U.S. patent application number 11/006468 was filed with the patent office on 2006-02-09 for compositions, methods and products comprising human papillomavirus for detecting and treating a cancer.
Invention is credited to Paul L. Hermonat, V. Suzanne Klimberg, Yong Liu.
Application Number | 20060029943 11/006468 |
Document ID | / |
Family ID | 25454937 |
Filed Date | 2006-02-09 |
United States Patent
Application |
20060029943 |
Kind Code |
A1 |
Hermonat; Paul L. ; et
al. |
February 9, 2006 |
Compositions, methods and products comprising human papillomavirus
for detecting and treating a cancer
Abstract
Methods for screening a patient for a cancer wherein the methods
comprise detecting an HPV in a biopsy from a patient are disclosed.
Also disclosed are compositions and products for screening and for
treating cancer in a patient, as well as methods of treating a
patient afflicted with a cancer.
Inventors: |
Hermonat; Paul L.; (Little
Rock, AR) ; Klimberg; V. Suzanne; (Little Rock,
AR) ; Liu; Yong; (Little Rock, AR) |
Correspondence
Address: |
GILBRETH & Associates, P.C.
P.O. Box 2428
Bellaire
TX
77402-2428
US
|
Family ID: |
25454937 |
Appl. No.: |
11/006468 |
Filed: |
December 6, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09927585 |
Aug 9, 2001 |
6884605 |
|
|
11006468 |
Dec 6, 2004 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/91.2 |
Current CPC
Class: |
G01N 2333/025 20130101;
A61B 10/0041 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Claims
1. A method of screening a patient for cancer, the method
comprising: a) assaying for the presence of human papillomavirus in
a sample from a biopsy taken from a patient, wherein the patient
has been determined to be negative for CIN III, wherein said
assaying comprises amplifying human papillomavirus gene sequences
by use of a pair of primers to produce an amplified product, and
probing said amplified product for the presence of a portion of an
HPV sequence, wherein said pair of primers is either SEQ ID NO: 1
and SEQ ID NO: 2, or SEQ ID NO: 3 and SEQ ID NO: 4, and wherein
presence of said HPV is indicative of breast cancer in said
patient.
2. The method of claim 1 wherein the biopsy is obtained by
performing the technique of ductal lavage on a breast of a
patient.
3. The method of claim 1 wherein the patient is a human, wherein
the cancer is in any stage of development, and wherein the cancer
is selected from the group consisting of breast, dermal, oral,
penile, vulvar cancer, and any combination thereof.
4. The method of claim 1 wherein said amplifying is achieved by use
of polymerase chain reaction amplification.
5. The method of claim 1 wherein said amplifying is achieved by use
of reverse-transcription polymerase chain reaction
amplification.
6. The method of claim 1 wherein said HPV sequence is selected from
the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45,
HPV58.
7. The method of claim 1 wherein said probing is for the presence
of at least two HPV sequences selected from the group consisting of
HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
8. The method of claim 7 wherein one of the at least two HPV
sequences is HPV18.
9. The method of claim 1 wherein said probing is for the presence
of a portion of HPV16 and at least one HPV sequence selected from
the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45,
HPV58.
10. A method of screening a patient for cancer, the method
comprising: a) assaying for the presence of human papillomavirus in
a sample from a biopsy taken from a patient wherein said assaying
comprises amplifying human papillomavirus gene sequences by use of
a pair of primers to produce an amplified product, and probing said
amplified product for the presence of a portion of an HPV sequence
selected from the group consisting of HPV18, HPV31, HPV 33, HPV35,
HPV45, HPV58, wherein said pair of primers is either SEQ ID NO: 1
and SEQ ID NO: 2, or SEQ ID NO: 3 and SEQ ID NO: 4, and wherein
presence of said HPV is indicative of breast cancer in said
patient.
11. The method of claim 9 wherein said probing is for the presence
of at least two HPV sequences selected from the group consisting of
HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
12. The method of claim 9 wherein the patient is a human, wherein
the cancer is in any stage of development, and wherein the cancer
is selected from the group consisting of breast, dermal, oral,
penile, vulvar cancer, and any combination thereof.
13. The method of claim 9 wherein the biopsy is obtained by
performing the technique of ductal lavage on a breast of a
patient.
14. The method of claim 9 wherein said amplifying is achieved by
use of polymerase chain reaction amplification.
15. The method of claim 9 wherein said amplifying is achieved by
use of reverse-transcription polymerase chain reaction
amplification.
16-20. (canceled)
21. A method of screening a patient for a cancer, the method
comprising: a) contacting cellular material with an HPV specific
probe, wherein the cellular material is extracted from a biopsy
taken from a patient, and wherein the patient has been determined
to test negative for CIN III.
22. The method of claim 21 wherein the cellular material is derived
from cells obtained by performing the technique of ductal lavage on
a breast of a patient.
23. The method of claim 21 wherein the cellular material comprises
nucleic acid, polypepetides, or a combination thereof.
24. The method of claim 21 wherein the probe is an HPV DNA or RNA
oligonucleotide sequence complementary to the plus strand of an HPV
DNA sequence.
25. The method of claim 21 wherein the probe is an HPV DNA or RNA
oligonucleotide sequence complementary to a portion of an HPV mRNA
sequence.
26. The method of claim 21 wherein the probe is an HPV DNA or RNA
oligonucleotide sequence complementary to a portion of an HPV
ribosomal RNA sequence.
27. The method of claim 21 wherein the probe is an antibody
specific to an epitope of an HPV protein.
28. The method of claim 27 wherein the protein is HPV16 E6 or HPV16
E7.
29. The method of claim 21 wherein the HPV is selected from the
group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
30. The method of claim 21 wherein step a) further comprises
contacting the cellular material with a second HPV specific probe,
wherein the first and second HPV are different from one another and
are selected from the group consisting of HPV18, HPV31, HPV 33,
HPV35, HPV45, HPV58.
31. The method of claim 21 wherein step a) further comprises
contacting the cellular material with a second HPV specific probe,
wherein the first HPV specific probe is specific to HPV 16 and the
second HPV specific probe is specific to at least one HPV selected
from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45,
HPV58.
32. The method of claim 21 wherein the cancer is in any stage of
development, and wherein the cancer is selected from the group
consisting of breast, dermal, oral, penile, vulvar cancer, and any
combination thereof.
33. A method of screening a patient for a cancer, the method
comprising: a) contacting cellular material with a probe specific
to a first HPV, and a second probe specific to a second HPV,
wherein the cellular material is extracted from a biopsy taken from
a patient and wherein the first HPV is HPV 16, and the second HPV
is selected from the group consisting of HPV18, HPV31, HPV 33,
HPV35, HPV45, HPV58.
34. The method of claim 33 wherein the cellular material is derived
from cells obtained by performing the technique of ductal lavage on
a breast of a patient.
35. The method of claim 33 wherein the cellular material comprises
nucleic acid, polypepetides, or a combination thereof.
36. The method of claim 33 wherein the probe is an HPV DNA or RNA
oligonucleotide sequence complementary to the plus strand of an HPV
DNA sequence.
37. The method of claim 33 wherein the probe is an HPV DNA or RNA
oligonucleotide sequence complementary to a portion of an HPV mRNA
sequence.
38. The method of claim 33 wherein the probe is an HPV DNA or RNA
oligonucleotide sequence complementary to a portion of an HPV
ribosomal RNA sequence.
39. The method of claim 33 wherein the probe is an antibody
specific to an epitope of an HPV protein.
40. The method of claim 36 wherein the protein is HPV16 E6 or HPV16
E7.
41. The method of claim 33 wherein the cancer is in any stage of
development, and wherein the cancer is selected from the group
consisting of breast, dermal, oral, penile, vulvar cancer, and any
combination thereof.
42. A method of screening a patient for a cancer, the method
comprising: a) contacting cellular material with a probe specific
to a HPV selected from the group consisting of HPV18, HPV31, HPV
33, HPV35, HPV45, HPV58, wherein the cellular material is extracted
from a biopsy taken from a patient.
43. The method of claim 37 wherein step a) further comprises a
second probe specific to a second HPV selected from the group
consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58, wherein
the first and second HPV are different from one another.
44. The method of claim 42 wherein the cellular material is derived
from cells obtained by performing the technique of ductal lavage on
a patient.
45. The method of claim 42 wherein the cellular material comprises
nucleic acid, polypepetides, or a combination thereof.
46. The method of claim 42 wherein the probe is an HPV DNA or RNA
oligonucleotide sequence complementary to the plus strand of an HPV
DNA sequence.
47. The method of claim 42 wherein the probe is an HPV DNA or RNA
oligonucleotide sequence complementary to a portion of an HPV mRNA
sequence.
48. The method of claim 42 wherein the probe is an HPV DNA or RNA
oligonucleotide sequence complementary to a portion of an HPV
ribosomal RNA sequence.
49. The method of claim 42 wherein the probe is an antibody
specific to an epitope of an HPV protein.
50. The method of claim 42 wherein the cancer is in any stage of
development, and wherein the cancer is selected from the group
consisting of breast, dermal, oral, penile, vulvar cancer, and any
combination thereof.
51. A method of treating a patient comprising: a) administering a
composition comprising an effective amount of an antisense HPV
sequence to a patient.
52. The method of claim 51 wherein administering comprises delivery
of the composition into a milk duct of a breast of the patient by
insertion of a microcatheter into a nipple surface orifice of said
breast.
53. The method of claim 51 wherein the HPV is selected from the
group consisting of HPV16, HPV18, HPV31, HPV 33, HPV35, HPV45,
HPV58, and any combination thereof.
54. The method of claim 51 wherein the antisense HPV sequence is
expressed from a viral expression vector.
55. The method of claim 51 wherein the patient is human and has a
cancer in any stage of development.
56. The method of claim 51 wherein the cancer is breast, dermal,
oral, penile, or vulvar cancer, or any combination thereof.
57. A method of treating a patient comprising: a) administering an
effective amount of a composition to a patient, wherein the
composition comprises an agent that inhibits expression of at least
one HPV gene.
58. The method of claim 57 wherein administering comprises delivery
of the composition into a milk duct of a breast of the patient by
insertion of a microcatheter into a nipple surface orifice of said
breast.
59. The method of claim 57 wherein the agent is an oligonucleotide
comprising antisense HPV DNA, RNA or ribosomal RNA.
60. The method of claim 57 wherein the agent is an oligonucleotide
comprising sequences complementary to the plus or minus strand of
HPV DNA.
