsc70 directed diagnostics and therapeutics for multidrug resistant neoplastic disease.
This patent application is currently assigned to Aurelium BioPharma Inc.. Invention is credited to Anne-Marie Bonneau, Frederic Dallaire, Elias Georges, Lucile Serfass.
Application Number | 20090004102 11/801415 |
Document ID | / |
Family ID | 32713265 |
Filed Date | 2009-01-01 |
United States Patent
Application |
20090004102 |
Kind Code |
A1 |
Georges; Elias ; et
al. |
January 1, 2009 |
HSC70 directed diagnostics and therapeutics for multidrug resistant
neoplastic disease
Abstract
Disclosed are methods for detecting neoplastic or damaged cells
and for detecting multidrug resistance in neoplastic or damaged
cells by detecting an increase in the cell surface expression of a
heat shock cognate (HSC70) protein 70 on the surface of such a
multidrug resistant neoplastic or damaged cells as compared to the
level of expression of the HSC70 protein on the surface of a normal
cell.
Inventors: |
Georges; Elias; (Laval,
CA) ; Bonneau; Anne-Marie; (Laval, CA) ;
Dallaire; Frederic; (Montreal, CA) ; Serfass;
Lucile; (Brussels, BE) |
Correspondence
Address: |
WILMERHALE/BOSTON
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Aurelium BioPharma Inc.
Montreal
QC
|
Family ID: |
32713265 |
Appl. No.: |
11/801415 |
Filed: |
May 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10737350 |
Dec 15, 2003 |
7226748 |
|
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11801415 |
|
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60438012 |
Jan 3, 2003 |
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Current U.S.
Class: |
424/1.11 ;
424/178.1; 424/185.1; 424/9.1; 435/29; 435/7.23; 530/391.3;
530/391.7 |
Current CPC
Class: |
A61K 47/51 20170801;
A61P 35/00 20180101; G01N 2800/52 20130101; G01N 33/5011 20130101;
G01N 2800/44 20130101; G01N 33/57492 20130101 |
Class at
Publication: |
424/1.11 ;
435/29; 435/7.23; 424/9.1; 530/391.3; 530/391.7; 424/185.1;
424/178.1 |
International
Class: |
A61K 51/00 20060101
A61K051/00; C12Q 1/02 20060101 C12Q001/02; G01N 33/53 20060101
G01N033/53; A61K 39/00 20060101 A61K039/00; A61P 35/00 20060101
A61P035/00; A61K 39/395 20060101 A61K039/395; C07K 16/00 20060101
C07K016/00; A61K 49/00 20060101 A61K049/00 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. A method for detecting a multidrug resistant cell in a patient
comprising: (a) administering to the patient, a HSC70 binding agent
operably linked to a detectable label; and (b) detecting the label
operably linked to the HSC70 binding agent, wherein the HSC70
binding agent specifically binds to cell surface-expressed HSC70
present on a multidrug resistant cell in the patient.
11. The method of claim 10, wherein the HSC70 binding agent is an
antibody or fragment thereof.
12. The method of claim 10, wherein the HSC70 binding agent is
selected from the group consisting of Alzheimer's tau protein,
BAG-1, small glutamine-rich tetratricopeptide repeat-containing
protein (SGT), (aa 642-658) of rotavirus VP5 protein, auxilin, and
the immunosuppressant 5-deoxyspergualin (DSG).
13. The method of claim 10, wherein the HSC70 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 HSC70; 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 HSC70; 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 20 or 21, wherein the probe for detecting
HSC70 is an anti-HSC70 antibody.
23. The kit of claim 20 or 21, wherein the probe for detecting
HSC70 is an HSC70 ligand selected from the group consisting of
Alzheimer's tau protein, BAG-1, small glutamine-rich
tetratricopeptide repeat-containing protein (SGT), (aa 642-658) of
rotavirus VP5 protein, auxilin, and the immunosuppressant
5-deoxyspergualin (DSG).
24. The kit of claim 20, wherein the second probe is selected from
the group consisting of a nucleophosmin antibody and a vimentin
antibody.
25. The kit of claim 20, wherein the second probe is selected from
the group consisting of a nucleophosmin ligand and a vimentin
ligand.
26. The kit of claim 20 or 21, wherein the first probe detects
HSC70 present on the surface of the test neoplastic cell.
27. The kit of claim 20 or 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 HSC70 in situ detection probe for the detection
of cell surface HSC70 in a patient, comprising a HSC70 binding
component and a detectable label for detection in situ.
30. The cell surface HSC70 in situ detection probe of claim 29,
wherein the HSC70 binding component is an antibody.
31. The cell surface HSC70 in situ detection probe of claim 29,
wherein the detectable label is Technetium.
32. A cell surface HSC70-targeted agent for treating or preventing
a multi-drug resistant neoplasm, comprising a HSC70 binding
component and a therapeutic component, wherein the HSC70 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 HSC70 binding component is
an anti-HSC70 antibody.
34. The agent of claim 32, wherein the HSC70 binding component is
selected from the group consisting of Alzheimer's tau protein,
BAG-1, small glutamine-rich tetratricopeptide repeat-containing
protein (SGT), (aa 642-658) of rotavirus VP5 protein, auxilin, and
the immunosuppressant 5-deoxyspergualin (DSG).
35. The agent of claim 32, wherein said HSC70 binding component is
selected from the group consisting of natural ligands, synthetic
small molecules, chemicals, nucleic acids, peptides, proteins,
antibodies, and HSC70 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 any of claims 32-41, wherein the HSC70 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 HSC70 polypeptide, or HSC70 polypeptide
subsequence thereof, and at least one pharmaceutically acceptable
vaccine component.
44. The vaccine of claim 43, wherein the HSC70 polypeptide or
polypeptide subsequence is a human HSC70 polypeptide sequence of
SEQ ID NO.: 1.
45. The vaccine of claim 43, wherein the HSC70 polypeptide
subsequence is at least eight amino acids long.
46. The vaccine of claim 45, wherein the HSC70 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
HSC70-targeted therapeutic agent of any of claims 32-41.
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 HSC70 vaccine of
any of claims 43-48.
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. A method for detecting whether a test cell is neoplastic
comprising a) measuring a level of cell surface-expressed HSC70
protein in the test cell of a given origin or cell type, and b)
comparing the level of cell surface-expressed HSC70 protein in the
test cell to the level of cell surface-expressed HSC70 in a
normeoplastic cell of the same origin or cell type, wherein the
test cell is neoplastic if the level of cell surface-expressed
HSC70 in the test cell is greater than the level of cell
surface-expressed HSC70 in the normeoplastic cell of the same
origin or cell type.
60. The method of claim 59, wherein measuring the level of cell
surface-expressed HSC70 in the test cell comprises isolating a
cytoplasmic membrane fraction from the cell and measuring the level
of HSC70 in the cytoplasmic membrane fraction.
61. The method of claim 59, wherein measuring the level of cell
surface-expressed HSC70 in the test cell comprises contacting said
cell with an anti-HSC70 antibody and measuring the level of
antibody bound to cell surface HSC70.
62. The method of claim 61, wherein measuring the level of antibody
bound to cell surface HSC70 is by immunofluorescence emission.
63. The method of claim 61, wherein measuring the level of antibody
bound to cell surface HSC70 is by radiolabel.
64. The method of claim 59, wherein 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.
65. The method of claim 59, wherein the normeoplastic 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.
66. A method for detecting a neoplastic cell in a patient
comprising: (a) administering to the patient, a HSC70 binding agent
operably linked to a detectable label; and (b) detecting the label
operably linked to the HSC70 binding agent, wherein the HSC70
binding agent specifically binds to cell surface-expressed HSC70
present on a neoplastic cell in the patient.
67. The method of claim 66, wherein the HSC70 binding agent is an
antibody or fragment thereof.
68. The method of claim 66, wherein the HSC70 binding agent is
selected from the group consisting of Alzheimer's tau protein,
BAG-1, small glutamine-rich tetratricopeptide repeat-containing
protein (SGT), (aa 642-658) of rotavirus VP5 protein, auxilin, and
immunosuppressant 5-deoxyspergualin (DSG).
69. The method of claim 66, wherein the HSC70 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 HSC70; 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 HSC70 is
an anti-HSC70 antibody or binding fragment thereof.
77. The kit of claim 75, wherein the probe for detecting HSC70 is a
HSC70 ligand selected from the group consisting of Alzheimer's tau
protein, BAG-1, small glutamine-rich tetratricopeptide
repeat-containing protein (SGT), (aa 642-658) of rotavirus VP5
protein, auxilin, and the immunosuppressant 5-deoxyspergualin
(DSG).
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 a vimentin
ligand.
80. The kit of claim 75, wherein the first probe detects HSC70
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 HSC70-targeted agent for treating a cancerous
neoplastic cell growth comprising a HSC70 binding component and a
therapeutic component, wherein the HSC70 binding component targets
the therapeutic component to the neoplastic cell growth and thereby
treats the cancer.
83. The agent of claim 82, wherein the HSC70 binding component is
an anti-HSC70 antibody.
84. The agent of claim 82, wherein the HSC70 binding component is
selected from the group consisting of Alzheimer's tau protein,
BAG-1, small glutamine-rich tetratricopeptide repeat-containing
protein (SGT), (aa 642-658) of rotavirus VP5 protein, auxilin, and
the immunosuppressant 5-deoxyspergualin (DSG).
85. The agent of claim 82, wherein said HSC70 binding component is
selected from the group consisting of natural ligands, synthetic
small molecules, chemicals, nucleic acids, peptides, proteins,
antibodies, and HSC70 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 any of claims 82-91, wherein the HSC70 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
HSC70 polypeptide, or HSC70 polypeptide subsequence thereof, and at
least one pharmaceutically acceptable vaccine component.
94. The vaccine of claim 93, wherein the HSC70 polypeptide or
polypeptide subsequence is a human HSC70 polypeptide sequence set
forth in SEQ ID NO.: 1.
95. The vaccine of claim 93, wherein the HSC70 polypeptide
subsequence is at least eight amino acids long.
96. The vaccine of claim 95, wherein the HSC70 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 HSC70-targeted therapeutic
agent of any of claims 82-91.
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 HSC70 vaccine of any of claims
93-98.
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.
Description
[0001] This Application claims the benefit of priority to U.S.
Provisional Application No. 60/438,012, filed Jan. 3, 2003, 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] A commonly used treatment for diseases, such as cancer or
those caused by pathogen-infection, is the administration of drugs,
e.g., chemotherapeutics and antibiotics. In order to kill the
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). For
example, some cancer cells in a tumor evade being killed by
chemotherapeutic drugs by becoming multidrug resistant to a broad
spectrum of chemotherapeutic drugs, including drugs that were not
originally used for treatment.
[0004] Patient cross-resistance to different anti-microbial and
anti-cancer agents, which are structurally and functionally
distinct, can cause problems for both cancer patients and diseased
non-cancer patients. Thus, MDR can involve cancer cells, as well as
damaged, non-cancerous cells (e.g., cells infected with pathogens
including virus and bacteria). 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). 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 multifactorial. The classic MDR
mechanism involves alterations in the gene by gene amplification
for the highly evolutionarily conserved plasma membrane protein
(P-glycoprotein or MDR 1) that actively transports (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
overexpression of P-glycoprotein (both messenger RNA and protein)
due to amplification of the gene encoding P-glyocoprotein. 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 where 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 Publication
No. WO9962547). Some other atypical markers for MDR include MRP5,
which is a novel mammalian efflux pump for nucleoside analog drugs
(see Fridland and Schuetz, PCT Publication No. WO0058471) and
certain sphinogoglycolipids (see U.S. Pat. No. 6,090,565).
[0007] Heat shock proteins (HSPs, also referred to as molecular
chaperones or chaperonins) are a family of highly evolutionarily
conserved proteins that are normally intracellular in location
(reviewed in Kusmierczyk, Martin J., FEBS Lett 505: 343-7, 2001).
The heat shock response is thought to be an intrinsic cellular
defense mechanism against external stressors from various sources,
playing a crucial role in proper protein assembly, folding, and
transport. Upregulation of the synthesis of heat shock proteins
upon environmental stress (i.e., elevated temperature (heat shock),
inflammation, heavy metals, certain drugs, amino acid analogs,
environmental toxic pollutants, infections) allow cells to adapt to
gradual changes in their environment and to survive otherwise
lethal conditions. The events of cell stress and cell death are
linked and heat shock proteins induced in response to stress appear
to function at key regulatory points in the control of apoptosis
(programmed cell death).
[0008] HSPs include anti-apoptotic proteins that interact with a
variety of cellular proteins. Their expression level can determine
the fate of the cell in response to a death stimulus, and
apoptosis-inhibitory HSPs, in particular HSP27 and HSP70, may
participate in carcinogenesis (reviewed in Gamido, et al., Biochem.
Biophys. Res. Commun., 286: 433-42, 2001). For example, HSP70
interacts with the cellular p63 tumor suppressor protein and breast
cancer cells sometimes express high levels of several HSPs.
Increased HSP70 is an ominous prognostic sign in node-negative
breast tumors while HSP27 increases specific resistance to
doxorubicin in breast cancer cell lines (Fugua, Breast Cancer Res.
Treat., 32: 67-71, 1994).
[0009] HSP70 is normally an intracellular protein and not found on
the cell surface of non-cancerous cells (Kiang, The Pharmacol.
Ther., 80: 183-201, 1998). Some types of human tumors do express
HSP70 on their cell surface. For example, HSP70 can be found on the
cell surface of primary tumor biopsy material of carcinomas of the
lung, colorectum, neurons, and pancreas, as well as liver
metastases, and leukemic blasts of patients with acute myelogenous
leukemia. However, SP70 is not found on the cell surface of cells
from fresh biopsy material of mammary carcinomas (Hantschel, et
al., Cell Stress Chaperones, 5: 438-42, 2000).
[0010] HSP70 genes form a large evolutionarily conserved
superfamily, with multiple different similar genes encoding similar
proteins (isoforms). While the classic bacterial and mammalian
HSP70-type protein is inducible by stress (e.g., elevated
temperature, chemicals, pathogen infection), one isoform, heat
shock cognate, HSC70, is constitutively expressed (Huang, et al.,
J. Biol. Chem. 266: 7537-41, 1991). The bacterial homolog of HSC70
is DnaK. Like HSP70, HSC70 and DnaK function as molecular
chaperones and are involved in mediating correct protein folding,
preventing premature protein folding or aggregation, and
facilitating protein translocation through the cell membrane and
secretion (reviewed in Feldman, Del., Frydman J. Curr. Opin.
