U.S. patent application number 10/618088 was filed with the patent office on 2005-08-11 for mesothelin vaccines and model systems.
This patent application is currently assigned to The Johns Hopkins University. Invention is credited to Hruban, Ralph, Hung, Chien-Fu, Jaffee, Elizabeth, Wu, Tzyy-Choou.
Application Number | 20050175625 10/618088 |
Document ID | / |
Family ID | 30119338 |
Filed Date | 2005-08-11 |
United States Patent
Application |
20050175625 |
Kind Code |
A1 |
Jaffee, Elizabeth ; et
al. |
August 11, 2005 |
Mesothelin vaccines and model systems
Abstract
Mesothelin can be used as an immunotherapeutic target. It
induces a cytolytic T cell response. Portions of mesothelin which
induce such responses are identified. Vaccines can be either
polynucleotide- or polypeptide-based. Carriers for raising a
cytolytic T cell response include bacteria and viruses. A mouse
model for testing vaccines and other anti-tumor therapeutics and
prophylactics comprises a strongly mesothelin-expressing,
transformed peritoneal cell line.
Inventors: |
Jaffee, Elizabeth;
(Lutherville, MD) ; Wu, Tzyy-Choou; (Stevenson,
MD) ; Hung, Chien-Fu; (Timonium, MD) ; Hruban,
Ralph; (Baltimore, MD) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
The Johns Hopkins
University
Baltimore
MD
21201
|
Family ID: |
30119338 |
Appl. No.: |
10/618088 |
Filed: |
July 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60475783 |
Jun 5, 2003 |
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60414931 |
Sep 30, 2002 |
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60398217 |
Jul 24, 2002 |
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60395556 |
Jul 12, 2002 |
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Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
A61K 2039/57 20130101;
A61K 2039/6031 20130101; A61K 39/001168 20180801; A61K 2039/53
20130101; Y02A 50/30 20180101; A61P 35/00 20180101; A61K 2039/523
20130101 |
Class at
Publication: |
424/185.1 |
International
Class: |
A61K 039/00 |
Goverment Interests
[0002] This invention was made using funds from the U.S.
government. The terms of grants NCI CA62924, NCI RO1 CA72631, NCI
RO1 CA71806, U19 CA72108-02, and NCDDG RFA CA-95-020 mandate that
the U.S. government retain certain rights in the invention.
Claims
We claim:
1. A method of inducing a T-cell response to a tumor that
overexpresses mesothelin relative to normal tissue from which the
tumor is derived, said method comprising: administering to a
patient who has said tumor or who has had said tumor removed, a
vaccine comprising a polypeptide comprising an MHC Class I-binding
epitope of mesothelin, wherein the epitope binds to an allelic form
of MHC class I which is expressed by the patient, whereby a T-cell
response to mesothelin is induced, wherein the vaccine does not
comprise whole tumor cells.
2. The method of claim 1 wherein the tumor is selected from the
group consisting of ovarian cancer, pancreatic cancer,
mesothelioma, and squamous cell carcinoma.
3. The method of claim 1 wherein the tumor is a pancreatic
cancer.
4. The method of claim 1 wherein the tumor is an ovarian
cancer.
5. The method of claim 1 wherein epitope is selected from the group
consisting of: SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID NO: 2);
ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY (SEQ ID NO: 4); FYPGYLCSL (SEQ
ID NO: 5); and LYPKARLAF (SEQ ID NO: 6).
6. The method of claim 1 wherein the polypeptide is mature
mesothelin.
7. The method of claim 1 wherein the polypeptide is the primary
translation product of mesothelin.
8. The method of claim 1 wherein a mixture of said polypeptides is
administered.
9. The method of claim 8 wherein said polypeptides bind to a
plurality of allelic forms of MHC Class I molecules.
10. The method of claim 8 wherein said polypeptides bind to a
single allelic form of MHC Class I molecules.
11. The method of claim 1 wherein the polypeptide is selected as
being an MHC class I-binding epitope using an algorithm.
12. The method of claim 1 wherein the polypeptide is selected as
being an MHC class I-binding epitope using two algorithms.
13. The method of claim 1 wherein the T-cell response is induction
of specific CD8.sup.+ T cells.
14. The method of claim 1 wherein the vaccine is acellular.
15. The method of claim 1 wherein the vaccine comprises a bacterium
selected from the group consisting of: Shigella flexneri, E. coli,
Listeria monocytogenes, Yersinia enterocolitica, Salmonella
typhimurium, Salmonella typhi, and mycobacterium.
16. The method of claim 1 wherein the vaccine is administered in
sufficient amount to induce tumor regression.
17. The method of claim 1 wherein the vaccine is administered in
sufficient amount to keep the patient tumor-free after removal of
the tumor.
18. A method of inducing a T-cell response to a tumor that
overexpresses mesothelin relative to normal tissue from which the
tumor is derived, said method comprising: administering to a
patient who has said tumor or who has had said tumor removed, a
vaccine comprising a polypeptide comprising an MHC Class II-binding
epitope of mesothelin, wherein the epitope binds to an allelic form
of MHC class II which is expressed by the patient, whereby a T-cell
response to mesothelin is induced, wherein the vaccine does not
comprise whole tumor cells.
19. A method of inducing a T-cell response to tumor cells that
overexpress mesothelin relative to normal cells from which the
tumor cells are derived, said method comprising: administering to a
patient who is at risk of developing a tumor that overexrpresses
mesothelin a vaccine comprising a polypeptide comprising an MHC
class I-binding epitope of mesothelin or an MHC class II-binding
epitope of mesothelin, wherein the epitope binds to an allelic form
of MHC class I or class II which is expressed by the patient,
whereby a T-cell response to mesothelin is induced, wherein the
vaccine does not comprise whole tumor cells.
20. The method of claim 19 wherein the patient has been exposed to
a carcinogen which is known to induce tumors which overexpress
mesothelin relative to normal tissue from which the tumor is
derived.
21. The method of claim 20 wherein the carcinogen is asbestos.
22. A method of inducing a T-cell response to a tumor which
overexpresses mesothelin relative to normal tissue from which it is
derived, said method comprising: administering to a patient who has
said tumor or who has had said tumor removed, a vaccine comprising
a polynucleotide encoding a polypeptide comprising an MHC Class
I-binding epitope of mesothelin, wherein the epitope binds to an
allelic form of MHC class I which is expressed by the patient,
whereby a T-cell response to mesothelin is induced, wherein the
vaccine does not comprise whole tumor cells.
23. The method of claim 22 wherein the tumor is selected from the
group consisting of ovarian cancer, pancreatic cancer,
mesothelioma, and squamous cell carcinoma.
24. The method of claim 22 wherein the tumor is a pancreatic
cancer.
25. The method of claim 22 wherein the tumor is an ovarian
cancer.
26. The method of claim 22 wherein epitope is selected from the
group consisting of: SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID
NO: 2); ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY (SEQ ID NO: 4);
FYPGYLCSL (SEQ ID NO: 5); and LYPKARLAF (SEQ ID NO: 6).
27. The method of claim 22 wherein the polypeptide is mature
mesothelin.
28. The method of claim 22 wherein the polypeptide is primary
translation product of mesothelin.
29. The method of claim 22 wherein the vaccine comprises one or
more polynucleotides encoding a mixture of said polypeptides.
30. The method of claim 29 wherein said polypeptides bind to a
plurality of allelic forms of MHC Class I molecules.
31. The method of claim 29 wherein said polypeptides bind to a
single allelic form of MHC Class I molecules.
32. The method of claim 22 wherein the polypeptide is selected as
being an MHC class I-binding epitope using an algorithm.
33. The method of claim 22 wherein the polypeptide is selected as
being an MHC class I-binding epitope using two algorithms.
34. The method of claim 22 wherein the T-cell response is induction
of specific CD8.sup.+ T cells.
35. The method of claim 22 wherein the vaccine is acellular.
36. The method of claim 22 wherein the vaccine comprises a
bacterium selected from the group consisting of: Shigella flexneri,
E. coli, Listeria monocytogenes, Yersinia enterocolitica,
Salmonella typhimurium, Salmonella typhi, and mycobacterium.
37. The method of claim 22 wherein the vaccine is administered in
sufficient amount to induce tumor regression.
38. The method of claim 22 wherein the vaccine is administered in
sufficient amount to keep the patient tumor-free after removal of
the tumor.
39. A method of inducing a T-cell response to a tumor that
overexpresses mesothelin relative to normal tissue from which the
tumor is derived, said method comprising: administering to a
patient who has said tumor or who has had said tumor removed, a
vaccine comprising a polynucleotide encoding a polypeptide
comprising an MHC Class II-binding epitope of mesothelin, wherein
the epitope binds to an allelic form of MHC class II which is
expressed by the patient, whereby a T-cell response to mesothelin
is induced, wherein the vaccine does not comprise whole tumor
cells.
40. A method of inducing a T-cell response to tumor cells that
overexpress mesothelin relative to normal cells from which the
tumor cells are derived, said method comprising: administering to a
patient who is at risk of developing a tumor that overexrpresses
mesothelin a vaccine comprising a polynucleotide encoding a
polypeptide comprising an MHC class I-binding epitope of mesothelin
or an MHC class II-binding epitope of mesothelin, wherein the
epitope binds to an allelic form of MHC class I or class II which
is expressed by the patient, whereby a T-cell response to
mesothelin is induced, wherein the vaccine does not comprise whole
tumor cells.
41. The method of claim 40 wherein the patient has been exposed to
a carcinogen which is known to induce tumors which overexpress
mesothelin relative to normal tissue from which the tumor is
derived.
42. The method of claim 41 wherein the carcinogen is asbestos.
43. A method of identifying immunogens useful as candidates for
anti-tumor vaccines, comprising: selecting a protein which is
expressed by a tumor and which is minimally or not expressed by
normal tissue from which the tumor is derived; testing lymphocytes
of humans who have been vaccinated with a vaccine which comprises
said protein to determine if said lymphocytes comprise CD8+ T cells
or CD4+ T cells which are specific for said protein, wherein the
presence of said CD8+ T cells or CD4+ T cells indicates that the
protein is a candidate for use as an anti-tumor vaccine.
44. The method of claim 43 wherein said humans have exhibited an
anti-tumor immune response.
45. The method of claim 43 wherein the vaccine comprises whole
tumor cells.
46. The method of claim 44 wherein the anti-tumor immune response
results in prolonged disease-free survival post-surgical tumor
removal relative to a similar population which has not been
vaccinated.
47. The method of claim 44 wherein the anti-tumor immune response
results in tumor regression.
48. The method of claim 44 wherein the anti-tumor immune response
results in prolonged survival time.
49. The method of claim 44 wherein the anti-tumor immune response
is delayed type hypersensitivity to autologous tumor cells.
50. The method of claim 43 wherein said lymphocytes are also tested
to determine if they comprise CD8+ T cells or CD4+ T cells specific
for an antigen not expressed by the vaccine.
51. The method of claim 43 wherein the humans are divided into two
groups based on their response to the vaccine, wherein a first
group comprises responders and a second group comprises
non-responders, wherein if said CD8+ T cells or CD4+ T cells are
found more frequently in responders than in non-responders then the
protein is identified as more likely to be useful in an anti-tumor
vaccine.
52. The method of claim 51 wherein responders display a DTH
response to autologous tumor cells but non-responders do not
display the response.
53. The method of claim 51 wherein responders have a longer period
of disease free survival than non-responders.
54. A method of predicting future response to a tumor vaccine
comprising at least one T-cell epitope of mesothelin in a patient
who has received the vaccine, comprising: testing lymphocytes of
the patient to determine if the lymphocytes comprise CD8+ T cells
or CD4+ T cells which are specific for mesothelin, wherein the
presence of said CD8+ T cells or CD4+ T cells predicts a longer
survival time than the absence of said CD8.sup.+ T cells.
55. The method of claim 54 wherein the vaccine comprises whole
tumor cells.
56. The method of claim 54 wherein the vaccine comprises pancreatic
tumor cells and the antigen is mesothelin.
57. The method of claim 54 wherein the vaccine comprises ovarian
tumor cells and the antigen is mesothelin.
58. The method of claim 54 wherein the vaccine comprises
mesothelioma cells and the antigen is mesothelin.
59. A vaccine which induces a CD8.sup.+ T cell or CD4.sup.+ T cell
response, comprising: a polypeptide comprising an MHC Class I- or
Class II-binding epitope of mesothelin, wherein the epitope binds
to an allelic form of MHC class I of class II which is expressed by
the patient, whereby a CD8.sup.+ T cell or CD4.sup.+ T-cell
response to mesothelin is induced, wherein the vaccine does not
comprise whole tumor cells; and a carrier for stimulating a
CD8.sup.+ T cell or CD4.sup.+ T cell immune response.
60. The vaccine of claim 59 wherein the polypeptide comprises an
MHC Class I-binding epitope.
61. The vaccine of claim 59 wherein the polypeptide comprises
between 6 and 20 amino acid residues.
62. The vaccine of claim 59 wherein the polypeptide comprises an
epitope selected from the group consisting of SLLFLLFSL (SEQ ID NO:
1); VLPLTVAEV (SEQ ID NO: 2); ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY
(SEQ ID NO: 4); FYPGYLCSL (SEQ ID NO: 5); and LYPKARLAF (SEQ ID NO:
6).
63. The vaccine of claim 59 wherein the carrier is CD40/CD40
ligand.
64. The vaccine of claim 59 wherein the carrier is OX-40/OX-40
ligand.
65. The vaccine of claim 59 wherein the carrier is a CTLA-4
antagonist.
66. The vaccine of claim 59 wherein the carrier is GM-CSF.
67. A vaccine which induces a CD8.sup.+ T cell or CD4.sup.+ T cell
response, comprising: a polynucleotide encoding a polypeptide
comprising an MHC Class I- or Class II-binding epitope of
mesothelin, wherein the epitope binds to an allelic form of MHC
class I or Class II which is expressed by the patient, whereby a
CD8.sup.+ T cell or CD4.sup.+ T-cell response to mesothelin is
induced, wherein the vaccine does not comprise whole tumor cells;
and a carrier for stimulating a CD8.sup.+ T cell or CD4.sup.+ T
cell immune response.
68. The vaccine of claim 67 wherein the carrier is CD40/CD40
ligand.
69. The vaccine of claim 67 wherein the carrier is OX-40/OX-40
ligand.
70. The vaccine of claim 67 wherein the carrier is a CTLA-4
antagonist.
71. The vaccine of claim 67 wherein the carrier is GM-CSF.
72. The vaccine of claim 67 wherein the polypeptide comprises an
epitope selected from the group consisting of SLLFLLFSL (SEQ ID NO:
1); VLPLTVAEV (SEQ ID NO: 2); ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY
(SEQ ID NO: 4); FYPGYLCSL (SEQ ID NO: 5); and LYPKARLAF (SEQ ID NO:
6).
73. The vaccine of claim 59 which comprises a bacterium.
74. The vaccine of claim 67 which comprises a bacterium.
75. The vaccine of claim 73 wherein the bacterium is selected from
the group consisting of: Shigella flexneri, E. coli, Listeria
monocytogenes, Yersinia enterocolitica, Salmonella typhimurium,
Salmonella typhi, and mycobacterium.
76. The vaccine of claim 74 wherein the bacterium is selected from
the group consisting of: Shigella flexneri, E. coli, Listeria
monocytogenes, Yersinia enterocolitica, Salmonella typhimurium,
Salmonella typhi, and mycobacterium.
77. An isolated polypeptide of 9 to 25 amino acid residues
comprising an epitope selected from the group consisting of
SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID NO: 2); ELAVALAQK (SEQ
ID NO: 3); ALQGGGPPY (SEQ ID NO: 4); FYPGYLCSL (SEQ ID NO: 5); and
LYPKARLAF (SEQ ID NO: 6).
78. A fusion protein comprising a first and a second portion,
wherein the first portion comprises a polypeptide of 9 to 25 amino
acid residues comprising an epitope selected from the group
consisting of SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID NO: 2);
ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY (SEQ ID NO: 4); FYPGYLCSL (SEQ
ID NO: 5); and LYPKARLAF (SEQ ID NO: 6), and the second portion
comprises a segment of at least 6 amino acid residues, wherein the
sequence of said second portion is not in mesothelin.
79. An expression vector which encodes a polypeptide of 9 to 25
amino acid residues comprising an epitope selected from the group
consisting of SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID NO: 2);
ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY (SEQ ID NO: 4); FYPGYLCSL (SEQ
ID NO: 5); and LYPKARLAF (SEQ ID NO: 6).
80. A bacterium which comprises the expression vector of claim
79.
81. The bacterium of claim 80 which is selected from the group
consisting of Shigella flexneri, E. coli, Listeria monocytogenes,
Yersinia enterocolitica, Salmonella typhimurium, Salmonella typhi,
and mycobacterium.
82. An expression vector which encodes the fusion protein of claim
78.
83. A bacterium which comprises the expression vector of claim
82.
84. The bacterium of claim 83 which is selected from the group
consisting of Shigella flexneri, E. coli, Listeria monocytogenes,
Yersinia enterocolitica, Salmonella typhimurium, Salmonella typhi,
and mycobacterium.
85. An isolated antibody that binds to an epitope selected from the
group consisting of SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID NO:
2); ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY (SEQ ID NO: 4); FYPGYLCSL
(SEQ ID NO: 5); and LYPKARLAF (SEQ ID NO: 6).