61. The method of claim 57 wherein the HPV is selected from the
group consisting of HPV16, HPV18, HPV31, HPV33, HPV35, HPV45,
HPV58, and any combination thereof.
62. The method of claim 57 wherein the patient is human and has a
cancer in any stage of development.
63. The method of claim 57 wherein the cancer is breast, dermal,
oral, penile, or vulvar cancer, or any combination thereof.
64. A method of treating a patient comprising: a) administering an
effective amount of a composition comprising an agent that
specifically inhibits the HPV16 E6 protein or the HPV16 E7
protein.
65. The method of claim 64 wherein administering comprises delivery
of the composition into a milk duct of a breast of the patient by
insertion of a microcatheter into a nipple surface orifice of said
breast.
66. The method of claim 64 wherein the agent is an antibody
specific for the HPV16 E6 protein or HPV16 E7 protein.
67. The method of claim 64 wherein the patient is human and has a
cancer in any stage of development.
68. The method of claim 64 wherein the cancer is breast, dermal,
oral, penile, or vulvar cancer, or any combination thereof.
69. A method of treating a patient comprising: a) transfecting
dendritic precursor cells of a patient with a recombinant viral
vector that drives expression of an HPV antigen; b) treating the
dendritic precursor cells with a cytokine to produce dendritic
cells stably expressing the HPV antigen; c) contacting T cells
together with the dendritic cells stably expressing the HPV antigen
to produce primed T cells; and d) administering to the patient an
effective amount of either the primed T cells, dendritic cells, or
a combination thereof.
70. The method of claim 69 wherein the cytokine is selected from
the group consisting of interluekins, GM-CSF, TNF, and any
combination thereof.
71. The method of claim 69 wherein the patient is human, and
wherein the patient has a cancer in any stage of development.
72. The method of claim 71 wherein the cancer is breast, dermal,
oral, penile, or vulvar cancer, or any combination thereof.
73. The method of claim 69 wherein the recombinant viral vector is
an adeno-associated viral vector.
74. The method of claim 69 wherein the HPV is selected from the
group consisting of HPV16, HPV18, HPV31, HPV 33, HPV35, HPV45,
HPV58, and any combination thereof.
75. The method of claim 69 wherein the HPV antigen is HPV E6 or HPV
E7.
76. A kit for screening a patient for a cancer, the kit comprising:
a) a probe specific for detection of an HPV.
77. The kit of claim 77 wherein the probe is a single-stranded
olidonucleotide sequence, a double-stranded oligonucletide
sequence, a polypeptide, or any combination thereof.
78. The kit of claim 77 wherein the HPV is selected from the group
consisting of HPV16, HPV18, HPV31, HPV35, HPV45, HPV58, and any
combination thereof.
79. The kit of claim 77 wherein the patient is human, wherein the
cancer is in any stage of development, and wherein the cancer is
selected from the group consisting of breast, dermal, oral, penile,
vulvar cancer, and any combination thereof.
80. A composition for treating a patient having a cancer, the
composition comprising: an effective amount of an HPV sequence.
81. The composition of claim 80 wherein the sequence is selected
from the group consisting of single-stranded nucleic acids,
double-stranded nucleic acids, polypeptides, and any combination
thereof.
82. The composition of claim 80 wherein the HPV sequence is
selected from the group consisting of HPV 16, HPV 18, HPV 31, HPV
33, HPV 35, HPV 45, HPV58, and any combinations thereof.
83. The composition of claim 80 wherein the HPV sequence is HPV16
and any one of the group consisting of HPV 18, HPV 31, HPV 33, HPV
35, HPV 45, HPV58, and any combinations thereof.
84. The composition of claim 80 wherein the HPV sequence is HPV 18
and any one of the group consisting of HPV 16, HPV 31, HPV 33, HPV
35, HPV 45, HPV58, and any combinations thereof.
85. The composition of claim 80 wherein the HPV sequence is a
combination of HPV 16 and HPV 18.
86. The composition of claim 80 wherein the HPV sequence is a
combination of HPV 16 and HPV 18 and at least any one of the group
consisting of HPV 31, HPV 33, HPV 35, HPV 45, HPV58, and any
combinations thereof.
87. The composition of claim 80 wherein the HPV sequence is a
combination of HPV 16, HPV 18 and HPV 33, and at least any one of
the group consisting of HPV 31, HPV 35, HPV 45, HPV58, and any
combinations thereof.
88. A method of screening a patient for breast cancer, the method
comprising: a) assaying for the presence of human papillomavirus in
a sample from a biopsy taken from a patient, wherein said assaying
comprises amplifying human papillomavirus gene sequences by use of
a pair of primers to produce an amplified product, and probing said
amplified product for the presence of HPV 16 and a second human
papillomavirus selected from the group consisting of HPV18, HPV31,
HPV 33, HPV35, HPV45, HPV58, wherein said pair of primers is SEQ ID
NO: 3 and SEQ ID NO: 4, and wherein presence of HPV 16 and said
second human papillomavirus is indicative of breast cancer in said
patient.
89. The method of claim 88 wherein the patient is a human, and
wherein the cancer is in any stage of development.
90. The method of claim 88 wherein the biopsy is obtained by
performing the technique of ductal lavage on a breast of a
patient.
91. The method of claim 88 wherein said amplifying is achieved by
use of polymerase chain reaction amplification.
92. The method of claim 88 wherein said amplifying is achieved by
use of reverse-transcription polymerase chain reaction
amplification.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to products, compositions,
methods and apparatus for identification of cancers and
pre-cancerous cellular changes. In another aspect, the present
invention relates to products, compositions, methods and apparatus
for identification of breast cancers or pre-cancerous cellular
changes in breast tissues. In even another aspect, the present
invention relates to products, compositions, methods and apparatus
for treatment of cancers and pre-cancerous cellular changes. In
still another aspect, the present invention relates to products,
compositions, methods and apparatus for treatment of breast cancer
and pre-cancerous cellular changes in breast tissues.
[0003] 2. Description of the Related Art
[0004] Breast cancer is the most common form of cancer in women in
the United States. It is estimated that in the year 2000, 182,800
new cases of female invasive breast cancer will be diagnosed, and
40,800 women will die from the disease. All women are at risk for
breast cancer, with this risk increasing as a woman ages. Women are
generally considered to be at increased risk for developing breast
cancer if they have one or more of the following risk factors: a) a
family history of breast cancer, b) a previous diagnosis of a
malignant breast tumor or other gynecological cancers, c) hormonal
factors, or d) not having had any children or having the first
child later in their child bearing years. Even so, the majority of
all breast cancers occur in women who apparently do not have
identifiable risk factors.
[0005] Breast cancer cannot currently be prevented. But detecting
and treating it at an early stage, when the tumor is small and has
not spread beyond the breast, can increase the chances of survival
significantly. However, not all breast cancers are currently
detected at this early stage. Therefore, screening for breast
cancer has become a critical aspect in the overall management of
this disease.
[0006] The techniques currently used to screen for breast cancer
and other breast conditions include monthly breast self
examination, mammography, and clinical breast examination. Also,
genetic testing can be performed for BRCA1 and BRCA2 genes in women
who have a strong family history of breast cancer, since these
genes are associated with approximately 5 to 10 percent of breast
cancer cases. In spite of this genetic knowledge, the genetic
changes involved in the vast majority of breast cancers remains
largely undetermined.
[0007] Human papillomaviruses (HPV) are strongly linked to cervical
and other cancers. Cervical cancer (CX CA) is the second most
prevalent female cancer world-wide. HPV 16 DNA is present in 65% of
CX CAs, and with the other HPV types, more than 90% of CX CAs
contain HPV DNA. The E6 and E7 genes of HPV 16s can cause
contact-inhibited cells to lose this phenotype. Furthermore, E6 and
E7 interact with the cellular anti-oncogenes RB.sup.105 and p53,
respectively, leading to their inactivation. Thus, it is widely
regarded that HPV-16 is a central etiologic agent and risk factor
in the development of cervical/genital cancer. The E7 protein, and
possibly E6 as well, also function as transcriptional
transactivators of heterologous genes. HPVs have also been found in
oral, penile, and vulvar cancer.
[0008] It appears that whatever tissue site HPVs are known to
infect, they cause pathology. Usually the pathology is limited to a
tissue hyperplasia or papilloma. However, there is a significant
risk that this higher than normal active cell growth may become an
outright malignancy.
[0009] However, as recent studies have indicated, the relationship
between HPV infection and breast cancer is controversial.
[0010] Hennig et al. (1999) have reported that of women studied in
Norway having concomitant advanced genital HPV infection (cervical
intraepithelial neoplasia III, "CIN III") in addition to breast
cancer, 46% of the breast cancers also contained HPV 16. However,
of the control study of eight patients having breast cancer
diagnosed before the CIN III lesions, none had HPV positive breast
carcinomas. Additionally no cases in the study were positive for
HPV 11, 18 or 33.
[0011] Yu et al. (1999) report that HPV 33 is associated in
pre-malignant and malignant breast lesions in Chinese and Japanese
populations, and further suggest that HPV 16 and HPV 18 are not
involved in breast hyperplastic lesion, especially breast
cancer.
[0012] In spite of the advancements in the art, there is a need in
the art for improved compositions, methods and products for
screening a patient for cancer and/or pre-cancerous cellular
changes.
[0013] There is another need in the art for improved compositions,
products and methods for screening a patient for breast cancer
and/or pre-cancerous cellular changes in the breast.
[0014] There is even another need in the art for improved
compositions, products and methods for treating a patient afflicted
with a cancer in any stage of development.
[0015] There is still another need in the art for improved
compositions, products and methods for treating a patient afflicted
with breast cancer and/or a pre-cancerous cellular changes in the
breast in any stage of development.
[0016] These and other needs in the art will become apparent to
those of skill in the art upon review of this specification,
including its drawings, claims and appendix.
SUMMARY OF THE INVENTION
[0017] It is an object of the present invention to provide
compositions, methods and products for screening a patient for
cancer and/or pre-cancerous cellular changes.
[0018] It is another object of the present invention to provide for
compositions, products and methods for screening a patient for
breast cancer and pre-cancerous cellular changes in the breast.
[0019] It is even another object of the present invention to
provide compositions, products and methods for treating a patient
afflicted with a cancer and/or pre-cancerous cellular changes in
any stage of development
[0020] It is still another object of the present invention to
provide compositions, products and methods for treating a patient
afflicted with breast cancer and/or a pre-cancerous cellular
changes in the breast in any stage of development.