Struct. Biol. 10: 13-5, 2000).
[0011] Thus, there remains a need in both humans and animals for
treating, detecting, preventing, and/or reversing the development
of both classical and atypical MDR phenotypes in cancer cells and
damaged non-cancerous 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 MDR
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
[0012] The invention is based, in part, upon the discovery that
full-length HSC70, 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 HSC70 are expressed on the cell surface of
drug-sensitive neoplastic cells, in contrast to other cell surface
MDR markers (such as P-glycoprotein), HSC70 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 HSC70
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 HSC70 without
detrimental side effects, since the only non-HSC70 cells that are
being killed are drug-sensitive neoplastic cells or damaged cells;
normal cells remain unharmed.
[0013] 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
HSC70 protein in the test neoplastic cell of a given origin or cell
type, and comparing it to the level of cell surface-expressed HSC70
in a nonresistant neoplastic cell of the same origin or cell type.
If the level of cell surface-expressed HSC70 in the test neoplastic
cell is greater than the level of cell surface-expressed HSC70 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 HSC70 in the test neoplastic cell is
measured by isolating a cytoplasmic membrane fraction from the cell
and measuring the level of HSC70 in the cytoplasmic membrane
fraction. In other embodiments, the level of cell surface-expressed
HSC70 in the test neoplastic cell is measured by contacting the
cell with an anti-HSC70 antibody and measuring the level of
antibody bound to cell surface HSC70. For example, the level of
antibody bound to cell surface HSC70 may be measured by
immunofluorescence emission or radiolabel.
[0014] 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.
[0015] 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 HSC70 binding agent operably linked
to a detectable label. The label is operably linked to the HSC70
binding agent, which specifically binds to cell surface-expressed
HSC70 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 HSC70 binding agent used is an antibody or
fragment thereof. In other embodiments, the HSC70 binding agent is
a 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 particular
embodiments, the HSC70 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.
[0016] 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.
[0017] 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 HSC70 and a
second probe for the detection of another multidrug resistance
marker such as nucleophosmin or vimentin. In certain embodiment,
these kits of the invention include a first probe for the detection
of HSC70 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-HSC70 antibody as the
probe for detecting HSC70. In other embodiments, the kits include a
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 further embodiments,
the kits include a nucleophosmin antibody or a vimentin antibody as
probes for detecting the second, non-HSC70 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 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).
[0018] In another embodiment of this aspect of the invention, the
kit includes a probe that detects HSC70 present on the surface of
the test neoplastic cell. In other embodiments, the kit includes a
second probe that detects another (non-HSC70) 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.
[0019] In another aspect, the invention provides a cell surface
HSC70 in situ detection probe for the detection of cell surface
HSC70 in a patient. This cell surface HSC70 probe has a HSC70
binding component and a detectable label for detection in situ
(e.g., a Technetium label). In some embodiments, the HSC70 binding
component is an antibody.
[0020] In yet another aspect, the invention provides a cell surface
HSC70-targeted agent for treating or preventing a multi-drug
resistant neoplasm. This HSC70-targeted agent includes both a HSC70
binding component and a therapeutic component which act together
such that the HSC70 binding component targets the therapeutic
component to the multi-drug resistant neoplasm and thereby treats
the multi-drug resistant neoplasm. In certain embodiments, the
HSC70 binding component is an anti-HSC70 antibody. In other
embodiments, the HSC70 binding component is a HSC70 ligand such as
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 particular
embodiments, the HSC70 binding component is a natural ligand,
synthetic small molecule, chemical, nucleic acid, peptide or
protein.
[0021] 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.
[0022] In other embodiments, the therapeutic component is a
radioisotope such as .sup.90Y, .sup.125I, .sup.131I, .sup.211At or
.sup.213Bi.
[0023] 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.
[0024] In particularly useful embodiments, the HSC70 binding
component of the cell surface HSC70-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.
[0025] 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 HSC70-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.
[0026] In yet another aspect, the invention provides vaccines for
treating or preventing multi-drug resistant neoplasms, comprising a
HSC70 polypeptide, or HSC70 polypeptide subsequence thereof, and at
least one pharmaceutically acceptable vaccine component. In certain
embodiments, the HSC70 polypeptide or polypeptide subsequence is a
human HSC70 polypeptide sequence having an amino acid sequence of
SEQ ID NO.: 1. In particular embodiments, the HSC70 polypeptide
subsequence is at least eight amino acids long, and, in certain
embodiments, functions as a hapten.
[0027] 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.
[0028] In another aspect, the invention provides a method of
treating or preventing a multidrug resistant neoplasm in a subject
by administering any of the HSC70 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.
[0029] 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 HSC70 protein in the test cell of a given
origin or cell type, and comparing it to the level of cell
surface-expressed HSC70 in a normeoplastic cell of the same origin
or cell type. If the level of cell surface-expressed HSC70 in the
test cell is greater than the level of cell surface-expressed HSC70
in the normeoplastic cell of the same given origin or cell type,
then the test cell is neoplastic.
[0030] In certain embodiments, the level of cell surface-expressed
HSC70 in the test cell is measured by isolating a cytoplasmic
membrane fraction from the cell and measuring the level of HSC70 in
the cytoplasmic membrane fraction. In other embodiments, the level
of cell surface-expressed HSC70 in the test cell is measured by
contacting the cell with an anti-HSC70 antibody and measuring the
level of antibody bound to cell surface HSC70. For example, the
level of antibody bound to cell surface HSC70 may be measured by
immunofluorescence emission or radiolabel.
[0031] 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.
[0032] In another aspect, the invention provides a method for
detecting a neoplastic cell or cells in a patient by administering
to the patient, a HSC70 binding agent operably linked to a
detectable label. The label is operably linked to the HSC70 binding
agent, which specifically binds to cell surface-expressed HSC70
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 HSC70 binding
agent used is an antibody or fragment thereof. In other
embodiments, the HSC70 binding agent is a HSC70 ligand 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 particular
embodiments, the HSC70 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.
[0033] 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).
[0034] In another aspect, the invention provides kits for
diagnosing or detecting a neoplasm, which include at least one
probe for detecting HSC70, and at least one other probe for
detecting another neoplastic marker such as nucleophosmin or
vimentin. In one embodiment, the probe for detecting HSC70 is an
anti-HSC70 antibody or a binding fragment thereof. In other
embodiments, the probe for detecting HSC70 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).
[0035] In certain embodiments, the second probe for detecting a
non-HSC70 neoplastic marker is a nucleophosmin antibody or an HSC70
antibody. In other embodiments, the second probe for detecting a
non-HSC70 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-HSC70
neoplastic marker 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.
[0036] In particularly useful embodiments, the kit includes a first
probe which detects HSC70 present on the surface of the test cell
if it is neoplastic, and a second probe which detects another
(non-HSC70) marker present of the surface of the test cell if it is
neoplastic.
[0037] In yet another aspect, the invention provides a cell surface
HSC70-targeted agent for treating a cancerous neoplastic cell
growth. The cell surface HSC70-targeted agent generally includes a
HSC70 binding component and a therapeutic component. The HSC70
binding component targets the therapeutic component to the
neoplastic cell growth and thereby treats the cancer. The HSC70
binding component and the therapeutic component, therefore, act
together such that the HSC70 binding component targets the
therapeutic component to the neoplasm to treat the neoplasm.
[0038] In certain embodiments, the HSC70 binding component is an
anti-HSC70 antibody. In other embodiments, the HSC70 binding
component is a 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 particular
embodiments, the HSC70 binding component is a natural ligands,
synthetic small molecules, chemicals, nucleic acids, peptides or
protein.
[0039] 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.
[0040] In other embodiments, the therapeutic component is a
radioisotope such as .sup.90Y, .sup.125I, .sup.131I, .sup.211At or
.sup.213Bi.
[0041] 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.
[0042] In particularly useful embodiments, the HSC70 binding
component of the cell surface HSC70-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.
[0043] In another aspect, the invention provides a method of
treating a neoplasm in a subject by administering any of the cell
surface HSC70-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.
[0044] In yet another aspect, the invention provides vaccines for
treating or preventing a neoplasm. These vaccines of the invention
include a HSC70 polypeptide, or HSC70 polypeptide subsequence
thereof, and at least one pharmaceutically acceptable vaccine
component. In certain embodiments, the HSC70 polypeptide or
polypeptide subsequence is a human HSC70 polypeptide sequence
having an amino acid sequence of SEQ ID NO: 1. In particular
embodiments, the HSC70 polypeptide subsequence is at least eight
amino acids long, and, in certain embodiments, functions as a
hapten.
[0045] 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.
[0046] In another aspect, the invention provides a method of
treating or preventing a neoplasm in a subject by administering any
of the HSC70 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.
[0047] 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 HSC70 protein in the
test cell of a given origin or cell type, and comparing it to the
level of cell surface-expressed HSC70 in a nondamaged cell of the
same origin or cell type. If the level of cell surface-expressed
HSC70 in the test cell is greater than the level of cell
surface-expressed HSC70 in the nondamaged cell of the same given
origin or cell type, then the test cell is damaged (e.g.,
infected).
[0048] In certain embodiments, the damaged cell is infected with a
pathogen. In particular embodiments, the level of cell
surface-expressed HSC70 in the test cell is measured by isolating a
cytoplasmic membrane fraction from the cell and measuring the level
of HSC70 in the cytoplasmic membrane fraction. In certain
embodiments, the level of cell surface-expressed HSC70 in the test
cell is measured with an anti-HSC70 antibody. In particular
embodiments, the anti-HSC70 antibody measures the level of cell
surface HSC70 present on the intact test cell. For example, the
level of antibody bound to cell surface HSC70 may be measured by
immunofluorescence emission or radiolabel.
[0049] 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.
[0050] 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.
[0051] 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 HSC70 binding agent
operably linked to a detectable label. The label is operably linked
to the HSC70 binding agent, which specifically binds to cell
surface-expressed HSC70 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 HSC70 binding agent used is an antibody or fragment thereof. In
other embodiments, the HSC70 binding agent is a 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 particular embodiments, the HSC70
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.
[0052] 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 HSC70 and a second probe for
the detection of another marker of damage (e.g., pathogen
infection) such as nucleophosmin or vimentin. In particular
embodiments, the kits include anti-HSC70 antibody as the probe for
detecting HSC70. In other embodiments, the kits include a 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 further embodiments, the kits include a
nucleophosmin antibody or an HSC70 antibody as probes for detecting
the second, non-HSC70 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 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 certain
embodiments, the HSC70 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.
[0053] In yet another aspect, the invention provides cell surface
HSC70-targeted agents for treating infection by a pathogen. The
HSC70-targeted agent includes a HSC70 binding component and a
therapeutic component. The HSC70 binding component targets the
therapeutic component to the pathogen infected cell and thereby
treats the infection. In certain embodiments, the HSC70 binding
agent is an anti-HSC70 antibody. In other embodiments the HSC70
binding component is a 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 HSC70 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 particular embodiments, the therapeutic component is an
antibacterial, antiviral or antiparasitic agent. In certain
embodiments, the HSC70 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.
[0055] In yet another aspect, the invention provides vaccines for
treating or preventing infection by a pathogen. These vaccines
include a HSC70 polypeptide or polypeptide subsequence at least one
pharmaceutically acceptable vaccine component. In certain
embodiments, the HSC70 polypeptide is a human HSC70 polypeptide
sequence having an amino acid sequence of SEQ ID NO: 1. In
particular embodiments, the HSC70 polypeptide subsequence is at
least eight amino acids long, and, in certain embodiments,
functions as a hapten.
[0056] 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.
[0057] In another aspect, the invention provides a method of
treating or preventing an infection in a subject by administering
any of the HSC70 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
[0058] FIG. 1A is a photographic representation of a 2-D gel
analysis from CEM cells from plasma membrane cell extracts.
[0059] FIG. 1B is a photographic representation of a 2-D gel
analysis from multidrug resistant CEM/VLB cells from plasma
membrane cell extracts.
[0060] FIGS. 2A and B are representations of printed reports from
MALDI mass spectrum analysis of the spots corresponding to isoform
2 (NC) from the 2-D gel shown in FIG. 1B.
[0061] FIG. 2C is a schematic representation of sequence data
obtained for 12 tryptic peptides.
[0062] FIG. 3 is a schematic representation of the amino acid
sequence of HSC70 protein showing tryptic peptide sequences from
MALDI analysis from CEM/VLB cells.
[0063] FIG. 4A is a photographic representation of a 2-D gel of
Gelcode Blue stained biotinylated HL60 total cell extract.
[0064] FIG. 4B is a photographic representation of a 2-D gel of
GelCode Blue stained biotinylated HL60/AR total cell extract.
[0065] FIGS. 5A and B are representations of printed reports from
MALDI mass spectrum analysis of the spots corresponding to HSC70
from the 2-D gel spots isolated from HL60/AR cells.
[0066] FIG. 5C is a schematic representation of sequence data
obtained for 12 tryptic peptides.
[0067] FIG. 6 is a schematic representation of the amino acid
sequence of HSC70 protein showing tryptic peptide sequences from
MALDI analysis from HL60/AR cells.
[0068] FIG. 7 is a photographic representation of SDS-PAGE resolved
membrane fractions from HL60 and HL60/AR cells immunoblotted with a
specific anti-HSC70 rat monoclonal antibody.
[0069] FIG. 8A is a photographic representation of Western blotting
analysis of 10% SDS-polyacrylamide gel resolved biotinylated total
cell extracts prepared from HL60 and HL60/AR cells and probed with
anti-HSC70 antibody.
[0070] FIG. 8B is a photographic representation of Western blotting
analysis of 10% SDS polyacrylamide gel resolved streptavidin
purified extracts prepared from streptavidin purified total cell
extracts from HL60 and HL60/AR cells and probed with anti-HSC70
antibody.
