U.S. patent application number 15/148674 was filed with the patent office on 2016-11-10 for diagnostic biomarkers and therapeutic targets for pancreatic cancer.
The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Robert Anders, Elizabeth A. Jaffee, Darshil T. Jhaveri.
Application Number | 20160327560 15/148674 |
Document ID | / |
Family ID | 50883901 |
Filed Date | 2016-11-10 |
United States Patent
Application |
20160327560 |
Kind Code |
A1 |
Jaffee; Elizabeth A. ; et
al. |
November 10, 2016 |
DIAGNOSTIC BIOMARKERS AND THERAPEUTIC TARGETS FOR PANCREATIC
CANCER
Abstract
We identified >40 proteins that elicited at least a 2-fold
increase in antibody response post-pancreatic-cancer vaccination,
from each of three patients' sera. The antibody responses detected
against these proteins in patients with >3 years disease-free
survival indicates the anti-tumor potential of targeting these
proteins. We found that tissue expression of proteins PSMC5, TFRC
and PPP1R12A increases during tumor development from normal to
pre-malignant to pancreatic tumor. In addition, these proteins were
shown to be pancreatic cancer-associated antigens that are
recognized by post-vaccination antibodies in the sera of patients
that received the vaccine and have demonstrated a favorable disease
free survival.
Inventors: |
Jaffee; Elizabeth A.;
(Lutherville, MD) ; Jhaveri; Darshil T.; (Towson,
MD) ; Anders; Robert; (Parkville, MD) |
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Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
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|
Family ID: |
50883901 |
Appl. No.: |
15/148674 |
Filed: |
May 6, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14649248 |
Jun 3, 2015 |
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PCT/US2013/072592 |
Dec 2, 2013 |
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15148674 |
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61732402 |
Dec 3, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/916 20130101;
G01N 2333/4703 20130101; G01N 33/57438 20130101; G01N 2333/79
20130101; G01N 2333/705 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
No. P50CA62924 awarded by the National Institutes of
Health/National Cancer Institute. The government has certain rights
in the invention.
Claims
1. A method for detecting pancreatic cancer in a body sample from a
human, comprising: contacting the body sample with at least one
antibody that specifically binds to a protein selected from the
group consisting of: Transferrin receptor (TFRC), regulatory
subunit 12A of protein phosphatase 1 (PPP1R12A), and regulatory
subunit 8 of the 26S proteasome (PSMC5); detecting amount of
antigen bound to the antibody or cellular localization of the
antigen, wherein an increased amount of antigen bound to the
antibody relative to an amount bound to a control sample or an
altered cellular localization indicates the presence of a
pancreatic cancer.
2. The method of claim 1 wherein the body sample is a tissue
sample.
3. The method of claim 1 wherein the body sample is a blood or
urine sample.
4. The method of claim 1 wherein the step of detecting is performed
using immunohistochemistry.
5. The method of claim 1 wherein the step of detecting is performed
using an ELISA.
6. The method of claim 1 wherein at least two of said antibodies
are contacted and detected.
7. The method of claim 1 wherein at least three of said antibodies
are contacted and detected.
8. The method of claim 1 wherein the steps of contacting and
detecting are further performed using at least one antibody that
specifically binds to an antigen selected from the group consisting
of: mesothelin, annexin A2, and galectin 3.
9. A method for monitoring progression of pancreatic cancer in a
body sample from a human, comprising: contacting the body sample
with at least one antibody that specifically binds to a protein
selected from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5); detecting
amount of antigen bound to the antibody, wherein an increased
amount of antigen bound to the antibody relative to an amount bound
to a sample taken at a prior time indicates progression of the
pancreatic cancer and a decreased amount of antigen bound to the
antibody relative to amount bound to a sample taken at a prior time
indicates responsiveness to an anti-cancer treatment.
10. The method of claim 9 wherein the body sample is a tissue
sample.
11. The method of claim 9 wherein the body sample is a blood or
urine sample.
12. The method of claim 9 wherein the step of detecting is
performed using immunohistochemistry.
13. The method of claim 9 wherein the step of detecting is
performed using an ELISA.
14. The method of claim 9 wherein at least two of said antibodies
are contacted and detected.
15. The method of claim 9 wherein at least three of said antibodies
are contacted and detected.
16. The method of claim 9 wherein the steps of contacting and
detecting are further performed using at least one antibody that
specifically binds to an antigen selected from the group consisting
of: mesothelin, annexin A2, and galectin 3.
17. A method to predict response to a pancreatic cancer vaccine in
a human, comprising: contacting a body sample of the human with at
least one antibody that specifically binds to a protein selected
from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5); detecting
amount of antigen bound to the antibody, wherein a decreased amount
of antigen bound to the antibody relative to an amount bound to a
control sample prior to vaccination predicts long term disease-free
survival.
18. The method of claim 17 wherein the body sample is a tissue
sample.
19. The method of claim 17 wherein the body sample is a blood or
urine sample.
20. The method of claim 17 wherein the step of detecting is
performed using immunohistochemistry.
21. The method of claim 17 wherein the step of detecting is
performed using an ELISA.
22. The method of claim 17 wherein at least two of said antibodies
are contacted and detected.
23. The method of claim 17 wherein at least three of said
antibodies are contacted and detected.
24. The method of claim 17 wherein the steps of contacting and
detecting are further performed using at least one antibody that
specifically binds to an antigen selected from the group consisting
of: mesothelin, annexin A2, and galectin 3.
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. A method to predict response to a pancreatic cancer vaccine in
a human, comprising: contacting a sample of the human comprising
antibodies with at least one protein selected from the group
consisting of: Transferrin receptor (TFRC), regulatory subunit 12A
of protein phosphatase 1 (PPP1R12A), and regulatory subunit 8 of
the 26S proteasome (PSMC5); detecting amount of antibody bound to
the at least one protein, wherein an increased amount of antibody
bound to the at least one protein relative to an amount bound to a
control sample prior to vaccination predicts long term disease-free
survival.
30. The method of claim 29 wherein the body sample is a tissue
sample.
31. The method of claim 29 wherein the body sample is a blood or
urine sample.
32. The method of claim 29 wherein the step of detecting is
performed using immunohistochemistry.
33. The method of claim 29 wherein the step of detecting is
performed using an ELISA.
34. The method of claim 29 wherein at least two of said antibodies
are contacted and detected.
35. The method of claim 29 wherein at least three of said
antibodies are contacted and detected.
36. The method of claim 29 wherein the steps of contacting and
detecting are further performed using at least one antibody that
specifically binds to an antigen selected from the group consisting
of: mesothelin, annexin A2, and galectin 3.
37.-90. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/649,248, filed on Jun. 3, 2015, which is a national stage
entry of International Application No. PCT/US2013/072592, filed on
Dec. 2, 2013, which claims priority to U.S. Provisional Patent
Application No. 61/732,402, filed on Dec. 3, 2012, each of which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention is related to the area of cancer diagnostics,
prognostics, and therapeutics. Moreover, it relates to the area of
immunotherapeutics.
BACKGROUND OF THE INVENTION
[0004] Pancreatic ductal adenocarcinoma is the fourth leading cause
of cancer-related death in the U.S. (1). It is notably the most
aggressive and debilitating malignant disease with a median
survival of less than 6 months. Only 1% to 4% of patients have an
overall survival of more than 5 years (1). Inadequate early
diagnosis, resistance to current therapies, and ineffective
treatment account for these low survival statistics. Alternative
treatment approaches are desperately needed for this disease; the
compelling need for superior treatment options has propelled the
development of new, better-targeted therapies. We have developed an
allogeneic, granulocyte-macrophage colony-stimulating factor
(GM-CSF)-secreting pancreatic cancer vaccine, which has recently
completed phase II clinical trial (2). This promising vaccine is
used in combination with chemoradiation. The observation of
favorable clinical and immunological responses in patients has
testified to the success of the vaccine (2-4). It was shown that
the induction of mesothelin-specific T cell responses only in
patients with a DFS>3 years, which suggests the vaccine induces
immunologically relevant T cell responses (2). Functional genomic
approaches were utilized to identify antigens recognized by T cells
(5). However, finding T cell antigens is limited by the need for
large amounts of patient lymphocytes and the lack of reagents for
each patient-specific HLA (6).