[0021] These and other objects in the art will become apparent to
those of skill in the art upon review of this specification,
including its drawings, claims and appendix.
[0022] According to one embodiment of the present invention there
is provided a method of screening a patient for a cancer. The
method generally comprises performing an amplification technique on
a sample from a biopsy taken from a patient. The sample comprises
nucleic acid, and the amplification technique is directed to
specific amplification of a portion of a human papillomavirus (HPV)
sequence contained therein. The method further includes probing for
the presence of an HPV sequence in the amplified sequence using an
HPV specific probe.
[0023] According to another embodiment of the present invention
there is provided a method of screening a patient for a cancer. The
method generally comprises contacting cellular material together
with an HPV specific probe. The cellular material is generally
extracted from a sample, such as a biopsy, taken from a patient.
The cellular material may be any purified or non-purified cellular
material such as, for example, deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), polypeptides, or a combination thereof. The
cellular material may be purified, either partially or wholly,
using any of the methods well known in the art.
[0024] The probe used in the screening methods of the invention may
be specific to any HPV selected from the group consisting of HPV18,
HPV31, HPV 33, HPV35, HPV45, HPV58. In a preferred embodiment, the
screening method further comprises contacting the cellular material
with a second HPV specific probe, wherein the first and second HPV
are different from one another and are selected from the group
consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
Alternatively, in another embodiment, the screening method further
comprises contacting the cellular material with a second HPV
specific probe, wherein the first HPV specific probe is specific to
HPV 16 and the second HPV specific probe is specific to at least
one HPV selected from the group consisting of HPV18, HPV31, HPV 33,
HPV35, HPV45, HPV58.
[0025] According to even another embodiment of the present
invention there is provided a method of treating a patient. The
method generally comprises administering a composition comprising
an effective amount of an antisense HPV DNA sequence to a patient.
Preferably, the antisense HPV DNA sequence is expressed from a
viral expression vector, such as an adeno-associated vector. The
HPV may be any member of the HPV family, such as, for example,
HPV16, HPV18, HPV31, HPV35, HPV45, HPV58, and any combination
thereof.
[0026] According to still another embodiment of the present
invention there is provided a method of treating a patient. The
treatment method generally comprises administering an effective
amount of a composition to a patient, wherein the composition
comprises an agent that inhibits expression of at least one HPV
gene.
[0027] According to yet another embodiment of the present invention
there is provided a method of treating a patient. The treatment
method generally comprises administering an effective amount of a
composition to a patient, wherein the composition comprises an
agent that specifically inhibits an HPV protein. Examples of HPV
proteins to target for inhibition include the HPV16 E6 protein and
the HPV16 E7 protein. Inhibition of a protein can be by any of the
methods known in the art, such as, targeting with an antibody,
inhibition of post-translation modification, inhibition of protein
stability and half-life. A preferred agent for use in the treatment
method of the present invention is an antibody specific for
interaction with an epitope of an HPV protein, such as HPV16 E6
protein or HPV16 E7 protein.
[0028] According to even still another embodiment of the present
invention there is provided a method of treating a patient. The
method comprises transfecting dendritic cells (DCs) into a patient,
wherein the dendritic cells have been altered to stably produce an
HPV antigen. Preferably, a recombinant retrovirus such as for
example an adeno-associated virus (AAV) that has been genetically
manipulated to comprise a portion of an HPV antigen-encoding gene
is used to infect monocyte precursors which are then induced to
differentiate into DCs. Differentiation of monocytes in DCs may be
accomplished by treating the monocytes with at least one
cytokine.
[0029] According to even yet another embodiment of the present
invention there is provided for a kit useful for screening a
patient for a cancer. Generally the kit comprises a probe that is
specific for the detection of an HPV family member. The
HPV-specific probe may be a single-stranded oligonucleotide
sequence, a double-stranded oligonucleotide sequence, a
polypeptide, or any combination thereof. The HPV may be any HPV
family member including, HPV16, HPV18, HPV31, HPV35, HPV45, HPV58,
and any combination thereof. In a preferred embodiment the HPV is
HPV16 or HPV18. The probe may be used on any sample derived from a
patient.
[0030] These and other embodiments of the present invention will
become apparent to those of skill in the art upon review of this
specification, including its drawings and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 is a Polymerase Chain Reaction (PCR)/Dot Blot
Hybridization analysis for HPV-16/18/31 "super" probe.
[0032] FIG. 2 provides results from a PCR/Dot blot analysis for
HPVs using an L1 targeting primer set and probing with HPV-16
sequences.
[0033] FIG. 3 is a Polymerase Chain Reaction (PCR)/Dot Blot
Hybridization analysis for HPVs in breast cancer specimens using an
L1 targeting primer set and probing with HPV-18 probe.
[0034] FIG. 4 is a Polymerase Chain Reaction (PCR)/Dot Blot
Hybridization analysis for HPVs in breast cancer specimens using an
L1 targeting primer set and probing with HPV-31 probe.
[0035] FIG. 5 provides results from a PCR/Dot blot analysis for
HPVs using an E6-E7 junction targeting primer set and probing with
HPV-16 sequences.
[0036] FIG. 6 provides results from a PCR/Dot blot analysis for
HPVs using an E6-E7 junction targeting primer set and probing with
HPV-18 sequences.
[0037] FIG. 7 is a PCR/Dot Blot Hybridization analysis for
contaminating plasmids by using a primer set which targets the
pBR322/ColE1 ori region.
[0038] FIG. 8A shows a structural map of the AAV/NE6/NEO (a.k.a.
d16-95/E6.sup.p5/NEO.sup.SV40) virus with the names of the
components at the top.
[0039] FIG. 8B shows the analysis of various 293/vector producer
cell lines.
[0040] FIG. 8C shows a titering analysis of the AAV/E6/Neo virus
stock used in this study.
[0041] FIG. 8D shows a graphic description of the experimental
protocol.
[0042] FIG. 9 provides E6 mRNA expression in infected DC.
[0043] FIG. 10 provides the efficiency of Mo/DC-pulsing analyzed by
intracellular staining.
[0044] FIG. 11 shows the amount of chromosomal integration by
AAV/E6/Neo in DC.
[0045] FIG. 12 shows the early appearance of priming rosettes
during AAV-mediated priming.
[0046] FIG. 13 shows the cytotoxic response resulting from AAV
vector and DOTAP-protein lipofection after 7 days of priming.
[0047] FIG. 14 is a two-color flow cytometric characterization of
primed cell populations.
[0048] FIG. 15 is the characterization of DC at day 7 under
different conditions.
[0049] FIG. 16 provides the characterization of CD80 in pulsed
DC.
DETAILED DESCRIPTION OF THE INVENTION
[0050] According to one embodiment of the present invention there
is provided a method of screening a patient for a cancer. The
method generally comprises performing an amplification technique on
a sample from a biopsy taken from a patient. The sample comprises
cellular material, preferably nucleic acid, and the amplification
technique is directed to specific amplification of a portion of a
marker for a cancer. In a preferred embodiment, the marker is a
human papillomavirus (HPV) sequence. In a more preferred
embodiment, the cancer is breast cancer in any stage of
development. The method may further include probing for the
presence of an HPV sequence in the amplified sequence using an HPV
specific probe.
[0051] The methods of the invention may comprise amplification of
at least one HPV sequence selected from the group consisting of
HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58, or any combination
comprising at least HPV sequences. Suitable combinations include:
HPV16 and at least any one of the group consisting of HPV18, HPV31,
HPV 33, HPV35, HPV45, HPV58; HPV18 and at least any one of the
group consisting of HPV16, HPV31, HPV 33, HPV35, HPV45, HPV58; and
at least any two of the group consisting of HPV 18, HPV31, HPV 33,
HPV35, HPV45, HPV58.
[0052] With respect to probing for a specific HPV sequence, the
methods of the invention may comprise probing for at least one HPV
sequence selected from the group consisting of HPV18, HPV31, HPV
33, HPV35, HPV45, HPV58, or any combination comprising at least HPV
sequences. Suitable combinations include: HPV16 and at least any
one of the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45,
HPV58; HPV18 and at least any one of the group consisting of HPV16,
HPV31, HPV 33, HPV35, HPV45, HPV58; and at least any two of the
group consisting of HPV 18, HPV31, HPV 33, HPV35, HPV45, HPV58.
[0053] The samples used in the present invention may be obtained
from a biopsy from a cervical intraepithelial neoplasia III (CIN
III) positive patient or a CIN III negative patient. Preferably the
sample is obtained from a biopsy from a patient who is not
afflicted with CIN III (a non-CIN III patient). Thus the sample may
first be tested/assayed for the presence of any CIN III-marker
known in the art.
[0054] The sample may be derived from the patient by any method
known in the art, such as, for example, any well known method for
obtaining a biopsy, including the recently reported technique of
breast duct lavage (Dooley W. C. et al., Lancet, 2001,
357(9265):1335-6; Evron, E. et al., Obstetrics & Gynecology,
2001, 97(4):S2, both of which are incorporated herein by
reference). Numerous methods for obtaining a sample via biopsy are
known in the art and include for example bite, brush, cone,
cytological, aspiration, endoscopic, excisional, exploratory,
incisional, percutaneous, punch, and surface biopsy. Breast duct
lavage, also referred to as ductal lavage and intraductal lavage,
is a relatively non-invasive procedure and enables the retrieval of
breast epithelial cells that line the ductal/lobular systems of all
milk ducts. Whereas a needle is used in aspiration biopsy, the
technique of ductal lavage comprises use of a microcatheter which
is inserted into the milk ducts through the nipple surface
orifices. Saline is then flushed through the ducts to wash out
epithelial cells for collection and further evaluation.
[0055] Preparation of samples for amplification is well known in
the art, and any such technique may be used herein. Amplification
methods are well known in the art and include techniques such as,
for example, polymerase chain reaction (PCR) amplification and
reverse transcription PCR (RT-PCR), as well as others. The
amplified products may be detected and analyzed using any of the
numerous techniques well known in the art.
[0056] The amplification technique used herein may be specific for
amplification of a portion of at least one HPV sequence selected
from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45,
HPV58. The amplification technique used herein may be specific for
amplification of a portion of at least two HPV sequences selected
from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45,
HPV58. The amplification technique used herein may be specific for
amplification of a portion of HPV16 and at least one HPV sequence
selected from the group consisting of HPV18, HPV31, HPV 33, HPV35,
HPV45, HPV58.