[0071] FIG. 8C is a photographic representation of Western blotting
analysis of 10% SDS polyacrylamide gel resolved immunoprecipitates
of surface biotinylated total cell extracts from HL60 and HL60/AR
cells containing HSC70 and probed with anti-HSC70 antibody.
[0072] FIG. 8D is a photographic representation of Western blotting
analysis of 10% SDS polyacrylamide gel resolved immunoprecipitates
of surface biotinylated total cell extracts from HL60 and HL60/AR
cells containing HSC70 and probed with streptavidin-HRP.
[0073] FIG. 9A is a photographic representation of Western blotting
analysis of 10% SDS-polyacrylamide gel resolved biotinylated total
cell extracts prepared from CEM and CEM/VLB cells and probed with
anti-HSC70 antibody.
[0074] FIG. 9B is a photographic representation of Western blotting
analysis of 10% SDS polyacrylamide gel resolved streptavidin
purified extracts prepared from surface biotinylated total cell
extracts from CEM and CEM/VLB cells and probed with anti-HSC70
antibody.
[0075] FIG. 9C is a photographic representation of Western blotting
analysis of 10% SDS polyacrylamide gel resolved immunoprecipitates
of surface biotinylated total cell extracts from CEM and CEM/VLB
cells containing HSC70 and probed with anti-HSC70 antibody.
[0076] FIG. 9D is a photographic representation of Western blotting
analysis of 10% SDS polyacrylamide gel resolved immunoprecipitates
of surface biotinylated total cell extracts from CEM and CEM/VLB
cells containing HSC70 and probed with streptavidin-HRP.
[0077] FIG. 10A is a graphic representation showing the results of
FACS analysis for the surface expression of HSC70 using 10 .mu.g,
20 .mu.g and 40 .mu.g monoclonal anti-HSC70 antibody as primary
antibody on HL60 and HL60/AR cell lines.
[0078] FIG. 10B is a graphic representation showing the results of
FACS analysis for the surface expression of HSC70 at saturating
amounts of mouse monoclonal anti-HSC70 antibody on HL60 and HL60/AR
cell lines.
[0079] FIG. 11A is a graphic representation showing the results of
FACS analysis for the surface expression of HSC70 on CEM and
multidrug resistant CEM/VLB cell lines using 1 .mu.g, 2.5 .mu.g and
5 .mu.g of anti-HSC70 as primary antibody.
[0080] FIG. 11B is a graphical representation showing the results
of FACS analysis for the surface expression of HSC70 on HL60/AR and
CEM/VLB cell lines.
[0081] FIG. 1C is a graphic representation showing the number of
molecules of HSC70 on HL60/AR and CEM/VLB cell lines.
[0082] FIG. 12A is a graphic representation showing the results of
FACS analysis for the surface expression of HSC70 using 10 .mu.g
monoclonal anti-HSC70 antibody and 1 .mu.g P-glycoprotein (Pgp) on
MCF-7 and MCF-7/AR cell lines.
[0083] FIG. 12B is a graphic representation showing the results of
FACS analysis for the surface expression of HSC70 using 10 .mu.g
monoclonal anti-HSC70 antibody and 1 .mu.g Pgp on MDA and MDA/AR
cell lines.
[0084] FIG. 13 is a graphic representation showing the results of
FACS analysis for the surface expression of HSC70 using monoclonal
anti-HSC70 antibody on normal white blood cells, HL60, HL60/AR, CEM
and CEM/VLB0.1 cell lines.
[0085] FIG. 14A is a schematic representation of the polypeptide
sequence of a human HSC70 corresponding to GenBank Accession No.
AAK17898 (SEQ ID NO. 1).
[0086] FIG. 14B is a schematic representation of the nucleotide
sequence of a human HSC70-encoding nucleic acid sequence
corresponding to GenBank Accession No. AF352832 (SEQ ID NO. 2). The
initiation and termination codons of the vimentin protein open
reading frame are underlined.
[0087] FIGS. 15A and 15B are flow charts describing in a stepwise
fashion the protocol used for immunostaining of permeabilized and
non-permeabilized adherent cells.
[0088] FIG. 16 shows photographic representations of permeabilized
and non-permeabilized MCF-7 and MCF-7/AR cells immunostained with
anti-HSC70 antibody (rat IgG2a, Stressgen SPA-815) using the
procedure described in FIGS. 15A & 15B. Rat IgG2a was used as
negative control and didn't show any staining (not shown). Only
MCF-7/AR shows surface exposed HSC70.
[0089] FIG. 17 is a photographic representation of a western blot
analysis of various normal, sensitive and resistant cancer cell
extracts resolved by SDS-PAGE.
[0090] FIG. 18 is a graphic representation showing that levels of
HSC70 mRNA levels are increased in drug resistant cells compared to
their related sensitive cells (the fold increase in resistant
versus nonresistant cells is shown in each case).
4. DETAILED DESCRIPTION
[0091] 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.
[0092] 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. YY/XXXXXX, filed Dec. 15, 2003 and entitled "Vimentin 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. YY/XXXXXX, filed Dec. 15, 2003 and entitled
"Nucleophosmin Directed Therapeutics and Diagnostics for Multidrug
Resistant Neoplastic Disease."
[0093] 4.1 General
[0094] The invention provides methods and reagents for diagnosing,
detecting, preventing and/or treating cancer, and for diagnosing,
detecting, preventing and/or treating the development of both
naturally occurring and drug-induced MDR phenotypes of damaged,
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 and viral
infections.
[0095] Accordingly, an aspect 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 a
full-length heat shock cognate protein 70 (HSC70) on a test damaged
cell of a specific type; measuring the level of cell surface
expression of HSC70 protein on a drug-susceptible damaged cell of
the same cell type; and determining if the test damaged cell is
multidrug resistant if an increased level of cell surface-expressed
HSC70 is present compared to the level of cell surface-expressed
HSC70 present on the drug-susceptible damaged cell. In particular
embodiments, the level of cell surface-expressed HSC70 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 HSC70 present in the fraction of the cells containing the
cytoplasmic or plasma membrane.
[0096] In certain 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. In particular embodiments, the human
is suffering from a disease caused by the presence of the test
damaged cell.
[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 HSC70 on a test neoplastic cell
of a specific cell type; measuring the level of cell
surface-expressed HSC70 protein of a drug-susceptible neoplastic
cell of the same cell type; and determining that the test
neoplastic cell is multidrug resistant if an increased level of
cell surface-expressed HSC70 is present compared to the level of
cell surface-expressed HSC70 on the drug-susceptible neoplastic
cell. In particular embodiments, the level of cell
surface-expressed HSC70 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 HSC70
present in the fraction of the cells containing the cytoplasmic or
plasma membrane of the cells.
[0098] Exemplary neoplastic cells include, but are not limited to,
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, and a carcinoma cell. 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, gastro-intestinal tract,
eye, breast, prostate, or ovary.
[0099] In certain embodiments, the test neoplastic cell is from a
human. In particular embodiments, the human is suffering from a
cancer caused by the presence of the test neoplastic cell.
[0100] 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 HSC70
protein operably linked to a detectable label, and detecting
increased binding of the binding agent specifically bound to HSC70
protein on the surface of a multidrug resistant cell in the patient
compared to the binding agent bound to HSCS70 protein on the
surface of a drug-susceptible cell. In this embodiment 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.
[0101] Exemplary detectable labels include, but are not limited to,
fluorophores, chemical dyes, radioactive compounds,
chemiluminescent compounds, magnetic compounds, paramagnetic
compounds, enzymes that yield a colored product, enzymes that yield
a chemiluminescent product and enzymes that yield a magnetic
product. In certain embodiments, the patient is human. In some
embodiments, the multidrug resistant cell is a damaged cell or a
neoplastic cell. In particular 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.
[0102] In particular embodiments, the neoplastic cell is selected
from the group consisting of breast cancer cells, ovarian cancer
cells, lymphoma cancer cells, melanoma cancer cells, sarcoma cancer
cells, leukemia cancer cells, retinoblastoma cells, hepatoma cancer
cells, glioma cancer cells, mesothelioma cancer cells and carcinoma
cancer cells. 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.
[0103] The invention also provides a method for detecting a
neoplastic cell. The method includes measuring the level of cell
surface-expressed HSC70 protein on a test cell of a specific cell
type suspected of being neoplastic, and determining that the test
cell is neoplastic if an increased level of cell surface-expressed
HSC70 protein is present compared to the level of cell
surface-expressed HSC70 protein present on a normal cell of the
same type. In some embodiments, the test cell is a blood cell
(e.g., white blood cell, red blood cell, dendritic cell, or natural
killer cell), a liver cell, a kidney cell, a brain cell, a skin
cell, a cell from the gastrointestinal tract, an eye cell, a breast
cell, an ovarian cell, or a prostate cell.
[0104] In some embodiments, the cell surface-expressed HSC70 is
measured by separating the cellular components of the test cell and
the normal cell into fractions, and measuring the level of HSC70
present in the cytoplasmic or plasma membrane fraction of the
cells.
[0105] 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 one embodiment, the
test cell is from a human.
[0106] In some embodiments, the cellular components of the test
cell are contacted with a detectable binding agent, followed by
detection of the binding agent to determine if an increased level
of cell surface-expressed HSC70 is present on the test cell
compared to the level of cell surface-expressed HSC70 present on a
normal cell of the same type. In particular embodiments, the intact
suspected neoplastic cell is contacted with the detectable binding
agent.
[0107] 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 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 or leukemia), 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.
[0108] The present invention stems from the realization that cell
surface expressed HSC70 is useful as a marker for multidrug
resistance of a cell. An important advantage of the HSC70 protein
cell surface marker is that it is found intracellularly in normal
cells of the body and is not expressed on their cell surface. This
expression profile is in contrast to the situation with other known
MDR markers such as P-glycoprotein and 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 Metabolism & Disposition, 30: 4-6, 2002). As a
consequence, cytotoxic agents directed against MDR cancer cell
markers such as P-glycoprotein and MRP have been limited by the
adverse effects of killing 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 problem because the
HSC70 MDR marker is expressed at high levels on the surface of MDR
cancer cells and MDR non-cancerous damaged cells (e.g., cells
infected with a virus), at moderate levels on the cell surface of
drug-sensitive cancer cells compared to the very low or negligible
levels on drug-sensitive non-cancerous normal cells. Thus, the
invention provides cytotoxic agents directed toward cell surface
expressed HSC70 which kills MDR neoplastic or damaged and
drug-sensitive neoplastic and damaged cells, and leaves normal
cells unscathed.
[0109] It should be noted that the same MDR neoplastic or damaged
cell may express more than one MDR marker (e.g., may express both
HSC70 and P-glycoprotein) simultaneously, or may express an MDR
marker independently. Joint expression of different markers 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 HSC70 can be combined with a sub-lethal dosage of a
binding agent that specifically binds to P-glycoprotein. Since
normal cells do not express HSC70 on their cell surface, these
cells will not be harmed by the binding agent that specifically
binds to HSC70. Rather, only MDR cells that express both
P-glycoprotein and HSC70 on their cell surface will be killed by
this combination therapy.
[0110] 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 chemotherapeutics 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.
[0111] 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). Moreover, 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 a drug).
[0112] 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).
[0113] 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.
[0114] Cell surface expressed HSC70 is a superior marker for use in
therapies that kill MDR neoplastic cells and MDR damaged cells
bearing the HSC70 marker on their cell surface (such as immunotoxin
therapy), since normal cells are 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 HSC70 marker are more sensitive and
accurate, and provide fewer false positives compared to diagnosis
and imaging of MDR neoplastic and damaged cells using the MDR
markers such as P-glycoprotein or MRP that are also expressed on
normal tissues. Moreover, cell surface HSC70 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 HSC70 on their cell surface.
[0115] The present invention also allows the identification of
those patients whose neoplastic or damaged 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 or leukemia 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 with which the patient had
never originally been treated. 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).
[0116] The invention allows for early diagnosis of multidrug
resistance by detecting the cell surface expression of HSC70 on the
patient's neoplastic or damaged cells. 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. Thus, the drug non-responders can be treated
with a more effective treatment. Further, the HSC70 marker can
occur together with, or independently of, other MDR markers on the
same cancerous or damaged cells, allowing for the possibility of
combination therapy directed simultaneously against HSC70 and other
MDR cancer and damage markers (e.g. P-glycoprotein and MRP).
[0117] In addition, diagnostic procedures using HSC70 cell surface
expression may also be used to follow the development and emergence
of MDR neoplastic or damaged 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 Res. Human Retroviruses 8: 1839-1844, 1992).
[0118] In another example, diagnostic assays for HSC70 cell surface
expression are useful for selecting patients in clinical studies
involving therapy for treatment of neoplastic or damaged cells.
Hence, the presence of HSC70 on the cell surface of a patient's
cells either qualifies or disqualifies that patient from being
included in a given clinical study.
[0119] 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 HSC70 protein on the surface of the
test damaged cell of a specific cell type; measuring the level of
cell surface-expressed HSC70 protein on a drug-susceptible damaged
cell and determining that the test damaged cell is multidrug
resistant if an increased level of cell surface-expressed HSC70 is
present compared to the level of cell surface-expressed HSC70 on
the drug-susceptible damaged cell.
[0120] 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 HSC70 protein on the test
neoplastic cell if a specific cell type; measuring the level of
cell surface-expressed HSC70 protein on a drug-susceptible
neoplastic cell of the same type; and that determining that the
test neoplastic cell is multidrug resistant if an increased level
of cell surface-expressed HSC70 is present compared to the level of
cell surface-expressed HSC70 present on the drug-susceptible
neoplastic cell.
[0121] In another aspect, the invention provides a method for
detecting cancer in a test cell suspected of being cancerous (i.e.
neoplastic). The method includes measuring the cell
surface-expressed HSC70 protein on the test cell and on a normal
cell (known not to be cancerous), and determining that the test
cell is cancerous if a level of cell surface-expressed HSC70 is
increased compared to the level of cell surface-expressed HSC70
present on a normal, nucleated cell of the same cell type.
[0122] In some embodiments, the test damaged cell or test
neoplastic cell expresses an amount of HSC70 on its cell surface
that is at least two-fold higher than the level of cell surface
expression of the heat shock cognate protein 70 on a normal cell or
on a non-MDR damaged cell or non-MDR neoplastic cell, respectively.