[0005] In contrast to T cells, antibodies hold potential as a high
throughput way of identifying antigens. Antibodies can also mount
an effective response against cancer cells through opsonizing,
antigen presentation to T-cells, and mediating cell toxicity via
natural killer cells or the complement system (7). Thus, the
application of seroproteomic approaches has recently gained ground
in the identification of new cancer biomarkers. These cancer
biomarkers are beneficial for both early detection and the
determination of new targets for the development of biologically
relevant therapies (7-12). The most well-known proteomic approaches
utilize sera from untreated cancer patients or individuals with
known genetic susceptibilities for cancer, to screen for
cancer-associated proteins that elicit an antibody response. These
approaches identify oncoproteins that elicit an antibody response
due to differences in expression levels or post-translational
modifications (11). GM-CSF secreting cancer vaccines can also
instigate a broad range of antibody responses, as seen in early
clinical studies (13). Through the study of the immunological
responses in vaccinated patients, we can discover the mechanisms
behind favorable vaccine-induced clinical responses. Identifying
cancer associated proteins will enhance our efforts of identifying
biologically relevant proteins. These proteins have high potential
as future targets for effective pancreatic cancer treatment. This
translational approach will advance the development of new drugs,
vaccines and antibody-based therapies that will halt the
progression and metastasis of the disease. This approach can also
help characterize new proteins that will serve as surrogate
biomarkers, prediction tools of the vaccine's success, and
biomarkers for early diagnosis of pancreatic cancer.
[0006] Common proteomic approaches to identify immunogenic proteins
are: Serological Screening of cDNA Expression Library (SEREX),
2-dimensional electrophoresis (2-DE) followed by mass-spectrometry
analysis and protein arrays (7). Proteins found using SEREX and
2-DE approaches are now shown to also elicit T cell responses (6,
13, 14). This provides evidence that antibodies can aid in the
identification of T cell antigens, which further testifies to the
advantages in studying antibodies. SEREX, however, utilizes
proteins expressed in Escherichia coli, which does not account for
human post-translational modifications (12). Contrastingly, the
approach utilizing 2-DE analysis can use human proteins as the
proteome. However, this process has an inherent bias towards
identifying proteins that are abundantly expressed (11). 2D-PAGE
has a lower threshold out of the throughput methods and does not
effectively identify proteins that are very acidic, very basic,
small in size (<15 kDa), or hydrophobic (15). Therefore, this
process is inadequate for detecting membrane-associated proteins,
the most relevant category of proteins as potential biomarkers.
Membrane proteins constitute about 30% of all cellular proteins and
are functionally key regulators (16). In addition, in 2D-PAGE, each
band cut holds several similar molecular weight proteins. This
process is inefficient in separating single proteins, which
obscures which protein instigates the antibody response.
Furthermore, low abundant antigens are generally overshadowed by
high abundant proteins with the same molecular weight in this
process. Both SEREX and SERPA identify linear epitopes, are
relatively low throughput and semi-quantitative (11).
[0007] Protein arrays come in many forms. Some protein arrays use
tumor cell lysate fractions, which identify proteins in their
native conformation (11). However, these arrays do not identify
which specific protein in the fraction instigates the immune
response and there also issues with fractionation. The protein
arrays with printed recombinant proteins do not contain human
post-translational modifications because the proteins are expressed
in E. coli or yeast (12). In addition, if a known protein panel is
printed, tumor antigen discovery can be prevented because the
proteome is biased. The protein arrays utilizing printed monoclonal
antibodies are potentially limited by reagent availability thereby
preventing an unbiased proteome being used because a high affinity
and highly specific monoclonal antibody is needed for each protein
to be probed.
[0008] There is a continuing need in the art to provide better
methods of early diagnosis, monitoring, prognosing, and treating
pancreatic cancer.
SUMMARY OF THE INVENTION
[0009] According to one embodiment of the invention a method
detects pancreatic cancer in a body sample from a human. A body
sample is contacted with at least one antibody that specifically
binds to a protein selected from the group consisting of:
Transferrin receptor (TFRC), regulatory subunit 12A of protein
phosphatase 1 (PPP1R12A), and regulatory subunit 8 of the 26S
proteasome (PSMC5). The amount of antigen bound to the antibody is
detected or cellular localization of the antigen is detected. An
increased amount of antigen bound to the antibody relative to an
amount bound to a control sample or an altered cellular
localization indicates the presence of a pancreatic cancer.
[0010] According to another embodiment a method monitors
progression of pancreatic cancer in a body sample from a human. A
body sample is contacted with at least one antibody that
specifically binds to a protein selected from the group consisting
of: Transferrin receptor (TFRC), regulatory subunit 12A of protein
phosphatase 1 (PPP1R12A), and regulatory subunit 8 of the 26S
proteasome (PSMC5). The amount of antigen bound to the antibody is
detected. An increased amount of antigen bound to the antibody
relative to an amount bound to a sample taken at a prior time
indicates progression of the pancreatic cancer. A decreased amount
of antigen bound to the antibody relative to amount bound to a
sample taken at a prior time indicates responsiveness to an
anti-cancer treatment.
[0011] According to another embodiment a method predicts response
to a pancreatic cancer vaccine in a human. A body sample of the
human is contacted with at least one antibody that specifically
binds to a protein selected from the group consisting of:
Transferrin receptor (TFRC), regulatory subunit 12A of protein
phosphatase 1 (PPP1R12A), and regulatory subunit 8 of the 26S
proteasome (PSMC5). The amount of antigen bound to the antibody is
detected. A decreased amount of antigen bound to the antibody
relative to an amount bound to a control sample prior to
vaccination predicts long term disease-free survival.
[0012] According to another embodiment a kit is provided for
detecting or monitoring pancreatic cancer disease or therapy. The
kit contains at least one antibody that specifically binds to an
antigen selected from the group consisting of: Transferrin receptor
(TFRC), regulatory subunit 12A of protein phosphatase 1 (PPP1R12A),
and regulatory subunit 8 of the 26S proteasome (PSMC5). The kit
further contains a detection means for detecting binding complexes
of the antibody and antigens in a test sample.
[0013] According to another embodiment a method predicts response
to a pancreatic cancer vaccine in a human A sample of the human
comprising antibodies is contacted with at least one protein
selected from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5). The amount of
antibody bound to the at least one protein is detected. An
increased amount of antibody bound to the at least one protein
relative to an amount bound to a control sample obtained prior to
vaccination predicts long term disease-free survival.
[0014] According to another embodiment a kit is provided for
detecting or monitoring pancreatic cancer disease or therapy. The
kit comprises at least one protein selected from the group
consisting of: Transferrin receptor (TFRC), regulatory subunit 12A
of protein phosphatase 1 (PPP1R12A), and regulatory subunit 8 of
the 26S proteasome (PSMC5). The kit further comprises a detection
means for detecting binding complexes of the protein with an
antibody in a test sample.
[0015] According to one embodiment of the invention a method tests
a body sample from a human A body sample is contacted with at least
one antibody that specifically binds to a protein selected from the
group consisting of: Transferrin receptor (TFRC), regulatory
subunit 12A of protein phosphatase 1 (PPP1R12A), and regulatory
subunit 8 of the 26S proteasome (PSMC5). The amount of antigen
bound to the antibody is detected or cellular localization of the
antigen is detected. An increased amount of antigen bound to the
antibody relative to an amount bound to a control sample or an
altered cellular localization is detected.
[0016] According to another embodiment a method tests a body sample
of a human with pancreatic cancer. A first body sample is contacted
with at least one antibody that specifically binds to a protein
selected from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5). The amount of
antigen bound to the antibody is detected. The amount detected in
the first body sample is compared to the amount detected in a
second body sample taken from the human at a second time.
[0017] According to another embodiment a method tests a body sample
of a human. A first and second body samples of the human are
contacted with at least one antibody that specifically binds to a
protein selected from the group consisting of: Transferrin receptor
(TFRC), regulatory subunit 12A of protein phosphatase 1 (PPP1R12A),
and regulatory subunit 8 of the 26S proteasome (PSMC5). The amount
of antigen bound to the antibody is detected in each sample. The
amount detected in each sample is compared to the other. The first
and second body samples of the human are collected at a first and
second time, wherein the first time is before and the second time
is after the human is vaccinated with a pancreatic cancer vaccine
composition.
[0018] According to another embodiment a method tests a body sample
of a human who has received a pancreatic cancer vaccine. A sample
of the human which comprises antibodies is contacted with at least
one protein selected from the group consisting of: Transferrin
receptor (TFRC), regulatory subunit 12A of protein phosphatase 1
(PPP1R12A), and regulatory subunit 8 of the 26S proteasome (PSMC5).
