U.S. patent application number 12/456055 was filed with the patent office on 2010-01-07 for in situ detection of early stages and late stages hpv infection.
Invention is credited to Shuling Cheng.
Application Number | 20100003704 12/456055 |
Document ID | / |
Family ID | 41415135 |
Filed Date | 2010-01-07 |
United States Patent
Application |
20100003704 |
Kind Code |
A1 |
Cheng; Shuling |
January 7, 2010 |
IN SITU detection of early stages and late stages HPV infection
Abstract
Embodiments of the invention provide methods, polyclonal
antibodies, monoclonal antibodies, assays, and kits for detecting
HPV infection, including infection by various HPV genotypes, early
and/or late HPV-associated or HPV-specific proteins or antibodies.
Mononoclonal antibodies are used to detect oncogenic high risk and
low risk HPV types in a single assay, which is not limited to assay
type or format. Useful tools for specific detection of invasive
cervical cancer are provided. Cervical cancer biomarkers are
identified and can be used in a detection method for early stage
precancerous lesions as well as late stage cancer progression.
Inventors: |
Cheng; Shuling; (Fremont,
CA) |
Correspondence
Address: |
SHULING CHENG
47853 Warm Springs BLVD
Fremont
CA
94539
US
|
Family ID: |
41415135 |
Appl. No.: |
12/456055 |
Filed: |
June 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61131991 |
Jun 13, 2008 |
|
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61192912 |
Sep 22, 2008 |
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Current U.S.
Class: |
435/7.21 |
Current CPC
Class: |
G01N 2469/10 20130101;
C07K 16/084 20130101; G01N 2333/025 20130101; G01N 33/56983
20130101; C12Q 1/708 20130101; G01N 33/571 20130101 |
Class at
Publication: |
435/7.21 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A method of detecting papillomavirus infection in a human
subject, comprising: conducting an immunological assay on a
clinical sample of the human subject; and staining of a nuclear
portion of a human cell from the clinical sample using one or more
monoclonal antibodies capable of binding to HPV early viral
proteins.
2. The method of claim 1, wherein positive staining of the nuclear
portion correlates with the progression to a disease stage selected
from the group consisting of an early dysplasia stage, low-grade
squamous intracervical lesion (LSIL), high-grade squamous
intracervical lesion (HSIL), cervical intraneoplasm (CIN1, CIN2,
CIN3) and combinations thereof.
3. The method of claim 1, wherein positive staining of a
cytoplasmic portion of the human cell indicates papillomavirus
infection being progressed into a disease stage selected form the
group consisting of a late dysplasia stage, cervical intraneoplasm
(CIN3), invasive cervical cancer, squamous cell carcinoma (SCC),
adenocarcinoma, and combinations thereof.
4. The method of claim 1, wherein the one or more monoclonal
antibodies are generated against one or more purified recombinant
papillomavirus proteins.
5. The method of claim 1, wherein the one or more monoclonal
antibodies are also capable of binding to the HPV late viral
proteins.
6. The method of claim 1, wherein the one or more monoclonal
antibodies further comprises an antibody capable of binding to the
HPV late viral proteins.
7. A method of detecting papillomavirus infection in a human
subject, comprising: conducting an immunohistochemistry assay on
humans cells from a clinical sample of the human subject using one
or more antibodies generated against one or more purified
recombinant papillomavirus proteins; and comparing the staining of
the nuclear portion with the staining of the cytoplasmic portion of
the human cell from the clinical sample.
8. The method of claim 7, wherein the one or more monoclonal
antibodies are selected from the group consisting of anti-HPV E6
monoclonal antibodies, anti-HPV E7 monoclonal antibodies, anti-HPV
L1 monoclonal antibodies, and combinations thereof.
9. The method of claim 7, wherein more positive staining being
observed in the nuclear portion than the cytoplasmic portion of the
human cell from the clinical sample indicates papillomavirus
infection in the human subject at an early disease stage selected
form the group consisting of an early dysplasia stage, low-grade
squamous intracervical lesion (LSIL), high-grade squamous
intracervical lesion (HSIL), cervical intraneoplasm (CIN1, CIN2,
CIN3), and combinations thereof.
10. The method of claim 7 wherein more positive staining being
observed in cytoplasmic portion than nuclear portion of the human
cell of the clinical sample indicates papillomavirus infection in
the human subject at a late disease stage selected from the group
consisting of a late dysplasia stage, CIN3, invasive cancer stage,
and combinations thereof.
11. The method of claim 7, further comprising performing a
hematoxylin and eosin stain on the clinical sample and comparing
the results of the hematoxylin and eosin stain with the results of
the one or more immunohistochemistry assays.
12. The method of claim 7, further comprising performing a
cytological papanicolaou smear assay on the clinical sample and
comparing the results of the cytological papanicolaou smear assay
with the results of the one or more immunohistochemistry
assays.
13. A method of detecting papillomavirus infection in a human
subject, comprising: conducting an immunohistochemistry assay on a
clinical sample of the human subject using two or more antibodies
capable of binding to a HPV viral protein selected form the group
consisting of E6, E7, L1 proteins, and combinations thereof; and
comparing the staining of the human cell by the two or more
antibodies, wherein positive staining of the human cell by at least
one of the two or more antibodies indicates papillomavirus
infection in the human subject.
14. The method of claim 13, further comprising comparing the
staining of the nuclear portion of the human cell from the clinical
sample with the staining of the cytoplasmic portion, wherein
positive staining of the cytoplasmic portion of the epithelial
tissue sample indicates dysplasia progression by HPV infection.
15. The method of claim 13, wherein the staining of the human cell
from the clinical sample is compared using the two or more
antibodies including an anti-HPV E6 antibody and an anti-HPV E7
antibody.
16. The method of claim 13, wherein the staining of the human cell
from the clinical sample is compared using the two or more
antibodies including an antibody capable of binding to an early HPV
viral protein and an antibody capable of binding to a late HPV
viral protein.
17. The method of claim 13, wherein the immunohistochemistry assay
is used to detect HPV infection at various disease stages, the
disease stage is selected from the group consisting of early stage
HPV infection, late stage HPV infection, early stage cervical cell
lesion, late stage cervical cell lesion, low grade of squamous
intraepithelial lesion (LSIL), high grade of squamous
intraepithelial lesion (HSIL), atypical squamous cells of
undetermined significance (ASCUS), cervical intraneoplasm stage 1,
(CIN1), cervical intraneoplasm stage 2 (CIN2), cervical
intraneoplasm stage 3 (CIN3), developed cervical cancer,
adenocarcinoma (ADC), squamous cell carcinoma (SCC), and
combinations thereof.
18. A kit for detecting papillomavirus infection in a human
subject, comprising: an anti-HPV monoclonal antibody for performing
an immunological assays on a clinical sample of the human subject,
capable of staining a nuclear portion of one or more human cells
from the clinical sample to compare the staining of the nuclear
portion with the staining of the cytoplasmic portion of the human
cells.
19. The kit of claim 18, wherein the anti-HPV antibody is selected
from the group consisting of an anti-HPV E7 monoclonal antibody, an
anti-HPV E6 monoclonal antibody, a combination of an anti-HPV L1
antibody and anti-HPV E7 monoclonal antibody, a combination of an
anti-HPV L1 antibody and anti-HPV E6 monoclonal antibody, a
combination of an anti-HPV E6 antibody and anti-HPV E7 monoclonal
antibody, and combinations thereof.
20. The kit of claim 18, wherein the immunological assay is
selected from the group consisting of ELISA (enzyme linked
immunoabsorbant assays), antigen assays for papillomavirus
proteins, antibody assays for antibodies against papillomavirus
proteins, assays for papillomavirus immunocomplexes, protein chip
assays, radioimmunoprecipitation assays, rapid membrane
immunochromatographic assays, rapid stick immunochromatographic
assays, immunohistochemistry for tissues and/or cervical cells, and
immunocytological assays followed by flow cytometry, and
combinations thereof.
21. The kit of claim 18, wherein the anti-HPV monoclonal antibody
is used to detect HPV infection at various disease stages, the
disease stage is selected from the group consisting of early stage
HPV infection, late stage HPV infection, early stage cervical cell
lesion, late stage cervical cell lesion, low grade of squamous
intraepithelial lesion (LSIL), high grade of squamous
intraepithelial lesion (HSIL), atypical squamous cells of
undetermined significance (ASCUS), cervical intraneoplasm stage 1,
(CIN1), cervical intraneoplasm stage 2 (CIN2), cervical
intraneoplasm stage 3 (CIN3), developed cervical cancer,
adenocarcinoma (ADC), squamous cell carcinoma (SCC), and
combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. provisional patent
application Ser. No. 61/131,991, filed Jun. 13, 2008, and U.S.
provisional patent application Ser. No. 61/192,912, filed Sep. 22,
2008. Each of the aforementioned related patent applications is
herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Infection by human papillomaviruses (HPV) at specific
epithelium cells to induce epithelial proliferations plays an
important role for cervical carcinogenesis. About 99 percent of
confirmed cervical cancer cases are found to be associated with HPV
infection with biopsy-confirmed squamous intraepithelial lesions
(SIL) or cervical intraepithelial neoplasia (CIN). The incidence of
HPV infection, primarily transmitted through sexual contact, is
highest among young women and about 20 millions of sexually active
men and women worldwide are currently infected. Approximately 1% of
the population has genital warts and 4% of women have cervical
precancerous lesions, such as low grade of squamous intraepithelial
lesion (LSIL) or high grade of squamous Intraepithelial lesion
(HSIL) or atypical squamous cells of undetermined significance
(ASCUS).
[0003] The presence of these lesions, preferentially observed in
women aged 35-40 yrs, are at high risk of progression toward
invasive cervical cancer. It is general thought that persistent
infection of human papillomavirus (HPV) is essential for developing
precancerous epitheliual lesions. Infection of high-risk types HPV
for women with LSIL may or may not progress to HSIL. In fact,
remission occurs in majority of LSIL human subjects while some
progress to HSIL. Although 99.7% of cervical cancers are HPV
positive, integration of viral genome into the host genome is
required to facilitate the necessary genes to express for
developing into HSIL or cancer. In fact, only one in every 10 women
with persistent HPV infection may develop into higher grades of CIN
lesions, such as cervical intraepithelial neoplasia (CIN) grade 2
and grade 3 (CIN2, and CIN3, respectively), and a portion of these
epitheliual lesion cases may ultimately progress into cervical
cancer.
[0004] In the past, screening for cervical cancer is based on
conventional cytology screening tests, e.g., obtaining papanicolaou
(Pap) smears for cytological staining tests, and suspicious smears
are followed up with colposcopy, and/or histological biopsy. The
use of these cytological screening tests contributes to a reduction
in the mortality of cervical cancer. However, due to subjective
test criteria, there are various drawbacks for pap smear tests:
difficulty in obtaining samples, poor inter- and intra-observer
agreement, high rates of false negatives nd false positives,
requiring specialized labs staffed with highly trained personnel,
and inability to identify the majority of HPV-infected human
subjects. More reproducible assays are needed to improve the
current screening tests to avoid unnecessary medical intervention
and psychological distress for the affected women. The current
conventional cervical cytology screening tests have sensitivity
varied from about 30% to about 87%.
[0005] Detecting HPV infection by nucleic acid tests, such as "DNA
Hybrid Capture", has been developed with high assay sensitivity,
but are still not ideal, due to not only its high cost, assay
operation procedures, the requirements for facility, equipment, and
highly trained personnel, but also its very low positive predictive
value (PPV) in cervical intraepithelial neoplasia (CIN) testing
samples. Assay like PreTect HPV-Proofer.RTM. provides the detection
of E6/E7 mRNA with sensitivity equivalent to HPV Hybrid Capture
tests with higher positive predictive value; but cannot directly
detect E6/E7 oncoproteins in situ. In addition, DNA testing could
not differentiate disease stages after HPV infection nor the
diagnosis of different cell lesions (e.g., cannot differentiate
LSIL from HSIL, nor CIN lesions from non-transforming latent or
remissive viral infection). What is needed is a low cost, simple,
sensitive and specific assay that can be performed on routine
practice of a clinical lab or doctor office and capable of
detecting early stages of epithelial lesions, distinguish LSIL from
HSIL, or predicting the risk of progression into cervical
cancer.
[0006] Known protocols for producing monoclonal antibodies are
generally unsuitable for the production of anti-HPV monoclonal
antibodies and cannot be used in immunocytochemical diagnostic
tests performed on human subjects of general population. This is
because antibodies produced by these protocols will not necessarily
react with the naturally occurring HPV viral proteins in infected
human cells. It is thought that the epitopes recognized by
antibodies if generated by conventional protocols will not
necessarily be those epitopes which are resistant to the harsh
procedures involved in standard sampling, fixing and storing of
clinical specimens. In addition, three problems exist in clinical
HPV detection. One is that HPV proteins in clinical samples are
present in very small quantities. Secondly, there are too many HPV
types and most HPV types present in clinical samples are not known
or systemically identified due to the lack of available antibodies.
Third, HPV virus can not be cultured in labs by standard tissue
culture techniques. Thus, there is no available HPV proteins
purified to large quantities as immunogens for generating anti-HPV
antibodies, and there is no available HPV proteins or purified
anti-HPV antibodies to recognize anti-viral antibodies or viral
proteins present in clinical samples for clinical HPV
detection.
[0007] Infections by only about 15 HPV types (out of more than 100
available HPV types) are at high risk of developing into cervical
intraepithelial neoplasia (CIN) or cervical cancer. Among them,
around 70% of reported cervical cancer cases and 50% of reported
CIN 2 and CIN 3 cases are caused by two high risk HPV types, i.e.,
HPV type-16 and HPV type-18. However, some progressive cervical
cancer cases are reported to be infected by low risk HPV types,
while infection of some high risk HPV types will never progress
into cervical cancer. Infections by these two prevailing high risk
HPV types do not correlate with tumor development or cancer
progression. It seems important to identify those HPV-infected
human subjects that express particular oncogenic proteins rather
than just identify HPV infection by high risk types.