Such a determination of level of cell surface expression can be
made by any number of known methods including, without limitation,
those methods described below.
[0123] As used in accordance with the invention, a "damaged cell"
is used to mean 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 Plasmodium, Leishmania, and Taxoplasma. Such non-limiting
viruses include HIV, West Nile virus and Dengue virus; such
non-limiting bacteria include Mycobacteria, Rickettsia, and
Chlamydia.
[0124] 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.sup.+
T cells and macrophages; thus a damaged cell (e.g., a CD4.sup.+ T
cell infected with HIV) is from a tissue (i.e., blood or bone
marrow).
[0125] 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."
[0126] 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 melanoma, breast cancer, ovarian cancer, prostate cancer,
sarcoma, leukemic, retinoblastoma, hepatoma, myeloma, glioma,
mesothelioma, carcinoma, leukemia, lymphoma, Hodgkin lymphoma,
Non-Hodgkin lymphoma, myeloma, promyelocytic leukemia, T
lymphoblastoic, myelodysplastic syndrome, lymphoblastoma, and
thymoma cells.
[0127] 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, but are not limited to,
blood, bone marrow, spleen, lymph node, liver, thymus, spleen,
kidney, brain, skin, gastro-intestinal tract, eye, breast,
prostate, and ovary.
[0128] In accordance with the invention, a damaged cell is from a
patient, such as a human. In certain embodiments, the patient 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+
T cells which were infected by the HIV virus).
[0129] In accordance with the invention, the test neoplastic cell
is from a patient, such as a human. In certain embodiments, the
patient 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).
[0130] As used herein, a "patient suffering from a disease or
disorder" is meant 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.
[0131] According to the invention, those neoplastic cells that have
become cancerous are distinguishable from normal, nucleated cells
by the increased expression of full-length HSC70 protein on the
cell surface.
[0132] 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 HSC70 protein on the
cell surface of multidrug resistant cells. Representative
nucleotide and amino acid sequences of HSC70 are set forth in FIG.
14 (also see GI 5729877 and GI 1398297). Thus, when the cellular
components are separated, those cells that are multidrug resistant
contain HSC70 on their cytoplasmic or plasma membrane fraction.
Cell surface expression of HSC70 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.
[0133] In some embodiments, measuring the level of expression of a
HSC70 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 measuring the level of HSC70 in
the fraction of the cell containing the plasma or cytoplasmic
membrane.
[0134] Alternatively, measuring the level of expression of HSC70 on
the surface of the test damaged cell or test neoplastic cell
comprise separating the products of enzymatic digestion of cell
surface-expressed HSC70 protein from the test cell. In one
non-limiting example, intact cells from a patient are digested with
enzymes. The peptides from the digested, surface-exposed proteins
are isolated by quickly spinning down the cells and leaving the
digested peptides in the supernatant. These digested peptides can
be analyzed by various methods (e.g., immunological methods or mass
spectroscopy) to determine if HSC70 is expressed on the cell
surface.
[0135] 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).
[0136] In some embodiments, measuring the level of expression of a
HSC70 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 HSC70
protein. Thus, the detectable binding agent specifically binds to
cells which express HSC70 on their cell surface.
[0137] According to the invention, while normal cells express no or
negligible amounts of HSC70 on their cell surface, a neoplastic
cell or a damaged cell expresses more HSC70 on their cell surface.
Additionally, when such neoplastic or damaged cells become
multidrug resistant, they express even higher levels of the entire,
full length HSC70 protein on their cell surface. An HSC70-specific
binding agent specifically binds to any portion of the HSC70
protein since the entire protein is expressed on the cell surface
of multidrug resistant neoplastic or damaged cells.
[0138] Of course, since HSC70 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 HSC70 in normal and
drug-sensitive neoplastic or damaged cells.
[0139] In some embodiments, MDR neoplastic cells or MDR damaged
cells that express HSC70 on their cell surface are distinguishable
from other types of cells, in that the MDR neoplastic or damaged
cells express the full length HSC70 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 HSC70 on its cell surface than
does a normal T cell. Moreover, as described below, a MDR leukemic
T cell expresses at least twice as much HSC70 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 HSC70 on its cell surface as its
drug-sensitive counterpart (i.e., the drug-sensitive counterpart is
not multidrug resistant). If the cellular components of such cells
are separated (e.g., into membrane fraction and cytosolic
fraction), those MDR neoplastic or damaged cells that express HSC70
at their cell surface contain HSC70 in their membrane fractions at
two fold or higher levels than do other cells from the same tissue
that are not multidrug resistant, regardless whether the
non-multidrug resistant cell is normal, neoplastic, or damaged.
[0140] In another aspect, the invention provides a binding agent
that specifically binds to a HSC70 protein. As used herein,
"specifically binds" means that a binding agent (e.g., an antibody)
recognizes and binds to a HSC70 protein, but does not substantially
recognize and bind to other molecules in a sample. Thus, such a
binding agent of the invention binds to the surface of a MDR cell
that expresses HSC70 on its cell surface. A useful binding agent
that specifically binds to an HSC70 protein forms an association
with the HSC70 protein with an affinity of at least 10.sup.6
M.sup.-1, or at least 10.sup.7 M.sup.-1, or at least 10.sup.8
M.sup.-1, or at least 10.sup.9 M.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.
[0141] A "binding agent" need not be any particular size or have
any particular structure so long as it specifically binds to the
HSC70 protein. Thus, a "binding agent" is a molecule that attaches
to any region (e.g., three dimensional structure, amino acid
sequence, or particular small chemical groups) so long as it
preferentially binds to the HSC70 protein. 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."
[0142] In certain embodiments, the binding agent is an antibody.
Where the binding agent that specifically binds an HSC70 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 HSC70 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).
[0143] As used herein, by "detectably labeled" is meant that a
binding agent of the invention is operably linked to a moiety that
is detectable. By "operably linked" is meant 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).
[0144] In accordance with the invention, a detectably labeled
binding agent 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 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 an HSC70 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).
[0145] According to the invention, a detectable label is a moiety
that can be detected 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 by 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 HSC70 antibody) that is itself
specifically bound to 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).
[0146] In some embodiments, measuring the level of expression of a
HSC70 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 HSC70 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.
[0147] 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, Pseudomonas
exotoxin (PE), and ribonuclease A (Fizgerald D., Semin. Cancer
Biol. 7: 87-95, 1996).
[0148] 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).
[0149] In a further aspect, the invention provides a therapeutic
composition comprising a cytotoxic drug, a binding agent that
specifically binds to a HSC70 protein, 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.
[0150] 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.
[0151] 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 antimicrobial 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. Where the damaged cells are
infected by a pathogen, non-limiting examples of the drug differs
with the infecting pathogen but may include Acyclovir,
amphotericin, ampicillin, anthracyclin, b-lactam antibiotics,
cephalothin, chloramphenicol, chloroquine (CQ), cidofovir (CDV),
ciprofloxacin, erythromycin, fluconazole, 5 flucytosine,
fluoroquinolone, foscamet, 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).
[0152] 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).
[0153] 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 an
HSC70 protein. 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 HSC70 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. The drug and the
binding agent can be separately administered at different times in
any order or can administered together. In some embodiments, the
patient is a human.
[0154] In certain embodiments, the damaged cells of the patient are
infected with a pathogen. Such as a virus, a bacterium, or a
parasite.
[0155] In yet 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 an HSC70 protein. 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 HSC70 protein is expressed 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. 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.
[0156] 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.
[0157] In certain embodiments, the binding agent administered to
the patient according to the methods of the invention is an
antibody. In some embodiments, the binding agent is operably linked
to a toxin. Non-limiting examples of such toxins are described
above.
[0158] 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.
[0159] As a physician will determine, 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.
[0160] 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
an HSC70 protein to a patient suspected of comprising 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.
[0161] 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 improvements on these technologies (PET+CT,
spiral CT, single photon emission CT (SPECT), high resolution PET
(microPET), and immunoscintingraphy (using radiolabeled antibodies
(Czemin et al., Ann. Rev. Med. 53:89-112, 2002; Goldenberg, D. M.,
Cancer 80(12): 2431-2435, 1997; Langer, S. G. et al. World J. Surg.
25: 1428-1437, 2001; Middleton et al., 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 J. Surg.
25: 1428-1437, 2001; Nabi et al., Oncol. J. Nuclear Med. Technol.
30(1): 3-9, 2002); ultrasonic imaging (US) (Harvey, C. J. et al.,
Adv. Ultrasound Clin. Radiol. 57: 157-177, 2002; Langer, S. G. et
al., World 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. et al., Circulation 105: 2493-6, 2002).
[0162] In certain embodiments, the patient is a human. The patient
may be, for example, a patient 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 cells. 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.
[0163] 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, or damaged 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.
[0164] 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).
[0165] 4.2 HSC70 Antibodies
[0166] The invention provides antibodies directed against HSC70 for
use in detection, imaging and treatment of cancers and damaged
(e.g., pathogen-infected) cells. Anti-HSC70 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 HSC70 mouse monoclonal
and/or rabbit polyclonal antibodies.
[0167] The term "antibody" is used in the broadest sense and
specifically covers single anti-HSC70 monoclonal and polyclonal
antibodies, as well as anti-HSC70 antibody fragments (e.g., Fab,
F(ab).sub.2, and Fv) and anti-HSC70 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.
[0168] The monoclonal antibodies herein include hybrid and
recombinant antibodies produced by splicing a variable (including
hypervariable) domain of an anti-HSC70 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).sub.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.
[0169] "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).sub.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.
[0170] HSC70 or anti-HSC70 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 HSC70, 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-HSC70 antibodies or
fragments thereof can be used alone or in mixtures.
[0171] The novel antibodies, antibody fragments, mixtures or
derivatives thereof advantageously have a binding affinity for
HSC70 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).
[0172] 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.
[0173] The invention furthermore relates to cells that synthesize
HSC70 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 HSC70 and
isolation of their antibody-producing B cells, selecting these
cells for HSC70-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 HSC70.
[0174] The invention further includes derivates of these
anti-HSC70, which preferably retain their HSC70-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 antiHSC70 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.131I, .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.
[0175] 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.
[0176] The anti-HSC70 antibodies of the invention can be
administered orally or parenterally subcutaneously,
intramuscularly, intravenously or interperitoneally.
[0177] 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. HSC70 can be detected on a wide variety of cell
types--particularly neoplastic cells.
[0178] The human HSC70 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 HSC70
antibodies are also available and are included in the
invention.
[0179] First, a mammal is immunized with human HSC70. Purified
human HSC70 is commercially available from Sigma (St. Louis, Mo.,
catalog A6152), as well as other commercial vendors. Human HSC70
may be readily purified from human placental tissue. Furthermore,
methods of immunoaffinity purification for obtaining highly
purified HSC70 immunogen are known (see, e.g., Vladutiu et al.,
(1975) .delta.: 147-59 Prep. Biochem.). The mammal used for raising
anti-human HSC70 antibody is not restricted and may be a primate, a
rodent such as mouse, rat or rabbit, bovine, sheep, goat or
dog.
[0180] 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.,
p3.times.63-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.
[0181] 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 HSC70 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.
[0182] By the above-described operation, hybridomas, which produce
anti-human HSC70 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.
[0183] From among the selected anti-human HSC70 monoclonal
antibodies, those that have an ability to bind cell surface HSC70
are then chosen for further analysis and manipulation.
[0184] The monoclonal antibodies herein further include hybrid and
recombinant antibodies produced by splicing a variable (including
hypervariable) domain of an anti-HSC70 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).sub.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)).
[0185] 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.
[0186] "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).sub.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 immunoglobulin constant
region (Fc), typically that of a human immunoglobulin.
[0187] 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.
[0188] 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).
[0189] 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.
[0190] Human antibodies directed against HSC70 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.
[0191] 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).
[0192] 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).
[0193] 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).
[0194] 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.
[0195] By using the above-described monoclonal antibody of the
present invention, human HSC70 in a sample can be detected or
quantified. The detection or quantification of the human HSC70 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 HSC70. 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 HSC70 is
preferably employed.
[0196] 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.
[0197] 4.3 HSC70 Binding Agents
[0198] In another aspect, the invention provides a binding agent
that specifically binds to a HSC70 protein or fragment thereof. As
used herein, "specifically binds" means that a binding agent
recognizes and binds to a HSC70 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 HSC70 on its cell
surface. A useful binding agent forms an association with the HSC70
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 HSC70 protein or fragment
thereof.
[0199] A binding agent need not be any particular size or have any
particular structure so long as it specifically binds to the HSC70
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 HSC70
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.
[0200] There are a number of examples of non-antibody HSC70 binding
agents known in the art. There are a number of examples of
non-antibody HSC70 binding agents known in the art. For example,
Alzheimer's tau protein (Shimura et al. (2003) J. Biol. Chem. (Nov.
10, 2003 e-publication); BAG-1 (Takamura et al. (2003) Int. J.
Oncol. 23: 1301-8); small glutamine-rich tetratricopeptide
repeat-containing protein (SGT) (Tobaben et al. J. Biol. Chem. 77:
7254-60); (aa 642-658) of rotavirus VP5 protein (Zarate et al.
(2003) J. Virol. 77: 7254-60); auxilin (Jiang et al. (1997) J.
Biol. Chem. 272: 6141-5); and the immunosuppressant
5-deoxyspergualin (DSG) (Nadler et al. (1998) Biochem. Biophys.
Res. Comm. 253: 176-80).
[0201] 4.4 HSC70-Targeted Diagnostics
[0202] 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 HSC70
marker
[0203] The diagnostic applications of the invention include probes
and other detectable agents that are joined to a HSC70 binding
agent, such as an anti-HSC70 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).
[0204] 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
HSC70 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).
[0205] 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 HSC70 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).
[0206] In some embodiments, measuring the level of expression of a
HSC70 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 HSC70 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.
[0207] 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 improvements 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 (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 ML and Shell EG (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 Radiolopy 57:157-177; Langer, S. G. et al.
(2001) World Journal of Surgery 25:1428-1437); (c) fiber optic
endoscope (Shelhase Del. (2002) Curr Oipin 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).
[0208] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to HSC70 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
HSC70. The invention provides for the detection of aberrant
expression of cell surface HSC70 (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 HSC70 and
(b) comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
cell surface HSC70 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.