The amount of antibody bound to the at least one protein is
detected. The amount of antibody detected in the sample of the
human who has received a pancreatic cancer vaccine is compared to
the amount detected in a sample of the human before he or she
received the vaccine.
[0019] These and other embodiments which will be apparent to those
of skill in the art upon reading the specification provide the art
with methods and kits for better managing pancreatic cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. The vaccination schedule we used.
[0021] FIG. 2. Purification of human antibodies from serum of
vaccinated pancreatic cancer patient. Antibodies (A) were extracted
from the pre-vaccination and post-vaccination sera (S) using a
protein G column
[0022] FIG. 3. Outline of the SASI approach.
[0023] FIG. 4. Validation of mass-spectrometry derived SILAC data
using Western-blots. The fold-change detected by mass spectrometry
is shown to the right of each blot.
[0024] FIG. 5A-5C. Global changes in antibody recognition
post-vaccination compared to pre-vaccination of patients 9 (FIG.
5A), 27 (FIG. 5B), and 52 (FIG. 5C), respectively.
[0025] FIGS. 6A to 6C. Increased antibody response post-vaccination
correlates with improved survival. Solid arrow shows an increase
post-vaccination whereas a dotted arrow shows a decrease
post-vaccination in antibody response. FIG. 6A: PSMC5; FIG. 6B:
TFRC; FIG. 6C: PPP1R12A
[0026] FIG. 7A-7B. PSMC5 staining by immunohistochemistry (IHC). N:
Normal duct cells, C: Cancer cells
[0027] FIG. 8A-8C. PPP1R12A staining by IHC. N: Normal duct cells,
C: Cancer cells, I: Isotype control
[0028] FIG. 9. TFRC staining by IHC. N: Normal duct cells, C:
Cancer cells
[0029] FIG. 10. Tumor microarrays were scored for the percentage of
cells that express cytoplasmic PPP1R12A (FIG. 10A) or PSMC5 (FIG.
10B). The distribution of positive staining cells was classified
into <25%, 25%, 50%, 75%, or 100% of tumor cells present. The
frequency of each percentage is plotted in the above histograms.
Tumors with expression patterns significantly different than
pancreas are noted with an * indicated P<0.01.
[0030] FIG. 11. Correlation between patient tissue expression of
marker post-surgery and survival post-treatment with vaccine.
Higher PSMC5 expression correlates with improved survival
post-vaccination
[0031] FIG. 12. Correlation between patient tissue expression of
marker post-surgery and survival post-treatment with vaccine.
Higher PPP1R12A expression correlates with improved survival
post-vaccination.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The inventors have identified three different proteins that
are strongly overexpressed in pancreatic cancer whereas they are
either weakly or not expressed at all in pancreatic normal duct
cells. These proteins are also shown to be targets of a clinically
relevant antibody response induced with a vaccination. Thus, we
have identified candidate proteins as new biomarkers for screening,
and as new targets for therapeutic intervention. Samples which can
be tested include any body sample in which pancreatic cancer
proteins are expressed or shed. These include without limitation
blood, urine, stool, pancreatic tissue samples, metastatic tissue
samples, lymph, lymph nodes.
[0033] Any immunological detection technique can be used as is
convenient. These include without limitation ELISA,
immunoprecipitation, immunonblots, radioimmunoassays, protein
arrays, and antibody arrays.
[0034] Amounts of antigen can be detected by preparing and
comparing to a standard curve, for example. Amounts may also be
determined relatively, by comparing to a relevant control sample,
such as a sample of the same type obtained from the patient at a
different time, or obtained from a tissue known to be
non-cancerous, or a sample obtained from one or a population of
normal patients.
[0035] One, two, or three of the identified markers (Transferrin
receptor (TFRC), regulatory subunit 12A of protein phosphatase 1
(PPP1R12A), and regulatory subunit 8 of the 26S proteasome (PSMC5))
may be used as a panel. Additional markers including mesothelin,
annexin A2, and galectin 3 may be used. Other clinical parameters
may be used and combined to render a diagnosis or prognosis or
assessment of current or future response to a therapy.
[0036] The amount of protein (Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5)) in a sample can
be used as a measure of the disease. Alternatively, the amount of
antibody that a patient is producing to these proteins can be
determined as a measure of a specific and clinically relevant
immune response.
[0037] Any type of antibody can be used for measurement of protein
in a sample. L Antibodies which can be used to measure proteins may
be polyclonal, monoclonal, single chain, chimeric, or hybrid, for
example. Antibodies can be conjugated to other functionalities to
aid in the detection of the antibodies in an antigen-antibody
complex. Secondary antibodies or radiolabels can be used to detect
antibodies, for example.
[0038] Kits can be made with the antibodies or proteins
(Transferrin receptor (TFRC), regulatory subunit 12A of protein
phosphatase 1 (PPP1R12A), and regulatory subunit 8 of the 26S
proteasome (PSMC5)) useful in carrying out the various described
methods. The kits may have one, two, or three of the described
antibodies or proteins. Additional antibodies or proteins can also
be included for further refinements. Detection means such as
enzymes or radiolabels or secondary antibodies may also be
included. Buffers and other necessary reagents may be included.
Instructions may be included in the kits. The kits' components may
be in a divided or undivided container. A main container may
contain sub-containers.
[0039] For detection of antibodies in patient samples, preferably
the reagents used will be purified proteins (e.g., Transferrin
receptor (TFRC), regulatory subunit 12A of protein phosphatase 1
(PPP1R12A), and regulatory subunit 8 of the 26S proteasome
(PSMC5)), although they need not be. The proteins may be made in
recombinant cells or purified from a natural source. The proteins
or portions thereof may be made sythetically.
[0040] To overcome the drawbacks of current seroproteomic
technologies, we developed a novel functional proteomic approach
that utilizes high-throughput immunoprecipitation instead of
traditional immunoprecipitation which only utilizes monoclonal
antibodies. The Serum Antibodies based SILAC-Immunoprecipitation
(SASI) approach utilizes immunoprecipitation by serum antibodies,
which is then coupled to quantitative stable isotope labeling by
amino acids in cell culture (SILAC) to identify proteins that
elicit a changed antibody response. Despite the aggressive nature
of pancreatic cancer, seroproteomic approaches have not yet been
extensively applied to studying pancreatic ductal adenocarcinomas
(PDA) (9, 10). We utilized a vaccine tumor cell line as the
proteome to analyze immunized sera from pancreatic cancer patients
vaccinated with the GM-CSF vaccine (2). Our study focuses on
immunized sera from patients showing a mesothelin-specific
post-vaccination T cell response correlated with post-vaccination
prolonged disease free survival (2). Using mass spectrometric
analysis, the SASI approach comprehensively identified >45
proteins that elicited at least a 2-fold increase in antibody
response post-vaccination. We present the first large scale study
to identify and categorize proteins that are targeted by antibodies
in the human body. The high-throughput SASI approach identifies
both proteins that are of low abundance as well as in their native
state (conformational epitopes), and provides quantitative measure
of the antibody response, including all changes that would not be
apparent by traditional western blots.
[0041] This approach successfully identified a panel of 13
proteins. Three of these proteins were previously identified by us
using the more crude 2-D gel approach followed by mass spectrometry
analysis. This older approach identified 17 proteins, but only 2
were found to have biologic importance (Annexin A2 and Galectin-3).
As an example, Annexin A2, was found to be differentially expressed
by pancreatic cancers (6, 18). In addition, we showed that this
protein translocates from the cytosol to the transmembrane through
a tyrosine phosphorylation mechanism that confers metastatic
potential to pancreatic cancer cells (18). Finally, the antibodies
induced by this protein halted metastases. This data provides
evidence that antibody targets have biologic importance to cancer
(6, 18).
[0042] The SASI approach was able to identify proteins that were
not found by our prior analysis. Of these proteins, transferrin
receptor (TFRC), regulatory subunit 12A of protein phosphatase 1
(PPP1R12A) and regulatory subunit 8 of the 26S proteasome (PSMC5)
were shown to be pancreatic cancer associated antigens that are
recognized by antibodies in the sera of vaccinated patients who
have demonstrated favorable disease free survival. We further
analyzed PSMC5, TFRC and PPP1R12A for tissue expression in normal,
pre-malignant and pancreatic tumor specimens and found these
proteins increase in expression with tumor development. Overall,
our data demonstrates that the novel SASI approach can enable
identification of candidate proteins as new biomarkers for
screening, prediction tools of the vaccine's success, and novel
targets for therapeutic intervention.