[0008] Thus, there is a need for detect the expression of
HPV-related oncoproteins in clinical samples as these oncoproteins
may better serve as cervical cancer biomarkers to better predict
the risk in developing into high grade of cell lesions or cervical
cancer-related diseases. There is also a need to develop anti-HPV
antibodies and appropriate HPV immunoassays to detect the presence
of invasive cervical cancer and/or HPV-related oncoproteins as
cervical cancer biomarkers and predict the risk for malignant
transformation of epithelial lesions into cervical cancer.
SUMMARY OF THE INVENTION
[0009] Embodiments of the invention provide various immunoassays
for in situ detection of HPV proteins using various monoclonal
antibodies against recombinant HPV proteins such that infection by
high risk and/or low risk HPV types can be detected by a single
specific monoclonal antibody and/or a general pan antibody. In one
embodiment, a method of detecting papillomavirus infection in a
human subject includes conducting an immunological assay on a
clinical sample of the human subject; and staining of a nuclear
portion of a human cell from the clinical sample using one or more
monoclonal antibodies capable of binding to HPV early viral
proteins. In another embodiment, a method of detecting
papillomavirus infection in a human subject include conducting an
immunohistochemistry assay on humans cells from a clinical sample
of the human subject using one or more antibodies generated against
one or more purified recombinant papillomavirus proteins and
comparing the staining of the nuclear portion with the staining of
the cytoplasmic portion of the human cell from the clinical sample.
Another method of detecting papillomavirus infection in a human
subject includes conducting an immunohistochemistry assay on a
clinical sample of the human subject using two or more antibodies
capable of binding to a HPV viral protein selected form the group
consisting of E6, E7, L1 proteins, and combinations thereof, and
comparing the staining of the human cell by the two or more
antibodies, wherein positive staining of the human cell by at least
one of the two or more antibodies indicates papillomavirus
infection in the human subject.
[0010] In one aspect, the invention provides an anti-HPV monoclonal
antibody capable of being used in an immunological assay to stain a
cytoplasmic portion of a HPV-infected human cell from a human
subject with a late disease stage. In another aspect, the invention
provides an anti-HPV E7 monoclonal antibody and anti-HPV E6
monoclonal antibody capable of being used in an immunological assay
to stain a nuclear portion of a human cell from a human subject to
indicate HPV infection at a disease stage. The staining of the
nuclear portion of the HPV infected human cell indicates early
stage HPV infection, whereas the staining of the cytoplasmic
portion of the HPV infected human cell indicates dysplasia
progression to a late disease stage by HPV infection. The anti-HPV
monoclonal antibody used in the immunological assay may be an
anti-HPV E7 monoclonal antibody, anti-HPV E6 monoclonal antibody,
anti-HPV L1 monoclonal antibody, and combinations thereof. In
another aspect, the invention provides an anti-HPV E7 monoclonal
antibody and HPV E7 protein as a biomarker used for detection of
cervical cancer progression.
[0011] In addition, the invention provides methods of performing
one or more immunological assays, including immunohistochemistry
assays and immunocytological assays. Monoclonal antibodies highly
specific for HPV viral proteins are also provided to be used in the
HPV-detecting immnological assays. In one embodiment, a nuclear
portion of an epithelial tissue sample is stained with the anti-HPV
monoclonal antibody to indicate HPV infection in general. In
another embodiment, a cytoplasmic portion of an epithelial tissue
sample is stained with the anti-HPV monoclonal antibody to indicate
dysplasia progression by HPV infection.
[0012] In still another aspect, a kit for performing an
immunological assays on a clinical sample is provided and includes
an anti-HPV monoclonal antibody capable of staining a nuclear
portion of a HPV infected human cell from a human subject to
indicate HPV infection and capable of staining a cytoplasmic
portion of the HPV infected human cell to indicate HPV infection at
a late disease stage. In one aspect, a kit for detecting
papillomavirus infection in a human subject may include an anti-HPV
monoclonal antibody for performing an immunological assays on a
clinical sample of the human subject, capable of staining a nuclear
portion of one or more human cells from the clinical sample to
compare the staining of the nuclear portion with the staining of
the cytoplasmic portion of the human cells.
SUMMARY OF DRAWINGS
[0013] FIG. 1A shows the representative image of the
squamocarcinoma (SCC) tissue from tissue microarray stained by IHC
using an anti-E7 monoclonal antibody.
[0014] FIG. 1B shows the representative image of the normal
epithelium (15 mm away from the tumor tissue) of the SCC subject
from FIG. 1A.
[0015] FIG. 1C shows the representative image of another SCC sample
from tissue microarray stained by IHC using the same anti-E7
monoclonal antibody.
[0016] FIG. 1D shows the magnified representative image of the
tumor cells stained in cytoplasm from FIG. 1C.
[0017] FIG. 2A shows the representative image of the tumor cells of
adenocarcinoma (ADC) sample stained by IHC using an anti-E7
monoclonal antibody.
[0018] FIG. 2B shows the representative image of the corresponding
normal epithelium (15 mm away from the tumor) of the ADC sample
from FIG. 2A.
[0019] FIG. 2C shows the magnified representative image of the
cytoplasm staining of adenocarcinoma tumor cells from FIG. 2A.
[0020] FIG. 3A shows a representative staining image of the
dysplasia cells of a CIN3 tissue using a mouse monoclonal anti-HPV
E7 antibody in an IHC assay according to another embodiment of the
invention.
[0021] FIG. 3B shows the magnified representative image of the
dysplasia epithelium of FIG. 2A, indicating specific nuclear
staining of the CIN3 dysplasia.
[0022] FIG. 4A shows a representative staining image of the
dysplasia cells of CIN2 tissues using an anti-HPV E6 mouse
monoclonal antibody in an IHC assay according to one embodiment of
the invention.
[0023] FIG. 4B shows a representative staining image of the normal
epithelium adjacent to the dysplasia tissue of the CIN2 sample of
FIG. 1A using the same anti-HPV E6 mouse monoclonal antibody an IHC
assay.
[0024] FIG. 4C shows the staining results of dysplasia epithelium
of a CIN3 tissue using the same anti-HPV E6 mouse monoclonal
antibody as the one in FIG. 1A in an IHC assay according to another
embodiment of the invention.
[0025] FIG. 4D shows the staining results of dysplasia epithelium
of another CIN3 tissue using the same anti-HPV E6 mouse monoclonal
antibody as the one in FIG. 1C in an IHC assay according to another
embodiment of the invention.
[0026] FIG. 5: Sandwich assay/EIA Antigen test to detect the
presence of the E6, E7 oncoproteins, and L1 viral proteins in the
serum sample
DETAILED DESCRIPTION
[0027] Developing appropriate assays, such as HPV immunological
assays, is needed for detection of HPV oncoproteins and
identification of biomarkers for cervical cancer. Embodiments of
the invention provide immunoassays, and kits for performing HPV
immunoassays to detect HPV proteins in a biological sample. The
invention also provides the use of the HPV immunoassays to identify
suitable biomarkers for HPV related cancers, including cervical
cancer. It is contemplated that, as evidenced by performing the
immunological assays using the antibodies as described herein, the
presence of HPV proteins, such as E6/E7 oncoproteins in CIN 2 and
CIN 3 lesions could be used as indicator/biomarker to indicate high
risk of cancer progression. Because of limited antibody available
for the detection of these E6/E7 oncoprotein in situ, the invention
also provides immunoassays, such as immunohistochemistry (IHC)
assay, ELISA (enzyme linked immunoabsorbant assays), and kits for
performing immunohistochemistry assay to detect the presence of HPV
proteins in situ. In addition, cervical cancer biomarkers are
identified, and methods and kits for identifying suitable invasive
cervical cancer biomarkers and/or predicting the risk for malignant
transformation into cervical cancer.
[0028] In one embodiment, an immunological assay is conducted on a
clinical sample of a human subject to stain a nuclear portion of a
human cell from the clinical sample using one or more monoclonal
antibodies capable of binding to HPV early viral proteins. In
another embodiment, staining of the nuclear portion of the human
cells from the clinical sample is compared with the staining of the
cytoplasmic portion of the human cells. Accordingly, performing the
immunological assay of the invention provides evidence that
positive staining of the nuclear portion correlates with the
progression to a disease stage, including an early dysplasia stage,
low-grade squamous intracervical lesion (LSIL), high-grade squamous
intracervical lesion (HSIL), cervical intraneoplasm (CIN1, CIN2,
CIN3) and combinations thereof. In addition, a correlation is found
between positive staining of a cytoplasmic portion of the human
cell and papillomavirus infection progressed into a disease stage,
including a late dysplasia stage, cervical intraepithelial neoplasm
(CIN3), invasive cervical cancer, squamous cell carcinoma (SCC),
adenocarcinoma, and combinations thereof.
[0029] For example, a method of papillomavirus infection detecting
a human subject includes conducting an immunohistochemistry assay
on the slide containing a thin section of human tissue to detect in
situ one or more papillomavirus proteins from one or more
papillomavirus types present in the biological sample on the slide
and using one or more anti-HPV antibodies to stain the thin section
of human tissue. In one embodiment, the one or more anti-HPV
antibodies may be anti-HPV monoclonal antibodies, for example,
anti-HPV E6 monoclonal antibodies, anti-HPV E7 monoclonal
antibodies, anti-HPV L1 monoclonal antibodies, and combinations
thereof
[0030] In one aspect, the one or more monoclonal antibodies are
generated against one or more purified recombinant papillomavirus
proteins. In another aspect, the one or more anti-HPV antibodies
are capable of recognizing a papillomavirus early viral proteins.
In still another aspect, the anti-HPV antibody are capable of
binding to HPV late viral proteins. The papillomavirus early
protein may be, for example, HPV-16 E6 protein, HPV-16 E7 protein,
HPV-18 E6 protein, HPV-18 E7 protein, and combinations thereof. The
one or more purified recombinant papillomavirus proteins may
include, but are not limited to, recombinant papillomavirus E6
protein, recombinant papillomavirus E7 protein, recombinant
papillomavirus L1 protein, such as recombinant HPV-16 E6 proteins,
recombinant HPV-16 E7 proteins, recombinant HPV-18 E6 proteins,
recombinant HPV-18 E7 proteins, and HPV-16 L1 proteins, recombinant
HPV-18 L1 proteins, and combinations thereof.
[0031] As another example, an immunohistochemistry assay can be
performed on a clinical sample of the human subject using two or
more antibodies, where each antibody is capable of binding to a HPV
E6, E7, or L1 viral protein. As the expression levels of most HPV
viral proteins, including E6, E7, and L1 viral protein in infected
human subjects are extremely low, most clinical samples may escape
detection by a single anti-HPV antibody. Thus, two or more anti-HPV
antibodies can be used, and the staining of the human cells from
clinical human subjects by the two or more antibodies are compared
such that positive staining of the human cells by at least one of
the two or more antibodies indicates papillomavirus infection in
the human subjects. As an example, the anti-HPV antibodies pair may
be a pair of an anti-HPV E6 antibody and an anti-HPV E7 antibody.
As another example, the pair of two or more antibodies may be an
antibody capable of binding to an early HPV viral protein and an
antibody capable of binding to a late HPV viral protein.
[0032] Using the two or more anti-HPV antibodies, staining of the
nuclear portion of the human cells from the clinical samples can be
compared with the staining of the cytoplasmic portion, such that
positive staining of the cytoplasmic portion of the epithelial
tissue sample indicates dysplasia progression by HPV infection.
Thus, two or more anti-HPV antibodies can be used in an
immunohistochemistry assay to detect HPV infection at various
disease stages, such as early stage HPV infection, late stage HPV
infection, early stage cervical cell lesion, late stage cervical
cell lesion, low grade of squamous intraepithelial lesion (LSIL),
high grade of squamous intraepithelial lesion (HSIL), atypical
squamous cells of undetermined significance (ASCUS), cervical
intraepithelial neoplasm stage 1, (CIN1), cervical intraepithelial
neoplasm stage 2 (CIN2), cervical intraepithelial neoplasm stage 3
(CIN3), developed cervical cancer, adenocarcinoma (ADC), and
squamous cell carcinoma (SCC).
[0033] In addition, a cytological papanicolaou smear assay can also
be performed on the clinical sample to compare the results of the
cytological papanicolaou smear test with the results of the one or
more immunohistological assays. The resets of the papanicolaou
smear assay are usually score and designated as follows:
[0034] (1) NILM: negative for intraepithelial lesion or malignancy;
used when there is no cellular evidence of neoplasia; including
microorganisms and/or other non-neoplastic findings such as
reactive/ reparative changes;
[0035] (2) ASC-US: atypical squamous cells of undetermined
significance; cells are usually the size of intermediate or
superficial squamous cells and have nuclear changes that are
suggestive but not diagnostic of LSIL or SIL not otherwise
specified;
[0036] (3) ASC-H: atypical squamous cells cannot exclude HSIL;
cells are usually the size of metaplastic cells and may be seen
singly or in clusters; suggestive but not diagnostic of HSIL;
[0037] (4) LSIL: low grade squamous intraepithelial lesion,
encompassing HPV cytopathic effect/ mild dysplasia/CIN 1;
[0038] (5) HSIL: high grade squamous intraepithelial lesion,
encompassing: moderate dysplasia/CIN 2 and severe dysplasia/CIS/CIN
3 and HSIL with features suspicious for invasion. High grade
squamous intraepithelial lesion or HSIL or HGSIL indicates moderate
(CIN2) or severe (CIN3) cervical intraepithelial neoplasia or
carcinoma in situ. It is usually diagnosed following a pap test. In
some cases these lesions can lead to invasive cervical caner, if
not followed appropriately. HGSIL does not mean that cancer is
present. Of all women with HGSIL results, 2% or less have invasive
cervical cancer at that time, however about 20% would progress to
having invasive cervical cancer without treatment. To combat this
progression, HGSIL is usually followed by an immediate colposcopy
with biopsy to sample or remove the dysplastic tissue. This tissue
is sent for pathology testing to assign a histologic classification
that is more definitive than a Pap smear result. HGSIL generally
corresponds to the histological classification of CIN2 or CIN3.