[0209] With respect to cancer, the presence of a relatively high
amount of cell surface HSC70 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.
[0210] 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 HSC70 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.
[0211] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of
cell surface HSC70 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-HSC70 antibody, or
other HSC70 binding agent, which specifically binds to cell surface
HSC70 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.
[0212] A HSC70-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 99 mTc. The labeled antibody or antibody
fragment will then preferentially accumulate at the location of
cells which express cell surface HSC70 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.
[0213] 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.
[0214] 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.
[0215] Significantly, the presence of the labeled anti-HSC70
antibody or other HSC70 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.
[0216] For example, in a specific embodiment, the anti-HSC70
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-HSC70 antibody is labeled with a fluorescent compound and is
detected in the patient using a fluorescence responsive scanning
instrument. In another embodiment, the anti-HSC70 antibody is
labeled with a positron emitting metal and is detected in the
patent using positron emission-tomography. In yet another
embodiment, the anti-HSC70 antibody is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
[0217] 4.5 HSC70-Targeted Therapeutics
[0218] The invention takes advantage of the fact that HSC70 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 HSC70.
[0219] Therapeutic agents to be targeted to HSC70 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).
[0220] Anti-HSC70 Antibodies
[0221] In one approach, anti-HSC70 antibodies specific to cell
surface expressed HSC70 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-HSC70 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.
[0222] 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).
[0223] 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. Theray. 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).
[0224] 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).
[0225] 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
disrupting the cellular process responsible for proliferation
growth and metastasis (see Abou-Jawde et al. (2003) Clin. Therap.
25: 2121-37).
[0226] HSC70-Targeted Antibody and Ligand Conjugates
[0227] 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-HSC70
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.
[0228] The invention further includes HSC70-targeted agents made up
of a HSC70 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 HSC70, 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.
[0229] For example, in one embodiment, anti-HSC70 antibodies are
conjugated to cobra venom factor. In accordance with the invention,
HSC70 specific antibodies conjugated to cobra venom factor are used
to treat cancer, including especially multidrug resistant cancer in
a human.
[0230] 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).
[0231] 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.
[0232] Drug Attachment
[0233] 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.
[0234] The most used site is that of the .epsilon.-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).
[0235] 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).
[0236] 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) Bioconiug.
Chem. 10:279-288).
[0237] 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).
[0238] 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. Chromatojr.
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.
[0239] 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.
[0240] 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(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.
[0241] 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.
[0242] 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.
[0243] 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 (Srinivasachur, 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.
[0244] 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).
[0245] 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.
[0246] 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.
[0247] 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.
[0248] 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.
[0249] 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.
[0250] 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.
[0251] 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.
[0252] 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).
[0253] 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).
[0254] 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.
[0255] 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).
[0256] 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).
[0257] 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.
[0258] Immunoconjugates with 5-fluoro-2'-deoxyuridine (FUDR) were
constructed by reacting an active ester derivative of succinylated
FUDR with anti-1y1.2 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.
[0259] 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.
[0260] 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).
[0261] 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.
[0262] 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.
[0263] Immuntoxins
[0264] 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, Il-8, 11-9, Il-10,
IL-12, IL-15, granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0265] 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.
[0266] 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.
[0267] 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`.
[0268] 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).
[0269] 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.
[0270] 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).
[0271] 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) L Biol. Chem.
248:3838-3844).
[0272] 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).
[0273] 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).
[0274] 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.
[0275] Toxins can be targeted to cells without chemically
conjugating the ligand and the toxin if both are connected as one
polypeptide unit.
[0276] 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).
[0277] 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).
[0278] 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.
[0279] Targeted Radiotherapy
[0280] Radioisotopes may also be used as cytotoxic agents for
HSC70-targeted therapeutics. Anti-HSC70 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.188Re, .sup.211At, and .sup.212Bi. Carriers
specific for radionuclide agents, to facilitate attachment to the
HSC70 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.
[0281] 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).
[0282] 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 HSC70-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.
[0283] 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.
[0284] Targeted Gene Therapy
[0285] Gene vectors may also be used as cytotoxic agents for
HSC70-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 HSC70-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.
[0286] 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 HSC70-specific
antibody to the adenoviral vector will target the resulting
construct to HSC70-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 an 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 HSC70 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).
[0287] 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.
[0288] 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).
[0289] 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.
[0290] 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.
[0291] 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 110. The term canine adenoviruses includes but
is not limited to canine types 1 (strains CLL, Glaxo, R1261,
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.
[0292] 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).
[0293] 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.
[0294] The term "tumor suppressor gene" refers to a nucleotide
sequence, the expression of which in the target cell is capable of
suppressing 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.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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 deltal3-19 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).
[0300] 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.
[0301] 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 HSC70 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. HSC70 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.
[0302] Liposomal Formulations
[0303] Another strategy that may be employed for HSC70-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).
[0304] Alternatively, non-antibody HSC70 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) Bioconi.
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) Bioconi.
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).
[0305] 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.
[0306] 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).
[0307] 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).
[0308] 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).
[0309] Therapies
[0310] 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-HSC70 antibodies or
nucleic acid molecules encoding said antibodies. Examples of types
of cancer and proliferative disorders to be treated with the
HSC70-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-HSC70 antibodies, functionally active fragments or derivatives
thereof are used in the present invention.
[0311] The HSC70 therabpeutics 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-HSC70 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-HSC70 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-HSC70 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.
[0312] The anti-HSC70 antibodies, and other HSC70-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-HSC70
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-HSC70 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 M195, LymphoCide, Smart I D10, and Oncolym.
[0313] 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-HSC70 antibodies or
nucleic acid molecules encoding said antibodies or other
HSC70-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), pararnyxoviruses (e.g., morbillivirus virus, human
respiratory syncytial virus, and pneumovirus), adenoviruses,
bunyaviruses (e.g., hantavirus), comaviruses, 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.
[0314] The HSC70-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-HSC70 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.
[0315] In other examples, one or more HSC70-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-HSC70 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.
[0316] 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-HSC70-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.
[0317] HSC70-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 HSC70-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.
[0318] In certain instances, one or more HSC70-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 HSC70-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.
[0319] 4.6 Kits
[0320] 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 HSC70-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 HSC70, 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 HSC70 protein in a biological
sample; as well as means for determining the amount of cell surface
HSC70 in the sample; and means for comparing the amount of HSC70 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 HSC70 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 HSC70 protein.
[0321] 4.7 HSC70 Vaccines
[0322] Immunological compositions, including vaccines, and other
pharmaceutical compositions containing the HSC70 protein, or
portions thereof, are included within the scope of the present
invention. One or more of the HSC70 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.
[0323] 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.
[0324] The HSC70 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.
[0325] The invention includes the HSC70 polypeptide fragments, or
subsequences of the intact HSC70 polypeptide shown in FIG. 12A (SEQ
ID NO. 1). Such HSC70 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-HSC70 vaccines apply also to the use of HSC70 polypeptide
sequences for use as immunogens for generating anti-HSC70
polyclonal and monoclonal antibodies for use in the HSC70-based
diagnostics and therapeutics described herein.
[0326] 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_ccurrence/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.
[0327] 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 HSC70
sequence shown in FIG. 14A, several suitable sequence having a high
Hopp and Woods antigenic index of an adequate length for
immunogenicity were revealed. These include HSC70 amino acid
residues: 240-260 (i.e. HFIAEFKRKHKKDISENKRAY); and 480-500 (i.e.,
IDANGILNVSAVDKSTGKENK).
[0328] In addition, the present invention provides a composition
comprising the HSC70 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 HSC70
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 HSC70 protein or HSC70-encoding nucleic acid,
concurrent with the administration of the HSC70 protein or
HSC70-encoding nucleic acid or up to five days after the
administration of the HSC70 protein or HSC70-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.
[0329] 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 HSC70
protein or HSC70-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 HSC70 polypeptide with and
without the adjuvant or combination of adjuvants, using standard
procedures, as described herein.
[0330] Furthermore, the present invention provides a composition
comprising the HSC70 protein or HSC70-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.
[0331] The term "vaccine" as used herein includes DNA vaccines in
which the nucleic acid molecule encoding HSC70 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).
[0332] In another embodiment, the invention is a polynucleotide
which comprises contiguous nucleic acid sequences capable of being
expressed to produce a HSC70 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.
[0333] 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+ 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 HSC70 genes
and gene fragments for their vaccine potential.
[0334] 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.3 C, 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 HSC70 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 HSC70 portion of the fusion is, preferably, a
portion shown in routine screening to have a strong antigenic
potential.
[0335] 4.8 Pharmaceutical Formulations and Methods of Treatment
[0336] 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 HSC70 targeting component that is
capable of binding to cell surface HSC70 present on neoplastic, and
particularly multidrug resistant neoplastic, cells and which
compound is linked to a therapeutic component.
[0337] Examples of HSC70 targeting components include monoclonal
anti-HSC70 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 HSC70 expressing neoplastic, or multidrug resistant neoplastic,
cell by the HSC70 targeting component of the therapeutic compound
and, upon binding of cell surface HSC70 and uptake into the cell,
function to kill or block the growth of the neoplastic cell.
[0338] 4.8.1 Effective Dose
[0339] 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.
[0340] 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.
[0341] 4.8.2 Formulation and Use
[0342] 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.
[0343] 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 Remington'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.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] 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.
[0348] 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).
[0349] 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.
[0350] In clinical settings, a therapeutic and gene delivery system
for the HSC70-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
HSC70-targeted therapeutic can be introduced systemically, e.g., by
intravenous injection.
[0351] The pharmaceutical preparation of the HSC70-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.
[0352] 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
[0353] 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 71 kDa Protein in Membranes of Multidrug
Resistant Cancer Cells
[0354] 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.
[0355] A. Cells
[0356] The seven different cell lines used 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 and Aurelium BioPharma
Promyelocytic NB4 NB4/VLB Deutsche Sammlung leukemia NB4/DOX von
Miroorganismen und Zellkulturen GmbH (DSMZ, Germany) & Aurelium
BioPharma T lymphoblastoid CEM CEM/VLB ATCC and Dr. William CEM/DOX
Beck, Aurelium T lymphoblastoid HSB2 HSB2/VLB ATCC & Aurelium
HSB2/DOX BioPharma T lymphoblastoid Molt4 Molt4/DOX ATCC &
Aurelium Molt4/VLB BioPharma Breast epithelial MCF-7 MCF-7/AR ATCC
and McGill University 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).
[0357] 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 (MDR) cells (HL60/AR, NB4/VLB, NB4/DOX,
CEM/VLB, CEM/DOX, Molt4/VLB, Molt4/DOX, HSB2/VLB, HSB2/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 in
addition to HSC70.
[0358] B. Cell Preparations
[0359] 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 and HL60) and
multidrug resistant cells (CEM/VLB and HL60/AR) were biotinylated
with a membrane impermeable biotinylating agent,
Sulfo-NHS-LC-LC-Biotin (Pierce Chemicals, Rockford, Ill.). Cells
were biotinylated by washing 3.times. with 50 ml PBS, pH 8. Next,
Sulfo-NHS-LC-LC-Biotin (Pierce Chemicals, Rockford, Ill.) 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 one 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 protease 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 (Sonics, Newtown, Conn.) amplitude 40 setting #25 for
3.times.10 seconds with one minute on ice between shots. The
sonicated 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
protease inhibitors and incubated 5 minutes on ice. The cell lysate
was then 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: 265-75,
1951).
[0360] The use of this sulfo-LC-LC-biotinylating agent ensured the
modification of the .epsilon. 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.
[0361] 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 minutes 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 1X: 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 minutes 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 minutes 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 T-1250 rotor in the capped
tubes catalog no. 03989 S/L PA (35 ml) 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. 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 of 60% sucrose 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. Next, a 16% sucrose solution containing crude
plasma membranes was slowly poured on the top of the gradient and
the sample spun for 18-20 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 catalog no. 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. Plasma membrane extracts were prepared
similarly from non-biotinylated and biotinylated cells (shown in
Example II, FIG. 1).
[0362] Alternatively, total membrane extracts were prepared from
surface biotinylated or non-biotinylated cells. For each cell type,
cells were washed three times with 50 ml of ice-cold phosphate
buffered saline (PBS), and resuspended in 100 .mu.l PBS containing
protease inhibitors. Cells were sonicated three times for twenty
seconds each, and were spun at 4.degree. C. for thirty minutes at
20,000 rpm. The pellet was resolubilized in PBS containing 4% CHAPS
with protease inhibitors and stored at -80.degree. C. until
use.
[0363] C. Gel Electrophoresis
[0364] Equivalent amounts of protein from surface tiotinylated
total cell extracts (containing biotinylated and non-biotinylated
proteins) and plasma membrane or total membrane preparations from
each of the cell types (HL60, HL60/AR, NB4, NB4/DOX, CEM, CEM/VLB,
CEM/DOX, Molt4, Molt4/AR, Molt4/VLB, HSB2, HSB2/VLB, HSB2/DOX,
MCF7, MCF7/AR, MDA, MDA/AR, MDA/MITO) were analyzed by 2-D gel
electrophoresis and visualized by either blue or silver staining or
immunoblotting with anti-HSC70 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, the 13 cm strips
were rehydrated in a ceramic strip holder in 250 .mu.l rehydration
buffer containing the protein samples (0.5-2 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: [0365] 13 cm strips (pH 4-7): -500V for 500 Vh
[0366] -1000V for 1000 Vh [0367] -8000V for 16000 Vh The strips
were then slightly rinsed with water and equilibrated in 1% DTT in
equilibration buffer for 15 minutes, followed by 4% iodoacetamide
in equilibration buffer for 15 minutes.
[0368] For the second dimension, the above isoelectric strips were
patient 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.
[0369] The gels were next stained with blue or 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).
[0370] D. Results
[0371] As shown in FIG. 1, two proteins corresponding to two
isoforms of an approximately 71 kDa protein were present in plasma
membrane cell extracts of CEM and CEM/VLB cells. Isoform 1 was
initially named UR4 and isoforms 2 was initially named NC. The pI's
of isoforms 1 and 2 are 5.58 and 5.51 respectively. Multidrug
resistant T-lymphoblastoid CEM/VLB cells expressed at least 2-fold
higher levels of isoform 2 than did drug sensitive CEM cells.
