U.S. patent application number 10/212031 was filed with the patent office on 2003-01-16 for kits comprising heat shock protein-antigenic molecule complexes.
This patent application is currently assigned to Fordham University. Invention is credited to Chandawarkar, Rajiv Y., Srivastava, Pramod K..
Application Number | 20030012794 10/212031 |
Document ID | / |
Family ID | 25167905 |
Filed Date | 2003-01-16 |
United States Patent
Application |
20030012794 |
Kind Code |
A1 |
Srivastava, Pramod K. ; et
al. |
January 16, 2003 |
Kits comprising heat shock protein-antigenic molecule complexes
Abstract
The present invention relates to methods and compositions for
eliciting an immune response and the prevention-and treatment of
primary and metastatic neoplastic diseases and infectious diseases.
The methods of the invention comprise administering a composition
comprising an effective amount of a complex, in which the complex
consists essentially of a heat shock protein (hsp) noncovalently
bound to an antigenic molecule. Optionally, the methods further
comprise administering antigen presenting cells sensitized with
complexes of hsps noncovalently bound to an antigenic molecule.
"Antigenic molecule" as used herein refers to the peptides with
which the hsps are endogenously associated in vivo as well as
exogenous antigens/immunogens (i.e., with which the hsps are not
complexed in vivo) or antigenic/immunogenic fragments and
derivatives thereof. In a preferred embodiment, the complex is
autologous to the individual. In a specific embodiment, the
effective amounts of the complex are in the range of 0.1 to 9.0
micrograms for complexes comprising hsp70, 5 to 49 micrograms for
hsp90, and 0.1 to 9.0 micrograms for gp96.
Inventors: |
Srivastava, Pramod K.;
(Riverdale, NY) ; Chandawarkar, Rajiv Y.;
(Mineola, NY) |
Correspondence
Address: |
Pennie & Edmonds LLP
1155 Avenue of the Americas
New York
NY
10036-2711
US
|
Assignee: |
Fordham University
Bronx
NY
|
Family ID: |
25167905 |
Appl. No.: |
10/212031 |
Filed: |
August 2, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10212031 |
Aug 2, 2002 |
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09439686 |
Nov 15, 1999 |
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6436404 |
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09439686 |
Nov 15, 1999 |
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08796319 |
Feb 7, 1997 |
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6017540 |
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Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
A61K 2039/6043 20130101;
A61K 2039/622 20130101; Y02A 50/30 20180101; A61K 39/0011 20130101;
A61P 35/04 20180101; A61P 35/00 20180101; A61P 37/04 20180101 |
Class at
Publication: |
424/185.1 |
International
Class: |
A61K 039/00 |
Goverment Interests
[0001] This invention was made with government support under grant
numbers CA44786 and CA64394 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
What is claimed is:
1. A method of eliciting an immune response in a human individual
comprising administering to the individual a first composition
comprising an amount of a first complex of less than 10 micrograms
effective to elicit an immune response, said first complex
consisting essentially of a heat shock protein (hsp) 70
noncovalently bound to a first antigenic molecule.
2. A method of eliciting an immune response in a human individual
comprising administering to the individual a first composition
comprising an amount of a first complex of less than 50 micrograms
effective to elicit an immune response, said first complex
consisting essentially of an hsp90 noncovalently bound to a first
antigenic molecule.
3. A method of eliciting an immune response in a human individual
comprising administering to the individual a first composition
comprising an amount of a first complex of less than 10 micrograms
effective to elicit an immune response, said first complex
consisting essentially of a gp96 noncovalently bound to a first
antigenic molecule.
4. The method according to claims 1, 2 or 3 in which the individual
has liver cancer, colon cancer, or breast cancer.
5. The method according to claim 1 in which the amount of the first
complex is in the range of 0.1 to 9.0 micrograms.
6. The method according to claim 2 in which the amount of the first
complex is in the range of 5 to 49 micrograms.
7. The method according to claim 3 in which the amount of the first
complex is in the range of 0.1 to 9.0 micrograms.
8. The method according to claim 1 in which the amount of the first
complex is in the range of 0.5 to 2.0 micrograms.
9. The method according to claim 2 in which the amount of the first
complex is in the range of 5 to 40 micrograms.
10. The method according to claim 3 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
11. The method according to claim 1, 2 or 3, further comprising
administering to the individual an effective amount of a biological
response modifier selected from the group consisting of
interferon-.alpha., interferon-.gamma., interleukin-2,
interleukin-4, interleukin-6, and tumor necrosis factor.
12. The method according to claim 1, 2 or 3 in which said
administering step is repeated at weekly intervals.
13. The method according to claim 1, 2 or 3 in which said first
complex is administered intradermally.
14. The method according to claim 1, 2 or 3 in which said first
complex is administered mucosally.
15. The method according to claim 1, 2 or 3 in which said
administering step is repeated five times, the first administration
being on the left arm, the second administration being on the right
arm, the third administration being on the left belly, the fourth
administration being on the right belly, the fifth administration
being on the left thigh, and the sixth administration being on the
right thigh; said first through sixth administration being
intradermally.
16. A method of treating a human individual having cancer,
comprising administering to the individual a first-composition
comprising a therapeutically effective amount of a first complex of
less than 10 micrograms, said first complex consisting essentially
of an hsp70 noncovalently bound to a first antigenic molecule.
17. A method of treating a human individual having cancer,
comprising administering to the individual a first composition
comprising a therapeutically effective amount of a first complex of
less than 50 micrograms, said first complex consisting essentially
of an hsp90 noncovalently bound to a first antigenic molecule.
18. A method of treating a human individual having cancer,
comprising administering to the individual a first composition
comprising a therapeutically effective amount of a first complex of
less than 10 micrograms, said first complex consisting essentially
of a gp96 noncovalently bound to a first antigenic molecule.
19. The method according to claim 16, 17 or 18 in which the cancer
comprises a sarcoma or carcinoma, selected from the group
consisting of fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma; embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease.
20. The method according to claim 16 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
21. The method according to claim 17 in which the amount of the
first complex is in the range of 5 to 49 micrograms.
22. The method according to claim 18 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
23. The method according to claim 16 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
24. The method according to claim 17 in which the amount of the
first complex is in the range of 5 to 40 micrograms.
25. The method according to claim 18 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
26. The method according to claim 16, 17 or 18 in which the first
antigenic molecule is a peptide with which the hsp is endogenously
associated in vivo, and the first complex is prepared from
cancerous tissue autologous to the individual.
27. The method according to claim 16, 17 or 18 in which the first
antigenic molecule is a peptide with which the hsp is endogenously
associated in vivo, and the first complex is prepared from
cancerous tissue allogeneic to the individual.
28. The method according to claim 16, 17, or 18 in which the first
antigenic molecule is a peptide with which the hsp is endogenously
associated in vivo, and the first complex is prepared from
cancerous tissue.
29. The method according to claim 28 in which the cancerous tissue
is from the individual.
30. The method according to claim 16, 17, or 18 in which the first
complex of the hsp and first antigenic molecule is produced in
vitro.
31. The method according to claim 30 in which the first antigenic
molecule is a tumor-specific antigen.
32. The method according to claim 16, 17 or 18, further comprising
administering to the individual an effective amount of a biological
response modifier selected from the group consisting of
interferon-.alpha., interferon-.gamma., interleukin-2,
interleukin-4, interleukin-6, and tumor necrosis factor.
33. The method according to claim 16, 17 or 18 in which said
administering step is repeated at weekly intervals.
34. The method according to claim 16, 17 or 18 in which the first
complex is purified to 60 to 100 percent of total mg protein.
35. The method according to claim 16, 17 or 18 in which the first
complex is administered intradermally.
36. The method according to claim 16, 17 or 18 in which the first
complex is administered mucosally.
37. The method according to claim 23, 24 or 25 in which said
administering step is repeated five times, the first administration
being on the left arm, the second administration being on the right
arm, the third administration being on the left belly, the fourth
administration being on the right belly, the fifth administration
being on the left thigh, and the sixth administration being on the
right thigh; said first through sixth administration being
intradermally.
38. A method of treating a human individual having cancer
comprising: (a) administering to the individual a composition
comprising about 2 micrograms of a complex, said complex consisting
essentially of a gp96 noncovalently bound to a peptide, said
complex having been isolated from cancerous tissue of said
individual; and (b) repeating said administering of step (a) at
weekly intervals for five weeks, the first administration being on
the left arm, the second administration being on the right arm, the
third administration being on the left belly, the fourth
administration being on the right belly, the fifth administration
being on the left thigh, and the sixth administration being on the
right thigh; said first through sixth administration being
intradermally.
39. A method of preventing cancer in a human individual in whom
prevention of cancer is desired comprising administering to the
individual a first composition comprising an amount of a first
complex of less than 10 micrograms effective to inhibit cancer,
said first complex consisting essentially of an hsp70 noncovalently
bound to a first antigenic molecule.
40. A method of preventing cancer in a human individual in whom
prevention of cancer is desired, comprising administering to the
individual a first composition comprising an amount of a first
complex of less than 50 micrograms effective to inhibit cancer,
said first complex consisting essentially of an hsp90 noncovalently
bound to a first antigenic molecule.
41. A method of preventing cancer in a human individual in whom
prevention of cancer is desired, comprising administering to the
individual a first composition comprising an amount of a first
complex of less than 10 micrograms effective to inhibit cancer,
said first complex consisting essentially of a gp96 noncovalently
bound to a first antigenic molecule.
42. The method according to claim 39, in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
43. The method according to claim 40, in which the amount of the
first complex is in the range of 5 to 49 micrograms.
44. The method according to claim 41, in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
45. The method according to claim 39, in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
46. The method according to claim 40, in which the amount of the
first complex is in the range of 5 to 40 micrograms.
47. The method according to claim 41, in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
48. The method according to claim 39, 40 or 41 in which the first
antigenic molecule is a peptide with which the hsp is endogenously
associated in vivo.
49. The method according to claim 48 in which the first complex is
prepared from cancerous tissue.
50. The method according to claim 39, 40 or 41 in which the first
complex of the hsp and first antigenic molecule is produced in
vitro.
51. The method according to claim 50 in which the first antigenic
molecule is a tumor-specific antigen.
52. A method of treating or preventing an infectious disease in a
human individual in whom such treatment or prevention is desired
comprising administering to the individual a first composition
comprising an amount of a first complex of less than 10 micrograms
effective to treat or prevent infectious disease, said first
complex consisting essentially of an hsp70 noncovalently bound to a
first antigenic molecule.
53. A method of treating or preventing an infectious disease in a
human individual in whom such treatment or prevention is desired
comprising administering to the individual a first composition
comprising an amount of a first complex of less than 50 micrograms
effective to treat or prevent infectious disease, said first
complex consisting essentially of an hsp90 noncovalently bound to a
first antigenic molecule.
54. A method of treating or preventing an infectious disease in a
human individual in whom such treatment or prevention is desired
comprising administering to the individual a first composition
comprising an amount of a first complex of less than 10 micrograms
effective to treat or prevent infectious disease, said first
complex consisting essentially of a gp96 noncovalently bound to a
first antigenic molecule.
55. The method according to claim 52 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
56. The method according to claim 53 in which the amount of the
first complex is in the range of 5 to 49 micrograms.
57. The method according to claim 54 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
58. The method according to claim 52 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
59. The method according to claim 53 in which the amount of the
first complex is in the range of 5 to 40 micrograms.
60. The method according to claim 54 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
61. The method according to claim 52, 53 or 54 in which the first
antigenic molecule is a peptide with which the hsp is endogenously
associated in cells infected with an infectious agent that causes
the infectious disease.
62. The method according to claim 52, 53 or 54 in which the first
antigenic molecule is an antigen of an infectious agent that causes
the infectious disease.
63. The method according to claim 62 in which the infectious agent
is a virus, bacterium, protozoa, fungus, or parasite.
64. The method according to claim 1 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first composition.
65. The method according to claim 2 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first composition.
66. The method according to claim 3 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first composition.
67. The method according to claim 64, 65 or 66 in which said second
hsp is selected from the group consisting of hsp70, hsp90, gp96,
and combinations of the foregoing.
68. The method according to claim 64, 65 or 66 in which the first
and second complexes are the same.
69. The method according to claim 64, 65 or 66 in which the
individual has liver cancer, colon cancer, or breast cancer.
70. The method according to claim 64 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
71. The method according to claim 65 in which the amount of the
first complex is in the range of 5 to 49 micrograms.
72. The method according to claim 66 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
73. The method according to claim 64 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
74. The method according to claim 65 in which the amount of the
first complex is in the range of 5 to 40 micrograms.
75. The method according to claim 66 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
76. The method according to claim 64, 65 or 66 further comprising
administering to the individual an effective amount of a biological
response modifier selected from the group consisting of
interferon-.alpha., interferon-.gamma., interleukin-2,
interleukin-4, interleukin-6, and-tumor necrosis factor.
77. The method according to claim 64, 65 or 66 in which
administering the first composition is repeated at weekly
intervals.
78. The method according to claim 64, 65 or 66 in which
administering the second composition is repeated at weekly
intervals.
79. The method according to claim 64, 65 or 66 in which the first
complex is administered intradermally.
80. The method according to claim 64, 65 or 66 in which the first
complex is administered mucosally.
81. The method according to claim 64, 65 or 66 in which the
sensitized antigen presenting cells are administered
intravenously.
82. The method according to claim 64, 65 or 66 in which 10.sup.6 to
10.sup.12 antigen presenting cells are administered.
83. The method according to claim 16 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first complex.
84. The method according to claim 17 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first complex.
85. The method according to claim 18 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first complex.
86. The method according to claim 83, 84 or 85 in which said second
hsp is selected from the group consisting of hsp70, hsp90, gp96,
and combination of the foregoing.
87. The method according to claim 83, 84 or 85 in which the first
and second complexes are the same
88. The method according to claim 83, 84 or 85 in which the cancer
comprises a sarcoma or carcinoma, selected from the group
consisting of fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease.
89. The method according to claim 83 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
90. The method according to claim 84 in which the amount of the
first complex is in the range of 5 to 49 micrograms.
91. The method according to claim 85 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
92. The method according to claim 83 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
93. The method according to claim 84 in which the amount of the
first complex is in the range of 5 to 40 micrograms.
94. The method according to claim 85 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
95. The method according to claim 83, 84 or 85 in which said second
antigenic molecule is a peptide with which said second heat shock
protein is endogenously associated in vivo, and said second complex
is prepared from cancerous tissue autologous to the individual.
96. The method according to claim 83, 84 or 85 in which said second
antigenic molecule is a peptide with which said second heat shock
protein is endogenously associated in vivo, and said second complex
is prepared from cancerous tissue allogeneic to the individual.
97. The method according to claim 83, 84 or 85 in which said second
antigenic molecule is a peptide with which said second heat shock
protein is endogenously associated in vivo, and said second complex
is prepared from cancerous tissue.
98. The method according to claim 97 in which the cancerous tissue
is from the individual.
99. The method according to claim 83, 84 or 85 in which the second
complex of the second hsp and second antigenic molecule is produced
in vitro.
100. The method according to claim 99 in which the second antigenic
molecule is a tumor-specific antigen.
101. The method according to claim 83, 84 or 85 further comprising
administering to the individual an effective amount of a biological
response modifier selected from the group consisting of
interferon-.alpha., interferon-.gamma., interleukin-2,
interleukin-4, interleukin-6, and tumor necrosis factor.
102. The method according to claim 83, 84 or 85 in which
administering the first composition is repeated at weekly
intervals.
103. The method according to claim 83, 84 or 85 in which
administering the second composition is repeated at weekly
intervals.
104. The method according to claim 83, 84 or 85 in which the first
complex is administered intradermally.
105. The method according to claim 83, 84 or 85 in which the first
complex is administered mucosally.