[0057] Another embodiment of the present invention provides for a
method of screening a patient for a cancer. The method generally
comprises contacting cellular material together with an HPV
specific probe. The cellular material is generally extracted from a
sample, such as a biopsy, taken from a patient. The cellular
material may be any purified or non-purified cellular material such
as, for example, deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), polypeptides, or a combination thereof. The cellular
material may be purified, either partially or wholly, using any of
the purification methods well known in the art.
[0058] The HPV probes useful in the screening methods of the
present invention may be any type of probe useful in detecting the
presence of HPV. These probe types include, but are not limited to,
a single-stranded or double-stranded oligonucleotide sequence
complementary to the plus or minus strand of an HPV DNA sequence, a
single-stranded or double-stranded oligonucleotide sequence
complementary to a portion of an HPV mRNA sequence, and an antibody
specific to an epitope of an HPV protein. Suitable examples of HPV
proteins include, but are not limited to, the HPV16 E6 or HPV16 E7.
Oligonucleotide sequences specific to HPV sequences are known in
the art, such as, for example, those disclosed in Breast Cancer
Rch. Trtmt, 53;121-135, Anticancer Rch. 19;5057-5062, J. Gen.
Virol. 76:1057-1062, J. Pathol 165: 301-309, and J. Clin.
Microbiol. 34: 2095-2100, with all of these articles herein
incorporated by reference. Oligonucleotides may also be designed
according to the Los Alamos National Laboratory Database
nomenclature for the different HPV genomes, incorporated herein by
reference.
[0059] Methods of detecting targeted sequences with a probe are
well known in the art and are included herein. Any type of
hybridization method is suitable for use in the present
invention.
[0060] The screening methods of the present invention may be
performed on a sample from any organism/patient capable of
developing cancer. Preferably, the method of the present invention
is performed on a samples taken from a mammal, more preferably a
human. The patients on which the methods of the invention are used
may be CIN III positive or CIN III negative. In a preferred
embodiment, the patients are not afflicted with CIN III (CIN III
negative). The screening methods described herein are useful in
detecting numerous types of cancer, such as, for example, breast,
dermal, oral, penile, vulvar cancer, and any combination thereof.
In addition, the screening methods of the present invention are
useful in detecting a cancer in any stage of development.
[0061] Even another embodiment of the present invention provides
for a method of treating a patient afflicted with a cancer. The
method generally comprises administering a composition comprising
an effective amount of an antisense HPV sequence to a patient. The
size of the sequence is not limited and can range in size from that
of an oligonucleotide to that of a transcript. The antisense HPV
sequence may comprise DNA, RNA, ribosomal RNA, or any combination
thereof. The HPV may be any member of the HPV family, non-limiting
examples of which include, HPV16, HPV18, HPV31, HPV33, HPV35,
HPV45, HPV58, and any combination thereof. Preferred HPV's include
HPV 16 and HPV 18. Non-limiting examples of combinations include:
HPV 16 with at least one of HPV18, HPV31, HPV33, HPV35, HPV45, and
HPV58; HPV 18 with at least one of HPV16, HPV31, HPV33, HPV35,
HPV45, and HPV58; both HPV16 and HPV18 with at least one of HPV31,
HPV33, HPV35, HPV45, and HPV58, and at least any two of HPV16,
HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58.
[0062] The HPV sequence may be expressed from a recombinant
expression vector. Suitable vectors are known in the art and
include, for example, mammalian expression vectors and viral
vectors. Examples of viral vectors suitable for use in the present
invention include: retroviruses; adenoviruses;
adenoviral/retroviral chimeras; adeno-associated viruses; herpes
simplex virus I or II; parvovirus; and reticuloendotheliosis virus.
Other possible viral vectors may be derived from poliovirus,
papillomavirus, vaccinia virus, lentivirus, as well as chimeric
vectors incorporating favorable aspects of any two or more of the
above viruses. Preferably, the antisense HPV sequence is expressed
from a recombinant viral expression vector, such as an
adeno-associated vector.
[0063] Still another embodiment of the present invention provides
for a method of treating a patient afflicted with a cancer. The
treatment method generally comprises administering an effective
amount of a composition to a patient, wherein the composition
comprises an agent that inhibits expression of at least one HPV
gene. The patients of the present invention may be CIN III positive
or CIN III negative. Preferably, the patient is negative for CIN
III.
[0064] Mechanisms for inhibiting the expression of a gene are
numerous and well known in the art and include, but are not limited
to, inhibiting gene transcription, inhibiting the messenger RNA
(mRNA) of a gene, inhibiting translation of an mRNA, inhibiting
post-translational modification of a gene product, and inhibiting a
gene product. These inhibition methods may be direct or indirect.
Any of these mechanisms may be used in the present invention.
[0065] Agents that inhibit expression of at least one HPV gene
suitable for use in the present invention include, an
oligonucleotide or longer stretch of nucleic acid comprising
antisense HPV DNA, RNA or ribosomal RNA, and an oligonucleotide or
longer stretch of DNA, RNA, or ribosomal RNA comprising a sequence
complementary to the plus or the minus strand of HPV DNA. The HPV
targeted in the present invention may be any HPV family member,
non-limiting examples of which include, HPV16, HPV18, HPV31, HPV33,
HPV35, HPV45, HPV58, and any combination thereof. Preferred HPV's
include HPV 16 and HPV 18. Non-limiting examples of combinations
include: HPV 16 with at least one of HPV18, HPV31, HPV33, HPV35,
HPV45, and HPV58; HPV 18 with at least one of HPV16, HPV31, HPV33,
HPV35, HPV45, and HPV58; both HPV16 and HPV18 with at least one of
HPV31, HPV33, HPV35, HPV45, and HPV58, and at least any two of
HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58.
[0066] Administration of the compositions of the present invention
to a recipient may be by any method known in the art. Thus,
administration of the present invention to a recipient may be by a
route selected from oral, parenteral (including, subcutaneous,
intradermal, intramuscular, and intravenous) and rectal. For
increased efficacy, the compositions of the present invention may
be administered via localized delivery to the targeted tissue, such
as, for example, breast tissue in the case of breast cancer. A
modified breast duct lavage technique, also known as ductal lavage,
may be used for localized directly of the anticancer compounds and
compositions of the present invention to a breast duct and breast
epithelial cells. Similar to the technique for ductal lavage, a
microcatheter may be inserted into a nipple surface orifice and
subsequently into a milk duct. However, instead of simply flushing
the duct in order to obtain epithelial cells, as is done with
ductal lavage, the microcatheter may be used for localized delivery
of a composition of the invention directly to the breast duct and
breast epithelial cells.
[0067] Yet another embodiment of the present invention provides for
a method of treating a patient afflicted with a cancer. The cancer
may be any cancer in any stage of development. In a preferred
embodiment, the cancer is breast cancer. In addition, the patient
may be CIN III positive or CIN III negative. Preferably, the
patient is negative for CIN III. The treatment method generally
comprises administering an effective amount of a composition to a
patient, wherein the composition comprises an agent that
specifically inhibits an HPV protein. Examples of HPV proteins to
target for inhibition include the HPV16 E6 protein and the HPV16 E7
protein. Inhibition of a protein can be by any of the methods known
in the art, such as, inhibition of gene expression, targeting a
protein with an antibody, inhibition of post-translation
modification, and inhibition of protein stability and half-life. A
preferred agent for use in the treatment method of the present
invention is an antibody specific for interaction with an epitope
of an HPV protein, such as HPV16 E6 protein or HPV16 E7
protein.
[0068] Additional inhibitory agents suitable for use in the
compositions and methods of the invention include agents wherein
the agent is a DNA, cDNA, RNA, ribosomal RNA, or polypeptide
sequence. Suitable examples of such agents include, an antisense
HPV sequence which inhibits transcription or translation of a HPV
gene or gene product, transcription factors which decrease
expression of an HPV gene, factors which affect translation of an
HPV mRNA, factors which decrease the stability/half-life of an HPV
mRNA molecule, factors which decrease the stability/half-life of an
HPV polypeptide, and factors which interact with an HPV
polypeptide, such as a polypeptide encoding an antibody which
specifically interacts with an epitope of a HPV. The material and
methods for producing these types of inhibitors (DNA, cDNA, RNA and
polypeptide) are known in the art and are included in the present
invention.
[0069] For example, expression vectors expressing a sequence
inhibitory to transcription of a HPV gene or expressing a sequence
inhibitory to translation of a HPV mRNA are within the scope of the
HPV inhibitors defined herein. Expression vectors suitable for the
present invention may comprise an antisense HPV sequence, or a
sequence encoding a negative regulator of transcription of a HPV
gene.
[0070] Even still another embodiment of the present invention
provides for a method of treating a patient afflicted with a
cancer. The method generally comprises transfecting dendritic cells
(DCs), primed T cells or a combination thereof, into a patient,
wherein the dendritic cells have been altered to stably produce an
HPV antigen. The basis for using DC cells for human immunotherapy
has recently been established as described in Young, J. W., and
Inaba, K. (1996) DCs as adjuvants for class I manor
histocompatibility complex-restricted antitumor immunity. J. Exp.
Med. 183:7-11, and Santin, A. D., Hermonat, P. L., Ravaggi, Al,
Chiriva-Internati, M., Hiserotdt, J. C., Pecorelli, S., and Parham,
G. P. (1999) Kinetics of expression of surface antigens during the
differentiation of human dendritic cells versus macrophages.
Immunobiology 200, both of which are incorporated herein by
reference.
[0071] Briefly, DCs are presently believed to be the most effective
antigen presenting cells for activating naive T cells. Blood
monocytes can be induced to differentiate in to DCs by treatment
with at least one cytokine. DCs when pulsed with antigen (Ag)
results in a class I restricted cytotoxic response against the Ag.
In most DC-pulsing protocols of the art, antigen proteins to be
targeted are transfected into DCs for immune stimulation. These DC
protocols may be used herein. However, both the antigen to be
presented by the Dcs, and the cytokines used to generate the DCs
degrade with time as a reflection of their half-lives. Thus, a more
effective technique for pulsing/treating the DCs may be the in situ
generation/production of the antigen protein such as at least a
portion of an HPV protein, and/or a cytokine within and by the DC
itself. By delivering a tumor-antigen gene and/or cytokine gene
directly in the DC, a stable and continuous production of the
proteins may be achieved.