Example 2
5.2 Identification of the 71 kDa Protein of T-lymphoblastoic Cancer
Cells as HSC70
[0372] To discover the identity of the approximately 71 kDa protein
of T-lymphoblastoic CEM and CEM/VLB cells (spots pointed to by the
arrow on the gels shown in FIGS. 1A and 1B), 2-D gels were loaded
with CEM/VLB plasma membrane extracts (2.times.750 .mu.g, pI 4-7,
10% gel), the gels were silver-stained, and the 71 kDa spots were
excised. The spots were 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) according to manufacturer's
instructions. The protein spots of interest were excised with a
clean (acid washed) razor blade, cut into small pieces on a clean
glass plate and transferred into a 200 .mu.l PCR tube (MeOH
treated), 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 minutes at room
temperature without agitation. The wash was removed. Water was
added to the gel pieces, mixed, and incubated 10 minutes 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 rehydration in 30%
methanol/water for 5 minutes. Gel pieces were then washed twice in
water for ten minutes and twice in 25 mM Ambic (ammonium
bicarbonate)/30% acetonitrile followed by a gel drying step.
Tryptic digestion of the destained and washed gel pieces was
performed by adding one volume of trypsin solution (130 ng of
enzyme in 25 mM ammonium bicarbonate, 5 mM CaCl.sub.2) to one
volume of gel pieces and samples left on ice for 45 minutes. The
digestion was allowed to proceed for 15-16 hours 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 reextracted with 25 mM Ambic/50%
acetonitrile without sonication. Digested peptides were further
extracted with 5% formic acid/50% acetonitrile/45% water freshly
prepared for 15 minutes at room temperature with shaking. The mix
was completed with one volume of acetonitrile and the gel
pieces/solvent were sonicated 5 minutes and reextracted the same
way without sonication. The collected material was combined and
dried. The extracted peptides were resuspended 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, et al., Ann. Rev. Biochem., 70: 437-73,
2001).
[0373] The mass spectrogram of the 71 kDa isoform 2 spot (following
purification and protease digestion) consisted of over 20 tryptic
peptides, of which 12 peptides were from the 71 kDa protein, while
the remaining peptides were trypsin autodigestion products. The 71
kDa peptides were further analyzed using a sequence database search
shareware software program called ProFound.TM.. 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 (NCBInr)). The NCBInr database contains
translated protein sequences form the entire collection of DNA
sequences kept at Genbank, and also the protein sequences in the
PDB, SWISS-PROT and PIR databases.
[0374] As shown in FIG. 2A, using the ProFound.TM. program, the 71
kDa isoforms 2 protein was identified as HSC70 with a probability
Z-score of 2.36, which is in the 99.sup.th percentile, based on the
analysis of the 12 tryptic peptide sequences that covered 25% of
the HSC70 complete amino acid sequence (FIGS. 2B and C). Two values
are taken into account when evaluating a ProFound.TM. analysis
result: the Z probability score and the % 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
95.sup.th percentile and Z=2.43 means that the result is in the
99.9.sup.th percentile. Thus the Z score of 2.36 indicated that the
71 kDa spot peptide sequences corresponded to those of HSC70 with a
high degree of probability.
[0375] The amino acid sequence of HSC70 protein is shown in FIG. 3
which also shows the location of sequence of the 12 tryptic
peptides in bold font. The sequences of the 12 peptides were spread
throughout the HSC70 molecule and together corresponded to 25% of
the complete HSC70 protein amino acid sequence, hence the protein
identified was the full length protein and not a fragment or fusion
protein.
Example 3
5.3 The 71 kDa Protein is Expressed on the Cell Surface of
Multidrug Resistant Promyelocytic Leukemia Cancer Cells
[0376] To determine whether HSC70 was present on the inside or
outside of the cell membrane, intact promyelocytic leukemia tumor
cells (HL60 and HL60/AR) were treated as described above with a
membrane impermeable biotinylating reagent that reacts with
lysines, and total cell extracts from both drug sensitive (HL60)
and multidrug resistant (HL60/AR) were prepared.
[0377] Two equivalent sets of 2-D gels of HL60 and HL60/AR were
prepared and GelCode blue (Pierce #24592, Rockford, Ill.) stained.
FIG. 4 represents the area corresponding to the presumed location
of HSC70 on a 2D gel (MW=71.11 kDa, pI=5.4). There was a 2-fold
increase in expression of the approximately 71 kDa protein in total
extracts of multidrug resistant HL60/AR cells as compared to the
drug-sensitive HL60 cells (compare FIGS. 4 (A) and 4 (B)).
[0378] To discover the identity of this protein, the 71 kDa spot
(marked by arrows on FIGS. 4(A) and 4(B)) was excised and processed
to prepare a sample for MALDI analysis as described in Example II
above. The mass spectrogram of the 71 kDa spot (following tryptic
digestion and peptide purification) consisted of 20 tryptic
peptides, of which 12 peptides were from the 71 kDa protein while
the remaining peptides were trypsin autodigestion products. The 71
kDa peptides were further analyzed using the sequence database
search shareware software program called ProFound.TM..
[0379] As shown in FIGS. 5A and 5B, using the ProFound.TM. program,
the 71 kDa protein was identified as HSC70 with a probability
Z-score of 2.43, which is in the 99.9.sup.th percentile, based on
the analysis of the 12 tryptic peptide sequences that covered 26%
of its complete amino acid sequence (FIG. 5C). The Z score of 2.43
indicated that the 71 kDa spot peptide sequences corresponded to
those of HSC70 with the highest degree of probability. As shown in
FIG. 5C, 3 out of 12 peptides were biotinylated.
[0380] The amino acid sequence of HSC70 protein is shown in FIG. 6
which also shows the location and sequence of the nine tryptic
peptides that were not biotinylated (in bold font), as well as the
three peptides that were biotinylated (in italic font). The
sequences of the 12 peptides were spread throughout the HSC70
molecule and together corresponded to 26% of the complete HSC70
protein amino acid sequence; hence the protein identified was the
full-length protein and not a fragment or fusion protein. The
biotinylated region that is shown in italic font in FIG. 6 was
detected in two ways. First, the bound biotin moiety changed the
mass of the peptide containing it relative to the non-biotinylated
peptide, and second, biotinylated peptides bound to streptavidin
beads (immobilized streptavidin beads commercially available from
Pierce #20347), whereas non-biotinylated peptides did not.
[0381] To demonstrate that HSC70 is overexpressed on the surface of
HL60/AR cells, total membrane and plasma membrane extracts were
resolved by 1 D-PAGE, transferred onto nitrocellulose, and blotted
with anti-HSC70 (SPA-815, Stressgen). FIG. 7 shows that HSC70 was
overexpressed in the total membrane extracts as well as plasma
membrane extracts of multidrug resistant HL60/AR as compared to
HL60.
Example 4
5.4 HSC70 is Expressed on the Cell Surface of Multidrug Resistant
Promyelocytic Leukemia Cancer Cells
[0382] To confirm that HSC70 was present on the outside of the cell
membrane, intact promyelocytic leukemia tumor cells (HL60 and
HL60/AR) were treated as described above in Example I 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.
[0383] To do this, cells were biotinylated by washing 3.times. with
50 ml PBS, pH 8. Next, Sulfo-NHS-LC-LC-Biotin (Pierce Chemicals)
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 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).
[0384] 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). 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.
[0385] Equivalent amounts of protein from HL60 and HL60/AR
(promyelocytic leukemia) cell surface biotinylated total cell
extracts (FIG. 8A) and streptavidin purified cell surface
biotinylated extracts (FIG. 8B) were resolved by SDS-PAGE according
to the method of Laemmli (supra) and subjected to Western blotting
analysis and probed with either anti-HSC70 antibody (rat monoclonal
SPA-815, Stressgen) (FIGS. 8A and 8B), or with horseradish
peroxidase (HRP)-linked streptavidin (which specifically binds to
biotinylated proteins, Amersham RPN 1231) (FIG. 8D). 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. U.S.A.
76:4350-4354, 1979). The nitrocellulose membranes were then probed
with antibody or HPR-streptavidin. Binding of the antibody was
detected with peroxidase conjugated rabbit anti-rat secondary
antibody (Sigma, A5795, St. Louis, Mo.). 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.).
[0386] As shown in FIG. 8A anti-HSC70 antibody bound to HSC70
protein which was expressed at significantly higher levels in
surface biotinylated total cell extracts of multidrug resistant
HL60/AR cells compared to HL60 cells. HSC70 was also overexpressed
in streptavidin purified cell surface biotinylated proteins of
HL60/AR cells compared to HL60 cells (FIG. 8B).
[0387] To confirm that HSC70 was present on the cell surface was
indeed biotinylated, surface biotinylated total cell extracts from
HL60 and HL60/AR cells were immunoprecipated with anti-HSC70
antibody (FIGS. 8C and 8D), 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-HSC70
monoclonal antibody or HRP-labeled streptavidin.
[0388] The blots were probed with anti-HSC70 antibody (FIG. 8(C))
and with streptavidin-HRP (FIG. 8D). As expected, HSC70 was
detected in the immunoprecipitates from both cell types (FIG. 8C),
however, significantly more cell surface biotinylated HSC70 was
present in the anti-HSC70 immunoprecipitates from HL60/AR cells
compared with those from HL60 cells (FIG. 8(D)).
[0389] These results, taken together, suggest that translocation of
HSC70 across the plasma membrane to the cell surface, as well as
additional de novo synthesis of HSC70, was associated with
multidrug resistance in HL60/AR cells.
Example 5
5.5 HSC70 is Expressed on the Cell Surface of Multidrug Resistant
of T-lymphoblastoic Cancer cells
[0390] To determine whether HSC70 was present on the inside or
outside of the cell membrane, intact T-lymphoblastoic cancer cells
(CEM and CEM/VLB) were treated with a membrane impermeable
biotinylating reagent that reacts with the amino acid lysine, and
total cell extracts from both drug sensitive (CEM) and multidrug
resistant (CEM/VLB) were prepared. In addition, streptavidin
purified biotinylated proteins as well as anti-HSC70
immunoprecipates were prepared from the surface biotinylated total
cell extracts of CEM and CEM/VLB cells, resolved on SDS-PAGE and
transferred onto nitrocellulose as described above.
[0391] As shown in FIG. 9A anti-HSC70 antibody bound to HSC70
protein which was expressed at slightly higher levels in surface
biotinylated total cell extracts of multidrug resistant CEM/VLB
cells compared to CEM cells. HSC70 was also slightly overexpressed
in streptavidin purified cell surface biotinylated proteins of
CEM/VLB cells compared to CEM cells (FIG. 9B). In addition, the
immunoprecipitates blots were probed with anti-HSC70 antibody (FIG.
9C) and with streptavidin-HRP (FIG. 9D). As expected, significantly
more cell surface biotinylated HSC70 was present in the anti-HSC70
immunoprecipitates from CEM/VLB cells compared with those from CEM
cells (FIG. 9D).
[0392] These results, taken together, suggest that translocation of
HSC70 across the plasma membrane to the cell surface, as well as
additional de novo synthesis of HSC70, was associated with
multidrug resistance in CEM/VLB cells.
Example 6
5.6 Characterization of HSC70 Expression on the Cell Surface of
Multidrug Resistant Promyelocytic Leukemia Cancer Cells
[0393] HL60 and HL60/AR cells were analyzed by cell surface
staining and analysis to determine the difference in cell surface
expression of heat shock cognate protein on the two cell lines. To
do this, indirect immunofluorescence analysis was performed using
10.mu., 20.mu. and 40 .mu.g of anti-HSC70 as primary antibody (rat
anti-HSC70. Stressgen SPA-815, San Diego, Calif.), followed by
rabbit anti-rat IgG FITC-conjugated secondary antibody (Sigma,
F1763).
[0394] Cells were washed three times in 50 ml PBS, pH 7.4 and 0.1%
NaN.sub.3 and counted. 1.times.10.sup.6 cells per sample were
placed in 100 PI PBS and 0.1% NaN.sub.3 in 12.times.75 mm tubes or
deep 96 well plate. The first 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 minutes
(1000 rpm/200.times.g). The supernatant was discarded and the
pellet was resuspended in 100 PI PBS pH 7.4 and 0.1% NaN.sub.3.
[0395] The second Ab (FITC conjugated) was added. The mixture was
prepared by diluting half in PBS, pH 7.4 and 0.1% NaN.sub.3 and
spun at maximum speed for 30 minutes at 4.degree. C. Separate from
pellet and use a 1/10 for staining. Incubate 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 and the mixture spun 5 minutes (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
minutes (1000 rpm/200.times.g).
[0396] 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 minutes (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 minutes (1000 rpm/200.times.g) and
resuspended in 500 .mu.l PBS, pH 7.2/2% paraformaldehyde and store
in the dark at 4.degree. C. 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 rat IgG2a
isotype (Cymbus, #CBL605).
[0397] As shown in FIG. 10A, HSC70 was expressed on the surface of
multidrug resistant HL60/AR cells at a level approximately 3 to 10
fold higher than expressed on the surface of drug sensitive HL60
cells, depending on how much primary antibody was used.
[0398] The number of molecules of HSC70 expressed on the surface of
HL60 and HL60/AR cells was determined by FACS using a Quantum
Simply Cellular flow cytometry quantification kit (Sigma #QSC20
(6951-213369 molecules) at saturating amounts of antibody for the
beads and for the cells. 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. 60 .mu.g of mouse anti-HSC70
IgG2a (Santa-Cruz, sc-7298) were used in two independent
experiments, and 30 .mu.g of the antibody were tested with QSC
beads purchased from Sigma (6951-213369 molecules) in the same
conditions as with the cells. The calibration curve obtained with
the beads was used to convert the RFI into number of molecules. As
shown in FIG. 10B, the averaged number of molecules of HSC70 on the
surface of HL60 and HL60/AR was 2541.+-.1184 and 7189.+-.590
molecules for HL60 and HL60/AR, respectively.