[0043] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
Example 1
Materials and Methods
Patients, Serum and Tissue Samples
[0044] Patients were enrolled in a phase II study of an allogeneic
GM-CSF secreting whole cell pancreatic cancer vaccine in compliance
with the Johns Hopkins Medical Institution Institutional Review
Board (IRB)-approved J9988 protocol. Blood samples were collected
pre-vaccination, 14 days after 1.sup.St vaccination and 28 days
after each subsequent vaccination. Sera was collected by
centrifugation, aliquoted and stored at -80.degree. C. Pancreatic
tumor tissue samples were obtained from patients prior to
vaccination.
Antibody Purification
[0045] Antibodies were purified from pre- and post-3.sup.rd
vaccination sera using a protein G column (GE Healthcare,
Piscataway, N.J., USA) as per manufacturer's protocol.
Quantification of purified antibodies was done using NanoDrop
spectrophotometer (Thermo Fisher Scientific, Waltham, Mass.,
USA).
Sample Preparation
[0046] The human pancreatic cancer cell line, Panc 10.05 was grown
as previously described. For the SILAC procedure, Panc 10.05 cells
were grown in either light (.sup.12C.sub.6-Lys, .sup.12C.sub.6-Arg)
or heavy (.sup.13C.sub.6-Lys, .sup.13C.sub.6-Arg) RPMI1640 media
containing 10% fetal bovine serum and antibiotics in a humidified
incubator at 37.degree. C. with 5% CO2. Stable isotope containing
amino acids, .sup.13C.sub.6-arginine and .sup.13C.sub.6-lysine,
were purchased from Cambridge Isotope Laboratories (Andover, Mass.,
USA). Arginine and lysine-free RPMI1640 media, fetal bovine serum
(FBS) and antibiotics (penicillin and streptomycin) were purchased
from Invitrogen (Carlsbad, Calif., USA). The light and heavy cells
were washed with phosphate buffered saline and were harvested using
M-PER buffer (Thermo Fisher Scientific) in the presence of cocktail
protease inhibitors (Thermo Fisher Scientific). Protein was
quantified using the Lowry method.
Immunoprecipitation for Mass Spectrometry
[0047] Equal amounts of light and heavy cell lysates were incubated
overnight with purified pre- and post-vaccination antibodies,
respectively. On the following day, the two sets of lysate:antibody
mixture were each incubated with protein G beads (Invitrogen) and
washed using M-PER buffer. The immunoprecipitates were eluted by
boiling in NuPAGE.RTM. LDS sample buffer (Invitrogen). The light
and heavy eluted lysates were mixed 1:1. The mixture was
concentrated and resolved by 10% SDS-PAGE. The gel was stained
using a coomassie dye staining kit (Invitrogen).
Liquid Chromatography Tandem Mass Spectrometry and Data
Analysis
[0048] The stained gel was excised into 18 bands and each band was
destained in 40 mM ammonium bicarbonate/40% acetonitrile solution.
The samples were reduced with 5 mM dithiothreitol/20% acetonitrile
solution, alkylated with 100 mM iodoacetamide and digested with
trypsin. Sequencing grade modified porcine trypsin was purchased
from Promega (Madison, Wis., USA). The peptides were extracted,
desalted, dried and reconstituted in 0.1% formic acid. The peptides
were analyzed by reversed phase liquid chromatography tandem mass
spectrometry (LC-MS/MS). Briefly, the peptides in solution were
separated using an on-line reverse phase nano high-performance
liquid chromatography using a C18 column and the Eksigent Nano 2D
high-performance liquid chromatography (HPLC) pumping system
(Eksigent). The nano-HPLC is interfaced directly with the
LTQ-Orbitrap-XL (Thermo Electron) allowing for introduction of the
separated peptide solution into the mass spectrometer for tandem
mass spectrometric analysis. Isolated proteins from each band were
identified using an automated database search algorithm, MASCOT,
within the Proteome Discoverer software platform (Thermo Electron)
and processed by MaxQuant. Our data was searched at a mass
tolerance of 10 ppm for MS species and 1 Da for MS/MS with
carbamidomethylation of cysteine as a fixed modification and
oxidation of methionine as a variable modification. The proteolytic
enzyme indicated was trypsin and we allowed up to two missed
cleavage events.
Mass-Spectrometry Data Validation
[0049] Panc 10.05 cells grown in light RPMI1640 media were lysed in
M-PER buffer supplemented with protease inhibitor cocktail. The
lysate was immunoprecipitated with either the pre- or
post-vaccination purified antibodies using protein G beads. The
immunoprecipitates were eluted by boiling in NUPAGE LDS sample
buffer and resolved on a NuPAGE 4-12% Bis-Tris gel (Invitrogen).
Proteins in the gel were transferred onto nitrocellulose membrane
using a semi-dry apparatus (Invitrogen). The membrane was blocked
in 5% bovine serum albumin (BSA, Invitrogen) in 0.1% Tween 20-PBS
(PBS-T) buffer for 1 hour at room temperature and probed with the
relevant primary antibody overnight at 4.degree. C. Antibodies
against galectin-3 (sc-19283), E3 ubiquitin protein ligase
(sc-9561), mesencephalic astrocyte-derived neurotrophic factor
(sc-34560), epidermal growth factor receptor kinase substrate
8-like protein 2 (sc-100722), calpain-1 (sc-81171) were purchased
from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). The
membrane was incubated with the corresponding peroxidase conjugated
secondary antibodies (A8419, Sigma) and then ECL Western Blotting
Detection Reagents (GE Healthcare) was used for 1 minute at room
temperature for developing.
Western Blot for Detecting Antibody Responses in Patients
[0050] Purified recombinant proteins, PSMC5 (TP301251), PPP1R12A
(TP323540) and TFRC (TP300980) expressed in human HEK293 cells were
purchased from Origene (Rockville, Md., USA). One microgram of
purified protein was denatured by boiling in SDS-PAGE sample buffer
and resolved on a NuPAGE 4-12% Bis-Tris gel (Invitrogen). Proteins
in the gel were transferred onto nitrocellulose membrane using a
semi-dry apparatus (Invitrogen). The membrane was cut into
individual lanes and was blocked in 5% bovine serum albumin (BSA,
Invitrogen) in 0.1% Tween 20-PBS (PBS-T) buffer for 1 hour at room
temperature. After blocking, each individual lane was probed with
either pre-vaccination or post-vaccination serum of the various
patients at 1:1000 dilution. A lane was used as a control and
probed with mouse anti-DDK antibody (TA150030, Origene) overnight
at 4.degree. C. The membrane was incubated with the peroxidase
conjugated secondary antibodies; goat anti-human IgG antibody
(A8419, Sigma) for patient serum lanes or rabbit anti-mouse IgG
(A9044, Sigma) for control lane. ECL Western Blotting Detection
Reagents (GE Healthcare) was used for 1 minute at room temperature
for developing.
Immunohistochemistry
[0051] Immunohistochemistry was performed on formalin-fixed
paraffin-embedded embedded 5 .mu.m thick sections of pancreatic
tumor tissue samples for the available 46 of the 60 patients
enrolled in the study was obtained from the Department of Pathology
at Johns Hopkins Medical Institutions. Standard IHC protocol was
applied using Bond-Leica autostainer (Leica Microsystems,
Bannockburn, Ill.). Briefly, tissue sections were baked for 20
minutes at 65.degree. C. followed by deparaffinization, antigen
retrieval and primary antibody incubation at optimal conditions.
Bond polymer detection system was applied to develop the reaction.
3',3' diaminobenzidin (DAB) chromogen-substrate was utilized for
visualization of reaction as per manufacturer's instructions (Leica
Microsystems, Bannockburn, Ill.). All sections were then
counterstained with hematoxylin, dehydrated and cover slipped.
Antibody information is detailed in the table below.
TABLE-US-00001 Clone/animal Name species Dilution Source Anti-PSMC5
Rabbit 1:150 HPA017871, Sigma Anti-PPP1R12A Rabbit 1:500 HPA039443,
Sigma Mouse anti-Human Mouse 1:2000 136800, Invitrogen Transferrin
Receptor (Clone:H68.4)
Example 2
Design and Validation of Quantitative Proteomic Approach
[0052] 60 pancreatic cancer patients, who had their pancreas
surgically removed, were involved in the study (FIG. 1) (2). The
patients received their first vaccination 2 months after surgery.