[0072] Thus, a recombinant viral vector that has been genetically
manipulated to comprise a portion of a HPV gene and thus expresses
at least a portion of an HPV gene may be used to infect monocyte
precursors which have been acquired from a blood sample of a
patient. The infected monocytes are then induced to differentiate
into DCs by treatment with at least one cytokine. Examples of
suitable cytokines include, but are not limited to IL-2, IL-4,
other interluekins, GM-CSF, TNF, INF, and any combination thereof.
The dendritic cells may also stably produce a cytokine from a
recombinant vector.
[0073] Techniques for transferring genes are well known in the art,
and any of those techniques may be used to produce dendritic cells
that stably produce the antigen of choice. Preferably, a
recombinant retrovirus such as, for example, an adeno-associated
virus (AAV) that has been genetically manipulated to comprise a
portion of an HPV antigen-encoding gene is used to infect monocyte
precursors which are then induced to differentiate into DCs. In a
preferred embodiment, an AAV-HPV is used to infect monocytes which
are then induced to differentiate into DCs.
[0074] Materials and techniques for the design, generation and
production of recombinant retroviral vectors and genomes are well
known in the art. All such standard DNA materials, techniques and
methodologies are suitable for use herein. An example of a
recombinant retroviral vector suitable for use herein may comprise
the general purpose p5 transcriptional promoter to express the
antigen genes. Recombinant adeno-associated virus (rAAVs) may be
produced by first generating high producer cell lines to make
high-titer virus stocks, as known in the art. As also known by one
of skill in the art, these stocks may then be CsCl purified and
titered by quantitating the amount of encapsidated genomes (virus)
by Southern blot.
[0075] Suitable non-limiting examples of HPV's useful in the
present invention include, HPV16, HPV18, HPV31, HPV33, HPV35,
HPV45, HPV58, and any combination thereof. Preferred HPV's include
HPV 16 and HPV 18. Non-limiting examples of combinations include:
HPV 16 with at least one of HPV18, HPV31, HPV33, HPV35, HPV45, and
HPV58; HPV 18 with at least one of HPV16, HPV31, HPV33, HPV35,
HPV45, and HPV58; both HPV16 and HPV18 with at least one of HPV31,
HPV33, HPV35, HPV45, and HPV58, and at least any two of HPV16,
HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58. For example, for
HPV16 the DCs stably produce HPV 16 E6, HPV 16 E7 or both. Any
portion of the HPV, full-length or not, may be utilized herein.
[0076] The treatment methods of the present invention may be
performed on any organism having a cancer. Preferably, the methods
of the present invention are performed on a human. The patient may
be positive for CIN III or negative for CIN III. In a preferred
embodiment, the screening methods are carried out on patients who
have tested negative for CIN III, and/or are not afflicted with
cervical intraepithelial neoplasia (CIN III). The treatment methods
described herein may be useful in treating numerous types of
cancer, such as, for example, breast, dermal, oral, penile, vulvar
cancer, and any combination thereof. In addition, the treatment
methods of the present invention may be against a cancer in any
stage of development.
[0077] The compositions and methods of the present invention are
suitable for any individual afflicted with a cancer. Suitable
individuals include mammals such as, humans, dogs, cats, horses,
cows, sheep, goats, pigs, rats and mice. As mentioned, preferably
the patient is human. The compositions and methods of the present
invention are also suitable for use in any tissue or cell line that
serves as a model for the study of cancer. Thus the present
invention is useful to medical and health care professionals
including, medical doctors, and veterinarians, as well as research
scientists.
[0078] It should be noted that the present invention encompasses
any and all methods for screening a patient for a cancer, wherein
the method comprises detection of an HPV. Any and all methods for
treating a patient having a cancer, wherein the method comprises
inhibition of an HPV are also within the scope of the
invention.
[0079] The compositions useful in the methods of the present
invention further comprise a pharmaceutically acceptable
carrier/vehicle. Pharmaceutically acceptable carriers/vehicles are
known in the art and include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and the like,
propylene glycol, polyethylene glycol, vegetable oil, injectable
organic esters such as ethyloleate, water, saline solutions,
parenteral vehicles such as sodium chloride and Ringer's dextrose,
glycerol, lipids, alcohols.
[0080] Compositions of the present invention may be in any form
known in the art, such as an orally digestible form, a sterile
injectable form, forms suitable for delayed release, and forms that
are enterically coated. Compositions of the invention may be in
solid forms, including, for example, powders, tablets, pills,
granules, capsules, sachets and suppositories, or may be in liquid
forms including solutions, suspensions, gels and emulsions.
[0081] The compositions and methods of the present invention may be
administered to a recipient/patient as a single dose unit, or may
be administered in several dose units, for a period ranging from
one day to several years. The dose schedule is dependent upon at
least the severity of the glomerular disorder, as well as the mode
of administration. The effective dose of the compositions of the
present invention is further dependent upon the body weight (BW) of
the recipient/patient and also upon the chosen inhibitor. Generally
the compositions of the present invention are administered orally
or intravenously.
[0082] Even still another embodiment of the present invention
provides for a kit for screening a patient for a cancer. Generally
the kit comprises a probe that is specific for the detection of an
HPV family member. The HPV-specific probe may be a single-stranded
oligonucleotide sequence, a double-stranded oligonucleotide
sequence, a polypeptide, or any combination thereof. The HPV may be
any HPV family member, non-limiting examples of which include,
HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV58, and any
combination thereof. Preferred HPV's include HPV 16 and HPV 18.
Non-limiting examples of combinations include: HPV 16 with at least
one of HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58; HPV 18 with at
least one of HPV16, HPV31, HPV33, HPV35, HPV45, and HPV58; both
HPV16 and HPV18 with at least one of HPV31, HPV33, HPV35, HPV45,
and HPV58, and at least any two of HPV16, HPV18, HPV31, HPV33,
HPV35, HPV45, and HPV58.
[0083] The kit of the present invention is useful in screening any
organism capable of developing a cancer. Preferably the sample to
be screened is derived from a human patient. The patient may be CIN
III positive or CIN III negative, preferably the patient is CIN III
negative. The kit of the present invention may be useful in
detecting a cancer that is in any stage of development, and may be
useful in detecting any cancer, such as, for example breast,
dermal, oral, penile, vulvar cancer, and any combination
thereof.
[0084] Even yet another embodiment of the present invention
provides a composition for treating a patient having a cancer.
Generally, the composition comprises an effective amount of an HPV
sequence. The size of the sequence is not limited. The HPV sequence
of the composition may comprise single-stranded nucleic acids,
double-stranded nucleic acids, polypeptides, and any combination
thereof. The HPV sequence may be any one or any combination of HPV
family members. Suitable HPV family members include but are not
limited to, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 45, HPV58,
and any combinations thereof. Suitable combinations include: HPV16
and any one of the group consisting of HPV 18, HPV 31, HPV 33, HPV
35, HPV 45, and HPV58; HPV 18 and any one of the group consisting
of HPV 16, HPV 31, HPV 33, HPV 35, HPV 45, and HPV 58; HPV 16 and
HPV 18; HPV 16 and HPV 18 and any one of the group consisting of
HPV 31, HPV 33, HPV 35, HPV 45, HPV 58, and any combinations
thereof; HPV 16, HPV 18 and HPV 33, and any one of the group
consisting of HPV 31, HPV 35, HPV 45, HPV 58, and any combinations
thereof, and at least any two of the group consisting of HPV 16,
HVP 18, HPV 31, HPV 33, HPV 45 and HPV 58.
[0085] All references cited herein, including research articles,
all U.S. and foreign patents and patent applications, are
specifically and entirely incorporated by reference.
EXAMPLES
[0086] The following examples are provided to illustrate the
present invention. These examples are not intended to and do not
limit the scope of the claims of the present invention, and should
not be so interpreted.
Example 1
Analysis of Breast Cancer Tissue for Presence of HPV Via PCR
[0087] In the present example, total DNA was isolated from breast
cancer tissues and analyzed for the presence of HPV by use of PCR
amplification. The amplification targeted the L1 gene and was broad
spectrum, thus allowing for amplification of many different HPV
types.
[0088] Patients, Breast Cancer Specimens, and DNA Isolation
[0089] 17 women with breast cancer receiving examinations and
treatment at the University of Arkansas for Medical Sciences (UAMS)
from May 1999 to October 1999. Portions of needle biopsy tissue was
fixed in a Phosphate Buffered Saline: Ethanol (1:1) solution soon
after they were acquired. All of the specimens were stored at
-80.degree. C. The specimens were processed by grinding, and total
cellular DNA was isolated from the specimens by pelleting and
resuspending them in lysis buffer (0.5 mg/ml Proteinase K, 0.5%
SDS, 0.5 mM EDTA, 0.5 mM Tris-HCL, pH7.4). After incubation
overnight at 37.degree. C., the total cellular DNA was extracted by
phenol/chloroform twice, and then precipitated by ethanol. The DNA
was stored at -80.degree. C. before use. The research has been
approved by the UAMS Human Research Advisory Committee and
undertaken in a P2 laboratory.
[0090] Polymerase Chain Reaction (PCR) Primer Sets
[0091] An HPV L1-targeting consensus primer set has been described
elsewhere and was designed to amplify a 450-base segment of HPV L1
gene sequence (Bauer, H. M., et al., JAMA 1991; 265: 472-7). These
primers enable PCR amplification of most genital HPV types. A
second PCR primer set targeting E6-E7 region has also been
described by elsewhere (Fujinaga, Y, et al., J. Gen. Virol. 1991;
72: 1039-1044). These primers allowed for the amplification of most
of the cancer associated HPVs. PCR amplification was carried out as
described by Hermonat et al. (Hermonat P; et al., Virus Genes 1997;
14:13-17). Positive controls included various amounts of the
indicated cloned HPV genome, while negative controls included all
reagents except specimen DNA.
[0092] In order to identify potential contamination from
recombinant plasmids carrying HPV sequences, which are used in the
inventors' laboratories, PCR amplification was carried out with
pBR322 plasmid targeting primers. The primers were designed to
amplify a 414-base segment of pBR322 sequence (upstream primer
5'-ATACCTGTCCGCCTTTCTC-3', and downstream primer
5'-AATCTGCTGCTTGCAA AC-3'), containing the origin of replication
(ori). The controls included known quantities of pBR322, as low as
ten molecules.