86. A T-cell line that binds to an epitope selected from the group
consisting of SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID NO: 2);
ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY (SEQ ID NO: 4); FYPGYLCSL (SEQ
ID NO: 5); and LYPKARLAF (SEQ ID NO: 6).
87. The polypeptide of claim 77 which is bound to an MHC Class I
molecule.
88. The fusion protein of claim 78 which is bound to an MHC Class I
molecule.
89. The vaccine of claim 59 wherein the carrier is an MHC Class I
molecule.
90. The polypeptide of claim 87 wherein the MHC Class I molecule is
on a dendritic cell.
91. The fusion protein of claim 88 wherein the MHC Class I molecule
is on a dendritic cell.
92. The vaccine of claim 89 wherein the MHC Class I molecule is on
a dendritic cell.
93. The polypeptide of claim 87 wherein the MHC Class I molecule is
on an antigen presenting cell.
94. The polypeptide of claim 88 wherein the MHC Class I molecule is
on an antigen presenting cell.
95. The vaccine of claim 89 wherein the MHC Class I molecule is on
an antigen presenting cell.
96. A method of predicting future response to a tumor vaccine in a
patient who has received the vaccine, comprising: testing the
patient to determine if the patient has a delayed type
hypersensitivity (DTH) response to mesothelin, wherein the presence
of said response predicts a longer survival time than the absence
of said response.
97. The method of claim 96 wherein the vaccine comprises whole
tumor cells.
98. The method of claim 96 wherein the vaccine comprises pancreatic
tumor cells.
99. The method of claim 96 wherein the vaccine comprises ovarian
tumor cells.
100. The method of claim 96 wherein the vaccine comprises
mesothelioma cells.
101. A recombinant mouse cell line which comprises peritoneal cells
which have been transformed by HPV-16 E6 and E7 and an activated
oncogene wherein the cell line is capable of forming ascites and
tumors upon intraperitoneal injection into an immunocompetent
mouse.
102. The recombinant mouse cell line of claim 101 wherein the
activated oncogene is an activated c-Ha-ras.
103. The recombinant mouse cell line of claim 101 which expresses
mesothelin.
104. The recombinant mouse cell line of claim 101 which is
WF-3.
105. A mouse model comprising: a mouse which has been injected with
the recombinant mouse cell line of claim 101.
106. The mouse model of claim 105 which is immunocompetent.
107. A method of testing a substance to determine if it is a
potential drug for treating a cancer selected from the group
consisting of ovarian cancer, pancreatic cancer, mesothelioma, and
squamous cell carcinoma, comprising: contacting the mouse model of
claim 105 with a test substance; and determining if the test
substance causes regression of a tumor in the mouse model,
diminution of ascites volume in the mouse model, or longer survival
time in the mouse model.
108. A method of testing a substance to determine if it is a
potential drug for treating a cancer selected from the group
consisting of ovarian cancer, pancreatic cancer, mesothelioma, and
squamous cell carcinoma, comprising: contacting a mouse with a test
substance; injecting the mouse with the recombinant cell line of
claim 101, and determining if the test substance causes regression
of a tumor in the mouse, diminution of ascites volume in the mouse,
or longer survival time in the mouse.
109. The vaccine of claim 59, wherein the polypeptide is
mesothelin.
110. The method of claim 1, wherein the polypeptide is
mesothelin.
111. The method of claim 22, wherein the polypeptide is
mesothelin.
112. The vaccine of claim 67, wherein the polypeptide is
mesothelin.
Description
[0001] The contents of each of the following applications are
specifically incorporated herein: provisional U.S. Applications
Ser. No. 60/395,556, filed Jul. 12, 2002, 60/398,217, filed Jul.
24, 2002, Ser. No. 60/414,931, filed Sep. 30, 2002, and Ser. No.
60/475,783 filed Jun. 5, 2003.
[0003] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure, as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
[0004] The invention relates to the field of cancer therapeutics,
cancer prognosis, and anti-cancer drug development. In some aspects
it relates to mesothelin as a therapeutic target. In another aspect
it relates to developing other therapeutic targets.
BACKGROUND OF THE INVENTION
[0005] Transformation from a normal to a malignant cell involves
complex genetic and epigenetic changes, affecting a large number of
genes (1, 2). Many of these altered genes are translated into new,
altered, or overexpressed proteins that may represent candidate
targets for immune rejection. T cell screening of cDNA libraries
isolated from tumor cells, biochemical elution and purification of
major histocompatibility complex (MHC) bound antigens, and antibody
screening of phage display libraries (SEREX method) have greatly
facilitated the identification of tumor antigens, particularly
those expressed by malignant melanomas (3-13). As a result, there
are a number of antigen-specific vaccine approaches under clinical
development for this disease (3-6, 14). Unfortunately, these
antigen identification approaches have not been successful for
identifying antigens expressed by many other common cancers. The
major limitation has been the inability to generate patient-derived
T cell lines and clones that can be employed to identify immune
relevant tumor targets. Furthermore, T cell responses to specific
human tumor antigens have not yet been correlated with clinical
responses after immunotherapy.
[0006] The recent development of high throughput technologies that
can quantify gene expression in human tissues has led to the
identification of a large number of genes that are differentially
expressed in tumors relative to the normal tissue from which they
derive (15-18). These gene expression databases can be used as
initial filters upon which to apply a functional immune-based
screening strategy (19). A growing number of genes shown to be
differentially expressed in pancreatic adenocarcinomas using serial
analysis of gene expression (SAGE) have been tabulated and reported
(20-22). However, it is unclear which of these differentially
expressed genes are immunologically relevant for an anti-tumor
response. There is a need in the art for a way of identifying
immunologically relevant proteins among the proteins which are
differentially expressed in tumor and normal tissues.
BRIEF SUMMARY OF THE INVENTION
[0007] In a first embodiment a method is provided for inducing a
T-cell response to a tumor that overexpresses mesothelin relative
to normal tissue from which the tumor is derived. The tumor can be,
for example, an ovarian cancer, a pancreatic cancer, a
mesothelioma, or a squamous cell carcinoma. A vaccine comprising a
polypeptide comprising an MHC Class I- or Class II-binding epitope
of mesothelin is administered to a patient who has said tumor or
who has had said tumor removed. The patient can also be one who is
at risk of developing such a tumor. The epitope binds to an allelic
form of MHC class I or MHC class II which is expressed by the
patient. A T-cell response to mesothelin is thereby induced. The
vaccine does not comprise whole tumor cells. The polypeptide is
optionally mesothelin. The T-cell response may be a CD4.sup.+
T-cell response and/or a CD8.sup.+ T-cell response.
[0008] In a second embodiment a method is provided for inducing a
T-cell response to a tumor that overexpresses mesothelin relative
to normal tissue from which the tumor is derived. The tumor can be,
for example, an ovarian cancer, a pancreatic cancer, a
mesothelioma, or a squamous cell carcinoma. A vaccine comprising a
polynucleotide encoding a polypeptide comprising an MHC Class I- or
MHC Class II-binding epitope of mesothelin is administered to a
patient who has said tumor or who has had said tumor removed. The
patient can also be one who is at risk of developing such a tumor.
The epitope binds to an allelic form of MHC class I or class II
which is expressed by the patient. A T-cell response to mesothelin
is thereby induced. The vaccine does not comprise whole tumor
cells. The polypeptide encoded by the polynucleotide of the vaccine
is optionally mesothelin. The T-cell response may be a CD4.sup.+
T-cell response and/or a CD8.sup.+ T-cell response.
[0009] In a third embodiment a method is provided for identifying
immunogens useful as candidates for anti-tumor vaccines. A protein
is selected which is expressed by a tumor and which is minimally or
not expressed by normal tissue from which the tumor is derived.
Preferably the protein is expressed by a greater than 10% of tumor
isolates tested of a type of tumor. Lymphocytes of humans who have
been vaccinated with a vaccine which expresses the protein are
tested to determine if the lymphocytes comprise CD8.sup.+ T cells
or CD4.sup.+ T cells which are specific for the protein. The
presence of the CD8.sup.+ T cells or CD4.sup.+ T cells indicates
that the protein is a candidate for use as an anti-tumor
vaccine.
[0010] A fourth embodiment of the invention provides a method of
predicting future response to a tumor vaccine in a patient who has
received the tumor vaccine. Lymphocytes of the patient are tested
to determine if the lymphocytes comprise CD8.sup.+ T cells or
CD4.sup.+ T cells which are specific for an antigen in the vaccine.
The presence of said CD8.sup.+ T cells or CD4.sup.+ T cells
predicts a longer survival time than the absence of said CD8.sup.+
T cells or CD4.sup.+ T cells.
[0011] A fifth embodiment of the invention provides a vaccine which
induces a CD8.sup.+ T cell or CD4.sup.+ T cell response. The
vaccine comprises a polypeptide comprising an MHC Class I- or MHC
Class II-binding epitope of mesothelin. The epitope binds to an
allelic form of MHC class I or class II which is expressed by the
patient. A T-cell response to mesothelin is thereby induced. The
vaccine does not comprise whole tumor cells. The vaccine further
comprises a carrier for stimulating a T cell immune response. The
polypeptide is optionally mesothelin.
[0012] Another embodiment of the invention provides another vaccine
which induces a CD8.sup.+ T cell or CD4.sup.+ T cell response. The
vaccine comprises a polynucleotide encoding a polypeptide
comprising an MHC Class I- or MHC Class II-binding epitope of
mesothelin. The epitope binds to an allelic form of MHC class I or
class II which is expressed by the patient. A CD8.sup.+ T cell or
CD4.sup.+ T cell response to mesothelin is thereby induced. The
vaccine does not comprise whole tumor cells. The vaccine further
comprises a carrier for stimulating a T cell immune response. The
polypeptide encoded by the polynucleotide of the vaccine is
optionally mesothelin.
[0013] Another embodiment of the invention provides an isolated
polypeptide of 9 to 25 amino acid residues. The polypeptide
comprises an epitope selected from the group consisting of
SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID NO: 2); ELAVALAQK (SEQ
ID NO: 3); ALQGGGPPY (SEQ ID NO: 4); FYPGYLCSL (SEQ ID NO: 5); and
LYPKARLAF (SEQ ID NO: 6).
[0014] Yet another embodiment of the invention provides an antibody
that binds to an epitope selected from the group consisting of
SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID NO: 2); ELAVALAQK (SEQ
ID NO: 3); ALQGGGPPY (SEQ ID NO: 4); FYPGYLCSL (SEQ ID NO: 5); and
LYPKARLAF (SEQ ID NO: 6).
[0015] Yet another embodiment of the invention provides a CD8.sup.+
T cell or CD4.sup.+ T cell line that binds to MHC class I-peptide
complexes, wherein the peptide comprises an epitope selected from
the group consisting of SLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID
NO: 2); ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY (SEQ ID NO: 4);
FYPGYLCSL (SEQ ID NO: 5); and LYPKARLAF (SEQ ID NO: 6).
[0016] A tenth embodiment of the invention provides a method for
predicting future response to a tumor vaccine in a patient who has
received the vaccine. The tumor vaccine comprises at least one
T-cell epitope of mesothelin. The patient is tested to determine if
the patient has a delayed type hypersensitivity (DTH) response to
mesothelin, wherein the presence of said response predicts a longer
survival time than the absence of said response.
[0017] An eleventh embodiment of the invention provides a
recombinant mouse cell line which comprises peritoneal cells which
have been transformed by HPV-16 genes E6 and E7 and an activated
oncogene. The cell line is capable of forming ascites and tumors
upon intraperitoneal injection into an immunocompetent mouse.
[0018] Also provided is a mouse model which comprises a mouse which
has been injected with a recombinant mouse cell line. The
recombinant mouse cell line comprises peritoneal cells transfected
by HPV-16 genes E6 and E7 and an activated oncogene. The former
genes immortalize and the latter gene transforms. The cell line is
capable of forming ascites and tumors upon intraperitoneal
injection into an immunocompetent mouse.
[0019] Another aspect of the invention is a method of testing a
substance to determine if it is a potential drug for treating a
cancer. The cancer may be, for example, an ovarian cancer, a
pancreatic cancer, a mesothelioma, or a squamous cell carcinoma. A
test substance is contacted with a mouse model. The mouse model
comprises a mouse that has been injected with a recombinant mouse
cell line. The injection can be accomplished before or after the
test substance is contacted with the mouse. The recombinant mouse
cell line comprises peritoneal cells which have been transfected by
HPV-16 genes E6 and E7 and an activated oncogene. The cell line is
capable of forming ascites and tumors upon intraperitoneal
injection into an immunocompetent mouse. One determines whether the
test substance causes delay of tumor formation or regression of a
tumor in the mouse model, diminution of ascites volume in the mouse
model, or longer survival time in the mouse model. Any of these
effects indicates that the test substance is a potential drug for
treating cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A to 1F show a T2 binding assay that identifies
mesothelin and PSCA protein derived epitopes that bind to HLA-A2,
A3, and A24 molecules. T2 cells were pulsed with 100-400 micrograms
of peptide overnight at room temperature before analysis by flow
cytometry. FIG. 1A. T2 cells expressing HLA-A2 and pulsed with
either: no peptide (black line), a Mesothelin A1309-318 binding
peptide (green line), Mesothelin A220-29 (pink line), and
Mesothelin A2530-539 (blue line). Peptide pulsed cells were stained
with an unlabeled mouse anti-HLA class I molecule monoclonal
antibody W6/32 and a goat-anti-mouse FITC-labeled IgG2a secondary
antibody. FIG. 1B. T2 cells genetically modified to express A3 and
pulsed with either: no peptide (black line), Mesothelin A1309-318
binding peptide (green line), Mesothelin A383-92 (pink line), and
Mesothelin A3225-234 (blue line). Peptide pulsed cells were stained
with an unlabeled mouse anti-human HLA-A3 specific monoclonal
antibody GAPA3 and a FITC-labeled IgG2a secondary antibody. FIG.
1C. T2 cells genetically modified to express A24 and pulsed with
either: no peptide (black line), Mesothelin A1309-318 peptide
(green line), Mesothelin A24435-444 (pink line), and Mesothelin
A24475-484 (blue line). Peptide pulsed cells were stained with an
unlabeled pan-HLA antibody W6/32 and a FITC-labeled IgG2a secondary
antibody. FIG. 1D. T2 cells expressing HLA-A2 and pulsed with
either: Mesothelin A1309-318 binding peptide (green line), PSCA
A25-13 (pink line), PSCA A214-22 (blue line), PSCA A2108-116
(orange line) and PSCA A243-51 (red line). Peptide pulsed cells
were stained with an unlabeled mouse anti-HLA class I molecule
monoclonal antibody W6/32 and a goat-anti-mouse FITC-labeled IgG2a
secondary antibody. FIG. 1E. T2 cells genetically modified to
express A3 and pulsed with either: Mesothelin A1309-318 binding
peptide (green line), PSCA A399-107 (pink line), A35-13 (blue
line), A314-22 (orange line), A3109-117 (purple line), A343-51 (red
line), and PSCA A320-28 (yellow line). Peptide pulsed cells were
stained with an unlabeled mouse anti-human HLA-A3 specific
monoclonal antibody GAPA3 and a FITC-labeled IgG2a secondary
antibody. FIG. 1F. T2 cells genetically modified to express A24 and
pulsed with either: Mesothelin A1309-318 peptide (green line), PSCA
A2476-84 (pink line), PSCA A24108-116 (blue line), PSCA A2499-107
(orange line), PSCA A24109-117 (purple line), and PSCA A2477-85
(red line). Peptide pulsed cells were stained with an unlabeled
pan-HLA antibody W6/32 and a FITC-labeled IgG2a secondary
antibody.
[0021] FIGS. 2A to 2D shows an ELISPOT analysis of CD8+ T cells
from PBMCs which demonstrates post-vaccination induction of
mesothelin-specific T cells in three DTH responders but not in 11
non-DTH responders who received an allogeneic GM-CSF-secreting
tumor vaccine for pancreatic cancer. FIG. 2A. ELISPOT analysis of
PBL from two patients who were HLA-A3 positive; FIG. 2B. ELISPOT
analysis of PBL from two patients who were HLA-A 2 and HLA-A3
positive; FIG. 2C. ELISPOT analysis of PBL from two patients who
were HLA-A24 positive. FIG. 2D. ELISPOT analysis was performed on
PBL from all 14 patients who were treated on the phase I allogeneic
GM-CSF secreting pancreatic tumor vaccine study (28). ELISPOT
analysis for IFN-.gamma.-expressing cells was performed using PBMC
that were isolated on the day prior to vaccination or 28 days
following the first vaccination. Lymphocytes were isolated by
ficoll-hypaque separation and stored frozen in liquid nitrogen
until the day of assay. CD8+ T cell enrichment was performed prior
to analysis. T2-A3 cells were pulsed with the two mesothelin
derived epitopes MesoA3(83-92) (open squares), MesoA3(225-234)
(closed circle) and HIV-NEFA3 (94-103) (open triangle). T2-A2 cells
were pulsed with the two mesothelin derived epitopes MesoA2(20-29)
(closed squares), MesoA2(530-539) (open circle), and
HIV-GAG(77-85), (closed triangle). T2-A24 cells were pulsed with
the two mesothelin derived epitopes MesoA24 (435-444) (open
diamond), MesoA24(475-484) (closed diamond), and tyrosinase
A24(206-214) (star). All DTH responders are represented by red
lines, and DTH non-responders are represented by black lines. For
the detection of nonspecific background, the number of IFN-.gamma.
spots for CD8+ T cells specific for the irrelevant control peptides
were counted. The HLA-A2 binding HIV-GAG protein derived epitope
(SLYNTVATL; SEQ ID NO:7), the HLA-A3 binding HIV-NEF protein
derived epitope (QVPLRPMTYK; SEQ ID NO: 8), and the HLA-A24 binding
tyrosinase protein derived epitope (AFLPWHRLF; SEQ ID NO: 9) were
used as negative control peptides in these assays. Data represents
the average of each condition assayed in triplicate and standard
deviations were less than 5%. Plotted are the # of human interferon
gamma (hIFNg) spots per 105 CD8+ T cells. Analysis of each
patient's PBL was performed at least twice.