One month after the first vaccination, the patients underwent a
6-month course of chemoradiation. The second, third and fourth
vaccines were each administered at sequential one-month intervals
from the time of chemotherapy completion. The fifth, and final,
vaccination was received 6 months after the fourth vaccination.
Serum samples were obtained pre- and post-vaccination for all five
vaccinations (2). The 60 vaccinated patients were divided into 3
groups (A, B and C) based on length of disease free survival (DFS)
(2). Group A was composed of 12 patients who received all of the
scheduled vaccinations and demonstrated a DFS>3 years (prolonged
DFS as well as overall survival). The clinical time point cutoff
was determined to be 3 years because patients characterized with a
3-year DFS were less likely to have cancer recurrence. The 21
patients in Group B received at least 3 scheduled vaccinations, but
had a DFS<3 years. The 27 patients in Group C relapsed before
receiving their second scheduled vaccination.
Example 3
Identification of Proteins by the SASI Approach
[0053] To identify the proteins in the post-vaccination sera of
patients in Group A (DFS>3 years), we used the immunized sera
from three patients (patients 9, 27 and 52) who demonstrated other
evidence of post-vaccination immune responses. We identified a
total of 976 proteins for patient 9, 811 proteins for patient 27
and 727 proteins for patient 52 (FIG. 5). A broad range of
post-vaccination antibody response was observed; from a 16 fold
change increase post-vaccination to a 10 fold change decrease. The
majority of the proteins, as expected, had no change in response
post-vaccination. We identified 51 proteins for patient 9, 47
proteins for patient 27 and 54 proteins for patient 52 that had a 2
fold change in response. Through the SASI approach, we present the
first large scale study to identify and categorize proteins that
are targeted by antibodies in the human body.
[0054] Pre-vaccination and post-4th vaccination sera from 3
patients, 3.009, 3.027 and 3.052 from Group A was used in the
development of the SASI approach.
[0055] The SASI approach consists of 4 key components: (a) Antibody
purification, (b) SILAC labeling, (c) Immunoprecipitation, and (d)
Downstream Analysis.
Example 4
(a) Purification of IgGs from Serum
[0056] Using a Protein G column, we isolated immunoglobulin G (IgG)
from the serum (FIG. 2). After washing the column, the IgGs are
eluted with a low pH buffer. The eluted IgGs are collected and the
pH is neutralized. Thus, functional pancreatic cancer specific IgGs
were isolated from the immune sera (FIG. 2).
[0057] Table 1 shows a partial list of proteins determined to be
biologically relevant in our study. Fold change is defined as the
ratio of post-vaccination to pre-vaccination antibody response.
TABLE-US-00002 Average Gene fold Protein symbol change Protein
function Galectin 3 LGALS3 11.0 Regulator of T-cell functions 26S
proteasome, regulatory PSMC5 4.6 Confers ATP dependency and subunit
8 substrate specificity to the 26S complex MRP-1 CD9 4.1 Cell
adhesion and motility HDGF-2 HDGFRP2 3.2 Function unknown
Centrosomal protein of 170 kDa CEPl70 3.1 Microtubule organization
Prohibitin-2 PHB2 2.4 Mediator of transcriptional repression via
recruitment of histone deacetylases Phosphatidylinositol synthase
CDIPT 2.2 Phosphatidylinositol biosynthesis Retinol dehydrogenase
11 RDHll 2.0 Short-chain aldehyde metabolism Aspartate
aminotransferase GOT2 1.9 Amino acid metabolism Protein phosphatase
1, PPPlRl2A 1.7 Regulator of protein phosphatase regulatory subunit
12A 1C and mediates binding to myosin Transferrin receptor TFRC 1.7
Iron uptake via endocytosis of transferrin Pyruvate kinase PKM2 1.7
Glycolytic enzyme generating ATP Annexin A2 ANXA2 1.4 Cell
adhesion
[0058] Of these proteins, galectin-3, annexin A2 and pyruvate
kinase were identified previously by a 2-D proteomic approach (17).
Galectin-3 and annexin A2 are currently under investigation for
their role in pancreatic ductal adenocarcinomas pathogenesis and
progression (18). In our studies to discover biologically relevant
proteins in pancreatic cancer, we have identified the same proteins
through two different proteomic methods. Ongoing research has
already shown these proteins are promising targets involved in
signaling pathways important to the biology of pancreatic cancer
progression and metastasis (17, 18). Therefore, we essentially have
ascertained that our approach determines biologically relevant
proteins. Overall, the SASI approach comprehensively identified
more than 2500 proteins.
Example 5
(b) SILAC Labeling
[0059] The Panc10.05 cell line was utilized in SILAC labeling
experiments. Panc 10.05 is one of the two vaccine tumor cell lines
(the proteome), and its use for SILAC labeling would ensure the
antibody response is specific to human proteins and would contain
the correct post-translational modifications, including
glycosylation. Panc 10.05 was grown in both a heavy version form
and a light version form. Stable isotope labeling with amino acids
in cell culture (SILAC) is a quantitative proteomics method that
involves in vivo labeling of proteins followed by mass
spectrometric analysis. In this method, Panc 10.05 cells
incorporate nonradioactive heavy isotopes of lysines
(.sup.13C.sub.6-Lys) and arginines (.sup.13C.sub.6-Arg) into its
proteome instead of the "light" versions (.sup.12C.sub.6-Lys and
.sup.12C.sub.6-Arg) present in the commercially available media.
Panc 10.05 cells were grown in either "heavy" media containing
heavy amino acids or in "light" media containing normal amino
acids. After 9 passages, cells grown in heavy and light media were
lysed to give heavy and light lysates, respectively.
Example 6
(c) Immunoprecipitation
[0060] The light and heavy lysates were subjected to overnight
immunoprecipitation, using purified pre- and post-vaccination
antibodies, respectively (FIG. 3). The following day, Protein G
beads were added to capture the IgGs, which were bound to various
proteins from the lysates. Unbound proteins were removed from the
beads by a series of washing steps. Boiling the beads in sample
buffer allowed elution of the immunoprecipitated proteins and IgGs.
This process gave us two sets of samples. One sample consists the
eluted heavy proteins with the post-vaccination IgGs, whereas the
other sample is the eluted light proteins with the pre-vaccination
IgGs. These samples were mixed in a 1:1 ratio. By using equal
amounts of heavy and light protein as well as an equal amount of
antibodies for immunoprecipitation, we are able to infer that the
changes reflected in the heavy to light ratio equates to the
changes in the antibody constitution for each antigen. If a protein
showed increased antibody response post-vaccination, we would see
greater heavy protein to light protein ratio for that protein. If a
protein showed decreased antibody response post-vaccination, we
would see a lower heavy protein to light protein ratio for the
protein.
Example 7
(d) Downstream Analysis
[0061] The 1:1 heavy and light mixed samples were separated by gel
electrophoresis and stained with coomassie dye. 18 protein bands
were excised and digested with trypsin. The extracted peptides were
analyzed by LTQ-Orbitrap mass spectrometer. The proteins were
identified and quantified using Mascot and MaxQuant,
respectively.
[0062] We wanted to further validate the SILAC data derived from
mass-spectrometry analysis. We used pre-vaccination and
post-vaccination antibodies of patient 3.052 for
immunoprecipitation with light cell lysates in both cases (FIG. 4).
Our SILAC data using patient 52 had revealed that galectin-3, E3
ubiquitin-protein ligase UBRS and mesencephalic astrocyte-derived
neurotrophic factor had an increased antibody response
post-vaccination by 15.3, 4.0 and 3.9 fold respectively.
Contrastingly, this patient also showed a decreased antibody
response post-vaccination for calpain-1 and epidermal growth factor
receptor kinase substrate 8-like protein 2 by 2.5 and 10.0 fold
respectively. To validate our SILAC data, we conducted Western
blots for these proteins. The immunoprecipitated proteins were
separated by SDS-PAGE followed by western blot using antibodies
against the following proteins: galectin-3, E3 ubiquitin-protein
ligase UBRS, mesencephalic astrocyte-derived neurotrophic factor,
calpain-1 and epidermal growth factor receptor kinase substrate
8-like protein 2. We saw that there was a dramatic increase in
galectin-3 protein level in the post-vaccination blot, whereas E3
ubiquitin-protein ligase UBRS and mesencephalic astrocyte-derived
neurotrophic factor showed a modest increase in detection
post-vaccination. Similarly, calpain-1 showed a dramatic decrease
in detection whereas epidermal growth factor receptor kinase
substrate 8-like protein 2 showed a modest decrease in the blot
containing the post-vaccination immunoprecipitated proteins. The
western blot analysis, though not quantitative, mirrored the trends
we observed from our quantitative mass-spectrometry derived SILAC
ratios.