Example 7
5.7 Characterization of HSC70 Expression on the Cell Surface of
Multidrug Resistant Lymphocytic Leukemia Cancer Cells
[0399] CEM and CEM/VLB cells were analyzed by cell surface staining
and FACS analysis to determine the difference in cell surface
expression of HSC70 on the two cell lines. To do this, indirect
immunofluorescence analysis was performed using 1.mu., 2.5.mu. and
5 .mu.g of anti-HSC70 as primary antibody (rat monoclonal antibody,
Stressgen SPA-815) followed by FITC-conjugated secondary antibody.
Cells were washed three times in 50 ml PBS, pH 7.4 and 0.1%
NaN.sub.3 and counted. 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 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 minutes
(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.
[0400] The second Ab (FITC conjugated) was added. The mixture was
diluted by half in PBS, pH 7.4 and 0.1% NaN.sub.3 and spun at
maximum speed for 30 minutes at 4.degree. C. The supernatant was
separated from the pellet and 1/10 was used for staining. The
mixture was 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 and the
mixture spun 5 minutes (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 minutes (1000 rpm/200.times.g).
[0401] 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 minutes (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 minutes (1000 rpm/200.times.g).
Resuspended in 500 .mu.l PBS pH 7.2/2% paraformaldehyde and store
in the dark at 4.degree. C.
[0402] 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 rat IgG2a isotype (Cymbus, CBL605).
[0403] As shown in FIG. 11A, HSC70 was expressed on the surface of
multidrug resistant CEM/VLB cells at a level approximately two fold
higher than expressed on the surface of drug sensitive CEM
cells.
[0404] The number of molecules of HSC70 expressed on the surface of
CEM and CEM/VLB cells was determined by applying the fold
difference obtained when comparing CEM/VLB to HL60/AR (FIG. 11B),
to the number of molecules obtained for HL60/AR (see Example VI).
As seen in FIG. 11C, the number of molecules of HSC70 present on
the surface of CEM/VLB is 10972 molecules.
Example 8
5.8 Characterization of HSC70 Expression on the Cell Surface of
Multidrug Resistant Breast Cancer Cells
[0405] MCF-7, MCF-7/AR, MDA, and MDA/AR breast cancer cells were
analyzed by cell surface staining and FACS analysis to determine
the difference in cell surface expression of HSC70 on the two
multidrug resistant breast cancer cell lines. To do this, indirect
immunofluorescence analysis was performed using 10 .mu.g of rat
monoclonal anti-HSC70 as primary antibody (SPA-815-Stressgen),
followed by rabbit anti-rat IgG FITC-conjugated secondary antibody
(Sigma, F1763). In addition, indirect immunofluorescence analysis
was performed using 1 .mu.g of mouse monoclonal anti-Pgp
(#801-008-C150, clone MRK-16, Alexis Biochemicals), followed by
goat anti-mouse IgG FITC-conjugated secondary antibody (AP181F,
Chemicon). The multidrug resistance marker Pgp was tested in
parallel as a positive control.
[0406] The cells were prepared as described above for FACS
analysis. 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 10 .mu.g of rat IgG2a (Cymbus, #CBL605) or 1
.mu.g of mouse IgG2a (Sigma, M9144) isotype control (for HSC70 and
Pgp respectively).
[0407] The results of FACS analysis are represented in a bar graph
in FIGS. 12A and 12B. As can be seen, HSC70 was expressed on the
surface of MCF-7/AR cells at a level three fold higher than the
level of HSC70 expressed on the surface of MCF-7 cells (FIG. 12A).
FIG. 12B represents the FACS results for MDA/AR cells, and shows
that HSC70 was expressed on the surface of MDA/AR cells at a level
two folds higher than the level of HSC70 expressed on the surface
of MDA cells.
Example 9
5.9 HSC70 is Expressed on the Cell Surface of Hematological Cancer
Cells and at Higher Levels on MDR Hematological Cancer Cells, but
is Absent on Normal Cells
[0408] Normal white blood cells were next compared to hematological
cancer cells and MDR hematological cancer cells to determine the
difference in levels of cell surface expressed HSC70. To do this,
indirect immunofluorescence analysis was performed using 5 .mu.g of
rat monoclonal anti-HSC70 as primary antibody (SPA-815-Stressgen),
followed by rabbit anti-rat IgG FITC-conjugated secondary antibody
(Sigma, F1763).
[0409] 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). Specifically, 15 ml of
blood was diluted one-half in pH 7.4 and put over equal volume of
Ficoll gradient. Cells were separated by centrifugation 400.times.
g for 30 minutes 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 then red blood cells (at
the bottom of the tube) were collected. All cells types were washed
with PBS two times by spinning 10 minutes at 250 g at 4.degree. C.
and counted. Leucocytes were then resuspended at 1.times.10.sup.7
cells/ml, and 100 .mu.l (1.times.10.sup.6 cells) aliquots were used
for flow cytometry (FACS) analysis.
[0410] The cells were prepared as described above for FACS
analysis. 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 of rat IgG2a (Cymbus, #CBL605).
[0411] 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.
[0412] When the expression levels of HSC70 on normal WBC were
compared to the expression levels of HSC70 on various hematological
cancer or MDR hematological cancer cells in comparison to the
hematological cancer and MDR hematological cancer cells, normal
cells do not express HSC70 on their cell surface (see FIG.
13B).
Example 10
5.10 Use of HSC70 as an Antigen for a Vaccine Against MDR
Hematological Cancer Cells
[0413] Example 10 HSC70-Based Vaccine Protection Against MDR
Hematological Cancer Cells and MDR Mammary Adenocarcinoma Cells
[0414] To determine whether or not the full length HSC70 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 HSC70 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.
[0415] 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 HSC70
protein; some receive the hematological tumor cell injection after
being immunized with purified full length murine HSC70 protein.
Note that the purified HSC70 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 HSC70
protein; another receives only the hematological tumor cell
injection).
[0416] 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
HSC70 prior to injection of the tumor cells are found to have
tumors that are smaller after treatment than those that were not
vaccinated with HSC70 prior to injection of the tumor cells.
[0417] In further studies, the efficacy of HSC70 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 HSC70 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-HSC70 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 HSC70-Targeted Therapy Against MDR Hematological Cancer
Cells
[0418] In order to determine whether targeting a therapeutic to
cell surface HSC70 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 HSC70 protein, where the binding agent is operably linked
to ricin toxin.
[0419] 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.
[0420] In further studies, the efficacy of a HSC70-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-HSC70
alone (100 ug-1 mg/kg) or both vincristine or doxorubicin and
anti-HSC70 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 Characterization of Heat shock cognate protein (HSC70)
Expression on the Cell Surface of Multidrug Resistant Breast Cancer
Cells
[0421] MCF-7 and MCF-7/AR cells were analyzed by immunostaining to
determine the difference in cell surface expression of heat shock
cognate protein 70 on the two cell lines. To do this,
immunofluorescence analysis was performed using 0.5 ug of
anti-HSC70 as primary antibody (rat anti-HSC70. Stressgen SPA-815)
on permeabilized and non-permeabilized cells. Ethidium monoazide
(EMA) was used to stain the nucleus of permeabilized or damaged
cells.
[0422] FIGS. 15A & B provides flow charts depicting the steps
taken to stain cells. The control cells were MCF-7 and MCF-7/AR
stained with 0.5 ug rat IgG2a and no staining was observed in the
control cells in either the permeabilized or non-permeabilized
cells (data not shown). As shown in FIG. 16, HSC70 was clearly
expressed on the surface of intact multidrug resistant MCF-7/AR
cells when MCF-7 didn't show any staining. Permeabilized cells
showed equivalent total pool of HSC70 which was confirmed by
western blot (FIG. 19).
[0423] A comparison of total pool of HSC70 for various sensitive
and resistant cancer cells as well as normal tissue is shown in
FIG. 19. Equivalent amounts of each extract were separated on a 10%
SDS gel. Extracts of normal tissues were obtained from BD (brain
#635301, lung #635304, ovary #635344, mammary gland #635308,
prostate #635366) and extracts of white blood cells were prepared
from cells purified from blood obtained from healthy donors
(procedure for WBC purification is given in example IX of previous
draft). All extracts (except ones purchased from BD) were prepared
in 50 mM Tris pH8, 50 mM NaCl, 4% CHAPS supplemented with protease
inhibitors and phosphatase inhibitors cocktails. Lysed cells were
sonicated on ice, treated with DNAseI and lysates brought to 8M
urea. FIG. 19 shows that HSC70 is overexpressed in all sensitive
and resistant cancer cell lines tested, whereas normal cells
express very little to undetectable HSC70. It is noteworthy that
HSC70's increase in surface exposure measured for MCF-7 and
MCF-7/AR cells isn't triggered by an increase in the intracellular
pool of the protein since the total pool doesn't change
significantly between sensitive and resistant cell lines.
Example 13
5.13 Qualitative Analysis of Cell Surface HSC70 Expression in MDR
Breast Cancer Cells
[0424] MCF-7 and MCF-7/AR cells are analyzed by immunostaining to
determine the difference in cell surface expression of HSC70 on the
two cell lines. To do this, immunofluorescence analysis is
performed using 0.5 mg of anti-HSC70 as primary antibody on
permeabilized and non-permeabilized cells. Ethidium monoazide (EMA)
is used to stain the nucleus of permeabilized or damaged cells.
[0425] The control cells are MCF-7 and MCF-7/AR stained with 0.5 mg
mouse IgG1 and no staining is observed in either the permeabilized
or non-permeabilized cells (data not shown). HSC70 is clearly
expressed on the surface of intact multidrug resistant MCF-7/AR
cells while MCF-7 didn't show any staining.
Example 14
5.14 Quantitative Analysis of Cell Surface HSC70 Expression in MDR
Promyelocytic Leukemia Cancer Cells
[0426] HL60 and HL60/AR cells are analyzed for surface exposure of
HSC70 by direct binding of 125-iodine labeled anti-HSC70 to the
surface of the cells. To do this, anti-HSC70, mouse IgG1 (isotype
matching negative control, Sigma, M-9035) and anti-CD33 (positive
control, Serotec, #MCA1271) are iodinated using IODO-GEN.RTM.
precoated iodination tubes (Pierce, #28601) following the procedure
given by the manufacturer (average activity obtained: 7.5
mCi/mg).
[0427] Cells are washed twice with RPMI 1640 and resuspended at
10.sup.6 cells/100 mL in the same media. Viability is assessed by
trypan blue staining and is less than 5%. Cells (1.times.10.sup.6)
are aliquoted into borosilicate tubes and incubated for 1 hour on
ice in the presence of 1 mg of radiolabeled anti-HSC70, IgG1 or
anti-CD33. After incubation, cells are washed 3 times with 1 ml
RPMI 1640 and the cell pellet is counted in a gamma-counter.
[0428] The results of the intact cell radioimmunoassay are
represented assessed as the number of counts per minute obtained
for the cell pellet, from which the IgG1 background has been
subtracted. HSC70 is expressed on the surface of the resistant
HL60/AR cells at a higher level than the level of HSC70 expressed
on the surface of drug-sensitive HL60 cells.
Example 15
5.15 Cytotoxic Effect of Radioiodinated Anti-HSC70 on HL60 and
HL60/AR Cells
[0429] Cells are washed twice with RPMI 1640 and resuspended at 106
cells/100 ml in the same media. Viability is assessed by trypan
blue staining and is less than 5%. Cells (1.times.10.sup.6) are
aliquoted into borosilicate tubes and incubated for 4 hour at
37.degree. C. (0.5% CO.sub.2) in the presence of 5 mg of sterile
filtered radiolabeled anti-HSC70, IgG1 or anti-CD33 (iodination
procedure is described in example above). After incubation, cells
are 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 is assessed after 72
hours in an MTT based assay. Anti-CD33 is used a positive control
for surface binding of the antibody and 120 nM doxorubicin (Doxo)
is used as positive control for cytotoxicity.
[0430] The results of the cytotoxicity assay are represented as
values expressing percent viability. Values for percent survival
with anti-HSC70 and anti-CD33 antibody-1251 conjugates are
normalized against the values obtained for the radiolabeled IgG1
(non-significant background binding, 100% viability). Values
obtained with the anticancer drug doxorubicin are normalized
against the values obtained for cells non-treated with drug.
[0431] The results show that radiolabeled anti-HSC70 has a
cytotoxic effect on HL60 and HL60/AR which both express HSC70 on
their surface.
Example 16
5.16 Molecular Quantitation of Cell Surface HSC70 in Drug Sensitive
and Drug Resistant Breast and Ovarian Cancer Cell Lines
[0432] 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 are analyzed for surface exposure of HSC70 by direct
binding of 125-iodine labeled anti-HSC70 to the surface of the
cells. To do this, anti-HSC70 and mouse IgG1 (isotype matching
negative control Sigma, M-9035) are iodinated using IODO-GEN.RTM.
precoated iodination tubes (Pierce, #28601) following the procedure
given by the manufacturer (average activity obtained: 7.5
mCi/mg).
[0433] Cells are 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% CO2), wells are gently washed
with 100 ml media containing 1% FBS and incubated for 1 hour at
37.degree. C. (0.5% CO2) in 100 mL media containing 1% FBS and 0.1%
sodium azide as well as 100 ng of radioiodinated anti-HSC70 (or
IgG1). Mortality is checked prior to incubation by trypan blue
staining and is typically less than 1%. After incubation, media is
discarded, wells washed twice with 350 mL media containing 1% FBS,
and individually counted in a gamma-counter.
[0434] Binding studies are performed on MDA/mito cells using
increasing amounts of radiolabeled anti-HSC70 in an intact cell
radioimmunoassay. Scatchard analysis is used to calculate the
apparent dissociation constant (Kd) and the number of molecules of
antibody bound per cell. The average number of molecules of
anti-HSC70 bound per cell is obtained from the average of 5
independent Scatchard determinations.
[0435] The average number of HSC70 epitopes present on the surface
of MDA/mito cells is determined from the Scatchard plot.
[0436] The number of HSC70 epitopes present on MCF-7, MCF-7/AR,
MDA, SKOV3, SKOV/T320, 2008 and 2008/T320 is calculated from the
fold difference in surface exposure between these cell lines and
MDA/mito. This fold is 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-HSC70. The results show that MDR cell lines express more cell
surface HSC70 epitopes than do non-MDR neoplastic cell lines of the
same cell type or origin.