[0022] FIG. 3 shows an ELISPOT analysis performed to assess the
recognition of the influenza matrix protein HLA-A2 binding epitope
M1 (GILGFVFTL; SEQ ID NO: 10) on PBL from all 5 patients on the
study who were HLA-A2 positive (4 non-DTH responders and 1 DTH
responder). This analysis was performed on the same PBL samples
described for FIGS. 2A to 2D above. The DTH responders are
represented by red lines, and the DTH non-responders are
represented by black lines. For the detection of nonspecific
background, the number of IFN-.gamma. spots for CD8+ T cells
specific for the irrelevant control peptides were counted. The
HLA-A2 binding HIV-GAG protein derived epitope (SLYNTVATL; SEQ ID
NO: 7), the HLA-A3 binding HIV-NEF protein derived epitope
(QVPLRPMTYK; SEQ ID NO: 8), and the HLA-A24 binding melanoma
tyrosinase protein derived epitope (AFLPWHRLF; SEQ ID NO: 9) were
used as negative control peptides in these assays. Data represents
the average of each condition assayed in triplicate and standard
deviations were less than 5%. Plotted are the # of human interferon
gamma (hIFNg) spots per 105 CD8+ T cells. Analysis of each
patient's PBL was performed at least twice and all ELISPOT assays
were performed in a blinded fashion.
[0023] FIG. 4A to 4D shows an ELISPOT analysis of CD8+ T cells from
PBMCs. No post-vaccination induction was observed of PSCA-specific
T cells in DTH responders or non-DTH responders who received an
allogeneic GM-CSF-secreting tumor vaccine for pancreatic cancer.
FIG. 4A. ELISPOT analysis of PBL from two patients who were HLA-A3
positive; FIG. 4B. ELISPOT analysis of PBL from two patients who
were HLA-A 2 and HLA-A3 positive; FIG. 4C. ELISPOT analysis of PBL
from two-patients who were HLA-A24 positive. FIG. 4D. ELISPOT
analysis of PBL from eight patients who were non-responders.
ELISPOT analysis for IFN-.gamma.-expressing cells was performed
using PBMC that were isolated on the day prior to vaccination or 28
days following each of the vaccination. Lymphocytes were isolated
by ficoll-hypaque separation and stored frozen in liquid nitrogen
until the day of assay. CD8+ T cell enrichment was performed prior
to analysis. T2-A3 cells were pulsed with the six PSCA derived
epitopes: PSCAA3(7-15) (closed squares), PSCAA3(52-60) (closed
diamond), PSCAA3(109-117) (closed triangle), PSCAA3(43-51) (open
square), PSCAA3(20-28) (open diamond), and PSCAA3(99-107) (open
triangle). Negative HIV-NEFA3 (94-103) values were subtracted out.
T2-A2 cells were pulsed with the three PSCA derived epitopes:
PSCAA2(5-13) (closed squares), PSCAA2(14-22) (closed diamonds),
PSCAA2(108-116) (closed triangles). Negative HIV-GAG(77-85) values
were subtracted out. T2-A24 cells were pulsed with the five PSCA
derived epitopes: PSCAA24(76-84) (closed diamond), PSCAA24(77-85)
(star), PSCAA24(109-117) (closed triangles), PSCAA24(108-116)
(closed circle), and PSCAA24(99-107) (open triangle). Negative
Tyrosinase A24(206-214) values were subtracted. All DTH responders
are represented by red lines, and DTH non-responders are
represented by black lines. For the detection of nonspecific
background, the number of IFN-.gamma. spots for CD8+ T cells
specific for the irrelevant control peptides were counted. The
HLA-A2 binding HIV-GAG protein derived epitope (SLYNTVATL; SEQ ID
NO: 7), the HLA-A3 binding HIV-NEF protein derived epitope
(QVPLRPMTYK; SEQ ID NO: 8), and the HLA-A24 binding tyrosinase
protein derived epitope (AFLPWHRLF; SEQ ID NO: 9) were used as
negative control peptides in these assays. Data represents the
average of each condition assayed in triplicate and standard
deviations were less than 5%. The number of human interferon gamma
(hIFNg) spots per 105 CD8+ T cells is plotted. Analysis of each
patient's PBL was performed at least twice.
[0024] FIG. 5 shows expression of surface Mesothelin and PSCA on
Panc 6.03 and Panc 10.05 vaccine lines. The pancreatic tumor
vaccine lines Panc 6.03 (top two panels) and Panc 10.05 (bottom two
panels) were analyzed by flow cytometry for their levels of surface
mesothelin and PSCA using the mesothelin specific monoclonal
antibody CAK1 (left panels) and the PSCA specific monoclonal
antibody 1G8 (right panels) as the primary antibody and goat
anti-mouse IgG FITC as the secondary antibody. The solid line
represents the isotype control, the green shaded area represents
mesothelin staining, and the pink shaded area PSCA staining.
[0025] FIGS. 6A to 6C show that mesothelin-specific CD8+ T cells
are detected following multiple vaccinations with an allogeneic
GM-CSF secreting tumor vaccine in DTH-responders but not in non-DTH
responders. FIG. 6A. ELISPOT analysis of PBL from two patients who
were HLA-A3 positive; FIG. 6B. ELISPOT analysis of PBL from two
patients who were HLA-A 2 and HLA-A3 positive; FIG. 6C. ELISPOT
analysis of PBL from two patients who were HLA-A24 positive.
ELISPOT analysis for IFN-.gamma.-expressing cells was performed
using PBMC that were isolated on the day prior to vaccination or 28
days following each vaccination as described in FIG. 2A to 2D. Each
peptide has the same symbol code as described for FIG. 2A to 2D.
The DTH responders are represented by the red lines and the DTH
non-responders are represented by the black lines. For the
detection of nonspecific background, the number of IFN-.gamma.
spots for CD8+ T cells specific for the irrelevant control peptides
were counted. The HLA-A2 binding HIV-GAG protein derived epitope
(SLYNTVATL; SEQ ID NO: 7), the HLA-A3 binding HIV-NEF protein
derived epitope (QVPLRPMTYK; SEQ ID NO: 8), and the HLA-A24 binding
melanoma tyrosinase protein derived epitope (AFLPWHRLF; SEQ ID NO:
9) were used as negative control peptides in these assays. Data
represent the average of each condition assayed in triplicate and
standard deviations were less than 5%. Plotted are the number of
human interferon gamma (hIFNg) spots per 105 CD8+ T cells. Analysis
of each patient's PBL was performed at least twice.
[0026] FIGS. 7A to 7C show the generation and characterization of
an ascitogenic ovarian tumor cell line (WF-3) in mice. WF-3 tumor
cells were injected into C57BL/6 mice intraperitoneally at a dose
of 1.times.10.sup.5 cells/mouse. Mice were euthanized 4 weeks after
tumor challenge (7A) Representative gross picture to demonstrate
ascites formation in mice. Note: Mice developed significant ascites
with an increase in abdominal girth 4 weeks after tumor challenge.
(7B) Hematoxylin and eosin staining of the explanted tumors viewed
at 90.times. magnification. The tumors displayed a papillary
configuration, morphologically consistent with tumors derived from
the peritoneum or ovaries. Tumors viewed at 400.times.
magnification. The inset displays the features of a WF-3 tumor cell
in greater detail.
[0027] FIGS. 8A and 8B show MHC class I (FIG. 8A) and MHC class II
(FIG. 8B) presentation on the mouse WF-3 tumor cells. WF-3 tumor
cells were harvested, trypsinized, washed, and resuspended in
FACSCAN buffer. Anti-H2 Kb/H-2D monoclonal antibody or anti-I-Ab
monoclonal antibody was added, followed by flow cytometry analysis
to detect MHC class I and class II expression on WF-3 tumor cells.
(8A) WF-3 tumor cells were positive for MHC class I presentation
(thick line) compared to the MHC class I-negative control (thin
line). (8B) WF-3 tumor cells were negative for MHC class II
presentation. The thin line indicates staining of the MHC class
II-negative control.
[0028] FIGS. 9A to 9B show the effect of WF-3 tumor dose on ascites
formation in two independent trials shown in FIG. 9A and FIG. 3B.
WF-3 tumor cells were injected into C57BL/6 mice intraperitoneally
at various doses (1.times.10.sup.4, 5.times.10.sup.4,
1.times.10.sup.5, and 1.times.10.sup.6 cells/mouse). Mice were
monitored twice a week for ascites formation and tumor growth.
Note: All of the mice injected with 5.times.10.sup.4,
1.times.10.sup.5, and 1.times.10.sup.6 cells intraperitoneally,
developed ascites and tumor growth within 30 days. 20% of mice
injected with 1.times.10.sup.4 cells were tumor-free without
ascites formation after 90 days of tumor injection. The data are
from one representative experiment of two performed.
[0029] FIG. 10 shows expression of murine mesothelin in WF-3 tumor
cells demonstrated by RT-PCR with gel electrophoresis. FIG. 10.
RT-PCR. RT-PCR was performed using the Superscript One-Step.RT-PCR
Kit (Gibco, BRL) and a set of primers:
5'-CCCGAATTCATGOCCTTGCCAACAGCTCGA-3' (SEQ ID NO: 11) and
5'-TATGAATCCGCTCAGCCTTAAAGCTGGGAG-3' (SEQ ID NO: 12). Lane 1, size
marker. Lane 2, RNA from W-3 cells and Lane 3, RNA from
mesothelin-negative B 16 tumor cells. Specific amplification
(indicated by an arrow) was observed in Lane 2 (WF-3 cells) but not
in the Lane 3 (B16 cells).
[0030] FIG. 11 shows in vivo tumor protection experiments against
WF-3 tumor growth using mesothelin-specific DNA vaccines. Mice
received a booster with the same dose one week later, followed by
intraperitoneal challenge with 5.times.10.sup.4 WF-3 cells/mouse
one week afterward. Ascites, formation in mice was monitored by
palpation and inspection. Mice were, sacrificed at day 90. Note:
Vaccination with pcDNA3-mesothelin DNA resulted in a significantly
higher percentage of tumor-free mice than vaccination with other
DNA. (P<0.00 1). Results shown here are from one representative
experiment of two performed.
[0031] FIG. 12 shows CTL assays which demonstrate specific lysis
induced by vaccination with mesothelin-specific DNA vaccines. Mice
(5 per group) were imunized with various DNA vaccines
intradermally. Mice received a booster with the same dose one week
later. Splenocytes from mice were pooled 14 days after vaccination.
To perform the cytotoxicity assay, splenocytes were cultured with
mesothelin protein-for 6 days and used as effector cells. WF-3
tumor cells served as target cells. WF-3 cells were mixed with
splenocytes at various E:T ratios. Cytolysis was determined by
quantitative measurements of LDH. Note: The pcDNA3-mesothelin DNA
vaccine generated a significantly higher percentage of specific
lysis than the other DNA vaccines (P<0.001). The data presented
in this figure are from one representative experiment of two
performed.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The recent development of high-throughput technologies that
quantify gene expression has led to the identification of many
genes that are differentially expressed in human cancers. However,
differential expression does not, on its own, indicate that an
antigen is a therapeutic target. Therefore, a functional
immunologic screen was applied to a SAGE gene expression database
in order to identify immunologically relevant tumor antigens. We
previously reported the association of prolonged disease-free
survival and in vivo induction of anti-tumor immunity in three of
fourteen patients receiving a pancreatic tumor vaccine. Here we
identify mesothelin as a tumor antigen recognized by uncultured
CD8+ T cells isolated from these vaccinated patients. Moreover, the
induction of mesothelin-specific T cells was not found in the
eleven other patients who received the same vaccine but relapsed.
To validate mesothelin as a tumor antigen, we show that none of the
patients respond to another differentially expressed gene product,
prostate stem cell antigen. These data identify mesothelin as an in
vitro marker of vaccine induced immune responses that correlate
with clinical anticancer responses. The inventors also describe a
functional genomic approach for identifying and validating other
immunologically relevant human tumor antigens.
[0033] The vaccines of the present invention can be administered by
any means known in the art for inducing a T cell cytolytic
response. These means include oral administration, intravenous
injection, percutaneous scarification, subcutaneous injection,
intramuscular injection, and intranasal administration. The
vaccines can be administered intradermally by gene gun. Gold
particles coated with DNA may be used in the gene gun. Other
inoculation routes as are known in the art can be used.
[0034] Additional agents which are beneficial to raising a
cytolytic T cell response may be used as well. Such agents are
termed herein carriers. These include, without limitation, B7
costimulatory molecule, interleukin-2, interferon-.gamma., GM-CSF,
CTLA-4 antagonists, OX-40/OX-40 ligand, CD40/CD40 ligand,
sargramostim, levamisol, vaccinia virus, Bacille Calmette-Guerin
(BCG), liposomes, alum, Freund's complete or incomplete adjuvant,
detoxified endotoxins, mineral oils, surface active substances such
as lipolecithin, pluronic polyols, polyanions, peptides, and oil or
hydrocarbon emulsions. Carriers for inducing a T cell immune
response which preferentially stimulate a cytolytic T cell response
versus an antibody response are preferred, although those that
stimulate both types of response can be used as well. In cases
where the agent is a polypeptide, the polypeptide itself or a
polynucleotide encoding the polypeptide can be administered. The
carrier can be a cell, such as an antigen presenting cell (APC) or
a dendritic cell. Antigen presenting cells include such cell types
aas macrophages, dendritic cells and B cells. Other professional
antigen-presenting cells include monocytes, marginal zone Kupffer
cells, microglia, Langerhans' cells, interdigitating dendritic
cells, follicular dendritic cells, and T cells. Facultative
antigen-presenting cells can also be used. Examples of facultative
antigen-presenting cells include astrocytes, follicular cells,
endothelium and fibroblasts. The carrier can be a bacterial cell
that is transformed to express the polypeptide or to deliver a
polynucleoteide which is subsequently expressed in cells of the
vaccinated individual. Adjuvants, such as aluminum hydroxide or
aluminum phosphate, can be added to increase the ability of the
vaccine to trigger, enhance, or prolong an immune response.
Additional materials, such as cytokines, chemokines, and bacterial
nucleic acid sequences, like CpG, are also potential adjuvants.
Other representative examples of adjuvants include the synthetic
adjuvant QS-21 comprising a homogeneous saponin purified from the
bark of Quillaja saponaria and Corynebacterium parvum (McCune et
al., Cancer, 1979; 43:1619). It will be understood that the
adjuvant is subject to optimization. In other words, the skilled
artisan can engage in routine experimentation to determine the best
adjuvant to use.
[0035] Further additives, such as preservatives, stabilizers,
adjuvants, antibiotics, and other substances can be used as well.
Preservatives, such as thimerosal or 2-phenoxy ethanol, can be
added to slow or stop the growth of bacteria or fungi resulting
from inadvertent contamination, especially as might occur with
vaccine vials intended for multiple uses or doses. Stabilizers,
such as lactose or monosodium glutamate (MSG), can be added to
stabilize the vaccine formulation against a variety of conditions,
such as temperature variations or a freeze-drying process.
[0036] Viral vectors can be used to administer polynucleotides
encoding a polypeptide comprising a mesothelin epitope. Such viral
vectors include vaccinia virus and avian viruses, such as Newcastle
disease virus. Others may be used as are known in the art.
[0037] One particular method for administering polypeptide vaccine
is by pulsing the polypeptide onto an APC or dendritic cell in
vitro. The polypeptide binds to MHC molecules on the surface of the
APC or dendritic cell. Prior treatment of the APCs or dendritic
cells with interferon-.gamma. can be used to increase the number of
MHC molecules on the APCs or dendritic cells. The pulsed cells can
then be administered as a carrier for the polypeptide. Peptide
pulsing is taught in Melero et al., Gene Therapy 7:1167 (2000).
[0038] Naked DNA can be injected directly into the host to produce
an immune response. Such naked DNA vaccines may be injected
intramuscularly into human muscle tissue, or through transdermal or
intradermal delivery of the vaccine DNA, typically using
biolistic-mediate gene transfer (i.e., gene gun). Recent reviews
describing the gene gun and muscle injection delivery strategies
for DNA immunization include Tuting, Curr. Opin. Mol. Ther. (1999)
1: 216-25, Robinson, Int. J. Mol. Med. (1999) 4: 549-55, and Mumper
and Ledbur, Mol. Biotechnol. (2001) 19: 79-95. Other possible
methods for delivering plasmid DNA includes electroporation and
iontophoreses.
[0039] Another possible gene delivery system comprises ionic
complexes formed between DNA and polycationic liposomes (see, e.g.,
Caplen et al. (1995) Nature Med. 1: 39). Held together by
electrostatic interaction, these complexes may dissociate because
of the charge screening effect of the polyelectrolytes in the
biological fluid. A strongly basic lipid composition can stabilize
the complex, but such lipids may be cytotoxic. Other possible
methods for delivering DNA includes electroporation and
iontophoreses.