106. The method according to claim 83, 84 or 85 in which the
sensitized antigen presenting cells are administered
intravenously.
107. The method according to claim 83, 84 or 85 in which 10.sup.6
to 10.sup.12 antigen presenting cells are administered.
108. The method according to claim 39 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first complex.
109. The method according to claim 40 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first complex.
110. The method according to claim 41 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first complex.
111. The method according to claim 108, 109 or 110 in which said
second hsp is selected from the group consisting of hsp70, hsp90,
gp96 and combinations of the foregoing.
112. The method according to claim 108, 109 or 110 in which the
first and second complexes are the same.
113. The method according to claim 108 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
114. The method according to claim 109 in which the amount of the
first complex is in the range of 5 to 49 micrograms.
115. The method according to claim 110 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
116. The method according to claim 108 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
117. The method according to claim 109 in which the amount of the
first complex is in the range of 5 to 40 micrograms.
118. The method according to claim 110 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
119. The method according to claim 108, 109 or 110 in which said
second antigenic molecule is a peptide with which said second heat
shock protein is endogenously associated in vivo.
120. The method according to claim 52 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first complex.
121. The method according to claim 53 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first complex.
122. The method according to claim 54 which further comprises
administering to the individual a second composition comprising
antigen presenting cells sensitized in vitro with a sensitizing
amount of a second complex of a second hsp noncovalently bound to a
second antigenic molecule in which said sensitized antigen
presenting cells are administered before, concurrently or after
administration of the first complex.
123. The method according to claim 120, 121 or 122 in which said
second hsp is selected from the group consisting of hsp70, hsp90,
gp96 and combinations of the foregoing.
124. The method according to claim 120, 121 or 122 in which the
first and second complexes are the same.
125. The method according to claim 120 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
126. The method according to claim 121 in which the amount of the
first complex is in the range of 5 to 49 micrograms.
127. The method according to claim 122 in which the amount of the
first complex is in the range of 0.1 to 9.0 micrograms.
128. The method according to claim 120 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
129. The method according to claim 121 in which the amount of the
first complex is in the range of 5 to 40 micrograms.
130. The method according to claim 122 in which the amount of the
first complex is in the range of 0.5 to 2.0 micrograms.
131. The method according to claim 120, 121 or 122 in which said
second antigenic molecule is a peptide with which said second heat
shock protein is endogenously associated in cells infected with an
infectious agent that causes the infectious disease.
132. The method according to claim 120, 121 or 122 in which said
second antigenic molecule is an antigen of an infectious agent that
causes the infectious disease.
133. The method according to claim 132 in which the infectious
agent is a virus, bacterium, protozoa, fungus, or parasite.
134. The method according to claim 64, 65 or 66 in which the
antigen presenting cells comprise macrophages.
135. The method according to claim 83, 84 or 85 in which the
antigen presenting cells comprise macrophages.
136. The method according to claim 108, 109 or 110 in which the
antigen presenting cells comprise macrophages.
137. The method according to claim 120, 121 or 122 in which the
antigen presenting cells comprise macrophages.
138. A kit comprising in a container a composition comprising an
amount of a complex of less than 10 micrograms effective to induce
an immune response or treat or prevent cancer or infectious disease
in a mammal, said complex consisting essentially of an hsp70
noncovalently bound to an antigenic molecule.
139. The kit of claim 138 which further comprises in a second
container human antigen presenting cells.
140. A kit comprising in a container a composition comprising an
amount of a complex of less than 50 micrograms effective to induce
an immune response or treat or prevent cancer or infectious disease
in a mammal, said complex consisting essentially of an hsp9o
noncovalently bound to an antigenic molecule.
141. The kit of claim 140 which further comprises in a second
container human antigen presenting cells.
142. A kit comprising in a container a composition comprising an
amount of a complex of less than 10 micrograms effective to induce
an immune response or treat or prevent cancer or infectious disease
in a mammal, said complex consisting essentially of a gp96
noncovalently bound to an antigenic molecule.
143. The kit of claim 142 which further comprises in a second
container human antigen presenting cells.
144. A kit comprising a plurality of containers, each container
having a composition comprising an amount of a complex of less than
10 micrograms effective to induce an immune response or treat or
prevent cancer or infectious disease, said complex consisting
essentially of an hsp70 noncovalently bound to an antigenic
molecule.
145. The kit of claim 138 in which the amount of the complex is in
the range of 0.1 to 9.0 micrograms.
146. The kit of claim 138 in which the amount of the complex is in
the range of 0.5 to 2.0 micrograms.
147. The kit of claim 145 which further comprises in a second
container human antigen presenting cells.
148. The kit of claim 146 which further comprises in a second
container human antigen presenting cells.
149. The kit of claim 140 in which the amount of the complex is in
the range of 5 to 49 micrograms.
150. The kit of claim 140 in which the amount of the complex is in
the range of 5 to 40 micrograms.
151. The kit of claim 149 which further comprises in a second
container human antigen presenting cells.
152. The kit of claim 150 which further comprises in a second
container human antigen presenting cells.
153. The kit of claim 142 in which the amount of the complex is in
the range of 0.1 to 9.0 micrograms.
154. The kit of claim 142 in which the amount of the complex is in
the range of 0.5 to 2.0 micrograms.
155. The kit of claim 153 which further comprises in a second
container human antigen presenting cells.
156. The kit of claim 154 which further comprises in a second
container human antigen presenting cells.
157. The method according to claim 1, 2, or 3 in which the first
complex is purified to apparent homogeneity as detected by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis.
158. A pharmaceutical composition comprising an amount of a complex
of less than 10 micrograms effective to induce an immune response
or treat or prevent cancer or infectious disease in a mammal, said
complex consisting essentially of an hsp70 noncovalently bound to
an antigenic molecule; and a pharmaceutically acceptable
carrier.
159. A pharmaceutical composition comprising an amount of a complex
of less than 50 micrograms effective to induce an immune response
or treat or prevent cancer or infectious disease in a mammal, said
complex consisting essentially of an hsp90 noncovalently bound to
an antigenic molecule; and a pharmaceutically acceptable
carrier.
160. A pharmaceutical composition comprising an amount of a complex
of less than 10 micrograms effective to induce an immune response
or treat or prevent cancer or infectious disease in a mammal, said
complex consisting essentially of an gp96 noncovalently bound to an
antigenic molecule; and a pharmaceutically acceptable carrier.
Description
1 INTRODUCTION
[0002] The present invention relates to methods and compositions
for the prevention and treatment of infectious diseases, primary
and metastatic neoplastic diseases, including, but not limited to
human sarcomas and carcinomas. In the practice of the prevention
and treatment of infectious diseases and cancer, compositions of
complexes of heat shock/stress proteins (hsps) including, but not
limited to, hsp70, hsp90, gp96 alone or in combination with each
other, noncovalently bound to antigenic molecules, are used to
augment the immune response to genotoxic and nongenotoxic factors,
tumors and infectious agents. In the practice of the invention,
hsp-antigenic molecule complexes may be administered alone or in
combination with the administration of antigen presenting cells
sensitized with an hsp-antigenic molecule complex.
2 BACKGROUND OF THE INVENTION
[0003] The era of tumor immunology began with experiments by Prehn
and Main, who showed that antigens on the methylcholanthrene
(MCA)-induced sarcomas were tumor specific in that transplantation
assays could not detect these antigens in normal tissue of the mice
(Prehn, R. T., et al., 1957, J. Natl. Cancer Inst. 18:769-778).
This notion was confirmed by further experiments demonstrating that
tumor specific resistance against MCA-induced tumors can be
elicited in the autochthonous host, that is, the mouse in which the
tumor originated (Klein, G., et al., 1960, Cancer Res.
20:1561-1572).
[0004] In subsequent studies, tumor specific antigens were also
found on tumors induced with other chemical or physical carcinogens
or on spontaneous tumors (Kripke, M. L., 1974, J. Natl. Cancer
Inst. 53:1333-1336; Vaage, J., 1968, Cancer Res. 28:2477-2483;
Carswell, E. A., et al., 1970, J. Natl. Cancer Inst. 44:1281-1288).
Since these studies used protective immunity against the growth of
transplanted tumors as the criterion for tumor specific antigens,
these antigens are also commonly referred to as "tumor specific
transplantation antigens" or "tumor specific rejection antigens."
Several factors can greatly influence the immunogenicity of the
tumor induced, including, for example, the specific type of
carcinogen involved, immunocompetence of the host and latency
period (Old, L. J., et al., 1962, Ann. N.Y. Acad. Sci. 101:80-106;
Bartlett, G. L. 1972, J. Natl. Cancer Inst. 49:493-504).
[0005] Most, if not all, carcinogens are mutagens which may cause
mutation, leading to the expression of tumor specific antigens
(Ames, B. N., 1979, Science 204:587-593; Weisburger, J. H., et al.,
1981, Science 214:401-407). Some carcinogens are immunosuppressive
(Malmgren, R. A., et al., 1952, Proc. Soc. Exp. Biol. Med. 79
:484-488). Experimental evidence suggests that there is a constant
inverse correlation between immunogenicity of a tumor and latency
period (time between exposure to carcinogen and tumor appearance)
(Old, L. J., et al., 1962, Ann. N.Y. Acad. Sci. 101:80-106; and
Bartlett, G. L., 1972, J. Natl. Cancer Inst. 49:493-504). Other
studies have revealed the existence of tumor specific antigens that
do not lead to rejection, but, nevertheless, can potentially
stimulate specific immune responses (Roitt, I., Brostoff, J and
Male, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps.
17.1-17.12).
2.1. Tumor-Specific Immunogenicities of Heat Shock/Stress Proteins
hsp70, hsp90 and gp96
[0006] Srivastava et al. demonstrated immune response to
methylcholanthrene-induced sarcomas of inbred mice (1988, Immunol.
Today 9:78-83). In these studies it was found that the molecules
responsible for the individually distinct immunogenicity of these
tumors were identified as cell-surface glycoproteins of 96 kDa
(gp96) and intracellular proteins of 84 to 86 kDa (Srivastava, P.
K., et al., 1986, Proc. Natl. Acad. Sci. USA 83:3407-3411; Ullrich,
S. J., et al., 1986, Proc. Natl. Acad. Sci. USA 83:3121-3125.
Immunization of mice with gp96 or p84/86 isolated from a particular
tumor rendered the mice immune to that particular tumor, but not to
antigenically distinct tumors. Isolation and characterization of
genes encoding gp96 and p84/86 revealed significant homology
between them, and showed that gp96 and p84/86 were, respectively,
the endoplasmic reticular and cytosolic counterparts of the same
heat shock proteins (Srivastava, P. K., et al., 1988,
Immunogenetics 28:205-207; Srivastava, P. K., et al., 1991, Curr.
Top. Microbiol. Immunol. 167:109-123). Further, hsp70 was shown to
elicit immunity to the tumor from which it was isolated but not to
antigenically distinct tumors. However, hsp70 depleted of peptides
was found to lose its immunogenic activity (Udono, M., and
Srivastava, P. K., 1993, J. Exp. Med. 178:1391-1396). These
observations suggested that the heat shock proteins are not
immunogenic per se, but are carriers of antigenic peptides that
elicit specific immunity to cancers (Srivastava, P. K., 1993, Adv.
Cancer Res. 62:153-177).
2.2. Pathobiology of Cancer
[0007] Cancer is characterized primarily by an increase in the
number of abnormal cells derived from a given normal tissue,
invasion of adjacent tissues by these abnormal cells, and lymphatic
or blood-borne spread of malignant cells to regional lymph nodes
and to distant sites (metastasis) Clinical data and molecular
biologic studies indicate that cancer is a multistep process that
begins with minor preneoplastic changes, which may under certain
conditions progress to neoplasia.
[0008] Pre-malignant abnormal cell growth is exemplified by
hyperplasia, metaplasia, or most particularly, dysplasia-(for
review of such abnormal growth conditions, see Robbins and Angell,
1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia,
pp. 68-79.) Hyperplasia is a form of controlled cell proliferation
involving an increase in cell number in a tissue or organ, without
significant alteration in structure or function. As but one
example, endometrial hyperplasia often precedes endometrial cancer.
Metaplasia is a form of controlled cell growth in which one type of
adult or fully differentiated cell substitutes for another type of
adult cell. Metaplasia can occur in epithelial or connective tissue
cells. Atypical metaplasia involves a somewhat disorderly
metaplastic epithelium. Dysplasia is frequently a forerunner of
cancer, and is found mainly in the epithelia; it is the most
disorderly form of non-neoplastic cell growth, involving a loss in
individual cell uniformity and in the architectural orientation of
cells. Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation, and
is often found in the cervix, respiratory passages, oral cavity,
and gall bladder.
[0009] The neoplastic lesion may evolve clonally and develop an
increasing capacity for invasion, growth, metastasis, and
heterogeneity, especially under conditions in which the neoplastic
cells escape the host's immune surveillance (Roitt, I., Brostoff, J
and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps.
17.1-17.12).
2.3. Immunotherapy
[0010] Four basic cell types whose function has been associated
with antitumor cell immunity and the elimination of tumor cells
from the body are: i) B-lymphocytes which secrete immunoglobulins
into the blood plasma for identifying and labeling the nonself
invader cells; ii) monocytes which secrete the complement proteins
which are responsible for lysing and processing the
immunoglobulin-coated target invader cells; iii) natural killer
lymphocytes having two mechanisms for the destruction of tumor
cells-antibody-dependent cellular cytotoxicity and natural killing;
and iv) T-lymphocytes possessing antigen-specific receptors and
each T-lymphocyte clone having the capacity to recognize a tumor
cell carrying complementary marker molecules (Schreiber, H., 1989,
in Fundamental Immunology (ed). W. E. Paul, pp. 923-955).
[0011] Several factors can influence the immunogenicity of tumors
induced. These factors include dose of carcinogen, immunocompetence
of the host, and latency period. Immunocompetence of the host
during the period of cancer induction and development can allow the
host to respond to immunogenic tumor cells. This may prevent the
outgrowth of these cells or select far less immunogenic escape
variants that have lost their respective rejection antigen.
Conversely, immunosuppression or immune deficiency of the host
during carcinogenesis or tumorigenesis may allow growth of highly
immunogenic tumors (Schreiber, H., 1989, in Fundamental Immunology
(ed). W. E. Paul, pp. 923-955).
[0012] Three major types of cancer immunotherapy are currently
being explored: i) adoptive cellular immunotherapy, ii) in vivo
manipulation of patient plasma to remove blocking factors or add
tumoricidal factors, and iii) in vivo administration of biological
response modifiers (e.g., interferons (IFN; IFN-alpha and
IFN-gamma), interleukins (IL; IL-2, IL-4 and IL-6),
colony-stimulating factors, tumor necrosis factor (TNF), monoclonal
antibodies and other immunopotentiating agents, such as
corynebacterium parvum (C. parvum) (Kopp, W. C., et al., 1994,
Cancer Chemotherapy and Biol. Response Modifiers 15:226-286). There
is little doubt that immunotherapy of cancer as it stands is
falling short of the hopes invested in it. Although numerous
immunotherapeutic approaches have been tested, few of these
procedures have proved to be effective as the sole or even as an
adjunct form of cancer prevention and treatment.
2.3.1. Adoptive Cellular Immunotherapy
[0013] Adoptive immunotherapy of cancer refers to a therapeutic
approach in which immune cells with an antitumor reactivity are
administered to a tumor-bearing host, with the aim that the cells
mediate either directly or indirectly, the regression of an
established tumor. Transfusion of lymphocytes, particularly T
lymphocytes, falls into this category and investigators at the
National Cancer Institute (NCI) have used autologous reinfusion of
peripheral blood lymphocytes or tumor-infiltrating lymphocytes
(TIL), T cell cultures from biopsies of subcutaneous lymph nodules,
to 20 treat several human cancers (Rosenberg, S. A., U.S. Pat. No.