[0093] PCR-Dot Blot Hybridization
[0094] Amplification of DNA samples was carried out in 100 .mu.l
reactions using approximately 5 .mu.g of the total cellular DNA,
0.2 mM of each dNTP, 1 .mu.M of each primers, and 2.5 U of Taq
iPolymerase according to the suppliers instructions (Fisher
Scientific Co., Pittsburgh, Pa.) instructions. After 5 minutes at
94.degree. C., each sample was subjected to the following
amplification cycle: 55 seconds at 94.degree. C., 1 minutes at
60.degree. C., and 50 seconds at 72.degree. C. for 35 cycles, then
10 minutes at 72.degree. C. in the final cycle.
[0095] The dot blotting was carried out with all of the PCR
products as described by Hermonat et al. (Hermonat P; et al., Virus
Genes 1997; 14:13-17). Briefly, 5 .mu.l of PCR products was first
denatured by the addition of 10 .mu.l of 0.4 N NaOH, incubated for
10 minutes, and then ice-bath was done for 5 minutes. After the
samples were reneutralized by the mixing of 200 .mu.l Tris-HCL,
pH7.0, 1.5 M NaCL, the samples were added immediately to a dot blot
apparatus under suction. Multiple nylon membranes were generated to
be analyze by one of several .sup.32P-labeled probes. The HPV
probes were made with Primer-a-Gene Labeling System (Promega Co.),
a random prime labeling kit according to the supplier=s
instructions, which templates were HPV 16, 18 and 31 genomic DNA
respectively.
[0096] The membranes were analyzed with the radiolabeled full
length HPV or pBR322 DNA sequences as indicated. The membranes were
soaked in hybridization solution (100 .mu.g of denatured salmon
sperm DNA, 1% SDS, 1M NaCL, 10% dextran sulfate) and incubated at
65.degree. C. overnight. After hybridization, the membranes were
first washed by 2.times.SSC twice at room temperature for 10
minutes, and then washed by 2.times.SSC and 1% SDS twice at
55.degree. C. (super probe) or 65.degree. C. (all others) for 30
minutes.
[0097] FIG. 1 provides the results of an assay in which PCR
products were dot blotted and probed with an HPV-16/18/31
"super"-probe. As seen in FIG. 1, of the breast cancer specimens,
six (B2, B4, B10, B13, B15, B17) were positive for HPV.
[0098] Next, the PCR products were probed with a probe specific for
HPV-16 only (FIG. 2), a probe specific for HPV-18 only (FIG. 3),
and a probe specific for HPV-31 (FIG. 4) only. As can be seen in
FIG. 2, the results for the HPV-16-specific probe revealed that
specimens B2, B4, and B13 gave a strong signal, suggesting that B2,
B4, and B13 are most likely HPV-16. The results from the HPV-18
probe (FIG. 3) revealed that all six specimens were positive for
HPV-18 (B2, B4, B10, B13, B15, B17), but that B15, in particular,
gave a very strong signal suggesting that this specimen was most
likely HPV-18. The results for HPV-31 probing (FIG. 4) revealed
that all of the six known HPV-positive specimens were weakly
positive. This verifies that the six specimens are HPV positive,
but also further suggests that the specimens are most likely not
HPV-31.
[0099] The results from a PCR/Dot blot analysis for HPV-16 using an
E6-E7 junction targeting primer set and probing with HPV-16
sequences are shown in FIG. 5. The dot blotted PCR products were
probed for HPV-16 and the membrane washed at 65.degree. C. Positive
controls are at the top. An unlabeled negative control is one row
below on the far left. Note that this primer/probe combination
appears to pick up the same specimens as identified by the L12
primer set and HPV-16 probe (compare to FIG. 2) (B2, B4, B13).
[0100] The results from a PCR/Dot blot analysis for HPV-18 using an
E6-E7 junction targeting primer set and probing with HPV-18
sequences are shown in FIG. 6. Positive controls are at the top. An
unlabeled negative control is one row below on the far left. Note
that this probe identifies all of the specimens identified by the
super probe and the L1 products, however B10, and B15 are
identified most strongly.
[0101] Experiments were then carried out in order to eliminate the
possibility of false positive signal from the tissue sample due to
any type of plasmid contamination. As shown in FIG. 7, these
samples were further analyzed for plasmid sequences using PCR
primers which targeted the ColE1 plasmid origin of replication
(ori) region. Essentially all plasmids in use today and in our
laboratory are based upon this ori. A dot blot hybridization
analysis, probed with .sup.32P-pBR322 is shown in FIG. 7. As can be
seen no contamination from plasmid DNA was observed in any of the
samples.
[0102] In cervical cancer the HPV DNA is often chromosomally
integrated. To determine the state of the HPV DNA in breast cancer,
10 .mu.gs of genomic DNA was digested with Bam HI or Xho I. HPV-16
and HPV contain a single Bam HI site, and no Xho I sites. The
restricted DNA, along with undigested DNA, were agarose gel
electrophoresed, Southern blotted and probed with .sup.32P-HPV-16
DNA. The Southern Blot revealed hybridization of the probe with an
8 kb band, consistent with episomal DNA. Only in the cervical swab
specimen C2 was there significant evidence of chromosomal
integration of the HPV DNA.
Example 2
Construction of the AAV/E6/Neo Genome, Generation of Virus Stocks,
and Titering of Virus Stocks
[0103] The AAV/E6/Neo genome was constructed as a plasmid, in a
similar manner to the construction of the AAV/GM-CSF/Neo viral
genome as described by Liu (Liu. Y., et al., J. Inf. Cytok. Res.
2000; 20:21-30), incoporated herein by reference. However, instead
of the GM-CSF gene, the HPV-16 E6 open reading frame was cloned by
PCR amplification using Pfu polymerase and ligated into the vector.
A structural map of the AAV/E6/Neo vector used in this study is
shown in FIG. 8A. In this construct the E6 gene is expressed from
the AAV p5 promoter, which is known to be active in DC. An
AAV/E7/Neo vector was also made in this study (not shown). In the
E7 construct, the E7 gene is expressed from the AAV p5
promoter.
[0104] High titer rough (non-purified) rAAV virus stocks were
generated in a two-step process, using the complementor plasmid
ins96-0.8, and titered as described previously by Hermonat et al.,
and Li et al. (Hermonat, P., et al., FEBS Let. 1997: 407:78-84; and
Liu. Y., et al., J. Inf. Cytok. Res. 2000; 20:21-30), incoporated
herein by reference.) In order to generate purified rAAV virus, the
technique described by Auricchio et al. was used (Human Gene
Therapy 2001; 12:71-76). Briefly, the virus solution treated by
DNase I (Promega Co.) was incubated with 0.5% deoxycholic acid
(Sigma Co.) for 30 minutes at 37.degree. C. After filteration the
solution was applied on a heparin-agarose column (Sigma Co.). The
matrix was washed twice with 25 ml of 0.254M NaCl-PBS, pH 7.4, and
then eluated with 15 ml of 0.554M NaCl-PBS, pH 7.4. The eluate was
then concentrated to about 1 ml using a Millipore Biomax-100K NMWL
filter device and cetrifugation..sup.50. Purity of the viral
preparation (100 ul) was assessed on 4-20% SDS-polyacrylamide gel
run. The proteins were detected by Coomassie staining. The titer of
purified virus was calculated by dot blot and determined to be
1.times.10.sup.11 encapsidated genomes per ml.
[0105] AAV/E6/Neo virus stock was generated by the two step process
mentioned above, and a comparison of various G418 resistant
producer cell lines, by dot blot hybridization, is shown in FIG.
8B. The titering of the non-purified virus stock, in encapsidated
genomes (eg) per ml of this virus stock, by dot blot hybridization
is shown in FIG. 8C (about 10.sup.11 eg/ml).
[0106] Cells Used in this Study
[0107] The primary cervical cancer cell lines, CA1 (patient 1) and
CA2 (patient 2), have been described previously, both containing
HPV-16 DNA, and were approximately 10 passages from initial
isolation (Santin, A. D., et al., J. Virol., 1999: 73: 5402-5410).
These cells were grown in Keratinocyte-SFM supplemented with
epidermal growth factor and bovine pituitary extract (Gibco
BRL/Life Technologies). Human Leucocyte Antigen (HLA) typing of
these cells gave haplotypes of HLA A1 for both CA1 and CA2,
respectively. Mo and DC were derived from peripheral blood
mononuclear cells (PBMC). PBMC were separated by routine Ficoll
gradient method from fresh blood drawn from healthy persons. The
normal donor had a haplotype of HLA A1, compatible with the target
cancer cells. The PBMCs were inoculated into six-well culture
plates and incubated with two milliliters of AIM-V medium for two
hours at 37.degree. C. and 5% CO.sub.2. At that time non-adherent
cells were removed by carefully washing the monolayer three times
with phosphate buffered saline (PBS, pH 7.0).
[0108] Infection of Mo/DC with AAV Virus and Treatment with
Cytokines.
[0109] Immediately after the removal of the non-adherent cells, the
adherent Mo were infected (pulsed) with 0.5 ml of virus stock
(.about.5.times.10.sup.10 encapsidated genomes) when using the
non-purified virus, or 10.sup.7 encapsidated genomes when using the
purified virus. After two hours incubation the medium/virus
solution was removed, the cells were washed with AIM-V, and finally
fed with AIM-V medium. The infection protocol is outlined in FIG.
8D. The Mo/DC precursors were infected with 0.5 ml of virus stock
or lysate at days 0, 3, and 5. Throughout this time period the
Mo/DC culture was treated with human GM-CSF (LEUKINE.RTM., Immunex
Corporation, 1.4.times.10.sup.6 IU/250 .mu.gs) at a final
concentration of 800 IU/ml. At day three, to induce the maturation
of Mo into DC, human interleukin 4 (IL-4, R & D SYSTEMS Co.) at
1000 IU/ml was added to the medium. Finally, at day 5, recombinant
human interleukin 2 (IL-2, R & D SYSTEMS Co.) at 10 U/ml was
added.
[0110] Generation of Bacterial E6 Protein and Lipofection of
Mo/DC.
[0111] GST-E6 protein was generated in a similar manner to previous
generation of GST-E7..sup.34 The Mo/DC were lipofected (pulsed)
with 15 .mu.g of GST-E6 on day 5 as previously described (Santin,
A. D., et al., J. Virol., 1999: 73: 5402-5410). The treatment of
the protein pulsed Mo/DC with cytokines was the same as the virus
infected DC.
[0112] mRNA Isolation and RT-PCR Analysis for E6 Expression.