Therefore, it is helpful to provide HPV IHC assay along with HE
stain (hematoxylin and eosin stain) or HPV Immunocytological (ICC)
assay along with the pap smear test for detecting HPV proteins in
situ, particularly helpful in CIN2/CIN3 samples;
[0039] (6) Squamous cell carcinoma (SCC); cancer of the cervix,
locally invasive into neighboring tissues, blood vessels, lymph
channels and lymph nodes. In its advanced stages it can be
difficult to treat and may prove fatal. Depending on the stage or
degree of invasion, invasive cancer of the cervix may be treated
with local excision, hysterectomy, radical hysterectomy, radiation,
and chemotherapy;
[0040] (7) Adenocarcinoma: While most cancer of the cervix comes
from the squamous cells making up the exterior skin, there is an
occasional cancer that arises from the mucous-producing cells which
line the endocervical canal leading up into the uterus. This
glandular-type is called "adenocarcinoma" as opposed to "squamous
cell carcinoma." Adenocarcinoma can be difficult to detect. Unlike
squamous cell cancer: Adenocarcinoma precursers, when present, can
be difficult to identify on Pap smears. The slow progression of
squamous cell dysplasia into squamous cell cancer of the cervix is
not as uniform in adenocarcinoma.
[0041] Further, nucleic acid hybridization assay can also be
performed on the clinical sample from a human subject to detect the
presence of a papillomavirus genome in the clinical sample.
Further, a hematoxylin and eosin staining assay may also be
performed on the same set of clinical samples and the results of
the immunohistochemistry assays or any additional immunological
assays as described herein are compared with the results of the
hematoxylin and eosin stainin assay.
[0042] One embodiment of the invention provides various novel
monoclonal antibodies against HPV proteins, useful as biomarkers
and useful tools for detecting HPV viral proteins, HPV
oncoproteins, early screening of cervical cancer, and diagnosing
CIN and/or invasive cervical and other cancers, are provided. The
tools as provided herein can also be used in early clinical
screening for HPV infection and general diagnosis for cervical
cancer and other cancers, specific detection of invasive cervical
cancer, detection of other HPV related cancers, early stage
precancerous lesions as well as late stage cancer progression.
[0043] A kit for performing HPV IHC assay is also provided. The kit
may include an pre-antibody blocking solution, post-antibody
blocking solution, an anti-HPV antibody as the primary antibody, an
anti-mouse or anti-rabbit immunoglobulins conjugated with HRP or
biotin, or other agents as secondary antibody, a solution
containing appropriate agents used as substrate for the secondary
antibody to be detected. The anti-HPV antibody may be, for example,
an anti-HPV E7 monoclonal antibody, an anti-HPV E6 monoclonal
antibody, a combination of an anti-HPV L1 antibody and anti-HPV E7
monoclonal antibody, a combination of an anti-HPV L1 antibody and
anti-HPV E6 monoclonal antibody, a combination of an anti-HPV E6
antibody and anti-HPV E7 monoclonal antibody. Such a kit can be
used to perform an immunological assay, including, but not limited
to, ELISA (enzyme linked immunoabsorbant assays), antigen assays
for papillomavirus proteins, antibody assays for antibodies against
papillomavirus proteins, assays for papillomavirus immunocomplexes,
protein chip assays, radioimmunoprecipitation assays, rapid
membrane immunochromatographic assays, rapid stick
immunochromatographic assays, immunohistochemistry for tissues
and/or cervical cells, and immunocytological assays followed by
flow cytometry, and combinations thereof.
[0044] One of embodiment provides various monoclonal antibodies
against HPV viral proteins such that infection by high risk and low
risk HPV types can be detected by a single monoclonal antibody. The
invention also provides HPV type specific monoclonal antibodies for
detecting only the high risk HPV types. The one or more
papillomavirus types include high risk HPV types, low risk HPV
types, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45,
HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, and HPV-68, HPV-6, HPV-11,
HPV-42, HPV-43, HPV-44, HPV-53, HPV-54, HPV-55, and HPV-56, and
combinations thereof.
[0045] In another embodiment, the immunological assay is used to
detect a diseased stage caused by HPV infection. The disease stage
may be, for example, an early stage HPV infection; a late stage HPV
infection; low grade of squamous intraepithelial lesion (LSIL);
high grade of squamous intraepithelial lesion (HSIL); Atypical
squamous cells of undetermined significance (ASCUS); Atypical
squamous cells without excluding HSIL (ASC-H); Atypical glandular
cells (AGC); ervical intraneoplasm CIN1, CIN2, CIN3 representing a
mild, moderate, or severe cell dysplasia. Respectively; invasive
cervical cancer; adenocarcinoma (ADC); or squamous cell carcinoma
(SCC).
[0046] The binding of the antibody with the one or more proteins
from one or more papillomavirus types present in the biological
sample were examined under a microscope, detecting the presence of
an agent reacting with the tagged one or more antibodies, wherein
the agent consists of a colormetric agent, a fluorescent chromogen,
and combinations thereof. The biological sample consists of
cervical cells, cervical tissues, cervical swabs, body fluids,
serum, blood, tumors, cell cultures, biopsies, and combination
thereof. The biological sample can be obtained from a group of
people as general population for routine screening of cervical
cancer.
[0047] Methods of producing the monoclonal antibody are provided
herein to obtain monoclonal antibodies recognizing one or more
common epitopes of HPV proteins among various HPV types. In
addition, some of the monoclonal antibodies obtained herein are HPV
type-specific while some of the monoclonal antibodies obtained
herein are non-HPV type-specific. The non-HPV type-specific
antibodies recognize most of the prevalent HPV types present in
clinical samples; as a result, these monoclonal antibodies are
suitable to be used in an assay screening for HPV infection in one
or more clinical samples. Epitope mapping of these non-HPV type
specific antibodies identifies and allocates the common epitope of
the HPV specific proteins for binding of these monoclonal
antibodies with most of prevalent HPV types in the clinical
samples. These monoclonal antibodies are suitable for use as
potential drug candidates for treatment of most HPV infections or
cervical cancer. Furthermore, the common epitope(s) of these
particular antibodies also present(s) the potential binding site to
target E6 or E7 oncoproteins, or L1 viral proteins in drug design,
drug screening and/or drug development.
[0048] For example, a number of cervical biopsy samples are tested
in an immunohistohistochemistry (IHC) assay concurrently as a
tissue microarray format using a monoclonal antibody to detect HPV
proteins from a variety of HPV types (as confirmed by HPV DNA
genotyping). Using a monoclonal antibody against HPV oncoprotein
E7, the invention provides detection of the presence of HPV E7
protein in clinical samples having either single HPV infection or
multiple HPV infections. A single anti-E7 monoclonal antibody as
described herein can detect single HPV infection by at least HPV-6,
HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, etc., which are
cancer-related HPV types (either high risk HPV types or low risk
HPV types). A single anti-E7 monoclonal antibody can detect HPV
infection by two or more HPV types, such as the combination of
HPV-16, HPV-18, HPV-52, HPV-58, HPV-44, HPV-51, HPV-39, HPV-59,
etc., which include high risk, low risk, and non-oncogenic
.alpha.-papillomaviruses.
[0049] In addition, the monoclonal antibodies generated using
methods of the invention are useful to detect infection by
oncogenic HPVs, such as infection by high risk HPV types and/or low
risk HPV types. As an example, antibodies raised against a
recombinant protein HPV16 E6 oncoprotein generated by the method of
invention are able to recognize E6 proteins present inside the
cells of clinical samples due to single or multiple HPV infection,
and react with E6 proteins from high risk HPV types (such as
HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, HPV-58, etc.) or
low risk HPV types (HPV-6, etc.). In addition, a single anti-E6
monoclonal antibody can detect multiple HPV infection in a clinical
sample, having two or more HPV types, such as the combination of
HPV-16, HPV-18, HPV-51, HPV-52, HPV-58, among others.
[0050] As another example, antibodies raised against a recombinant
protein HPV16 L1 capsid protein generated by the method of
invention are able to recognize L1 proteins present inside the
cells of clinical samples due to HPV infection, and react with L1
proteins from high risk HPV types (such as HPV-16, HPV-18, HPV-31,
HPV-33, HPV-45, HPV-52, HPV-58, etc.) or low risk HPV types (HPV-6,
etc.). In addition, a single anti-L1 monoclonal antibody can detect
multiple HPV infection in a clinical sample, having two or more HPV
types, such as the combination of HPV-16, HPV-18, HPV-51, HPV-52,
HPV-58, among others.
[0051] In another embodiment, various monoclonal antibodies against
HPV proteins, E6, E7 or L1 are provided, including those monoclonal
antibodies specific for detecting HPV types correlated with the
immunogens that the antibodies were raised, and other non-HPV
type-specific monoclonal antibodies. In still another embodiment,
monoclonal antibodies are provided in a screening/diagnosis assay
with high positive predictive value (PPV) and high negative
predictive value (NPV) for HPV associated CIN and invasive cervical
cancer diagnosis. The high positive predictive value (PPV) in an
assay represents statistically the possibility of relating CIN or
invasive cancer to the detection of HPV associated proteins E6, E7,
or L1 in the assay is very high, and vice versa. The high negative
predictive value (NPV) in an assay represents statistically the
possibility of relating CIN or invasive cancer to the non-detection
of HPV associated proteins E6, E7, or L1 in the assay is very high,
and vice versa. The antibodies of the invention includes, but are
not limited to anti-E6, anti-E7, and anti-L1 antibodies, etc., and
are used in one or more immunological assays to result in high PPV
and/or high NPV values on clinical samples. For examples, samples
confirmed with various grades of epithelial lesions (CIN2, CIN3,
LSIL, HSIL, ASCUS) as well as different cervical cancers, squamous
cell carcinoma (SCC, a type of common cervical cancer) and
adenocarcinoma (ADC, a type of gland cancer) can be tested.
[0052] The PPV (positive predictive values) of the anti-E6
antibodies in an immunoassay as performed herein range from about
57% to about 100% for CIN2 samples, about 68% to about 100% for
CIN3 samples, about 62% to about 100% for SCC samples, and about
63% to about 100% for ADC samples. The NPV vlaues of the antibodies
of the invention range from about 54% to about 61% for CIN2
samples, about 60% to about 68% for CIN3 samples, about 60% to
about 100% for SCC samples, and about 75% to about 100% for ADC
samples. In addition, the specificity of the antibodies of the
invention ranges from about 36% to about 100% for CIN2 samples,
about 77% to about 100% for CIN3 samples, about 55% to about 100%
for SCC samples, and about 50% to about 100% for ADC samples. The
sentitivity of the antibodies of the invention ranges from about
17% to about 72% for CIN2 samples, about 30% to about 57% for CIN3
samples, about 67% to about 100% for SCC samples, and about 75% to
about 100% for ADC samples
[0053] For example, one or more anti-E7 monoclonal antibody as
provided herein are useful for detecting HPV infection and
predicting HPV associated CIN or invasive cervical cancer in an
immunoassay with a positive predictive value (PPV) ranging from
about 57% to about 100%. The obtained monoclonal antibodies can be
very useful in screening clinical samples for invasive cervical
cancer. In an IHC assay on cervical biosy samples, the best results
using the monoclonal antibodies of the invention result in about
100% positive predictive value (PPV) for invasive cervical cancer
or at least more than 90%, considering the non-result from
difficulties due to experimental procedural error. Also, monoclonal
antibodies with about 100% positive predictive value (PPV) for IHC
staining of CIN 2 or CIN3 clinical samples can also be
obtained.
[0054] In another embodiment, a negative predictive value (NPV)
ranging from more than about 50% to about 100% for clinical samples
can be observed in an IHC assay for one or more anti-E7 monoclonal
antibody. NPV value of about 100% for SCC and ADC clinical biopy
samples as observed for some of the antibodies provided herein
supports the use of these antibodies for diagnozing and screening
HPV associated CIN or invasive cervical cancer.
[0055] On the cellular level, the HPV proteins can be observed in
the nucleus and cytoplasm, but not in the membrane of a cell. It is
found that the HPV proteins are present in the nucleus and/or
cytoplasm of dysplasia epithelium from most of the samples tested.
The staining of dysplasia cells by the antibodies of the invention
results in diffused staining for the full layer of epithelium (the
whole layer in thickness). However, there is focused nucleus
staining in the basal, parabasal, and intermediate layer of the
adjacent normal epithelium. It indicates that HPV proteins present
in the peripheral of epithelium where HPV infection occurs adjacent
to the dysplasia cells. However, HPV proteins are localized
differently in adjacent normal epithelium and dysplasia
epithelium.
[0056] For invasive cancer like SCC and ADC described here in this
invention, staining of the dysplasia epithelium by anti-HPV protein
antibodies displays distinct high levels of staining on both the
nucleus and cytoplasm as compared to the staining of the adjacent
normal epithelium. For HSIL like CIN 3 cases, about 90% to 100% of
CIN 3 cases display distinct high levels of staining on the
nucleus, while about 60% of those display distainct cytoplasmic
staining. These data confirm that E6 or E7 expression is detected
in most of HSIL like CIN3 and invasive cancer. We have also found
that staining of the cytoplasm is only present in the dysplasia
cells, but not in the adjacent normal epithilia.