4,690,914, issued Sep. 1, 1987; Rosenberg, S. A., et al., 1988, N.
England J. Med. 319:1676-1680). For example, TIL expanded in vitro
in the presence of interleukin (IL)-2 have been transferred to
cancer patients, resulting in tumor regression in select patients
with metastatic melanoma. Melanoma TIL grown in IL-2 have been
identified as activated T lymphocytes CD3.sup.+ HLA-DR.sup.+, which
are predominantly CD8.sup.+ cells with unique in vitro antitumor
properties. Many long-term melanoma TIL cultures lyse autologous
tumors in a specific MHC class I- and T cell antigen receptor
dependent manner (Topalian, S. L., et al., 1989, J. Immunol.
142:3714). However, studies of TIL derived from other types of
tumors have revealed only scant evidence for cytolytic or
proliferative antitumor immune specificity (Topalian, S. L. et al.,
1990, in Important Advances in Oncology, V. T. DeVita, S. A.
Hellman and S. A. Rosenberg, eds. J. B. Lippincott, Philadelphia,
pp. 19-41). In addition, the toxicity of the high-dose IL-2
+activated lymphocyte treatment advocated by the NCI group has been
considerable, including high fevers, severe rigors, hypotension,
damage to the endothelial wall due to capillary leak syndrome, and
various adverse cardiac events such as arrhythmias and myocardial
infarction (Rosenberg S. A., et al., 1988, N. England J. Med.
319:1676-1680).
2.3.2. Interleukins (IL-2, IL-4 and IL-6)
[0014] IL-2 has significant antitumor activity in a small
percentage of patients with renal cell carcinoma and melanoma.
Investigators continue to search for IL-2 based regimens that will
increase the response rates in IL-2 responsive tumors, but, for the
most part, have neither defined new indications nor settled
fundamental issues, such as whether dose intensity is important in
IL-2 therapy (Kopp, W. C., et al., 1994, Cancer Chemotherapy and
Biol. Response Modifiers 15:226-286). Numerous reports have
documented IL-2 associated toxicity involving increased nitrate
levels and the syndrome of vascular leak and hypotension, analogous
to septic shock. In addition, an increased incidence of
nonopportunistic bacterial infections and autoimmune complications
are frequently accompanied by the antitumor response of IL-2 (Kopp,
W. C., et al., 1994, Cancer Chemotherapy and Biol. Response
Modifiers 15:226-286).
[0015] IL-4 and IL-6 are also being tested as antitumor agents
either directly or through immunomodulating mechanisms.
Dose-limiting toxicities have been observed with both agents in
Phase I clinical trials (Gilleece, M. H., et al., 1992, Br. J.
Cancer 66:204-210, Weber, J., et al., 1993, J. Clin. Oncol.
11:499-506).
2.3.3. Tumor Necrosis Factor
[0016] The toxicity of systemically administered TNF seriously
limits its use for the treatment of cancer. TNF has been most
effective when used for regional therapy, in which measures, such
as limb isolation for perfusion, are taken to limit the systemic
dose and hence the toxicity of TNF. Dose-limiting toxicity of TNF
consist of thrombocytopenia, headache, confusion and hypotension
(Mittleman, A., et al., 1992, Inv. New Drugs 10:183-190)
2.3.4. Interferons
[0017] The activity of IFN-.alpha. has been described as being
modest in a number of malignancies, including renal cell carcinoma,
melanoma, hairy cell leukemia low-grade non-Hodgkin's lymphoma, and
others. Higher doses of IFN-.alpha. are usually associated with
higher response rates in some malignancies, but also cause more
toxicity. In addition, more and more reports indicate that relapses
after successful interferon therapy coincide with formation of
neutralizing antibodies against interferon (Ouesada, J. R., et al.,
1987, J. Interferon Res. 67:678.
2.4. Pharmacokinetic Models for Anticancer Chemotherapeutic and
Immunotherapeutic Drugs: Extrapolation and Scaling of Animal Data
to Humans
[0018] The ethical and fiscal constraints which require the use of
animal models for most toxicology research also impose the
acceptance of certain fundamental assumptions in order to estimate
dose potency in humans from dose-response data in animals.
Interspecies dose-response equivalence is most frequently estimated
as the product of a reference species dose and a single scaling
ratio based on a physiological parameter such as body weight, body
surface area, maximum lifespan potential, etc. Most frequently,
exposure is expressed as milligrams of dose administered in
proportion to body mass in kilograms (mg kg.sup.-1). Body mass is a
surrogate for body volume, and therefore, the ratio milligrams per
kilogram is actually concentrations in milligrams per liter
(Hirshaut, Y., et al., 1969, Cancer Res. 29:1732-1740). The key
assumptions which accompany this practice and contribute to its
failure to accurately estimate equipotent exposure among various
species are: i) that the biological systems involved are
homogeneous, "well-stirred volumes" with specific gravity equal to
1.0; ii) that the administered compounds are instantly and
homogeneously distributed throughout the total body mass; and iii)
that the response of the biological systems is directly
proportional only to the initial concentration of the test material
in the system. As actual pharmacokinetic conditions depart from
these assumptions, the utility of initial concentration scaling
between species declines.
[0019] Through pharmacokinetics, one can study the time course of a
drug and its metabolite levels in different fluids, tissues, and
excreta of the body, and the mathematical relationships required to
develop models to interpret such data. It, therefore, provides the
basic information regarding drug distribution, availability, and
the resulting toxicity in the tissues and hence, specifies the
limitation in the drug dosage for different treatment schedules and
different routes of drug administration. The ultimate goal of the
pharmacokinetic studies of anticancer drugs is thus to offer a
framework for the design of optimal therapeutic dosage regimens and
treatment schedules for individual patients.
[0020] The currently utilized guidelines for prescription have
evolved qradually without always having a complete and explicit
justification. In 1966, Freireich and co-workers proposed the use
of surface area proportions for interspecies extrapolation of the
acute toxicity of anticancer drugs. This procedure has become the
method of choice for many risk assessment applications (Freireich,
E. J., et al., 1966, Cancer Chemotherapy Rep. 50:219-244). For
example, surface area scaling is the basis of the National Cancer
Institute's interspecies extrapolation procedure for anti-cancer
drugs (Schein, P. S., et al., 1970, Clin. Pharmacol. Therap.
11:3-40; Goldsmith, M. A., et al., 1975, Cancer Res. 35:1354-1364).
In accepting surface area extrapolation, the tenuous basis for
initial concentration scaling has been replaced by an empirical
approach. The basic formula used for estimating prescription of
cancer chemotherapy per body surface area (BSA) is
BSA=k.times.kg.sup.2/3, in which k is a constant that differs for
each age group and species. For example, the k value for adult
humans is 11, while for mice it is 9 (See Quiring, P., 1955,
Surface area determination, in Glasser E. (ed.) Medical Physics I
Chicago: Medical Year Book, p. 1490 and Vriesendorp, H. M., 1985,
Hematol. (Supplm. 16) 13:57-63). The major attraction of expressing
cancer chemotherapy per m.sup.2 BSA appears to be that it offers an
easily remembered simplification, i.e., equal doses of drug per
m.sup.2 BSA will produce approximately the same effect in comparing
different species and age groups. However, simplicity is not proof
and alternative methods for estimating prescription of anticancer
drugs appear to have a better scientific foundation, with the added
potential for a more effective use of anticancer agents (Hill, J.
A., et al., 1989, Health Physics 57:395-401).
[0021] The effectiveness of an optimal dose of a drug used in
chemotherapy and/or immunotherapy can be altered by various
factors, including tumor growth kinetics, drug resistance of tumor
cells, total-body tumor cell burden, toxic effects of chemotherapy
and/or immunotherapy on cells and tissues other than the tumor, and
distribution of chemotherapeutic agents and/or immunotherapeutic
agents within the tissues of the patient. The greater the size of
the primary tumor, the greater the probability that a large number
of cells (drug resistant and drug sensitive) have metastasized
before diagnosis and that the patient will relapse after the
primary.
[0022] Some metastases arise in certain sites in the body where
resistance to chemotherapy is based on the limited tissue
distribution of chemotherapeutic drugs administered in standard
doses. Such sites act as sanctuaries that shield the cancer cells
from drugs that are circulating in the blood; for example, there
are barriers in the brain and testes that impede drug diffusion
from the capillaries into the tissue. Thus, these sites may require
special forms of treatment such as immunotherapy, especially since
immunosuppression is characteristic of several types of neoplastic
diseases.
3 SUMMARY OF THE INVENTION
[0023] The methods of the invention comprise methods of eliciting
an immune response in an individual in whom the treatment or
prevention of cancer or infectious disease is desired by
administering, preferably intradermally or mucosally, a composition
comprising an effective amount of a complex in which the complex
consists essentially of heat shock protein(s) (hsp(s))
noncovalently bound to antigenic molecule(s). The amounts of the
complex that are administered are within ranges of effective
dosages, discovered by the present inventor to be effective, and
which are surprisingly smaller than those amounts predicted to be
effective by extrapolation by prior art methods from dosages used
in animal studies. In a preferred embodiment, the complex is
autologous to the individual; that is, the complex is isolated from
the cancer cells of the individual himself (e.g., preferably
prepared from tumor biopsies of the patient). Alternatively, the
hsp and or the antigenic molecule can be isolated from the
individual or from others or by recombinant production methods
using a cloned hsp originally derived from the individual or from
others. "Antigenic molecule" as used herein refers to the peptides
with which the hsps are endogenously associated in vivo (e.g., in
precancerous or cancerous tissue), as well as exogenous
antigens/immunogens (i.e., with which the hsps are not complexed in
vivo) or antigenic/immunogenic fragments and derivatives thereof.
Such exogenous antigens and fragments and derivatives (both peptide
and non-peptide) thereof for use in complexing with hsps, can be
selected from among those known in the art, as well as those
readily identified by standard immunoassays known in the art by
detecting the ability to bind antibody or MHC molecules
(antigenicity) or generate immune response (immunogenicity).
[0024] In the practice of the invention, therapy by administration
of hsp-peptide complexes using any convenient route of
administration may optionally be in combination with adoptive
immunotherapy involving the administration of antigen-presenting
cells that have been sensitized in vitro with complexes of hsp(s)
noncovalently bound to antigenic molecules. The methods for
adoptive immunotherapy of cancer and infectious diseases have the
goal of enhancing the host's immunocompetence and activity of
immune effector cells. Adoptive immunotherapy with macrophages
and/or other antigen-presenting cells (APC), for example, dendritic
cells and B cells (B lymphocytes), that have been sensitized in
vitro with noncovalent complexes of an hsp noncovalently bound to
an antigenic molecule, induces specific immunity to tumor cells
and/or antigenic components, promoting regression of the tumor mass
or treatment of immunological disorders or infectious diseases, as
the case may be.
[0025] In a specific embodiment, the present invention relates to
methods and compositions for prevention and treatment of primary
and metastatic neoplastic diseases.
[0026] Specific therapeutic regimens, pharmaceutical compositions,
and kits are provided by the invention. In contrast to the prior
art, the dosages of the hsp-antigenic molecule complex are not
based on, and are smaller than those dosages based on, body weight
or surface area of the patient. The present inventor has discovered
that a dosage substantially equivalent to or smaller than that seen
to be effective in smaller non-human mammals (e.g., mice) is
effective for human intradermal administration, optionally subject
to a correction factor not exceeding a fifty fold increase, based
on the relative lymph node sizes in such mammals and in humans. The
present inventor has discovered that effective intradermal dosages
are about tenfold smaller even than the surprisingly small doses
effective in subcutaneous administration in humans. (See U.S.
patent application Ser. No. 08/527,391, filed Sep. 13, 1995, which
is incorporated by reference herein in its entirety.)
Pharmaceutical formulations are provided, based on these
newly-discovered effective dose ranges for humans, comprising
compositions of complexes of antigenic molecules and heat
shock/stress proteins, including but not limited to hsp70, hsp90,
gp96 either alone or in combination. Specifically, interspecies
dose-response equivalence for hsp noncovalently bound to antigenic
molecules for a human intradermal or mucosal dose is estimated as
the product of the therapeutic dosage observed in mice and a single
scaling ratio, not exceeding a fifty fold increase.
[0027] The present invention encompasses methods for prevention and
treatment of cancer by enhancing the host's immune competence and
activity of immune effector cells. Furthermore, the invention
provides methods for evaluating the efficacy of drugs in enhancing
immune responses for treatment and monitoring the progress of
patients participating in clinical trials for the treatment of
primary and metastatic neoplastic diseases.
[0028] Immunotherapy using the therapeutic regimens of the
invention, by administering such complexes of heat shock/stress
proteins noncovalently bound to antigenic molecules, can induce
specific immunity to tumor cells, and leads to regression of the
tumor mass. Cancers which are responsive to specific immunotherapy
by administering the heat shock/stress proteins of the invention
include but are not limited to human sarcomas and carcinomas. In a
specific embodiment, the hsp-antigenic molecule complexes are
allogeneic to the patient; in a preferred embodiment, the
hsp-antigenic molecule complexes are autologous to (derived from)
the patient to whom they are administered.
[0029] Particular compositions of the invention and their
properties are described in the sections and subsections which
follow. A preferred composition comprises hsp-peptide complexes
isolated from the tumor biopsy of the patient to whom the
composition is to be administered. Such a composition that
comprises hsp70, hsp90 and/or gp96 demonstrates strong inhibition
of a variety of tumors in mammals. Moreover, the therapeutic doses
that are effective in the corresponding experimental model in
rodents as described infra, in Section 6 can be used to inhibit the
in vivo growth of colon and liver cancers in human cancer patients
as described in Sections 7 and 8, infra. Preferred compositions
comprising hsp70, hsp90 and/or gp96 which preferably exhibit no
toxicity when administered to human subjects are also
described.
[0030] In another embodiment, the methods further optionally
comprise administering biological response modifiers, e.g.,
IFN-.alpha., IFN-.gamma., IL-2, IL-4, IL-6, TNF, or other cytokine
growth factors affecting the immune cells, in combination with the
hsp complexes.
[0031] In addition to cancer therapy, the complexes of hsps
noncovalently bound to antigenic molecules can be utilized for the
prevention of a variety of cancers, e.g., in individuals who are
predisposed as a result of familial history or in individuals with
an enhanced risk to cancer due to environmental factors.
[0032] The Examples presented in Sections 6, 7 and 8 below, detail
the use according to the methods of the invention of hsp-peptide
complexes in cancer immunotherapy in experimental tumor models and
in human patients suffering from advanced colon and liver
cancer.
4 BRIEF DESCRIPTION OF FIGURES
[0033] FIGS 1A-C. Effect of intradermal administration of gp96 on
retardation of tumor growth measured as average tumor diameter
(mm).
[0034] FIG. 1A: Mice were injected intradermally in different sites
with buffer solution, twice at weekly intervals. One week after the
second injection, the mice were challenged with 1.times.10.sup.5
Meth A sarcoma cells.
[0035] FIG. 1B: Mice were injected intradermally in different sites
with 1 microgram of gp96-antigenic molecule complex derived from
Meth A sarcoma cells, twice at weekly intervals. One week after the
second injection, the mice were challenged with 1.times.10.sup.5
Meth A sarcoma cells.
[0036] FIG. 1C: Mice were injected intradermally in different sites
with 5 micrograms of gp96-antigenic molecule complex derived from
Meth A sarcoma cells, twice at weekly intervals. One week after the
second injection, the mice were challenged with 1.times.10.sup.5
Meth A sarcoma cells.
5 DETAILED DESCRIPTION OF THE INVENTION
[0037] Methods and compositions for the prevention and treatment of
primary and metastatic neoplastic diseases and infectious diseases
and for eliciting an immune response in a human individual, are
described. The invention is based, in part, on a newly discovered
dosage regimen for administration of compositions comprising
complexes of hsps noncovalently bound to antigenic molecules. The
present inventor has discovered that a dosage substantially
equivalent to or smaller than that seen to be effective in smaller
non-human animals (e.g., mice) is effective for human intradermal
administration, such as described in Section 5.1, below.
