U.S. patent application number 15/425856 was filed with the patent office on 2017-05-25 for methods for the treatment of infections and tumors.
This patent application is currently assigned to Emory University. The applicant listed for this patent is Dana-Farber Cancer Institute, Inc., Emory University. Invention is credited to Rafi Ahmed, Rama Amara, Gordon Freeman, Kehmia Titanji, Vijayakumar Velu.
Application Number | 20170146520 15/425856 |
Document ID | / |
Family ID | 42226387 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170146520 |
Kind Code |
A1 |
Ahmed; Rafi ; et
al. |
May 25, 2017 |
METHODS FOR THE TREATMENT OF INFECTIONS AND TUMORS
Abstract
PD-1 antagonists are disclosed that can be used to reduce the
expression or activity of PD-1 in a subject. An immune response
specific to an infectious agent or to tumor cells can be enhanced
using these PD-1 antagonists in conjunction with an antigen from
the infectious agent or tumor. Thus, subjects with infections, such
as persistent infections can be treated using PD-1 antagonists. In
addition, subjects with tumors can be treated using the PD-1
antagonists. In several examples, subjects can be treated by
transplanting a therapeutically effective amount of activated T
cells that recognize an antigen of interest and by administering a
therapeutically effective amount of a PD-1 antagonist. Methods are
also disclosed for determining the efficacy of a PD-1 antagonist in
a subject administered the PD-1 antagonist. In some embodiments,
these methods include measuring proliferation of memory B cells in
a sample from a subject administered the PD-1 antagonist.
Inventors: |
Ahmed; Rafi; (Atlanta,
GA) ; Amara; Rama; (Decatur, GA) ; Velu;
Vijayakumar; (Tucker, GA) ; Titanji; Kehmia;
(Atlanta, GA) ; Freeman; Gordon; (Brookline,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Emory University
Dana-Farber Cancer Institute, Inc. |
Atlanta
Boston |
GA
MA |
US
US |
|
|
Assignee: |
Emory University
Atlanta
GA
Dana-Farber Cancer Institute, Inc.
Boston
MA
|
Family ID: |
42226387 |
Appl. No.: |
15/425856 |
Filed: |
February 6, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12626848 |
Nov 27, 2009 |
9598491 |
|
|
15425856 |
|
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|
|
61118570 |
Nov 28, 2008 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/001102 20180801;
A61P 37/00 20180101; C07K 2317/76 20130101; Y02A 50/41 20180101;
A61P 1/16 20180101; A61K 39/39 20130101; A61P 31/22 20180101; A61K
39/39541 20130101; A61P 31/12 20180101; A61K 39/12 20130101; C07K
16/2827 20130101; C12N 15/1138 20130101; C07K 2317/74 20130101;
C12N 2310/14 20130101; C12N 2760/10034 20130101; C07K 16/2818
20130101; A61K 2039/507 20130101; C07K 16/2803 20130101; G01N
33/56966 20130101; A61K 39/0011 20130101; A61K 2039/505 20130101;
A61K 2039/55516 20130101; C07K 2317/24 20130101; G01N 33/5052
20130101; Y02A 50/30 20180101; A61P 35/00 20180101; G01N 2333/52
20130101; A61K 31/7105 20130101; A61P 31/10 20180101; G01N 33/5088
20130101; A61P 31/18 20180101; G01N 2800/26 20130101; A61K 39/39541
20130101; A61K 2300/00 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; A61K 39/00 20060101 A61K039/00; A61K 39/12 20060101
A61K039/12; C07K 16/28 20060101 C07K016/28; C12N 15/113 20060101
C12N015/113 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
numbers RO1 AI057029, RO1 AI071852 and RO1 AI074417 awarded by the
National Institutes of Health. The government has certain rights in
the invention.
Claims
1. A method for determining the dose of a PD-1 antagonist that is
useful to treat a subject, comprising measuring
CD20.sup.+CD27.sup.+CD21.sup.- memory B cell proliferation and
CD20.sup.+CD21.sup.+CD27.sup.- naive B cell proliferation in a
first sample from the subject administered the first dose of the
PD-1 antagonist; comparing proliferation of the
CD20.sup.+CD27.sup.+CD21.sup.- memory B cells to the proliferation
of CD20.sup.+CD21.sup.+CD27.sup.- naive B cells; and identifying
the subject has having a significant increase in the proliferation
of the CD20.sup.+CD27.sup.+CD21.sup.- memory B cells and an absence
of a significant increase in proliferation of
CD20.sup.+CD21.sup.+CD27.sup.- naive B cells from the first sample,
thereby determining that the dose of the PD-1 antagonist is
effective for treating the subject, or identifying the subject as
having an absence of a significant increase in the proliferation of
the CD20.sup.+CD27.sup.+CD21.sup.- memory B cells and an absence of
a significant increase in proliferation of
CD20.sup.+CD21.sup.+CD27.sup.- naive B cells from the first sample,
thereby determining that the dose of the PD-1 antagonist is
insufficient for treating the subject.
2. The method of claim 1, further comprising measuring
proliferation of CD20.sup.+CD27.sup.+CD21.sup.- memory B cells and
proliferation of CD20.sup.+CD21.sup.+CD27.sup.- naive B cells in a
second sample from the subject following the administration of the
second dose; identifying the subject as having an absence of a
significant_increase in the proliferation of
CD20.sup.+CD27.sup.+CD21.sup.- memory B cells and an absence of a
significant increase in proliferation of
CD20.sup.+CD21.sup.+CD27.sup.- naive B cells from the second
sample.
3. The method of claim 2, wherein there is the absence of a
significant alteration in the proliferation of memory B cells in
the first sample as compared to the control, and wherein the second
dose is higher than the first dose.
4. The method of claim 2, wherein there is an increase in the
proliferation of memory B cells from the first sample as compared
to a control, and wherein the second dose is lower than the first
dose.
5. A method for treating a subject with a PD-1 antagonist,
comprising administering the PD-1 antagonist to the subject;
isolating CD20.sup.+CD27.sup.+CD21.sup.- memory B cells from a
sample from the subject administered the PD-1 antagonist wherein
said isolating comprises detecting expression of CD20, CD27 and
CD21; isolating CD20.sup.+CD21.sup.+CD27.sup.- naive B cells from a
sample from the subject administered the PD-1 antagonist, wherein
said isolating comprises detecting expression of CD20, CD21 and
CD27; measuring proliferation of the memory B cells and the naive B
cells; identifying the subject as having an absence of in
proliferation of the memory B cells as compared to proliferation of
the naive B cells, thereby determining that the PD-1 antagonist was
not efficacious for inducing the immune response in the subject;
and administering a second dose of the PD-1 antagonist to the
subject.
6. The method of claim 5, wherein measuring proliferating memory B
cells comprises measuring the expression of Ki67, measuring the
incorporation of bromodeoxyuridine, or the use of fluorescence
activated cells sorting (FACS).
7. The method of claim 5, wherein the subject has a persistent
viral infection or a tumor.
8. The methods of claim 5, wherein the subject has the persistent
viral infection and wherein the subject is also administered a
viral antigen.
9. The method of claim 8, wherein the viral infection is an
infection with a hepatitis virus, a human immunodeficiency virus
(HIV), a human T-lymphotrophic virus (HTLV), a herpes virus, an
Epstein-Barr virus, or a human papilloma virus.
10. The method of claim 5, wherein the subject has the tumor and
wherein the subject is also administered a tumor antigen.
11. The method of claim 1, wherein the PD-1 antagonist is an
antibody that specifically binds PD-1, an antibody that
specifically binds PD-L1, or an antibody that specifically binds
PD-L2.
12. The method of claim 11, wherein the antibody that specifically
binds PD-1, the antibody that specifically binds PD-L1, or the
antibody that specifically binds PD-L2 is (1) a monoclonal antibody
or a functional fragment thereof, (2) a humanized antibody or a
functional fragment thereof, or (3) an immunoglobulin fusion
protein.
13. A method of inducing an immune response in a human subject,
comprising: administering to the subject a therapeutically
effective amount of a PD-1 antagonist; and isolating
CD20.sup.+CD27.sup.+CD21.sup.- memory B cells from a sample from
the subject, wherein said isolating comprises detecting expression
of CD20, CD27 and CD21; isolating CD20.sup.+CD21.sup.+CD27.sup.-
naive B cells from a sample from the subject administered the PD-1
antagonist, wherein said isolating comprises detecting expression
of CD20, CD21 and CD27 and comprises the use of fluorescence
activated cell sorting; and quantifying proliferation of the memory
B cells and the naive B cells; and identifying the subject as
having an increase in the proliferation of the
CD20.sup.+CD27.sup.+CD21.sup.- memory B cells and an absence of a
significant increase in the proliferation of
CD20.sup.+CD21.sup.+CD27.sup.- naive B cells, thereby producing an
immune response to the antigen of interest in the mammalian
subject, wherein the mammalian subject has a persistent infection
with a virus or a tumor.
14. The method of claim 13, wherein the subject has the persistent
viral infection.
15. The method of claim 13, wherein the subject is
immunosuppressed.
16. The method of claim 13, wherein the subject has a hepatitis
infection.
17. The method of claim 16, wherein the hepatitis infection is a
hepatitis B infection.
18. The method of claim 13, wherein the PD-1 antagonist is an
antibody that specifically binds PD-1, an antibody that
specifically binds PD-L1, an antibody that specifically binds
PD-L2, a small inhibitory anti-PD-1 RNAi, a small inhibitory
anti-PD-L1 RNA, an small inhibitory anti-PD-L2 RNAi, an anti-PD-1
antisense RNA, an anti-PD-L1 antisense RNA, an anti-PD-L2 antisense
RNA, a dominant negative PD-1 protein, a dominant negative PD-L1
protein, a dominant negative PD-L2 protein, a small molecule
inhibitor of PD-1, or combinations thereof.
19. A method of selecting a PD-1 antagonist of use, comprising:
contacting a population of cells comprising
CD20.sup.+CD27.sup.+CD21.sup.- memory B cells with an agent in
vitro; and detecting the proliferation of
CD20.sup.+CD27.sup.+CD21.sup.- memory B cells and/or the
differentiation of CD20.sup.+CD27.sup.+CD21.sup.- memory B cells
into antibody secreting cells, wherein an increase of the
proliferation of CD20.sup.+CD27.sup.+CD21.sup.- memory B cells
and/or an increase in the differentiation of
CD20.sup.+CD27.sup.+CD21.sup.- memory B cells into antibody
secreting cells indicates that the agent is a PD-1 antagonist.
20. The method of claim 18, wherein the PD-1 antagonist is an
antibody that specifically binds PD-1, an antibody that
specifically binds PD-L1, an antibody that specifically binds
PD-L2, a small inhibitory anti-PD-1 RNAi, a small inhibitory
anti-PD-L1 RNA, an small inhibitory anti-PD-L2 RNAi, an anti-PD-1
antisense RNA, an anti-PD-L1 antisense RNA, an anti-PD-L2 antisense
RNA, a dominant negative PD-1 protein, a dominant negative PD-L1
protein, a dominant negative PD-L2 protein, a small molecule
inhibitor of PD-1, or combinations thereof.
Description
PRIORITY CLAIM
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/626,848, filed Nov. 27, 2009, which claims
the benefit of U.S. Provisional Patent Application No. 61/118,570
filed Nov. 28, 2008, both of which are incorporated by reference
herein in their entirety.
FIELD
[0003] This application relates to the use of antagonists,
specifically to the use of PD-1 antagonists for the treatment of
persistent infections and tumors, and to methods for determining an
effective dose of a PD-1 antagonist.
BACKGROUND
[0004] Immunosuppression of a host immune response plays a role in
persistent infection and tumor immunosuppression. Persistent
infections are infections in which the virus is not cleared but
remains in specific cells of infected individuals. Persistent
infections often involve stages of both silent and productive
infection without rapidly killing or even producing excessive
damage of the host cells. There are three types of persistent
virus-host interaction: latent, chronic and slow infection. Latent
infection is characterized by the lack of demonstrable infectious
virus between episodes of recurrent disease. Chronic infection is
characterized by the continued presence of infectious virus
following the primary infection and can include chronic or
recurrent disease. Slow infection is characterized by a prolonged
incubation period followed by progressive disease. Unlike latent
and chronic infections, slow infection may not begin with an acute
period of viral multiplication. During persistent infections, the
viral genome can be either stably integrated into the cellular DNA
or maintained episomally. Persistent infection occurs with viruses
such as human T-Cell leukemia viruses, Epstein-Barr virus,
cytomegalovirus, herpesviruses, varicella-zoster virus, measles,
papovaviruses, xenotropic murine leukemia virus-related virus
(XMRV), prions, hepatitis viruses, adenoviruses, parvoviruses and
papillomaviruses.
[0005] The mechanisms by which persistent infections are maintained
can involve modulation of virus and cellular gene expression and
modification of the host immune response. Reactivation of a latent
infection may be triggered by various stimuli, including changes in
cell physiology, superinfection by another virus, and physical
stress or trauma. Host immunosuppression is often associated with
reactivation of a number of persistent virus infections.
[0006] Many studies show defective immune responses in patients
diagnosed with cancer. A number of tumor antigens have been
identified that are associated with specific cancers. Many tumor
antigens have been defined in terms of multiple solid tumors: MAGE
1, 2, & 3, defined by immunity; MART-1/Melan-A, gp100,
carcinoembryonic antigen (CEA), HER-2, mucins (i.e., MUC-1),
prostate-specific antigen (PSA), and prostatic acid phosphatase
(PAP). In addition, viral proteins such as hepatitis B (HBV),
Epstein-Barr (EBV), and human papilloma (HPV) have been shown to be
important in the development of hepatocellular carcinoma, lymphoma,
and cervical cancer, respectively. However, due to the
immunosuppression of patients diagnosed with cancer, the innate
immune system of these patients often fails to respond to the tumor
antigens.
[0007] Both passive and active immunotherapy has been proposed to
be of use in the treatment of tumors. Passive immunity supplies a
component of the immune response, such as antibodies or cytotoxic T
cells to the subject of interest. Active immunotherapy utilizes a
therapeutic agent, such as a cytokine, antibody or chemical
compound to activate an endogenous immune response, where the
immune system is primed to recognize the tumor as foreign. The
induction of both passive and active immunity have been successful
in the treatment of specific types of cancer.
[0008] In general, a need exists to provide safe and effective
therapeutic methods and to establish safe dosing of agents to treat
disease, for example, autoimmune diseases, inflammatory disorders,
allergies, transplant rejection, cancer, immune deficiency, viral
infections and other immune system-related disorders. There also
remains a need for methods for determining if a particular dose of
a therapeutic agent, such as a PD-1 antagonist, is effectively
treating a subject.
SUMMARY
[0009] PD-1 antagonists reduce the expression and/or activity of
PD-1. Subjects with infections, such as persistent infections can
be treated using PD-1 antagonists. Subject with tumors can also be
treated using PD-1 antagonists. Additionally, subjects can be
treated by transplanting a therapeutically effective amount of
activated T cells that recognize an antigen of interest in
conjunction with a therapeutically effective amount of a PD-1
antagonist.
[0010] An immune response can be measured in the mammalian
recipient. In some embodiments the method of treatment disclosed
herein includes measuring B cells. In some embodiments, the methods
include measuring the proliferation of memory B cells in a sample
from the subject.
[0011] In some embodiments, methods are disclosed for determining
the efficacy of a PD-1 antagonist in a subject administered the
PD-1 antagonist. These methods include measuring proliferation of
memory B cells in a sample from a subject administered the PD-1
antagonist, wherein an increase in proliferation of memory B cells
from the sample as compared to a control indicates that the PD-1
antagonist is efficacious for treating the subject.
[0012] Methods for determining the dose of a PD-1 antagonist that
is useful to treat a subject are also disclosed herein. These
methods include administering to the subject a first dose of a PD-1
antagonist, and determining the proliferation of memory B cells in
a first sample from the subject. An increase in the proliferation
of memory B cells from the first sample as compared to a control
indicates that the first dose of the PD-1 antagonist is of use
treating the subject. An absence of a significant alteration in the
proliferation of memory B cells as compared to the control
indicates that the first dose of the PD-1 antagonist is not
sufficient to treat the subject.
[0013] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIGS. 1A-1C. FIG. 1A is a bar graph showing the levels of
PD-1 mRNA in D.sup.bGP33-41 and/or D.sup.bGP276-286 specific T
cells from naive transgenic mice, lymphocytic choriomeningitis
virus (LCMV) Armstrong immune (approximately 30 days
post-infection) infected mice, or CD4-depleted LCMV-Cl-13 infected
mice (approximately 30 days post-infection), as measured by gene
array analysis. FIG. 1B is a series of images of a flow cytometry
experiment showing PD-1 surface expression on CD8+ tetramer+ T
cells in LCMV Armstrong immune and CD4 depleted LCMV-C1-13 infected
mice approximately 60 days post-infection. Anergic CD8+ T cells
express high levels of PD-1 polypeptide on the cell surface
approximately 60 days after chronic infection with LCMV-C1-13 virus
(labeled "chronic"), but virus-specific CD8+ T cells do not express
PD-1 polypeptide after clearance of an acute LCMV Armstrong
infection (labeled "immune"). FIG. 1C is a series of images of a
flow cytometry experiment demonstrating the presence of PD-L1 on
splenocytes from chronically infected and uninfected mice. It
demonstrates that PD-L1 expression is the highest on the
splenocytes that are infected by the virus.
[0015] FIGS. 2A-2E. FIG. 2A is a series of scatter plots showing
that when Cl-13 infected mice are treated from day 23 to 37
post-infection there was approximately a 3 fold increase in the
number of DbNP396-404 specific and DbGP33-41 specific CD8+ T cells
compared to the untreated controls. In order to determine any
changes in function IFN-.gamma. and TNF-.alpha. production was
measured in response to 8 different LCMV epitopes. FIG. 2B is a
scatter plot showing that when all the known CD8+ T cell
specificities are measured there is a 2.3 fold increase in total
number of LCMV specific CD8+ T cells. FIG. 2C is a series of flow
cytometry graphs showing IFN-.gamma. and TNF-.alpha. production in
response to eight different LCMV epitopes. FIG. 2D is a scatter
plot showing that more virus specific CD8+ T cells in treated mice
have the ability to produce TNF-.alpha.. FIG. 2E is a series of bar
charts showing that PD-L1 blockade also resulted in increased viral
control in the spleen liver lung and serum.
[0016] FIGS. 3A-3D. FIG. 3A is a graph demonstrating the increase
in DbGP33-41 and DbGP276-286 specific CD8+ T cells (labeled "GP33"
and "GP276") in CD4-depleted C1-13 infected mice treated with
anti-PD-L1 (labeled ".alpha.PD-L") from day 46 to day 60
post-infection versus control (labeled "untx"), which demonstrates
that mice treated with anti-PD-L1 contained approximately 7 fold
more DbGP276-286 specific splenic CD8+ T cells and approximately 4
fold more DbGP33-41 specific splenic CD8+ T cells than untreated
mice. FIG. 3B is a series of images of a flow cytometry experiment
demonstrating the increased frequency of DbGP33-41 and DbGP276-286
specific CD8+ T cells in the spleen of CD4-depleted C1-13 infected
mice treated with anti-PD-L1 (labeled ".alpha.PD-L1 Tx") from day
46 to day 60 post-infection versus control (labeled "untx"). FIG.
3C is a series of images of a flow cytometry experiment
demonstrating increased proliferation of DbGP276-286 specific CD8+
T cells in anti-PD-L-treated mice, as measured by BrdU
incorporation and Ki67 expression. FIG. 3D is a chart showing that
mice having high levels of CD8+ T cell expansion demonstrate an
appreciable response in peripheral blood mononuclear cells (PBMC),
as shown by comparing DbGP276-286 specific CD8+ T cells in the PBMC
as compared to DbGP276-286 specific CD8+ T cells in the spleen.
[0017] FIGS. 4A-4E. FIG. 4A is a series of charts demonstrating the
increase in IFN-.gamma. producing DbGP276-286 and DbGP33-41
specific CD8+ T cells in anti-PD-L-treated mice, as compared to
controls. Higher frequencies of DbNP396-404, KbNP205-212,
DbNP166-175, and DbGP92-101 specific CD8+ T cells were also
detected in anti-PD-L-treated mice. FIG. 4B is a chart
demonstrating that in anti-PD-L-treated mice, 50% of DbGP276-286
specific CD8+ T cells produce IFN-.gamma., as compared to 20% of
DbGP276-286 specific CD8+ T cells in control mice. FIG. 4C is a
series of images of a flow cytometry experiment demonstrating that
anti-PD-L-treated chronically infected mice produce higher levels
of TNF-.alpha. than untreated chronically infected mice, but still
produce lower levels of TNF-.alpha. than immune mice infected with
LCMV Armstrong virus. FIG. 4D is a chart demonstrating that
treatment of LCMV-C1-13 infected mice with anti-PD-L1 renews ex
vivo lytic activity of the virus-specific T cells, as compared to
untreated infected mice, measured using a .sup.51Cr release assay.