Example 8
PSMC5, PPP1R12A and TFRC are Antibody Targets of Immune Response
Against PDA
[0063] Our interest focused on proteins that had greater than 1.5
fold change response. Previous proteomic approaches had identified
annexin A2 as biologically relevant. In the SASI approach, annexin
A2 revealed a 1.4 fold change in response post vaccination. From
there, we set an average 1.5 fold change post-vaccination with at
least one of the 3 sera tested showing a 2 fold change as our
benchmark for a biologically relevant response. However, some of
these proteins had an increased post-vaccination response in 2 or
all of the sera tested by the SASI approach. We further decided to
test if there was a correlation between the increased
post-vaccination antibody response and disease free status.
[0064] Using purified recombinant proteins, we examined the
post-vaccination response in patients with favorable DFS. For this
experiment, we used the serum before the first vaccination as the
pre-vaccination serum, while the serum after the 3.sup.rd
vaccination was designated the post-vaccination serum. PSMC5,
PPP1R12A and TFRC showed elevated antibody titers in patients with
favorable DFS (FIG. 6). PSMC5 elicited an increased antibody
response in 8 of 12 patients. TFRC elicited an increased antibody
response in 8 of 12 patients. PPP1R12A elicited an increased
antibody response in 9 of 12 patients. Interestingly, these 3
proteins also demonstrated an increased antibody response in each
of the 3 patients who were tested in the SASI approach. Although,
we cannot correlate the quantitative SASI approach data with the
qualitative Western blot results, the overall trends were similar.
This observation further provided validation of our SASI
results.
[0065] We then wanted to compare the patients with DFS>3 years
to those with DFS<3 years (FIG. 6). For this comparison, we
selected 12 out of the 21 patients in the group with DFS<3
years. The selection was based on the level of vaccinations
completed. Each of these 12 patients had received at least 3
vaccinations, allowing us to compare the post-vaccination serum to
the pre-vaccination serum. Western blot analysis showed an increase
in antibody response post-vaccination to recombinant both PSMC5 and
TFRC in only 2 of the 12 patients that showed DFS<3 years
(compared to 8 of the 12 patients with DFS>3 years). Western
blot analysis showed an increase in antibody response
post-vaccination to recombinant PPP1R12A in 5 of the 12 patients
that showed DFS<3 years (compared to 9 of the 12 patients with
DFS>3 years). Interestingly, 4 of the 12 patients with DFS<3
years showed a decreased antibody response to PPP1R12A
post-vaccination, whereas only 1 of the 12 patients with DFS>3
years showed a decreased response post-vaccination. Similarly, both
PSMC5 and TFRC demonstrated a decreased antibody response in 2 out
of the 12 patients DFS<3 years (compared to only 1 patient with
DFS>3 years showing a decreased response). These results imply
that the vaccine-induced antibody response to PSMC5, PPP1R12A and
TFRC have strong correlations to clinical benefit. A decreased
response post-vaccination for these proteins is comparable to a
shorter DFS. Data suggests that these proteins are antigenic
targets of vaccine-induced humoral responses in pancreatic cancer
patients. Most significantly, the antibody responses detected
against these proteins in patients with >3 years disease-free
survival suggests an anti-tumor potential of targeting these
proteins.
Example 9
Increased PSMC5, PPP1R12A, TFRC Tissue Expression Correlates with
PDA Development
[0066] Next, we wanted to examine the cause behind the antibody
response. There are 3 main reasons for how these self proteins
could induce an altered antibody response in the patients:
difference in expression levels, difference in localization, or
post-translational modifications. The reports on levels of PSMC5
and PPP1R12A in pancreatic cancer or other cancers are very
preliminary with no extensive information.
[0067] First, we analyzed the expression levels and localization of
PSMC5 (FIG. 7) in normal as well as cancer tissues by
immunohistochemistry (IHC). The resected tumors for this study came
from 46 of the 60 patients who were treated in our Phase II study
and were available for staining. With immunohistochemistry, we
found that normal pancreatic epithelial ductal cells display weak
cytoplasmic staining for PSMC5. However, ductal carcinoma cells
display strong cytoplasmic staining. PSMC5 is overexpressed in
pancreatic cancer compared to normal tissue. Specifically, 85% of
pancreatic tumor cells have increased expression of PSMC5. PSMC5 is
a part of the 26S proteasome, which is present in all cells;
however, normal cell level of PSMC5 is very low. Normal duct tissue
stained very weakly for PSMC5 (only 15%) in the cytoplasm, with
almost no nuclear staining observed. Additionally, we observed that
PSMC5 localizes to the nucleus in cancer cells, which is shown by
the intense staining in the nucleus of cancer cells. The
cytoplasmic and nuclear expression increases with the progression
from pancreatic intraepithelial neoplasia (PanINs) to PDA. Our data
shows that 50% of the pancreatic tumor cells have increased nuclear
staining of PSMC5. Contrastingly, only 5% of normal duct cells,
acinar cells, blood vessels show nuclear staining of PSMC5. The
isotype controls demonstrated complete negative staining in the 10
slides examined. This data provides evidence that PSMC5 is
overexpressed in PDA and furthermore, the nuclear expression of
PSCM5 increases from normal to cancer tissue. The cancer-specific
increase in PSMC5 provides support to the idea that the protein is
a potential immunologic target.
Example 10
Abnormal Subcellular Localization
[0068] PPP1R12A or MYPT1 is part of the Rho Kinase pathway
component. We analyzed the expression levels as well as
localization of PPP1R12A (FIG. 8) in normal and cancer tissues by
immunohistochemistry (IHC). Through immunohistochemistry, we found
that normal pancreatic epithelial ductal cells display weak
cytoplasmic staining Contrastingly, the ductal carcinoma cells
displayed strong cytoplasmic staining. PPP1R12A was found to be
overexpressed in pancreatic cancer compared to normal tissue.
Specifically, 82% of the cancer cells have increased expression of
PPP1R12A. Only 2% of normal duct cells stained very weakly for
PPP1R12A, with no membrane staining seen in these cells. We also
observed PPP1R12A to be localized to the membrane and stained
strongly and intensely in the cancer cells. The membrane
localization was only observed in PDA cells. We showed that about
20% of PDA cells have increased membrane staining of PPP1R12A. On
the contrary, the normal duct cells, acinar cells, blood vessels
showed no membrane staining of PPP1R12A. This data provides support
that PPP1R12A is overexpressed in PDA and that membrane expression
of PPP1R12A is a unique feature of cancer. Thus, PPP1R12A is a
potential immunologic target. TFRC staining were similar to those
of PPP1R12A. 74% of PDA cells stained strongly for TFRC whereas
only 1% of the normal duct cells showed very weak staining (FIG.
9). We also observed some membrane TFRC staining in only the PDA
cells. Similar to PSMC5, both PPP1R12A and TFRC showed increased
staining as the normal duct cells progressed to the PanIN stages to
the full blown PDA disease.
[0069] Thus, the SASI approach has been able to successfully
identify biologically relevant proteins, all 3 of which could be
extensively validated. We saw that each of the 3 markers, PSMC5,
PPP1R12A and TFRC, increases in expression when we compare normal
to cancer cells. Furthermore, there is evidence of mislocalization
of these proteins in cancer. In cancer, PSMC5 is found abnormally
in the nucleus, and PPP1R12A and TFRC are also found on the cell
membrane. Both overexpression and mislocalization in the cancer
cells help explain why an antibody response was targeted towards
these proteins. PSMC5, PPP1R12A, and TFRC have great potential,
both as immunologic targets as well as diagnostic biomarkers. The
heterogeneous nature of both the cancer as well as the antibody
responses illustrates a need for a biomarker panel in order not
only to cover more patients but also retain high specificity.