[0038] "Antigenic molecule" as used herein refers to the peptides
with which the hsps are endogenously associated in vivo (e.g., in
infected cells or precancerous or cancerous tissue) as well as
exogenous antigens/immunogens (i.e., with which the hsps are not
complexed in vivo) or antigenic/immunogenic fragments and
derivatives thereof.
[0039] The methods of the invention comprise methods of eliciting
an immune response in an individual in whom the treatment or
prevention of infectious diseases or cancer is desired by
administering, preferably intradermally or mucosally, a composition
comprising an effective amount of a complex, in which the complex
consists essentially of an hsp noncovalently bound to an antigenic
molecule.
[0040] In the practice of the invention, therapy by administration
of hsp-antigenic molecule complexes using any convenient mode of
administration may optionally be in combination with adoptive
immunotherapy. The APC can be selected from among those antigen
presenting cells known in the art, including but not limited to
macrophages, dendritic cells, B lymphocytes, and a combination
thereof, and are preferably macrophages. The hsp-antigenic
molecule-sensitized APC may be administered concurrently or before
or after administration of the hsp-antigenic molecule complexes.
The hsp-antigenic molecule complex that is administered to the
patient can be the same or different from the hsp-antigenic
molecule complex used to sensitize the APC that are administered to
the patient. In a specific embodiment wherein the APC and
hsp-antigenic molecule complexes are administered concurrently, the
APC and hsp-antigenic molecule complexes can be present in a single
composition or different composition for administration. Adoptive
immunotherapy according to the invention allows activation of
immune antigen presenting cells by incubation with hsp-antigenic
molecule complexes. Preferably, prior to use of-the cells in vivo
measurement of reactivity against the tumor or infectious agent in
vitro is done. This in vitro boost followed by clonal selection
and/or expansion, and patient administration constitutes a useful
therapeutic/prophylactic strategy.
[0041] In a preferred embodiment, the hsp-antigenic molecule
complex is autologous to the individual; that is, the complex is
isolated from either the infected cells or the cancer cells or
precancerous cells of the individual himself (e.g., preferably
prepared from infected tissues or tumor biopsies of the patient).
Alternatively, the complex is produced in vitro (e.g., wherein a
complex with an exogenous antigenic molecule is desired).
Alternatively, the hsp and/or the antigenic molecule can be
isolated from the individual or from others or made by recombinant
production methods using a cloned hsp originally derived from the
individual or from others. Exogenous antigens and fragments and
derivatives (both peptide and non-peptide) thereof for use in
complexing with hsps, can be selected from among those known in the
art, as well as those readily identified by standard immunoassays
known in the art by the ability to bind antibody or MHC molecules
(antigenicity) or generate immune response (immunogenicity).
Complexes of hsps and antigenic molecules can be isolated from
cancer or precancerous tissue of a patient, or from a cancer cell
line, or can be produced in vitro (as is necessary in the
embodiment in which an exogenous antigen is used as the antigenic
molecule).
[0042] The hsps of the present invention that can be used include
but are not limited to, hsp70, hsp90, gp96 alone or in combination.
Preferably, the hsps are human hsps.
[0043] Heat shock proteins, which are also referred to
interchangeably herein as stress proteins, useful in the practice
of the instant invention can be selected from among any cellular
protein that satisfies any one of the following criteria. It is a
protein whose intracellular concentration increases when a cell is
exposed to a stressful stimuli, it is capable of binding other
proteins or peptides, it is capable of releasing the bound proteins
or peptides in the presence of adenosine triphosphate (ATP) or low
pH, or it is a protein showing at least 35% homology with any
cellular protein having any of the above properties.
[0044] The first stress proteins to be identified were the heat
shock proteins (hsps). As their name implies, hsps are synthesized
by a cell in response to heat shock. To date, three major families
of hsp have been identified based on molecular weight. The families
have been called hsp60, hsp70 and hsp90 where the numbers reflect
the approximate molecular weight of the stress proteins in
kilodaltons. Many members of these families were found subsequently
to be induced in response to other stressful stimuli including, but
not limited to, nutrient deprivation, metabolic disruption, oxygen
radicals, and infection with intracellular pathogens. (See Welch,
May 1993, Scientific American 56-64; Young, 1990, Annu. Rev.
Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903; Gething, et
al., 1992, Nature 355:33-45; and Lindquist, et al., 1988, Annu.
Rev. Genetics 22:631-677), the disclosures of which are
incorporated herein by reference. It is contemplated that
hsps/stress proteins belonging to all of these three families can
be used in the practice of the instant invention.
[0045] The major hsps can accumulate to very high levels in
stressed cells, but they occur at low to moderate levels in cells
that have not been stressed. For example, the highly inducible
mammalian hsp70 is hardly detectable at normal temperatures but
becomes one of the most actively synthesized proteins in the cell
upon heat shock (Welch, et al., 1985, J. Cell. Biol.
101:1198-1211). In contrast, hsp90 and hsp60 proteins are abundant
at normal temperatures in most, but not all, mammalian cells and
are further induced by heat (Lai, et al., 1984, Mol. Cell. Biol.
4:2802-10; van Bergen en Henegouwen, et al., 1987, Genes Dev.
1:525-31).
[0046] Heat shock proteins are among the most highly conserved
proteins in existence. For example, DnaK, the hsp70 from E. coli
has about 50% amino acid sequence identity with hsp70 proteins from
excoriates (Bardwell, et al., 1984, Proc. Natl. Acad. Sci.
81:848-852). The hsp60 and hsp90 families also show similarly high
levels of intrafamilies conservation (Hickey, et al., 1989, Mol.
Cell. Biol. 9:2615-2626; Jindal, 1989, Mol. Cell. Biol.
9:2279-2283). In addition, it has been discovered that the hsp60,
hsp70 and hsp90 families are composed of proteins that are related
to the stress proteins in sequence, for example, having greater
than 35% amino acid identity, but whose expression levels are not
altered by stress. Therefore it is contemplated that the definition
of heat shock protein or stress protein, as used herein, embraces
other proteins, muteins, analogs, and variants thereof having at
least 35% to 55%, preferably 55% to 75%, and most preferably 75% to
85% amino acid identity with members of the three families whose
expression levels in a cell are enhanced in response to a stressful
stimulus. The purification of stress proteins belonging to these
three families is described below.
[0047] The immunogenic hsp-peptide complexes of the invention may
include any complex containing an hsp and a peptide --that is
capable of inducing an immune response in a mammal. The peptides
are preferably noncovalently associated with the hsp. Preferred
complexes may include, but are not limited to, hsp60-peptide,
hsp70-peptide and hsp90-peptide complexes. For example, an hsp
called gp96 which is present in the endoplasmic reticulum of
eukaryotic cells and is related to the cytoplasmic hsp90's can be
used to generate an effective vaccine containing a gp96-peptide
complex.
[0048] Although the hsps can be allogeneic to the patient, in a
preferred embodiment, the hsps are autologous to (derived from) the
patient to whom they are administered. The hsps and/or antigenic
molecules can be purified from natural sources, chemically
synthesized, or recombinantly produced.
[0049] The invention provides combinations of compositions which
enhance the immunocompetence of the host individual and elicit
specific immunity against infectious agents or specific immunity
against preneoplastic and neoplastic cells. The therapeutic
regimens and pharmaceutical compositions of the invention are
described below. These compositions have the capacity to prevent
the onset and progression of infectious diseases and prevent the
development of tumor cells and to inhibit the growth and
progression of tumor cells indicating that such compositions can
induce specific immunity in infectious diseases and cancer
immunotherapy.
[0050] Hsps appear to induce an inflammatory reaction at the tumor
site and ultimately cause a regression of the tumor burden in the
cancer patients treated. Cancers which can be treated with
complexes of hsps noncovalently bound to antigenic molecules
include, but are not limited to, human sarcomas and carcinomas.
Human sarcomas and carcinomas are also responsive to adoptive
immunotherapy by the hsp complex-sensitized macrophages and/or
APC.
[0051] Accordingly, the invention provides methods of preventing
and treating cancer in an individual comprising administering
hsp-antigenic molecule complexes, optionally in combination with
APC sensitized by such complexes, which stimulates the
immunocompetence of the host individual and elicits specific
immunity against the preneoplastic and/or neoplastic cells. As used
herein, "preneoplastic" cell refers to a cell which is in
transition from a normal to a neoplastic form; and morphological
evidence, increasingly supported by molecular biologic studies,
indicates that preneoplasia progresses through multiple steps.
Non-neoplastic cell growth commonly consists of hyperplasia,
metaplasia, or most particularly, dysplasia (for review of such
abnormal growth conditions (See Robbins and Angell, 1976, Basic
Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79).
Hyperplasia is a form of controlled cell proliferation involving an
increase in cell number in a tissue or organ, without significant
alteration in structure or function. As but one example,
endometrial hyperplasia often precedes endometrial cancer.
Metaplasia is a form of controlled cell growth in which one type of
adult or fully differentiated cell substitutes for another type of
adult cell. Metaplasia can occur in epithelial or connective tissue
cells. Atypical metaplasia involves a somewhat disorderly
metaplastic epithelium. Dysplasia is frequently a forerunner of
cancer, and is found mainly in the epithelia; it is the most
disorderly form of non-neoplastic cell growth, involving a loss in
individual cell uniformity and in the architectural orientation of
cells. Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation, and
is often found in the cervix, respiratory passages, oral cavity,
and gall bladder. Although preneoplastic lesions may progress to
neoplasia, they may also remain stable for long periods and may
even regress, particularly if the inciting agent is removed or if
the lesion succumbs to an immunological attack by its host.
[0052] The therapeutic regimens and pharmaceutical compositions of
the invention may be used with additional immune response enhancers
or biological response modifiers including, but not limited to, the
cytokines IFN-.alpha., IFN-.gamma., IL-2, IL-4, IL-6, TNF, or other
cytokine affecting immune cells. In accordance with this aspect of
the invention, the complexes of the hsp and antigenic molecule are
administered in combination therapy with one or more of these
cytokines.
[0053] The invention further relates to administration of complexes
of hsp-antigenic molecules, optionally in combination with APC
sensitized by such complexes, to individuals at enhanced risk of
cancer due-to familial history or environmental risk factors.
5.1. Dosage Regimens
[0054] It was established in experimental tumor models (Blachere et
al., 1993, J. Immunotherapy 14:352-356) that the lowest dose of hsp
noncovalently bound to peptide complexes which produced tumor
regression in mice was between 10 and 25 microgram/mouse weighing
20-25g which is equal to 25 .mu.g/25 g =1 mg/kg. Prior art methods
extrapolate to human dosages based on body weight and surface area.
For example, prior art methods of extrapolating human dosage based
on body weight can be carried out as follows: since the conversion
factor for converting the mouse dosage to human dosage is Dose
Human per kg=Dose Mouse per kg.times.12 (See Freireich, E. J., et
al., 1966, Cancer Chemotherap. Rep. 50:219-244), the effective dose
of hsp-peptide complexes in humans weighing 70 kg should be 1
mg/kg.div.12.times.70, i.e., about 6 mg (5.8 mg).
[0055] Drug doses are also given in milligrams per square meter of
body surface area because this method rather than body weight
achieves a good correlation to certain metabolic and excretionary
functions (Shirkey, H. C., 1965, JAMA 193:443). Moreover, body
surface area can be used as a common denominator for drug dosage in
adults and children as well as in different animal species as
indicated below in Table 1 (Freireich, E. J., et al., 1966, Cancer
Chemotherap. Rep. 50:219-244).
1TABLE 1 REPRESENTATIVE SURFACE AREA TO WEIGHT RATIOS (km) FOR
VARIOUS SPECIES.sup.1 Body Weight Surface Area Species (kg) (Sq m)
km Factor Mouse 0.02 0.0066 3.0 Rat 0.15 0.025 5.9 Monkey 3.0 0.24
12 Dog 8.0 0.40 20 Human, Child 20 0.80 25 Adult 60 1.6 37 Example:
To express a mg/kg dose in any given species as the equivalent
mg/sq m dose, multiply the dose by the appropriate km factor. In an
adult human, 100 mg/kg is equivalent to 100 mg/kg .times. 37 kg/sq
m = 3700 mg/sq m. .sup.1Freireich, et al., 1966, Cancer
Chemotherap. Rep. 50: 219-244.
[0056] In contrast to both of the above-described prior art methods
of determining dosage levels, the present invention provides
dosages of the purified complexes of hsps and antigenic molecules
that are much smaller than the dosages estimated by prior art
methods. For example, according to a preferred embodiment of the
invention, an amount of hsp70- and/or gp96-antigenic molecule
complexes is administered that is in the range of about 0.1
micrograms to about 60 micrograms for a human patient. In another
specific embodiment, the therapeutically effective amount of hsp70-
and/or gp96-antigenic molecule complexes is less than 10
micrograms, e.g., in the range of 0.1 to 9 micrograms; the
preferred human dosage being substantially equivalent to or smaller
than the dosage used in a 25 g mouse, e.g., in the range of 0.5 to
2.0 micrograms. The preferred dosage for hsp90-antigenic molecule
complexes in a human patient provided by the present invention is
in the-range of about 5 to 500 micrograms. In a specific
embodiment, the therapeutically effective amount of hsp90-antigenic
molecule complexes is less than 50 micrograms, e.g., in the range
of 5 to 49 micrograms; the preferred dosage being in the range of 5
to 40 micrograms.
[0057] The doses recited above are preferably administered
intradermally or mucosally. By way of example, the doses can be
administered, preferably intradermally, every other day for a total
of 5 injections. In a preferred embodiment, the doses recited above
are given once weekly for a period of about 4 to 6 weeks, and the
mode of site of administration is preferably varied with each
administration. In a preferred example, intradermal administrations
are given, with each site of administration varied sequentially.
Thus, by way of example and not limitation, the first injection may
be given intradermally on the left arm, the second on the right
arm, the third on the left belly, the fourth on the right belly,
the fifth on the left thigh, the sixth on the right thigh, etc. The
same site may be repeated after a gap of one or more injections.
Also, split injections may be given. Thus, for example, half the
dose may be given in one site and the other half in another site on
the same day.
[0058] After 4-6 weeks, further injections are preferably given at
two-week intervals over a period of time of one month. Later
injections may be given monthly. The pace of later injections may
be modified, depending upon the patient's clinical progress and
responsiveness to the immunotherapy. Alternatively, the mode of
administration is sequentially varied, e.g., weekly administrations
are given in sequence intradermally or mucosally.
[0059] In an embodiment wherein adoptive immunotherapy is also
employed, the above regimens for administration of hsp-antigenic
molecule complexes may occur before, during or after administration
of the hsp-antigen molecule-sensitized APC. For example, the mode
of therapy is sequentially varied, e.g., hsp-antigenic molecule
complexes may be administered at one time and hsp-antigenic
molecule-sensitized APC another time. Preferably the hsp-antigenic
molecule-sensitized APC and the hsp-antigenic molecule complexes
are administered to the patient within 1 week of each other.
[0060] The invention is illustrated by non-limiting examples in
Sections 6, 7 and 8.
5.2. Therapeutic Compositions Comprising Purified Hsp-Peptide
Complexes, for Eliciting Immune Responses to Cancer or Infectious
Disease, and for In Vitro Sensitization of APC
[0061] The compositions comprising hsp noncovalently bound to
antigenic molecules are administered to elicit an effective
specific immune response to the complexed antigenic molecules (and
not to the hsp). In accordance with the methods described herein,
the hsp-antigenic molecule complexes are preferably purified in the
range of 60 to 100 percent of the total mg protein, or at least
70%, 80% or 90% of the total mg protein. In another embodiment, the
hsp-antigenic molecule complexes are purified to apparent
homogeneity; as assayed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis.