FIG. 4E is a series of charts demonstrating the reduction of viral
titers in various organs following treatment of LCMV-C1-13 infected
mice with .alpha.-PD-L1. Viral titers decreased approximately 3
fold in the spleen, 4 fold in the liver, 2 fold in the lung, and 2
fold in serum after 2 weeks of anti-PD-L1 treatment, as compared to
untreated mice.
[0018] FIGS. 5A-5D. FIG. 5A is a series of images of a flow
cytometry experiment showing PD-1 surface expression using 10 HIV
tetramers specific for dominant epitopes targeted in chronic clade
C HIV infection. The percentages indicate the percentage of
tetramer.sup.+ cells that are PD-1.sup.+. FIG. 5B is a series of
charts demonstrating that the percentage and MFI of PD-1 is
significantly upregulated on HIV-specific CD8+ T cells compared to
the total CD8+ T cell population (p<0.0001) in antiretroviral
therapy naive individuals, and PD-1 is increased on the total CD8+
T cell population in HIV-infected versus HIV-seronegative controls
(p=0.0033 and p<0.0001, respectively). 120 HIV tetramer stains
from 65 HIV-infected individuals and 11 HIV seronegative controls
were included in the analysis. FIG. 5C is a series of charts
showing the median percentage and MFI of PD-1 expression on
tetramer.sup.+ cells by epitope specificity. FIG. 5D is a chart
depicting the variation in the percentage of PD-1.sup.+ cells on
different epitope-specific populations within individuals with
multiple detectable responses. Horizontal bars indicate the median
percentage of PD-1.sup.+ HIV tetramer.sup.+ cells in each
individual.
[0019] FIGS. 6A-6D. FIG. 6A is a series of charts demonstrating
that there is no correlation between the number of HIV-specific
CD8+ T cells, as measured by tetramer staining, and plasma viral
load, whereas there is a positive correlation between both the
percentage and MFI of PD-1 on tetramer.sup.+ cells and plasma viral
load (p=0.0013 and p<0.0001, respectively). FIG. 6B is a series
of charts showing that there is no correlation between the number
of HIV tetramer.sup.+ cells and CD4 count, whereas there is an
inverse correlation between the percentage and MFI of PD-1 on HIV
tetramer.sup.+ cells and CD4 count (p=0.0046 and p=0.0150,
respectively). FIG. 6C is a series of charts demonstrating that the
percentage and MFI of PD-1 on the total CD8+ T cell population
positively correlate with plasma viral load (p=0.0021 and
p<0.0001, respectively). FIG. 6D is a series of charts depicting
the percentage and MFI of PD-1 expression on the total CD8+ T cell
population is inversely correlated with CD4 count (p=0.0049 and
p=0.0006, respectively).
[0020] FIGS. 7A-7B. FIG. 7A is a series of images of a flow
cytometry experiment showing representative phenotypic staining of
B*4201 TL9-specific CD8+ T cells from subject SK222 in whom 98% of
B*4201 TL9-specific CD8+ T cells are PD-1.sup.+. FIG. 7B is a chart
illustrating a summary of phenotypic data from persons in whom
>95% of HIV-specific CD8+ T cells are PD-1.sup.+. Seven to 19
samples were analyzed for each of the indicated phenotypic markers.
The horizontal bar indicates median percentage of tetramer.sup.+
PD-1.sup.+ cells that were positive for the indicated marker.
[0021] FIGS. 8A-8C. FIG. 8A is a series of images of a flow
cytometry experiment showing the representative proliferation assay
data from a B*4201 positive subject. After a 6-day stimulation with
peptide, the percentage of B*4201 TL9-specific CD8+ T cells
increased from 5.7% to 12.4% in the presence of anti-PD-L1 blocking
antibody. FIG. 8B is a line graph depicting the summary
proliferation assay data indicating a significant increase in
proliferation of HIV-specific CD8+ T cells in the presence of
anti-PD-L1 blocking antibody (n=28, p=0.0006, paired t-test). FIG.
8C is a bar graph showing the differential effects of PD-1/PD-L1
blockade on proliferation of HIV-specific CD8+ T cells on an
individual patient basis. White bars indicate fold increase of
tetramer.sup.+ cells in the presence of peptide alone, black bars
indicate the fold increase of tetramer.sup.+ cells in the presence
of peptide plus anti-PD-L1 blocking antibody. Individuals in whom
CFSE assays were performed for more than one epitope are indicated
by asterisk, square, or triangle symbols.
[0022] FIGS. 9A-9D are a diagram and a set of graphs showing the
synergistic effect of therapeutic vaccine combined with PD-L1
blockade on antigen-specific CD8-T cell frequency and viral titer
in chronically infected mice. FIG. 9A is a schematic diagram of an
experimental protocol. LCMV clone-13 (CL-13)-infected mice were
vaccinated with wild-type vaccinia virus (VV/WT) or LCMV GP33-41
epitope-expressing vaccinia virus (VV/GP33) at 4 (week)
post-infection. At the same time, the mice were treated 5 times
every three days with or without anti-PD-L1. FIG. 9B is a series of
images of a flow cytometry experiment showing the frequency of
GP33- and GP276-specific CD8-T cells in PBMC at 1-wk post-therapy.
The number represents frequency of tetramer-positive cells per
CD8-T cells. Data are representative of three experiments. FIGS.
9C-9D are graphs of the frequency of GP33- and GP276-specific CD8-T
cells (FIG. 9C) and viral titers (FIG. 9D) in the blood
post-therapy. Changes in the numbers of tetramer-positive CD8-T
cells and the viral titers were monitored in the blood by tetramer
staining and plaque assay, respectively, at the indicated time
points. The numbers of tetramer-positive CD8-T cells and viral
titers are shown for individual (upper four panels) and multiple
(lower panel) mice following infection with VV/WT or VV/GP33
(straight line) and treatment with anti-PD-L1 (shade region).
Dashed lines represent virus detection limit. Results are pooled
from three experiments.
[0023] FIGS. 10A-10D are graphs and digital images showing
increased antigen-specific CD8-T cells and enhanced viral control
in different tissues of the mice given therapeutic vaccine combined
with PD-L1 blockade. FIG. 10A is a series of images of a flow
cytometry experiment showing the frequency of GP33-specific CD8-T
cells in different tissues at 4-wk post-therapy. The number
represents frequency of GP33 tetramer-positive cells per CD8-T
cells. Data are representative of two experiments. FIG. 10B is a
graph of GP33-specific CD8 T-cell numbers in different tissues at
4-wk post-therapy. FIG. 10C is a set of bar graphs showing viral
titers in the indicated tissues at 2 (filled)- and 4 (blank)-wk
post-therapy. Dashed lines represent virus detection limit. n=6
mice per group. Results are pooled from two experiments. FIG. 10D
is a digital image of immuno-staining of spleen with aLCMV antigens
(red) at 2-wk post-therapy. Magnification, .times.20.
[0024] FIGS. 11A-11D are plots and graphs showing enhanced
restoration of function in exhausted CD8-T cells by therapeutic
vaccine combined with PD-L1 blockade. FIG. 11A is a series of
images of a flow cytometry experiment showing IFN-.gamma.
production and degranulation by splenocytes of the vaccinated mice
at 4-wk post-therapy. Splenocytes were stimulated with the
indicated peptides in the presence of .alpha.CD107a/b antibodies
and then co-stained for IFN-.gamma.. The shown plots are gated on
CD8-T cells and are the representative of two independent
experiments. FIG. 11B is a graph showing the percentage of
IFN-.gamma..sup.+CD107.sup.+ cells per CD8-T cells specific for
each of LCMV peptides from FIG. 11A are summarized for multiple
mice (n=6 for each response). Results are pooled from two
experiments. FIG. 11C is a set of plots showing TNF-.alpha.
production from CD8-T cells capable of producing IFN-.gamma. in the
vaccinated mice. After stimulation of splenocytes with GP33-41 or
GP276-286 peptide, IFN-.gamma.-producing CD8-T cells were gated and
then plotted by IFN-.gamma. (x-axis) versus TNF-.alpha. (y-axis).
The upper and lower numbers on plots indicate frequency of
TNF-.alpha..sup.+ cells among IFN-.gamma..sup.+ cells and mean
fluorescent intensity (MFI) of IFN-.gamma..sup.+ cells,
respectively. The data are representative of two independent
experiments. FIG. 11D is a graph showing the percentage of
TNF-.alpha..sup.+ cells per IFN-.gamma..sup.+ cells for GP33-41 or
GP276-286 peptide from FIG. 11C are summarized for multiple mice
(n=6 for each response).
[0025] FIGS. 12A-12B are a set of plots showing the effect of a
therapeutic vaccine combined with PD-L1 blockade changes phenotype
of antigen-specific CD8-T cells of chronically infected mice. FIG.
12A is a set of plots showing the phenotype of GP33
tetramer-specific CD8-T cells in PBMC at the indicated times
post-therapy. Histograms were gated on GP33.sup.+ CD8-T cells.
Frequency of population expressing high-level of CD27 or CD127 is
indicated by percent on plots. The numbers on histograms of
Granzyme B represent MFI of expression. The data are representative
of three independent experiments. FIG. 12B is a set of plots
showing phenotypic changes of GP33 tetramer-specific CD8-T cells in
different tissues at 4-wk post-therapy. Histograms were gated on
GP33.sup.+CD8-T cells. Frequency of population expressing
high-level of CD127 or PD-1 is indicated by percent on plots. The
numbers on histograms of Granzyme B and Bcl-2 represent MFI of
expression. The data are representative of two independent
experiments.
[0026] FIGS. 13A-13E are a schematic diagram, plots and graphs
showing the synergistic effect of therapeutic vaccine combined with
PD-L1 blockade on restoration of function in `helpless` exhausted
CD8 T cells. FIG. 13A is a schematic diagram of the protocol. Mice
were depleted of CD4 T cells and then infected with LCMV clone-13.
Some mice were vaccinated with wild-type vaccinia virus (VV/WT) or
LCMV GP33-41 epitope-expressing vaccinia virus (VV/GP33) at 7-wk
post-infection. At the same time, the mice were treated 5 times
every three days with .alpha.PD-L1 or its isotype. Two weeks after
initial treatment of antibodies, mice were sacrificed for analysis.
FIG. 13B is a series of images of a flow cytometry experiment and a
bar graph showing the frequency of GP33-specific CD8-T cells in the
indicated tissues at 4-weeks post-therapy. The number represents
frequency of GP33 tetramer-positive cells per CD8-T cells.
Frequency of GP33-specific cells per CD8 T-cells in different
tissues at 2-weeks post-therapy is also summarized. FIG. 13C is a
series of images of a flow cytometry experiment showing the results
from experiments wherein splenocytes stimulated with GP33 peptide
in the presence of .alpha.CD107a/b antibodies and then co-stained
for IFN-.gamma.. The shown plots are gated on CD8-T cells. The
percentage of IFN-.gamma..sup.+CD107.sup.+ cells per CD8-T cells
specific for GP33 peptide are summarized for multiple mice. FIG.
13D is a bar graph of the percentage of IFN-.gamma..sup.+ cells
after stimulation with GP33 peptide per cells positive for
Db-restricted GP33-41 tetramer are summarized for multiple mice.
FIG. 13E is a bar graph of viral titers in the indicated tissues at
2-wk post-therapy. All plots are representative of two experiments
and all summarized results are pooled from two experiments (n=6
mice per group).
[0027] FIGS. 14A-14B are a set of plots and graphs showing that
blockade of the PD1/PD-L1 signaling pathway increases the total
number of antigen-specific T cells following adoptive transfer into
congenital carrier mice. Whole splenocytes were adoptively
transferred into congenital carrier mice with or without therapy
with anti-PD-L1. FIG. 14A is a set of representative flow cytometry
plots from specific time-points gated on CD8+ T cells. FIG. 14B are
graphs showing the kinetics of Db GP33-specific CD8 T cell
expansion in peripheral blood from two independent experiments (n=4
animals per group)
[0028] FIGS. 15A-15E are plots and graphs showing that blockade of
the PD-1/PDL1 pathway following adoptive T cell immunotherapy
enhances cytokine production in antigen specific CD8 T cells.
Splenocytes were isolated at day 17 post-transfer and analyzed for
cytokine expression upon stimulation with antigenic peptide. FIG.
15A is a set of representative flow plots are shown for the
expression of IFN.gamma. assessed by intracellular cytokine
staining following 5 hours of stimulation with defined CD8 epitopes
or no peptide controls. FIGS. 15B and 15D are representative plots
are shown for the dual expression of TNF.alpha. or 107ab and
IFN.gamma. (quadrant stats are percentage of CD8 gate). FIGS. 15C
and 15E are graphs of the percentage of IFN.gamma. producing cells
also producing TNF.alpha. or 107ab (n=3 animals per group)
[0029] FIGS. 16A-16B are a graph and plots showing increased levels
of Antibody Secreting cells in LCMV Clone-13 infected mice. Total
ASC levels were measured in chronic LCMV infected mice following
.alpha.PD-L1 treatment by ELISPOT and CD138 staining. FIG. 16A is a
graph of total number of splenic ASC, summary of results from three
independent experiments. FIG. 16B is a set of plots showing an
increase in antibody secreting cells (ASC) in the spleen can be
measured by the marker CD138. Showing one representative plot, ASC
are CD138+ and B220 low/intermediate (gated on lymphocytes).
[0030] FIG. 17 is a graph showing treatment of chronic LCMV
infected mice with anti-PD-L1 does not lead to elevated levels of
bone marrow ASC. Total numbers of ASC were enumerated from the
spleen and bone marrow of chronic LCMV infected mice 14 days post
anti (.alpha.)PD-L1 treatment by ELISPOT. Line represents geometric
mean within the group.
[0031] FIG. 18 is a graph showing that co-administration of
.alpha.PD-L1 and .alpha.CTLA-4 leads to synergistic increases in
splenic ASC. Chronic LCMV infected mice were administered
.alpha.PD-L1, .alpha.CTLA-4, or both for 14 days and ASC in the
spleen was enumerated by ELISPOT. Line represents geometric mean
within treatment group.
[0032] FIGS. 19A-19B are plots showing enhanced B cell and CD4 T
cell proliferation and germinal center activity in .alpha.PD-L1
treated mice. FIG. 19A is a plot of flow cytometric analysis of CD4
T cells and B cells shows elevated Ki-67 levels following
.alpha.PD-L1 treatment. Results are gated on either CD4 or B cells
as listed above each column. FIG. 19B is a set of plots showing an
increased frequency of B cells expressing PNA and high levels of
FAS, which indicate enhanced germinal center activity in mice
treated with .alpha.PD-L1. Plots are one representative graph
summarizing the results of two separate experiments.
[0033] FIGS. 20A-20C are plots and graphs showing PD-1 expression
on CD8 and CD4 T cell subsets. FIG. 20A is a series of images of a
flow cytometry experiment showing co-expression of PD-1 and various
phenotypic markers among CD8+/CD3+ lymphocytes in blood. FIG. 20B
is a set of plots of the percentage of various CD8+/CD3+ and (D)
CD4+/CD3+ T cell subsets that express PD-1. Horizontal bars
indicate mean percentage of PD-1 on T cells that are positive
(hollow circles) and negative (solid triangles) for the indicated
marker. FIG. 20C is a set of plots representing the phenotypic data
pf PD-1 expressing CD4+ T cells from one subject.
[0034] FIGS. 21A-21B are plots and graphs demonstrating that PD-1
is more highly expressed among CD8 T cells specific for chronic
infections. FIG. 21A is a series of images of a flow cytometry
experiment showing representative PD-1 staining of Ebstein Bar
Virus (EBV), Cylomegalovirus (CMV), influenza and vaccinia
virus-specific CD8 T cells. Geometric mean fluorescence intensity
(GMFI) of PD-1 expression among tetramer+ cells is indicated. FIG.
21B is a plot showing a summary of PD-1 GMFI on EBV, CMV, influenza
and vaccinia virus-specific CD8 T cells from healthy volunteers
(n=35).
[0035] FIGS. 22A-22C are plots and graphs demonstrating that
anti-PD-L1 blockade increases in vitro proliferation of CD8 T cells
specific for chronic infections. FIG. 22A is a series of images of
a flow cytometry experiment showing lymphocytes that were labeled
with CFSE, then cultured for 6 days under the indicated conditions.
The images show representative staining from EBV and CMV positive
subjects. FIG. 22B is a bar graph of EBV, CMV, influenza and
vaccinia virus antigen-specific responses following blockade with
anti-PD-L1 blocking antibody. The bars indicate fold increase of
tetramer+ cells in the presence of peptide plus anti-PD-L1 blocking
antibody compared to peptide alone. FIG. 22C is a line graph
showing the relationship between the fold-increase in tetramer+
cells following anti-PD-L1 antibody blockade and PD-1 expression
(prior to culture).
[0036] FIGS. 23A-23C are plots and graphs showing hepatitis C virus
(HCV) specific CD8+ T cells express PD-1 in human chronic HCV
infection. FIG. 23A are representative plots from five patients
with chronic HCV infection showing the expression of PD-1 on HCV
specific CD8+ T cells. Numbers in bold identify the frequency of
PD-1 expression (x-axis) on HCV specific CD8+ T cells (y-axis).
Numbers in italics within the plots identify the frequency of
tetramer positive cells among total CD8+ T cells. On the y-axis,
1073 and 1406, identify the HCV epitope specificity of the
tetramer. Patients are identified by "Pt" followed by the patient
number. Cells were gated on CD8+ lymphocytes. Plots are on a
logarithmic scale. FIG. 23B is a comparison of PD-1 expression on
CD8+ T cells from healthy donors (CD8 Healthy), HCV infected
patients (CD8 HCV) and on CD8+ HCV specific T cells (HCV tet+).
FIG. 23C is a graph of PD-1 expression on CD8+ T cells specific for
influenza virus (Flu tet+) from HCV infected (HCV+) and healthy
donors (Healthy) compared with PD-1 expression on CD8+ T cells
specific for HCV (HCV tet+). An unpaired t test was used to compare
differences in expression of PD-1 within the same patient on total
CD8+ T cells versus HCV specific CD8+ T cells.
[0037] FIGS. 24A-24D are plots and graphs showing the frequency of
PD-1 expressing CD8+ T cells from the liver is greater than in the
peripheral blood. FIG. 24A is representative plots from five
patients with chronic HCV infection showing the expression of PD-1
on total CD8+ T cells from the peripheral blood versus the liver.
Numbers in bold within the plots identify the frequency of cells
with PD-1 expression among total CD8+ T cells in the lymphocyte
gate. Plots are on a logarithmic scale. FIG. 24B is a comparison of
PD-1 expression on CD8+ T cells from peripheral blood versus liver
in HCV chronically infected patients. A paired t test was used to
compare the difference in PD-1 expression within the same patients.
FIG. 24C is a comparison of PD-1 expression on the CD8+ Effector
Memory (T.sub.EM) cells from peripheral blood versus the liver.
Memory subsets were identified by differential expression of CD62L
and CD45RA. Bold numbers in the top plots represent the frequency
of cells in each quadrant. Cells were gated on CD8+ lymphocytes.
The T.sub.EM subset was gated (boxes) and the expression of PD-1 is
shown in the histogram plots below. The dotted line shows PD-1
expression on naive CD8+ T cells (used as the negative population).
The numbers in the histogram plots represent the frequency of cells
expressing PD-1. Comparison of the frequency of PD-1 expression on
CD8+ T.sub.EM cells for ten patients with chronic HCV infection is
summarized below the histogram plots. A paired t test was used to
compare the difference in PD-1 expression on CD8+ T.sub.EM from the
peripheral blood versus the liver within the same patient. FIG. 24D
are representative plots from two patients with chronic HCV
infection showing the difference in CD127 expression on total CD8+
T cells from the peripheral blood versus the liver. Numbers in bold
identify the frequency of CD127 expression on total CD8+ T cells.