[0057] For CIN2 cases, about 60% to 70 or 80% of CIN 2 cases
display distinct high levels of staining by anti-HPV proteins
antibodies on the nucleus, while about only 40% to 50% of those
diaplay distinct cytoplasmic staining. Since CIN2 cases represent
moderate HSIL, it's reasonable to explain CIN2 has lower rate of
dysplasia progression, thus the cytoplasm staining by anti-E6 or E7
antibodies has less positive rate compared to CIN3 or invasive
cancer. This method thus provides tool to distinguish dysplasia
progression in HSIL. Therefore, staining of cytoplasm by anti-HPV
oncoproteins antibody identifies progression of HSIL dysplasia
cells.
[0058] Since 100% of invasive samples are positive in both
cytoplasmic and nucleus staining by anti-HPV proteins antibodies,
staining of both nucleus and cytoplasmic of dysplasia cells from
CIN2/3 could indicate possibility of further progression. It is
contemplated that the cytoplasmic staining is a unique staining
pattern in localization of HPV proteins for those with potential to
progress further to severe dysplasia or invasive cervical cancer.
Thus, the cytoplasmic staining of dysplasia cells is a good tool to
screen HPV associated cancer pathogenesis and cancer
progression.
[0059] Comparing various HPV proteins related to HPV early genes
and late genes, it is found that HPV E7 oncoprotein is present at
high level than other HPV proteins such that it may be relatively
easier for antibodies of the invention to detect HPV E7. In
addition, HPV E7 can be served as a cervical cancer biomarker and
the invention provides various antibodies tested to high PPV and
NPV values, high specificity and sensitivity for clinical samples
form all stages of CIN or cervical cancer development such that it
is possible to use one single monoclonal antibody to detect HPV E7
proteins present in various early precancerous lesions as well as
late stage invasive cancer progression. For example, the invention
provides antibodies useful for in situ detection screening of
clinical samples with early stage epithelium lesions, including
CIN1 ASCUS, LSIL, HSIL, among others. In addition, the antibodies
are also very successful in screening CIN2 and CIN 3 lesions as
well as various types of cervical cancers during cancer
development.
[0060] Detection of HPV DNAs, genomes, early viral proteins, late
viral proteins, oncoproteins, and/or capsid proteins from various
HPV genotypes can be performed by the method and detection assays
as described herein and can be very useful in general clinical
screening for HPV infection. Detection of HPV antibodies and/or
oncoproteins by immunological assays can be used in early clinical
screening for HPV infection and general diagnosis for cervical
cancer and can be performed in a single rapid test or in
multiplexed test. Comparative detection of altered levels of HPV
proteins and host proteins can be performed in the same or
different assays. It can also be used in diagnosing HPV-associated
carcinomas of the uterine cervix, as well as those cases associated
with epithelial cell abnormalities induced by HPV infection,
pre-malignant and malignant HPV-associated epithelial cell lesions,
and those at risk of developing HPV-associated cervical carcinoma
and adenocarcinoma. The methods as described herein can be used
independently or as an adjunct screening tool to convention
cytological papanicolaou smear tests or histological tests and the
results thereof can be compared for follow-up patient
management.
[0061] In one embodiment, a method of screening a human subject of
papillomavirus infection includes obtaining a clinical sample from
the human subject, and conducting one or more immunological assays
on the clinical sample from the human subject using various HPV
recombinant proteins and lab-generated antibodies specific for HPV
oncoproteins in order to detect and screen for the presence of HPV
infection from the presence of HPV proteins and HPV antibodies in
the human subject. In another embodiment, the HPV proteins in the
human subject are detected using antibodies raised against HPV
recombinant proteins, including but not limiting to various
polyclonal and monoclonal antibodies against various HPV early and
late proteins.
[0062] The one or more immunological assays as developed herein
lend themselves to the high quality and properly purified
recombinant proteins encoded by HPV early and late genes, as well
as high quality polyclonal and monoclonal antibodies, resulting in
immunological assays with very high sensitivity and specificity for
screening HPV infection. The one or more immunological assays
include, but are not limited to, protein chip assays, antigen
assays for papillomavirus proteins, antibody assays for antibodies
against papillomavirus proteins ELISA (enzyme linked
immunoabsorbant assays), assays for papillomavirus immunocomplexes,
radioimmunoprecipitation assays, rapid membrane
immunochromatographic assays, rapid stick immunochromatographic
assays, immunohistochemistry for tissues and/or cervical cells
among others, and immunocytological assays followed by flow
cytometry. The one or more immunological assays may be non-invasive
with minimal or no additional instrument required.
[0063] The basic techniques for conducting the immunological assays
can be found in "Antibodies: A Laboratory Manual", Harlow and Lane,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. The basic
techniques for conducting the immunological assays can be found in
"Antibodies: A Laboratory Manual", Harlow and Lane, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y. 1989; "Molecular
Cloning", A Laboratory Manual, eds. Sambrook, Fritsch and Maniatis,
Cold Spring Harbor Laboratory Press, 1989, and others books and
manuals known in the art. The related immunological assays,
immunohistochemistry for tissues and/or cervical cells, and/or
immunocytological assays followed by flow cytometry can also be
found in co-pending U.S. patent applications: Ser. No. 11/559,366,
filed on Nov. 13, 2006, titled "Detection method for human
papillomavirus (HPV) and its application in cervical cancer"; U.S.
Ser. No. 12/082,740, filed Apr. 14, 2008, titled "Protein chips for
HPV detection"; Ser. No. 61/131,991, filed Jun. 13, 2008 titled
"Antibodies and assays for HPV detection"; Ser. No. 61/192,912
Filed on Sep. 22, 2008, titled "Novel monoclonal antibodies against
HPV proteins useful for early stage and late stage detection,
screening, and diagnosis of HPV related cervical cancer"; Ser. No.
______ (NEOD/0004), filed concurrently as this application, titled
"Novel monoclonal antibodies against HPV proteins"; Ser. No. ______
(NEOD/0005.01), filed concurrently as this application, titled "in
situ detection of early stages and late stages HPV infection"; Ser.
No. ______ (NEOD/0005.03), filed concurrently as this application,
titled "Detection of early stages and alte stages HPV infection".
All of the above referenced applications are herein incorporated by
reference.
EXAMPLES
[0064] 1. Expression, Purification, and Preparation of HPV
Recombinant Proteins to be used as Immunogens for Generating
Antiserum and Anti-HPV Antibodies, and Screening Hybridoma Cell
Lines for Monoclonal Antibodies
[0065] HPV recombinant proteins can be any kinds of HPV proteins,
HPV proteins of early genes and/or late genes, including, but not
limited to, E2, E6, E7, L1, L2 and can be from various HPV types.
Full-length E6, E7, and/or L1 polypeptide sequence, which have been
found very difficult to obtain and purify due to undesirable
aggregation during protein purification, protein instability, low
levels of expression, low immunogenic responses of purified
proteins. For example, many early E6 oncoproteins contain many
cysteine amino acids and thus the correct topography of the E6
oncoproteins requires formation of many disulfide bonds properly.
In addition, it was known that certain immunological assays using
small peptides of early E6 and E7 proteins results in extremely low
assay specificity and sensitivity and thus unsuitable as tools for
clinical in vitro diagnostics. Thus, the invention provides
recombinant proteins, such as recombinant hybrid proteins
containing a partial sequence or a full length sequence of HPV
oncogenic proteins.
[0066] 1). Cloning and production of various recombinant proteins
encoded by HPV16 E6 and HPV18 E6 gene. An exemplary oncogenic E6
early gene from an exemplary HPV type, HPV-16, was clone. The HPV
16 E6 gene cloned herein is a 474 base pair (b.p.) DNA fragment
containing the 157 amino acid coding region of the whole HPV-16 E6
gene and obtained by polymerase chain reaction (PCR) amplification.
The DNA sequence of the isolated DNA fragment was confirmed by
comparing with the sequence from Gene Bank database. Recombinant
HPV-18 E6 protein was also obtained. All cloning procedures are
carried out according to the protocols described in "Molecular
Cloning", A Laboratory Manual, eds. Sambrook, Fritsch and Maniatis,
Cold Spring Harbor Laboratory Press, 1989. In addition, HPV18 E6
gene was also cloned and the DNA sequence was confirmed.
[0067] 2). Cloning and production of various recombinant proteins
encoded by HPV16 E7 and HPV18 E7 gene. An exemplary oncogenic E7
early gene from an exemplary HPV type, HPV-16, was cloned. A 294
base pair (b.p.) DNA fragment containing the 99 amino acid coding
region of the entire HPV-16 E7 gene was obtained by polymerase
chain reaction (PCR) amplification. The DNA sequence of the
isolated DNA fragment was confirmed by comparing with the sequence
from Gene Bank database. Recombinant HPV-18 E7 protein was also
obtained. In addition, E7 DNA fragments from different HPV types
can also be cloned from different clinical samples or sources.
[0068] 3). Cloning and Production of various recombinant Proteins
encoded by HPV16 L1 and HPV18 L1 gene. An exemplary late gene from
an exemplary HPV type, HPV-16, was cloned. A 1596 base pair (b.p.)
DNA fragment containing the 531 amino acid coding region of the
HPV-16 L1 gene was obtained by polymerase chain reaction (PCR)
amplification. The DNA sequence of the isolated DNA fragment was
confirmed by comparing with the sequence from Gene Bank database.
In addition, L1 DNA fragments from different HPV types can also be
cloned from different clinical samples or sources.
[0069] A recombinant N-terminal fragment of HPV 16 L1 protein was
also obtained from a His-tagged expression system. The molecular
weight of the HPV-16 L1 N-terminal recombinant protein ais bout 34
KD. L1 C-terminal fragments can also be obtained. Recombinant
HPV-18 L1 protein was also obtained and used as immunogens for
generating antiserum, polyclonal and monoclonal antibodies.
[0070] The one or more recombinant proteins as described herein can
be expressed in various suitable systems, such as bacterial
expression systems, viral expression systems, yeast expression
systems, mammalian expression systems, e.g., in E coli, yeast,
baculovirus, and/or mammalian cell cultures, generally known in the
field. Although the polypeptides could be obtained by other means,
embodiments of the invention provide one or more recombinant
proteins mostly in (or close to) their native forms, which may be a
much desirable conformation for binding with antibodies from
tissues of human subjects with HPV infection in an immunological
assay. For example, GST, MBP, or His tagged-HPV16-E6, HPV18 E6,
HPV16 E7, HPV18 E7, HPV16 L1, and HPV18 L1 recombinant proteins
were expressed in E. coli BL21(DE3) using IPTG driven induction.
After induction of protein expression, tagged-HPV recombinant
proteins were obtained from soluble fraction after lysis of the
cultured cells and purified to a final concentration of about 0.1
to 1 mg/ml or higher. The purity of the recombinant HPV proteins
was estimated to be>90% based on PAGE analysis. Recombinant HPV
proteins were used to detect the presence of HPV antibody on
clinical samples and was also used as immunogens for producing
polyclonal antiserum and monoclonal antibodies.
[0071] The cell culture containing various recombinant
papillomavirus proteins in various expression vectors as described
herein are then scaled up to 1 liter or 10 liter, or 100 liters or
higher to obtain high quantity of soluable recombinant protein for
purification. The soluble fraction after cell lysis was passed
through various chromatography columns with appropriate expression
systems to bind to the tag expressed along with the HPV recombinant
proteins. The tag-HPV recombinant proteins were then eluted from
the column and concentrated down to 100 ml or 10 ml to 1 ml. The
purified soluble recombinant HPV proteins were further concentrated
and dailysed with buffers at neutral pH or PBS buffers to be used
as immunogen to generate antiserum against the HPV proteins. The
soluble recombinant HPV proteins were thus purified from soluble
fractions and folded close to their native folding states as in
vivo natural conditions.
[0072] Obtaining high quality purified recombinant HPV proteins is
critical in generating various types of monoclonal antibodies that
recognizing common epitopes or specific epitopes for detecting HPV
infection. The purified recombinant HPV proteins were tested to
confirm its binding to the HPV antibody from the HPV infected
clinical samples. Thus, such purified recombinant HPV proteins are
suitable for use as immunogen to raise antiserum and generate
antibodies that can recognize natural HPV viral proteins in
vivo.
2. Anti-HPV Polyclonal Antibody Production:
[0073] Recombinant HPV E6, E7 or L1 proteins expressed in E coli
was purified, concentrated, and dialyzed with PBS to be used as
immunogens. Immunization was followed by standard protocol. Titer
of each serum obtained was tested by ELISA assays followed by
periodical boosting and bleeding. Production bleed from optimal
titer was collected; processed serum was used to do immunoglobulin
(Ig) purification via protein A columns or affinity columns.
Purified IgG was used as anti-HPV antibodies for HPV
immunoassays.
[0074] Monoclonal antibodies, polyclonal antibodies, and antiserum
were obtained, purified, and tested herein to be able to detect HPV
infection regardless of the pathogenesis of HPV infection, cell
lesions, inflammatory, or cancer disease development. Other
researchers have tried to develop anti-HPV monoclonal antibodies
but have failed because they failed to generate sufficient HPV
proteins for monoclonal antibodies production; they failed to
generate monoclonal antibodies with high specificity because the
immunogens were not immunogenic; or the generated antibodies were
not able to recognize native forms of HPV proteins present in
clinical samples with early stage HPV infection. Some antibodies
raised against mutant peptides were only able to recognize late
stage cervical cancer, but are not sure whether their antibodies
would recognize wild type HPV native proteins or any early stage
HPV infection. In addition, late stage HPV detection is too late
for disease intervention and treatment.