[0062] In a preferred embodiment, non-covalent complexes of hsp70,
hsp90 and gp96 with peptides are prepared and purified
postoperatively from tumor cells obtained from the cancer
patient.
[0063] In accordance with the methods described herein, immunogenic
or antigenic peptides that are endogenously complexed to hsps or
MHC antigens can be used as antigenic molecules. For example, such
peptides may be prepared that stimulate cytotoxic T cell responses
against different tumor antigens (e.g., tyrosinase, gp100, melan-A,
gp75, mucins, etc.) and viral proteins including, but not limited
to, proteins of immunodeficiency virus type I (HIV-I), human
immunodeficiency virus type II (HIV-II), hepatitis type A,
hepatitis type B, hepatitis type C, influenza, Varicella,
adenovirus, herpes simplex type I (HSV-I), herpes simplex type II
(HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory
syncytial virus, papilloma virus, papova virus, cytomegalovirus,
echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus,
measles virus, rubella virus and polio virus. In the embodiment
wherein the antigenic molecules are peptides noncovalently
complexed to hsps in vivo, the complexes can be isolated from
cells, or alternatively, produced in vitro from purified
preparations each of hsps and antigenic molecules.
[0064] In another specific embodiment, antigens of cancers (e.g.,
tumors) or infectious agents (e.g., viral antigen, bacterial
antigens, etc.) can be obtained by purification from natural
sources, by chemical synthesis, or recombinantly, and, through in
vitro procedures such as that described below, noncovalently
complexed to hsps.
[0065] In an embodiment wherein the hsp-antigenic molecule complex
to be used is a complex that is produced in vivo in cells,
exemplary purification procedures such as described in Sections
5.2.1-5.2.3 below can be employed. Alternatively, in an embodiment
wherein one wishes to use antigenic molecules by complexing to hsps
in vitro hsps can be purified for such use from the endogenous
hsp-peptide complexes in the presence of ATP or low pH (or
chemically synthesized or recombinantly produced). The protocols
described herein may be used to isolate hsp-peptide complexes, or
the hsps alone, from any eukaryotic cells for example, tissues,
isolated cells, or immortalized eukaryote cell lines infected with
a preselected intracellular pathogen, tumor cells or tumor cell
lines.
5.2.1. Preparation and Purification of Hsp70-peptide Complexes
[0066] The purification of hsp70-peptide complexes has been
described previously, see, for example, Udono et al., 1993, J. Exp.
Med. 178:1391-1396. A procedure that may be used, presented by way
of example but not limitation, is as follows:
[0067] Initially, tumor cells are suspended in 3 volumes of
1.times.Lysis buffer consisting of 5 mM sodium phosphate buffer, pH
7, 150 mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2 and 1 mM phenyl
methyl sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on
ice, until >99% cells are lysed as determined by microscopic
examination. As an alternative to sonication, the cells may be
lysed by mechanical shearing and in this approach the cells
typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mM
PMSF, incubated on ice for 20 minutes and then homogenized in a
Dounce homogenizer until >95% cells are lysed.
[0068] Then the lysate is centrifuged at 1,000 g for 10 minutes to
remove unbroken cells, nuclei and other cellular debris. The
resulting supernatant is recentrifuged at 100,000 g for 90 minutes,
the supernatant harvested and then mixed with Con A Sepharose
equilibrated with phosphate buffered saline (PBS) containing 2 mM
Ca.sup.2+ and 2 mM Mg.sup.2+. When the cells are lysed by
mechanical shearing the supernatant is diluted with an equal volume
of 2.times.lysis buffer prior to mixing with Con A Sepharose. The
supernatant is then allowed to bind to the Con A Sepharose for 2-3
hours at 4.degree. C. The material that fails to bind is harvested
and dialyzed for 36 hours (three times, 100 volumes each time)
against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM
PMSF. Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34
rotor) for 20 minutes. Then the resulting supernatant is harvested
and applied to a Mono Q FPLC column equilibrated in 20 mM
Tris-Acetate pH 7.5, 20 mM NaCl, 0.1 mM EDTA and 15 mM
2-mercaptoethanol. The column is then developed with a 20 mM to 500
mM NaCl gradient and then eluted fractions fractionated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
characterized by immunoblotting using an appropriate anti-hsp70
antibody (such as from clone N27F3-4, from StressGen).
[0069] Fractions strongly immunoreactive with the anti-hsp70
antibody are pooled and the hsp70-peptide complexes precipitated
with ammonium sulfate; specifically with a 50%-70% ammonium sulfate
cut. The resulting precipitate is then harvested by centrifugation
at 17,000 rpm (SS34 Sorvall rotor) and washed with 70% ammonium
sulfate. The washed precipitate is then solubilized and any
residual ammonium sulfate removed by gel filtration on a
Sephadex.RTM. G25 column (Pharmacia). If necessary the hsp70
preparation thus obtained can be repurified through the Mono Q FPLC
Column as described above.
[0070] The hsp70-peptide complex can be purified to apparent
homogeneity using this method. Typically 1 mg of hsp70-peptide
complex can be purified from 1 g of cells/tissue.
[0071] An improved method for purification of hsp70-peptide
complexes comprises contacting cellular proteins with ADP or a
nonhydrolyzable analog of ATP affixed to a solid substrate, such
that hsp70 in the lysate can bind to the ADP or nonhydrolyzable ATP
analog, and eluting the bound hsp70. A preferred method uses column
chromatography with ADP affixed to a solid substratum (e.g.,
ADP-agarose). The resulting hsp70 preparations are higher in purity
and devoid of contaminating peptides. The hsp70 yields are also
increased significantly by about more than 10 fold. Alternatively,
chromatography with nonhydrolyzable analogs of ATP, instead of ADP,
can be used for purification of hsp70-peptide complexes. By way of
example but not limitation, purification of hsp70-peptide complexes
by ADP-agarose chromatography can be carried out as follows:
[0072] Meth A sarcoma cells (500 million cells) are homogenized in
hypotonic buffer and the lysate is centrifuged at 100,000 g for 90
minutes at 4.degree. C. The supernatant is applied to an
ADP-agarose column. The column is washed in buffer and is eluted
with 5 column volumes of 3 mM ADP The hsp70-peptide complexes elute
in fractions 2 through 10 of the total 15 fractions which elute.
The eluted fractions are analyzed by SDS-PAGE. The hsp70-peptide
complexes can be purified to apparent homogeneity using this
procedure.
5.2.2. Preparation and Purification of Hsp90-peptide Complexes
[0073] A procedure that can be used, presented by way of example
and not limitation, is as follows:
[0074] Initially, tumor cells are suspended in 3 volumes of
1.times.Lysis buffer consisting of 5 mM sodium phosphate buffer
(pH7), 150 mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2 and 1 mM
phenyl methyl sulfonyl fluoride (PMSF). Then, the pellet is
sonicated, on ice, until >99% cells are lysed as determined by
microscopic examination. As an alternative to sonication, the cells
may be lysed by mechanical shearing and in this approach the cells
typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mM
PMSF, incubated on ice for 20 minutes and then homogenized in a
Dounce homogenizer until >95% cells are lysed.
[0075] Then the lysate is centrifuged at 1,000 g for 10 minutes to
remove unbroken cells, nuclei and other cellular debris. The
resulting supernatant is recentrifuged at 100,000 g for 90 minutes,
the supernatant harvested and then mixed with Con A Sepharose
equilibrated with PBS containing 2 mM Ca.sup.2+ and 2 mM Mg.sup.2+.
When the cells are lysed by mechanical shearing the supernatant is
diluted with an equal volume of 2.times.Lysis buffer prior to
mixing with Con A Sepharose. The supernatant is then allowed to
bind to the Con A Sepharose for 2-3 hours at 4.degree. C. The
material that fails to bind is harvested and dialyzed for 36 hours
(three times, 100 volumes each time) against 10 mM Tris-Acetate pH
7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF. Then the dialyzate is
centrifuged at 17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then
the resulting supernatant is harvested and applied to a Mono Q FPLC
column equilibrated with lysis buffer. The proteins are then eluted
with a salt gradient of 200 mM to 600 mM NaCl.
[0076] The eluted fractions are fractionated by SDS-PAGE and
fractions containing the hsp90-peptide complexes identified by
immunoblotting using an anti-hsp90 antibody such as 3G3 (Affinity
Bioreagents). Hsp90-peptide complexes can be purified to apparent
homogeneity using this procedure. Typically, 150-200 .mu.g of
hsp90-peptide complex can be purified from 1 g of cells/tissue.
5.2.3. Preparation and Purification of gp96-peptide Complexes
[0077] A procedure that can be used, presented by way of example
and not limitation, is as follows:
[0078] A pellet of tumors is resuspended in 3 volumes of buffer
consisting of 30 mM sodium bicarbonate buffer (pH 7.5) and 1 mM
PMSF and the cells allowed to swell on ice 20 minutes. The cell
pellet is then homogenized in a Dounce homogenizer (the appropriate
clearance of the homogenizer will vary according to each cell type)
on ice until >95% cells are lysed.
[0079] The lysate is centrifuged at 1,000 g for 10 minutes to
remove unbroken cells, nuclei and other debris. The supernatant
from this centrifugation step is then recentrifuged at 100,000 g
for 90 minutes. The gp96-peptide complex can be purified either
from the 100,000 pellet or from the supernatant.
[0080] When purified from the supernatant, the supernatant is
diluted with equal volume of 2.times.lysis buffer and the
supernatant mixed for 2-3 hours at 4.degree. C. with Con A
Sepharose equilibrated with PBS containing 2 mM Ca.sup.2+ and 2 mM
Mg.sup.2+. Then, the slurry is packed into a column and washed with
1.times.lysis buffer until the OD.sub.280 drops to baseline. Then,
the column is washed with 1/3 column bed volume of 10%
.alpha.-methyl mannoside (.alpha.-MM) dissolved in PBS containing 2
mM Ca.sup.2+ and 2 mM Mg.sup.2+, the column sealed with a piece of
parafilm, and incubated at 37.degree. C. for 15 minutes. Then the
column is cooled to room temperature and the parafilm removed from
the bottom of the column. Five column volumes of the .alpha.-MM
buffer are applied to the column and the eluate analyzed by
SDS-PAGE. Typically the resulting material is about 60-95% pure,
however this depends upon the cell type and the tissue-to-lysis
buffer ratio used. Then the sample is applied to a Mono Q FPLC
column (Pharmacia) equilibrated with a buffer containing 5 mM
sodium phosphate, pH 7. The proteins are then eluted from the
column with a 0-1M NaCl gradient and the gp96 fraction elutes
between 400 mM and 550 mM NaCl.
[0081] The procedure, however, may be modified by two additional
steps, used either alone or in combination, to consistently produce
apparently homogeneous gp96-peptide complexes. One optional step
involves an ammonium sulfate precipitation prior to the Con A
purification step and the other optional step involves
DEAE-Sepharose purification after the Con A purification step but
before the Mono Q FPLC step.
[0082] In the first optional step, described by way of example as
follows, the supernatant resulting from the 100,000 g
centrifugation step is brought to a final concentration of 50%
ammonium sulfate by the addition of ammonium sulfate. The ammonium
sulfate is added slowly while gently stirring the solution in a
beaker placed in a tray of ice water. The solution is stirred from
about 1/2 to 12 hours at 4.degree. C. and the resulting solution
centrifuged at 6,000 rpm (Sorvall SS34 rotor). The supernatant
resulting from this step is removed, brought to 70% ammonium
sulfate saturation by the addition of ammonium sulfate solution,
and centrifuged at 6,000 rpm (Sorvall SS34 rotor). The resulting
pellet from this step is harvested and suspended in PBS containing
70% ammonium sulfate in order to rinse the pellet. This mixture is
centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the pellet
dissolved in PBS containing 2 mM Ca.sup.2+ and Mg.sup.2+.
Undissolved material is removed by a brief centrifugation at 15,000
rpm (Sorvall SS34 rotor). Then, the solution is mixed with Con A
Sepharose and the procedure followed as before.
[0083] In the second optional step, described by way of example as
follows, the gp96 containing fractions eluted from the Con A column
are pooled and the buffer exchanged for 5 mM sodium phosphate
buffer, pH 7, 300 mM NaCl by dialysis, or preferably by buffer
exchange on a Sephadex G25 column. After buffer exchange, the
solution is mixed with DEAE-Sepharose previously equilibrated with
5 mM sodium phosphate buffer, pH 7, 300 mM NaCl. The protein
solution and the beads are mixed gently for 1 hour and poured into
a column. Then, the column is washed with 5 mM sodium phosphate
buffer, pH 7, 300 mM NaCl, until the absorbance at 280 nm drops to
baseline. Then, the bound protein is eluted from the column with
five volumes of 5 mM sodium phosphate buffer, pH 7, 700 mM NaCl.
Protein containing fractions are pooled and diluted with 5 mM
sodium phosphate buffer, pH 7 in order to lower the salt
concentration to 175 mM. The resulting material then is applied to
the Mono Q FPLC column (Pharmacia) equilibrated with 5 mM sodium
phosphate buffer, pH 7 and the protein that binds to the Mono Q
FPLC column (Pharmacia) is eluted as described before.
[0084] It is appreciated, however, that one skilled in the art may
assess, by routine experimentation, the benefit of incorporating
the second optional step into the purification protocol. In
addition, it is appreciated also that the benefit of adding each of
the optional steps will depend upon the source of the starting
material.
[0085] When the gp96 fraction is isolated from the 100,000 g
pellet, the pellet is suspended in 5 volumes of PBS containing
either 1% sodium deoxycholate or 1% oxtyl glucopyranoside (but
without the Mg.sup.2+ and Ca.sup.2+) and incubated on ice for 1
hour. The suspension is centrifuged at 20,000 g for 30 minutes and
the resulting supernatant dialyzed against several changes of PBS
(also without the Mg.sup.2+ and Ca.sup.2+) to remove the detergent.
The dialysate is centrifuged at 100,000 g for 90 minutes, the
supernatant harvested, and calcium and magnesium are added to the
supernatant to give final concentrations of 2 mM , respectively.
Then the sample is purified by either the unmodified or the
modified method for isolating gp96-peptide complex from the 100,000
g supernatant, see above.
[0086] The gp96-peptide complexes can be purified to apparent
homogeneity using this procedure. About 10-20 .mu.g of gp96 can be
isolated from 1 g cells/tissue.
Infectious Disease
[0087] In an alternative embodiment wherein it is desired to treat
a patient having an infectious disease, the above-described methods
in Sections 5.2.1-5.2.3 are used to isolate hsp-peptide complexes
from cells infected with an infectious organism, e.g., of a cell
line or from a patient. Such infectious organisms include but are
not limited to, viruses, bacteria, protozoa, fungi, and parasites
as described in detail in Section 5.2.4 below.
5.2.4. Isolation of Antigenic/Immunogenic Components
[0088] It has been found that antigenic peptides and/or components
can be eluted from hsp-complexes either in the presence of ATP or
low pH. These experimental conditions may be used to isolate
peptides and/or antigenic components from cells which may contain
potentially useful antigenic determinants. Once isolated, the amino
acid sequence of each antigenic peptide may be determined using
conventional amino acid sequencing methodologies. Such antigenic
molecules can then be produced by chemical synthesis or recombinant
methods, purified, and complexed to hsps in vitro.
[0089] Similarly, it has been found that potentially immunogenic
peptides may be eluted from MHC-peptide complexes using techniques
well known in the art (Falk, K. et al., 1990 Nature 348:248-251;
Elliott, T., et al., 1990, Nature 348:195-197; Falk, K., et al.,
1991, Nature 351:290-296).