Cells were gated on CD8+ lymphocytes. Plots are on a logarithmic
scale. A summary of the comparison of CD127 expression on total
CD8+ T cells in the peripheral blood versus the liver is shown
below the FACS plots. A paired t test was used for statistical
analysis.
[0038] FIG. 25 is sets of graphs and plots showing HCV specific
CD8+ T cells in the liver express an exhausted phenotype.
Representative plots of PD-1 and CD127 expression on HCV specific
CD8+ T cells from the peripheral blood and the liver of two
patients with chronic HCV infection. The first row of plots
identifies the HCV tetramer positive population (boxes). The
numbers above the boxes represent the frequency of tetramer
positive cells among CD3+ lymphocytes. The epitope specificity of
the HCV tetramer is identified on the y-axis (1073). The second and
third row of plots shows PD-1 and CD127 expression on HCV specific
CD8+ T cells from the peripheral blood and liver of two patients
with chronic HCV infection. Numbers in bold represent the frequency
of PD-1 or CD127 expression on HCV specific CD8+ T cells. Plots are
on a logarithmic scale and gated on CD3+CD8+ lymphocytes. Below the
FACS plots, a summary of the comparison of PD-1 expression (left)
and CD127 expression (right) on total CD8+ T cells versus CD8+ HCV
specific T cells from the periphery (HCV tet+ PBMC) versus HCV
specific CD8+ T cells from the liver (HCV tet+ Liver) is shown.
Paired t tests were used to compare expression within the same
patient.
[0039] FIG. 26 is a set of plots showing blockade of the PD-1/PD-L1
pathway increases the expansion of antigen stimulated HCV-specific
T cells. CFSE labeled PBMCs from two separate HLA-A2 patients were
stimulated using the cognate peptide antigen for 6 days in the
presence of IL-2 and anti-PD-L1 antibody (top panel) or anti-PD-1
antibody (lower panel). An unstimulated control is also shown. The
percentage of proliferating CFSE low- and CFSE high-HCV-specific
HLA-A2+CD8+ T cells are shown in each quadrant.
[0040] FIGS. 27A-27D are plots and graphs showing elevated PD-1
expression on simian immunodeficiency virus (SIV) specific CD8 T
cells following SIV239 infection. FIG. 27A is a plot showing PD-1
expression on total CD8 T cells from a normal macaque. FIG. 27B is
a plot showing PD-1 expression on total and SIV gag-specific CD8 T
cells in a SIV239 infected macaque. Analysis was done on PBMC at 12
weeks following SIV-infection. FIG. 27C is a graph providing a
summary of PD-1 positive cells on total and SIV-specific CD8 T
cells from normal and SIV-infected macaques. Data for SIV-infected
macaques represent at 12 weeks following infection. FIG. 27D (last
panel) is a graph providing a summary of mean fluorescence
intensity (MFI) of PD-1 expression on total and SIV-specific CD8 T
cells from normal and SIV-infected macaques.
[0041] FIGS. 28A-28B are a plot and a graph, respectively, showing
in vitro blockade of PD-1 results in enhanced expansion of
SIV-specific CD8 T cells. PBMC from Mamu A*01 positive macaques
that were infected with SHIV89.6P were stimulated with P11C peptide
(0.1 .mu.g/ml) in the absence and presence of anti-PD-1 blocking Ab
(10 .mu.g/ml) for six days. After three days of stimulation, IL-2
(50 units/ml) was added. At the end of stimulation cells were
stained on the surface for CD3, CD8 and Gag-CM9 tetramer.
Unstimulated cells (nostim) served as negative controls. Cells were
gated on lymphocytes based on scatter then on CD3 and analyzed for
the expression of CD8 and tetramer. FIG. 28A is a representative
FACS plots. Numbers on the graph represent the frequency of
tetramer positive cells as a percent of total CD8 T cells. FIG. 28B
is a graph providing a summary of data from six macaques. Analyses
were performed using cells obtained at 12 weeks following
infection. Fold increase was calculated as a ratio of the frequency
of tetramer positive cells in P11C stimulated cultures and
unstimulated cells.
[0042] FIG. 29 is a set of plots showing the kinetics of PD-L1,
PD-L2, and PD-1 expression on different cell types after LCMV
infection. Mice were infected with 2.times.10.sup.6 pfu of clone-13
(CL-13). PD-L1, PD-L2, and PD-1 expression on different type of
cells was shown as a histogram at the indicated time points
post-infection. Mean fluorescence intensity (MFI) of PD-1
expression on the indicated type of cells is shown.
[0043] FIGS. 30A-30C are FACS plots showing in vivo PD-1 blockade
during chronic SIV infection increases the Gag-CM9-specific CD8 T
cells with improved functional quality in both blood and gut. FIG.
30A is a representative FACS plots for macaque RRk10. FIGS. 30B and
30C are FACS plots showing the magnitude and phenotype of
Gag-CM9-tetramer-positive CD8 T cells in blood (FIG. 30B) and gut
(colorectal mucosal tissue) (FIG. 30c). Representative FACS plots
are shown on the left and summary for all Mamu A*01-positive
animals is shown on the right. Numbers on the FACS plots represent
the frequency of tetramer-positive cells as a percent of total CD8
T cells. Arrows and vertical lines indicate anti-PD-1 antibody or
control antibody treatment.
[0044] FIGS. 31A-31B show that in vivo PD-1 blockade during chronic
SIV infection increases the polyfunctional virus-specific CD8 T
cells. FIG. 31a shows the frequency of Gag-specific
cytokine-secreting CD8 T cells as a percentage of total CD8 T
cells. Representative FACS plots are shown on the left and summary
for the group is shown on the right. Arrows and vertical lines
indicate anti-PD-1 antibody or control antibody treatment. Lines
represent anti-PD-1-antibody-treated macaques and red lines
represent control-antibody-treated macaques. FIG. 31B shows
cytokine co-expression subsets expressed as a percentage of total
cytokine-positive cells. Mean percentages for each group are
shown.
[0045] FIGS. 32A-32C show that in vivo PD-1 blockade during chronic
SIV infection enhances SIV-specific humoral immunity. FIG. 32A
shows expression of PD-1 on memory (CD20.sup.+CD27.sup.+CD21.sup.-)
and naive (CD20.sup.+CD27.sup.-CD21.sup.+) B cells in blood after
SIV infection and before in vivo PD-1 blockade. FIG. 32B shows
titres of anti-SIV Env-binding antibody in serum after blockade.
FIG. 32C shows Ki67 expression (marker for proliferation) on memory
and naive B cells after blockade. Numbers on the FACS plots
represent Ki67-positive cells as a percentage of respective total
cells. Macaques RAf11 and RJd1 were treated simultaneously with
anti-PD-1 antibody and anti-retroviral therapy at 22 weeks after
SIV infection.
[0046] FIGS. 33A-33E show that in vivo PD-1 blockade reduces plasma
viraemia and prolongs survival of SIV-infected macaques. Plasma
viral load in macaques treated with anti-PD-1 antibody during the
early chronic phase of infection (FIG. 33A), macaques treated with
anti-PD-1 antibody during the late chronic phase of infection (FIG.
33B), and macaques treated with control antibody during the
early/late chronic phase of SIV infection (FIG. 33C). An asterisk
indicates death of animal. FIG. 33D shows the fold reduction in
plasma viral load between day 0 and day 28 (early chronic study) or
day 0 and day 21 (late chronic study). FIG. 33E shows the survival
of SIV-infected macaques after PD-1 blockade.
SEQUENCE LISTING
[0047] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand.
The Sequence Listing is submitted as an ASCII text file
[6975_76374-98_Sequence_Lisiting, Feb. 6, 2017, 23.2 kb], which is
incorporated by reference herein.
In the accompanying sequence listing:
[0048] SEQ ID NO: 1 is an exemplary amino acid sequence of human
PD-1.
[0049] SEQ ID NO: 2 is an exemplary amino acid sequence of mouse
PD-1.
[0050] SEQ ID NO: 3 is an exemplary amino acid sequence of human
PD-L1.
[0051] SEQ ID NO: 4 is an exemplary amino acid sequence of human
PD-L2.
[0052] SEQ ID NOs: 5-12 are exemplary amino acid sequences of human
framework regions.
[0053] SEQ ID NOs: 13-35 are exemplary amino acid sequences of
antigenic peptides.
[0054] SEQ ID NOs: 36-43 are the amino acid sequences of major
histocompatibility peptides.
[0055] SEQ ID NO: 44 and SEQ ID NO: 45 are the amino acid sequence
of T cell epitopes.
[0056] SEQ ID NO: 46 is an exemplary amino acid sequence of a
variant human PD-L2.
[0057] SEQ ID NOs: 47-52 are exemplary amino acid sequences of
antigenic peptides.
[0058] SEQ ID NOs: 53-56 are the nucleic acid sequences of
primers.
DETAILED DESCRIPTION
[0059] This disclosure relates to the use of PD-1 antagonists for
the induction of an immune response, such as to a tumor or a
persistent viral infection. This disclosure also relates to methods
for determining the dose of a PD-1 antagonist that is effective for
treating a subject.
TERMS
[0060] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0061] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of specific terms are
provided:
[0062] Altering: A statistically significant change in a parameter
as compared to a control value for that parameter. In one example,
an "increase" is a statistically significant elevation in a
parameter, such as the number or proliferation of memory B cells,
as compared to a control. Suitable statistical analyses are well
known in the art, and include, but are not limited to, Student's T
test and ANOVA assays. In some examples, this is a p value
<0.05. In other examples, a significant alteration, such as an
increase or a decrease is a change that is two standard deviations
from the mean or greater. An "absence of a significant alteration"
means that a change in a value did not achieve statistical
significance, using the appropriate statistical test. In some
examples, this is a p value >0.05. In other examples, an
"absence of a significant alteration" is an increase or a decrease
that is less than two standard deviations from the mean. In some
embodiments, an "increase" or "elevation," such as in the
proliferation of memory B cells, is about a 20%, 30%, 40% 50%, 60%,
70%, 80%, 90% or a 2-fold, 3-fold, 4-fold or 5-fold increase. In
one example, a "decrease" or "reduction" is a statistically
significant decline in a parameter, such as the number or
proliferation of memory B cells, as compared to a control. Suitable
statistical analyses are well known in the art, and include, but
are not limited to, Student's T test and ANOVA assays. In some
embodiments, a "decrease," such as in the proliferation of memory B
cells, is about a 20%, 30%, 40% 50%, 60%, 70%, 80%, 90% or a
2-fold, 3-fold, 4-fold or 5-fold decrease.
[0063] Antisense, Sense, and Antigene: DNA has two antiparallel
strands, a 5'.fwdarw.3' strand, referred to as the plus strand, and
a 3'.fwdarw.5' strand, referred to as the minus strand. Because RNA
polymerase adds nucleic acids in a 5'.fwdarw.3' direction, the
minus strand of the DNA serves as the template for the RNA during
transcription. Thus, an RNA transcript will have a sequence
complementary to the minus strand, and identical to the plus strand
(except that U is substituted for T).
[0064] Antisense molecules are molecules that are specifically
hybridizable or specifically complementary to either RNA or the
plus strand of DNA. Sense molecules are molecules that are
specifically hybridizable or specifically complementary to the
minus strand of DNA. Antigene molecules are either antisense or
sense molecules directed to a DNA target. An antisense RNA (asRNA)
is a molecule of RNA complementary to a sense (encoding) nucleic
acid molecule.
[0065] Amplification: When used in reference to a nucleic acid,
this refers to techniques that increase the number of copies of a
nucleic acid molecule in a sample or specimen. An example of
amplification is the polymerase chain reaction, in which a
biological sample collected from a subject is contacted with a pair
of oligonucleotide primers, under conditions that allow for the
hybridization of the primers to nucleic acid template in the
sample. The primers are extended under suitable conditions,
dissociated from the template, and then re-annealed, extended, and
dissociated to amplify the number of copies of the nucleic acid.
The product of in vitro amplification can be characterized by
electrophoresis, restriction endonuclease cleavage patterns,
oligonucleotide hybridization or ligation, and/or nucleic acid
sequencing, using standard techniques. Other examples of in vitro
amplification techniques include strand displacement amplification
(see U.S. Pat. No. 5,744,311); transcription-free isothermal
amplification (see U.S. Pat. No. 6,033,881); repair chain reaction
amplification (see WO 90/01069); ligase chain reaction
amplification (see EP-A-320 308); gap filling ligase chain reaction
amplification (see U.S. Pat. No. 5,427,930); coupled ligase
detection and PCR (see U.S. Pat. No. 6,027,889); and NASBA.TM. RNA
transcription-free amplification (see U.S. Pat. No. 6,025,134).
[0066] Antibody: A polypeptide ligand comprising at least a light
chain or heavy chain immunoglobulin variable region which
specifically recognizes and binds an epitope (e.g., an antigen,
such as a tumor or viral antigen or a fragment thereof). This
includes intact immunoglobulins and the variants and portions of
them well known in the art, such as Fab' fragments, F(ab)'.sub.2
fragments, single chain Fv proteins ("scFv"), and disulfide
stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein
in which a light chain variable region of an immunoglobulin and a
heavy chain variable region of an immunoglobulin are bound by a
linker, while in dsFvs, the chains have been mutated to introduce a
disulfide bond to stabilize the association of the chains. The term
also includes genetically engineered forms such as chimeric
antibodies (e.g., humanized murine antibodies), heteroconjugate
antibodies (e.g., bispecific antibodies). See also, Pierce Catalog
and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.);
Kuby, J., Immunology, 3.sup.rd Ed., W.H. Freeman & Co., New
York, 1997.
[0067] Typically, an immunoglobulin has a heavy and light chain.
Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). In
combination, the heavy and the light chain variable regions
specifically bind the antigen. Light and heavy chain variable
regions contain a "framework" region interrupted by three
hypervariable regions, also called "complementarity-determining
regions" or "CDRs". The extent of the framework region and CDRs has
been defined (see, Kabat et al., Sequences of Proteins of
Immunological Interest, U.S. Department of Health and Human
Services, 1991, which is hereby incorporated by reference). The
Kabat database is now maintained online. The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a species. The framework region of an antibody,
that is the combined framework regions of the constituent light and
heavy chains, serves to position and align the CDRs in
three-dimensional space.
[0068] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located. Thus, a V.sub.H CDR3 is located in
the variable domain of the heavy chain of the antibody in which it
is found, whereas a V.sub.L CDR1 is the CDR1 from the variable
domain of the light chain of the antibody in which it is found.
[0069] References to "V.sub.H" or "VH" refer to the variable region
of an immunoglobulin heavy chain, including that of an Fv, scFv,
dsFv or Fab. References to "V.sub.L" or "VL" refer to the variable
region of an immunoglobulin light chain, including that of an Fv,
scFv, dsFv or Fab.
[0070] A "monoclonal antibody" is an antibody produced by a single
clone of B-lymphocytes or by a cell into which the light and heavy
chain genes of a single antibody have been transfected. Monoclonal
antibodies are produced by methods known to those of skill in the
art, for instance by making hybrid antibody-forming cells from a
fusion of myeloma cells with immune spleen cells. Monoclonal
antibodies include humanized monoclonal antibodies.
[0071] A "humanized" immunoglobulin is an immunoglobulin including
a human framework region and one or more CDRs from a non-human
(such as a mouse, rat, or synthetic) immunoglobulin. The non-human
immunoglobulin providing the CDRs is termed a "donor," and the
human immunoglobulin providing the framework is termed an
"acceptor." In one embodiment, all the CDRs are from the donor
immunoglobulin in a humanized immunoglobulin. Constant regions need
not be present, but if they are, they must be substantially
identical to human immunoglobulin constant regions, i.e., at least
about 85-90%, such as about 95% or more identical. Hence, all parts
of a humanized immunoglobulin, except possibly the CDRs, are
substantially identical to corresponding parts of natural human
immunoglobulin sequences. A "humanized antibody" is an antibody
comprising a humanized light chain and a humanized heavy chain
immunoglobulin. A humanized antibody binds to the same antigen as
the donor antibody that provides the CDRs. The acceptor framework
of a humanized immunoglobulin or antibody may have a limited number
of substitutions by amino acids taken from the donor framework.
Humanized or other monoclonal antibodies can have additional
conservative amino acid substitutions which have substantially no
effect on antigen binding or other immunoglobulin functions.
Humanized immunoglobulins can be constructed by means of genetic
engineering (e.g., see U.S. Pat. No. 5,585,089).
[0072] A "neutralizing antibody" is an antibody that interferes
with any of the biological activities of a polypeptide, such as a
PD-1 polypeptide. For example, a neutralizing antibody can
interfere with the ability of a PD-1 polypeptide to reduce an
immune response such as the cytotoxicity of T cells. In several
examples, the neutralizing antibody can reduce the ability of a
PD-1 polypeptide to reduce an immune response by about 50%, about
70%, about 90% or more. Any standard assay to measure immune
responses, including those described herein, may be used to assess
potentially neutralizing antibodies.
[0073] Antigen: A compound, composition, or substance that can
stimulate the production of antibodies or a T cell response in an
animal, including compositions that are injected or absorbed into
an animal. An antigen reacts with the products of specific humoral
or cellular immunity, including those induced by heterologous
immunogens. The term "antigen" includes all related antigenic
epitopes. "Epitope" or "antigenic determinant" refers to a site on
an antigen to which B and/or T cells respond. In one embodiment, T
cells respond to the epitope, when the epitope is presented in
conjunction with an MHC molecule. Epitopes can be formed both from
contiguous amino acids or noncontiguous amino acids juxtaposed by
tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5, about 9, or
about 8-10 amino acids in a unique spatial conformation. Methods of
determining spatial conformation of epitopes include, for example,
x-ray crystallography and 2-dimensional nuclear magnetic
resonance.
[0074] An antigen can be a tissue-specific antigen, or a
disease-specific antigen. These terms are not exclusive, as a
tissue-specific antigen can also be a disease specific antigen. A
tissue-specific antigen is expressed in a limited number of
tissues, such as a single tissue. Specific, non-limiting examples
of a tissue specific antigen are a prostate specific antigen, a
uterine specific antigen, and/or a testes specific antigen. A
tissue specific antigen may be expressed by more than one tissue,
such as, but not limited to, an antigen that is expressed in more
than one reproductive tissue, such as in both prostate and uterine
tissue. A disease-specific antigen is expressed coincidentally with
a disease process. Specific non-limiting examples of a
disease-specific antigen are an antigen whose expression correlates
with, or is predictive of, tumor formation. A disease-specific
antigen can be an antigen recognized by T cells or B cells.
[0075] Antigen-presenting cell (APC): A cell that can present
antigen bound to MHC class I or class II molecules to T cells. APCs
include, but are not limited to, monocytes, macrophages, dendritic
cells, B cells, T cells and Langerhans cells. A T cell that can
present antigen to other T cells (including CD4+ and/or CD8+ T
cells) is an antigen presenting T cell (T-APC).
[0076] B Cells: A subset of lymphocytes, that is, white blood cells
(leukocytes). Mature B cells differentiate into plasma cells, which
produces antibodies, and memory B cells. A "B cell progenitor" is a
cell that can develop into a mature B cell. B cell progenitors
include stem cells, early pro-B cells, late pro-B cells, large
pre-B cells, small pre-B cells, and immature B cells and
transitional B cells. Generally, early pro-B cells (that express,
for example, CD43 or B220) undergo immunoglobulin heavy chain
rearrangement to become late pro B and pre B cells, and further
undergo immunoglobulin light chain rearrangement to become an
immature B cells. Immature B cells include T1 and T2 B cells. For
example, in mice, immature B cells include T1 B cells that are
AA41.sup.hiCD23.sup.lo cells. Another example of a mouse immature B
cell is a T2 B that is an AA41.sup.hiCD23.sup.hi cell. In humans,
immature B cells (for example, immature peripheral transitional B
cells) include CD38.sup.hi, IgD.sup.+, CD10.sup.+, CD24.sup.hi,
CD44.sup.lo, CD23.sup.lo and CD1.sup.lo cells. Thus, immature B
cells include B220 (CD45R) expressing cells wherein the light and
the heavy chain immunoglobulin genes are rearranged. In one
embodiment, immature B cells express CD45R, class II, IgM, CD19 and
CD40. Immature B cells do not exhibit surrogate light chain
expression, but do express Ig .alpha..beta. and RAG. Immature B
cells can develop into mature B cells, which can produce
immunoglobulins (e.g., IgA, IgG or IgM). Mature B cells have
acquired surface IgM and IgD, are capable of responding to antigen,
and express characteristic markers such as CD21 and CD23
(CD23.sup.hiCD21.sup.hi cells). B cells can be activated by agents
such as lippopolysaccharide (LPS) or IL-4 and antibodies to IgM.