[0075] The clinical utility of the antibodies described herein was
validated by HPV immunoassays, such as ELISA assays,
immunocytochemistry assays, immunohistochemistry assays, using
appropriate clinical samples. The novel monoclonal antibodies and
antiserum, obtained from methods of this invention are able to
interact and bind HPV viral proteins present in clinical samples,
which have been confirmed to contain early stage cell lesions such
as cervical intraepithelial neoplasia (CIN) as well as late stage
HPV associated cervical cancer. The monoclonal antibodies and
antiserum as described herein provide powerful tools to detect and
screen HPV related pathogenesis and cervical cancer development in
both early stages and late stages; thus provides an avenue to
intervene disease progression and a chance to provide early
treatment.
3. HPV Monoclonal Antibody Development:
[0076] Recombinant HPV E6, E7 or L1 proteins expressed in E coli
was purified, concentrated, and dialyzed with PBS to be used as
immunogen. Immunization of mice was followed by standard procedure.
Titer of the obtained serum was tested by ELISA followed by
periodical boosting and bleeding. When the titer of the serum of
the mice reaches optimal, fusion of the spleen cells of the mice
with tumor cells was done by standard procedure. Clones of fused
cells, e.g., hybridoma cells, were further cultured.
[0077] 1). Hybridoma screening: To obtain anti-HPV antibody
producing hybridoma cells with pan and specific binding capability
to various HPV proteins as described in this invention, hybridoma
clones were screened with various proteins, including, not only the
original immunogens but also additional HPV proteins as positive
screening, and unrelated proteins as negative screenings. For
example, two or more purified HPV recombinant proteins were used to
screen against each hybridoma clone to screen and obtain monoclonal
antibody-producing hybridoma cell lines and to test and understand
the specificity of each antibody-producing hybridoma cell line thus
obtained.
[0078] As an example of hybridoma screening, antibody-producing
hybridoma cells were screened with two or more purified recombinant
human papillomavirus proteins such that the monoclonal antibody is
capable of reacting with the two or more purified recombinant human
papillomavirus proteins. The two or more purified recombinant human
papillomavirus proteins include, but are not limited to, HPV 16 E6
protein, HPV 16 E7 protein, HPV 16 L1 protein, HPV 18 E6 protein,
HPV18 E7 protein, HPV 18 L1 protein, and other HPV early proteins
and late proteins from various HPV types.
[0079] The antibody-producing hybridoma cells were screened with
positive reactivity to all of the two or more purified recombinant
human papillomavirus proteins and negative reactivity to non-HPV
proteins, including BSA, his.sub.6 tags, GST proteins, maltose
binding proteins (MBP), other tags or proteins used in recombinant
protein, and other readily available non-HPV proteins. As such, the
monoclonal antibodies generated form such hybridoma screening is
capable of binding to all of the two or more HPV viral proteins
(e.g., the HPV viral proteins present in clinical samples), which
correspond to the two or more purified recombinant human
papillomavirus proteins.
[0080] One example of the two or more purified recombinant human
papillomavirus proteins are HPV early proteins such that the
monoclonal antibody is capable of reacting with the two or more
human papillomavirus early proteins. For example, one hybridoma
cell line thus screened and obtained can produce a monoclonal
antibody recognizing a common epitope on both HPV16 E6 and HPV16 E7
proteins. Another hybridoma cell line thus screened and obtained
can produce a monoclonal antibody recognizing a common epitope on
both HPV18 E6 and HPV18 E7 proteins.
[0081] Another example of the two or more purified recombinant
human papillomavirus proteins includes a purified recombinant human
papillomavirus early protein and a purified recombinant human
papillomavirus late protein such that the monoclonal antibody
produced is capable of reacting with a common epitope on the
purified recombinant human papillomavirus early protein and the
purified recombinant human papillomavirus late protein. The
purified recombinant human papillomavirus early protein may be HPV
16 E6 protein, HPV 16 E7 protein, HPV 18 E6 protein, HPV18 E7
protein, and other HPV recombinant early proteins, and the purified
recombinant human papillomavirus late protein may be HPV 16 L1
protein, HPV 18 L1 protein, and other HPV recombinant late
proteins. For examples, hybridoma cell lines thus screened and
obtained can produce a monoclonal antibody recognizing a common
epitope on HPV16 E6, HPV16 E7, and HPV16 L1 proteins; or a
monoclonal antibody recognizing a common epitope on HPV16 E6 and
HPV18 E6 proteins; or monoclonal antibody recognizing a common
epitope on HPV16 E7 and HPV18 E7 proteins; or monoclonal antibody
recognizing a common epitope on HPV16 E6, HPV16 E7, HPV16 L1, HPV18
E6, and HPV18 E7 proteins. More examples are provided in the
drawings of this invention.
[0082] The antibody-producing hybridoma cells were also screened
with a first purified recombinant human papillomavirus protein from
a first HPV type and a second purified recombinant human
papillomavirus protein from a second HPV type such that the
monoclonal antibody is capable of reacting with a common epitope on
human papillomavirus proteins from two or more different HPV types.
The first and the second HPV types can be HPV 16, HPV 18, and other
HPV types. The two or more different HPV types can be, for example,
high risk HPV types, low risk HPV types, HPV-16, HPV-18, HPV-31,
HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58,
HPV-59, and HPV-68, HPV-6, HPV-11, HPV-42, HPV-43, HPV-44, HPV-53,
HPV-54, HPV-55, and HPV-56. As an example, the first and the second
purified recombinant human papillomavirus proteins may be
recombinant HPV 16 E6 protein, recombinant HPV 16 E7 protein,
recombinant HPV 16 L1 protein, recombinant HPV 18 E6 protein,
recombinant HPV18 E7 protein, and recombinant HPV 18 L1
protein.
[0083] As another example of hybridoma screening,
antibody-producing hybridoma cells were screened with positive
reactivity to some of the two or more purified recombinant human
papillomavirus proteins and negative reactivity to some of the two
or more recombinant human papillomavirus proteins and/or non-HPV
proteins. As such, the monoclonal antibodies generated form such
hybridoma screening is capable of binding to some HPV viral
proteins but not other HPV viral proteins.
[0084] For example, a monoclonal antibody is obtained by screening
antibody-producing hybridoma cells with a first purified
recombinant human papillomavirus protein from a first HPV type and
a second purified recombinant human papillomavirus protein from a
second HPV type such that the monoclonal antibody is capable of
reacting with a specific epitope on only one of the first and the
second purified recombinant human papillomavirus proteins and not
the other purified recombinant human papillomavirus protein.
Specific monoclonal antibodies obtained includes a monoclonal
antibody capable of binding to only HPV 16 E6 protein, but not any
other HPV proteins; a monoclonal antibody capable of binding to
only HPV 16 E7 protein, but not any other HPV proteins; a
monoclonal antibody capable of binding to only HPV 16 L1 protein,
but not any other HPV proteins; a monoclonal antibody capable of
binding to only HPV 18 E6 protein, but not any other HPV proteins;
and a monoclonal antibody capable of binding to only HPV 18 E7
protein, but not any other HPV proteins.
[0085] 2). Hybridoma cell line stocks: Hybridoma cell line clones
with desired positive reactivity and desired negative reactivity as
judged by an immunoassays (e.g., ELISA, EIA and other assays) were
selected and cloned down to single cell. Each single cell clone was
then grown up by tissue culture. When the cell numbers reach
millions of cells per ml, the cells were frozen down and kept at
-80.degree. C. or in liquid nitrogen as storage stocks.
[0086] 3). Ascites Production: Each hybridoma cell line was grown
in tissue culture and injected to mice for ascites production.
Ascites were collected and processed for immunoglobin purification
by protein G columns. Purified immunoglobin from each hybridoma
cell line was isotyped and used for HPV immunoassays.
4. HPV Immunohistochemistry (IHC) Assay:
1). HPV IHC Kit and the Assay:
[0087] In one embodiment, a kit for performing a HPV IHC assay is
provided. The kit may include an antigen retrieval agent, a
pre-antibody blocking solution, a post-antibody blocking solution,
an anti-HPV antibody as the primary antibody, an anti-mouse or
anti-rabbit immunoglobulins conjugated with HRP or biotin, or other
agents as secondary antibody, a solution containing appropriate
agents used as substrate for the secondary antibody to be
detected.
[0088] The antigen retrieval agent may contain a solution in low
pH, or neutral pH or high pH buffer. The pre-antibody blocking
solution may contain certain proteins or BSA, or serum or other
agents to block the cells from nonspecific binding of antibody. The
post blocking solution may contain similar solution as the
pre-antibody blocking solution with less proteins or serum to be
used along with primary antibody incubation. The solution
containing HPV antibodies may be in concentrated form, or may be in
diluted form as ready to use reagent. The anti-HPV antibodies may
also be directly tagged with HRP or biotin, or other agents to be
detected following appropriate agents used as substrate. The
solution containing secondary antibodies may be in concentrated
form, or may be in diluted form as ready to use reagent. The
solution containing appropriate agents used as substrate may
include DAB (3.3'-diaminobenzidine) as one component, or two
components, or AEC (3-Amino-9-Ethylcarbazole) substrate as one
component, or two components, or other substrates.
[0089] Once the cervical tissues are processed and fixed, the
Immunohistochemistry (IHC) assay is performed by boiling the
tissues on the slide with antigen retrieval buffer for a period of
time. The slides were then cool down to room temperature, blocked
with pre-antibody blocking solution for a period of time, then
incubated with the HPV antibodies. The slides were then washed 3 to
5 times with PBS or H2O, or other solution to remove any unbound
HPV antibody. Then the slides were incubated with the secondary
antibody, for example, anti-mouse IgG HRP, followed by appropriate
substrate for detection. As an example, DAB is oxidized in the
presence of peroxidase and hydrogen peroxide resulting in the
deposition of a brown, alcohol-insoluble precipitate at the site of
enzymatic activity. The precipitate may range in color from a light
golden brown to dark golden brown depending upon the amount of
enzyme present. The golden brown precipitate viewed under a
microscope indicates the specific binding of HPV antibodies with
HPV proteins present in the cells off the tissue section on the
slide. The assay can be performed at room temperature or higher
temperature to accelerate the binding reaction. This HPV IHC assay
can be performed manually, or operated by IHC automation, thus
provides a powerful tool to detect HPV infection and HPV
oncoproteins in situ. Therefore, the HPV IHC staining assay is very
useful as a confirmatory test. For the dysplasia cells identified,
HPV IHC staining may provide additional information for status of
HPV infection and/or expression of HPV oncoproteins. In addition,
overexpression of HPV E6 and E7 oncoproteins in various stage of
cervical dysplasia may indicate progression of CIN and/or cervical
cancer development.
[0090] 2). Sample selection and preparation: In order to analyze if
the anti-HPV antibody provided in this invention is able to detect
HPV proteins in situ from different stage of CIN or cancers, the
cervical tissues to be tested on the IHC assay include HSIL
consisting of CIN2 (stage 2 of Cervical Intraepithelial Neoplasia
with lesions appearing moderate) and CIN3 (stage 3 of Cervical
Intraepithelial Neoplasia with lesions appearing severe), and
invasive cancer consisting of squamous cell carcinoma (SCC, the
most common carcinoma in cervical cancer) and adenocarcinoma (ADC,
the gland type of carcinoma). Paraffin tissues blocks sectioned
into 4 microns were placed on slide and baked at 60 C overnight.
Deparaffin/hydrate sections were unmasked followed by standard IHC
staining procedures. Purified monoclonal antibody against HPV
proteins were diluted to use as the primary antibody. Staining
procedure is followed by secondary antibody solution, washing,
followed by appropriate substrate reagent to each section. As soon
as the sections develop, immerse slides in dH.sub.2O, counterstain
sections with hematoxylin, dehydrate and mount coverslips.
[0091] 3). Tissue Microarray: In order to perform homogeneous assay
for many samples in one reaction, tissue microarray was generated
to spot many samples on one slide. To process total of 84 samples
from CIN2, CIN3, or invasive cancers, three tissue microarrays were
prepared: One contains 30 individual CIN 2 and their peripheral
normal epithelia, One contains 30 individual CIN 3 and their
peripheral normal epithelia, One contains 12 cervical squamous cell
carcinomas and their normal epithelial counterparts, and 12
adenocarcinomas and their normal epithelial counterparts, vaginal
or cervical mucosa of at least 15 mm away from the gross tumor
border. One representative tissue spot for neoplasia and another
one spot representing its normal counterpart were taken for each
CIN case. For the case of invasive cancer, 2 spots of tumor tissue
and one spot of the normal counterpart were taken. A 2 mm round
tissue spot was retrieved from the corresponding paraffin-embedded
tissue block after taking a tissue slide for HPV DNA typing.
[0092] 4). HPV DNA test: HPV DNA typing of each case was identified
by a modified MY11/GP6+ PCR-based reverse-blot assay using
EasyChip.RTM. HPV blot or a HR-HPV chip, which contained 13
type-specific oligonucleotides on a nylon membrane. Total cellular
DNA was used as source of nucleic acid for amplification followed
by hybridization for detection.
[0093] 5). IHC score and data interpretation: The staining of each
dot on the tissue microarray slide was viewed by certified anatomy
pathologist under a microscope. Areas of tumor cells or dysplasia
cells were looked up to find the percentage of cells stained, with
staining intensity of score 0-3. Adjacent normal epithelium or
normal tissue 15 mm away from its corresponding dysplasia or tumors
was also scored. All data were scored by certified pathologist.
Stained percentage 10% was used as cut off to determine positive or
negative of the assay. All data were shown as Tables 1-17.