Example 11
Proteins Eliciting Antibody Responses in Vaccinated Pancreatic
Cancer Patients are Expressed by a Range of Adenocarcinomas
[0070] Background: Developing targets that identify patients for
appropriate therapies is a key goal of cancer research. A high
throughput proteomic screen identified two proteins, PPP1R12A and
PSMC5, which were found to enhance antibody responses in pancreatic
cancer patients participating in a phase II trial of an allogeneic,
GMCSF-secreting vaccine. Responses to these proteins correlated
with increased disease free survival in trial patients. We sought
to define PPP1R12A and PSMC5 expression in pancreatic and other
common solid malignancies.
[0071] Design: Tissue microarrays (TMA) of pancreatic, breast,
biliary, lung, liver, and colon carcinomas were stained for
PPP1R12A and PSCM5. The intensity of tumor cell expression was
scored for each protein from no specific (0); greater than
background (1) or strong (2) staining. The percentage of tumor
cells expressing each protein and the cellular compartment
(cytoplasmic, membranous) was recorded. Positive staining=a score
of 1-2 in >25% of cells.
[0072] Results: Expression of PPP1R12A was seen in pancreatic
(97%), biliary (58%), colon (46%) ER+ breast (37%) and HER-2+
breast (17%) adenocarcinomas. Minimal expression was seen in lung
(8%) and basal breast (4%) adenocarcinomas. A higher percentage of
pancreatic cancer expressed PPP1R12A compared to other tumors
(p<0.0001). Significantly more ER+ breast carcinomas expressed
PPP1R12A than HER-2+ or basal type (p<0.001). Membranous
PPP1R12A staining was observed only in pancreas (45%) and colon
(30%) cancers. PSMC5 expression was present in all tumors types:
pancreatic (57%), ER+ breast (97%), HER-2+ breast (82%), basal
breast (86%), liver (69%), biliary (24%), colon (58%), and lung
(74%). Breast tumors showed particularly high expression of PSMC5.
Additionally, HER-2+ tumors consistently showed expression by 100%
of cells within an individual TMA, which was significantly more
than either ER+ or basal type breast tumors (p<0.01).
[0073] Conclusions: Our study confirms strong expression of both
PPP1R12A and PMSC5 in pancreatic cancer. In addition, we identify a
range of adenocarcinomas with expression of PPP1R12A and/or PMSC5
including breast, biliary, lung, colon, and liver. This identifies
tumor types that might respond to GVAX immunotherapy and provides
rationale to direct therapy based on these proteins expression
patterns. Membranous expression of PPP121RA in pancreatic and colon
cancers is particularly attractive for therapeutic targeting.
Additional studies are needed to evaluate the relationship between
tumor evolution in these adenocarcinomas and the expression of
PPP1R12A and PMSC5.
CLAUSES
[0074] 1. A method for detecting pancreatic cancer in a body sample
from a human, comprising: contacting the body sample with at least
one antibody that specifically binds to a protein selected from the
group consisting of: Transferrin receptor (TFRC), regulatory
subunit 12A of protein phosphatase 1(PPP1R12A), and regulatory
subunit 8 of the 6S proteasome (PSMC5); [0075] detecting amount of
antigen bound to the antibody or cellular localization of the
antigen, wherein an increased amount of antigen bound to the
antibody relative to an amount bound to a control sample or an
altered cellular localization in dictates the presence of a
pancreatic cancer. [0076] 2. The method of clause 1 wherein the
body sample is a tissue sample. [0077] 3. The method of clause 1
wherein the body sample is a blood or urine sample. [0078] 4. The
method of clause 1 wherein the step of detecting is performed using
immunohistochemistry. [0079] 5. The method of clause 1 wherein the
step of detecting is performed using an ELISA. [0080] 6. The method
of clause 1 wherein at least two of said antibodies are contacted
and detected. [0081] 7. The method of clause 1 wherein at least
three of said antibodies are contacted and detected. [0082] 8. The
method of clause 1 wherein the steps of contacting and detecting
are further performed using at least one antibody that specifically
binds to an antigen selected from the group consisting of:
mesothelin, annexin A2, and galectin 3. [0083] 9. A method for
monitoring progression of pancreatic cancer in a body sample from a
human, comprising: contacting the body sample with at least one
antibody that specifically binds to a protein selected from the
group consisting of: Transferrin receptor (TFRC), regulatory
subunit 12A of protein phosphatase 1 (PPP1R12A), and regulatory
subunit 8 of the 26S proteasome (PSMC5); detecting amount of
antigen bound to the antibody, wherein an increased amount of
antigen bound to the antibody relative to an amount bound to a
sample taken at a prior time indicates progression of the
pancreatic cancer and a decreased amount of antigen bound to the
antibody relative to amount bound to a sample taken at a prior time
indicates responsiveness to an anti-cancer treatment. [0084] 10.
The method of clause 9 wherein the body sample is a tissue sample.
[0085] 11. The method of clause 9 wherein the body sample is a
blood or urine sample. [0086] 12. The method of clause 9 wherein
the step of detecting is performed using immunohistochemistry.
[0087] 13. The method of clause 9 wherein the step of detecting is
performed using an ELISA. [0088] 14. The method of clause 9 wherein
at least two of said antibodies are contacted and detected. [0089]
15. The method of clause 9 wherein at least three of said
antibodies are contacted and detected. [0090] 16. The method of
clause 9 wherein the steps of contacting and detecting are further
performed using at least one antibody that specifically binds to an
antigen selected from the group consisting of: mesothelin, annexin
A2, and galectin 3. [0091] 17. A method to predict response to a
pancreatic cancer vaccine in a human, comprising: contacting a body
sample of the human with at least one antibody that specifically
binds to a protein selected from the group consisting of:
Transferrin receptor (TFRC), regulatory subunit 12A of protein
phosphatase 1 (PPP1R12A), and regulatory subunit 8 of the 26S
proteasome (PSMC5); detecting amount of antigen bound to the
antibody, wherein a decreased amount of antigen bound to the
antibody relative to an amount bound to a control sample prior to
vaccination predicts long term disease-free survival. [0092] 18.
The method of clause 17 wherein the body sample is a tissue sample.
[0093] 19. The method of clause 17 wherein the body sample is a
blood or urine sample. [0094] 20. The method of clause 17 wherein
the step of detecting is performed using immunohistochemistry.
[0095] 21. The method of clause 17 wherein the step of detecting is
performed using an ELISA. [0096] 22. The method of clause 17
wherein at least two of said antibodies are contacted and detected.
[0097] 23. The method of clause 17 wherein at least three of said
antibodies are contacted and detected. [0098] 24. The method of
clause 17 wherein the steps of contacting and detecting are further
performed using at least one antibody that specifically binds to an
antigen selected from the group consisting of: mesothelin, annexin
A2, and galectin 3. [0099] 25. A kit for detecting or monitoring
pancreatic cancer disease or therapy, comprising: at least one
antibody that specifically binds to an antigen selected from the
group consisting of: Transferrin receptor (TFRC), regulatory
subunit 12A of protein phosphatase 1 (PPP1R12A), and regulatory
subunit 8 of the 26S proteasome (PSMC5); a detection means for
detecting binding complexes of the antibody and antigens in a test
sample. [0100] 26. The kit of clause 25 further comprising a solid
support for binding antibodies, antigens, or antibody-antigen
complexes. [0101] 27. The kit of clause 25 wherein the detection
means comprises an enzyme that can produce a colored reaction
product. [0102] 28. The kit of clause 25 wherein the detection
means comprises a second antibody that binds to said at least one
antibody. [0103] 29. A method to predict response to a pancreatic
cancer vaccine in a human, comprising: contacting a sample of the
human comprising antibodies with at least one protein selected from
the group consisting of: Transferrin receptor (TFRC), regulatory
subunit 12A of protein phosphatase 1 (PPP1R12A), and regulatory
subunit 8 of the 26S proteasome (PSMC5); detecting amount of
antibody bound to the at least one protein, wherein an increased
amount of antibody bound to the at least one protein relative to an
amount bound to a control sample prior to vaccination predicts long
term disease-free survival. [0104] 30. The method of clause 29
wherein the body sample is a tissue sample. [0105] 31. The method
of clause 29 wherein the body sample is a blood or urine sample.