Common biological sources of B cells and B cell progenitors include
bone marrow, peripheral blood, spleen and lymph nodes.
[0077] B cells that encounter antigen for the first time are known
as "naive" B cells; the cells have IgM and IgD on their cell
surfaces. After a B cell progenitor (e.g., a pre-committed small
lymphocyte) is stimulated by an antigen, it differentiates into a
blast cell, which differentiates into an immature plasma cell that
can differentiate into either a mature plasma cell or a memory B
cell. A mature plasma cell secretes immunoglobulins in response to
a specific antigen. A memory B cell is a B cell that undergoes
isotype switching and somatic hypermutation that is generally found
during a secondary immune response (a subsequent antigen exposure
following a primary exposure) but can also be detected during a
primary antigen response. The development of memory B cells takes
place in germinal centers (GC) of lymphoid follicles where
antigen-driven lymphocytes undergo somatic hypermutation and
affinity selection, presumably under the influence of helper T
cells. Memory B cells generally express CD27. Typically, memory B
cells also express high affinity antigen specific immunoglobulin (B
cell receptor) on their cell surface. Thus, memory B cells can be
CD20.sup.+CD27.sup.+, and include
CD20.sup.int/CD21.sup.+/CD27.sup.+ (resting memory),
CD20.sup.hi/CD21.sup.-/CD27.sup.+ (activated memory).
CD20.sup.hi/CD21.sup.-/CD27.sup.- cells are distinct
"unconventional or tissue memory" B cells.
[0078] Binding affinity: Affinity of an antibody for an antigen. In
one embodiment, affinity is calculated by a modification of the
Scatchard method described by Frankel et al., Mol. Immunol.,
16:101-106, 1979. In another embodiment, binding affinity is
measured by an antigen/antibody dissociation rate. In yet another
embodiment, a high binding affinity is measured by a competition
radioimmunoassay. In several examples, a high binding affinity is
at least about 1.times.10.sup.-8 M. In other embodiments, a high
binding affinity is at least about 1.5.times.10.sup.-8, at least
about 2.0.times.10.sup.-8, at least about 2.5.times.10.sup.-8, at
least about 3.0.times.10.sup.-8, at least about
3.5.times.10.sup.-8, at least about 4.0.times.10.sup.-8, at least
about 4.5.times.10.sup.-8, or at least about 5.0.times.10-8 M.
[0079] Binding or stable binding (oligonucleotide): An
oligonucleotide binds or stably binds to a target nucleic acid if a
sufficient amount of the oligonucleotide forms base pairs or is
hybridized to its target nucleic acid, to permit detection of that
binding. Binding can be detected by either physical or functional
properties of the target: oligonucleotide complex. Binding between
a target and an oligonucleotide can be detected by any procedure
known to one skilled in the art, including both functional and
physical binding assays. For instance, binding can be detected
functionally by determining whether binding has an observable
effect upon a biosynthetic process such as expression of a gene,
DNA replication, transcription, translation and the like.
[0080] Physical methods of detecting the binding of complementary
strands of DNA or RNA are well known in the art, and include such
methods as DNase I or chemical footprinting, gel shift and affinity
cleavage assays, Northern blotting, dot blotting and light
absorption detection procedures. For example, one method that is
widely used, because it is simple and reliable, involves observing
a change in light absorption of a solution containing an
oligonucleotide (or an analog) and a target nucleic acid at 220 to
300 nm as the temperature is slowly increased. If the
oligonucleotide or analog has bound to its target, there is a
sudden increase in absorption at a characteristic temperature as
the oligonucleotide (or analog) and the target disassociate from
each other, or melt.
[0081] The binding between an oligomer and its target nucleic acid
is frequently characterized by the temperature (T.sub.m) at which
50% of the oligomer is melted from its target. A higher (T.sub.m)
means a stronger or more stable complex relative to a complex with
a lower (T.sub.m).
[0082] Cancer or Tumor: A malignant neoplasm that has undergone
characteristic anaplasia with loss of differentiation, increase
rate of growth, invasion of surrounding tissue, and is capable of
metastasis. A reproductive cancer is a cancer that has its primary
origin in a reproductive tissue, such as in the uterus, testes,
ovary, prostate, fallopian tube, or penis. For example, prostate
cancer is a malignant neoplasm that arises in or from prostate
tissue, and uterine cancer is a malignant neoplasm that arises in
or from uterine tissue, and testicular cancer is a malignant
neoplasm that arises in the testes. Residual cancer is cancer that
remains in a subject after any form of treatment given to the
subject to reduce or eradicate thyroid cancer. Metastatic cancer is
a cancer at one or more sites in the body other than the site of
origin of the original (primary) cancer from which the metastatic
cancer is derived.
[0083] CD28 (Cluster of Differentiation 28): One of the molecules
expressed on T cells that provide co-stimulatory signals, which are
required for T cell activation. CD28 is the receptor for B7.1
(CD80) and B7.2 (CD86). When activated by Toll-like receptor
ligands, the B7.1 expression is upregulated in antigen presenting
cells (APCs). The B7.2 expression on antigen presenting cells is
constitutive. CD28 is the only B7 receptor constitutively expressed
on naive T cells.
[0084] Chemotherapy; chemotherapeutic agents: As used herein, any
chemical agent with therapeutic usefulness in the treatment of
diseases characterized by abnormal cell growth. Such diseases
include tumors, neoplasms and cancer as well as diseases
characterized by hyperplastic growth such as psoriasis. In one
embodiment, a chemotherapeutic agent is an agent of use in treating
neoplasms such as solid tumors. In one embodiment, a
chemotherapeutic agent is a radioactive molecule. One of skill in
the art can readily identify a chemotherapeutic agent of use (e.g.
see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in
Harrison's Principles of Internal Medicine, 14th edition; Perry et
al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2.sup.nd
ed., .COPYRGT. 2000 Churchill Livingstone, Inc; Baltzer L, Berkery
R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis,
Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds):
The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year
Book, 1993). The immunogenic polypeptides disclosed herein can be
used in conjunction with additional chemotherapeutic agents.
[0085] CD28: A cell surface antigen is known also as T90/44 antigen
or Tp44 that is expressed on T cells. CD28 is a receptor for
co-stimulatory proteins acting on T-cells. The natural ligand of
CD28 is a 44-54 kDa glycoprotein, called B7-1 or CD80. There is a
related molecule, B7-2. B7-1 is expressed on activated B cells and
other antigen-presenting cells. It is expressed by macrophages,
keratinocytes, T-cells, B-cells, peripheral blood dendritic and
Langerhans cells. B7-2 is found on blood dendritic and Langerhans
cells, B-cells, macrophages, Kupffer cells, activated monocytes and
various natural killer cell clones. Binding of B7 to CD28 on
T-cells delivers a costimulatory signal that triggers T-cell
proliferation
[0086] Control level (immune parameter): A baseline level of an
immune parameter. In some embodiments, and control level is the
level of a component of the immune system, such as memory B cells
or proliferating memory B cells, in the absence of a therapeutic
agent. A control level can be measured in a sample from a subject
that has not been treated with an agent of interest, or a sample
from a subject that has been treated with a control agent. The
control level can also be a standard value, such as a value
determined from an average of a large number of samples over time.
The control level can also be measured in a sample from a subject
treated with the specific dose of a therapeutic agent, wherein that
dose is not administered to the subject at the time the subject is
currently under evaluation. The control can be from the subject
under evaluation, or can be from a different subject.
[0087] Control level (polypeptide or nucleic acid): The level of a
molecule, such as a polypeptide or nucleic acid, normally found in
nature under a certain condition and/or in a specific genetic
background. In certain embodiments, a control level of a molecule
can be measured in a cell or specimen that has not been subjected,
either directly or indirectly, to a treatment. In some examples, a
control level can be the level in a cell not contacted with the
agent, such as a PD-1 antagonist. In additional examples, a control
level can be the level in a subject not administered the PD-1
antagonist.
[0088] DNA (deoxyribonucleic acid): DNA is a long chain polymer
which comprises the genetic material of most living organisms (some
viruses have genes comprising ribonucleic acid (RNA)). The
repeating units in DNA polymers are four different nucleotides,
each of which comprises one of the four bases, adenine, guanine,
cytosine and thymine bound to a deoxyribose sugar to which a
phosphate group is attached. Triplets of nucleotides (referred to
as codons) code for each amino acid in a polypeptide, or for a stop
signal. The term codon is also used for the corresponding (and
complementary) sequences of three nucleotides in the mRNA into
which the DNA sequence is transcribed.
[0089] Unless otherwise specified, any reference to a DNA molecule
is intended to include the reverse complement of that DNA molecule.
Except where single-strandedness is required by the text herein,
DNA molecules, though written to depict only a single strand,
encompass both strands of a double-stranded DNA molecule.
[0090] Detecting or detection (cell or biomolecule): Refers to
quantitatively or qualitatively determining the presence of a
biomolecule or specific cell type, such as a memory B cell, under
investigation. For example, quantitatively or qualitatively
determining the presence of memory B cells in a sample from a
subject, or detecting proliferating memory B cells. Generally,
detection of a biological molecule, such as a protein, nucleic
acid, or detecting a specific cell type or cell proliferation,
requires performing a biological assay and not simple observation.
For example, assays that utilize antibodies or nucleic acid probes
(which can both be labeled), or can be used to detect proteins or
cells, respectively. Diagnosing or diagnosis of the efficacy of
treatment with a PD-1 antagonist involves detecting a significant
change in a cell or biomolecule, such as the proliferation of
memory B cells.
[0091] Encode: A polynucleotide is said to encode a polypeptide if,
in its native state or when manipulated by methods well known to
those skilled in the art, it can be transcribed and/or translated
to produce the mRNA for and/or the polypeptide or a fragment
thereof. The anti-sense strand is the complement of such a nucleic
acid, and the encoding sequence can be deduced therefrom.
[0092] Expression: The process by which a gene's coded information
is converted into the structures present and operating in the cell.
Expressed genes include those that are transcribed into mRNA and
then translated into protein and those that are transcribed into
RNA but not translated into protein (for example, siRNA, transfer
RNA and ribosomal RNA). Thus, expression of a target sequence, such
as a gene or a promoter region of a gene, can result in the
expression of an mRNA, a protein, or both. The expression of the
target sequence can be inhibited or enhanced (decreased or
increased).
[0093] Expression Control Sequences: Nucleic acid sequences that
regulate the expression of a heterologous nucleic acid sequence to
which it is operatively linked. Expression control sequences are
operatively linked to a nucleic acid sequence when the expression
control sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus,
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signals, elements for
the maintenance of the correct reading frame of that gene to permit
proper translation of mRNA, and stop codons. The term "control
sequences" is intended to include, at a minimum, components whose
presence can influence expression, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences. Expression control
sequences can include a promoter.
[0094] A promoter is a minimal sequence sufficient to direct
transcription. Also included are those promoter elements which are
sufficient to render promoter-dependent gene expression
controllable for cell-type specific, tissue-specific, or inducible
by external signals or agents; such elements may be located in the
5' or 3' regions of the gene. Both constitutive and inducible
promoters are included (see e.g., Bitter et al., Methods in
Enzymology 153:516-544, 1987). For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like
can be used. In one embodiment, when cloning in mammalian cell
systems, promoters derived from the genome of mammalian cells (such
as the metallothionein promoter) or from mammalian viruses (such as
the retrovirus long terminal repeat; the adenovirus late promoter;
the vaccinia virus 7.5K promoter) can be used. Promoters produced
by recombinant DNA or synthetic techniques can also be used to
provide for transcription of the nucleic acid sequences.
[0095] Heterologous: Originating from separate genetic sources or
species. Generally, an antibody that specifically binds to a
protein of interest will not specifically bind to a heterologous
protein.
[0096] Host cells: Cells in which a vector can be propagated and
its DNA expressed. The cell may be prokaryotic or eukaryotic. The
cell can be mammalian, such as a human cell. The term also includes
any progeny of the subject host cell. It is understood that all
progeny may not be identical to the parental cell since there may
be mutations that occur during replication. However, such progeny
are included when the term "host cell" is used.
[0097] Immune response: A response of a cell of the immune system,
such as a B cell, T cell, or monocyte, to a stimulus. In one
embodiment, the response is specific for a particular antigen (an
"antigen-specific response"). In one embodiment, an immune response
is a T cell response, such as a CD4+ response or a CD8+ response.
In another embodiment, the response is a B cell response, and
results in the production of specific antibodies, or the
proliferation of memory B cells. A B cell response can be a memory
B cell response or a plasma B cell response. An example of a plasma
B cell response is the production of antibody. An example of a
response of a memory B cell is proliferation of memory B cells.
[0098] "Unresponsiveness" with regard to immune cells includes
refractivity of immune cells to stimulation, such as stimulation
via an activating receptor or a cytokine. Unresponsiveness can
occur, for example, because of exposure to immunosuppressants or
exposure to high doses of antigen. As used herein, the term
"anergy" or "tolerance" includes refractivity to activating
receptor-mediated stimulation. Such refractivity is generally
antigen-specific and persists after exposure to the tolerizing
antigen has ceased.
[0099] For example, anergy in T cells (as opposed to
unresponsiveness) is characterized by lack of cytokine production
(such as IL-2). T cell anergy occurs when T cells are exposed to
antigen and receive a first signal (a T cell receptor or CD-3
mediated signal) in the absence of a second signal (a costimulatory
signal). Under these conditions, re-exposure of the cells to the
same antigen (even if exposure occurs in the presence of a
costimulatory molecule) results in failure to produce cytokines
and, thus, failure to proliferate. Anergic T cells can, however,
mount responses to unrelated antigens and can proliferate if
cultured with cytokines (such as IL-2). For example, T cell anergy
can also be observed by the lack of IL-2 production by T
lymphocytes as measured by ELISA or by a proliferation assay using
an indicator cell line. Alternatively, a reporter gene construct
can be used. For example, anergic T cells fail to initiate IL-2
gene transcription induced by a heterologous promoter under the
control of the 5' IL-2 gene enhancer or by a multimer of the API
sequence that can be found within the enhancer (Kang et al. Science
257:1134, 1992). Anergic antigen specific T cells may have a
reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or even 100% in cytotoxic activity relative a corresponding
control antigen specific T cell.
[0100] Immunogenic peptide: A peptide which comprises an
allele-specific motif or other sequence such that the peptide will
bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL")
response, or a B cell response (e.g. antibody production or memory
B cell proliferation) specific to the antigen from which the
immunogenic peptide is derived.
[0101] In one embodiment, immunogenic peptides are identified using
sequence motifs or other methods, such as neural net or polynomial
determinations, known in the art. Typically, algorithms are used to
determine the "binding threshold" of peptides to select those with
scores that give them a high probability of binding at a certain
affinity and will be immunogenic. The algorithms are based either
on the effects on MHC binding of a particular amino acid at a
particular position, the effects on antibody binding of a
particular amino acid at a particular position, or the effects on
binding of a particular substitution in a motif-containing peptide.
Within the context of an immunogenic peptide, a "conserved residue"
is one which appears in a significantly higher frequency than would
be expected by random distribution at a particular position in a
peptide. In one embodiment, a conserved residue is one where the
MHC structure may provide a contact point with the immunogenic
peptide.
[0102] Immunogenic peptides can also be identified by measuring
their binding to a specific MHC protein (e.g. HLA-A02.01) and by
their ability to stimulate CD4 and/or CD8 when presented in the
context of the MHC protein.
[0103] Immunogenic composition: A composition comprising an
immunogenic polypeptide or a nucleic acid encoding the immunogenic
polypeptide that induces a measurable CTL response against cells
expressing the polypeptide, or induces a measurable B cell response
(such as production of antibodies that specifically bind the
polypeptide or proliferation of memory B cells) against the
polypeptide. For in vitro use, the immunogenic composition can
consist of the isolated nucleic acid, vector including the nucleic
acid/or immunogenic peptide. For in vivo use, the immunogenic
composition will typically comprise the nucleic acid, vector
including the nucleic acid, and or immunogenic polypeptide, in
pharmaceutically acceptable carriers, and/or other agents. An
immunogenic composition can optionally include an adjuvant, a PD-1
antagonist, a costimulatory molecule, or a nucleic acid encoding a
costimulatory molecule. A polypeptide, or nucleic acid encoding the
polypeptide, can be readily tested for its ability to induce a CTL
by art-recognized assays.
[0104] Immunologically reactive conditions (in vitro): Includes
"conditions sufficient to form an immune complex" which allow an
antibody raised against a particular epitope to bind to that
epitope to a detectably greater degree than, and/or to the
substantial exclusion of, binding to substantially all other
epitopes. Immunologically reactive conditions are dependent upon
the format of the antibody binding reaction and typically are those
utilized in immunoassay protocols (such as ELISA or
radioimmunoassay), FACS or those conditions encountered in vivo.
See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York (1988), for a description of
immunoassay formats and conditions. The immunologically reactive
conditions employed in the methods disclosed herein are
"physiological conditions" which include reference to conditions
(e.g., temperature, osmolarity, pH) that are typical inside a
living mammal or a mammalian cell. While it is recognized that some
organs are subject to extreme conditions, the intra-organismal and
intracellular environment normally lies around pH 7 (i.e., from pH
6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the
predominant solvent, and exists at a temperature above 0.degree. C.
and below 50.degree. C. Osmolarity is within the range that is
supportive of cell viability and proliferation.
[0105] Inhibiting or treating a disease: Inhibiting a disease, such
as tumor growth or a persistent infection, refers to inhibiting the
full development of a disease or lessening the physiological
effects of the disease process. In several examples, inhibiting or
treating a disease refers to lessening symptoms of a tumor or an
infection with a pathogen. For example, cancer treatment can
prevent the development of paraneoplastic syndrome in a person who
is known to have a cancer, or lessening a sign or symptom of the
tumor. In another embodiment, treatment of an infection can refer
to inhibiting development or lessening a symptom of the infection.
"Treatment" refers to a therapeutic intervention that ameliorates a
sign or symptom of a disease or pathological condition related to
the disease. Therapeutic vaccination refers to administration of an
agent to a subject already infected with a pathogen. The subject
can be asymptomatic, so that the treatment prevents the development
of a symptom. The therapeutic vaccine can also reduce the severity
of one or more existing symptoms, or reduce pathogen load.
[0106] Infectious disease: Any disease caused by an infectious
agent. Examples of infectious pathogens include, but are not
limited to: viruses, bacteria, mycoplasma and fungi. In a
particular example, it is a disease caused by at least one type of
infectious pathogen. In another example, it is a disease caused by
at least two different types of infectious pathogens. Infectious
diseases can affect any body system, be acute (short-acting) or
chronic/persistent (long-acting), occur with or without fever,
strike any age group, and overlap each other.
[0107] Viral diseases commonly occur after immunosupression due to
re-activation of viruses already present in the recipient.
Particular examples of persistent viral infections include, but are
not limited to, cytomegalovirus (CMV) pneumonia, enteritis and
retinitis; Epstein-Barr virus (EBV) lymphoproliferative disease;
chicken pox/shingles (caused by varicella zoster virus, VZV); HSV-1
and -2 mucositis; HSV-6 encephalitis, BK-virus hemorrhagic
cystitis; viral influenza; pneumonia from respiratory syncytial
virus (RSV); AIDS (caused by HIV); and hepatitis A, B or C.
[0108] Additional examples of infectious virus include:
Retroviridae; Picornaviridae (for example, polio viruses, hepatitis
A virus; enteroviruses, human coxsackie viruses, rhinoviruses,
echoviruses); Calciviridae (such as strains that cause
gastroenteritis); Togaviridae (for example, equine encephalitis
viruses, rubella viruses); Flaviridae (for example, dengue viruses,
encephalitis viruses, yellow fever viruses); Coronaviridae (for
example, coronaviruses); Rhabdoviridae (for example, vesicular
stomatitis viruses, rabies viruses); Filoviridae (for example,
ebola viruses); Paramyxoviridae (for example, parainfluenza
viruses, mumps virus, measles virus, respiratory syncytial virus);
Orthomyxoviridae (for example, influenza viruses); Bungaviridae
(for example, Hantaan viruses, bunga viruses, phleboviruses and
Nairo viruses); Arena viridae (hemorrhagic fever viruses);
Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);
Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae
(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);
Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex
virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus
(CMV), herpes viruses); Poxviridae (variola viruses, vaccinia
viruses, pox viruses); and Iridoviridae (such as African swine
fever virus); and unclassified viruses (for example, the
etiological agents of Spongiform encephalopathies, the agent of
delta hepatitis (thought to be a defective satellite of hepatitis B
virus), the agents of non-A, non-B hepatitis (class 1=internally
transmitted; class 2=parenterally transmitted (i.e., Hepatitis C);
Norwalk and related viruses, and astroviruses).