[0040] The use of intracellular and intercellular targeting
strategies in DNA vaccines may further enhance the
mesothelin-specific antitumor effect. Previously, intracellular
targeting strategies and intercellular spreading strategies have
been used to enhance MHC class I or MHC class II presentation of
antigen, resulting in potent CD8+ or CD4+ T cell-mediated antitumor
immunity, respectively. For example, MHC class I presentation of a
model antigen, HPV-16 E7, was enhanced using linkage of
Mycobacterium tuberculosis heat shock protein 70 (HSP70) (Chen, et
al., (2000), Cancer Research, 60: 1035-1042), calreticulin (Cheng,
et al., (2001) J Clin Invest, 108:669-678) or the translocation
domain (domain II) of Pseudomonas aeruginosa exotoxin A (ETA(dII))
(Hung, et al., (2001) Cancer Research, 61: 3698-3703) to E7 in the
context of a DNA vaccine. To enhance MHC class II antigen
processing, the sorting signals of the lysosome associated membrane
protein (LAMP-1) have been linked to the E7 antigen, creating the
Sig/E7/LAMP-1 chimera (Ji, et al, (1999), Human Gene Therapy, 10:
2727-2740). To enhance further the potency of naked DNA vaccines,
an intercellular strategy that facilitates the spread of antigen
between cells can be used. This improves the potency of DNA
vaccines as has been shown using herpes simplex virus (HSV-1) VP22,
an HSV-1 tegument protein that has demonstrated the remarkable
property of intercellular transport and is capable of distributing
protein to many surrounding cells (Elliot, et al., (1997) Cell, 88:
223-233). Such enhanced intercellular spreading of linked protein,
results in enhancement of antigen-specific CD8+ T cell-mediated
immune responses and antitumor effect. Any such methods can be used
to enhance DNA vaccine potency against mesothlin-expressing
tumors.
[0041] Mesothelin is known to be expressed in ovarian cancer,
pancreatic cancer, mesothelioma, and squamous cell carcinomas
carcinomas of the esophagus, lung, and cervix. Thus the vaccines of
the invention are useful for treating at least these types of
tumors. Other tumors which express mesothelin can also be treated
similarly.
[0042] In one embodiment, the vaccines of the present invention
comprise a polypeptide comprising at least one MHC Class I-binding
epitope of mesothelin or at least one MHC Class II-binding epitope
of mesothelin. Alternatively, the vaccines of the present invention
optionally comprise a polynucleotide encoding a polypeptide
comprising at least one MHC Class I-binding epitope of mesothelin
or at least one MHC Class II-binding epitope of mesothelin.
Optionally, the polypeptides of the vaccines (or the p olypeptides
encoded by the polynucleotides of the vaccines) comprise a
plurality of MHC Class I-binding epitopes of mesothelin and/or MHC
Class II-binding epitopes of mesothelin. The multiple epitopes of
the polypeptides may bind the same or different MHC allelic
molecules. In one embodiment, the epitopes of the polypeptide bind
a diverse variety of MHC allelic molecules.
[0043] While MHC Class I-binding epitopes are effective in the
practice of the present invention, MHC Class II-binding epitopes
can also be used. The former are useful for activating CD8.sup.+ T
cells and the latter for activating CD4.sup.+ T cells. Publicly
available algorithms can be used to select epitopes that bind to
MHC class I and/or class II molecules. For example, the predictive
algorithm "BIMAS" ranks potential HLA binding epitopes according to
the predictive half-time disassociation of peptide/HLA complexes
(23). The "SYFPEITHI" algorithm ranks peptides according to a score
that accounts for the presence of primary and secondary HLA-binding
anchor residues (25). Both computerized algorithms score candidate
epitopes based on amino acid sequences within a given protein that
have similar binding motifs to previously published HLA binding
epitopes. Other algorithms can also be used to identify candidates
for further biological testing.
[0044] Polypeptides for immunization to raise a cytolytic T cell
response are optionally from 8 to 25 amino acid residues in length.
Although nonamers are specifically disclosed herein, any 8
contiguous amino acids of the nonamers can be used as well. The
polypeptides can be fused to other such epitopic polypeptides, or
they can be fused to carriers, such as B-7, interleukin-2, or
interferon-.gamma.. The fusion polypeptide can be made by
recombinant production or by chemical linkage, e.g., using
heterobifunctional linking reagents. Mixtures of polypeptides can
be used. These can be mixtures of epitopes for a single allelic
type of an MHC molecule, or mixtures of epitopes for a variety of
allelic types. The polypeptides can also contain a repeated series
of an epitope sequence or different epitope sequences in a
series.
[0045] The effectiveness of an MHC Class I-binding epitope of
mesothelin or an MHC Class II-binding epitope of mesothelin as an
immunogen in a vaccine can be evaluated by assessing whether a
peptide comprising the epitope is capable of activating
T-lymphocytes from an individual having a successful immunological
response to a tumor that overexpresses mesothelin (relative to
normal tissue from which the tumor is derived), when the peptide is
bound to an MHC molecule on an antigen-presenting cell and
contacted with the T-lymphocytes under suitable conditions and for
a time sufficient to permit activation of T-lymphocytes. A specific
example of such an assessment is illustrated in Examples 1-4,
below.
[0046] Multiple groups have cloned cDNAs encoding mesothelin, and
the sequences of the cDNA clones, as well as the sequence of the
encoded mesothelin polypeptides, have been reported in U.S. Pat.
No. 6,153,430, Chang and Pastan, Proc. Natl. Acad. Sci. USA,
93:136-140 (1996), Kojima et al., J. Biol. Chem., 270:21984-21990
(1995), and U.S. Pat. No. 5,723,318. These references, including
the sequences of the mesothelin-encoding nucleic acids,
corresponding mesothelin polypeptides, and fragments described
therein, are incorporated by reference herein in their entirety.
Mesothelin cDNA encodes a protein with a molecular weight of
approximately 69 kD, i.e., the primary translation product. The 69
kD form of mesothelin is proteolytically processed to form a 40 kD
mature mesothelin protein that is membrane-bound (Chang and Pastan
(1996)). The term "mesothelin" as used herein encompasses all
naturally occurring variants of the mesothelin, regardless of the
cell or tissue in which the protein is expressed. In one
embodiment, the mesothelin protein comprises one or more of the
following amino acid sequences: SLLFLLFSL (SEQ ID NO:1); VLPLTVAEV
(SEQ ID NO:2); ELAVALAQK (SEQ ID NO:3); ALQGGGPPY (SEQ ID NO:4);
FYPGYLCSL (SEQ ID NO:5); and LYPKARLAF (SEQ ID NO:6). For instance,
the mesothelin protein optionally comprises one, two, three, four,
or five of these epitopes. In another embodiment, the mesothelin
protein comprises each of the following amino acid sequences:
SLLFLLFSL (SEQ ID NO:1); VLPLTVAEV (SEQ ID NO:2); ELAVALAQK (SEQ ID
NO:3); ALQGGGPPY (SEQ ID NO:4); FYPGYLCSL (SEQ ID NO:5); and
LYPKARLAF (SEQ ID NO:6).
[0047] The vaccines of the invention optionally comprise mesothelin
or a polynucleotide encoding mesothelin. For instance, the vaccine
may comprise or encode the mature form of mesothelin, the primary
translation product, or the full-length translation product of the
mesothelin gene. In one embodiment, the vaccine comprises the cDNA
of mesothelin. In addition to the use of naturally occurring forms
of mesothelin (or polynucleotides encoding those forms),
polypeptides comprising fragments of mesothelin, or polynucleotides
encoding fragments of mesothelin may be used in the vaccines. The
polypeptides in the vaccines or encoded by polynucleotides of the
vaccines are optionally at least about 95%, at least about 90%, at
least about 85%, at least about 80%, at least about 75%, at least
about 70%, at least about 65%, at least about 60%, at least about
55%, or at least about 50% identical to mesothelin.
[0048] In an alternative embodiment of the invention, the
polypeptide of the vaccine or the polypeptide encoded by the
polynucleotide of the vaccine is not a naturally-occurring
mesothelin protein, such as the mature mesothelin protein, the
primary translation product of mesothelin, or the mature
megakaryocyte potentiating factor.
[0049] In one embodiment, the MHC Class I-binding epitope of
mesothelin comprises less than 15 amino acids, less than 14 amino
acids, less than 13 amino acids, less than 12 amino acids, or less
than 11 amino acids in length. In another embodiment, the MHC Class
I-binding epitope of mesothelin comprises at least seven or at
least eight contiguous amino acids present in a peptide selected
from the group consisting of SLLFLLFSL (SEQ ID NO:1), VLPLTVAEV
(SEQ ID NO:2), ELAVALAQK (SEQ ID NO:3), ALQGGGPPY (SEQ ID NO:4),
FYPGYLCSL (SEQ ID NO:5), and LYPKARLAF (SEQ ID NO:6). The MHC Class
I-binding epitope of mesothelin is at least 7 amino acids in
length, at least 8 amino acids in length, or at least 9 amino acids
in length.
[0050] In addition, the MHC Class I-binding epitopes of mesothelin
and the MHC Class II binding epitopes of mesothelin used in
vaccines of the present invention need not necessarily be identical
in sequence to the naturally occurring epitope sequences within
mesothelin. The naturally occurring epitope sequences are not
necessarily optimal peptides for stimulating a CTL response. See,
for example, (Parkhurst, M. R. et al., J. Immunol., 157:2539-2548,
(1996); Rosenberg, S. A. et al., Nat. Med., 4:321-327, (1998)).
Thus, there can be utility in modifying an epitope, such that it
more readily induces a CTL response. Generally, epitopes may be
modified at two types of positions. The epitopes may be modified at
amino acid residues that are predicted to interact with the MHC
molecule, in which case the goal is to create a peptide sequence
that has a higher affinity for the MHC molecule than does the
parent epitope. The epitopes can also be modified at amino acid
residues that are predicted to interact with the T cell receptor on
the CTL, in which case the goal is to create an epitope that has a
higher affinity for the T cell receptor than does the parent
epitope. Both of these types of modifications can result in a
variant epitope that is related to a parent eptiope, but which is
better able to induce a CTL response than is the parent
epitope.
[0051] Thus, the MHC Class I-binding epitopes of mesothelin
identified in the Examples below (SEQ ID NO:1-6), or identified by
application of the methods of the invention, and the MHC Class
II-binding epitopes of mesothelin identified by application of the
methods of the invention can be modified by the substitution of one
or more residues at different, possibly selective, sites within the
epitope sequence. Such substitutions may be of a conservative
nature, for example, where one amino acid is replaced by an amino
acid of similar structure and characteristics, such as where a
hydrophobic amino acid is replaced by another hydrophobic amino
acid. Even more conservative would be replacement of amino acids of
the same or similar size and chemical nature, such as where leucine
is replaced by isoleucine. In studies of sequence variations in
families of naturally occurring homologous proteins, certain amino
acid substitutions are more often tolerated than others, and these
are often show correlation with similarities in size, charge,
polarity, and hydrophobicity between the original amino acid and
its replacement, and such is the basis for defining "conservative
substitutions."
[0052] Conservative substitutions are herein defined as exchanges
within one of the following five groups: Group 1--small aliphatic,
nonpolar or slightly polar residues (Ala, Ser, Thr, Pro, Gly);
Group 2--polar, negatively charged residues and their amides (Asp,
Asn, Glu, Gln); Group 3--polar, positively charged residues (His,
Arg, Lys); Group 4--large, aliphatic, nonpolar residues (Met, Leu,
lie, Val, Cys); and Group 4--large, aromatic residues (Phe, Tyr,
Trp). An acidic amino acid might also be substituted by a different
acidic amino acid or a basic (i.e., alkaline) amino acid by a
different basic amino acid. Less conservative substitutions might
involve the replacement of one amino acid by another that has
similar characteristics but is somewhat different in size, such as
replacement of an alanine by an isoleucine residue.
[0053] Plasmids and viral vectors, for example, can be used to
express a tumor antigen protein in a host cell. The host cell may
be any prokaryotic or eukaryotic cell. Thus, for example, a
nucleotide sequence derived from the cloning of mesothelin
proteins, encoding all or a selected portion of the full-length
protein, can be used to produce a recombinant form of a mesothelin
polypeptide via microbial or eukaryotic cellular processes. The
coding sequence can be ligated into a vector and the loaded vector
can be used to transform or transfect hosts, either eukaryotic
(e.g., yeast, avian, insect or mammalian) or prokaryotic
(bacterial) cells. Such techniques involve standard procedures
which are well known in the art.
[0054] Typically, expression vectors used for expressing a
polypeptide, in vivo or in vitro contain a nucleic acid encoding an
antigen polypeptide, operably linked to at least one
transcriptional regulatory sequence. Regulatory sequences are
art-recognized and can be selected to direct expression of the
subject proteins in the desired fashion (time and place).
Transcriptional regulatory sequences are described, for example, in
Goeddel, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990).
[0055] Suitable vectors for the expression of a polypeptide
comprising HLA-binding epitopes include plasmids of the types:
pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived
plasmids, pBTac-derived plasmids and pUC-derived plasmids for
expression in prokaryotic cells, such as E. coli. Mammalian
expression vectors may contain both prokaryotic and eukaryotic
sequences in order to facilitate the propagation of the vector in
bacteria, and one or more eukaryotic transcription units that can
be expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and selection
in both prokaryotic and eukaryotic cells. Alternatively,
derivatives of viruses such as the bovine papillomavirus (BPV-1),
or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used
for transient expression of proteins in eukaryotic cells. Vaccinia
and avian virus vectors can also be used. The methods which may be
employed in the preparation of vectors and transformation of host
organisms are well known in the art. For other suitable expression
systems for both prokaryotic and eukaryotic cells, as well as
general recombinant procedures, see Molecular Cloning: A Laboratory
Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring
Harbor Laboratory Press: 1989) Chapters 16 and 17.
[0056] Other types of expression cassettes can also be used. For
instance, the references described below in regard to viral,
bacterial, and yeast vectors illustrate additional expression
vectors which may be used in the present invention.
[0057] In another embodiment of the invention, a polypeptide
described herein, or a polynucleotide encoding the polypeptide, is
delivered to a host organism in an immunogenic composition
comprising yeast. The use of live yeast DNA vaccine vectors for
antigen delivery has been reviewed recently and reported to be
efficacious in a mouse model using whole recombinant Saccharomyces
cerevisiae yeast expressing tumor or HIV-1 antigens (see Stubbs et
al. (2001) Nature Medicine 7: 625-29).
[0058] The use of live yeast vaccine vectors is known in the art.
Furthermore, U.S. Pat. No. 5,830,463, the contents of which are
incorporated herein by reference, describes particularly useful
vectors and systems for use in the instant invention. The use of
yeast delivery systems may be particularly effective for use in the
tumor/cancer vaccine methods and formulations of the invention as
yeast appears to trigger cell-mediated immunity without the need
for an additional adjuvant. Particularly preferred yeast vaccine
delivery systems are nonpathogenic yeast carrying at least one
recombinant expression system capable of modulating an immune
response.
[0059] Bacteria can also be used as carriers for the epitopes of
the present invention. Typically the bacteria used are mutant or
recombinant. The bacterium is optionally attenuated. For instance,
a number of bacterial species have been developed for use as
vaccines and can be used in the present invention, including, but
not limited to, Shigella flexneri, E. coli, Listeria monocytogenes,
Yersinia enterocolitica, Salmonella typhimurium, Salmonella typhi
or mycobacterium. The bacterial vector used in the immunogenic
composition may be a facultative, intracellular bacterial vector.
The bacterium may be used to deliver a polypeptide described herein
to antigen-presenting cells in the host organism. The use of live
bacterial vaccine vectors for antigen delivery has been reviewed
recently (Medina and Guzman (2001) Vaccine 19: 1573-1580; Weiss and
Krusch, (2001) Biol. Chem. 382: 533-41; and Darji et al. (2000)
FEMS Immunol and Medical Microbiology 27: 341-9). Furthermore, U.S.
Pat. Nos. 6,261,568 and 6,488,926, the contents of which are
incorporated herein by reference, describe systems useful for
cancer vaccines.
[0060] Bacterially mediated gene transfer is particularly useful in
genetic vaccination by intramuscular, intradermal, or oral
administration of plasmids; such vaccination leads to antigen
expression in the vaccinee. Furthermore, bacteria can provide
adjuvant effects and the ability to target inductive sites of the
immune system. Furthermore, bacterial vaccine vectors have almost
unlimited coding capacity. The use of bacterial carriers is often
associated with still other significant benefits, such as the
possibility of direct mucosal or oral delivery. Other direct
mucosal delivery systems (besides live viral or bacterial vaccine
carriers) which can be used include mucosal adjuvants, viral
particles, ISCOMs, liposomes, and microparticles.
[0061] Both attenuated and commensal microorganisms have been
successfully used as carriers for vaccine antigens. Attenuated
mucosal pathogens which may be used in the invention include: L.
monocytogenes, Salmonella spp., V. cholorae, Shigella spp.,
mycobacterium, Y. enterocolitica, and B. anthracis. Commensal
strains which can be used in the invention include: S. gordonii,
Lactobacillus spp., and Staphylococcus spp. The genetic background
of the carrier strain used in the formulation, the type of mutation
selected to achieve attenuation, and the intrinsic properties of
the immunogen can be adjusted to optimize the extent and quality of
the immune response elicited. The general factors to be considered
to optimize the immune response stimulated by the bacterial carrier
include: selection of the carrier; the specific background strain,
the attenuating mutation and the level of attenuation; the
stabilization of the attenuated phenotype and the establishment of
the optimal dosage. Other antigen-related factors to consider
include: intrinsic properties of the antigen; the expression
system, antigen-display form and stabilization of the recombinant
phenotype; co-expression of modulating molecules and vaccination
schedules.