[0094] To demonstrate detection of HPV proteins in invasive
cervical cancer, tissues of squamous cell carcinoma (SCC),
appearing to be the most common cervical cancer, were processed to
be performed on HPV IHC assay. FIG. 1A-1D show IHC staining of
squamous cell carcinoma tissue using a mouse monoclonal HPV E7
antibody. FIG. 1A shows the representative image of the SCC tissue
from tissue microarray stained by IHC using an anti-HPV E7 mouse
monoclonal antibody. FIG. 1B shows the representative image of the
normal epithelium (15 mm away from the tumor tissue) of the SCC
subject from FIG. 1A. FIG. 1C shows the representative image of
another SCC sample from tissue microarray stained by IHC using the
same anti-HPV E7 antibody. FIG. 1D shows the magnified
representative image of the tumor cells stained by the same
anti-HPV E7 antibody in cytoplasm from FIG. 1C. Results indicate
expression of E7 oncoprotein can be detected in the tumor cells of
SCC tissue. Solid Black arrows indicate the specific staining of E7
protein in tumor cells, while empty clear arrows indicate the
normal cells with no staining. These results demonstrate that in
situ presence of HPV E7 protein from SCC cervical tissues can be
detected by the mouse monoclonal anti-HPV E7 antibody used in the
IHC assay.
[0095] To demonstrate detection of HPV proteins in another type of
invasive cervical cancer, FIGS. 2A-2C show IHC staining of cervical
adenocarcinoma using the same mouse monoclonal HPV E7 antibody.
Results indicate expression of E7 oncoprotein can be detected in
the tumor cells of adenocarcinoma tissue. Solid Black arrows
indicate the specific staining of E7 protein in tumor cells, while
empty clear arrows indicate the normal cells with no stain. FIG. 2A
shows the representative image of the tumor cells of adenocarcinoma
(ADC) sample stained by IHC using the same anti-HPV E7 monoclonal
antibody shown in FIG. 1. FIG. 2B shows the representative image of
the corresponding normal epithelium (15 mm away from the tumor) of
the ADC sample from FIG. 2A. FIG. 2C shows the magnified
representative image of the cytoplasm staining of adenocarcinoma
tumor cells from FIG. 2A. Highly magnified images indicate
localization of the E7 proteins expressed in the cytoplasm of tumor
cells, but not in the normal epithelium, or other cells including
stroma cells. These data demonstrate the IHC staining by the E7
monoclonal antibody can detect tumor cells of adenocarcinoma
showing similar staining pattern found in SCC.
TABLE-US-00001 TABLE 1 IHC staining results (stained %) and HPV DNA
typing for 12 SCC biopsy samples and 12 ADC biopsy samples (C:
Cytoplasmic; N: Nucleus; Dys: dysplasia or tumor cells). Anti-E7
Anti- Another Anti- Normal Another E6 anti-E6 L1 Dys epith. (%
anti-E7 Dys Dys. Dys. (% stained) stained) Dys (%) (%) (%) (%)
Sample # HPV type C N C N C C C C SCC-1 18 85 85 0 20 12.5 10 70 55
SCC-2 16, 52 90 85 0 25 15 15 10 55 SCC-3 16 60 65 0 40 5 0 10 20
SCC-4 16 92 50 0 40 5 0 10 85 SCC-5 16, 52, 58 92 55 0 50 20 5 15
88 SCC-6 18, 52, 58 90 60 25 18 10 70 SCC-7 16, 52 92 75 0 30 30 5
10 20 SCC-8 16, 58 10 10 0 5 0 0 10 50 SCC-9 no DNA 95 60 0 40 25 8
15 8 SCC-10 18 92 65 0 60 45 25 20 65 SCC-11 16, 58 0 80 5 0 0
SCC-12 33 95 90 0 0 30 1 20 55 ADE-1 16, 18 30 20 0 50 15 25 20 82
ADE-2 no DNA 62 40 0 30 35 70 35 78 ADE-3 16 20 30 0 20 35 55 60
ADE-4 16, 18 80 80 0 0 10 5 0 90 ADE-5 51, 52 95 80 0 50 10 70 15
92 ADE-6 11, 16, 52 0 40 5 0 0 15 ADE-7 18 50 40 0 60 25 20 20 75
ADE-8 18 85 60 0 40 15 50 15 82 ADE-9 45 82 55 0 30 30 2 20 40
ADE-10 18 15 10 0 40 15 15 5 70 ADE-11 18, 59 70 0 0 50 15 8 5 65
ADE-12 18 30
[0096] To analyze the HPV IHC results from each subject of invasive
cancer, Table 1 shows data from 24 cases of invasive cancer samples
with IHC score for staining of cytoplasm (C), and nucleus (N) using
C, or N followed by the % of staining using the anti-HPV E7
antibody shown in FIG. 1A-1D & FIG 2A-2C. Additional anti-HPV
antibodies including another anti-E7 antibody, Anti-HPV E6 antibody
like MAb1 and MAb 7 and anti-HPV L1 antibody were also also tested
on the same tissue microarray. To demonstrate the IHC staining by
various anti-HPV antibodies, IHC score from cytoplasm staining of
tumor cells using other anti-HPV antibodies was also shown in Table
1. Results of HPV DNA typing were also shown on the table for its
corresponding case.
[0097] As shown in Table 1, both nucleus and cytoplasmic staining
are found in all the subjects of tumor cells from SCC and ADE
stained by the anti-E7 antibody. However, there is more staining
(percentage stained) found in the cytoplasm of tumor cells
comparing the staining of nuclear of tumor cells. The detection of
HPV E7 protein in its adjacent normal epithelium cells was only
found in nucleus, but not found in the cytoplasm of the epithelial
cells. The staining of cytoplasm appears most distinguishable in
tumor cells compared to its corresponding normal adjacent cells.
These data demonstrate expression of HPV E7 proteins was detected
in the cytoplasm and nuclear of tumor cells of SCC and ADE tissues.
The localization of the E7 proteins expressed in the cytoplasm of
tumor cells, but not in the normal epithelium or stroma cells
appears tumor specific. HPV E7 proteins present in the nucleus of
normal adjacent epithelium and tumor cells detected by the anti-HPV
E7 antibody indicate HPV infection with oncoproteins expression.
Similar staining pattern was also found when used other anti-HPV
antibodies as shown in Table 1. Data indicate that the HPV IHC
assay as described herein can detect HPV early gene such as E6, E7,
and late gene such as L1 proteins present in the tumor cells of
cervical cancer tissues.
[0098] Comparing the results of HPV IHC to the HPV DNA typing, the
anti-E7 antibody reacts positively with all the HPV types present
in the samples tested. For example, the anti-E7 monoclonal antibody
as described herein can detect single HPV infection by at least
HPV-16, HPV-18, HPV-33, HPV-45, etc., which are cancer-related HPV
types (high risk HPV types). The single anti-E7 monoclonal antibody
can also detect HPV infection by two or more HPV types, such as the
combination of HPV 11, HPV-16, HPV-18, HPV-52, HPV-58, HPV-51,
HPV-59, etc., which include high risk, low risk, and non-oncogenic
.alpha.-papillomaviruses. However, infection by multiple HPV types
contains at least one type is high-risk HPV type. These data
indicate that the anti-E7 antibody described in this invention is
non-type specific, thus provides a powerful tool to detect HPV E7
proteins from most common high-risk HPV types in the cervical
cancer.
[0099] To demonstrate detection of HPV proteins in non-invasive
with severe dysplasia cervical tissue (HSIL; high grade squamous
intraneoplasm lesion, stage 3), FIG. 3 show IHC staining of CIN3
tissue using the same mouse monoclonal HPV E7 antibody. Results
indicate expression of E7 oncoprotein can be detected in the stage
3 of CIN tissue. Solid Black arrows indicate the specific staining
of E7 protein in dysplasia cells, while empty clear arrows indicate
the normal cells with no stain. FIG. 3A shows the representative
image of the dysplasia cells of a cervical intraepithelial neoplasm
(CIN3) tissue stained by IHC using the same anti-HPV E7 antibody
shown in FIG. 1 and FIG. 2 for invasive cancer tissue. FIG. 3B
shows the representative image of the adjacent normal epithelium of
the CIN3 tissue of FIG. 3A. These results demonstrate that in situ
presence of HPV E7 protein from CIN3 cervical tissues can be
detected by the mouse monoclonal anti-HPV E7 antibody used in the
IHC assay described herein.
[0100] To analyze the HPV IHC results from each subject of CIN3,
Tabel 2 shows data from 30 cases of CIN 3 samples with IHC score
for staining of cell membrane (M), cytoplasm (C), and nucleus (N)
using M, C, or N followed by the % of staining with the anti-E7
antibody. Additional anti-HPV antibodies including Anti-HPV E6
antibody like MAb1 and MAb 7 and anti-HPV L1 antibody were also
also tested on the same tissue microarray. To demonstrate the IHC
staining by various anti-HPV antibodies, IHC score from cytoplasm
staining of tumor cells using other anti-HPV antibodies was also
shown in Table 1. Results of HPV DNA typing were also shown on the
table for its corresponding case.
[0101] As shown in Table 2, nucleus staining are found in the
dysplasia cells of all the CIN3 samples tested while only certain
proportion of cases found staining of cytoplasm by the anti-E7
antibody. The results indicate that there is more staining found in
the cytoplasm than in the nuclear of dysplasia cells. As shown
previously in invasive cancer tissues, HPV E7 protein in its
adjacent normal epithelium cells was only found in nucleus, but not
found in the cytoplasm of the epithelial cells. The staining of
cytoplasm appears most distinguishable in dysplasia cells compared
to its corresponding normal adjacent cells. The localization of the
E7 proteins expressed in the cytoplasm of dysplasia cells, but not
in the normal epithelium or stroma cells appears HSIL specific.
These data demonstrate expression of HPV E7 proteins can be
detected in the cytoplasm and nuclear of dysplasia cells of CIN3
tissues. HPV E7 proteins present in the nucleus of normal adjacent
epithelium and dysplasia cells detected by the anti-HPV E7 antibody
indicate HPV infection with oncoproteins expression. For the cases
with high level expression of HPV E7 proteins detected in the
cytoplasm of dysplasia cells, it may suggest specific indication of
dysplasia progression. Similar staining pattern was also found when
used other anti-HPV antibodies as shown in Table 2. Data indicate
that the HPV IHC assay as described herein can detect HPV early
gene such as E6, E7, and late gene such as L1 proteins present in
the dysplasia cells of CIN3.
[0102] Comparing the results of HPV IHC to the HPV DNA typing, the
anti-E7 antibody reacts positively with all the HPV types present
in the samples tested. For example, the anti-E7 monoclonal antibody
as described herein can detect single HPV infection by at least
HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-58, etc., which are
cancer-related HPV types (high risk HPV types). The single anti-E7
monoclonal antibody can also detect HPV infection by two or more
HPV types, such as the combination of HPV-16, HPV-18, HPV-33,
HPV-39, HPV-52, HPV-58, etc., which include most common high-risk
HPV. These data indicate that the anti-E7 antibody described in
this invention is non-type specific, thus provides a powerful tool
to detect HPV E7 proteins from most common high-risk HPV types in
the CIN3 tissues.
TABLE-US-00002 TABLE 2 IHC staining results (stained % and score;
0-3) and HPV DNA typing of 30 CIN 3 samples (M: Membrane; C:
Cytoplasmic; N: Nucleus; Dys: Dysplasia). anti-E7 Dysplasia Normal
Anti-E6 Another Anti-L1 (% epithelium Dys. anti-E7 Dys. ID HPV
stained) (% stained) (%) Dys. (%) (%) # type M C N M C N Cyto Cyto
Cyto 31 33 0 80 80 0 0 50 70 40 80 32 16 0 80 80 60 0 0 5 33 16, 58
0 0 60 34 31 0 50 70 0 0 50 0 0 10 35 16, 39 0 70 90 0 0 40 0 10 30
36 31 0 70 60 0 0 50 0 20 20 37 39 0 0 40 0 0 0 0 0 0 38 16 0 0 40
39 16 0 60 70 0 0 40 0 0 40 58 0 90 90 0 0 50 50 0 30 41 16 0 0 50
0 0 50 0 20 20 42 16 0 70 70 0 0 30 0 0 43 33 0 0 90 0 0 50 0 0 5
44 52 0 70 80 0 0 50 70 10 50 45 51, 52 0 90 90 0 0 30 80 50 10 46
16 0 0 80 0 0 50 0 0 5 47 16 0 60 80 0 0 50 30 10 20 48 16, 58 0 0
80 0 0 50 0 0 10 49 31 0 80 60 50 70 40 40 50 16 0 0 60 0 0 30 0 20
20 51 6 0 0 20 0 52 16, 18, 0 0 20 0 0 30 0 0 0 33, 39 53 51, 52, 0
70 60 0 0 60 60 40 58 54 16, 45 0 0 70 0 0 50 0 20 20 55 16 0 0 75
0 0 50 0 0 0 56 33, 52 0 0 80 0 0 50 0 0 10 57 16 0 0 50 0 0 40 0 0
0 58 33 0 0 80 0 0 0 20 10 59 16 0 0 60 0 0 20 0 10 5 60 16, 52, 0
70 80 0 0 50 70 0 20 58
[0103] To demonstrate detection of HPV proteins in HSIL with
moderate dysplasia (stage 2 of CIN), FIG. 4 show IHC staining of
CIN2 tissue using the mouse monoclonal anti-HPV E6 antibody.
Results indicate expression of E6 oncoprotein can be detected early
in the stage of CIN2. FIG. 4A shows the representative image of the
dysplasia cells of cervical intraneoplasm (CIN2) tissues stained by
immunohistocytostaining (IHC) using the anti-E6 monolonal antibody.
FIG. 4B shows the representative image of the adjacent normal
epithelium from the dysplasia tissue of the CIN2 sample of FIG. 4A.