[0109] Examples of fungal infections include but are not limited
to: aspergillosis; thrush (caused by Candida albicans);
cryptococcosis (caused by Cryptococcus); and histoplasmosis. Thus,
examples of infectious fungi include, but are not limited to,
Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida
albicans.
[0110] Examples of infectious bacteria include: Helicobacter
pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria
sps (such as. M. tuberculosis, M. avium, M. intracellulare, M.
kansaii, M. gordonae), Staphylococcus aureus, Neisseria
gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,
Streptococcus pyogenes (Group A Streptococcus), Streptococcus
agalactiae (Group B Streptococcus), Streptococcus (viridans group),
Streptococcus faecalis, Streptococcus bovis, Streptococcus
(anaerobic sps.), Streptococcus pneumoniae, pathogenic
Campylobacter sp., Enterococcus sp., Haemophilus influenzae,
Bacillus anthracis, corynebacterium diphtheriae, corynebacterium
sp., Erysipelothrix rhusiopathiae, Clostridium perfringers,
Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae,
Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum,
Streptobacillus moniliformis, Treponema pallidium, Treponema
pertenue, Leptospira, and Actinomyces israelli. Other infectious
organisms (such as protists) include: Plasmodium falciparum: and
Toxoplasma gondii.
[0111] A "persistent infection" is an infection in which the
infectious agent (such as a virus, mycoplasma, bacterium, parasite,
or fungus) is not cleared or eliminated from the infected host,
even after the induction of an immune response. Persistent
infections can be chronic infections, latent infections, or slow
infections. Latent infection is characterized by the lack of
demonstrable infectious virus between episodes of recurrent
disease. Chronic infection is characterized by the continued
presence of infectious virus following the primary infection and
can include chronic or recurrent disease. Slow infection is
characterized by a prolonged incubation period followed by
progressive disease. Unlike latent and chronic infections, slow
infection may not begin with an acute period of viral
multiplication. While acute infections are relatively brief
(lasting a few days to a few weeks) and resolved from the body by
the immune system, persistent infections can last for example, for
months, years, or even a lifetime. These infections may also recur
frequently over a long period of time, involving stages of silent
and productive infection without cell killing or even producing
excessive damage to the host cells. Persistent infections often
involve stages of both silent and productive infection without
rapidly killing or even producing excessive damage of the host
cells. During persistent viral infections, the viral genome can be
either stably integrated into the cellular DNA or maintained
episomally. Persistent infection occurs with viruses such as human
T-Cell leukemia viruses, Epstein-Barr virus, cytomegalovirus,
herpesviruses, varicella-zoster virus, measles, papovaviruses,
prions, hepatitis viruses, adenoviruses, parvoviruses and
papillomaviruses.
[0112] The causative infectious agents may also be detected in the
host (such as inside specific cells of infected individuals) even
after the immune response has resolved, using standard techniques.
Mammals are diagnosed as having a persistent infection according to
any standard method known in the art and described, for example, in
U.S. Pat. Nos. 6,368,832, 6,579,854, and 6,808,710 and U.S. Patent
Application Publication Nos. 20040137577, 20030232323, 20030166531,
20030064380, 20030044768, 20030039653, 20020164600, 20020160000,
20020110836, 20020107363, and 20020106730, all of which are hereby
incorporated by reference.
[0113] "Alleviating a symptom of a persistent infection" is
ameliorating any condition or symptom associated with the
persistent infection. Alternatively, alleviating a symptom of a
persistent infection can involve reducing the infectious microbial
(such as viral, bacterial, fungal or parasitic) load in the subject
relative to such load in an untreated control. As compared with an
equivalent untreated control, such reduction or degree of
prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%,
or 100% as measured by any standard technique. Desirably, the
persistent infection is completely cleared as detected by any
standard method known in the art, in which case the persistent
infection is considered to have been treated. A patient who is
being treated for a persistent infection is one who a medical
practitioner has diagnosed as having such a condition. Diagnosis
may be by any suitable means. Diagnosis and monitoring may involve,
for example, detecting the level of microbial load in a biological
sample (for example, a tissue biopsy, blood test, or urine test),
detecting the level of a surrogate marker of the microbial
infection in a biological sample, detecting symptoms associated
with persistent infections, or detecting immune cells involved in
the immune response typical of persistent infections (for example,
detection of antigen specific T cells that are anergic and/or
functionally impaired). A patient in whom the development of a
persistent infection is being prevented may or may not have
received such a diagnosis. One in the art will understand that
these patients may have been subjected to the same standard tests
as described above or may have been identified, without
examination, as one at high risk due to the presence of one or more
risk factors (such as family history or exposure to infectious
agent).
[0114] Isolated: An "isolated" biological component (such as a
nucleic acid or protein or organelle) has been substantially
separated or purified away from other biological components in the
cell of the organism in which the component naturally occurs, i.e.,
other chromosomal and extra-chromosomal DNA and RNA, proteins and
organelles. Nucleic acids and proteins that have been "isolated"
include nucleic acids and proteins purified by standard
purification methods. The term also embraces nucleic acids and
proteins prepared by recombinant expression in a host cell as well
as chemically synthesized nucleic acids.
[0115] A "purified antibody" is at least 60%, by weight free from
proteins and naturally occurring organic molecules with which it is
naturally associated. In some examples the preparation is at least
about 75%, at least about 80%, at least about 90%, at least about
95%, or at least about 99%, by weight of antibody, such as a PD-1,
PD-L1, or PD-L2 specific antibody. A purified antibody can be
obtained, for example, by affinity chromatography using
recombinantly-produced protein or conserved motif peptides and
standard techniques.
[0116] Label: A detectable compound or composition that is
conjugated directly or indirectly to another molecule, such as an
antibody or a protein, to facilitate detection of that molecule.
Specific, non-limiting examples of labels include fluorescent tags,
enzymatic linkages, and radioactive isotopes. In one example, a
"labeled antibody" refers to incorporation of another molecule in
the antibody. For example, the label is a detectable marker, such
as the incorporation of a radiolabeled amino acid or attachment to
a polypeptide of biotinyl moieties that can be detected by marked
avidin (for example, streptavidin containing a fluorescent marker
or enzymatic activity that can be detected by optical or
colorimetric methods). Various methods of labeling polypeptides and
glycoproteins are known in the art and may be used. Examples of
labels for polypeptides include, but are not limited to, the
following: radioisotopes or radionucleotides (such as .sup.35S or
.sup.131I), fluorescent labels (such as fluorescein isothiocyanate
(FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as
horseradish peroxidase, beta-galactosidase, luciferase, alkaline
phosphatase), chemiluminescent markers, biotinyl groups,
predetermined polypeptide epitopes recognized by a secondary
reporter (such as a leucine zipper pair sequences, binding sites
for secondary antibodies, metal binding domains, epitope tags), or
magnetic agents, such as gadolinium chelates. In some embodiments,
labels are attached by spacer arms of various lengths to reduce
potential steric hindrance.
[0117] Lymphocytes: A type of white blood cell that is involved in
the immune defenses of the body. There are two main types of
lymphocytes: B cells and T cells.
[0118] Major Histocompatibility Complex (MHC): A generic
designation meant to encompass the histocompatibility antigen
systems described in different species, including the human
leukocyte antigens ("HLA").
[0119] Mammal: This term includes both human and non-human mammals.
Similarly, the term "subject" includes both human and veterinary
subjects.
[0120] Mean Fluorescence Intensity (flow cytometry): Flow cytometry
is concerned with the measurement of the light intensity of a cell
or particle, whether it be scattered laser light or fluorescence
emitted by a fluorochrome. Light is detected by a photomultiplier
tube (PMT) which converts it via an amplifier to a voltage that is
proportional to the original fluorescence intensity and the voltage
on the PMT. These voltages, which are a continuous distribution,
are converted to a discrete distribution by an Analog to Digital
converter (ADC) which places each signal into a specific channel
depending on the level of fluorescence. The greater the resolution
of the ADC, the closer this reflects the continuous
distribution.
[0121] Flow cytometric data can be displayed using either a linear
or a logarithmic scale. The use of a logarithmic scale is indicated
in most biological situations where distributions are skewed to the
right. In this case the effect is to normalize the distribution--it
is said to be Log Normal and the data has been log-transformed.
Linear signals come through a linear amplifier but the logarithmic
transformation may be achieved either by a logarithmic amplifier or
by the use of Look Up Tables (LUT). Most ADCs in analytical
cytometers are 10-bit, i.e., they divide data into 2e10 or 1024
channels, although there is a growing trend to use 12- or 14-bit
ADCs to give greater resolution of data.
[0122] Data from a single data channel (scatter or fluorescence) is
displayed as a histogram in which the x axis is divided into 1024
channels (for a 10-bit ADC). If the data is in a linear scale, the
channel number and the linear value for that channel will be easily
obtained. On a logarithmic scale, the x axis is still divided into
1024 channels but is displayed as a 4-log decade scale (in general
4 log decades are used).
[0123] To quantify flow cytometric data the measures of the
distribution of a population are utilized. Generally, the measures
of central tendency are the mean and the median. The mean is the
`average` and can be either arithmetic or geometric. The arithmetic
mean is calculated as Sigma(x)/n, and the geometric mean as n
root(a1.times.a2.times.a3 . . . an). In general, with log-amplified
data the geometric mean is used as it takes into account the
weighting of the data distribution, and the arithmetic mean is used
for linear data or data displayed on a linear scale. The median is
the central value, i.e., the 50th percentile, where half the values
are above and half below. A cell with "high" expression and "low"
expression can be determined relatively depending on the
fluorescence of the entire population; these parameters are readily
visualized on plots of flow cytometric data.
[0124] Neoplasm: An abnormal cellular proliferation, which includes
benign and malignant tumors, as well as other proliferative
disorders.
[0125] Neutralizing antibody: An antibody which reduces the
infectious titer of an infectious agent by binding to a specific
antigen on the infectious agent. In some examples the infectious
agent is a virus, bacteria or fungus.
[0126] Oligonucleotide: A linear polynucleotide sequence of up to
about 100 nucleotide bases in length.
[0127] Open reading frame (ORF): A series of nucleotide triplets
(codons) coding for amino acids without any internal termination
codons. These sequences are usually translatable into a
peptide.
[0128] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
[0129] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers of use are conventional. Remington's
Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co.,
Easton, Pa., 15th Edition (1975), describes compositions and
formulations suitable for pharmaceutical delivery of the fusion
proteins herein disclosed.
[0130] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(such as powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0131] A "therapeutically effective amount" is a quantity of a
composition or a cell to achieve a desired biological effect in a
subject being treated. For instance, this can be the amount of a
PD-1 antagonist necessary to induce an immune response, inhibit
tumor growth, induce memory B cell proliferation, or to measurably
alter outward symptoms of a tumor or persistent infection. When
administered to a subject, a dosage will generally be used that
will achieve target tissue concentrations (for example, in
lymphocytes) that has been shown to achieve an in vitro effect.
[0132] In particular examples, a therapeutically effective amount
is an amount of an agent, such as PD-1 antagonist, effective to
induce the proliferation of memory B cells. In another particular
example, a therapeutically effective amount is an amount of a PD-1
antagonist that alters a sign or a symptom of a disorder in a
subject, such as a disorder that can be improved by increasing a
memory B cell response and/or a T cell response.
[0133] An effective amount of an agent such as a PD-1 antagonist
can be administered in a single dose, or in several doses, for
example daily, during a course of treatment. However, the effective
amount of a PD-1 antagonist will be dependent on the subject being
treated, the severity and type of the condition being treated, and
the manner of administration. The methods disclosed herein have
equal application in medical and veterinary settings. Therefore,
the general term "subject being treated" is understood to include
all organisms (e.g., humans, apes, dogs, cats, horses, and cows)
that require an increase in the desired biological effect, such as
an enhanced immune response.
[0134] Polynucleotide: The term polynucleotide or nucleic acid
sequence refers to a polymeric form of nucleotide at least 10 bases
in length. A recombinant polynucleotide includes a polynucleotide
that is not immediately contiguous with both of the coding
sequences with which it is immediately contiguous (one on the 5'
end and one on the 3' end) in the naturally occurring genome of the
organism from which it is derived. The term therefore includes, for
example, a recombinant DNA which is incorporated into a vector;
into an autonomously replicating plasmid or virus; or into the
genomic DNA of a prokaryote or eukaryote, or which exists as a
separate molecule (e.g., a cDNA) independent of other sequences.
The nucleotides can be ribonucleotides, deoxyribonucleotides, or
modified forms of either nucleotide. The term includes single- and
double-stranded forms of DNA.
[0135] Polypeptide: Any chain of amino acids, regardless of length
or post-translational modification (e.g., glycosylation or
phosphorylation). A polypeptide can be between 3 and 30 amino acids
in length. In one embodiment, a polypeptide is from about 7 to
about 25 amino acids in length. In yet another embodiment, a
polypeptide is from about 8 to about 10 amino acids in length. In
yet another embodiment, a peptide is about 9 amino acids in length.
With regard to polypeptides, "comprises" indicates that additional
amino acid sequence or other molecules can be included in the
molecule, "consists essentially of" indicates that additional amino
acid sequences are not included in the molecule, but that other
agents (such as labels or chemical compounds) can be included, and
"consists of" indicates that additional amino acid sequences and
additional agents are not included in the molecule.
[0136] Proliferation: The division of a cell to produce progeny,
which can be measured in a number of ways known in the art. This
includes, but is not limited to, assays that count the total number
of cells, assays that count the number of cells of a specific cell
type, KI-67 assays, thymidine incorporation, and bromodeoxyuridine
assays.
[0137] Programmed Death (PD)-1: A protein that forms a complex with
PD-L1 or PD-L2 protein and is involved in an immune response, such
as the co-stimulation of T cells. Generally, PD-1 protein are
substantially identical to the naturally occurring (wild type) PD-1
(see, for example, Ishida et al. EMBO J. 11:3887-3895, 1992,
Shinohara et al. Genomics 23:704-706, 1994; and U.S. Pat. No.
5,698,520, all incorporated by reference herein in their entirety).
In several examples, PD-1 signaling reduces, for example, CD8+ T
cell cytoxicity by reducing T cell proliferation, cytokine
production, or viral clearance. Thus, a PD-1 polypeptide can reduce
CD8+ T cell cytotoxic activity by at least 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or more than 100% below control levels as
measured by any standard method.
[0138] As used herein, the term "activity" with respect to a PD-1
polypeptide or protein includes any activity which is inherent to
the naturally occurring PD-1 protein, such as the ability to
modulate an inhibitory signal in an activated immune cell, such as
by engaging a natural ligand on an antigen presenting cell. Such
modulation of an inhibitory signal in an immune cell results in
modulation of proliferation and/or survival of an immune cell
and/or cytokine secretion by an immune cell. PD-1 protein can also
modulate a costimulatory signal by competing with a costimulatory
receptor for binding of a B7 molecule. Thus, the term "PD-1
activity" includes the ability of a PD-1 polypeptide or protein to
bind its natural ligand(s), the ability to modulate immune cell
costimulatory or inhibitory signals, and the ability to modulate
the immune response.
[0139] "Reduce the expression or activity of PD-1" refers to a
decrease in the level or biological activity of PD-1 relative to
the level or biological activity of PD-1 protein in a control, such
as an untreated subject or sample. In specific examples, the level
or activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, or even greater than 100%, relative to an
untreated control. For example, the biological activity of PD-1
protein is reduced if binding of PD-1 protein to PD-L1, PD-L2, or
both is reduced, thereby resulting in a reduction in PD-1 signaling
and therefore resulting in an increase in CD8+ T cell
cytotoxicity.
[0140] A "PD-1 gene" is a nucleic acid that encodes a PD-1 protein.
A "PD-1 fusion gene" is a PD-1 coding region operably linked to a
second, heterologous nucleic acid sequence. A PD-1 fusion gene can
include a PD-1 promoter, or can include a heterologous promoter. In
some embodiments, the second, heterologous nucleic acid sequence is
a reporter gene, that is, a gene whose expression may be assayed;
reporter genes include, without limitation, those encoding
glucuronidase (GUS), luciferase, chloramphenicol transacetylase
(CAT), green fluorescent protein (GFP), alkaline phosphatase, and
.beta.-galactosidase.
[0141] Sample (Biological sample): Includes biological samples
containing fluids, tissues, cells, and subcomponents thereof, such
as DNA, RNA, and proteins. For example, common samples in the
context of the present invention include bone marrow, spleen, lymph
node, blood, e.g., peripheral blood (but can also include any other
source from which B cells or B cell progenitors can be isolated,
including: urine, saliva, tissue biopsy, surgical specimens, fine
needle aspirates, autopsy material, and the like).
[0142] Specific binding agent: An agent that binds substantially
only to a defined target. Thus a PD-1 specific binding agent is an
agent that binds substantially to a PD-1 polypeptide and not
unrelated polypeptides. In one embodiment, the specific binding
agent is a monoclonal or polyclonal antibody that specifically
binds the PD-1, PD-L1 OR PD-L2 polypeptide.
[0143] The term "specifically binds" refers, with respect to an
antigen such as PD-1, to the preferential association of an
antibody or other ligand, in whole or part, with a cell or tissue
bearing that antigen and not to cells or tissues lacking that
antigen. It is, of course, recognized that a certain degree of
non-specific interaction may occur between a molecule and a
non-target cell or tissue. Nevertheless, specific binding may be
distinguished as mediated through specific recognition of the
antigen. Although selectively reactive antibodies bind antigen,
they may do so with low affinity. Specific binding results in a
much stronger association between the antibody (or other ligand)
and cells bearing the antigen than between the antibody (or other
ligand) and cells lacking the antigen. Specific binding typically
results in greater than 2-fold, such as greater than 5-fold,
greater than 10-fold, or greater than 100-fold increase in amount
of bound antibody or other ligand (per unit time) to a cell or
tissue bearing the PD-1 polypeptide as compared to a cell or tissue
lacking the polypeptide. Specific binding to a protein under such
conditions requires an antibody that is selected for its
specificity for a particular protein. A variety of immunoassay
formats are appropriate for selecting antibodies or other ligands
specifically immunoreactive with a particular protein. For example,
solid-phase ELISA immunoassays are routinely used to select
monoclonal antibodies specifically immunoreactive with a protein.
See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York (1988), for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity.
[0144] T Cell: A white blood cell critical to the immune response.
T cells include, but are not limited to, CD4.sup.+ T cells and
CD8.sup.+ T cells. A CD4.sup.+ T lymphocyte is an immune cell that
carries a marker on its surface known as "cluster of
differentiation 4" (CD4). These cells, also known as helper T
cells, help orchestrate the immune response, including antibody
responses as well as killer T cell responses. CD8.sup.+ T cells
carry the "cluster of differentiation 8" (CD8) marker. In one
embodiment, a CD8+ T cell is a cytotoxic T lymphocyte. In another
embodiment, a CD8+ cell is a suppressor T cell. A T cell is
"activated" when it can respond to a specific antigen of interest
presented on an antigen presenting cells.
[0145] Transduced/Transfected: A transduced cell is a cell into
which has been introduced a nucleic acid molecule by molecular
biology techniques. As used herein, the term transduction
encompasses all techniques by which a nucleic acid molecule might
be introduced into such a cell, including transfection with viral
vectors, transformation with plasmid vectors, and introduction of
naked DNA by electroporation, lipofection, and particle gun
acceleration.
[0146] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. A vector may also
include one or more nucleic acids encoding a selectable marker and
other genetic elements known in the art. Vectors include plasmid
vectors, including plasmids for expression in gram negative and
gram positive bacterial cells. Exemplary vectors include those for
expression in E. coli and Salmonella. Vectors also include viral
vectors, such as, but are not limited to, retrovirus, orthopox,
avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha
virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus
vectors.
[0147] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. The term "comprises"
means "includes." All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including explanations of terms, will control. In addition, the
materials, methods, and examples are illustrative only and not
intended to be limiting.