[0062] Salmonella typhimurium can be used as a bacterial vector in
the immunogenic compositions of the invention. Use of this
bacterium as an effective vector for a vaccine has been
demonstrated in the art. For instance, the use of S. typhimurium as
an attenuated vector for oral somatic transgene vaccination has
been described (see Darji et al. (1997) Cell 91: 765-775; and Darji
et al. (2000) FEMS Immun and Medical Microbiology 27: 341-9).
Indeed most knowledge of bacteria-mediated gene transfer has been
acquired using attenuated S. typhimurium as carrier. Two
metabolically attenuated strains that have been used include S.
typhimurium aroA, which is unable to synthesize aromatic amino
acids, and S. typhimurium 22-11, which is defective in purine
metabolism. Several antigens have been expressed using these
carriers: originally, listeriolysin and actA (two virulence factors
of L. monocytogenes) and beta-galactosidase (.beta.-gal) of E. coli
were successfully tested. Cytotoxic and helper T cells as well as
specific antibodies could be detected against these antigens
following oral application of a single dose of the recombinant
salmonella. In addition, immunization with Salmonella carrying a
listeriolysin-encoding expression plasmid elicited a protective
response against a lethal challenge with L. monocytogenes. Oral
transgene vaccination methodology has now been extended to include
protective responses in herpes simplex virus 2 and hepatitis B
infection models, with cell-mediated immune responses detected at
the mucosal level.
[0063] In tumor models using .beta.-gal as a surrogate tumor
antigen, partial protective immunity against an aggressive
fibrosarcoma was induced by orally administering Salmonella
carrying a .beta.-gal-encoding plasmid (see Paglia et al. (1998)
Blood 92: 3172-76). In similar experiments using a
.beta.-gal-expressing transfectant of the murine renal cell
carcinoma line RENCA, Zller and Christ (Woo et al. (2001) Vaccine
19: 2945-2954) demonstrated superior efficacy when the
antigen-encoding plasmid was delivered in bacterial carriers as
opposed to using naked DNA. Interestingly, Salmonella can be used
to induce a tumor growth retarding response against the murine
melanoma B16; the Salmonella carry minigenes encoding epitopes of
the autologous tumor antigens gp100 and TRP2 fused to ubiquitin.
This suggests that under such circumstances peripheral tolerance
towards autologous antigens can be overcome. This was confirmed by
the same group (Lode et al. (2000) Med Ped Oncol 35: 641-646 using
similar constructs of epitopes of tyrosine hydroxylase as
autologous antigen in a murine neuroblastoma system. Furthermore,
these findings were recently extended by immunizing mice that were
transgenic for human carcinogenic antigen (hCEA) using a plasmid
encoding a membrane-bound form of complete hCEA. In this case, a
hCEA-expressing colon carcinoma system was tested and protection
against a lethal challenge with the tumor could be improved by
systemic application of interleukin 2 (IL-2) as adjuvant during the
effector phase (see Xiang et al. (2001) Clin Cancer Res 7:
856s-864s).
[0064] Another bacterial vector which may be used in the
immunogenic compositions described herein is Salmonella typhi. The
S. typhi strain commonly used for immunization--Ty21a galE--lacks
an essential component for cell-wall synthesis. Recently developed
improved strains include those attenuated by a mutation in guaBA,
which encodes an essential enzyme of the guanine biosynthesis
pathway (Pasetti et al., Infect. Immun. (2002) 70:4009-18; Wang et
al., Infect. Immun. (2001) 69:4734-41; Pasetti et al., Clin.
Immunol. (1999) 92:76-89). Additional references describing the use
of Salmonella typhi and/or other Salmonella strains as delivery
vectors for DNA vaccines include the following: Lundin, Infect.
Immun. (2002) 70:5622-7; Devico et al., Vaccine, (2002) 20:1968-74;
Weiss et al., Biol. Chem. (2001) 382:533-41; and Bumann et al.,
FEMS Immunol. Med. Microbiol. (2000) 27:357-64.
[0065] The vaccines and immunogenic compositions of the present
invention can employ Shigella flexneri as a delivery vehicle. S.
flexneri represents the prototype of a bacterial DNA transfer
vehicle as it escapes from the vacuole into the cytosol of the host
cell. Several attenuated mutants of S. flexneri have been used
successfully to transfer DNA to cell lines in vitro. Auxotrophic
strains were defective in cell-wall synthesis (Sizemore et al.
(1995) Science 270: 299-302 and Courvalin et al. (1995) C R Acad
Sci Ser III, 318: 1207-12), synthesis of aromatic amino acids
(Powell et al. (1996) Vaccines 96: Molecular Approaches to the
Control of Infectious Disease; Cold Spring Harbor Laboratory Press)
or synthesis of guanine nucleotides (Anderson et al. (2000) Vaccine
18: 2193-2202).
[0066] The vaccines and immunogenic compositions of the present
invention can comprise Listeria monocytogenes (Portnoy et al,
Journal of Cell Biology, 158:409-414 (2002); Glomski et al.,
Journal of Cell Biology, 156:1029-1038 (2002)). The ability of L.
monocytogenes to serve as a vaccine vector has been reviewed in
Wesikirch, et al., Immunol. Rev. 158:159-169 (1997). Strains of
Listeria monocytogenes have recently been developed as effective
intracellular delivery vehicles of heterologous proteins providing
delivery of antigens to the immune system to induce an immune
response to clinical conditions that do not permit injection of the
disease-causing agent, such as cancer (U.S. Pat. No. 6,051,237;
Gunn et al., J. Of Immunology, 167:6471-6479 (2001); Liau, et al.,
Cancer Research, 62: 2287-2293 (2002); U.S. Pat. No. 6,099,848; WO
99/25376; and WO 96/14087) and HIV (U.S. Pat. No. 5,830,702). A
recombinant L. monocytogenes vaccine expressing an lymphocytic
choriomeningitis virus (LCMV) antigen has also been shown to induce
protective cell-mediated immunity to the antigen (Shen et al.,
Proc. Natl. Acad. Sci. USA, 92: 3987-3991 (1995).
[0067] As a facultative intracellular bacterium, L. monocytogenes
elicits both humoral and cell-mediated immune responses. Following
entry of Listeria into a cell of the host organism, the Listeria
produces Listeria-specific proteins that enable it to escape from
the phagolysosome of the engulfing host cell into the cytosol of
that cell. Here, L. monocytogenes proliferates, expressing proteins
necessary for survival, but also expressing heterologous genes
operably linked to Listeria promoters. Presentation of peptides of
these heterologous proteins on the surface of the engulfing cell by
MHC proteins permit the development of a T cell response. Two
integration vectors that are useful for introducing heterologous
genes into the bacteria for use as vaccines include pL1 and pL2 as
described in Lauer et al., Journal of Bacteriology, 184: 4177-4186
(2002).
[0068] In addition, attenuated forms of L. monocytogenes useful in
immunogenic compositions have been produced. The ActA protein of L.
monocytogenes is sufficient to promote the actin recruitment and
polymerization events responsible for intracellular movement. A
human safety study has reported that oral administration of an
actA/plcB-deleted attenuated form of Listeria monocytogenes caused
no serious sequelae in adults (Angelakopoulos et al., Infection and
Immunity, 70:3592-3601 (2002)). Other types of attenuated forms of
L. monocytogenes have also been described (see, for example, WO
99/25376 and U.S. Pat. No. 6,099,848, which describe auxotrophic,
attenuated strains of Listeria that express heterologous
antigens).
[0069] Yersinia enterocolitica is another intraceullular bacteria
that can optionally be used as a bacterial vector in immunogenic
compositions of the present invention. The use of attenuated
strains of Yersini enterocolitica as vaccine vectors is described
in PCT Publication WO 02/077249.
[0070] In further embodiments of the invention, the immunogenic
compositions of the invention comprise mycobacterium, such as
Bacillus Calmette-Guerin (BCG). The Bacillus of Calmette and Guerin
has been used as a vaccine vector in mouse models (Gicquel et al.,
Dev. Biol. Stand 82:171-8 (1994)). See also, Stover et al., Nature
351: 456-460 (1991).
[0071] Alternatively, viral vectors can be used. The viral vector
will typically comprise a highly attenuated, non-replicative virus.
Viral vectors include, but are not limited to, DNA viral vectors
such as those based on adenoviruses, herpes simplex virus, avian
viruses, such as Newcastle disease virus, poxviruses such as
vaccinia virus, and parvoviruses, including adeno-associated virus;
and RNA viral vectors, including, but not limited to, the
retroviral vectors. Vaccinia vectors and methods useful in
immunization protocols are described in U.S. Pat. No. 4,722,848.
Retroviral vectors include murine leukemia virus, and lentiviruses
such as human immunodeficiency virus. Naldini et al. (1996) Science
272:263-267. Replication-defective retroviral vectors harboring a
polynucleotide of the invention as part of the retroviral genome
can be used. Such vectors have been described in detail. (Miller,
et al. (1990) Mol. Cell Biol. 10:4239; Kolberg, R. (1992) J. NIH
Res. 4:43; Cornetta, et al. (1991) Hum. Gene Therapy 2:215).
[0072] Adenovirus and adeno-associated virus vectors useful in this
invention may be produced according to methods already taught in
the art. (See, e.g., Karlsson, et al. (1986) EMBO 5:2377; Carter
(1992) Current Opinion in Biotechnology 3:533-539; Muzcyzka (1992)
Current Top. Microbiol. Immunol. 158:97-129; Gene Targeting: A
Practical Approach (1992) ed. A. L. Joyner, Oxford University
Press, NY). Several different approaches are feasible.
[0073] Alpha virus vectors, such as Venezuelan Equine Encephalitis
(VEE) virus, Semliki Forest virus (SFV) and Sindbis virus vectors,
can be used for efficient gene delivery. Replication-deficient
vectors are available. Such vectors can be administered through any
of a variety of means known in the art, such as, for example,
intranasally or intratumorally. See Lundstrom, Curr. Gene Ther.
2001 1:19-29.
[0074] Additional references describing viral vectors which could
be used in the methods of the present invention include the
following: Horwitz, M. S., Adenoviridae and Their Replication, in
Fields, B., et al. (eds.) Virology, Vol. 2, Raven Press New York,
pp. 1679-1721, 1990); Graham, F. et al., pp. 109-128 in Methods in
Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols,
Murray, E. (ed.), Humana Press, Clifton, N.J. (1991); Miller, et
al. (1995) FASEB Journal 9:190-199, Schreier (1994) Pharmaceutica
Acta Helvetiae 68:145-159; Schneider and French (1993) Circulation
88:1937-1942; Curiel, et al. (1992) Human Gene Therapy 3:147-154;
WO 95/00655; WO 95/16772; WO 95/23867; WO 94/26914; WO 95/02697
(Jan. 26, 1995); and WO 95/25071.
[0075] In another form of vaccine, DNA is complexed with liposomes
or ligands that often target cell surface receptors. The complex is
useful in that it helps protect DNA from degradation and helps
target plasmid to specific tissues. The complexes are typically
injected intravenously or intramuscularly.
[0076] Polynucleotides used as vaccines can be used in a complex
with a colloidal dispersion system. A colloidal system includes
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. The preferred colloidal system of
this invention is a lipid-complexed or liposome-formulated DNA. In
the former approach, prior to formulation of DNA, e.g., with lipid,
a plasmid containing a transgene bearing the desired DNA constructs
may first be experimentally optimized for expression (e.g.,
inclusion of an intron in the 5' untranslated region and
elimination of unnecessary sequences (Felgner, et al., Ann NY Acad
Sci 126-139, 1995). Formulation of DNA, e.g., with various lipid or
liposome materials, may then be effected using known methods and
materials and delivered to the recipient mammal. See, e.g.,
Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et
al, Am J Physiol 268; Alton et al., Nat Genet. 5:135-142, 1993 and
U.S. Pat. No. 5,679,647.
[0077] In addition, complex coacervation is a process of
spontaneous phase separation that occurs when two oppositely
charged polyelectrolytes are mixed in an aqueous solution. The
electrostatic interaction between the two species of macromolecules
results in the separation of a coacervate (polymer-rich phase) from
the supernatant (polymer-poor phase). This phenomenon can be used
to form microspheres and encapsulate a variety of compounds. The
encapsulation process can be performed entirely in aqueous solution
and at low temperatures, and has a good chance, therefore, of
preserving the bioactivity of the encapsulant. In developing an
injectable controlled release system, the complex coacervation of
gelatin and chondroitin sulfate to encapsulate a number of drugs
and proteins has been exploited (see Truong, et al. (1995) Drug
Delivery 2: 166) and cytokines have been encapsulated in these
microspheres for cancer vaccination (see Golumbek et al. (1993)
Cancer Res 53: 5841). Anti-inflammatory drugs have also been
incorporated for intra-articular delivery to the joints for
treating osteoarthritis (Brown et al. (1994) 331: 290). U.S. Pat.
Nos. 6,193,970, 5,861,159 and 5,759,582, describe compositions and
methods of use of complex coacervates for use as DNA vaccine
delivery systems of the instant invention. In particular, U.S. Pat.
No. 6,475,995, the contents of which are incorporated herein by
reference, teaches DNA vaccine delivery systems utilizing
nanoparticle coacervates of nucleic acids and polycations which
serve as effective vaccines when administered orally.
[0078] Antibodies can be isolated which are specific for a
particular MHC Class I- of Class II binding epitope of mesothelin.
These antibodies may be monoclonal or polyclonal. They can be used,
inter alia, for isolating and purifying polypeptides for use as
vaccines. T-cell lines that bind to an MHC class I or class
II-peptide complex comprising a particular MHC Class I- of Class II
binding epitope of mesothelin are useful for screening for T cell
adjuvants and immune response enhancers. Such cell lines can be
isolated from patients who have been immunized with a
mesothelin-containing vaccine and who have mounted an effective T
cell reseponse to mesothelin.
[0079] To test candidate cancer vaccines in the mouse model, the
candidate vaccine containing the desired tumor antigen can be
administered to a population of mice either before or after
challenge with the tumor cell line of the invention. Thus the mouse
model can be used to test for both therapeutic and prophylactic
effects. Vaccination with a candidate vaccine can be compared to
control populations that are either not vaccinated, vaccinated with
vehicle alone, or vaccinated with a vaccine that expresses an
irrelevant antigen. If the vaccine is a recombinant microbe, its
relative efficacy can be compared to a population of microbes in
which the genome has not been modified to ecxpress the antigen. The
effectiveness of candidate vaccine can be evaluated in terms of
effect on tumor or ascites volume or in terms of survival rates.
The tumor or ascites volume in mice vaccinated with candidate
vaccine may be about 5%, about 10%, about 25%, about 50%, about
75%, about 90% or about 100% less than the tumor volume in mice
that are either not vaccinated or are vaccinated with vehicle or a
vaccine that expresses an irrevelant antigen. The differential in
tumor or ascites volume may be observed at least about 10, at least
about 17, or at least about 24 days following the implantation of
the tumor cells into the mice. The median survival time in mice
vaccinated with a nucleic acid-modified microbe may be, for
example, at least about 2, at least about 5, at least about 7, or
at least about 10 days longer than in mice that are either not
vaccinated or are vaccinated with vehicle or a vaccine that
expresses an irrelevant antigen.
[0080] The mouse model can be used to test any kind of cancer
treatment known in the art. These may be conventional or
complementry medicines. These can be immunological agents or
cytotoxic agents. For example, the candidate cancer treatment may
be radiation therapy, chemotherapy, or surgery. The candidate
cancer treatment may be a combination of two or more therapies or
prophylaxes, including but not limited to anti-cancer agents,
anti-tumor vaccines, radiation therapy, chemotherapies, and
surgery.
[0081] Any oncogene known in the art can be used to make the
peritoneal or mesothelium cell line for making the mouse model.
Such oncogenes include without limitation, Ki-ras, Erb-B2, N-ras,
N-myc, L-myc, C-myc, ABL1, EGFR, Fos, Jun, c-Ha-ras, and SRC.
[0082] The vaccines, polynucleotides, polypeptides, cells, and
viruses of the present invention can be administered to either
human or other mammals. The other mammals can be domestic animals,
such as goats, pigs, cows, horses, and sheep, or can be pets, such
as dogs, rabbits, and cats. The other mammals can be experimental
subjects, such as mice, rats, rabbits, monkeys, or donkeys.
[0083] A reagent used in therapeutic methods of the invention is
present in a pharmaceutical composition. Pharmaceutical
compositions typically comprise a pharmaceutically acceptable
carrier, which meets industry standards for sterility, isotonicity,
stability, and non-pyrogenicity and which is nontoxic to the
recipient at the dosages and concentrations employed. The
particular carrier used depends on the type and concentration of
the therapeutic agent in the composition and the intended route of
administration. If desired, a stabilizing compound can be included.
Formulation of pharmaceutical compositions is well known and is
described, for example, in U.S. Pat. Nos. 5,580,561 and
5,891,725.
[0084] The determination of a therapeutically effective dose is
well within the capability of those skilled in the art. A
therapeutically effective dose refers to that amount of active
ingredient that increases anti-tumor cytolytic T-cell activity
relative to that which occurs in the absence of the therapeutically
effective dose.