FIG. 4C shows the representative image of the dysplasia epithelium
of another CIN2 sample stained by IHC using the same anti-E6
monolonal antibody. FIG. 4D shows the magnified representative
image of the dysplasia epithelium in FIG. 4C. Solid Black arrows
indicate the specific staining of E6 protein in dysplasia cells,
while empty clear arrows indicate the normal cells with no stain.
Similar staining pattern of CIN2 found in CIN3 indicate
localization of the E6 proteins expressed in the cytoplasm of
dysplasia cells, but not in the normal epithelium, or other cells
including stroma cells. These results demonstrate that in situ
presence of HPV E6 protein from CIN2 cervical tissues can be
detected by the mouse monoclonal anti-HPV E6 antibody used in the
IHC assay described herein.
[0104] To analyze the HPV IHC results from each subject of CIN2,
Table 3 shows data from 30 cases of CIN 2 samples with IHC score
for staining of cell membrane (M), cytoplasm (C), and nucleus (N)
using M, C, or N followed by the % of staining with the anti-E7
antibody. Additional anti-HPV antibodies including Anti-HPV E6
antibody like MAb1 and MAb 7 and anti-HPV L1 antibody were also
also tested on the same tissue microarray. To demonstrate the IHC
staining by various anti-HPV antibodies, IHC score from cytoplasm
staining of dysplasia cells using other anti-HPV antibodies was
also shown in Table 3. Results of HPV DNA typing were also shown in
the table for its corresponding case.
TABLE-US-00003 TABLE 3 IHC staining results (stained % and score;
0-3) and HPV DNA typing for 30 biopsy samples (CIN2). (M: membrane;
C: cytoplasmic; N: nucleus; Dys: dysplasia) Anti-E7 another
Dysplasia Normal Anti-E6 anti-E7 Anti-L1 (% epithelium Dys. Dys.
Dys. ID HPV stained) (% stained) (%) (%) (%) # type M C N M C N
Cyto Cyto Cyto 1 6 0 80 80 0 0 30 70 40 80 2 31 0 0 90 0 40 0 3 52
0 25 50 0 0 70 0 20 20 4 16 0 0 40 0 0 30 0 5 0 5 58 0 0 50 0 0 10
0 0 0 6 52 0 80 70 0 0 50 0 5 0 7 53 0 0 80 0 0 30 0 10 10 8 52 0
50 90 0 0 20 60 10 20 9 31 0 80 80 0 0 50 70 20 40 10 16 0 50 80 0
0 50 60 20 10 11 no DNA 0 0 50 0 0 70 0 0 10 12 33 0 60 60 0 0 50 0
10 30 13 no DNA 0 70 80 0 0 70 0 20 10 14 52 0 0 70 0 0 70 0 30 20
15 no DNA 0 0 70 0 0 50 0 20 5 16 52 0 0 10 0 0 30 0 0 5 17 52 0 0
60 0 0 80 0 0 5 18 16 0 50 60 0 0 30 50 10 20 19 16 0 50 70 0 10 20
20 52, 44 0 50 80 0 0 40 0 30 30 21 16 0 0 50 0 0 50 0 20 20 22 16,
18, 6 0 0 40 0 0 0 0 10 0 23 16, 31 0 0 30 0 0 60 0 0 24 6 0 0 80 0
0 50 0 10 5 25 16 0 0 10 0 0 60 0 0 0 26 58 0 0 40 0 0 40 0 10 5 27
16, 39, 52 0 0 70 0 28 6 0 0 50 0 0 70 0 10 5 29 16 0 0 70 0 0 5 0
10 20 30 66, 68, 0 0 30 0 0 60 0 10 0
[0105] As shown in Table 3, nucleus staining are found in the
dysplasia cells of all the CIN2 samples tested while only certain
proportion of cases found staining of cytoplasm by the anti-E6 or
anti-E7 antibody. The results indicate there is more staining of
nucleus than cytoplasm of dysplasia cells found in CIN2 samples. As
shown previously in SCC, ADC, and CIN3, HPV E7 protein in its
adjacent normal epithelium cells was only found in nucleus, but not
found in the cytoplasm of the epithelial cells. The staining of
cytoplasm in CIN2 using anti-E6 antibody appears most
distinguishable in dysplasia cells compared to its corresponding
normal adjacent cells. The localization of the E6 proteins
expressed in the cytoplasm of dysplasia cells, but not in the
normal epithelium or stroma cells appears HSIL specific. These data
demonstrate expression of HPV E6 proteins can be detected in the
cytoplasm and nuclear of dysplasia cells of CIN2 tissues. For the
cases with high level expression of HPV E6 proteins detected in the
cytoplasm of dysplasia cells, it may suggest dysplasia progression.
Similar staining pattern was also found when used other anti-HPV
antibodies as shown in Table 3. The HPV IHC assay as described
herein can be used to detect HPV early gene such as E6, E7, and
late gene such as L1 proteins present in the dysplasia cells of
CIN2.
[0106] Comparing the results of HPV IHC to the HPV DNA typing, the
anti-E7 antibody reacts positively with all the HPV types present
in the samples tested. For example, the anti-E7 monoclonal antibody
as described herein can detect single HPV infection by at least,
HPV-16, HPV-18, HPV-31, HPV-52, HPV-58, etc., which are
cancer-related HPV types (high risk HPV types) and HPV6, HPV 53
which are not high-risk HPV types. The single anti-E7 monoclonal
antibody can also detect HPV infection by two or more HPV types,
such as the combination of HPV6, HPV-16, HPV-18, HPV-31, HPV-39,
HPV-44, HPV-52, HPV-58, HPV-66, HPV-68, etc., which include most
common high-risk HPV as well as low risk HPV types. These data
indicate that the anti-E7 antibody described in this invention is
non-type specific, able to detect HPV E7 proteins from common
high-risk HPV types as well as low risk types in the CIN2 tissues.
It is possible that formation of dysplasia cells is resulted from
expression of oncoproteins, rather than genotyping of HPV types. It
explains regression may occur for those infection by high-risk
types with no detection of oncoproteins in cytoplasm. Thus, the HPV
IHC assay described herein provides additional clinical
information, not only for detection of HPV infection, but also for
indication of dysplasia progression.
[0107] Since the staining of cytoplasm is, only found in dysplasia
cells distinguishable to its corresponding normal cells,
cytoplasmic staining was used to demonstrate the assay performance
in dysplasia progression. To compare the specific cytoplasm
staining of dysplasia cells in different stages of CIN or cancer
tissues, data from Table 1-3 were further analyzed to obtain the
assay performance. Percentage of staining 10% or above from each
subject is considered positive, otherwise is negative for the
sample less than 10% stained. As shown in table 4, the positive
rate of the assay increases with the severity of CIN shown 38%, 52%
to 100% for CIN2, CIN3, and SCC or ADE respectively. Positive
predictive value (PPV) and negative predictive value (NPV) were
also shown in this table to demonstrate the assay specificity using
percentage staining of cytoplasm.
TABLE-US-00004 TABLE 4 Summary of the IHC staining results using
anti-E7 on various biopsy samples during cervical cancer
development and tissue lesions. dysplasia Normal or Tumor Cyto- IHC
Cyto. plasmic positive Speci- Anti-E7 stain stain rate ficity CIN2
Positive 11 0 38% 100% 100% PPV Negative 18 28 61% NPV CIN3
Positive 14 0 52% 100% 100% PPV Negative 13 28 68% NPV SCC Positive
11 0 100% 100% 100% PPV Negative 0 11 100% NPV ADC positive 10 0
100% 100% 100% PPV negative 0 10 100% NPV
TABLE-US-00005 TABLE 5 Summary of the IHC staining results using
another anti-E7 on various biopsy samples during cervical cancer
development and tissue lesions. dysplasia Normal or Tumor Cyto- IHC
Another Cyto. plasmic positive Speci- anti-E7 stain stain rate
ficity CIN2 Positive 21 16 72% 57% PPV negative 8 9 36% 53% NPV
CIN3 Positive 13 4 48% 76% PPV negative 14 25 86% 64% NPV SCC
Positive 8 5 67% 62% PPV negative 4 6 55% 60% NPV ADC Positive 10 6
83% 63% PPV negative 2 6 50% 75% NPV
[0108] To show the expression of HPV E7 oncoproteins can be
detected by another anti-E7 monoclonal antibodies, same tissue
microarray was tested and shown in Table 5 representing the assay
performance of another E7 antibody with positive rate of 72%, 48%,
67%, 83% for CIN2, CIN3, SCC and adenocarcinoma respectively.
Positive predictive value (PPV) and negative predicative value
(NPV) also shown in this table indicates the assay specificity
using this antibody is not as good.
[0109] To validate the expression of HPV E6 oncoproteins can be
detected by anti-E6 monoclonal antibodies, same tissue microarray
were tested and shown in Table 6 and Table 7, representing the
assay performance of two anti-E6 antibodies. E6 proteins are
expressed in the cytoplasm of dysplasia cells from CIN2, CIN3, as
well as tumor cells from invasive cancer samples can be detected by
the anti-E6 antibody described in this invention. The same trend
shown positive rate of the anti-E7 IHC assay increased with the
severity of CIN is also found in the assay using anti-E6 antibody.
The two anti-E6 antibodies show the same trend of increasing
positive rate of assay over severity of CIN although one anti-E6
may give better assay performance than the other one. It is
possible that MAb1 recognize different epitope from MAb7 does, thus
give different assay performance. However, both monoclonal
antibodies give high positive predictive value (PPV) and high
negative predictive value (NPV) as shown in the tables. The overall
positive rate of IHC assay using anti-E7 antibody is higher than
using anti-E6 antibody. It is possible that E7 proteins are
expressed earlier to serve as a biomarker for early detection of
cervical cancer.
TABLE-US-00006 TABLE 6 Summary of the IHC staining results using
anti-E6 on various biopsy samples during cervical cancer
development and tissue lesions. dysplasia Normal IHC or Tumor Cyto-
posi- Cyto. plasmic tive Speci- Anti-E6 stain stain rate ficity
CIN2 Positive 5 1 17% 83% PPV Negative 25 29 97% 54% NPV CIN3
Positive 17 7 57% 71% PPV Negative 13 23 77% 64% NPV SCC Positive 7
1 64% 88% PPV Negative 4 10 91% 71% NPV ADC Positive 9 0 75% 100%
PPV Negative 3 12 100% 80% NPV total Positive 38 9 46% 0 81% PPV
Negative 45 74 0 89% 62% NPV
[0110] To detect the expression of L1 viral proteins present in
different stage of CIN, the same tissues microarrays were also
tested on IHC assay using anti-L1 antibody. IHC score from L1
staining of cytoplasm was also obtained to analyze its positivity
rate on all the samples. To study the correlation of HPV early and
late proteins expressed in situ in different stage of CIN, Table 8
shows the positive rate of IHC assay with E7 expression and L1
expression in the cytoplasm for CIN2, CIN3, and invasive cancers.
Since E7 seems a good biomarker for early detection of cervical
caner, we compare the correlation of E7 and L1 expression in
various stages of CIN and invasive cancer tissues using the HPV IHC
assay described in this invention. For L1 IHC positive of CIN
samples, as data shown in Table 8, about 60% (9 out of 15) of CIN2,
or 58% (11 out of 19) of CIN3 show positive on E7 IHC assay. For L1
cytoplasmic positive of invasive cancer samples, 100% of both SCC
(11 out of 11) and adenocarcinoma (10 out of 10) are E7 cytoplasmic
positive, indicating 100% correlation of E7 expression with cancer
progression. Data indicate that both L1 and E7 cytoplasmic positive
of CIN2/3 may have higher risk in further dysplasia progression
compared to those L1 positive but E7 negative on IHC assay.
TABLE-US-00007 TABLE 7 Summary of the IHC staining results using
another anti-E6 on various biopsy samples during cervical cancer
development and tissue lesions. dysplasia Normal or Tumor Cyto- IHC
Another Cyto. plasmic positive Speci- anti-E6 stain stain rate
ficity CIN2 positive 5 0 17% 100% PPV negative 24 28 100% 54% NPV
CIN3 positive 8 0 30% 100% PPV negative 19 29 100% 60% NPV SCC
positive 11 0 92% 100% PPV negative 1 11 100% 92% NPV ADC positive
9 0 75% 100% PPV negative 3 12 100% 80% NPV total positive 33 0 41%
0 100% PPV negative 47 80 0 100% 63% NPV
TABLE-US-00008 TABLE 8 Summary of the IHC staining results using
Anti-E7 compared with Anti-L1 and Anti-E6 on various biopsy samples
during cervical cancer development and tissue lesions. CIN2 CIN3
SCC ADC Total No. of samples L1 (+) L1 (-) L1 (+) L1 (-) L1 (+) L1
(-) L1 (+) L1 (-) E7 positive 9 2 11 3 11 0 10 0 E7 negative 6 8 8
5 0 0 0 0 No. of samples E6 (+) E6 (-) E6 (+) E6 (-) E6 (+) E6 (-)
E6 (+) E6 (-) E7 positive 5 6 8 6 11 0 9 1 E7 negative 0 17 0 13 0
0 0 0
[0111] Table 8 also shows the correlation of sample positivity with
E7 expression and E6 expression in the cytoplasm. As data shown,
for E7 cytoplasmic positive samples, about 45% (5 out of 11) of
CIN2, or 57% (8 out of 14) of CIN3 show positive on E6 cytoplasmic
expression, while 100% (11 out of 11) of SCC or 90% (9 out of 10)
of ADE show E6 cytoplasmic expression. These data indicate that E6
may be expressed behind E7 during early dysplasia, but co-expressed
in the late stage of cervical cancer.