RELATED APPLICATIONS
[0148] The disclosed subject matter is also related to the subject
matter of PCT Application No. PCT/US2007/088851, filed Dec. 26,
2007, U.S. Provisional Application No. 60/688,872, filed Jun. 8,
2005, U.S. Utility application Ser. No. 11/449,919, filed Jun. 8,
2006, and PCT Application No. PCT/US2006/22423, Jun. 8, 2006. This
application is also related to U.S. Provisional Application No.
60/877,518, filed Dec. 27, 2006. These prior applications are
incorporated herein by reference in their entirety.
PD-1 Antagonists
[0149] The methods disclosed herein involve the use of inhibitors
of the PD-1 pathway (PD-1 antagonists). PD-1 molecules are members
of the immunoglobulin gene superfamily. The human PD-1 has an
extracellular region containing an immunoglobulin superfamily
domain, a transmembrane domain, and an intracellular region
including an immunoreceptor tyrosine-based inhibitory motif (ITIM)
((Ishida et al., EMBO J. 11:3887, 1992; Shinohara et al., Genomics
23:704, 1994; U.S. Pat. No. 5,698,520). These features also define
a larger family of molecules, called the immunoinhibitory
receptors, which also includes gp49B, PIR-B, and the killer
inhibitory receptors (KIRs) (Vivier and Daeron (1997) Immunol.
Today 18:286). Without being bound by theory, it is believed that
the tyrosyl phosphorylated ITIM motif of these receptors interacts
with the S112-domain containing phosphatase, which leads to
inhibitory signals. A subset of these immuno-inhibitory receptors
bind to major histocompatibility complex (MHC) molecules, such as
the KIRs, and CTLA4 binds to B7-1 and B7-2.
[0150] In humans, PD-1 is a 50-55 kDa type I transmembrane receptor
that was originally identified in a T cell line undergoing
activation-induced apoptosis. PD-1 is expressed on T cells, B
cells, and macrophages. The ligands for PD-1 are the B7 family
members PD-ligand 1 (PD-L1, also known as B7-H1) and PD-L2 (also
known as B7-DC).
[0151] In vivo, PD-1 is expressed on activated T cells, B cells,
and monocytes. Experimental data implicates the interactions of
PD-1 with its ligands in downregulation of central and peripheral
immune responses. In particular, proliferation in wild-type T cells
but not in PD-1-deficient T cells is inhibited in the presence of
PD-L1. Additionally, PD-1-deficient mice exhibit an autoimmune
phenotype.
[0152] An exemplary amino acid sequence of human PD-1 is set forth
below (see also Ishida et al., EMBO J. 11:3887, 1992; Shinohara et
al. Genomics 23:704, 1994; U.S. Pat. No. 5,698,520):
TABLE-US-00001 (SEQ ID NO: 1) mqipqapwpv vwavlqlgwr pgwfldspdr
pwnpptffpa llvvtegdna tftcsfsnts esfvlnwyrm spsnqtdkla afpedrsqpg
qdcrfrvtql pngrdfhmsv vrarrndsgt ylcgaislap kaqikeslra elrvterrae
vptahpspsp rpagqfqtlv vgvvggllgs lvllvwvlav icsraargti garrtgqplk
edpsavpvfs vdygeldfqw rektpeppvp cvpeqteyat ivfpsgmgts sparrgsadg
prsagplrpe dghcswpl
[0153] An exemplary amino acid sequence of mouse PD-1 is set forth
below:
TABLE-US-00002 (SEQ ID NO: 2) mwvrqvpwsf twavlqlswq sgwllevpng
pwrsltfypa wltvsegana tftcslsnws edlmlnwnrl spsnqtekqa afcnglsgpv
qdarfqiiql pnrhdfhmni ldtrrndsgi ylcgaislhp kakieespga elvvterile
tstrypspsp kpegrfqgmv igimsalvgi pvllllawal avfcstsmse argagskddt
lkeepsaapv psvayeeldf qgrektpelp tacvhteyat ivfteglgas amgrrgsadg
lqgprpprhe dghcswpl
[0154] Additional amino acid sequences are disclosed in U.S. Pat.
No. 6,808,710 and U.S. Patent Application Publication Nos.
2004/0137577, 2003/0232323, 2003/0166531, 2003/0064380,
2003/0044768, 2003/0039653, 2002/0164600, 2002/0160000,
2002/0110836, 2002/0107363, and 2002/0106730, which are
incorporated herein by reference. PD-1 is a member of the
immunoglobulin (Ig) superfamily that contains a single Ig V-like
domain in its extracellular region. The PD-1 cytoplasmic domain
contains two tyrosines, with the most membrane-proximal tyrosine
(VAYEEL (see amino acids 223-228 of SEQ ID NO: 2) in mouse PD-1)
located within an ITIM (immuno-receptor tyrosine-based inhibitory
motif). The presence of an ITIM on PD-1 indicates that this
molecule functions to attenuate antigen receptor signaling by
recruitment of cytoplasmic phosphatases. Human and murine PD-1
proteins share about 60% amino acid identity with conservation of
four potential N-glycosylation sites, and residues that define the
Ig-V domain. The ITIM in the cytoplasmic region and the ITIM-like
motif surrounding the carboxy-terminal tyrosine (TEYATI (see amino
acids 166-181 of SEQ ID NO: 2) in human and mouse, respectively)
are also conserved between human and murine orthologues.
[0155] PD-1 is a member of the CD28/CTLA-4 family of molecules
based on its ability to bind to PD-L1. In vivo, like CTLA4, PD-1 is
rapidly induced on the surface of T-cells in response to anti-CD3
(Agata et al. Int. Immunol. 8:765, 1996). In contrast to CTLA4,
however, PD-1 is also induced on the surface of B-cells (in
response to anti-IgM). PD-1 is also expressed on a subset of
thymocytes and myeloid cells (Agata et al. (1996) supra; Nishimura
et al. (1996) Int. Immunol. 8:773).
[0156] T cell anergy is concomitant with an induction in PD-1
expression. It is disclosed herein that T-cell cytoxicity can be
increased by contacting a T-cell with an agent that reduces the
expression or activity of PD-1. More specifically, it is disclosed
herein that an agent that reduces the expression or activity of
PD-1 can be used to increase an immune response, such as to a viral
antigen or a tumor antigen.
[0157] Without being bound by theory, reduction of PD-1 expression
or activity results in an increase in cytotoxic T cell activity,
increasing the specific immune response to the infectious agent. In
order for T cells to respond to foreign proteins, two signals must
be provided by antigen-presenting cells (APCs) to resting T
lymphocytes. The first signal, which confers specificity to the
immune response, is transduced via the T cell receptor (TCR)
following recognition of foreign antigenic peptide presented in the
context of the major histocompatibility complex (MHC). The second
signal, termed costimulation, induces T cells to proliferate and
become functional. Costimulation is neither antigen-specific, nor
MHC-restricted and is provided by one or more distinct cell surface
polypeptides expressed by APCs. If T cells are only stimulated
through the T cell receptor, without receiving an additional
costimulatory signal, they become nonresponsive, anergic, or die,
resulting in downmodulation of the immune response.
[0158] The CD80 (B7-1) and CD86 (B7-2) proteins, expressed on APCs,
are critical costimulatory polypeptides. While B7-2 plays a
predominant role during primary immune responses, B7-1 is
upregulated later in the course of an immune response to prolong
primary T cell responses or costimulating secondary T cell
responses. B7 polypeptides are capable of providing costimulatory
or inhibitory signals to immune cells to promote or inhibit immune
cell responses. For example, when bound to a costimulatory
receptor, PD-L1 (B7-4) induces costimulation of immune cells or
inhibits immune cell costimulation when present in a soluble form.
When bound to an inhibitory receptor, PD-L1 molecules can transmit
an inhibitory signal to an immune cell. Exemplary B7 family members
include B7-1, B7-2, B7-3 (recognized by the antibody BB-1), B7h
(PD-L1), and B7-4 and soluble fragments or derivatives thereof. B7
family members bind to one or more receptors on an immune cell,
such as CTLA4, CD28, ICOS, PD-1 and/or other receptors, and,
depending on the receptor, have the ability to transmit an
inhibitory signal or a costimulatory signal to an immune cell.
[0159] CD28 is a receptor that is constitutively expressed on
resting T cells. After signaling through the T cell receptor,
ligation of CD28 and transduction of a costimulatory signal induces
T cells to proliferate and secrete IL-2. CTLA4 (CD152), a receptor
homologous to CD28, is absent on resting T cells but its expression
is induced following T cell activation. CTLA4 plays a role in
negative regulation of T cell responses. ICOS, a polypeptide
related to CD28 and CTLA4, is involved in IL-10 production. PD-1,
the receptor to which PD-L1 and PD-L2 bind, is also rapidly induced
on the surface of T-cells. PD-1 is also expressed on the surface of
B-cells (in response to anti-IgM) and on a subset of thymocytes and
myeloid cells.
[0160] Engagement of PD-1 (for example by crosslinking or by
aggregation), leads to the transmission of an inhibitory signal in
an immune cell, resulting in a reduction of immune responses
concomitant with an increase in immune cell anergy. PD-1 family
members bind to one or more receptors, such as PD-L1 and PD-L2 on
antigen presenting cells. PD-L1 and PD-L2, both of which are human
PD-1 ligand polypeptides, are members of the B7 family of
polypeptides (see above). Each PD-1 ligand contains a signal
sequence, an IgV domain, an IgC domain, a transmembrane domain, and
a short cytoplasmic tail. In vivo, these ligands have been shown to
be expressed in placenta, spleen, lymph nodes, thymus, and heart.
PD-L2 is also expressed in the pancreas, lung, and liver, while
PD-L1 is expressed in fetal liver, activated T-cells and
endothelial cells. Expression of both PD-1 ligands are upregulated
on activated monocytes and dendritic cells.
[0161] An exemplary amino acid sequence for PD-L1 (GENBANK.RTM.
Accession No. AAG18508, as available Oct. 4, 2000) is set forth
below:
TABLE-US-00003 (SEQ ID NO: 3) mrifavfifm tywhllnaft vtvpkdlyvv
eygsnmtiec kfpvekqldl aalivyweme dkniiqfvhg eedlkvqhss yrqrarllkd
qlslgnaalq itdvklqdag vyrcmisygg adykritvkv napynkinqr ilvvdpvtse
heltcqaegy pkaeviwtss dhqvlsgktt ttnskreekl fnvtstlrin tttneifyct
frrldpeenh taelvipelp lahppnerth lvilgaillc lgvaltfifr lrkgrmmdvk
kcgiqdtnsk kqsdthleet
[0162] An exemplary PD-L2 precursor amino acid sequence
(GENBANK.RTM. Accession No. AAK15370, as available Apr. 8, 2002) is
set forth below:
TABLE-US-00004 (SEQ ID NO: 4) miflllmlsl elqlhqiaal ftvtvpkely
iiehgsnvtl ecnfdtgshv nlgaitaslq kvendtsphr eratlleeql plgkasfhip
qvqvrdegqy qciiiygvaw dykyltlkvk asyrkinthi lkvpetdeve ltcqatgypl
aevswpnvsv pantshsrtp eglyqvtsvl rlkpppgrnf scvfwnthvr eltlasidlq
sqmeprthpt wllhifipsc iiafifiatv ialrkqlcqk lysskdttkr pvtttkrevn
sai
[0163] An exemplary variant PD-L2 precursor amino acid sequence
(GENBANK.RTM. Accession No. Q9BQ51, as available Dec. 12, 2006) is
set forth below:
TABLE-US-00005 (SEQ ID NO: 46) miflllmlsl elqlhqiaal ftvtvpkely
iiehgsnvtl ecnfdtgshv nlgaitaslq kvendtsphr eratlleeql plgkasfhip
qvqvrdegqy qciiiygvaw dykyltlkvk asyrkinthi lkvpetdeve ltcqatgypl
aevswpnvsv pantshsrtp eglyqvtsvl rlkpppgrnf scvfwnthvr eltlasidlq
sqmeprthpt wllhifipfc iiafifiatv ialrkqlcqk lysskdttkr pvtttkrevn
sai
[0164] PD-1 antagonists include agents that reduce the expression
or activity of a PD ligand 1 (PD-L1) or a PD ligand 2 (PD-L2) or
reduces the interaction between PD-1 and PD-L1 or the interaction
between PD-1 and PD-L2. Exemplary compounds include antibodies
(such as an anti-PD-1 antibody, an anti-PD-L1 antibody, and an
anti-PD-L2 antibody), RNAi molecules (such as anti-PD-1 RNAi
molecules, anti-PD-L1 RNAi, and an anti-PD-L2 RNAi), antisense
molecules (such as an anti-PD-1 antisense RNA, an anti-PD-L1
antisense RNA, and an anti-PD-L2 antisense RNA), dominant negative
proteins (such as a dominant negative PD-1 protein, a dominant
negative PD-L1 protein, and a dominant negative PD-L2 protein), and
small molecule inhibitors.
[0165] An antagonist of PD-1 is any agent having the ability to
reduce the expression or the activity of PD-1 in a cell. PD-1
expression or activity is reduced by at least about 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to such expression
or activity in a control. Exemplary reductions in activity are at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, at least about 95%, or a complete
absence of detectable activity. In one example, the control is a
cell that has not been treated with the PD-1 antagonist. In another
example, the control is a standard value, or a cell contacted with
an agent, such as a carrier, known not to affect PD-1 activity.
PD-1 expression or activity can be determined by any standard
method in the art, including those described herein. Optionally,
the PD-1 antagonist inhibits or reduces binding of PD-1 to PD-L1,
PD-L2, or both.
A. Antibodies
[0166] Antibodies that specifically bind PD-1, PD-L1 or PD-L2 (or a
combination thereof) are of use in the methods disclosed herein.
Antibodies include monoclonal antibodies, humanized antibodies,
deimmunized antibodies, and immunoglobulin (Ig) fusion proteins.
Polyclonal anti-PD-1, anti-PDL1 or PD-L2 antibodies can be prepared
by one of skill in the art, such as by immunizing a suitable
subject (such as a veterinary subject) with a PD-1 ligand or PD-1
immunogen. The anti-PD-1, anti-PD-L1 or anti-PD-L2 antibody titer
in the immunized subject can be monitored over time by standard
techniques, such as with an enzyme linked immunosorbent assay
(ELISA) using immobilized a PD-1 ligand or PD-1 polypeptide.
[0167] In one example, the antibody molecules that specifically
bind PD-1, PD-L1 or PD-L2 (or combinations thereof) can be isolated
from the mammal (such as from serum) and further purified by
techniques known to one of skill in the art. For example,
antibodies can be purified using protein A chromatography to
isolate IgG antibodies.
[0168] Antibody-producing cells can be obtained from the subject
and used to prepare monoclonal antibodies by standard techniques
(see Kohler and Milstein Nature 256:495 49, 1995; Brown et al., J.
Immunol. 127:539 46, 1981; Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc., pp. 77 96, 1985; Gefter, M. L.
et al. (1977) Somatic Cell Genet. 3:231 36; Kenneth, R. H. in
Monoclonal Antibodies: A New Dimension In Biological Analyses.
Plenum Publishing Corp., New York, N.Y. (1980); Kozbor et al.
Immunol. Today 4:72, 1983; Lerner, E. A. (1981) Yale J. Biol. Med.
54:387 402; Yeh et al., Proc. Natl. Acad. Sci. 76:2927 31, 1976).
In one example, an immortal cell line (typically a myeloma) is
fused to lymphocytes (typically splenocytes) from a mammal
immunized with PD-1, PD-L1 or PD-L2, and the culture supernatants
of the resulting hybridoma cells are screened to identify a
hybridoma producing a monoclonal antibody that specifically binds
to the polypeptide of interest.
[0169] In one embodiment, to produce a hybridoma, an immortal cell
line (such as a myeloma cell line) is derived from the same
mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with a PD-1, PD-L1 or PD-L2 peptide with an immortalized mouse cell
line. In one example, a mouse myeloma cell line is utilized that is
sensitive to culture medium containing hypoxanthine, aminopterin
and thymidine ("HAT medium"). Any of a number of myeloma cell lines
can be used as a fusion partner according to standard techniques,
including, for example, P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or
Sp2/O-Ag14 myeloma lines, which are available from the American
Type Culture Collection (ATCC), Rockville, Md. HAT-sensitive mouse
myeloma cells can be fused to mouse splenocytes using polyethylene
glycol ("PEG"). Hybridoma cells resulting from the fusion are then
selected using HAT medium, which kills unfused (and unproductively
fused) myeloma cells. Hybridoma cells producing a monoclonal
antibody of interest can be detected, for example, by screening the
hybridoma culture supernatants for the production antibodies that
bind a PD-1, PD-L1 or PD-L2 molecule, such as by using an
immunological assay (such as an enzyme-linked immunosorbant
assay(ELISA) or radioimmunoassay (RIA).
[0170] As an alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody that specifically binds PD-1,
PD-L1 or PD-L2 can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (such as an
antibody phage display library) with PD-1, PD-L1 or PD-L2 to
isolate immunoglobulin library members that specifically bind the
polypeptide. Kits for generating and screening phage display
libraries are commercially available (such as, but not limited to,
Pharmacia and Stratagene). Examples of methods and reagents
particularly amenable for use in generating and screening antibody
display library can be found in, for example, U.S. Pat. No.
5,223,409; PCT Publication No. WO 90/02809; PCT Publication No. WO
91/17271; PCT Publication No. WO 92/18619; PCT Publication WO
92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO
92/01047; PCT Publication WO 93/01288; PCT Publication No. WO
92/09690; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978 7982,
1991; Hoogenboom et al., Nucleic Acids Res. 19:4133 4137, 1991.
[0171] The amino acid sequence of antibodies that bind PD-1 are
disclosed, for example, in U.S. Patent Publication No.
2006/0210567, which is incorporated herein by reference. Antibodies
that bind PD-1 are also disclosed in U.S. Patent Publication No.
2006/0034826, which is also incorporated herein by reference. In
several examples, the antibody specifically binds PD-1 or a PD-1 or
PD-2 ligand with an affinity constant of at least 10.sup.7
M.sup.-1, such as at least 10.sup.8 M.sup.-1 at least
5.times.10.sup.8 M.sup.-1 or at least 10.sup.9 M.sup.-1.
[0172] In one example the sequence of the specificity determining
regions of each CDR is determined. Residues are outside the SDR
(non-ligand contacting sites) are substituted. For example, in any
of the CDR sequences, at most one, two or three amino acids can be
substituted. The production of chimeric antibodies, which include a
framework region from one antibody and the CDRs from a different
antibody, is well known in the art. For example, humanized
antibodies can be routinely produced. The antibody or antibody
fragment can be a humanized immunoglobulin having complementarity
determining regions (CDRs) from a donor monoclonal antibody that
binds PD-1, PD-L1 or PD-L2, and immunoglobulin and heavy and light
chain variable region frameworks from human acceptor immunoglobulin
heavy and light chain frameworks. Generally, the humanized
immunoglobulin specifically binds to PD-1, PD-L1 or PD-L2 with an
affinity constant of at least 10.sup.7 M.sup.-1, such as at least
10.sup.8 M.sup.-1 at least 5.times.10.sup.8 M.sup.-1 or at least
10.sup.9 M.sup.-1.
[0173] Humanized monoclonal antibodies can be produced by
transferring donor complementarity determining regions (CDRs) from
heavy and light variable chains of the donor mouse immunoglobulin
(such PD-1, PD-L1 or PD-L2) into a human variable domain, and then
substituting human residues in the framework regions when required
to retain affinity. The use of antibody components derived from
humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of the constant regions of the
donor antibody. Techniques for producing humanized monoclonal
antibodies are described, for example, by Jones et al., Nature
321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et
al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci.
U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and
Singer et al., J. Immunol. 150:2844, 1993. The antibody may be of
any isotype, but in several embodiments the antibody is an IgG,
including but not limited to, IgG.sub.1, IgG.sub.2, IgG.sub.3 and
IgG.sub.4.
[0174] In one embodiment, the sequence of the humanized
immunoglobulin heavy chain variable region framework can be at
least about 65% identical to the sequence of the donor
immunoglobulin heavy chain variable region framework. Thus, the
sequence of the humanized immunoglobulin heavy chain variable
region framework can be at least about 75%, at least about 85%, at
least about 99% or at least about 95%, identical to the sequence of
the donor immunoglobulin heavy chain variable region framework.
Human framework regions, and mutations that can be made in
humanized antibody framework regions, are known in the art (see,
for example, in U.S. Pat. No. 5,585,089, which is incorporated
herein by reference).