[0113] E6 mRNA expression was measured by RT-PCR amplification
along with a cellular mRNA control. Total RNA was isolated from
mock (lysate) infected and AAV/E6/Neo infected Mo/DC using Trizol
reagent (GIBCO BRL Life Technologies Inc.), according to the
manufacturer's protocol and treated with 5 U/.mu.g of RNase-free
DNase I (Promega Co.) at 37.degree. C. for 1 hour. Messenger RNA
was then separated using the Oligotex mRNA Mini Kit (QIAGEN Inc.)
according to the supplier's instruction. The first-strand cDNA
synthesis was performed at 37.degree. C. for 1 hour in a final
volume of 25 .mu.l reaction buffer [1 .mu.g mRNA; 50 mM Tris-HCl,
pH8.3; 75 mM KCl; 3 mM MgCl.sub.2; 10 mM DTT; 0.5 .mu.g
oligo(dT).sub.15 (Promega Co.); 0.5 mM each of the four dNTPs; 30 U
of RNasin (Promega Co.) and 200 U of M-MLV Reverse Transcriptase
RNase H Minus (Promega Co.)]. PCR amplification of the cDNA was
performed in 100 .mu.l reaction volume which contained 2.5 U Taq
DNA polymerase (Fisher Scientific Co.); 10 mM Tris-HCl.sub.1,
pH8.3; 50 mM KCl; 2 mM MgCl.sub.2; 0.2 mM each of the four dNTPs; 1
.mu.M of each upstream and downstream primer specific for the cDNA
template and 10 .mu.l cDNA template. The E6 primer set used was
5'-ACCACAGTTATGCACAGAGC-3' and 5-AGGACACAGTGGCTTTTGAC-3', which
targeted amplification of the HPV-16 sequences from nt 139 to 420.
A control RT-PCR analysis of expression of the housekeeping gene
TFIIB was also undertaken with the primer set
5'-GTGAAGATGGCGTCTACCAG-3' and 5'-GCCTCAATTTATAGCTGTGG-3', which
amplified nt 356-1314 of that mRNA. To insure that DNA wasn't
contributing to the results, a direct PCR was also undertaken. The
products were then analyzed on an agarose gel, stained with
ethidium bromide, and visualized by ultraviolet light.
[0114] In these studies three infections were undertaken as
indicated in FIG. 8D. After AAV infection and GM-CSF treatment, at
day three, the cells were finally treated with IL-4 to induce
differentiation into DC. The transduction of the Mo/DC population
was analyzed by observing RNA expression of the E6 transgene.
Polyadenylated RNA was isolated from AAV/E6/Neo infected and
mock-infected DC cultures at day 10 (after IL-4 introduction and
differentiation into DC) and was analyzed for E6 mRNA by RT-PCR.
Expression of the cellular TF.sub.IIB gene was also undertaken as a
control. The results, shown in FIG. 9 demonstrate that E6
expression only takes place in the virally infected DC. In FIG. 9,
the positive contro was the PCR product resulting from the
Aav/E6/NEO vector plasmid as a template. Another control was PCR
analysis of RNA from cells infected by AAV/E6/Neo virus. A final
control included the analysis of cellular TF.sub.IIB RNA. Note that
only RNA from cells infected with AAV/E6/Neo virus resulted in an
appropriate E6 RT-PCR sized product, while mock and PCR
amplification of the RNA from cells infected by AAV/E6/Neo virus
did not give a product, indicating lack of contaminating DNA.
[0115] Intracellular Staining for E6.
[0116] The protocol used was adapted from that described by Pala et
al., (Immunology 2000; 100:209-216). The Mo/DC were infected with
virus or lipofected with protein as described above. Cells were
then treated with IL-4 and GM-CSF under standard conditions. Seven
days after infection/lipofection the cells were harvested, washed
and fixed with 2% paraformaldehyde in PBS for 20 min at room
temperature. The cells were washed and permeabilized with PBS/1%
BSA/0.5% saponin (S-7900, Sigma) for 10 min at room temperature.
Cells were stained with anti-HPV-16/18 E6 (Chemicon Inc., Temecula,
Calif.; Cat no MAB874) plus FITC-anti-mouse-Ig (Becton Dickinson
Inc., cat no 554001) and analyzed by flow cytometry.
[0117] The efficiency of virus/gene and lipofection/protein-pulsing
of Mo/DC was analyzed and compared by intracellular staining. The
cells were analyzed four days after pulsing. The results, shown in
FIG. 10, demonstrate that AAV/E6/Neo infection of Mo resulted in a
much higher percentage of cells containing intracellular E6 protein
than direct protein-pulsing (72% to 29%). FIG. 10A provides the
results from lipofection, 10B provides the results from infection.
Mo were pulsed (infection or lipofection) as indicated, treated
with cytokines, and analyzed for E6 protein by intracellular
staining on day 4 as described in the Materials and Methods
section. Note that AAV/E6/Neo infection/pulsing gave the highest
levels of E6 positive cells compared to protein lipofection (72%
versus 29%)
[0118] Detection of Viral Integration by PCR/Southern Blot
Analysis.
[0119] Chromosomal integration of the AAV/E6/Neo genome was
undertaken by vector-chromosome junction PCR amplification and
Southern blot analysis as previously described (Liu. Y., et al., J.
Inf. Cytok. Res. 2000; 20:21-30). Chromosomal integration of the
AAV/E6/Neo vector in DC was observed. Chromosomal integration,
while not essential for gene expression from AAV vectors, does
signifies a permanent genetic alteration of the DC, and is a
desirable "gold standard" for viral transduction. Chromosomal
integration was demonstrated by PCR amplification of
vector-chromosome junctions using primers complementary to the SV40
promoter within the vector and Alu I repetitive chromosomal
elements. DC were similarly treated as in the RNA analysis
experiments of FIG. 9. Junction products were analyzed by agarose
gel electrophoresis, Southern blotted, and probed for Neo
sequences. Briefly, total cellular DNA from the infected, CD83+
selected cells and uninfected cells served as template in PCR
amplification assays using primers targeting the SV40 early
promoter of the vector and the cellular repetitive Alu I element.
The products were Southern blotted and probed with .sup.32P-Neo
DNA, shown in FIG. 11. The positive control lane contained 100 ng
of Eco RV digested AAV/GM-CSF/Neo plasmid (6.7 & 1.3 Kb). The
negative control lane contained products from a PCR reaction with
DNA mock infected cells. Note that multiple Neo-positive bands
result from the infected DC population indicating chromosomal
integration by the vector, and that multiple vector-positive cell
clones are present in the population. As shown in FIG. 11, multiple
vector-chromosomal junction products were observed in the
AAV/E6/Neo infected DC, but not in mock infected DC. Unfortunately,
the vector must integrate close to an Alu I element in order to be
identified by this technique. In any case, these data indicate that
at least some of the viral genomes are able to chromosomally
integrate in the DC population.
[0120] Chromium Release Assay of CTL Activity.
[0121] Non-adherent PBMCs (T cells and B cells) were washed and
resuspended in AIM-V at 10-20.times.10.sup.6 cells/well in 6-well
culture plates (Costar, Cambridge, Mass.) with rAAV or GST-E6
pulsed DC (ratios from 20:1, responders:dendritic). The cultures
were supplemented with recombinant human GM-CSF (500 U/ml) and
recombinant human IL-2 (10 U/ml). At 7 days post-addition of the
PBMC, the cells were assayed for tumor cell killing activity in a
6-hour chromium-51 release assay as previously described (Santin,
A. D., et al., J. Virol., 1999: 73: 5402-5410). One of two HLA
class I A1 compatible primary cervical tumor cells was
.sup.51Cr-labeled and used as targets as previously described
(Santin, A. D., et al., J. Virol., 1999: 73: 5402-5410). To
determine the structures on the target cells involved in lysis,
monoclonal anti-Class I Mabs were used to block cytotoxicity. The
.sup.51Cr-labeled tumor targets were pre-incubated with Mabs
specific for monomorphic HLA class I W6/32 (50 ug/ml) (hybridoma
obtained from the ATCC, Rockville, Md.). The effector cells and
.sup.51Cr-labeled targets were then incubated in a final volume of
200 ul for 6 hours at 37.degree. C. with 5% CO.sub.2.
[0122] Cell Surface Marker Analysis of T Cells and DC by
Flourescent Antibody Cell Sorting (FACS).
[0123] For the analysis of T cells, at day 12 of the experiment the
primed T cell populations were analyzed for surface markers. A
panel of mAbs recognizing the following antigens was used:
anti-CD4, anti-CD8, anti-CD56 (Pharmingen, San Diego, Calif.).
Control irrelevant isotype-matched FITC- or PE-conjugated mAbs were
obtained from Becton-Dickinson. These cells were greater than 95%
viable as assessed by trypan blue exclusion. Cell suspensions were
counted and distributed into 12.times.75 mm tubes. Mouse monoclonal
antibodies were diluted in cold assay buffer and the final pellet
was resuspended in 500-.mu.l volume. Tubes were incubated for 30
minutes followed by two washes with assay buffer and the final cell
pellet was resuspended in 500 .mu.ls of assay buffer for subsequent
analysis. Cells were analyzed with a fluorescence activated cell
sorter (FACS; Becton-Dickinson) with a 15 mW argon laser with an
excitation of 488 nm. Fluorescent signals were gated on the basis
of cell dimension (i.e. forward and right angle light scattering
typical of PBL activated. Gated signals (5,000-10,000) were
detected at 585 BP filter and analyzed using Cell Quest software
(Becton-Dickinson).
[0124] For the analysis of DC, a panel of mAbs recognizing the
following antigens was used: anti-CD40 (Immunotech, Marseille,
France); anti-CD14, anti-DR, anti-CD80 (Becton-Dickinson),
anti-CD86 (Pharmingen, San Diego, Calif., USA), anti-CD83 (Coulter,
Miami, Fla., USA). Control irrelevant isotype-matched FITC- or
PE-conjugated mAb were obtained from Becton-Dickinson. Briefly,
nonadherent cells were harvested by washing the plates with
phosphate buffered saline (PBS pH 7.2, Gibco). Adherent cells were
recovered by incubating the plates at room temperature for 15-20
minutes in the presence of Cal+ and Mg'+-free PBS, followed by
gentle scraping. These cells were >95% viable as assessed by
trypan blue exclusion. Cell suspensions were counted and
distributed into 12.times.75 mm tubes. Mouse monoclonal antibodies
were diluted in cold assay buffer (PBS, pH 7.2, supplemented with
0.I % FBS) and added in a 50 .mu.l volume. For direct fluorescence,
tubes were incubated for 30 min followed by two washes with assay
buffer and the final cell pellet was resuspended in 500 .mu.l assay
buffer for subsequent analysis.