[0112] In order to have homogeneous assay conditions for all stages
of samples in one reaction, additional tissue microarray was
generated to spot many samples from CIN2, CIN3, and invasive
cancers on the same slide. Two tissue microarrays were prepared:
One contains 10 individuals each for CIN2, CIN3, and SCC and their
peripheral normal epithelia. One contains 10 individuals each for
CIN2, CIN3, and ADE and their peripheral normal epithelia. To
confirm the HPV IHC assay is specific to HPV related dysplasia or
cervical cancer, additional tissue microarray containing more than
90 samples from various normal human tissues were also tested to
use as the negative control of the HPV IHC assay. The same HPV IHC
assays using anti-E6 and anti-E7 antibody were applied on these
tissue microarray to obtain IHC staining percentage as described
above. IHC staining of 10% or above shown in nucleus and/or
cytoplasm was scored as positive of the assay.
[0113] All together, the IHC data from all the tissue microarrays
tested herein were shown in Table 9-14. Table 9 shows IHC staining
results using a mouse anti-HPVE7 antibody on various biopsy samples
during cervical cancer development and tissue lesions. Data in
Table 9 indicate that the presence of HPV E7 proteins in situ can
be detected from various stages of cervical tissues with increasing
positivity rate of assay from, CIN2, CIN3 to invasive cancer tissue
like squamous cell carcinoma (SCC) or adenocarcinoma (AD). There is
about 72%, and 90% positive rate for samples with CIN2 and CIN3
respectively. For cancer tissues (SCC and AD), 100% of samples
stains positively by IHC using anti-HPV E7 antibody, indicating
100% of cancers expressing HPV oncogenic proteins. These data
indicate the IHC assay using the HPV E7 antibody described in this
invention provides a powerful tool to confirm the diagnosis of
cervical cancer from the tissues in various grade.
TABLE-US-00009 TABLE 9 IHC staining results using a monoclonal
anti-HPVE7 antibody on various biopsy samples during cervical
cancer development and tissue lesions. IHC CIN2 CIN3 SCC ADC total
Anti-HPV E7 34 43 22 23 122 positive Anti-HPV E7 13 5 0 0 18
negative Positive rate 72% 90% 100% 100% 87%
[0114] To obtain assay performance, data from table 9 were further
analyzed. Table 10 shows summary of the IHC staining results from
Table 9 using normal human tissues as CIN2 negative samples. Data
indicate that the IHC staining method using the anti-HPV E7
antibody provides IHC assay sensitivity of 87% for CIN2+ with
specificity of 92%. These data suggest this assay can be useful to
detect HPV proteins for confirming of cervical lesion CIN2 or
above.
TABLE-US-00010 TABLE 10 Summary of the immunohistochemistry
staining results using a mouse monoclonal anti-HPVE7 antibody on
CIN2+ lesions compared to CIN negative samples. CIN2+ CIN negative
HPVE7 positive 122 7 95% PPV HPVE7 negative 18 85 83% NPV
Sensitivity 87% Specificity 92%
[0115] To further analyze the data, Table 11 shows summary of the
immunohistochemistry staining results from Table 9 and Table 10
indicating that using the anti-HPV E7 antibody described in this
invention provides immunohistochemistry assay for CIN3+ (including
CIN3, and invasive cancer) with 95% sensitivity, 92% specificity
and 93% of positive predictive value, and 95% of negative
predictive value. These data suggest this assay can be useful for
clinical application to detect HPV proteins confirming cervical
lesion in different stages.
TABLE-US-00011 TABLE 11 Summary of the immunohistochemistry
staining results using a mouse monoclonal anti-HPVE7 antibody on
CIN3+ lesions compared to CIN negative samples. CIN3+ CIN negative
HPVE7 positive 88 7 93% PPV HPVE7 negative 5 85 94% NPV sensitivity
95% Specificity 92%
[0116] To confirm if expression of E6 protein can be detected by
immunohistochemistry assay using the anti-E6 antibody described in
this invention, the same tissues microarrays were performed on IHC
assay using an anti-E6 antibody. As an example of another HPV
immunohistochemistry assay, Table 12-14 show results of
immunohistochemistry staining using anti-HPV E6 antibody. As data
shown, HPV anti-E6 gives comparable immunohistochemistry results as
HPV anti-E7.
TABLE-US-00012 TABLE 12 Immunohistochemistry staining results using
a monoclonal anti-HPVE6 antibody on various biopsy samples during
cervical cancer development and tissue lesions. IHC CIN2 CIN3 SCC
AD total HPVE6 positive 32 35 23 17 107 HPVE6 negative 17 15 1 7 40
sensitivity 65% 70% 96% 71% 73%
[0117] Table 12 shows immunohistochemistry staining results using a
mouse anti-HPVE6 antibody on various biopsy samples during cervical
cancer development and tissue lesions. Data in Table 12 indicate
that HPV E6 protein can be detected by the mouse monoclonal
anti-HPV E6 antibody used in the immunohistochemistry assay
described in this invention. The presence of HPV E6 proteins in
situ can be detected from various stages of cervical tissues. As
data shown, HPV E6 proteins are present in the samples with
increasing positivity rate from, CIN2, CIN3 to cancer tissue like
squamous cell carcinoma (SCC) or adenocarcinoma (AD). There is
about 65%, and 70% positive rate for samples with CIN2 and CIN3
respectively. For cancer tissues, 96% of SCC samples stain
positively by immunohistochemistry using anti-HPV E6 antibody,
while only 71% of AD samples stain positively, indicating HPV E6
oncoproteins expressing more predominantly in SCC than in AD. These
data indicate the immunohistochemistry assay using the HPV E6
antibody described in this invention provides a tool to confirm the
diagnosis of cervical cancer from the tissues in various grade.
TABLE-US-00013 TABLE 13 Summary of the immunohistochemistry
staining results using a mouse monoclonal anti-HPVE6 antibody on
CIN2+ lesions compared to CIN negative samples. CIN2+ CIN negative
HPVE6 positive 107 12 90% PPV HPVE6 negative 40 80 67% NPV
Sensitivity 73% Specificity 87%
[0118] To further analyze the data, Table 13 shows summary of the
IHC staining results from Table 11 and Table 12. As data indicated,
using the anti-HPV E6 antibody provides immunohistochemistry assay
sensitivity of 73% for. CIN2+ with specificity of 87%. These data
suggest this assay can be useful for clinical application to detect
HPV proteins confirming cervical lesion in different stages.
TABLE-US-00014 TABLE 14 Summary of the immunohistochemistry
staining results using a mouse monoclonal anti-HPVE7 antibody on
CIN3+ lesions compared to CIN negative samples. CIN3+ CIN negative
HPVE6 positive 75 7 91% PPV HPVE6 negative 23 85 79% NPV
sensitivity 77% Specificity 92%
[0119] To further analyze the data, Table 14 shows summary of the
immunohistochemistry staining results from Table 13 indicating the
immunohistochemistry staining method using the anti-HPV E6 antibody
described in this invention provides immunohistochemistry assay for
CIN3 or above with sensitivity of 77% specificity of 92% and 91%
PPV, and 79% NPV. These data suggest this assay can be useful for
clinical application to detect HPV proteins confirming cervical
lesion in different stages.
[0120] The one or more immunological assays using antibodies and
purified recombinant proteins derived from HPV early and/or late
genes as obtained herein serve as reliable indicators whether HPV
infection has occurred. In addition, HPV associated malignancy or
pre-malignant cell transformation can be assayed. One of the most
useful aspects of the invention is in diagnosing cervical
carcinoma, both squamous cell and adenocarcinoma as well as any
epithelial cell abnormality associated with oncogenic HPV infection
including koilocytosis; hyperkerotosis; precancerous conditions
encompasssing intraepithelial neoplasias or intraepithelial lesion;
high-grade dysplasias; and invasive or malignant cancers.
[0121] In one embodiment, the early gene that can be used herein
may include papillomavirus E6 genes, papillomavirus E7 genes, among
others. In another embodiment, the late gene that can be used
herein may include papillomavirus L1 genes, papillomavirus L2
genes, among others.
[0122] One aspect of the invention provides recombinant proteins,
such as a recombinant hybrid protein containing a full length
sequence of HPV oncogenic proteins, e.g., full-length E6, E7,
and/or L1 polypeptide sequence, which have been found very
difficult to obtain and purify due to undesirable aggregation
during protein purification, protein instability, low levels of
expression, low immunogenic responses of purified proteins. For
example, many early E6 oncoproteins contain many cysteine amino
acids and thus the correct topography of the E6 oncoproteins
requires formation of many disulfide bonds properly. In addition,
it was known that certain immunological assays using small peptides
of early E6 and E7 proteins results in extremely low assay
specificity and sensitivity and thus unsuitable as commercialized
diagnostic tools.
[0123] In high grade CIN lesions, E6 and E7 are strongly expressed
in host basal epithelial cells and interfere substantially with
cell cycle control of these replication competent host cells.
Expression of HPV oncoproteins interfers with G1-S-Phase regulation
in host cells. The HPV E6 and E7 proteins target a plethora of
cellular interactions, such as the inactivation of pRB by E7 and
the degradation of p53 by E6. High level of HPV E7 proteins
inactivates pRB and leads to disruption of E2F-Rb binding. Usually,
binding of pRB to E2F blocks E2F driven cell cycle activation. In
replicating cells, E2F is regulated by phosphorylation of RB. Rb
phosphorylation is normally mediated by cyclin dependent kinases
(CDK4, CDK6) that are controlled by several kinase inhibitors
(INKs).
[0124] As a result of the loss of Rb/E2F repression and the strong
activation by free E2F, the expression of a host cell protein,
p16INK4a, is strongly overexpressed. In addition, S-phase genes are
continuously activated since the p16INK4a mediated repression of
Cdk4/6 has no downstream effect on pRb host cell protein. Since
E7-dependent E2F release is not mediated by phosphorylation of pRb,
the counter-regulatory p16INK4a expression has no effect on the
activated cell cycle. Under physiological conditions p16INK4a is
expressed when cells undergo a genomic stress situation such as
substantial shortening of telomeres in ageing tissues. Also,
apoptosis is abrogated by HPV E6 mediated degradation of p53. The
overexpression of the cyclin dependent kinase (CDK) inhibitor,
p16INK4a, is a direct consequence of deregulated HPV oncogene
expression.
[0125] In addition, host cell proteins important for proliferation
and host cell genome replication may be overexpressed as a result
of HPV infection. These host cell proteins include, ki67 (MIB-1),
MYC cellular oncogene, Cyclin proteins (e.g., cyclin A, B, E,
etc.), CDKN2A/p16INK4a, telomerase (e.g., TERC), replication
complex proteins (e.g., MCM5, CDC6, topoisomerase II alpha (TOP2A),
MCM2, minchromosome maintenance proteins 2, 4, and 5, etc.).
[0126] As an example, the immunological assays for detection of HPV
proteins, such as E6, E7, L1, etc., or immune response thereof due
to HPV infection can be performed in high throughput ELISA
screening assays, rapid immunological screening assays, and
additional multiplexed protein chip assays, etc., and combinations
thereof. Embodiments of the invention provides one or more assays,
including an antibody, antigen, or immunocomplex assays developed
to detect HPV viral proteins encoded by early genes (e.g., E6 and
E7) and late genes (e.g., L1). In addition, the developed antibody,
antigen, or immunocomplex assays for E6, E7, L1, protein or their
antibodies thereof in one format, for example, a microplate format,
can be adapted into a one-step immunochromatographic assay for the
direct measurement of E6, E7, L1 proteins or antibodies induced by
HPV infection.
[0127] The one or more protein chip assays, immunological assays,
nucleic acid assays, as provided herein aims to employ user
friendly procedures with simple instrument or no additional
instrument to perform in a short period of time. Comparison of the
results of the various immunological assays, nucleic acid
hybridization assays with cytological and histological data for the
human subjects as well as demographic information serve to validate
the correlation and accuracy in diagnosing HPV infection and/or
cervical cancer.
[0128] Another example of a method of screening a human subject
infected with a human papillomavirus may include obtaining a
clinical sample from the human subject, conducting a nucleic acid
hybridization assay on the clinical sample, detecting the presence
of a papillomavirus genome in the clinical sample from the human
subject, conducting one or more immunological assays on the
clinical sample, detecting the presence of an antibody to an early
papillomavirus viral protein or the presence of the early
papillomavirus viral protein in the clinical sample using a first
recombinant protein of the early papillomavirus viral protein, and
detecting the presence of an antibody to a late papillomavirus
viral protein or the presence of the papillomavirus late viral
protein in the clinical sample using a second recombinant protein
of the late papillomavirus viral protein.
[0129] The one or more diagnostic immunological assays as described
therein may also include obtaining polyclonal antibodies,
monoclonal antibodies, and/or antiserum specific against the one or
more recombinant proteins as obtained and described herein, taking
a clinical sample likely to contain HPV associated proteins and/or
antigens, reacting it with the obtained polyclonal antibodies,
monoclonal antibodies, and/or antiserum specific for the one or
more recombinant proteins, and assaying for the presence of any
antibody-antigen complexes by suitable detection systems. Suitable
detection system may employ various colormetric, chemilumenescent,
flourescent substrates, etc., specific for a secondary antibody
used in each immunological assay.
[0130] Early diagnosis of HPV infection is important for successful
prevention and treatment of cervical cancer. Strategies to prevent
cervical cancer requires improved HPV testing/screening to cover a
broad range of the worldwide population in addition to closely
follow-up those subjects with past or present HPV infection and/or
pre-cancerous lesions. Importantly, it is known that infection in
women for 12-15 years with HPV is required before invasive cancer
to develop. It is thus important to be able to assay biomarkers for
HPV infection as described herein to pre-screen women early, such
that it will be possible to treat HPV infection early and prevent
cervical cancer development, rather than having to rely on
chemotherapy or radiation to treat cancer malignancy.
* * * * *