[0175] Exemplary human antibodies are LEN and 21/28 CL. The
sequences of the heavy and light chain frameworks are known in the
art. Exemplary light chain frameworks of human MAb LEN have the
following sequences:
TABLE-US-00006 (SEQ ID NO: 5) FR1: DIVMTQS PDSLAVSLGERATINC (SEQ ID
NO: 6) FR2: WYQQKPGQPPLLIY (SEQ ID NO: 7) FR3:
GVPDRPFGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 8) FR4:
FGQGQTKLEIK
[0176] Exemplary heavy chain frameworks of human MAb 21/28' CL have
the following sequences:
TABLE-US-00007 (SEQ ID NO: 9) FR1: QVQLVQSGAEVKKPQASVKVSCKASQYTFT
(SEQ ID NO: 10) FR2: WVRQAPGQRLEWMG (SEQ ID NO: 11) FR3:
RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 12) FR4:
WGQGTLVIVSS.
[0177] Antibodies, such as murine monoclonal antibodies, chimeric
antibodies, and humanized antibodies, include full length molecules
as well as fragments thereof, such as Fab, F(ab').sub.2, and Fv
which include a heavy chain and light chain variable region and are
capable of binding specific epitope determinants. These antibody
fragments retain some ability to selectively bind with their
antigen or receptor. These fragments include:
[0178] (1) Fab, the fragment which contains a monovalent
antigen-binding fragment of an antibody molecule, can be produced
by digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain;
[0179] (2) Fab', the fragment of an antibody molecule can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody
molecule;
[0180] (3) (Fab').sub.2, the fragment of the antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds;
[0181] (4) Fv, a genetically engineered fragment containing the
variable region of the light chain and the variable region of the
heavy chain expressed as two chains; and
[0182] (5) Single chain antibody (such as scFv), defined as a
genetically engineered molecule containing the variable region of
the light chain, the variable region of the heavy chain, linked by
a suitable polypeptide linker as a genetically fused single chain
molecule.
[0183] Methods of making these fragments are known in the art (see
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988). In several examples, the
variable region includes the variable region of the light chain and
the variable region of the heavy chain expressed as individual
polypeptides. Fv antibodies are typically about 25 kDa and contain
a complete antigen-binding site with three CDRs per each heavy
chain and each light chain. To produce these antibodies, the
V.sub.H and the V.sub.L can be expressed from two individual
nucleic acid constructs in a host cell. If the V.sub.H and the
V.sub.L are expressed non-contiguously, the chains of the Fv
antibody are typically held together by noncovalent interactions.
However, these chains tend to dissociate upon dilution, so methods
have been developed to crosslink the chains through glutaraldehyde,
intermolecular disulfides, or a peptide linker. Thus, in one
example, the Fv can be a disulfide stabilized Fv (dsFv), wherein
the heavy chain variable region and the light chain variable region
are chemically linked by disulfide bonds.
[0184] In an additional example, the Fv fragments comprise V.sub.H
and V.sub.L chains connected by a peptide linker. These
single-chain antigen binding proteins (scFv) are prepared by
constructing a structural gene comprising DNA sequences encoding
the V.sub.H and V.sub.L domains connected by an oligonucleotide.
The structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
scFvs are known in the art (see Whitlow et al., Methods: a
Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et
al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al.,
Bio/Technology 11:1271, 1993; and Sandhu, supra).
[0185] Antibody fragments can be prepared by proteolytic hydrolysis
of the antibody or by expression in E. coli of DNA encoding the
fragment. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an
Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No.
4,331,647, and references contained therein; Nisonhoff et al.,
Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119,
1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422,
Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10
and 2.10.1-2.10.4).
[0186] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0187] One of skill will realize that conservative variants of the
antibodies can be produced. Such conservative variants employed in
antibody fragments, such as dsFv fragments or in scFv fragments,
will retain critical amino acid residues necessary for correct
folding and stabilizing between the V.sub.H and the V.sub.L
regions, and will retain the charge characteristics of the residues
in order to preserve the low pI and low toxicity of the molecules.
Amino acid substitutions (such as at most one, at most two, at most
three, at most four, or at most five amino acid substitutions) can
be made in the V.sub.H and the V.sub.L regions to increase yield.
Conservative amino acid substitution tables providing functionally
similar amino acids are well known to one of ordinary skill in the
art. The following six groups are examples of amino acids that are
considered to be conservative substitutions for one another:
[0188] 1) Alanine (A), Serine (S), Threonine (T);
[0189] 2) Aspartic acid (D), Glutamic acid (E);
[0190] 3) Asparagine (N), Glutamine (Q);
[0191] 4) Arginine (R), Lysine (K);
[0192] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0193] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Thus, one of skill in the art can readily review the amino acid
sequence of an antibody of interest, locate one or more of the
amino acids in the brief table above, identify a conservative
substitution, and produce the conservative variant using well-known
molecular techniques.
[0194] Effector molecules, such as therapeutic, diagnostic, or
detection moieties can be linked to an antibody that specifically
binds PD-1, PD-L1 or PD-L2, using any number of means known to
those of skill in the art. Both covalent and noncovalent attachment
means may be used. The procedure for attaching an effector molecule
to an antibody varies according to the chemical structure of the
effector. Polypeptides typically contain a variety of functional
groups; such as carboxylic acid (COOH), free amine (--NH.sub.2) or
sulfhydryl (--SH) groups, which are available for reaction with a
suitable functional group on an antibody to result in the binding
of the effector molecule. Alternatively, the antibody is
derivatized to expose or attach additional reactive functional
groups. The derivatization may involve attachment of any of a
number of linker molecules such as those available from Pierce
Chemical Company, Rockford, Ill. The linker can be any molecule
used to join the antibody to the effector molecule. The linker is
capable of forming covalent bonds to both the antibody and to the
effector molecule. Suitable linkers are well known to those of
skill in the art and include, but are not limited to, straight or
branched-chain carbon linkers, heterocyclic carbon linkers, or
peptide linkers. Where the antibody and the effector molecule are
polypeptides, the linkers may be joined to the constituent amino
acids through their side groups (such as through a disulfide
linkage to cysteine) or to the alpha carbon amino and carboxyl
groups of the terminal amino acids.
[0195] Nucleic acid sequences encoding the antibodies can be
prepared by any suitable method including, for example, cloning of
appropriate sequences or by direct chemical synthesis by methods
such as the phosphotriester method of Narang et al., Meth. Enzymol.
68:90-99, 1979; the phosphodiester method of Brown et al., Meth.
Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of
Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phase
phosphoramidite triester method described by Beaucage &
Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using
an automated synthesizer as described in, for example,
Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984;
and, the solid support method of U.S. Pat. No. 4,458,066. Chemical
synthesis produces a single stranded oligonucleotide. This can be
converted into double stranded DNA by hybridization with a
complementary sequence, or by polymerization with a DNA polymerase
using the single strand as a template. One of skill would recognize
that while chemical synthesis of DNA is generally limited to
sequences of about 100 bases, longer sequences may be obtained by
the ligation of shorter sequences.
[0196] Exemplary nucleic acids encoding sequences encoding an
antibody that specifically binds PD-1, PD-L1 or PD-L2 can be
prepared by cloning techniques. Examples of appropriate cloning and
sequencing techniques, and instructions sufficient to direct
persons of skill through many cloning exercises are found in
Sambrook et al., supra, Berger and Kimmel (eds.), supra, and
Ausubel, supra. Product information from manufacturers of
biological reagents and experimental equipment also provide useful
information. Such manufacturers include the SIGMA Chemical Company
(Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia
Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo
Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life
Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika
Analytika (Fluka Chemie A G, Buchs, Switzerland), Invitrogen (San
Diego, Calif.), and Applied Biosystems (Foster City, Calif.), as
well as many other commercial sources known to one of skill.
[0197] Nucleic acids can also be prepared by amplification methods.
Amplification methods include polymerase chain reaction (PCR), the
ligase chain reaction (LCR), the transcription-based amplification
system (TAS), the self-sustained sequence replication system (3SR).
A wide variety of cloning methods, host cells, and in vitro
amplification methodologies are well known to persons of skill.
[0198] In one example, an antibody of use is prepared by inserting
the cDNA which encodes a variable region from an antibody that
specifically binds PD-1, PD-L1 or PD-L2 into a vector which
comprises the cDNA encoding an effector molecule (EM). The
insertion is made so that the variable region and the EM are read
in frame so that one continuous polypeptide is produced. Thus, the
encoded polypeptide contains a functional Fv region and a
functional EM region. In one embodiment, cDNA encoding a detectable
marker (such as an enzyme) is ligated to a scFv so that the marker
is located at the carboxyl terminus of the scFv. In another
example, a detectable marker is located at the amino terminus of
the scFv. In a further example, cDNA encoding a detectable marker
is ligated to a heavy chain variable region of an antibody that
specifically binds PD-1, PD-L1 or PD-L2, so that the marker is
located at the carboxyl terminus of the heavy chain variable
region. The heavy chain-variable region can subsequently be ligated
to a light chain variable region of the antibody that specifically
binds PD-1, PD-L1 or PD-L2 using disulfide bonds. In a yet another
example, cDNA encoding a marker is ligated to a light chain
variable region of an antibody that binds PD-1, PD-L1 or PD-L2, so
that the marker is located at the carboxyl terminus of the light
chain variable region. The light chain-variable region can
subsequently be ligated to a heavy chain variable region of the
antibody that specifically binds PD-1, PD-L1 or PD-L2 using
disulfide bonds.
[0199] Once the nucleic acids encoding the antibody or functional
fragment thereof are isolated and cloned, the protein can be
expressed in a recombinantly engineered cell such as bacteria,
plant, yeast, insect and mammalian cells. One or more DNA sequences
encoding the antibody or functional fragment thereof can be
expressed in vitro by DNA transfer into a suitable host cell. The
cell may be prokaryotic or eukaryotic. The term also includes any
progeny of the subject host cell. It is understood that all progeny
may not be identical to the parental cell since there may be
mutations that occur during replication. Methods of stable
transfer, meaning that the foreign DNA is continuously maintained
in the host, are known in the art.
[0200] Polynucleotide sequences encoding the antibody or functional
fragment thereof can be operatively linked to expression control
sequences. An expression control sequence operatively linked to a
coding sequence is ligated such that expression of the coding
sequence is achieved under conditions compatible with the
expression control sequences. The expression control sequences
include, but are not limited to appropriate promoters, enhancers,
transcription terminators, a start codon (i.e., ATG) in front of a
protein-encoding gene, splicing signal for introns, maintenance of
the correct reading frame of that gene to permit proper translation
of mRNA, and stop codons.
[0201] The polynucleotide sequences encoding the antibody or
functional fragment thereof can be inserted into an expression
vector including, but not limited to a plasmid, virus or other
vehicle that can be manipulated to allow insertion or incorporation
of sequences and can be expressed in either prokaryotes or
eukaryotes. Hosts can include microbial, yeast, insect and
mammalian organisms. Methods of expressing DNA sequences having
eukaryotic or viral sequences in prokaryotes are well known in the
art. Biologically functional viral and plasmid DNA vectors capable
of expression and replication in a host are known in the art.
[0202] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method using procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired, or by electroporation.
[0203] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate coprecipitates, conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or virus vectors may be used.
Eukaryotic cells can also be cotransformed with polynucleotide
sequences encoding the antibody of functional fragment thereof and
a second foreign DNA molecule encoding a selectable phenotype, such
as the herpes simplex thymidine kinase gene. Another method is to
use a eukaryotic viral vector, such as simian virus 40 (SV40) or
bovine papilloma virus, to transiently infect or transform
eukaryotic cells and express the protein (see for example,
Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman
ed., 1982). One of skill in the art can readily use expression
systems such as plasmids and vectors of use in producing proteins
in cells including higher eukaryotic cells such as the COS, CHO,
HeLa and myeloma cell lines.
[0204] Isolation and purification of recombinantly expressed
polypeptide can be carried out by conventional means including
preparative chromatography and immunological separations. Once
expressed, the recombinant antibodies can be purified according to
standard procedures of the art, including ammonium sulfate
precipitation, affinity columns, column chromatography, and the
like (see, generally, R. Scopes, Protein Purification,
Springer-Verlag, N.Y., 1982). Substantially pure compositions of at
least about 90 to 95% homogeneity are disclosed herein, and 98 to
99% or more homogeneity can be used for pharmaceutical purposes.
Once purified, partially or to homogeneity as desired, if to be
used therapeutically, the polypeptides should be substantially free
of endotoxin.
[0205] Methods for expression of single chain antibodies and/or
refolding to an appropriate active form, including single chain
antibodies, from bacteria such as E. coli have been described and
are well-known and are applicable to the antibodies disclosed
herein. See, Buchner et al., Anal. Biochem. 205:263-270, 1992;
Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science
246:1275, 1989 and Ward et al., Nature 341:544, 1989, all
incorporated by reference herein.
[0206] Often, functional heterologous proteins from E. coli or
other bacteria are isolated from inclusion bodies and require
solubilization using strong denaturants, and subsequent refolding.
During the solubilization step, as is well known in the art, a
reducing agent must be present to separate disulfide bonds. An
exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M
guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of
the disulfide bonds can occur in the presence of low molecular
weight thiol reagents in reduced and oxidized form, as described in
Saxena et al., Biochemistry 9: 5015-5021, 1970, incorporated by
reference herein, and especially as described by Buchner et al.,
supra.
[0207] Renaturation is typically accomplished by dilution (for
example, 100-fold) of the denatured and reduced protein into
refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M
L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
[0208] As a modification to the two chain antibody purification
protocol, the heavy and light chain regions are separately
solubilized and reduced and then combined in the refolding
solution. An exemplary yield is obtained when these two proteins
are mixed in a molar ratio such that a 5 fold molar excess of one
protein over the other is not exceeded. It is desirable to add
excess oxidized glutathione or other oxidizing low molecular weight
compounds to the refolding solution after the redox-shuffling is
completed.
[0209] In addition to recombinant methods, the antibodies and
functional fragments thereof that are disclosed herein can also be
constructed in whole or in part using standard peptide synthesis.
Solid phase synthesis of the polypeptides of less than about 50
amino acids in length can be accomplished by attaching the
C-terminal amino acid of the sequence to an insoluble support
followed by sequential addition of the remaining amino acids in the
sequence. Techniques for solid phase synthesis are described by
Barany & Merrifield, The Peptides: Analysis, Synthesis,
Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp.
3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, and
Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce
Chem. Co., Rockford, Ill., 1984. Proteins of greater length may be
synthesized by condensation of the amino and carboxyl termini of
shorter fragments. Methods of forming peptide bonds by activation
of a carboxyl terminal end (such as by the use of the coupling
reagent N, N'-dicycylohexylcarbodimide) are well known in the
art.
B. Inhibitory Nucleic Acids
[0210] Inhibitory nucleic acids that decrease the expression and/or
activity of PD-1, PD-L1 or PD-L2 can also be used in the methods
disclosed herein. One embodiment is a small inhibitory RNA (siRNA)
for interference or inhibition of expression of a target gene.
Nucleic acid sequences encoding PD-1, PD-L1 and PD-L2 are disclosed
in GENBANK.RTM. Accession Nos. NM_005018, AF344424, NP_079515, and
NP_054862.
[0211] Generally, siRNAs are generated by the cleavage of
relatively long double-stranded RNA molecules by Dicer or DCL
enzymes (Zamore, Science, 296:1265-1269, 2002; Bernstein et al.,
Nature, 409:363-366, 2001). In animals and plants, siRNAs are
assembled into RISC and guide the sequence specific ribonucleolytic
activity of RISC, thereby resulting in the cleavage of mRNAs or
other RNA target molecules in the cytoplasm. In the nucleus, siRNAs
also guide heterochromatin-associated histone and DNA methylation,
resulting in transcriptional silencing of individual genes or large
chromatin domains. PD-1 siRNAs are commercially available, such as
from Santa Cruz Biotechnology, Inc.
[0212] The present disclosure provides RNA suitable for
interference or inhibition of expression of a target gene, which
RNA includes double stranded RNA of about 15 to about 40
nucleotides containing a 0 to 5-nucleotide 3' and/or 5' overhang on
each strand. The sequence of the RNA is substantially identical to
a portion of an mRNA or transcript of a target gene, such as PD-1,
PD-L1 or PD-L2) for which interference or inhibition of expression
is desired. For purposes of this disclosure, a sequence of the RNA
"substantially identical" to a specific portion of the mRNA or
transcript of the target gene for which interference or inhibition
of expression is desired differs by no more than about 30 percent,
and in some embodiments no more than about 10 percent, from the
specific portion of the mRNA or transcript of the target gene. In
particular embodiments, the sequence of the RNA is exactly
identical to a specific portion of the mRNA or transcript of the
target gene.
[0213] Thus, siRNAs disclosed herein include double-stranded RNA of
about 15 to about 40 nucleotides in length and a 3' or 5' overhang
having a length of 0 to 5-nucleotides on each strand, wherein the
sequence of the double stranded RNA is substantially identical to
(see above) a portion of a mRNA or transcript of a nucleic acid
encoding PD-1, PD-L1 or PD-L2. In particular examples, the double
stranded RNA contains about 19 to about 25 nucleotides, for
instance 20, 21, or 22 nucleotides substantially identical to a
nucleic acid encoding PD-1, PD-L1 or PD-L2. In additional examples,
the double stranded RNA contains about 19 to about 25 nucleotides
100% identical to a nucleic acid encoding PD-1, PD-L1 or PD-L2. It
should be not that in this context "about" refers to integer
amounts only. In one example, "about" 20 nucleotides refers to a
nucleotide of 19 to 21 nucleotides in length.
[0214] Regarding the overhang on the double-stranded RNA, the
length of the overhang is independent between the two strands, in
that the length of one overhang is not dependent on the length of
the overhang on other strand. In specific examples, the length of
the 3' or 5' overhang is 0-nucleotide on at least one strand, and
in some cases it is 0-nucleotide on both strands (thus, a blunt
dsRNA). In other examples, the length of the 3' or 5' overhang is
1-nucleotide to 5-nucleotides on at least one strand. More
particularly, in some examples the length of the 3' or 5' overhang
is 2-nucleotides on at least one strand, or 2-nucleotides on both
strands. In particular examples, the dsRNA molecule has 3'
overhangs of 2-nucleotides on both strands.
[0215] Thus, in one particular provided RNA embodiment, the
double-stranded RNA contains 20, 21, or 22 nucleotides, and the
length of the 3' overhang is 2-nucleotides on both strands. In
embodiments of the RNAs provided herein, the double-stranded RNA
contains about 40-60% adenine+uracil (AU) and about 60-40%
guanine+cytosine (GC). More particularly, in specific examples the
double-stranded RNA contains about 50% AU and about 50% GC.
[0216] Also described herein are RNAs that further include at least
one modified ribonucleotide, for instance in the sense strand of
the double-stranded RNA. In particular examples, the modified
ribonucleotide is in the 3' overhang of at least one strand, or
more particularly in the 3' overhang of the sense strand. It is
particularly contemplated that examples of modified ribonucleotides
include ribonucleotides that include a detectable label (for
instance, a fluorophore, such as rhodamine or FITC), a
thiophosphate nucleotide analog, a deoxynucleotide (considered
modified because the base molecule is ribonucleic acid), a
2'-fluorouracil, a 2'-aminouracil, a 2'-aminocytidine, a
4-thiouracil, a 5-bromouracil, a 5-iodouracil, a
5-(3-aminoallyl)-uracil, an inosine, or a 2'O-Me-nucleotide
analog.
[0217] Antisense and ribozyme molecules for PD-1, PD-L1 and PD-L2
are also of use in the method disclosed herein. Antisense nucleic
acids are DNA or RNA molecules that are complementary to at least a
portion of a specific mRNA molecule (Weintraub, Scientific American
262:40, 1990). In the cell, the antisense nucleic acids hybridize
to the corresponding mRNA, forming a double-stranded molecule. The
antisense nucleic acids interfere with the translation of the mRNA,
since the cell will not translate an mRNA that is double-stranded.
Antisense oligomers of about 15 nucleotides are preferred, since
they are easily synthesized and are less likely to cause problems
than larger molecules when introduced into the target cell
producing PD-1, PD-L1 or PD-L2. The use of antisense methods to
inhibit the in vitro translation of genes is well known in the art
(see, for example, Marcus-Sakura, Anal. Biochem. 172:289,
1988).
[0218] An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An
antisense nucleic acid can be constructed using chemical synthesis
and enzymatic ligation reactions using procedures known in the art.