Example 17
5.17 Induction of Cell Surface Exposure of HSC70 in Breast and
Ovarian Cancer Cells
[0437] It is of further interest to determine whether these drugs
are capable of inducing surface exposure of HSC70 in MDA and SKOV3
cell lines. To do this, 20,000 cells are seeded into a 96
Stripwell.TM. plate (Costar, #9102). After 5 h, the culture media
is removed and replaced with culture media containing various drugs
and incubated for 12 to 16 hours at 37.degree. C. (0.5% CO2). After
incubation, wells are gently washed with 100 mL media containing 1%
FBS and incubated for 1 hour at 37.degree. C. (0.5% CO2) in 100 mL
media containing 1% FBS and 0.1% sodium azide as well as 100 ng of
radioiodinated anti-HSC70 (or IgG1) (labeling procedure is given in
example above). Mortality is checked prior to incubation by trypan
blue staining and is typically less than 1%. After incubation,
media is discarded, wells washed twice with 350 mL media containing
1% FBS, and individually counted in a gamma-counter. The surface
exposure of HSC70 when MDA cells are incubated with 1 or 10 mM
taxol, 1 or 10 mM doxorubicin, or 0.1 or 1 mM mitoxanthrone is
thereby determined. Values are corrected for non-specific binding
with IgG1.
Example 18
5.18 Internalization of .sup.125I-labeled Anti-HSC70 by MCF-7/AR
MDA MDA/AR and MDA/mito Cells
[0438] Internalization of cell surface HSC70 is measured on breast
cancer cells kept in suspension or cells adhered in a 96 well
plate. 106 cells are subcultured with a dissociation buffer (Gibco,
#13150-016), and then transferred to a borosilicate tube, washed in
PBS and resuspended in 200 ml alpha-MEM, 3% BSA containing 1 mg of
radioiodinated anti-HSC70, or 1 mg of anti-Mucin-1 as positive
control for internalization or 1 mg 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 are washed twice and further
incubated additional 4 hours at 4.degree. C. or 37.degree. C. Cell
viability is 100% according to trypan blue staining of cells prior
to incubation with radiolabeled IgGs. After incubation, cells are
washed and the radiolabel determined by counting samples in a
gamma-counter. Cells are 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 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. is
determined. The % internalization is obtained from the difference
in internalization measured at 4 and 37.degree. C. and is
significant.
[0439] The results show that anti-HSC70 antibodies attached to a
radionuclide therapeutic agent (or diagnostic probe) bind to cell
surface HSC70 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-1251 conjugate is actively taken up by
endocytosis. Regardless of the mechanism of uptake, the results
indicate that anti-HSC70 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. Therefore, these results further support the usefulness
of anti-HSC70 antibodies for the targeting and uptake of linked
therapeutic and diagnostic agents for both neoplastic, and MDR
neoplastic cells.
EQUIVALENTS
[0440] 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
271646PRTHomo sapiens 1Met Ser Lys Gly Pro Ala Val Gly Ile Asp Leu
Gly Thr Thr Tyr Ser1 5 10 15Cys Val Gly Val Phe Gln His Gly Lys Val
Glu Ile Ile Ala Asn Asp20 25 30Gln Gly Asn Arg Thr Thr Pro Ser Tyr
Val Ala Phe Thr Asp Thr Glu35 40 45Arg Leu Ile Gly Asp Ala Ala Lys
Asn Gln Val Ala Met Asn Pro Thr50 55 60Asn Thr Val Phe Asp Ala Lys
Arg Leu Ile Gly Arg Arg Phe Asp Asp65 70 75 80Ala Val Val Gln Ser
Asp Met Lys His Trp Pro Phe Met Val Val Asn85 90 95Asp Ala Gly Arg
Pro Lys Val Gln Val Glu Tyr Lys Gly Glu Thr Lys100 105 110Ser Phe
Tyr Pro Glu Glu Val Ser Ser Met Val Leu Thr Lys Met Lys115 120
125Glu Ile Ala Glu Ala Tyr Leu Gly Lys Thr Val Thr Asn Ala Val
Val130 135 140Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala
Thr Lys Asp145 150 155 160Ala Gly Thr Ile Ala Gly Leu Asn Val Leu
Arg Ile Ile Asn Glu Pro165 170 175Thr Ala Ala Ala Ile Ala Tyr Gly
Leu Asp Lys Lys Val Gly Ala Glu180 185 190Arg Asn Val Leu Ile Phe
Asp Leu Gly Gly Gly Thr Phe Asp Val Ser195 200 205Ile Leu Thr Ile
Glu Asp Gly Ile Phe Glu Val Lys Ser Thr Ala Gly210 215 220Asp Thr
His Leu Gly Gly Glu Asp Phe Asp Asn Arg Met Val Asn His225 230 235
240Phe Ile Ala Glu Phe Lys Arg Lys His Lys Lys Asp Ile Ser Glu
Asn245 250 255Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg
Ala Lys Arg260 265 270Thr Leu Ser Ser Ser Thr Gln Ala Ser Ile Glu
Ile Asp Ser Leu Tyr275 280 285Glu Gly Ile Asp Phe Tyr Thr Ser Ile
Thr Arg Ala Arg Phe Glu Glu290 295 300Leu Asn Ala Asp Leu Phe Arg
Gly Thr Leu Asp Pro Val Glu Lys Ala305 310 315 320Leu Arg Asp Ala
Lys Leu Asp Lys Ser Gln Ile His Asp Ile Val Leu325 330 335Val Gly
Gly Ser Thr Arg Ile Pro Lys Ile Gln Lys Leu Leu Gln Asp340 345
350Phe Phe Asn Gly Lys Glu Leu Asn Lys Ser Ile Asn Pro Asp Glu
Ala355 360 365Val Ala Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu Ser
Gly Asp Lys370 375 380Ser Glu Asn Val Gln Asp Leu Leu Leu Leu Asp
Val Thr Pro Leu Ser385 390 395 400Leu Gly Ile Glu Thr Ala Gly Gly
Val Met Thr Val Leu Ile Lys Arg405 410 415Asn Thr Thr Ile Pro Thr
Lys Gln Thr Gln Thr Phe Thr Thr Tyr Ser420 425 430Asp Asn Gln Pro
Gly Val Leu Ile Gln Val Tyr Glu Gly Glu Arg Ala435 440 445Met Thr
Lys Asp Asn Asn Leu Leu Gly Lys Phe Glu Leu Thr Gly Ile450 455
460Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu Val Thr Phe Asp
Ile465 470 475 480Asp Ala Asn Gly Ile Leu Asn Val Ser Ala Val Asp
Lys Ser Thr Gly485 490 495Lys Glu Asn Lys Ile Thr Ile Thr Asn Asp
Lys Gly Arg Leu Ser Lys500 505 510Glu Asp Ile Glu Arg Met Val Gln
Glu Ala Glu Lys Tyr Lys Ala Glu515 520 525Asp Glu Lys Gln Arg Asp
Lys Val Ser Ser Lys Asn Ser Leu Glu Ser530 535 540Tyr Ala Phe Asn
Met Lys Ala Thr Val Glu Asp Glu Lys Leu Gln Gly545 550 555 560Lys
Ile Asn Asp Glu Asp Lys Gln Lys Ile Leu Asp Lys Cys Asn Glu565 570
575Ile Ile Asn Trp Leu Asp Lys Asn Gln Thr Ala Glu Lys Glu Glu
Phe580 585 590Glu His Gln Gln Lys Glu Leu Glu Lys Val Cys Asn Pro
Ile Ile Thr595 600 605Lys Leu Tyr Gln Ser Ala Gly Gly Met Pro Gly
Gly Met Pro Gly Gly610 615 620Phe Pro Gly Gly Gly Ala Pro Pro Ser
Gly Gly Ala Ser Ser Gly Pro625 630 635 640Thr Ile Glu Glu Val
Asp64521941DNAHomo sapiens 2atgtccaagg gacctgcagt tggtattgat
cttggcacca cctactcttg tgtgggtgtt 60ttccagcacg gaaaagtcga gataattgcc
aatgatcagg gaaaccgaac cactccaagc 120tatgtcgcct ttacggacac
tgaacggttg atcggtgatg ccgcaaagaa tcaagttgca 180atgaacccca
ccaacacagt ttttgatgcc aaacgtctga ttggacgcag atttgatgat
240gctgttgtcc agtctgatat gaaacattgg ccctttatgg tggtgaatga
tgctggcagg 300cccaaggtcc aagtagaata caagggagag accaaaagct
tctatccaga ggaggtgtct 360tctatggttc tgacaaagat gaaggaaatt
gcagaagcct accttgggaa gactgttacc 420aatgctgtgg tcacagtgcc
agcttacttt aatgactctc agcgtcaggc taccaaagat 480gctggaacta
ttgctggtct caatgtactt agaattatta atgagccaac tgctgctgct
540attgcttacg gcttagacaa aaaggttgga gcagaaagaa acgtgctcat
ctttgacctg 600ggaggtggca cttttgatgt gtcaatcctc actattgagg
atggaatctt tgaggtcaag 660tctacagctg gagacaccca cttgggtgga
gaagattttg acaaccgaat ggtcaaccat 720tttattgctg agtttaagcg
caagcataag aaggacatca gtgagaacaa gagagctgta 780agacgcctcc
gtactgcttg tgaacgtgct aagcgtaccc tctcttccag cacccaggcc
840agtattgaga tcgattctct ctatgaagga atcgacttct atacctccat
tacccgtgcc 900cgatttgaag aactgaatgc tgacctgttc cgtggcaccc
tggacccagt agagaaagcc 960cttcgagatg ccaaactaga caagtcacag
attcatgata ttgtcctggt tggtggttct 1020actcgtatcc ccaagattca
gaagcttctc caagacttct tcaatggaaa agaactgaat 1080aagagcatca
accctgatga agctgttgct tatggtgcag ctgtccaggc agccatcttg
1140tctggagaca agtctgagaa tgttcaagat ttgctgctct tggatgtcac
tcctctttcc 1200cttggtattg aaactgctgg tggagtcatg actgtcctca
tcaagcgtaa taccaccatt 1260cctaccaagc agacacagac cttcactacc
tattctgaca accagcctgg tgtgcttatt 1320caggtttatg aaggcgagcg
tgccatgaca aaggataaca acctgcttgg caagtttgaa 1380ctcacaggca
tacctcctgc accccgaggt gttcctcaga ttgaagtcac ttttgacatt
1440gatgccaatg gtatactcaa tgtctctgct gtggacaaga gtacgggaaa
agagaacaag 1500attactatca ctaatgacaa gggccgtttg agcaaggaag
acattgaacg tatggtccag 1560gaagctgaga agtacaaagc tgaagatgag
aagcagaggg acaaggtgtc atccaagaat 1620tcacttgagt cctatgcctt
caacatgaaa gcaactgttg aagatgagaa acttcaaggc 1680aagattaacg
atgaggacaa acagaagatt ctggacaagt gtaatgaaat tatcaactgg
1740cttgataaga atcagactgc tgagaaggaa gaatttgaac atcaacagaa
agagctggag 1800aaagtttgca accccatcat caccaagctg taccagagtg
caggaggcat gccaggagga 1860atgcctgggg gatttcctgg tggtggagct
cctccctctg gtggtgcttc ctcagggccc 1920accattgaag aggttgatta a
1941310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Met Val Asn His Phe Ile Ala Glu Phe Lys1 5
10410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Phe Glu Glu Leu Asn Ala Asp Leu Phe Arg1 5
10511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Met Val Asn His Phe Ile Ala Glu Phe Lys Arg1 5
10612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Ala Arg Phe Glu Glu Leu Asn Ala Asp Leu Phe Arg1
5 10713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu
Arg1 5 10816PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 8Ser Thr Ala Gly Asp Thr His Leu Gly Gly
Glu Asp Phe Asp Asn Arg1 5 10 15917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Ile
Ile Asn Glu Pro Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys1 5 10
15Lys1016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Asn Gln Val Ala Met Asn Pro Thr Asn Thr Val Phe
Asp Ala Lys Arg1 5 10 151117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Leu Asp Lys Ser Gln Ile His
Asp Ile Val Leu Val Gly Gly Ser Thr1 5 10 15Arg1218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Asp
Asn Asn Leu Leu Gly Lys Phe Glu Leu Thr Gly Ile Pro Pro Ala1 5 10
15Pro Arg1318PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 13Thr Val Thr Asn Ala Val Val Thr Val
Pro Ala Tyr Phe Asn Asp Ser1 5 10 15Gln Arg1424PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Gln
Thr Gln Thr Phe Thr Thr Tyr Ser Asp Asn Gln Pro Gly Val Leu1 5 10
15Ile Gln Val Tyr Glu Gly Glu Arg201512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Asp
Ala Gly Thr Ile Ala Gly Leu Asn Val Leu Arg1 5 101610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Phe
Glu Glu Leu Asn Ala Asp Leu Phe Arg1 5 101711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Met
Val Asn Arg Phe Ile Ala Glu Phe Lys Arg1 5 101812PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Ala
Arg Phe Glu Glu Ile Asn Ala Asp Leu Phe Arg1 5 101913PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Thr
Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu Arg1 5
102016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Ser Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp
Phe Asp Asn Arg1 5 10 152117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Leu Asp Lys Ser Gln Thr His
Asp Ile Val Leu Val Gly Gly Ser Thr1 5 10 15Arg2214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Gly
Thr Leu Asp Pro Val Glu Lys Ala Leu Arg Asp Ala Lys1 5
102318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Thr Val Thr Asn Ala Val Val Thr Val Pro Ala Tyr
Phe Asn Asp Ser1 5 10 15Gln Arg2416PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Met
Val Gln Phe Ala Glu Lys Tyr Lys Ala Glu Asp Glu Lys Gln Arg1 5 10
152517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Asn Gln Val Ala Ala Met Asn Pro Thr Asn Thr Val
Phe Asp Ala Lys1 5 10 15Arg2624PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 26Gln Thr Gln Thr Phe Thr Thr
Tyr Ser Asp Asn Gln Pro Gly Val Leu1 5 10 15Ile Gln Val Tyr Glu Gly
Glu Arg202718PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 27Asn Ser Leu Glu Ser Tyr Ala Phe Asn
Met Lys Ala Thr Val Glu Asp1 5 10 15Glu Lys
* * * * *
References