[0090] Thus, potentially immunogenic or antigenic peptides may be
isolated from either endogenous stress protein-peptide complexes or
endogenous MHC-peptide complexes for use subsequently as antigenic
molecules, by complexing in vitro to hsps. Exemplary protocols for
isolating peptides and/or antigenic components from either of the
these complexes are set forth below in Sections 5.2.4.1 and
5.2.4.2.
5.2.4.1. Peptides From Stress Protein-Peptide Complexes
[0091] Two methods may be used to elute the peptide from a stress
protein-peptide complex. One approach involves incubating the
stress protein-peptide complex in the presence of ATP. The other
approach involves incubating the complexes in a low pH buffer.
[0092] Briefly the complex of interest is centrifuged through a
Centricon 10 assembly (Millipore) to remove any low molecular
weight material loosely associated with the complex. The large
molecular weight fraction may be removed and analyzed by SDS-PAGE
while the low molecular weight may be analyzed by HPLC as described
below. In the ATP incubation protocol, the stress protein-peptide
complex in the large molecular weight fraction is incubated with 10
mM ATP for 30 minutes at room temperature. In the low pH protocol,
acetic acid or trifluoroacetic acid (TFA) is added to the stress
protein-peptide complex to give a final concentration of 10%
(vol/vol) and the mixture incubated at room temperature or in a
boiling water bath or any temperature in between, for 10 minutes
(See, Van Bleek, et al., 1990, Nature 348:213-216; and Li, et al.,
1993, EMBO Journal 12:3143-3151).
[0093] The resulting samples are centrifuged through a Centricon 10
assembly as mentioned previously. The high and low molecular weight
fractions are recovered. The remaining large molecular weight
stress protein-peptide complexes can be reincubated with ATP or low
pH to remove any remaining peptides.
[0094] The resulting lower molecular weight fractions are pooled,
concentrated by evaporation and dissolved in 0.1% TFA. The
dissolved material is then fractionated by reverse phase high
pressure liquid chromatography (HPLC) using for example a VYDAC C18
reverse phase column equilibrated with 0.1% TFA. The bound material
is then eluted at a flow rate of about 0.8 ml/min by developing the
column with a linear gradient of 0 to 80% acetonitrile in 0.1% TFA.
The elution of the peptides can be monitored by OD.sub.210 and the
fractions containing the peptides collected.
5.2.4.2. Peptides from MHC-peptide Complexes
[0095] The isolation of potentially immunogenic peptides from MHC
molecules is well known in the art and so is not described in
detail herein (See, Falk, et al., 1990, Nature 348:248-251;
Rotzsche, at al., 1990, Nature 348:252-254; Elliott, et al., 1990,
Nature 348:191-197; Falk, et al., 1991, Nature 351:290-296; Demotz,
et al., 1989, Nature 343:682-684; Rotzsche, et al., 1990, Science
249:283-287), the disclosures of which are incorporated herein by
reference.
[0096] Briefly, MHC-peptide complexes may be isolated by a
conventional immunoaffinity procedure. The peptides then may be
eluted from the MHC-peptide complex by incubating the complexes in
the presence of about 0.1% TFA in acetonitrile. The eluted peptides
may be fractionated and purified by reverse phase HPLC, as
before.
[0097] The amino acid sequences of the eluted peptides may be
determined either by manual or automated amino acid sequencing
techniques well known in the art. Once the amino acid sequence of a
potentially protective peptide has been determined the peptide may
be synthesized in any desired amount using conventional peptide
synthesis or other protocols well known in the art.
[0098] Peptides having the same amino acid sequence as those
isolated above may be synthesized by solid-phase peptide synthesis
using procedures similar to those described by Mierrifield, 1963,
J. Am. Chem. Soc., 85:2149. During synthesis, N-.alpha.-protected
amino acids having protected side chains are added stepwise to a
growing polypeptide chain linked by its C-terminal and to an
insoluble polymeric support i.e., polystyrene beads. The peptides
are synthesized by linking an amino group of an
N-.alpha.-deprotected amino acid to an .alpha.-carboxy group of an
N-.alpha.-protected amino acid that has been activated by reacting
it with a reagent such as dicyclohexylcarbodiimide. The attachment
of a free amino group to the activated carboxyl leads to peptide
bond formation. The most commonly used N-.alpha.-protecting groups
include Boc which is acid labile and Fmoc which is base labile.
[0099] Briefly, the C-terminal N-.alpha.-protected amino acid is
first attached to the polystyrene beads. The N-.alpha.-protecting
group is then removed. The deprotected .alpha.-amino group is
coupled to the activated .alpha.-carboxylate group of the next
N-.alpha.-protected amino acid. The process is repeated until the
desired peptide is synthesized. The resulting peptides are then
cleaved from the insoluble polymer support and the amino acid side
chains deprotected. Longer peptides can be derived by condensation
of protected peptide fragments. Details of appropriate chemistries,
resins, protecting groups, protected amino acids and reagents are
well known in the art and so are not discussed in detail herein
(See, Atherton, et al., 1989, Solid Phase Peptide Synthesis: A
Practical Approach, IRL Press, and Bodanszky, 1993, Peptide
Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag).
[0100] Purification of the resulting peptides is accomplished using
conventional procedures, such as preparative HPLC using gel
permeation, partition and/or ion exchange chromatography. The
choice of appropriate matrices and buffers are well known in the
art and so are not described in detail herein.
5.2.5. Exogenous Antigenic Molecules
[0101] Antigens or antigenic portions thereof can be selected for
use as antigenic molecules, for complexing to hsps, from among
those known in the art or determined by immunoassay to be able to
bind to antibody or MHC molecules (antigenicity) or generate immune
response (immunogenicity). To determine immunogenicity or
antigenicity by detecting binding to antibody, various immunoassays
known in the art can be used, including but not limited to
competitive and non-competitive assay systems using techniques such
as radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in vivo immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example),
western blots, immunoprecipitation reactions, agglutination assays
(e.g., gel agglutination assays, hemagglutination assays),
complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary
antibody. In another embodiment, the primary antibody-is-detected
by detecting binding of a secondary antibody or reagent to the
primary antibody. In a further embodiment, the secondary antibody
is labelled. Many means are known in the art for detecting binding
in an immunoassay and are envisioned for use. In one embodiment for
detecting immunogenicity, T cell-mediated responses can be assayed
by standard methods, e.g., in vitro cytoxicity assays or in vivo
delayed-type hypersensitivity assays.
[0102] Potentially useful antigens or derivatives thereof for use
as antigenic molecules can also be identified by various criteria,
such as the antigen's involvement in neutralization of a pathogen's
infectivity (wherein it is desired to treat or prevent infection by
such a pathogen) (Norrby, 1985, Summary, in Vaccines 85, Lerner, et
al. (eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., pp. 388-389), type or group specificity, recognition by
patients' antisera or immune cells, and/or the demonstration of
protective effects of antisera or immune cells specific for the
antigen. In addition, where it is desired to treat or prevent a
disease caused by pathogen, the antigen's encoded epitope should
preferably display a small or no degree of antigenic variation in
time or amongst different isolates of the same pathogen.
[0103] Preferably, where it is desired to treat or prevent cancer,
known tumor-specific antigens or fragments or derivatives thereof
are used. For example, such tumor specific or tumor-associated
antigens include but are not limited to KS 1/4 pan-carcinoma
antigen (Perez and Walker, 1990, J. Immunol. 142:3662-3667; Bumal,
1988, Hybridoma 7(4):407-415); ovarian carcinoma antigen (CA125)
(Yu, et al., 1991, Cancer Res. 51(2) :468-475); prostatic acid
phosphate (Tailer, et al., 1990, Nucl. Acids Res. 18 (16) :4928);
prostate specific antigen (Henttu and Vihko, 1989, Biochem.
Biophys. Res. Comm. 160(2) :903-910; Israeli, et al., 1993, Cancer
Res. 53:227-230); melanoma-associated antigen p97 (Estin, et al.,
1989, J. Natl. Cancer Inst. 81 (6) :445-446); melanoma antigen gp75
(Vijayasardahl, et al., 1990, J. Exp. Med. 171(4):1375-1380); high
molecular weight melanoma antigen (Natali, et al., 1987, Cancer
59:55-63) and prostate specific membrane antigen.
[0104] In a specific embodiment, an antigen or fragment or
derivative thereof specific to a certain tumor is selected for
complexing to hsp and subsequent administration to a patient having
that tumor.
[0105] Preferably, where it is desired to treat or prevent viral
diseases, molecules comprising epitopes of known viruses are used.
For example, such antigenic epitopes may be prepared from viruses
including, but not limited to, hepatitis type A, hepatitis type B,
hepatitis type C, influenza, varicella, adenovirus, herpes simplex
type I (HSV-I), herpes simplex type II (HSV-II), rinderpest,
rhinovirus, echovirus, rotavirus, respiratory syncytial virus,
papilloma virus, papova virus, cytomegalovirus, echinovirus,
arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,
rubella virus, polio virus, human immunodeficiency virus type I
(HIV-I), and human immunodeficiency virus type II (HIV-II).
[0106] Preferably, where it is desired to treat or prevent
bacterial infections, molecules comprising epitopes of known
bacteria are used. For example, such antigenic epitopes may be
prepared from bacteria including, but not limited to, mycobacteria
rickettsia, mycoplasma, neisseria and legionella.
[0107] Preferably, where it is desired to treat or prevent
protozoal infections, molecules comprising epitopes of known
protozoa are used. For example, such antigenic epitopes may be
prepared from protozoa including, but not limited to, leishmania,
kokzidioa, and trypanosoma.
[0108] Preferably, where it is desired to treat or prevent
parasitic infections, molecules comprising epitopes of known
parasites are used. For example, such antigenic epitopes may be
from parasites including, but not limited to, chlamydia and
rickettsia.
5.2.6. In Vitro Production of Stress Protein-Antigenic Molecule
Complexes
[0109] In an embodiment in which complexes of hsps and the peptides
with which they are endogenously associated in vivo are not
employed, complexes of hsps to antigenic molecules are produced in
vitro. As will be appreciated by those skilled in the art, the
peptides either isolated by the aforementioned procedures or
chemically synthesized or recombinantly produced may be
reconstituted with a variety of purified natural or recombinant
stress proteins in vitro to generate immunogenic non-covalent
stress protein-antigenic molecule complexes. Alternatively,
exogenous antigens or antigenic/immunogenic fragments or
derivatives thereof can be noncovalently complexed to stress
proteins for use in the immunotherapeutic or prophylactic vaccines
of the invention. A preferred, exemplary protocol for noncovalently
complexing a stress protein and an antigenic molecule in vitro is
discussed below.
[0110] Prior to complexing, the hsps are pretreated with ATP or low
pH to remove any peptides that may be associated with the hsp of
interest. When the ATP procedure is used, excess ATP is removed
from the preparation by the addition of apyranase as described by
Levy, et al., 1991, Cell 67:265-274. When the low pH procedure is
used, the buffer is readjusted to neutral pH by the addition of pH
modifying reagents.
[0111] The antigenic molecules (1 .mu.g) and the pretreated hsp (9
.mu.g) are admixed to give an approximately 5 antigenic molecule: 1
stress protein molar ratio. Then, the mixture is incubated for 15
minutes to 3 hours at 40 to 45.degree. C. in a suitable binding
buffer such as one containing 20 mM sodium phosphate, pH 7.2, 350
mM NaCl, 3 mM MgCl.sub.2 and 1 mM phenyl methyl sulfonyl fluoride
(PMSF). The preparations are centrifuged through a Centricon 10
assembly (Millipore) to remove any unbound peptide. The association
of the peptides with the stress proteins can be assayed by
SDS-PAGE. This is the preferred method for in vitro complexing of
peptides isolated from MHC-peptide complexes of peptides
disassociated from endogenous hsp-peptide complexes.
[0112] In an alternative embodiment of the invention, preferred for
producing complexes of hsp70 to exogenous antigenic molecules such
as proteins, 5-10 micrograms of purified hsp is incubated with
equimolar quantities of the antigenic molecule in 20 mM sodium
phosphate buffer pH 7.5, 0.5M NaCl, 3 mM, MgCl.sub.2 and 1 mM ADP
in a volume of 100 microliter at 37.degree. C. for 1 hr. This
incubation mixture is further diluted to 1 ml in phosphate-buffered
saline.
[0113] In an alternative embodiment of the invention, preferred for
producing complexes of gp96 or hsp90 to peptides, 5-10 micrograms
of purified gp96 or hsp90 is incubated with equimolar or excess
quantities of the antigenic peptide in a suitable buffer such as
one containing 20 mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 3nM
MgCl2 at 60-65.degree. C. for 5-20 min. This incubation mixture is
allowed to cool to room temperature and centrifuged one or more
times if necessary, through a Centricon 10 assembly (Millipore) to
remove any unbound peptide.
[0114] Following complexing, the immunogenic stress
protein-antigenic molecule complexes can optionally be assayed in
vitro using for example the mixed lymphocyte target cell assay
(MLTC) described below. Once immunogenic complexes have been
isolated they can be optionally characterized further in animal
models using the preferred administration protocols and excipients
discussed below.
5.2.7. Determination of Immunogenicity of Stress Protein-Peptide
Complexes
[0115] The purified stress protein-antigenic molecule complexes can
be assayed for immunogenicity using the MLTC assay well known in
the art.
[0116] By way of example but not limitation, the following
procedure can be used. Briefly, mice are injected, preferably
intradermally or mucosally, with the candidate stress
protein-antigenic molecule complexes. Other mice are injected with
either other stress protein peptide complexes or whole infected
cells which act as positive controls for the assay. The mice are
injected twice, 7-10 days apart. Ten days after the last
immunization, the spleens are removed and the lymphocytes released.
The released lymphocytes may be restimulated subsequently in vitro
by the addition of dead cells that expressed the complex of
interest.
[0117] For example, 8.times.10.sup.6 immune spleen cells may be
stimulated with 4.times.10.sup.4 mitomycin C treated or
.gamma.-irradiated (5-10,000 rads) infected cells (or cells
transfected with an appropriate gene, as the case may be) in 3 ml
RPMI medium containing 10% fetal calf serum. In certain cases 33%
secondary mixed lymphocyte culture supernatant may be included in
the culture medium as a source of T cell growth factors (See,
Glasebrook, et al., 1980, J. Exp. Med. 151:876). To test the
primary cytotoxic T cell response after immunization, spleen cells
may be cultured without stimulation. In some experiments spleen
cells of the immunized mice may also be restimulated with
antigenically distinct cells, to determine the specificity of the
cytotoxic T cell response.
[0118] Six days later the cultures are tested for cytotoxicity in a
4 hour .sup.51Cr-release assay (See, Palladino, et al., 1987,
Cancer Res. 47:5074-5079 and Blachere, at al., 1993, J.
Immunotherapy 14: 352-356). In this assay, the mixed lymphocyte
culture is added to a target cell suspension to give different
effector:target (E:T) ratios (usually 1:1 to 40:1). The target
cells are prelabelled by incubating 1.times.10.sup.6 target cells
in culture medium containing 20 mCi .sup.51Cr/ml for one hour at
37.degree. C. The cells are washed three times following labeling.
Each assay point (E:T ratio) is performed in triplicate and the
appropriate controls incorporated to measure spontaneous .sup.51Cr
release (no lymphocytes added to assay) and 100% release (cells
lysed with detergent). After incubating the cell mixtures for 4
hours, the cells are pelletted by centrifugation at 200 g for 5
minutes. The amount of .sup.51Cr released into the supernatant is
measured by a gamma counter. The percent cytotoxicity is measured
as cpm in the test sample minus spontaneously released cpm divided
by the total detergent released cpm minus spontaneously released
cpm.
[0119] In order to block the MHC class I cascade a concentrated
hybridoma supernatant derived from K-44 hybridoma cells (an
anti-MHC class I hybridoma) is added to the test samples to a final
concentration of 12.5%.