[0125] AAV-Mediated Pulsing of DC Results in Rapid and Effective T
Cell Priming.
[0126] With strong evidence of DC transduction and expression by
the AAV/E6/Neo vector, the ability of the AAV/E6/Neo vector to
antigenically pulse DCs was analyzed. Adherent Mo were mock,
GST-E6, or AAV/E6/Neo virus pulsed as before (FIG. 8D). Each of
these cultures were then treated with GM-CSF and IL-4 as prescribed
by Sallusto and Lanzavecchia (1994) and Romani et al. (1994) for
generating DC. At day 5 the resulting DCs were then incubated with
non-adherent peripheral blood lymphocytes. At day two of IL-4
treatment, representative pictures were taken of the cultures at
low and high power. Note that the virus treated DC-T cell cultures
exhibited much higher levels of rosetted cell clusters, suggesting
stronger DC-T cell interaction.
[0127] As AAV/antigen pulsing is novel, the cultures were observed
on a daily basis. It was almost immediately noticed that cell
clustering was taking place in an enhanced manner in the AAV-pulsed
DC cultures relative to the protein-pulsed or lysate control
cultures (FIG. 12). Upon examining the cell morphology present, the
present inventors believe these clusters to be due to T cell-DC
resetting. This led to the speculation by the inventors that the
AAV-pulsing of DC might be allowing for more rapid priming of the T
cells. After 7 days of priming the rosettes were dispersing. This
change was interpreted as signaling the completion of priming and
the subsequent death of the DC as a target. This is in sharp
contrast to the protein- and lysate pulsed cultures which still did
not show extensive resetting. Normally DC-T cell incubation and
priming require 2 to 3 weeks to allow for significant cytotoxic T
lymphocyte activity (CTL). AAV-pulsing of DCs may require only 7
days of priming.
[0128] The ability of these 7 day-primed T cells to carry out class
I restricted recognition and killing of HLA A1 matched primary
cervical cancer cells was tested in chromium release assays. The
previously characterized primary cervical cancer cell line 1, CA-1,
was used as a target. Our impressions of rapid priming was fully
borne out by the level of target killing. A representative
experiment is shown in FIG. 13. FIG. 13A Shows a representative
experiment of cytotoxic response resulting from the indicated
pulsing techniques Mo/DC, and T cells from a normal individual
against HLA A1 matched primary cervical cancer cells (CA1) and a
primary multiple myeloma (MM). Note that the addition of the class
I blocking antibody W632 greatly inhibits killing. Also note lack
of killing activity against the primary MM. FIG. 13B shows a
representative experiment of cytotoxic response against a second
HLA A1 matched primary cervical cancer (CA2). Again, note that the
addition of the class I blocking antibody W632 greatly inhibits
killing.
[0129] Normally, protein/DOTAP pulsing of DC results in significant
target killing. However, by allowing only a short period of
priming, the percent killing resulting from this technique was only
slightly above the cell lysate control (mock). In sharp contrast,
the AAV/E6/Neo-pulsed-DC-primed T cells resulted in much higher
killing activity than the protein primed and lysate controls. These
same AAV/E6/Neo primed cells were unable to lysis an unrelated
multiple myeloma target. Finally, the addition of the anti-class I
antibody W632 significantly blocked killing, indicating class I
restriction of killing. We next assayed for class I restricted
killing of a second HLA A1 matched, previously characterized
primary cervical cancer cell line CA-2 (Santin, A. D., et al., J.
Virol. 1999; 73: 5402-5410). The resulting killing was very similar
to that against CA-1 (FIG. 13). Again, the addition of the
anti-class I antibody W632 significantly blocked killing,
indicating class I restriction of killing. Finally, because
non-purified virus was utilized in these experiments which
contained lysed cellular components, the CTL experiment was
repeated using a heparin column-purified virus stock. DC were
pulsed with 10.sup.7 purified encapsidated genomes of AAV/E6/Neo
(approximately 10.sup.-3 virus used in the FIG. 13 experiments). A
CTL assay was then carried out on the CA1 cervical cancer primary
target as described in FIG. 13. The resulting killing for
AAV/E6/Neo, AAV/E6/Neo plus anti-class I antibodies, GST-E6, and
lysate control pulsing of DC was 36.1+/-1.4%, 0.4+/-0.3%,
3.1+/-1.4%, and 2.0+/-0.5%, respectively. These data indicate that
the CTL activity in all of these experiments was in fact due to
AAV/E6/Neo viral transduction/pulsing of DC.
Example 3
Higher CD8/CD4 and Lower CD56/CD8 Cell Ratios Result with
AAV-Mediated Pulsing/Priming
[0130] The makeup of the T cell populations, which resulted from
AAV-transduction or protein lipofection, was observed. An effective
CTL response, while requiring CD8+ T cells as an effector of lysis,
also requires CD4+ helper T cells. Flow cytometric analysis was
used to determine the phenotype of the population of the lysate,
GST-E6 pulsed, and AAV/E6/Neo pulsed T cell populations.
[0131] FIG. 14A shows the CD8 and CD4 prevalence within the primed
population resulting from three different techniques as indicated
(on the right), as well as an FL1-H, FL2-H control (left). FIG. 14B
shows the CD56 and CD8 ratios in the same experimental situations
as A.
[0132] As shown in FIG. 14A, in the mock case, one sees a normal
ratio of CD8 to CD4 postive cells (1.21:1). In the GST-E6 pulsing
case, the D8/CD4 ratio remains the same (1.23:1). In sharp contrast
in the AAV/E6/Neo pulsed case, the ratio of CD8/CD4 changes
dramatically to 7.0:1. Normally, one would not expect this high
ratio to arise until three weeks of DC/T cell priming suggesting
that the AAV-Ag pulsing of DC results in not only quicker
activation, but also higher killing activity, HLA class I
restricted, on a per CD8+ T cell basis. The high killing is
consistent with a Type 1 (Th 1) response. This is important because
recent studies have suggested that progression to cervical cancer
from precursor lesions may be associated with a preferential Type 2
(Th 2) T cell response, along with significant dysfunction of Type
1 T cell response in patients with high grade cervical
intraepithelial lesions and invasive cervical cancer (Cleric M., et
al., J. Natl. Can. Inst. 1997; 90: 261-3).
[0133] The expression of CD56 was also observed. Some consider this
marker as being specific for natural killer T cells. However,
others have reported that some CD8+ T cells do express CD56 and do
exhibit HLA class I restricted killing. In spite of this confusion,
all of T cell populations tested herein exhibited low CD56
expression. However, what is noteworthy in this analysis is the
very high level of CD8+ T cells in the case of AAV/E6/Neo pulsing,
confirming the CD8/CD4 analysis (FIG. 14A). Taken together, these
data indicate a very different resulting primed T cell population
when AAV-antigen pulsing was used, and suggests that the increased
killing activity may be a result of these changes in the T cell
population These data also indicate that CD8 is a specific lineage
marker in HLA class I restriction in our system.
Example 4
AAV/E6 Vector-Pulsing Results in DC with Higher CD80, and Lower
CD86 Expression
[0134] Finally, the DC resulting from the various pulsing
techniques was characterized to observe if significant differences
were discernable. Flow cytometric analysis was used to determine
the phenotype of untreated, lysate-pulsed, GST-E6-pulsed, and
AAV/E6/Neo-pulsed DC populations. The results, shown in FIGS. 15
and 16, demonstrate that the DC generated from all for techniques
share all of the common DC markers. However, AAV/E6/Neo pulsed DC
did express significantly higher levels of CD80 and lower levels of
CD86 than the GST-E6-pulsed DC. In FIG. 15, Mo were treated as
indicated, treated with GM-CSF and IL-4 and analyzed by FACS for
mean flourescent intensity (MFI) on day 7. "-" no detectable MFI
staining, "+"=MFI 10.sup.1-10.sup.2. "++"=MFI 10.sup.2-10.sup.3.
"+++"=MFI 10.sup.3-10.sup.4. Representative of three experiments.
Note AAV-pulsing results in higher CD80 and lower CD86 levels
compared to protein pulsing. FIG. 16 provides histograms of CD80
expression complied in FIG. 13. FIG. 16A provides the size analysis
of general DC populations, and FIG. 16B provides the DC CD80
expression under different conditions.
[0135] While the illustrative embodiments of the invention have
been described with particularity, it will be understood that
various other modifications will be apparent to and can be readily
made by those skilled in the art without departing from the spirit
and scope of the invention. Accordingly, it is not intended that
the scope of the claims appended hereto be limited to the examples
and descriptions set forth herein but rather that the claims be
construed as encompassing all the features of patentable novelty
which reside in the present invention, including all features which
would be treated as equivalents thereof by those skilled in the art
to which this invention pertains.
Sequence CWU 1
1
10 1 20 DNA Artificial Sequence Based on HPV E6 sequence 1
tgtcaaaaac cgttgtgtcc 20 2 20 DNA Artificial Sequence Based on HPV
E7 sequence 2 tgctaattcg gtgctacctg 20 3 20 DNA Artificial Sequence
Based on HPV-L1 sequence; m=a or c; r=a or g; w=a or t; y=c or t 3
gcmcagggwc ataayaatgg 20 4 20 DNA Artificial Sequence Based on
HPV-L1 sequence; m=a or c; r=a or g; w=a or t; y=c or t 4
cgtccmarrg gawactgatc 20 5 19 DNA Artificial Sequence Based on
plasmid pBR322 sequence 5 atacctgtcc gcctttctc 19 6 16 DNA
Artificial Sequence Based on plasmid pBR322 sequence 6 aatctgctgc
ttgcaa 16 7 20 DNA Artificial Sequence Based on HPV-16 E6 gene 7
accacagtta tgcacagagc 20 8 20 DNA Artificial Sequence Based on
HPV-16 E6 gene 8 aggacacagt ggcttttgac 20 9 20 DNA Artificial
Sequence Based on transcription factor TFIIB sequence 9 gtgaagatgg
cgtctaccag 20 10 20 DNA Artificial Sequence Based on transcription
factor TFIIB sequence 10 gcctcaattt atagctgtgg 20
* * * * *