[0085] For any substance, the therapeutically effective dose can be
estimated initially either in cell culture assays or in animal
models, usually mice, rabbits, dogs, or pigs. The animal model also
can be used to determine the appropriate concentration range and
route of administration. Such information can then be used to
determine useful doses and routes for administration in humans.
[0086] Therapeutic efficacy and toxicity, e.g., ED50 (the dose
therapeutically effective in 50% of the population) and LD50 (the
dose lethal to 50% of the population), can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals. The dose ratio of toxic to therapeutic effects is the
therapeutic index, and it can be expressed as the ratio,
LD50/ED50.
[0087] Pharmaceutical compositions that exhibit large therapeutic
indices are preferred. The data obtained from cell culture assays
and animal studies is used in formulating a range of dosage for
human use. The dosage contained in such compositions is preferably
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage varies within this range
depending upon the dosage form employed, sensitivity of the
patient, and the route of administration.
[0088] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active ingredient or to maintain the desired effect. Factors
that can be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions can be
administered every 3 to 4 days, every week, or once every two weeks
depending on the half-life and clearance rate of the particular
formulation.
[0089] Normal dosage amounts can vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc. Effective in vivo dosages of
polynucleotides and polypeptides are in the range of about 100 ng
to about 200 ng, 500 ng to about 50 mg, about 1 .mu.g to about 2
mg, about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g to about
100 .mu.g.
[0090] Desirable immunogens for use as anti-tumor vaccines are
those which are highly differentially expressed between tumors and
their corresponding normal tissues. Expression differences are
preferably at least 2-fold, 3-fold, 4-fold, 5-fold, or even 10
fold. Expression can be measured by any means known in the art,
including but not limited to SAGE, microarrays, Northern blots, and
Western blots. Interest in such proteins as immunogens is enhanced
by determining that humans respond to immunization with the protein
(or gene encoding it) by generating CD4 or CD8 T cells which are
specifically activated by the protein. Testing for such activation
can be done, inter alia, using TAP deficient cell lines such as the
human T2 cell line to present potential antigens in an MHC complex.
Activation can be measured by any assay known in the art. One such
assay is the ELISPOT assay. See references 33-35.
[0091] Future responses to tumor vaccines can be predicted based on
the response of CD8+ and or CD4+ T cells. If the tumor vaccine
comprises mesothelin or at least one T cell epitope of mesothelin,
then monitoring of the of CD8+ and or CD4+ response to mesothelin
provides useful prognostic information. A robust CD8+ and or CD4+
response indicates that the patient has mounted an effective
immunological response and will survive significantly longer than
those who have not mounted such a response. The tumor vaccine may
comprise whole tumor cells, particularly pancreatic, ovarian or
mesothelioma cells. The tumor vaccine may comprise a polyethylene
glycol fusion of tumor cells and dendritic cells. The tumor vaccine
may comprise apoptotic or necrotic tumor cells which have been
incubated with dendritic cells. The tumor vaccine may comprise mRNA
or whole RNA which has been incubated wioth dendritic cells. The T
cell responses to mesothelin can be measured by any assay known in
the art, including an ELISPOT assay. Alternatively, future response
to such a tumor vaccine can be monitored by assaying for a delayed
type hypersensitivity respone to mesothelin. Such a response has
been identified as a positive prognostic indicator.
[0092] Test substances which can be tested for use as a potential
drug or immune enhancing agent can be any substance known in the
art. The substance can be previously known for another purpose, or
it can be previously unknown for any purpose. The substance can be
a purified compound, such as a single protein, nucleic acid, or
small molecule, or it can be a mixture, such as an extract from a
natural source. The substance can be a natural product, or it can
be a synthetic product. The substance can be specifically and
purposefully synthesized for this purpose or it can be a substance
in a library of compounds which can be screened.
[0093] While the invention has been described with respect to
specific examples including presently preferred modes of carrying
out the invention, those skilled in the art will appreciate that
there are numerous variations and permutations of the above
described systems and techniques that fall within the spirit and
scope of the invention as set forth in the appended claims.
EXAMPLES
Example 1
[0094] To identify genes that can serve as potential immune targets
for the majority of pancreatic adenocarcinoma patients, we focused
only on those genes that were non-mutated, overexpressed by the
majority of pancreatic cancer patients, and overexpressed by the
vaccine cell lines. One gene at the top of this list was mesothelin
(20, 21). For comparison and validation purposes we also looked at
prostate stem cell antigen (PSCA). SAGE data demonstrated PSCA to
be expressed by pancreatic tumors at similar levels to that of
mesothelin (22).
[0095] We used the combination of two public use computer
algorithms (23-25) to predict peptide nonamers that bind to three
common human leukocyte antigen (HLA)-class I molecules. All 14
patients treated with the allogeneic GM-CSF vaccine express at
least one of these HLA-Class I molecules (Table 2). The predictive
algorithm "BIMAS", ranks potential HLA binding epitopes according
to the predictive half-time disassociation of peptide/HLA complexes
(23). The "SYFPEITHI" algorithm ranks peptides according to a score
that accounts for the presence of primary and secondary HLA-binding
anchor residues (25). Both computerized algorithms score candidate
epitopes based on amino acid sequences within a given protein that
have similar binding motifs to previously published HLA binding
epitopes. We synthesized the top two ranking mesothelin epitopes
for HLA-A2, HLA-A3, and HLA-A24 and the top six PSCA epitopes for
each MHC molecule favored by both algorithms (Table 1), since at
least one of these three HLA class I molecules is expressed by the
14 patients that were treated in our vaccine study (Table 2).
1TABLE 1 Mesothelin peptides predicted to bind to HLA A2, A3, and
A24. Amino Acid HLA-Restriction Sequence Amino Acid Position in
Protein HLA-A2 SLLFLLFSL Mesothelin A2.sub.(20-28) HLA-A2 VLPLTVAEV
Mesothelin A2.sub.(530-538) HLA-A2 LLALLMAGL PSCA A2.sub.(5-13)
HLA-A2 ALQPGTALL PSCA A2.sub.(14-22) HLA-A2 ALLPALGLL PSCA
A2.sub.(108-116) HLA-A3 ELAVALAQK Mesothelin A3.sub.(83-92) HLA-A3
ALQGGGPPY Mesothelin A3.sub.(225-234) HLA-A3 ALQPAAAIL PSCA
A3.sub.(99-107) HLA-A3 LLALLMAGL PSCA A3.sub.(5-13) HLA-A3
ALQPGTALL PSCA A3.sub.(14-22) HLA-A3 LLPALGLLL PSCA
A3.sub.(109-117) HLA-A3 QLGEQCWTA PSCA A3.sub.(43-51) HLA-A3
ALLCYSCKA PSCA A3.sub.(20-28) HLA-A24 FYPGYLCSL Mesothelin
A24.sub.(435-444) HLA-A24 LYPKARLAF Mesothelin A24.sub.(475-484)
HLA-A24 DYYVGKKNI PSCA A24.sub.(76-84) HLA-A24 ALLPALGLL PSCA
A24.sub.(108-116) HLA-A24 ALQPAAAIL PSCA A24.sub.(99-107) HLA-A24
LLPALGLLL PSCA A24.sub.(109-117) HLA-A24 YYVGKKNIT PSCA
A24.sub.(77-85)
[0096] The three peptides, HIV-gag A2.sub.77-85 (SLYNTVATL) (48),
HIV-NEF A3.sub.94-103 (QVPLRPMTYK) (49), and tyrosinase
A242.sub.206-214 (AFLPWHRLF) (50), are previously published
epitopes that were used as control peptides for HLA-A2, A3, and A24
binding, respectively. The Mesothelin Al.sub.309-318 binding
epitope (EIDESLIFY) was used as a negative control peptide for all
binding studies. The M1 peptide (GILGFVFTL).sub.58-66 (Gotch et al
1988) was used as a positive control for all of the HLA-A2
studies.
2TABLE 2 Selected Characteristics of the 14 patients treated with
an allogeneic GM-CSF secreting pancreatic tumor vaccine. Post-
vaccine increase in Disease- Dose .times. # of total HLA Class I
DTH to free Overall Disease Status 10.sup.7 vaccines Expression at
Auto Survival Survival Pt # T (cm) LN Margins Cells received the A
Locus.sup.1 Tumor.sup.2 (months) (months) 1 3.0 5/17 - 1 2 A1, A2 0
11 14 2 2.7 3/17 + 1 1 A2, A3 0 6 14 3 2.5 3/11 + 1 1 A1, A3 0 9 18
4 2.5 3/14 - 5 1 A2, A29 0 8 10 5 2.7 2/23 - 5 4 A3, A3 0 15 39 6
2.5 2/17 - 5 3 A2, A24 0 13 27 7 4.0 4/13 + 10 1 A31, A24 0 16 18 8
2.7 5/18 + 10 2 A2, A3 252 mm 60+ 60+ 9 1.2 2/11 - 10 1 A3, A31 0 8
21 10 2.0 11/27 - 50 1 A3, A30 0 9 17 11 3.5 2/32 + 50 1 A1, A3 N/A
9 13 12 2.5 2/11 - 50 1 A3, A33 N/A 11 13 13 3.0 2/14 - 50 4 A3,
A23 100 mm 60+ 60+ 14 3.0 0/14 + 50 4 A1, A24 110 mm 60+ 60+
Abbreviations: Pt = patient, # = number, T = tumor size at surgery,
LN = number of positive lymph nodes/total number of lymph nodes
sampled, HLA = human leukocyte antigen, DTH = delayed type
hypersensitivity testing, Auto = autologous, N/A = not assessed due
to unavailability of DTH cell reagents, + = still alive and
disease-free. .sup.1HLA typing was performed serologically and
confirmed molecularly. .sup.2Delayed type hypersensitivity
reactions to autologous tumor cells was assessed using unpassaged
autologous tumor cells. 10.sup.6 autologous tumor cells were placed
pre-vaccination, and at 28 days post-vaccination. Reported are the
post-vaccination change in the product of the perpendicular
diameters (measured in mm) of the observed induration at 48 hours
after cell placement.
[0097] Binding of these epitopes to their respective HLA class I
molecule was tested by pulsing TAP deficient T2 cells that
expressed the corresponding HLA class I molecule (T2-A2, T2-A3, or
T2-A24 cells). As shown in FIG. 1A, pulsing of two
mesothelin-derived epitopes predicted to bind to HLA-A2 allows for
detection of HLA-A2 on the cell surface of T2-A2 cells by flow
cytometry following staining with the HLA class I specific
antibody, W6/32. In contrast, unpulsed T2 cells or T2 cells pulsed
with an mesothelin epitope predicted to bind to HLA-A1 do not stain
with the same antibody. Binding of T2 cells pulsed with two
candidate mesothelin derived HLA-A3 and two candidate HLA-A24
epitopes are shown in FIG. 1B and FIG. 1C, respectively. A similar
binding experiment was done with the PSCA derived peptides for
HLA-A2, HLA-A3, and HLA-A24. (FIG. 1D, FIG. 1E and FIG. 1F).
[0098] Materials and Methods: Identification of candidate genes and
epitope selection. SAGE was used to identify mesothelin as one of
the genes overexpressed in pancreatic cancer cell lines and fresh
tissue as previously reported (20, 21). Two computer algorithms
that are available to the general public and accessible through the
internet were used to predict peptides that bind to HLA A2, A3, and
A24 molecules. "BIMAS" was developed by K. C. Parker and
collaborators http://bimas.dcrt.nih.gov/ (NIH) that determined the
optimal binding for the most common HLA class I molecule types
(23). "SYFPEITHI" was developed by Rammensee et al. and ranks the
peptides according to a score that takes into account the presence
of primary and secondary MHC-binding anchor residues
http://www.uni-tuebingen.de/uni/kxi (24).
[0099] Materials and Methods: Peptides and T2 cell lines. Peptides
were synthesized by Macromolecular Resources (Fort Collins, Colo.)
according to published sequences: M1 peptide GILGFVFTL (SEQ ID NO:
10), derived from influenza matrix protein (amino acid positions
58-66) (28), Mesothelin A2 peptides and PSCA A2 peptides listed in
table 1 were identified using the available databases, HIV-gag A2
peptide SLYNTVATL (SEQ ID NO: 7) (amino acid positions 75-83) (29)
contain an HLA-A2 binding motif. Mesothelin A3 peptides and PSCA A3
peptides and HIV-NEF A3 peptide QVPLRPMTYK (SEQ ID NO: 8) (amino
acid positions 94-103) (30) contain an HLA-A3 binding motif.
Mesothelin A24 peptides and PSCA A24 peptides and Tyrosinase
peptide AFLPWHRLF (SEQ ID NO: 9) (amino acid positions 206-214)
(31) contain an HLA-A24 binding motif. Stock solutions (1 mg/ml) of
each peptide were prepared in 10% DMSO (JTBaker, Phillippsburg,
N.J.) and further diluted in cell culture medium to yield a final
peptide concentration of 10 ng/ml for each assay. The control M1
peptide was initially dissolved in 100% DMSO and further diluted in
cell culture medium using the same stock and final concentrations.
The T2 cells are a human B and T lymphoblast hybrid that only
express the HLA-A*0201 allele (26). The human T2 cell line is a TAP
deficient cell line that fails to transport newly digested HLA
class I binding epitopes from the cytosol into the endoplasmic
reticulum where these epitopes would normally bind to nascent HLA
molecules and stabilize them for expression on the cell surface
(26). The T2-A3 are T2 cells genetically modified to express the
HLA-A301 allele and were a gift from Dr. Walter Storkus (University
of Pittsburgh) (32). T2-A24 are T2 cells genetically modified to
express the HLA-A24 allele. The HLA-A24 gene was a gift from Dr.
Paul Robbins (Surgery Branch, National Cancer Institute) (31). T2
cells were grown in suspension culture in RPMI-1640 (Gibco, Grand
Island, N.Y.), 10% serum (Hyclone, Logan, Utah) supplemented with
200 .mu.M L-Glutamine (Gibco, Grand Island, N.Y.), 50
units-.mu.g/ml Pen/Strep (Gibco), 1% NEAA (Gibco), and 1%
Na-Pyruvate (Gibco).
[0100] Materials and Methods: Peptide/MHC binding Assays. T2 cells
expressing the HLA molecule of interest were resuspended in AimV
serum free media (Gibco) to a concentration of 2.5.times.10.sup.5
cells/ml and pulsed with 100-200 micrograms of peptide at room
temperature overnight. Pulsing at room temperature allows for
optimizing the number of empty HLA molecules available for binding
each epitope (30). The cells were washed and resuspended at
1.times.10.sup.5 cells/ml. Peptide binding was determined by FACS
(Beckon Dickenson, San Jose, Calif.) analysis.
Example 2
[0101] To determine if mesothelin and PSCA are recognized by CD8+ T
cells, we screened antigen-pulsed T2 cells with CD8+ T cell
enriched PBL from patients that have received an allogeneic GM-CSF
secreting pancreatic tumor vaccine. We previously reported the
association of in vivo post-vaccination delayed type
hypersensitivity (DTH) responses to autologous tumor in three of
eight patients receiving the highest two doses of vaccine. These
"DTH responders" (each of whom had poor prognostic indicators at
the time of primary surgical resection (27) are the only patients
who remain clinically free of pancreatic cancer >4 years after
diagnosis ((27), Table 2). PBL obtained prior to vaccination and 28
days after the first vaccination were initially analyzed. T2-A3
cells pulsed with the two A3 binding epitopes were incubated
overnight with CD8+ T cell enriched lymphocytes isolated from the
peripheral blood of patient 10 (non-DTH responder who relapsed 9
months after diagnosis) and 13 (DTH responder who remains
disease-free) and analyzed using a gamma interferon (IFN-.gamma.)
ELISPOT assay. The ELISPOT assay was chosen because it requires
relatively few lymphocytes, is among the most sensitive in vitro
assays for quantitating antigen-specific T cells, and correlates
number of antigen-specific T cells with function (cytokine
expression) (33-35). The number of IFN-.gamma. spots per
1.times.105 CD8+ positive T cells detected in the peripheral blood
of the two patients prior to vaccination and twenty-eight days
following the first vaccination in response to the two HLA-A3
binding mesothelin peptides are shown in FIG. 2A.
[0102] Induction of mesothelin-specific T cells was detected
twenty-eight days following vaccination in patient 13 a DTH
responder, but not in patient 10, a non-DTH responder. Similarly,
post-vaccination induction of mesothelin-specific CD8+ T cells was
observed in two other disease-free DTH responders (patient 8 and
patient 14), but not for two other non-DTH responders when tested
with T2-A2 and T2-A24 cells pulsed with the A2 (FIG. 2B) and A24
(FIG. 2C) binding epitopes, respectively. A summary of the ELISPOT
results analyzing all 14 patients treated with the allogeneic
vaccine on this study for the induction of mesothelin-specific CD8+
T cells following the first vaccination are shown in FIG. 2D. These
data demonstrate that there is a direct correlation between
observed post-vaccination in vivo DTH responses to autologous
tumor, long term disease-free survival, and post-vaccination
induction of mesothelin-specific T cell responses in this clinical
trial. Specifically, each of the three DTH responders demonstrated
a post-vaccination induction in T cell response to every mesothelin
peptide that matched their respective HLA type, whereas only one of
eleven DTH non-responders had an increased post-vaccination
mesothelin-specific T cell response and only to a single peptide.
Thus, the in vitro measurement of mesothelin-specific T cells
responses represents a new candidate in vitro immune marker for
predicting which patients will respond to this vaccine therapy.