5.3. Combination With Adoptive Immunotherapy
[0120] Adoptive immunotherapy refers to a therapeutic approach for
treating cancer or infectious diseases in which immune cells are
administered to a host with the aim that the cells mediate either
directly or indirectly specific immunity to tumor cells and/or
antigenic components or regression of the tumor or treatment of
infectious diseases, as the case may be. (See U.S. patent
application Ser. No. 08/527,546, filed Sep. 13, 1995, which is
incorporated by reference herein in its entirety.) As an optional
step, in accordance with the methods described herein, APC are
sensitized with hsps noncovalently complexed with antigenic (or
immunogenic) molecules and used in adoptive immunotherapy.
[0121] In a specific embodiment, therapy by administration of
hsp-peptide complexes, using any desired route of administration,
may optionally be combined with adoptive immunotherapy using APC
sensitized with hsp-antigenic molecule complexes. As described in
Section 5 herein, the hsp-peptide complex-sensitized APC can be
administered alone, in combination with hsp-peptide complexes, or
before or after administration of hsp-peptide complexes.
Furthermore, the mode of administration can be varied, including
but not limited to, e.g., subcutaneously, intravenously or
intramuscularly, although intradermally or mucosally is
preferred.
5.3.1. Obtaining Macrophages and Antigen-Presenting Cells
[0122] The antigen-presenting cells, including but not limited to
macrophages, dendritic cells and B-cells, are preferably obtained
by production in vitro from stem and progenitor cells from human
peripheral blood or bone marrow as described by Inaba, K., et al.,
1992, J. Exp. Med. 176:1693-1702.
[0123] APC can be obtained by any of various methods known in the
art. In a preferred aspect human macrophages are used, obtained
from human blood cells. By way of example but not limitation,
macrophages can be obtained as follows:
[0124] Mononuclear cells are isolated from peripheral blood of a
patient (preferably the patient to be treated), by Ficoll-Hypaque
gradient centrifugation and are seeded on tissue culture dishes
which are pre-coated with the patient's own serum or with other AB+
human serum. The cells are incubated at 37.degree. C. for 1 hour,
then non-adherent cells are removed by pipetting. To the adherent
cells left in the dish, is added cold (4.degree. C.) 1 mM EDTA in
phosphate-buffered saline and the dishes are left at room
temperature for 15 minutes. The cells are harvested, washed with
RPMI buffer and suspended in RPMI buffer. Increased numbers of
macrophages may be obtained by incubating at 37.degree. C. with
macrophage-colony stimulating factor (M-CSF); increased numbers of
dendritic cells may be obtained by incubating with
granulocyte-macrophage-colony stimulating factor (GM-CSF) as
described in detail by Inaba, K., et al., 1992, J. Exp. Med.
176:1693-1702.
5.3.2. Sensitization of Macrophages and Antigen Presenting Cells
With Hsp-Peptide Complexes
[0125] APC are sensitized with hsp noncovalently bound to antigenic
molecules preferably by incubating the cells in vitro with the
complexes. The APC are sensitized with complexes of hsps and
antigenic molecules by incubating in vitro with the hsp-complex at
37.degree. C. for 15 minutes to 24 hours. By way of example but not
limitation, 4.times.10.sup.7 macrophages can be incubated with 10
microgram gp96-peptide complexes per ml or 100 microgram
hsp90-peptide complexes per ml at 37.degree. C. for 15 minutes-24
hours in 1 ml plain RPMI medium. The cells are washed three times
and resuspended in a physiological medium preferably sterile, at a
convenient concentration (e.g., 1.times.10.sup.7/ml) for injection
in a patient. Preferably, the patient into which the sensitized
APCs are injected is the patient from which the APC were originally
isolated (autologous embodiment).
[0126] Optionally, the ability of sensitized APC to stimulate, for
example, the antigen-specific, class I-restricted cytotoxic
T-lymphocytes (CTL) can be monitored by their ability to stimulate
CTLs to release tumor necrosis factor, and by their ability to act
as targets of such CTLs.
5.3.3. Reinfusion of Sensitized APC
[0127] The hsp-antigenic molecule-sensitized APC are reinfused into
the patient systemically, preferably intravenously, by conventional
clinical procedures. These activated cells are reinfused,
preferentially by systemic administration into the autologous
patient. Patients generally receive from about 10.sup.6 to about
10.sup.12 sensitized macrophages, depending on the condition of the
patient. In some regimens, patients may optionally receive in
addition a suitable dosage of a biological response modifier
including but not limited to the cytokines IFN-.alpha.,
IFN-.gamma., IL-2, IL-4, IL-6, TNF or other cytokine growth
factor.
5.4. Formulation, Administration & Kits
[0128] Hsp-antigenic molecule complexes of the invention may be
formulated into pharmaceutical preparations for administration to
mammals, preferably humans, for treatment or prevention of cancer
or infectious diseases. Compositions comprising a compound of the
invention formulated in a compatible pharmaceutical carrier may be
prepared, packaged, and labelled for treatment of the indicated
tumor(s), such as human sarcomas and carcinomas, e.g.,
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and
acute myelocytic leukemia (myeloblastic, promyelocytic,
myelomonocytic, monocytic and erythroleukemia); chronic leukemia
(chronic myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease. Alternatively, it can
be labeled for treatment of the appropriate infectious disease.
Alternatively, pharmaceutical compositions may be formulated for
treatment of appropriate infectious diseases.
[0129] Drug solubility and the site of absorption are factors which
should be considered when choosing the route of administration of a
therapeutic agent. In an embodiment of the invention, hsp-antigenic
molecule complexes may be administered using any desired route of
administration, and preferably intradermally or mucosally.
Advantages of intradermal or mucosal administration include use of
lower doses and rapid absorption, respectively. Mucosal routes of
administration include, but are not limited to, oral, rectal and
nasal administration. Preparations for mucosal administrations are
suitable in various formulations as described below.
[0130] If the complex is water-soluble, then it may be formulated
in an appropriate buffer, for example, phosphate buffered saline or
other physiologically compatible solutions, preferably sterile.
Alternatively, if the resulting complex has poor solubility in
aqueous solvents, then it may be formulated with a non-ionic
surfactant such as Tween, or polyethylene glycol. Thus, the
compounds and their physiologically acceptable solvates may be
formulated for administration by inhalation or insufflation (either
through the mouth or the nose) or oral, buccal, parenteral, or
rectal administration or, in the case of tumors, directly injected
into a solid tumor.
[0131] For oral administration, the pharmaceutical preparation may
be in liquid form, for example, solutions, syrups or suspensions,
or may be presented as a drug product for reconstitution 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.,
almond oil, oily esters, or fractionated vegetable oils) ; and
preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic
acid). 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., pregelatinized maize starch, polyvinyl pyrrolidone 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 sulphate). The tablets may be
coated by methods well-known in the art.
[0132] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0133] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0134] 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.
[0135] 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.
[0136] 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. Liposomes and emulsions are well known examples of delivery
vehicles or carriers for hydrophilic drugs.
[0137] 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 nebulizer, 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.
[0138] The compositions may, if desired, be presented in a pack or
dispenser device which 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.
[0139] The invention also provides kits for carrying out the
therapeutic regimens of the invention. Such kits comprise in one or
more containers therapeutically or prophylactically effective
amounts of the hsp-antigenic molecule complexes, preferably
purified, in pharmaceutically acceptable form. The kits optionally
further comprise in a second container the sensitized APC of the
invention, preferably purified. The hsp-antigenic molecule complex
in a vial of a kit of the invention may be in the form of a
pharmaceutically acceptable solution, e.g., in combination with
sterile saline, dextrose solution, or buffered solution, or other
pharmaceutically acceptable sterile fluid. Alternatively, the
complex may be lyophilized or desiccated; in this instance, the kit
optionally further comprises in a container a pharmaceutically
acceptable solution (e.g., saline, dextrose solution, etc.),
preferably sterile, to reconstitute the complex to form a solution
for injection purposes.
[0140] In another embodiment, a kit of the invention further
comprises a needle or syringe, preferably packaged in sterile form,
for injecting the complex, and/or a packaged alcohol pad.
Instructions are optionally included for administration of
hsp-antigenic molecule complexes by a clinician or by the
patient.
5.5. Target Infectious Diseases
[0141] Infectious diseases that can be treated or prevented by the
methods of the present invention are caused by infectious agents
including, but not limited to, viruses, bacteria, fungi protozoa
and parasites.
[0142] Viral diseases that can be treated or prevented by the
methods of the present invention include, but are not limited to,
those caused by hepatitis type A, hepatitis type B, hepatitis type
C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I),
herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,
rotavirus, respiratory syncytial virus, papilloma virus, papova
virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsackie virus; mumps virus, measles virus, rubella virus, polio
virus, human immunodeficiency virus type I (HIV-I), and human
immunodeficiency virus type II (HIV-II).
[0143] Bacterial diseases that can be treated or prevented by the
methods of the present invention are caused by bacteria including,
but not limited to, mycobacteria rickettsia, mycoplasma, neisseria
and legionella.
[0144] Protozoal diseases that can be treated or prevented by the
methods of the present invention are caused by protozoa including,
but not limited to, leishmania, kokzidioa, and trypanosoma.
[0145] Parasitic diseases that can be treated or prevented by the
methods of the present invention are caused by parasites including,
but not limited to, chlamydia and rickettsia.
5.6. Target Cancers
[0146] Cancers that can be treated or prevented by the methods of
the present invention include, but are not limited to human
sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and
acute myelocytic leukemia (myeloblastic, promyelocytic,
myelomonocytic, monocytic and erythroleukemia); chronic leukemia
(chronic myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease. Specific examples of
such cancers are described in the sections below.
[0147] In a specific embodiment the cancer is metastatic. In
another specific embodiment, the patient having a cancer is
immunosuppressed by reason of having undergone anti-cancer therapy
(e.g., chemotherapy radiation) prior to administration of the
hsp-antigenic molecule complexes or administration of the
hsp-sensitized APC.
5.6.1. Colorectal Cancer Metastatic to the Liver
[0148] In 1992, approximately 150,000 Americans were diagnosed with
colorectal cancer and more than 60,000 died as a result of
colorectal metastases. At the time of their deaths, 80 percent of
patients with colorectal cancer have metastatic disease involving
the liver, and one-half of these patients have no evidence of other
(extrahepatic) metastases. Most metastatic tumors of the liver are
from gastrointestinal primaries. Unfortunately, the natural history
of metastatic liver lesions carries a grave prognosis and systemic
chemotherapy regimens have been unable to induce significant
response rates or alter length of survival (Drebin, J. A., et al.,
in Current Therapy In Oncology, ed. J. E. Niederhuber, B. C.
Decker, Mosby, 1993, p.426).
[0149] Colorectal cancer initially spreads to regional lymph nodes
and then through the portal venous circulation to the liver, which
represents the most common visceral site of metastasis. The
symptoms that lead patients with colorectal cancer to seek medical
care vary with the anatomical location of the lesion. For example,
lesions in the ascending colon frequently ulcerate, which leads to
chronic blood loss in the stool.
[0150] Radical resection offers the greatest potential for cure in
patients with invasive colorectal cancer. Before surgery, the CEA
titer is determined. Radiation therapy and chemotherapy are used in
patients with advanced colorectal cancer. Results with
chemotherapeutic agents (e.g., 5-fluorouracil) are mixed and fewer
than 25 percent of patients experience a greater than 50 percent
reduction in tumor mass (Richards, 2d., F., et al., 1986, J. Clin.
Oncol. 4:565).
[0151] Patients with widespread metastases have limited survival
and systemic chemotherapy has little impact in this group of
patients. In addition, systemically administered chemotherapy is
often limited by the severity of toxicities associated with the
various agents, such as severe diarrhea, mucositis and/or
myelosuppression. Other techniques, including hepatic radiation,
systemic chemotherapy, hepatic arterial ligation, tumor
embolization and immunotherapy have all been explored, but, for the
most part, have proven ineffectual in prolonging patient
survival.
[0152] In a specific embodiment, the present invention provides
compositions and methods for enhancing tumor specific immunity in
individuals suffering from colorectal cancer metastasized to the
liver, in order to inhibit the progression of the neoplastic
disease. Preferred methods of treating these neoplastic diseases
comprise administering a composition of autologous hsp
noncovalently bound to peptide complexes, which elicits
tumor-specific immunity against the tumor cells. Most specifically,
the use of a composition of the invention, comprising gp96, can
result in nearly complete inhibition of liver cancer growth in
cancer patients, without inducing toxicity and thus providing a
dramatic therapeutic effect.
[0153] Accordingly, as an example of the method of the invention,
gp96-antigenic molecule complexes are administered to a patient
diagnosed with colorectal cancer, with or without liver metastasis,
via one of many different routes of administration, the preferred
route being intradermally at different anatomical sites, e.g., left
arm, right arm, left belly, right belly, left thigh, right thigh,
etc. The site of injection is varied for each weekly injection as
described in Sections 7 and 8. Exemplary primary and metastatic
cancers that can be prevented or treated according to the methods
of the invention are described in detail in the sections which
follow and by way of example, infra.
5.6.2. Hepatocellular Carcinoma
[0154] Hepatocellular carcinoma is generally a disease of the
elderly in the United States. Although many factors may lead to
hepatocellular carcinoma, the disease is usually limited to those
persons with preexisting liver disease. Approximately 60 to 80
percent of patients in the United States with hepatocellular
carcinoma have a cirrhotic liver and about four percent of
individuals with a cirrhotic liver eventually develop
hepatocellular carcinoma (Niederhuber, J. E., (ed.), 1993, Current
Therapy in Oncology, B. C. Decker, Mosby). The risk is highest in
patients whose liver disease is caused by inherited hemochromatosis
or hepatic B viral infection (Bradbear, R. A., et al., 1985, J.
Natl. Cancer Inst. 75:81; Beasley, R. P., et al., 1981, Lancet
2:1129). Other causes of cirrhosis that can lead to hepatocellular
carcinoma include alcohol abuse and hepatic fibrosis caused by
chronic administration of methotrexate. The most frequent symptoms
of hepatocellular carcinoma are the development of a painful mass
in the right upper quadrant or epigastrium, accompanied by weight
loss. In patients with cirrhosis, the development of hepatocellular
carcinoma is preceded by ascites, portal hypertension and
relatively abrupt clinical deterioration. In most cases, abnormal
values in standard liver function tests such as serum
aminotransferase and alkaline phosphatase are observed.
[0155] CT scans of the liver are used to determine the anatomic
distribution of hepatocellular carcinoma and also provide
orientation for percutaneous needle biopsy. Approximately 70
percent of patients with hepatocellular carcinoma have an elevated
serum alpha-fetoprotein concentration (McIntire, K. R., et al.,
1975, Cancer Res. 35:991) and its concentration correlates with the
extent of the disease.
[0156] Radical resection offers the only hope for cure in patients
with hepatocellular carcinoma. Such operative procedures are
associated with five-year survival rates of 12 to 30 percent. Liver
transplantation may improve survival of some younger individuals.
However, most patients are not surgical candidates because of
extensive cirrhosis multifocal tumor pattern or scarcity of
compatible donor organs. Chemotherapeutic agents have been
administered either by intravenous route or through an intrahepatic
arterial catheter. Such therapy has sometimes been combined with
irradiation to the liver. Reductions in the size of measurable
tumors of 50% or more have been reported in some patients treated
with either systemic doxorubicin or 5-fluorouracil. However,
chemotherapy often induces immunosuppression and rarely causes the
tumor to disappear completely and the duration of response is
short. The prognosis for patients with hepatocellular carcinoma is
negatively correlated with cirrhosis and metastases to the lungs or
bone. Median survival for patients is only four to six months. In
another specific embodiment, the present invention provides
compositions and methods for enhancing specific immunity in
individuals suffering from hepatocellular carcinoma in order to
inhibit the progression of the neoplastic disease and ultimately
irradiate all preneoplastic and neoplastic cells.