[0106] 32. The method of clause 29 wherein the step of detecting is
performed using immunohistochemistry. [0107] 33. The method of
clause 29 wherein the step of detecting is performed using an
ELISA. [0108] 34. The method of clause 29 wherein at least two of
said antibodies are contacted and detected. [0109] 35. The method
of clause 29 wherein at least three of said antibodies are
contacted and detected. [0110] 36. The method of clause 29 wherein
the steps of contacting and detecting are further performed using
at least one antibody that specifically binds to an antigen
selected from the group consisting of: mesothelin, annexin A2, and
galectin 3. [0111] 37. A kit for detecting or monitoring pancreatic
cancer disease or therapy, comprising: at least one protein
selected from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5); a detection
means for detecting binding complexes of the protein with an
antibody in a test sample. [0112] 38. The kit of clause 37 further
comprising a solid support for binding antibodies, antigens, or
antibody-antigen complexes. [0113] 39. The kit of clause 37 wherein
the detection means comprises an enzyme that can produce a colored
reaction product. [0114] 40. The kit of clause 37 wherein the
detection means comprises a second antibody that binds to said at
least one antibody. [0115] 41. The method of clause 2, 10, 18, or
30 wherein the tissue sample is selected from the group consisting
of pancreas, breast, biliary, lung, colon, and liver. [0116] 42. A
method of treating a human with a tumor selected from the group
consisting of pancreas, breast, biliary, lung, colon, and liver,
comprising: administering a pancreatic cancer vaccine composition
to the human whereby an immune response to PPP1R12A and/or PMSC5 is
raised in the human. [0117] 43. The method of clause 42 wherein
prior to said step of administering a sample of the tumor is tested
and expression of PPP1R12A and/or PMSC5 on cell membranes of the
tumor is detected. [0118] 44. The method of clause 42 wherein the
vaccine is GVAX. [0119] 45. A method for testing a tumor sample,
comprising: testing a sample of a tumor and detecting expression of
PPP1R12A and/or PMSC5 on cell membranes of the tumor. [0120] 46.
The kit of clause 25 wherein the at least one antibody comprises at
least 30% of the antibodies in the kit. [0121] 47. The kit of
clause 25 wherein the at least one antibody comprises at least 50%
of the antibodies in the kit. [0122] 48. The kit of clause 25
wherein the at least one antibody comprises at least 70% of the
antibodies in the kit. [0123] 49. The kit of clause 25 wherein the
at least one antibody comprises at least 90% of the antibodies in
the kit. [0124] 50. The kit of clause 37 wherein the at least one
protein comprises at least 30% of the proteins in the kit. [0125]
51. The kit of clause 37 wherein the at least one protein comprises
at least 50% of the proteins in the kit. [0126] 52. The kit of
clause 37 wherein the at least one protein comprises at least 70%
of the proteins in the kit. [0127] 53. The kit of clause 37 wherein
the at least one protein comprises at least 90% of the proteins in
the kit. [0128] 54. A method of testing a body sample from a human,
comprising: contacting the body sample with at least one antibody
that specifically binds to a protein selected from the group
consisting of: transferrin receptor (TFRC), regulatory subunit 12A
of protein phosphatase 1 (PPP1R12A), and regulatory subunit 8 of
the 26S proteasome (PSMC5); detecting either amount of antigen
bound to the antibody or cellular localization of the antigen.
[0129] 55. The method of clause 54 wherein the body sample is a
tissue sample. [0130] 56. The method of clause 54 wherein the body
sample is a blood or urine sample. [0131] 57. The method of clause
54 wherein the step of detecting is performed using
immunohistochemistry. [0132] 58. The method of clause 54 wherein
the step of detecting is performed using an ELISA. [0133] 59. The
method of clause 54 wherein at least two of said antibodies are
contacted and detected. [0134] 60. The method of clause 54 wherein
at least three of said antibodies are contacted and detected.
[0135] 61. The method of clause 54 wherein the steps of contacting
and detecting further comprise using at least one antibody that
specifically binds to an antigen selected from the group consisting
of: mesothelin, annexin A2, and galectin 3. [0136] 62. A method of
testing a human with a pancreatic cancer, comprising: contacting a
first and a second body sample of the human collected at two
distinct times with at least one antibody that specifically binds
to a protein selected from the group consisting of: Transferrin
receptor (TFRC), regulatory subunit 12A of protein phosphatase 1
(PPP1R12A), and regulatory subunit 8 of the 26S proteasome (PSMC5);
detecting amount of antigen bound to the antibody; and comparing
the amount of antigen bound to the first and second body samples.
[0137] 63. The method of clause 62 wherein the body sample is a
tissue sample. [0138] 64. The method of clause 62 wherein the body
sample is a blood or urine sample. [0139] 65. The method of clause
62 wherein the step of detecting is performed using
immunohistochemistry. [0140] 66. The method of clause 62 wherein
the step of detecting is performed using an ELISA. [0141] 67. The
method of clause 62 wherein at least two of said antibodies are
contacted and detected. [0142] 68. The method of clause 62 wherein
at least three of said antibodies are contacted and detected.
[0143] 69. The method of clause 62 wherein the steps of contacting
and detecting further comprise using at least one antibody that
specifically binds to an antigen selected from the group consisting
of: mesothelin, annexin A2, and galectin 3. [0144] 70. A method to
test a body sample of a human, comprising: contacting a first and a
second body sample of the human with at least one antibody, wherein
the body samples are collected at a first and second time, wherein
the first time is before and the second time is after the human is
vaccinated with a pancreatic cancer vaccine composition, wherein
the at least one antibody specifically binds to a protein selected
from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5); detecting
amount of antigen in the first and second body sample that bound to
the antibody; and comparing the amounts of antigen in the first and
second body sample. [0145] 71. The method of clause 70 wherein the
body sample is a tissue sample. [0146] 72. The method of clause 70
wherein the body sample is a blood or urine sample. [0147] 73. The
method of clause 70 wherein the step of detecting is performed
using immunohistochemistry. [0148] 74. The method of clause 70
wherein the step of detecting is performed using an ELISA. [0149]
75. The method of clause 70 wherein at least two of said antibodies
are contacted and detected. [0150] 76. The method of clause 70
wherein at least three of said antibodies are contacted and
detected. [0151] 77. The method of clause 70 wherein the steps of
contacting and detecting are further performed using at least one
antibody that specifically binds to an antigen selected from the
group consisting of: mesothelin, annexin A2, and galectin 3. [0152]
78. A method to test a sample of a human who has been vaccinated
with a pancreatic cancer vaccine, comprising: contacting a first
and a second sample comprising antibodies of the human with at
least one protein selected from the group consisting of:
transferrin receptor (TFRC), regulatory subunit 12A of protein
phosphatase 1 (PPP1R12A), and regulatory subunit 8 of the 26S
proteasome (PSMC5), wherein the first sample was obtained from the
human prior to vaccination and the second sample was obtained from
the human post-vaccination; detecting in the first and second
samples amount of antibody bound to the at least one protein;
comparing the amount of antibody detected in the first and second
sample. [0153] 79. The method of clause 78 wherein the body sample
is a tissue sample. [0154] 80. The method of clause 78 wherein the
body sample is a blood or urine sample. [0155] 81. The method of
clause 78 wherein the step of detecting is performed using
immunohistochemistry. [0156] 82. The method of clause 78 wherein
the step of detecting is performed using an ELISA. [0157] 83. The
method of clause 78 wherein at least two of said antibodies are
contacted and detected. [0158] 84. The method of clause 78 wherein
at least three of said antibodies are contacted and detected.
[0159] 85. The method of clause 78 wherein the steps of contacting
and detecting further comprises using at least one antibody that
specifically binds to an antigen selected from the group consisting
of: mesothelin, annexin A2, and galectin 3.
[0160] 86. The method of clause 55, 63, 71, or 83 wherein the
tissue sample is selected from the group consisting of pancreas,
breast, biliary, lung, colon, and liver. [0161] 87. A method of
treating a human with a tumor selected from the group consisting of
pancreas, breast, biliary, lung, colon, and liver, comprising:
administering a pancreatic cancer cell vaccine composition to the
human whereby an immune response to PPP1R12A and/or PMSC5 is raised
in the human. [0162] 88. The method of clause 87 wherein prior to
said step of administering a sample of the tumor is tested and
expression of PPP1R12A and/or PMSC5 on cell membranes of the tumor
is detected. [0163] 89. The method of clause 87 wherein the vaccine
is GVAX. [0164] 90. A method for testing a tumor sample,
comprising: testing a sample of a tumor and detecting expression of
PPP1R12A and/or PMSC5 on cell membranes of the tumor.
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