[0103] Materials and Methods: Peripheral blood lymphocytes (PBL)
and donors. Peripheral blood (100 cc pre-vaccination and 28 days
after each vaccination) were obtained from all fourteen patients
who received an allogeneic GM-CSF secreting pancreatic tumor
vaccine as part of a previously reported phase I vaccine study
(27). Informed consent for banking lymphocytes to be used for this
antigen identification study was obtained at the time of patient
enrollment into the study. Pre and post-vaccine PBL were isolated
by density gradient centrifugation using Ficoll-Hypaque (Pharmacia,
Uppsala, Sweden). Cells were washed twice with serum free
RPMI-1640. PBL were stored frozen at -180.degree. C. in 90% AIM-V
media containing 10% DMSO.
[0104] Materials and Methods: Enrichment of PBL for CD8+ T cells.
CD8+ T cells were isolated from thawed PBL using Magnetic Cell
Sorting of Human Leukocytes as per the manufacturers
directions(MACS, Miltenyi Biotec, Auburn, Calif.). Cells were
fluorescently stained with CD8-PE antibody (Becton Dickenson, San
Jose, Calif.) to confirm that the positive population contained
CD8+ T cells and analyzed by flow cytometry. This procedure
consistently yielded >95% CD8+ T cell purity.
[0105] Materials and Methods: ELISPOT assay. Multiscreen ninety-six
well filtration plates (Millipore, Bedford, Mass.) were coated
overnight at 4.degree. C. with 60 .mu.l/well of 10 .mu.g/ml
anti-hIFN-.gamma. mouse monoclonal antibody (Mab) 1-D1K (Mabtech,
Nacka, Sweden). Wells were then washed 3 times each with
1.times.PBS and blocked for 2 hours with T cell media. 1.times.105
T2 cells pulsed with peptide (10 ng/ml) in 100 .mu.l of T cell
media were incubated overnight with 1.times.105 thawed PBL that are
purified to select CD8+ T cells in 100 .mu.l T-cell media on the
ELISPOT plates in replicates of six. The plates were incubated
overnight at 37.degree. C. in 5% CO2. Cells were removed from the
ELISPOT plates by washing six times with PBS+0.05% Tween 20 (Sigma,
St. Louis, Mo.). Wells were incubated for 2 hours at 37.degree. C.
in 5% CO2 using 60 .mu.l/well of 2 .mu.g/ml biotinylated Mab
anti-hIFNgamma 7-B6-1 (Mabtech, Nacka, Sweden). The avidin
peroxidase complex (Vectastain ELITE ABC kit, Vetcor Laboratories,
Burlingame, Calif.) was added after washing six times with
PBS/Tween 0.05% at 100 .mu.l per well and incubated for one hour at
room temperature. AEC-substrate solution (3-amino-9-ethylcarbazole)
was added at 100 .mu.l/well and incubated for 4-12 minutes at room
temperature. Color development was stopped by washing with tap
water. Plates were dried overnight at room temperature and colored
spots were counted using an automated image system ELISPOT reader
(Axioplan2, Carl Zeiss Microimaging Inc., Thornwood, N.Y.).
Example 3
[0106] The above data clearly demonstrate a correlation of in vivo
DTH response to autologous tumor and long term disease-free
survival with the post-vaccination induction of mesothelin-specific
CD8+ T cell responses. It is possible, however, that this
correlation represents generalized immune suppression (in the
patients who failed to demonstrate post-vaccination DTH responses
to their autologous tumor and who had disease progression), rather
than a vaccine specific induction of T cell responses to mesothelin
in the DTH responder patients who remain disease-free. To
demonstrate that the post-vaccination induction of
mesothelin-specific CD8+ T cells is tumor antigen-specific, we
evaluated each HLA-A2 positive patient for T cell responses to the
HLA-A2-binding influenza matrix peptide, M1 (28). We chose the
influenza M1 peptide because most patients on the vaccine study had
received an influenza vaccine sometime prior to enrollment. As
shown in FIG. 3, all HLA-A2 positive patients demonstrated similar
pre- and post-vaccination T cell responses to the M1 peptide.
Pre-vaccination responses ranged from 19 to 50 IFN-.gamma. spots
per 105 total CD8+ T cells, and post-vaccination responses remained
about the same in each patient (FIG. 3). A similar study was not
done for HLA-A3 and A24 positive patients because there are no
published influenza M1 epitopes known to bind these HLA
molecules.
[0107] We evaluated the lymphocytes from the same 14 patients for
the post-vaccination induction of CD8+ T lymphocytes directed
against a second overexpressed antigen, PSCA. In contrast to
mesothelin, PSCA did not elicit an immune response in the 3 DTH
responders. Again, we synthesized the top two ranking epitopes for
HLA-A2, HLA-A3, and HLA-A24 favored by both algorithms and analyzed
these according to the same protocols used in the mesothelin
experiments. We did not see any post-vaccination induction of
PSCA-specific T cells in any of the patients; therefore, we
synthesized 4 additional PSCA peptides for each HLA class I
molecule to ensure that we had not missed the immunogenic epitope.
Analysis of these peptides also failed to demonstrate a
post-vaccination induction of PSCA-specific CD8+ T cell responses
(FIGS. 4A, 4B, and 4C, respectively). PSCA specific responses could
not be demonstrated in the eight non-responders as well (FIG. 4d).
This result further supports our finding that mesothelin is a
relevant pancreatic tumor antigen because there were no vaccine
induced immune responses to PSCA even though they are similarly
overexpressed in pancreatic cancer on SAGE analysis. In addition,
the PSCA data demonstrate that overexpression of a protein in a
tumor is insufficient to predict the protein's utility as a vaccine
target.
[0108] Flow cytometry analysis of mesothelin and PSCA expression by
the two allogeneic vaccine cell lines is shown in FIG. 5.
Interestingly, mesothelin is expressed equally by both vaccine cell
lines whereas PSCA is only expressed by one of the vaccine cell
lines (Panc 6.03).
[0109] Materials and Methods: CD8+ M1 specific T cell lines. M1
specific T cell lines were generated by repeated in vitro
stimulation of HLA-A201 positive PBL initially with irradiated
autologous dendritic cells followed by irradiated autologous
Ebstein Barr Virus (EBV) transformed B cells, both pulsed with the
HLA-A201 restricted epitope. This line was stimulated biweekly
using autologous EBV cells that were pulsed with 10 .mu.g
peptide/ml of their respective peptides at 37.degree. C. for 2
hours, washed twice with RPMI-1640, and irradiated with 10,000
rads. T cells were stimulated at a 1:2 T cell to EBV cell ratio in
T cell media (RPMI-1640, 10% human serum (pooled serum collected at
the Johns Hopkins Hemapheresis Unit) containing 200 .mu.M
L-Glutamine, 50 units-.mu.g/ml Pen/Strep, 1% NEAA, and 1%
Na-Pyruvate) supplemented with 20 cetus units IL-2/well and 10
ng/well IL-7. This line was used a positive control T cell line in
all assays.
[0110] Materials and Methods: Flow cytometry. The expression of
mesothelin and PSCA on the vaccine lines was evaluated by flow
cytometry analysis. The vaccine lines were washed twice and
resuspended in "FACS" buffer (HBSS supplemented with 1% PBS, 2%
FBS, and 0.2% sodium azide), then stained with mouse monoclonal
mesothelin (CAK1) (Signet Laboratories, Dedham, Mass.) or mouse
monoclonal to PSCA (clone 1G8, obtained from R.E.R.) followed by
FITC-labeled goat antimouse IgG (BD PharMingen, San Jose, Calif.)
for flow analysis in a FACScan analyzer (BD Immunocytometry
Systems).
[0111] These data demonstrate that mesothelin-specific CD8+ T cells
are detected following a single vaccination with an allogeneic
GM-CSF secreting tumor vaccine in DTH responders but not in non-DTH
responders. The patients treated on the reported vaccine study
received an initial vaccination 8-10 weeks following
pancreaticoduodenectomy and 4 weeks prior to receiving a six month
course of adjuvant chemoradiation (27). Six of these patients
remained disease-free at the end of the six months and received up
to 3 more vaccinations given one month apart. Repeat ELISPOT
studies were performed on serial CD8+ T cell enriched PBL samples
from these six patients following multiple vaccine treatments to
assess the effect of chemoradiation and multiple vaccinations on
mesothelin-specific T cell responses.
[0112] As shown in FIG. 6, two of the three DTH responders
demonstrated decreased mesothelin-specific T cell responses
following the second vaccination. In both patients,
mesothelin-specific T cell responses returned to levels achieved
after the initial vaccination by the fourth vaccination. The
suppressed mesothelin-specific T cell responses that were observed
following the second vaccine are likely the result of the
chemotherapy that each patient received between the first and
second vaccination. Interestingly, one of the three patients
demonstrated similar mesothelin-specific T cell responses after the
first and second vaccination. This DTH responder only received two
vaccines because she subsequently developed a late autoimmune
antibody mediated complication attributed to the Mitomycin-C that
required medical intervention and withdrawal from the vaccine
study. In contrast, repeated vaccination failed to induce
mesothelin-specific T cell responses in those patients who did not
demonstrate an initial mesothelin-specific T cell response
following the first vaccination (FIG. 6).
[0113] These data describing CD8.sup.+ T cell responses induced by
an allogeneic GM-CSF-secreting pancreatic tumor vaccine support the
following conclusions. First, mesothelin can serve as an in vitro
biomarker of vaccine-induced immune responses that correlate with
in vivo responses in patients with pancreatic adenocarcinoma.
Second, the recognition of mesothelin and lack of recognition of
another overexpressed gene product, PSCA, by uncultured
post-vaccination CD8.sup.+ T cells from patients that demonstrated
evidence of in vivo immune responses in association with clinical
responses validates this antigen identification approach as a rapid
functional genomic-based approach for identifying immune relevant
tumor targets of CD8.sup.+ T cells.
[0114] Mesothelin is a 40 kilodalton transmembrane glycoprotein
member of the mesothelin/megakaryocyte potentiating factor (MPF)
family expressed by most pancreatic adenocarcinomas (36), (37-39).
It has also been reported to be expressed by ovarian cancers,
mesotheliomas, and some squamous cell carcinomas (37-39).
Mesothelin is known to be attached to the cell membrane by a
glycosylphosphatidyl-inositol anchor and is postulated to function
in cell adhesion (36). Mesothelin and other members of its gene
family exhibit limited normal tissue expression. It therefore meets
three important criteria that strongly favor its potential use as
an immunogen in the future development of antigen-based vaccines
for patients with pancreatic adenocarcinoma and other tumor types
that overexpress mesothelin: it is widely shared by most pancreatic
and ovarian cancers, it has a limited expression in normal tissues,
and it induces CD8.sup.+ T cell responses following vaccination
with tumor cells that express this antigen.
[0115] The identification of shared, biologically relevant tumor
antigens provides the opportunity to design antigen-based vaccines
that have the potential to be more efficient at inducing anti-tumor
immunity than current whole cell vaccines. In addition, scale up of
recombinant antigen-based approaches is technically more feasible
than currently employed whole tumor cell vaccines. However,
recombinant antigen-based vaccines require identification of
antigens that are both broadly expressed by patients and that are
immunogenic. Until now, T cell screening of cDNA libraries,
antibody screening of phage display libraries, or the biochemical
elution and purification of antigens bound to MHC have identified
the majority of known tumor antigens, many of which appear to
derive from shared, non-mutated genes that are either overexpressed
by or reactivated in tumor cells relative to normal tissue (3-13).
Unfortunately, this expanding list of tumor associated antigens
recognized by T cells is limited mostly to melanoma because of the
technical difficulty of isolating and propagating T cell lines and
clones from vaccinated patients with other types of cancer. The
tumor antigen identification approach disclosed herein is feasible
because it only requires a database of differentially expressed
genes within a given tumor, and banked, uncultured bulk PBL from
vaccinated patients. Therefore, this antigen identification
approach is rapid and can be generalized to most types of cancer.
In addition, the use of uncultured lymphocytes rather than T cell
lines and clones that have been in long term culture provide the
advantage of identifying new biologically relevant immune
targets.
[0116] PSCA is a second gene product that was found to be
overexpressed in our SAGE pancreatic gene expression database. In
fact, PSCA was shown to be overexpressed at higher levels than even
mesothelin. However, post-vaccination PSCA specific T cell
responses were not detected in the DTH responders and DTH
non-responder patients. It is unclear at this time why a GM-CSF
secreting allogeneic vaccine induces T cell responses to one
overexpressed antigen and not to a second similarly overexpressed
antigen. It is possible that these two antigens are differently
processed and presented during the initial priming event (58).
[0117] In this study we also demonstrate that mesothelin-specific T
cells can be induced against at least six different peptides
presented by three different HLA-A locus alleles. This finding
provides further support that mesothelin can serve as a shared
antigen. In this study, the highest ranking antigenic epitopes
predicted to be the best HLA-A allele binding epitopes based on
their motif, bound to their respective HLA alleles and were also
recognized by mesothelin-specific T cells. Reports analyzing other
tumor antigens have found that the highest ranking epitopes do not
necessarily correlate with optimal recognition by T cells (25). We
also performed the computer algorithms on two melanoma antigens,
tyrosinase and MAGE 1, to determine how their published HLA-A2
binding peptides rank by this method. We found that our HLA-A2
binding mesothelin epitopes were given similar scores as the known
tyrosinase and MAGE 1 HLA-A2 binding epitopes. This was also true
for the published HLA-A2 HIV GAG and HLA-A3 HIV NEF epitopes that
were used as control antigens in our analyses. Choosing epitopes
that rank high by both algorithms appears to be an important
predictor of the probability of binding to the respective HLA
molecule.
[0118] We have developed a functional genomic approach that
identified a candidate pancreatic tumor antigen. This approach to
antigen identification facilitates the identification of other
human cancer antigens that are biologically relevant immune
targets. The correlation of in vitro T cell responses with in vivo
measures of response validates the biologic importance of this
approach. This approach is rapid and feasible and can easily be
adapted to identify antigens expressed by other cancer types. This
in turn, should accelerate the development of recombinant
antigen-based vaccines for most human cancer treatment.
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Example 5
[0187] Construction of Mouse Tumor Cells by Co-transformation with
HPV-16 E6 and E7 and Activated ras Oncogene. Primary peritoneal
cells of C57BL/6 mice were immortalized by HPV-16 E6 and E7 and
then transformed with pEJB expressing activated human c-Ha-ras
gene. This co-transformation produced a tumorigenic cell line.
[0188] C57BL/6 mouse peritoneal cells were collected and washed
with 1.times. HBSS. The primary single cell suspension was cultured
in vitro in RPMI1640, supplemented with 10% fetal calf serum, 50
units/ml penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium
pyruvate, and 2 mM nonessential amino acids, and grown at 370 C
with 5% CO2. Transduction of HPV-16 E6 and E7 genes into primary
peritoneal cells was performed using the LXSN16E6E7 retroviral
vector, kindly provided by Denise A. Galloway (Fred Hutchinson
Cancer Research Center, Seattle, Wash.) (Halbert, et al., (1991) J
Virol, 65:473-478). HPV-16 E6- and E7-containing LXSN16E6E7 was
used to infect CRIP cells to generate recombinant virus with a wide
host range. Primary peritoneal cells were immortalized by
transduction as described previously (Halbert, et al., (1991) J
Virol, 65:473-478). Following transduction, the retroviral
supernatant was removed, and cells were grown in G418 (0.4 mg/ml)
culture medium for an additional 3 days to allow for integration
and expression of recombinant retroviral genes. The immortalized
lung (E6+E7+) cells were then transduced with pVEJB expressing
activated human c-Ha-ras gene, kindly provided by Chi V. Dang (The
John Hopkins Hospital, Baltimore, Md.), and selected with G418 (0.4
mg/ml) and hygromycin (0.2 mg/ml).
Example 6
[0189] Characterization of Histological and Pathological Features
of WF-3. 5.times.104 WF-3 tumor cells were injected into C57BL/6
mice intraperitoneally. 4 weeks later, mice were sacrificed to
examine the formation of ascites and tumors. Removed organs were
fixed with 4% buffered formaldehyde and histological sections were
made, followed by routine hematoxylin-eosin staining. Slides were
observed under a light microscope.
[0190] Mice were injected with 5.times.104 WF-3 tumor cells
intraperitoneally and sacrificed 4 weeks later. This cell line was
capable of generating ascites in mice challenged with tumor cells
intraperitoneally (see FIG. 7A). Morphologically, WF-3 tumor cells
showed a papillary architecture resembling serous tumors found in
the human ovary/peritoneum (FIG. 1B). Furthermore, the tumor showed
a high level of mitotic activity, pleiomorphic nuclei, abnormal
mitosis, and a high nuclear/cytoplasmic ratio, consistent with a
highly malignant neoplasm (see FIG. 7C).
[0191] FIG. 7 shows the generation and characterization of an
ascitogenic ovarian tumor cell line (WF-3). WF-3 tumor cells were
injected into C57BL/6 mice intraperitoneally at a dose of
1.times.105 cells/mouse. Mice were euthanized 4 weeks after tumor
challenge (see FIG. 7A) Representative gross picture to demonstrate
ascites formation in mice. Note: Mice developed significant ascites
with an increase in abdominal girth 4 weeks after tumor challenge.
FIG. 7B shows hematoxylin and eosin staining of the explanted
tumors viewed at 100.times. magnification. The tumors displayed a
papillary configuration, morphologically consistent with tumors
derived from the peritoneum or ovaries. FIG. 7C shows tumors viewed
at 400.times. magnification. The inset displays the features of a
WF-3 tumor cell in greater detail.