5.6.3. Breast Cancer
[0157] Another specific aspect of the invention relates to the
treatment of breast cancer. The American Cancer Society estimated
that in 1992 180,000 American women were diagnosed with breast
cancer and 46,000 succumbed to the disease (Niederhuber, J. E. ed.
Current Therapy in Oncology B. C. Decker, Mosby, 1993). This makes
breast cancer the second major cause of cancer death in women,
ranking just behind lung cancer. A disturbing fact is the
observation that breast cancer has been increasing at a rate of 3
percent per year since 1980 (Niederhuber, J. E., ed. Current
Therapy in Oncology, B. C. Decker, Mosby, (1993)). The treatment of
breast cancer presently involves surgery, radiation, hormonal
therapy and/or chemotherapy. Consideration of two breast cancer
characteristics, hormone receptors and disease extent, has governed
how hormonal therapies and standard-dose chemotherapy are sequenced
to improve survival and maintain or improve quality of life. A wide
range of multidrug regimens have been used as adjuvant therapy in
breast cancer patients, including, but not limited to combinations
of 2 cyclophosphamide, doxorubicin, vincristine methotrexate,
5-fluorouracil and/or leucovorin. In a specific embodiment, the
present invention provides hsp compositions and methods for
enhancing specific immunity to preneoplastic and neoplastic mammary
cells in women. The present invention also provides compositions
and methods for preventing the development of neoplastic cells in
women at enhanced risk for breast cancer, and for inhibiting cancer
cell proliferation and metastasis. These compositions can be
applied alone or in combination with each other or with biological
response modifiers.
5.7. Autologous Embodiment
[0158] The specific immunogenicity of hsps derives not from hsps
per se, but from the peptides bound to them. In a preferred
embodiment of the invention directed to the use of autologous
complexes of hsp-peptides as cancer vaccines, two of the most
intractable hurdles to cancer immunotherapy are circumvented. First
is the possibility that human cancers, like cancers of experimental
animals, are antigenically distinct. In an embodiment of the
present invention, hsps chaperone antigenic peptides of the cancer
cells from which they are derived and circumvent this hurdle.
Second, most current approaches to cancer immunotherapy focus on
determining the CTL-recognized epitopes of cancer cell lines. This
approach requires the availability of cell lines and CTLs against
cancers. These reagents are unavailable for an overwhelming
proportion of human cancers. In an embodiment of the present
invention directed to the use of autologous complexes of
hsp-peptides, cancer immunotherapy does not depend on the
availability of cell lines or CTLs nor does it require definition
of the antigenic epitopes of cancer cells. These advantages make
autologous hsps noncovalently bound to peptide complexes attractive
immunogens against cancer.
5.8. Prevention and Treatment of Primary and Metastatic Neoplastic
Diseases
[0159] There are many reasons why immunotherapy as provided by the
present invention is desired for use in cancer patients. First, if
cancer patients are immunosuppressed, surgery with anesthesia and
subsequent chemotherapy may worsen the immunosuppression. With
appropriate immunotherapy in the preoperative period, this
immunosuppression may be prevented or reversed. This could lead to
fewer infectious complications and to accelerated wound healing.
Second, tumor bulk is minimal following surgery and immunotherapy
is most likely to be effective in this situation. A third reason is
the possibility that tumor cells are shed into the circulation at
surgery and effective immunotherapy applied at this time can
eliminate these cells.
[0160] The preventive and therapeutic methods of the invention are
directed at enhancing the immunocompetence of the cancer patient
either before surgery, at or after surgery, and to induce
tumor-specific immunity to cancer cells, with the objective being
inhibition of cancer, and with the ultimate clinical objective
being total cancer regression and eradication.
5.9. Monitoring of Effects During Cancer Prevention and
Immunotherapy with Hsp-peptide Complexes
[0161] The effect of immunotherapy with hsp-antigenic molecule
complexes on development and progression of neoplastic diseases can
be monitored by any methods known to one skilled in the art,
including but not limited to measuring: a) delayed hypersensitivity
as an assessment of cellular immunity; b) activity of cytolytic
T-lymphocytes in vitro; c) levels of tumor specific antigens, e.g.,
carcinoembryonic (CEA) antigens; d) changes in the morphology of
tumors using techniques such as a computed tomographic (CT) scan;
and e) changes in levels of putative biomarkers of risk for a
particular cancer in individuals at high risk, and f) changes in
the morphology of tumors using a sonogram.
5.9.1. Delayed Hypersensitivity Skin Test
[0162] Delayed hypersensitivity skin tests are of great value in
the overall immunocompetence and cellular immunity to an antigen.
Inability to react to a battery of common skin antigens is termed
anergy (Sato, T., et al., 1995, Clin. Immunol. Pathol.
74:35-43).
[0163] Proper-technique of skin testing requires that the antigens
be stored sterile at 4.degree. C., protected from light and
reconstituted shorted before use. A 25- or 27-gauge needle ensures
intradermal, rather than subcutaneous, administration of antigen.
Twenty-four and 48 hours after intradermal administration of the
antigen, the largest dimensions of both erythema and induration are
measured with a rule. Hypoactivity to any given antigen or group of
antigens is confirmed by testing with higher concentrations of
antigen or, in ambiguous circumstances, by a repeat test with an
intermediate test.
5.9.2. Activity of Cytolytic T-lymphocytes In Vitro
[0164] 8.times.10.sup.6 peripheral blood derived T lymphocytes
isolated by the Ficoll-Hypaque centrifugation gradient technique,
are restimulated with 4.times.10.sup.4 mitomycin C treated tumor
cells in 3 ml RPMI medium containing 10% fetal calf serum. In some
experiments, 33% secondary mixed lymphocyte culture supernatant or
IL-2, is included in the culture medium as a source of T cell
growth factors.
[0165] In order to measure the primary response of cytolytic
T-lymphocytes after immunization, T cells are cultured without the
stimulator tumor cells. In other experiments, T cells are
restimulated with antigenically distinct cells. After six days, the
cultures are tested for cytotoxicity in a 4 hour .sup.51Cr-release
assay. The spontaneous .sup.51Cr-release of the targets should
reach a level less than 20%. For the anti-MHC class I blocking
activity, a tenfold concentrated supernatant of W6/32 hybridoma is
added to the test at a final concentration of 12.5% (Heike M., et
al., J. Immunotherapy 15:15-174).
5.9.3. Levels of Tumor Specific Antigens
[0166] Although it may not be possible to detect unique tumor
antigens on all tumors, many tumors display antigens that
distinguish them from normal cells. The monoclonal antibody
reagents have permitted the isolation and biochemical
characterization of the antigens and have been invaluable
diagnostically for distinction of transformed from nontransformed
cells and for definition of the cell lineage of transformed cells.
The best-characterized human tumor-associated antigens are the
oncofetal antigens. These antigens are expressed during
embryogenesis, but are absent or very difficult to detect in normal
adult tissue. The prototype antigen is carcinoembryonic antigen
(CEA), a glycoprotein found on fetal gut and human colon cancer
cells, but not on normal adult colon cells. Since CEA is shed from
colon carcinoma cells and found in the serum, it was originally
thought that the presence of this antigen in the serum could be
used to screen patients for colon cancer. However, patients with
other tumors, such as pancreatic and breast cancer, also have
elevated serum levels of CEA. Therefore, monitoring the fall and
rise of CEA levels in cancer patients undergoing therapy has proven
useful for predicting tumor progression and responses to
treatment.
[0167] Several other oncofetal antigens have been useful for
diagnosing and monitoring human tumors, e.g., alpha-fetoprotein, an
alpha-globulin normally secreted by fetal liver and yolk sac cells,
is found in the serum of patients with liver and germinal cell
tumors and can be used as a marker of disease status.
5.9.4. Computed Tomographic (CT) Scan
[0168] CT remains the choice of techniques for the accurate staging
of cancers. CT has proved more sensitive and specific than any
other imaging techniques for the detection of metastases.
5.9.5. Measurement of Putative Biomarkers
[0169] The levels of a putative biomarker for risk of a specific
cancer are measured to monitor the effect of hsp noncovalently
bound to peptide complexes. For example, in individuals at enhanced
risk for prostate cancer, serum prostate-specific antigen (PSA) is
measured by the procedure described by Brawer, M. K., et al., 1992,
J. Urol. 147:841-845, and Catalona, W. J., et al., 1993, JAMA
270:948-958; or in individuals at risk for colorectal cancer, CEA
is measured as described above in Section 4.5.3; and in individuals
at enhanced risk for breast cancer, 16-.alpha.-hydroxylation of
estradiol is measured by the procedure described by Schneider, J.
et al., 1982, Proc. Natl. Acad. Sci. ISA 79:3047-3051.
5.9.6. Sonogram
[0170] A sonogram remains an alternative choice of technique for
the accurate staging of cancers.
6 EXAMPLE: METHYLCHOLANTHRENE (METH A)-INDUCED SARCOMA MODEL
[0171] Gp96-antigenic molecule complexes, administered
intradermally in low doses, can prevent development of cancer and
can mediate therapy of pre-existing cancers.
6.1. Prevention Modality
[0172] (a) Materials and Methods.
[0173] Gp96-antigenic molecule complexes were derived from Meth A
sarcoma cells as described in Section 5.2.3.
[0174] Five groups of BALB/cJ mice (from The Jackson Laboratories,
Bar Harbor, Me.) were given the following-treatments: A)
Intradermal injection of buffer solution; B) Intradermal injection
of 1 microgram gp96-antigenic molecule complexes derived from Meth
A sarcoma cells; and C) Intradermal injection of 5 microgram
gp96-antigenic molecule complexes derived from Meth A sarcoma
cells.
[0175] The above treatments were administered twice, at different
sites, at weekly intervals before injecting intradermally, 1 week
after the second injection 1.times.10.sup.5 Meth A sarcoma cells.
Tumor growth was monitored by measuring the average tumor
diameter.
[0176] (b) Results.
[0177] Tumor growth was comparable in groups A and C, i.e., mice
receiving the control buffer solution or the 5 microgram dose of
gp95-peptide complexes derived from Meth A sarcoma cells. In mice
treated with 1 microgram gp96-peptide complexes (B), tumor growth
was markedly inhibited compared with the mice receiving the buffer
control or the 5 microgram gp96-antigenic molecule complex (FIGS.
1A-C). The most preferred dose of gp96-antigenic molecule complex
per administration was 0.5 to 2.0 micrograms (data not shown).
[0178] Thus, intradermal administration of low doses of antigenic
molecule complexes, described herein, represents an approach to
prevention of cancer with potential applicability to a wide range
of cancers, infectious diseases or immunological disorders.
7 EXAMPLES: ADOPTIVE TRANSFER OF SENSITIZED MACROPHAGES, ALONE OR
IN COMBINATION WITH ADMINISTRATION OF HSP-PEPTIDE COMPLEXES
[0179] Autologous human macrophages are sensitized with autologous
human gp96 noncovalently bound to an antigenic/immunogenic
molecule. The sensitized macrophages are administered to the human
patient at approximately the same time as, or before, or after the
administration of the gp96-antigenic molecule complex.
7.1. Materials and Methods
[0180] Macrophages are obtained as follows: mononuclear cells are
isolated from peripheral blood of the human patient to be treated,
by Ficoll-Hypaque gradient centrifugation and are seeded on tissue
culture dishes which are pre-coated with the patient's own serum or
with other AB+ human serum. The cells are incubated at 37.degree.
C. for 1 hour, then non-adherent cells are removed by pipetting. To
the adherent cells left in the dish, is added cold (4.degree. C.) 1
mM EDTA in phosphate-buffered saline and the dishes are left at
room temperature for 15 minutes. The cells are harvested, washed
with RPMI buffer and suspended in RPMI buffer. Increased numbers of
macrophages may be obtained by incubating at 37.degree. C. with
macrophage-colony stimulating factor (M-CSF); increased numbers of
dendritic cells may be obtained by incubating with
granulocyte-macrophage-colony stimulating factor (GM-CSF) as
described in detail by Inaba, K., et al., 1992, J. Exp. Med.
176:1693-1702.
[0181] The macrophages (4.times.10.sup.7) are then incubated at
37.degree. C. for 3 hour in 1 ml RPMI containing 50 .mu.g
gp96-peptide complexes derived from the autologous tumor or from
autologous liver, using methods as described in Section 5.2.3. The
macrophages are then washed 3 times and resuspended at a
concentrate of 1.times.10.sup.7/ml in RPMI medium. 200 microliters
of this suspension is administered as described in the experimental
protocol below.
7.2. Treatment of Hepatocellular Carcinoma
[0182] Five groups of human patients with hepatocellular carcinoma
are injected with autologous macrophages sensitized with
hsp-peptide complexes derived from their own tumors post surgery.
Treatment with hsp-peptide complexes is started any time after
surgery. However, if the patient-has received chemotherapy,
sensitized macrophages alone or in combination with an hsp-peptide
complexes are usually administered after an interval of four weeks
or more so as to allow the immune system to recover. The
immunocompetence of the patient is tested by procedures described
in sections 5.7 above.
[0183] The preferred therapeutic regimen includes weekly injections
of the sensitized macrophages in combination with an hsp-peptide
complex dissolved in saline or other physiologically compatible
solution. Sensitized macrophages may be administered at
approximately the same time with an hsp-peptide complex or one may
be administered prior to administration of the other.
[0184] The dosage used for hsp70 or gp96 is in the range of 0.1 to
9 micrograms, with the preferred dosage being 0.5-2.0 micrograms.
The dosage used for hsp90 is in the range of 5 to 500 micrograms,
with the preferred dosage being about 10 micrograms.
[0185] The site of injection is varied each time, for example, the
first injection is given intradermally on the left arm, the second
injection on the right arm, the third injection on the left
abdominal region, the fourth injection on the right abdominal
region, the fifth injection on the left thigh, the sixth injection
on the right thigh, etc. The same site is repeated after a gap of
one or more injections. In addition, injections are split and each
half of the dose is administered at a different site on the same
day.
[0186] Overall, the first four to six injections are given at
weekly intervals. Subsequently, two injections are given at
two-week intervals; followed by a regimen of injections at monthly
intervals. The effect of therapy is monitored by measuring: a)
delayed hypersensitivity as an assessment of cellular immunity; b)
activity of cytolytic T-lymphocytes in vitro; c) levels of tumor
specific antigens, e.g., carcinoembryonic (CEA) antigens; d)
changes in the morphology of tumors using techniques such as a
computed tomographic (CT) scan; and e) changes in putative
biomarkers of risk for a particular cancer in individuals at high
risk.
[0187] Depending on the results obtained, as described above in
Section 5.10, the therapeutic regimen may be modified to maintain
and/or boost the immunological responses of the patient, with the
ultimate goal of achieving tumor regression and complete
eradication of cancer cells.
8 EXAMPLE: ADMINISTRATION OF HSP-PEPTIDE COMPLEXES IN THE TREATMENT
OF COLORECTAL CANCER
[0188] Hsp-peptide complexes (gp96, hsp70, hsp90 or a combination
thereof) are administered as adjuvant therapy and as prophylactic
adjuvant therapy in patients after complete reduction of colorectal
cancer to eliminate undetectable micrometastases and to improve
survival.
[0189] The therapeutic and prophylactic regimens used in patients
suffering from colorectal cancer are the same as those described in
Section 7 above for patients recovering with hepatocellular
carcinoma. The methods of monitoring of patients under clinical
evaluation for prevention and treatment of colorectal cancer is
done by procedures described in Section 5.7. Specifically, CEA
levels are measured as a useful monitor of tumor regression and/or
recurrence (Mayer, R. J. , et al., 1978, Cancer 42:1428).
[0190] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0191] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
* * * * *