Example 7
[0192] MHC Class I and Class II Presentation of WF-3 Tumor Cells.
WF-3 tumor cells were harvested and prepared for flow cytometry
analysis. Anti-H-2 Kb/H-2 Db monoclonal antibody or anti-I-Ab
monoclonal antibody was added for the detection of MHC class I and
class II expression on WF-3 tumor cells.
[0193] WF-3 tumor cells were harvested, trypsinized, washed, and
resuspended in FACScan buffer. Anti-H-2 Kb/H-2 Db monoclonal
antibody (Clone 28-8-6, PharMingen, San Diego, Calif.) or anti-I-Ab
monoclonal antibody (Clone 25-9-17, PharMingen, San Diego, Calif.)
was added and incubated for 30 min on ice. After washing twice in
FACScan buffer, FITC-conjugated goat anti-mouse antibody (Jackson
ImmunoResearch Lab. Inc., West Grove, Pa.) was added and incubated
for 20 min on ice. Samples were resuspended in FACScan buffer.
Analysis was performed on a Becton Dickinson FACScan with CELLQuest
software (Becton Dickinson Immunocytometry System, Mountain View,
Calif.).
[0194] Our data indicate that WF-3 is positive for MHC class I
expression (FIG. 8A) but negative for MHC class II expression (FIG.
8B). In particular, FIG. 8 shows the MHC class I and II
presentation on WF-3 tumor cells. WF-3 tumor cells were harvested,
trypsinized, washed, and resuspended in FACSCAN buffer. Anti-H-2
Kb/H-2 Db monoclonal antibody or anti-I-Ab monoclonal antibody was
added, followed by flow cytometry analysis to detect MHC class I
and class II expression on WF-3 tumor cells. FIG. 8A shows WF-3
tumor cells which were positive for MHC class I presentation (thick
line) compared to the MHC class I-negative control (thin line).
FIG. 8B shows the WF-3 tumor cells which were negative for MHC
class II presentation. The thin line indicates staining of the MHC
class II-negative control.
Example 8
[0195] Determination of Minimal Tumor Dose of WF-3 Tumor Cells to
Lead to Formation of Lethal Ascites. WF-3 tumor cells were injected
into C57BL/6 mice intraperitoneally at various doses (1.times.104,
5.times.104, 1.times.105, and 1.times.106 cells/mouse). Mice were
monitored twice a week for formation of ascites and tumors and
sacrificed after 90 days. For survival following tumor challenge,
mice were challenged intraperitoneally with various doses of WF-3
(1.times.104, 5.times.104, 1.times.105, and 1.times.106
cells/mouse) and monitored for their survival after tumor
challenge.
[0196] As shown in FIG. 9A, all of the mice injected with
5.times.104, 1.times.105, and 1.times.106 cells intraperitoneally
formed ascites within 30 days. Meanwhile, 20% of mice injected with
1.times.104/mouse were tumor-free and without ascites formation 90
days after tumor challenge. All of the mice injected with a dose of
5.times.104 tumor cells or greater died within 50 days of tumor
challenge (FIG. 9B). These data suggest that WF-3 tumor cells are
able to lead to formation of ascites and solid tumors in the
peritoneum of mice and eventually kill the injected mice at a
certain tumor challenge dose.
Example 9
[0197] Mesothelin is Highly Expressed in the WF-3 Preclinical
Ovarian Cancer Model. We have performed microarray analysis (Incyte
Genomics Corporation, Palo Alto, Calif.) to characterize the gene
expression profile of WF-3 compared to pre-WF0. The pre-WF0 cell
line was generated by immortalizing mouse primary peritoneal cells
with a retroviral vector carrying HPV-16 E6 and E7 genes using a
previously described method (Lin, et al., (1996) Cancer Research,
56:21-26). We have chosen pre-WF0 as a reference cell line in order
to identify genes in WF-3 that are relevant to tumorigenicity in
later stages of ovarian cancer. Table 4 (below) summarizes highly
expressed genes present in WF-3 relative to pre-WF0. As shown in
Table 4, below, mesothelin is among the top 10 up-regulated genes
in WF-3, suggesting that WF-3 may be a suitable preclinical model
for developing mesothelin-specific cancer immunotherapy against
ovarian cancer.
3TABLE 4 Summary of Specifically Expressed Genes in WF-3 Sequence
Balanced differential Marker / Antigen Accession # expression
EGF-containing fibulin-like AI156278 6.5 extracellular matrix
protein 1 Mesothelin AA673869 3.8 alpha-2-HS-glycoprotein AI386037
3.3 Protein kinase, cGMP-dependent, AA771678 3.2 type II sema
domain, immunoglobulin AA241390 3.2 domain (Ig), short basic
domain, secreted (semaphorin) 3E Ankyrin-like repeat protein
AA792499 2.8 RIKEN cDNA 1300019103 gene AA600596 2.6 Matrix
gamma-carboxyglutamate W88093 2.4 (gla) protein serine (or
cysteine) proteinase AA727967 2.3 inhibitor clade F (alpha-2
antiplasmin, pigment epithelium Dervied factor). member 1 RIKEN
cDNA1200011C15 gene AA608330 2.2
Example 10
[0198] Expression of Mesothelin mRNA and Protein in WF-3 Tumor
Cells. We further confirmed the expression of mesothelin by the
WF-3 cell line using RT-PCR.
[0199] RNA was extracted from WF-3 tumor cells using RNAzol (Gibco
BRL, Gaithersburg, Md.) according to the manufacturer's
instructions. RNA concentration was measured and 1 mg of total
cellular RNA was reverse transcribed in a 20 ml volume using
oligo(dT) as a primer and Superscript reverse transcriptase (Gibco
BRL). One ml of cDNA was amplified by the PCR using a set of
primers (5'-CCCGAATTCATGGCCTTGCCAACAGCTCGA-3' and
5'-TATGGATCCGCTCAGCCTTAAAGCTGGGAG-3'; SEQ ID NOS: 11 and 12,
respectively). The primer was derived from the published murine
mesothelin cDNA sequence (Kojima, et al., (1995) J Biol Chem,
270:21984-21990). PCR was performed in a 50 ml reaction mixture
with 250 mM of each dNTP, 100 nM of primers, 5 ml of 10.times.
buffer (New England Biolabs, Berverly, Mass.), and 1 U of Vent DNA
polymerase (New England Biolabs) using 30 cycles (94.degree. C.,
1-min denaturation; 55.degree. C., 1-min annealing; and 72.degree.
C., 2-min extension). The reaction mixture (10 ml samples) was
analyzed using agarose gel electrophoresis (1%) in TAE buffer
containing 0.2 mg/ml ethidium bromide.
[0200] Murine mesothelin protein shares about 65% similarity with
human mesothelin protein. As shown in FIG. 10, we were able to
detect mRNA expression of murine mesothelin in WF-3 tumor by RT-PCR
(lane 2) but not in the control, B-16 tumor cells (lane 3). Western
blot analysis was performed to determine expression of mesothelin
protein in WF-3 tumor cells. Tumor cells were stained with
anti-mesothelin mouse polyclonal antibodies. Results of the Western
blot analysis confirmed that WF-3 was positive for mesothelin
protein while B16 melanoma cells were mesothelin-negative (data not
shown). Thus, our results indicate that WF-3 cells express
mesothelin mRNA and protein.
[0201] FIG. 10 shows expression of murine mesothelin in WF-3 tumor
cells as demonstrated by RT-PCR with gel electrophoresis. Western
blot analysis was also performed to confirm expression (not shown).
As shown in FIG. 10, RT-PCR was performed using the Superscript
One-Step RT-PCR Kit (Gibco, BRL) and a set of primers:
5'-CCCGAATTCATGGCCTTGCCAA-CAGCTCGA-3' and 5'-TATGGATCCGCTCA
GCCTTAAAGCTGGGAG-3' (SEQ ID NOS: 11 and 12, respectively). Western
blot analysis was also used to demonstrate the expression of
mesothelin protein in WF-3 tumor cells. Tumor cells were stained
with anti-mesothelin mouse polyclonal antibody followed by
FITC-conjugated goat anti-mouse IgG secondary antibody (data not
shown).
Example 11
[0202] Mesothelin DNA Cancer Vaccine Immunotherapy. Using the
peritoneal tumor model described above we demonstrated the ability
of a DNA vaccine encoding mesothelin to generate
mesothelin-specific cytotoxic T lymphocyte responses and antitumor
effects greater than empty plasmid DNA. These data indicate that a
DNA tumor vaccine targeting mesothelin can be used in treating or
controlling ovarian carcinomas and other cancers in which
mesothelin is highly expressed.
[0203] Plasmid DNA Construction. With the availability of the
mesothelin-expressing tumor cell line, WF-3, we created DNA
vaccines encoding mesothelin to test their antitumor effect against
WF-3 in C57BL/6 mice. We used a mammalian cell expression vector,
pcDNA3, to generate a DNA vaccine encoding murine full-length
mesothelin protein (total length: 625 aa).
[0204] For construction of pcDNA3-mesothelin, a DNA fragment
encoding mesothelin was first amplified from WF-3 extracted RNA and
a set of primers (5'-CCCGAATTCATGGCCTTGCCAACAGCTCGA-3' and
5'-TATGGATCCGCTCAGCCTTA- AAGCTGGGAG-3'; SEQ ID NOS: 11 and 12,
respectively) by RT-PCR using the Superscript One-Step RT-PCR Kit
(Gibco, BRL) and cloned into the EcoRI/BamHI sites of pcDNA3. The
primer was derived from the published murine mesothelin cDNA
sequence (11). The accuracy of DNA constructs was confirmed by DNA
sequencing.
[0205] Vaccination with a DNA Vaccine Encoding Mesothelin Protein
Protects Against Challenge with Mesothelin-Expressing Ovarian
Tumors. We tested the ability of this pcDNA3-mesothelin DNA vaccine
to protect against tumor challenge with WF-3 cells. Preparation of
DNA-coated gold particles and gene gun particle-mediated DNA
vaccination using a helium-driven gene gun (Bio-rad, Hercules,
Calif.) was performed according to a previously described protocol
(Chen, et al., (2000) Cancer Research, 60: 1035-1042. DNA-coated
gold particles (1 mg DNA/bullet) were delivered to the shaved
abdominal region of C57BL/6 mice using a helium-driven gene gun
(Bio-rad, Hercules, Calif.) with a discharge pressure of 400
p.s.i.
[0206] For the tumor protection experiment, mice (ten per group)
were vaccinated intradermally with 2 mg of pcDNA3-mesothelin DNA.
One week later, mice received a booster with the same dose. Mice
were challenged one week after booster with a lethal injection of
5.times.104 WF-3 tumor cells intraperitoneally. Mice were monitored
for evidence of ascites formation by palpation and inspection twice
a week; the mice were sacrificed at day 90. The percentage of
ascites-free mice in each vaccination group was determined.
[0207] Our data indicated that pcDNA3-mesothelin generated a high
degree of protection (60%) against WF-3 tumor challenge. Controls
were vaccinated with pcDNA3 vector alone (0%) or were not
vaccinated (0%). FIG. 11 shows in vivo tumor protection experiments
against WF-3 tumor growth using mesothelin-specific DNA
vaccines.
Example 12
[0208] Vaccination with pcDNA3-mesothelin Generate
Mesothelin-Specific Cytotoxic Immune Responses. CD8+ T lymphocytes
are important effector cells for mediating antitumor immunity.
Cytotoxic T lymphocyte (CTL) assays were performed to determine the
cytotoxic effect of mesothelin-specific CD8+ T cells generated by
the pcDNA3-mesothelin DNA vaccine. Splenocytes from vaccinated mice
served as effector cells after being cultured with cell lysates
containing mesothelin protein. WF-3 tumor cells served as target
cells.
[0209] Generation of Mesothelin-Containing Cell Lysates from
Transfected 293 Db,Kb Cells. To generate mesothelin containing cell
lysates to pulse splenocytes for the CTL assays, a total of 20 mg
of pcDNA3-mesothelin or empty plasmid DNA was transfected into
5.times.10.sup.6 Db,Kb cells with lipofectamine 2000 (Life
Technologies) according to the manufacturer's protocol. The
transfected 293 Db,Kb cells were collected 40-44 h after
transfection, then treated with three cycles of freeze-thaw. The
protein concentration was determined using the Bio-Rad protein
assay (Bio-Rad, Hercules, Calif.) according to vendor's protocol.
Cell lysates containing mesothelin were used to pulse splenocytes
obtained from the various vaccinated mice as described below.
[0210] Cytotoxic T Lymphocyte (CTL) Assays. Cytolysis was
determined by quantitative measurements of lactate dehydrogenase
(LDH) using CytoTox96 non-radioactive cytotoxicity assay kits
(Promega, Madison, Wis.) according to the manufacturer's protocol.
Briefly, splenocytes were harvested from vaccinated mice (5 per
group) and pooled 1 week after the last vaccination. Splenocytes
were pulsed with 20 mg of cell lysates in a total volume of 2 ml of
RPMI 1640, supplemented with 10% (vol/vol) fetal bovine serum, 50
units/ml penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium
pyruvate, 2 mM nonessential amino acids in a 24-well tissue culture
plate for 6 days as effector cells. WF-3 tumor cells were used as
target cells. WF-3 cells were mixed with splenocytes at various
effector/target (E:T) ratios. After 5 hr incubation at 370 C, 50
.mu.l of the cultured media were collected to assess the amount of
LDH in the cultured media according to the manufacturer's protocol.
The percentage of lysis was calculated from the following equation:
100.times.(A-B)/(C-D), where A is the reading of
experimental-effector signal value, B is the effector spontaneous
background signal value, C is maximum signal value from target
cells, D is the target spontaneous background signal value.
[0211] Statistical Analysis. Statistical determinations were made
using the Student's t-test. Two-sided P values are presented in all
experiments, and significance was defined as P<0.05. No mice
were excluded from statistical evaluations.
[0212] As shown in FIG. 12, vaccination with pcDNA3-mesothelin
generated a significant percentage of specific lysis compared to
vaccination with pcDNA3 or no vaccination (P<0.001, one-way
ANOVA). These results indicate that vaccination with
pcDNA3-mesothelin DNA is capable of generating mesothelin-specific
T cell-mediated specific lysis of WF-3.
[0213] Cytotoxic T Lymphocyte (CTL) assays which demonstrate
specific lysis induced by vaccination with mesothelin-specific DNA
vaccines. Mice (5 per group) were immunized with various DNA
vaccines intradermally. Mice received a booster with the same dose
one week later. Splenocytes from mice were pooled 14 days after
vaccination. To perform the cytotoxicity assay, splenocytes were
cultured with mesothelin protein for 6 days and used as effector
cells. WF-3 tumor cells served as target cells. WF-3 cells were
mixed with splenocytes at various E:T ratios. Cytolysis was
determined by quantitative measurements of LDH. Note: The
pcDNA3-mesothelin DNA vaccine generated a significantly higher
percentage of specific lysis than the other DNA vaccines
(P<0.001). The data presented in this figure are from one
representative experiment of two performed.
Example 13
[0214] In this example, we utilize an attenuated strain of
Salmonella typhimurium as a vehicle for oral genetic immunization.
PcDNA3.1/myc-His(-) vectors expressing a myc-tagged version of
mesothelin were constructed. Following immunization with the
recombinant S. typhimurium aroA strain harboring the mesothelin
expression vector, we are able to detect high levels of expression
of the mesothelin/myc fusion protein using an anti-myc antibody by
immunoassay. The S. typhimurium auxotrophic aroA strain SL7202 S.
typhimurium 2337-65 derivative hisG46, DEL407 [aroA::Tn10(Tc-s)]),
is used as carrier for these in vivo studies (see Darji et al.
(1997) Cell 91: 761-775; Darji et al. (2000) FEMS Immunology and
Medical Microbiology 27: 341-9). This S. typhimurium-based
mesothelin DNA vaccine delivery system is then used to test whether
this vaccine can protect ovarian cancer cells challenge using our
WF-3 tumor model system.
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Sequence CWU 1
1
12 1 9 PRT Homo sapiens 1 Ser Leu Leu Phe Leu Leu Phe Ser Leu 1 5 2
9 PRT Homo sapiens 2 Val Leu Pro Leu Thr Val Ala Glu Val 1 5 3 9
PRT Homo sapiens 3 Glu Leu Ala Val Ala Leu Ala Gln Lys 1 5 4 9 PRT
Homo sapiens 4 Ala Leu Gln Gly Gly Gly Pro Pro Tyr 1 5 5 9 PRT Homo
sapiens 5 Phe Tyr Pro Gly Tyr Leu Cys Ser Leu 1 5 6 9 PRT Homo
sapiens 6 Leu Tyr Pro Lys Ala Arg Leu Ala Phe 1 5 7 9 PRT Homo
sapiens 7 Ser Leu Tyr Asn Thr Val Ala Thr Leu 1 5 8 10 PRT Homo
sapiens 8 Gln Val Pro Leu Arg Pro Met Thr Tyr Lys 1 5 10 9 9 PRT
Homo sapiens 9 Ala Phe Leu Pro Trp His Arg Leu Phe 1 5 10 9 PRT
Homo sapiens 10 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 11 30 DNA
Mus musculus 11 cccgaattca tggccttgcc aacagctcga 30 12 30 DNA Mus
musculus 12 tatggatccg ctcagcctta aagctgggag 30
* * * * *
References