For example, an antisense nucleic acid molecule can be chemically
synthesized using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological stability
of the molecules or to increase the physical stability of the
duplex formed between the antisense and sense nucleic acids, such
as phosphorothioate derivatives and acridine substituted
nucleotides can be used. Examples of modified nucleotides which can
be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridin-e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, amongst others.
[0219] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the bloomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. and Dev. 1(3):227, 1991;
Helene, C., Anticancer Drug Design 6(6):569), 1991. This type of
inhibitory oligonucleotide is also of use in the methods disclosed
herein.
[0220] Ribozymes, which are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases, are also of use. Through the
modification of nucleotide sequences which encode these RNAs, it is
possible to engineer molecules that recognize specific nucleotide
sequences in an RNA molecule and cleave it (Cech, J. Amer. Med.
Assn. 260:3030, 1988). A major advantage of this approach is that,
because they are sequence-specific, only mRNAs with particular
sequences are inactivated.
[0221] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-base
recognition sequences are preferable to shorter recognition
sequences.
[0222] Various delivery systems are known and can be used to
administer the siRNAs and other inhibitory nucleic acid molecules
as therapeutics. Such systems include, for example, encapsulation
in liposomes, microparticles, microcapsules, nanoparticles,
recombinant cells capable of expressing the therapeutic molecule(s)
(see, e.g., Wu et al., J. Biol. Chem. 262, 4429, 1987),
construction of a therapeutic nucleic acid as part of a retroviral
or other vector, and the like.
C. Small Molecule Inhibitors
[0223] PD-1 antagonists include molecules that are identified from
large libraries of both natural product or synthetic (or
semi-synthetic) extracts or chemical libraries according to methods
known in the art. The screening methods that detect decreases in
PD-1 activity (such as detecting cell death) are useful for
identifying compounds from a variety of sources for activity. The
initial screens may be performed using a diverse library of
compounds, a variety of other compounds and compound libraries.
Thus, molecules that bind PD-1, PD-L1 or PD-L2, molecules that
inhibit the expression of PD-1, PD-L1 and/or PD-L2, and molecules
that inhibit the activity of PD-1, PD-L1 and/or PD-L2 can be
identified. These small molecules can be identified from
combinatorial libraries, natural product libraries, or other small
molecule libraries. In addition, PD-1 antagonist can be identified
as compounds from commercial sources, as well as commercially
available analogs of identified inhibitors.
[0224] The precise source of test extracts or compounds is not
critical to the identification of PD-1 antagonists. Accordingly,
PD-1 antagonists can be identified from virtually any number of
chemical extracts or compounds. Examples of such extracts or
compounds that can be PD-1 antagonists include, but are not limited
to, plant-, fungal-, prokaryotic- or animal-based extracts,
fermentation broths, and synthetic compounds, as well as
modification of existing compounds. Numerous methods are also
available for generating random or directed synthesis (e.g.,
semi-synthesis or total synthesis) of any number of chemical
compounds, including, but not limited to, saccharide-, lipid-,
peptide-, and nucleic acid-based compounds. Synthetic compound
libraries are commercially available from Brandon Associates
(Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). PD-1
antagonists can be identified from synthetic compound libraries
that are commercially available from a number of companies
including Maybridge Chemical Co. (Trevillet, Cornwall, UK),
Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.),
and Microsource (New Milford, Conn.). PD-1 antagonists can be
identified from a rare chemical library, such as the library that
is available from Aldrich (Milwaukee, Wis.). PD-1 antagonists can
be identified in libraries of natural compounds in the form of
bacterial, fungal, plant, and animal extracts are commercially
available from a number of sources, including Biotics (Sussex, UK),
Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft.
Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Natural
and synthetically produced libraries and compounds are readily
modified through conventional chemical, physical, and biochemical
means.
[0225] Useful compounds may be found within numerous chemical
classes, though typically they are organic compounds, including
small organic compounds. Small organic compounds have a molecular
weight of more than 50 yet less than about 2,500 daltons, such as
less than about 750 or less than about 350 daltons can be utilized
in the methods disclosed herein. Exemplary classes include
heterocycles, peptides, saccharides, steroids, and the like. The
compounds may be modified to enhance efficacy, stability,
pharmaceutical compatibility, and the like. In several embodiments,
compounds of use has a Kd for PD-1, PD-L1 or PD-L2 of less than 1
nM, less than 10 nm, less than 1 .mu.M, less than 10 .mu.M, or less
than 1 mM.
D. PD-1 Peptide Variants as Antagonists
[0226] In one embodiment, variants of a PD-1 protein which function
as an antagonist can be identified by screening combinatorial
libraries of mutants, such as point mutants or truncation mutants,
of a PD-1 protein to identify proteins with antagonist activity. In
one example, the antagonist is a soluble PD-1 protein.
[0227] Thus, a library of PD-1 variants can be generated by
combinatorial mutagenesis at the nucleic acid level and is encoded
by a variegated gene library. A library of PD-1 variants can be
produced by, for example, by enzymatically ligating a mixture of
synthetic oligonucleotides into gene sequences such that a
degenerate set of potential PD-1 sequences is expressible as
individual polypeptides, or alternatively, as a set of larger
fusion proteins (such as for phage display) containing the set of
PD-1 sequences.
[0228] There are a variety of methods which can be used to produce
libraries of potential PD-1 variants from a degenerate
oligonucleotide sequence. Chemical synthesis of a degenerate gene
sequence can be performed in an automatic DNA synthesizer, and the
synthetic gene then ligated into an appropriate expression vector.
Use of a degenerate set of genes allows for the provision, in one
mixture, of all of the sequences encoding the desired set of
potential PD-1 antagonist sequences. Methods for synthesizing
degenerate oligonucleotides are known in the art (see, for example,
Narang, et al., Tetrahedron 39:3, 1983; Itakura et al. Annu. Rev.
Biochem. 53:323, 1984; Itakura et al. Science 198:1056, 1984).
[0229] In addition, libraries of fragments of a PD-1 protein coding
sequence can be used to generate a population of PD-1 fragments for
screening and subsequent selection of variants of a PD-1
antagonist. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a PD-1 coding sequence with a nuclease under conditions
wherein nicking occurs only about once per molecule, denaturing the
double stranded DNA, renaturing the DNA to form double stranded DNA
which can include sense/antisense pairs from different nicked
products, removing single stranded portions from reformed duplexes
by treatment with S1 nuclease, and ligating the resulting fragment
library into an expression vector. By this method, an expression
library can be derived which encodes N-terminal, C-terminal and
internal fragments of various sizes of PD-1.
[0230] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of PD-1 proteins. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis
(REM) can be used in combination with the screening assays to
identify PD-1 antagonists (Arkin and Youvan, Proc. Natl. Acad. Sci.
USA 89:7811 7815, 1992; Delagrave et al., Protein Eng. 6(3):327
331, 1993).
[0231] In one embodiment, cell based assays can be exploited to
analyze a library of PD-1 variants. For example, a library of
expression vectors can be transfected into a cell line which
ordinarily synthesizes and secretes PD-1. The transfected cells are
then cultured such that PD-1 and a particular PD-1 variant are
secreted. The effect of expression of the mutant on PD-1 activity
in cell supernatants can be detected, such as by any of a
functional assay. Plasmid DNA can then be recovered from the cells
wherein endogenous PD-1 activity is inhibited, and the individual
clones further characterized.
[0232] Peptidomimetics can also be used as PD-1 antagonists.
Peptide analogs are commonly used in the pharmaceutical industry as
non-peptide drugs with properties analogous to those of the
template peptide. These types of non-peptide compounds and are
usually developed with the aid of computerized molecular modeling.
Peptide mimetics that are structurally similar to therapeutically
useful peptides can be used to produce an equivalent therapeutic or
prophylactic effect. Generally, peptidomimetics are structurally
similar to a paradigm polypeptide (for example, polypeptide that
has a PD-1 biological activity), but has one or more peptide
linkages optionally replaced by a --CH.sub.2NH--, --CH.sub.2S--,
--CH.sub.2--CH.sub.2--, --CH.=.CH-- (cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO-- linkages.
These peptide linkages can be replaced by methods known in the art
(see, for example, Morley, Trends Pharm. Sci. pp. 463 468, 1980;
Hudson et al. Int. J. Pept. Prot. Res. 14:177 185, 1979; Spatola,
Life Sci. 38:1243 1249, 1986; Holladay, et al. Tetrahedron Lett.
24:4401 4404, 1983). Peptide mimetics can be procured economically,
be stable, and can have increased half-life or absorption. Labeling
of peptidomimetics usually involves covalent attachment of one or
more labels, directly or through a spacer (such as by an amide
group), to non-interfering position(s) on the peptidomimetic that
are predicted by quantitative structure-activity data and/or
molecular modeling. Such non-interfering positions generally are
positions that do not form direct contacts with the
macromolecules(s) to which the peptidomimetic binds to produce the
therapeutic effect. Derivitization of peptidomimetics should not
substantially interfere with the desired biological or
pharmacological activity of the peptidomimetic.
[0233] A dominant negative protein or a nucleic acid encoding a
dominant negative protein that interferes with the biological
activity of PD-1 (i.e. binding of PD-1 to PD-L1, PD-L2, or both)
can also be used in the methods disclosed herein. A dominant
negative protein is any amino acid molecule having a sequence that
has at least 50%, 70%, 80%, 90%, 95%, or even 99% sequence identity
to at least 10, 20, 35, 50, 100, or more than 150 amino acids of
the wild type protein to which the dominant negative protein
corresponds. For example, a dominant-negative PD-L1 has mutation
such that it binds PD-1 more tightly than native (wild-type) PD-1
but does not activate any cellular signaling through PD-1.
[0234] The dominant negative protein may be administered as an
expression vector. The expression vector may be a non-viral vector
or a viral vector (e.g., retrovirus, recombinant adeno-associated
virus, or a recombinant adenoviral vector). Alternatively, the
dominant negative protein may be directly administered as a
recombinant protein systemically or to the infected area using, for
example, microinjection techniques.
[0235] Polypeptide antagonists can be produced in prokaryotic or
eukaryotic host cells by expression of polynucleotides encoding the
amino acid sequence, frequently as part of a larger polypeptide (a
fusion protein, such as with ras or an enzyme). Alternatively, such
peptides can be synthesized by chemical methods. Methods for
expression of heterologous proteins in recombinant hosts, chemical
synthesis of polypeptides, and in vitro translation are well known
in the art (see Maniatis er al. Molecular Cloning: A Laboratory
Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and
Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular
Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.;
Kaiser et al., Science 243:187, 1989; Merrifield, Science 232:342,
1986; Kent, Annu. Rev. Biochem. 57:957, 1988).
[0236] Peptides can be produced, such as by direct chemical
synthesis, and used as antagonists of a PD-1 interaction with a
ligand. Peptides can be produced as modified peptides, with
nonpeptide moieties attached by covalent linkage to the N-terminus
and/or C-terminus. In certain preferred embodiments, either the
carboxy-terminus or the amino-terminus, or both, are chemically
modified. The most common modifications of the terminal amino and
carboxyl groups are acetylation and amidation, respectively.
Amino-terminal modifications such as acylation (for example,
acetylation) or alkylation (for example, methylation) and
carboxy-terminal-modifications such as amidation, as well as other
terminal modifications, including cyclization, can be incorporated
into various embodiments. Certain amino-terminal and/or
carboxy-terminal modifications and/or peptide extensions to the
core sequence can provide advantageous physical, chemical,
biochemical, and pharmacological properties, such as: enhanced
stability, increased potency and/or efficacy, resistance to serum
proteases, desirable pharmacokinetic properties, and others.
[0237] Method of Treatment: Administration of a PD-1 Antagonist to
a Subject
[0238] Methods are provided herein to treat a variety of infections
and cancers. In these methods, the infection or cancer is treated,
prevented or a symptom is alleviated by administering to a subject
a therapeutically effective amount of a PD-1 antagonist. The
subject can be any mammal such as human, a primate, mouse, rat,
dog, cat, cow, horse, and pig. In several examples, the subject is
a primate, such as a human. In additional examples, the subject is
a murine subject, such as a mouse. In some embodiments, the method
includes measuring memory B cell proliferation in a sample from the
subject (see below). In some examples, the methods also include
measuring naive B cells in a sample from the subject. In additional
examples, the methods include measure T cells that express CD28
(CD28+) cells.
[0239] In several embodiments, the subject is at risk of developing
infection. A subject at risk of developing infection is a subject
that does not yet have the infection, but can be infected by the
infectious agent of interest. In additional examples, the subject
has an infection, such as a persistent infection, for example a
chronic infection. A subject with a persistent infection, such as a
chronic infection, can be identified by standard methods suitable
by one of skill in the art, such as a physician.
[0240] In several examples, the subject has a persistent infection
with a bacteria virus, fungus, or parasite. Generally, persistent
infections, in contrast to acute infections are not effectively
cleared by the induction of a host immune response. The infectious
agent and the immune response reach equilibrium such that the
infected subject remains infectious over a long period of time
without necessarily expressing symptoms. Persistent infections
include for example, latent, chronic and slow infections.
Persistent infection occurs with viruses such as human T-Cell
leukemia viruses, Epstein-Barr virus, cytomegalovirus,
herpesviruses, varicella-zoster virus, measles, papovaviruses,
prions, hepatitis viruses, adenoviruses, XMRV, polyoma JC virus,
parvoviruses and papillomaviruses.
[0241] In a chronic infection, the infectious agent can be detected
in the body at all times. However, the signs and symptoms of the
disease may be present or absent for an extended period of time.
Examples of chronic infection include hepatitis B (caused by
heptatitis B virus (HBV)) and hepatitis C (caused by hepatitis C
virus (HCV)) adenovirus, cytomegalovirus, Epstein-Barr virus,
herpes simplex virus 1, herpes simplex virus 2, human herpesvirus
6, varicella-zoster virus, hepatitis B virus, hepatitis D virus,
papilloma virus, parvovirus B19, polyomavirus K, polyomavirus JC,
XMRV, measles virus, rubella virus, human immunodeficiency virus
(HIV), human T cell leukemia virus I, and human T cell leukemia
virus II. Parasitic persistent infections may arise as a result of
infection by Leishmania, Toxoplasma, Trypanosoma, Plasmodium,
Schistosoma, and Encephalitozoon.
[0242] In a latent infection, the infectious agent (such as a
virus) is seemingly inactive and dormant such that the subject does
always exhibit signs or symptoms. In a latent viral infection, the
virus remains in equilibrium with the host for long periods of time
before symptoms again appear; however, the actual viruses cannot be
detected until reactivation of the disease occurs. Examples of
latent infections include infections caused by herpes simplex virus
(HSV)-1 (fever blisters), HSV-2 (genital herpes), and varicella
zoster virus VZV (chickenpox-shingles).
[0243] In a slow infection, the infectious agents gradually
increase in number over a very long period of time during which no
significant signs or symptoms are observed. Examples of slow
infections include AIDS (caused by HIV-1 and HIV-2), lentiviruses
that cause tumors in animals, and prions.
[0244] In addition, persistent infections often arise as late
complications of acute infections. For example, subacute sclerosing
panencephalitis (SSPE) can occur following an acute measles
infection or regressive encephalitis can occur as a result of a
rubella infection.
[0245] In one non-limiting example, a subject may be diagnosed as
having a persistent Chlamydial infection following the detection of
Chlamydial species in a biological sample from this individual
using PCR analysis. Mammals need not have not been diagnosed with a
persistent infection to be treated according to this disclosure.
Microbial agents capable of establishing a persistent infection
include viruses (such as papilloma virus, hepatitis virus, human
immune deficiency virus, and herpes virus), bacteria (such as
Escherichia coli and Chlamydia spp.), parasites, (such as
Leishmania spp., Schistosoma spp., Trypanosoma spp., Toxoplasma
spp.) and fungi.
[0246] In addition to the compound that reduces PD-1 expression or
activity, the subject being treated may also be administered a
vaccine. In one example, the vaccine can include an adjuvant. In
another example, the vaccine can include a prime booster
immunization. The vaccine can be a heat-killed vaccine, an
attenuated vaccine, or a subunit vaccine. A subject already
infected with a pathogen can be treated with a therapeutic vaccine,
such as a PD-1 antagonist and an antigen. The subject can be
asymptomatic, so that the treatment prevents the development of a
symptom. The therapeutic vaccine can also reduce the severity of
one or more existing symptoms, or reduce pathogen load.
[0247] In several examples of treatment methods, the subject is
administered a therapeutically effective amount of a PD-1
antagonist in conjunction with a viral antigen. Non-limiting
examples of suitable viral antigens include: influenza HA, NA, M,
NP and NS antigens; HIV p24, pol, gp41 and gp120; Metapneumovirus
(hMNV) F and G proteins; Hepatitis C virus (HCV) E1, E2 and core
proteins; Dengue virus (DEN1-4) E1, E2 and core proteins; Human
Papilloma Virus L1 protein; Epstein Barr Virus gp220/350 and
EBNA-3A peptide; Cytomegalovirus (CMV) gB glycoprotein, gH
glycoprotein, pp65, IE1 (exon 4) and pp150; Varicella Zoster virus
(VZV) IE62 peptide and glycoprotein E epitopes; Herpes Simplex
Virus Glycoprotein D epitopes, polyoma JC virus polypeptides, XMRV
polypeptides, among many others. The antigenic polypeptides can
correspond to polypeptides of naturally occurring animal or human
viral isolates, or can be engineered to incorporate one or more
amino acid substitutions as compared to a natural (pathogenic or
non-pathogenic) isolate. Exemplary antigens are listed below:
TABLE-US-00008 TABLE 1 Exemplary antigens of interest (target
antigens) Exemplary Antigen Sequences from the SEQ ID Antigens of
interest NO: Viral Antigens BK TLYKKMEQDVKVAHQ 13 GNLPLMRKAYLRKCK
14 TFSRMKYNICMGKCI 15 JC SITEVECFL 16 Epstein-Barr (EBV) QPRAPIRPI
17 cytomegalovirus NLVPMVATV 18 (CMV) HPV YMLDLQPET(T) 19 Influenza
A GILGFVFTL 20 Fungal Antigen Blastomyces CELDNSHEDYNWNLWFKWCSGHGR
47 dermatitidis TGHGKHFYDCDWDPSHGDYSWYLW 48
DPSHGDYSWYLWDYLCGNGHHPYD 49 DYLCGNGHHPYDCELDNSHEDYSW 50
DPYNCDWDPYHEKYDWDLWNKWCN 51 KYDWDLWNKWCNKDPYNCDWDPYH 52
[0248] In additional embodiments, the subject has a tumor. The
method includes administering to the subject a therapeutically
effective amount of a PD-1 antagonist, thereby treating the tumor.
In several examples, a therapeutically effective amount of a tumor
antigen, or a nucleotide encoding the tumor antigen, is also
administered to the subject. The PD-1 antagonist and the tumor
antigen, or nucleotide encoding the tumor antigen, can be
administered simultaneously or sequentially.
[0249] Administration of the PD-1 antagonist results in a decrease
in size, prevalence, or metastatic potential of a tumor in a
subject. Assessment of cancer is made using standard clinical
protocols. Efficacy is determined in association with any known
method for diagnosing or treating the particular tumor.
[0250] Tumors (also called "cancers") include solid tumors and
leukemias. Exemplary tumors include those listed in table 2 (along
with known tumor antigens associated with these cancers).
TABLE-US-00009 TABLE 2 Exemplary tumors and their tumor antigens
Tumor Tumor Antigens Acute myelogenous leukemia Wilms tumor 1
(WT1), preferentially expressed antigen of melanoma (PRAME), PR1,
proteinase 3, elastase, cathepsin G Chronic myelogenous WT1, PRAME,
PR1, proteinase 3, elastase, leukemia cathepsin G Myelodysplastic
syndrome WT1, PRAME, PR1, proteinase 3, elastase, cathepsin G Acute
lymphoblastic leukemia PRAME Chronic lymphocytic leukemia Survivin
Non-Hodgkin's lymphoma Survivin Multiple myeloma New York
esophageous 1 (NY-Eso1) Malignant melanoma MAGE, MART, Tyrosinase,
PRAME, GP100 Breast cancer WT1, herceptin Lung cancer WT1 Prostate
cancer Prostate-specific antigen (PSA) Colon cancer
Carcinoembryonic antigen (CEA) Renal cell carcinoma (RCC)
Fibroblast growth factor 5 (FGF-5)
TABLE-US-00010 TABLE 3 Exemplary tumor antigens of interest include
those listed below Table 3: Tumor Antigens and their derivative
peptides PRAME LYVDSLFFL 21 WT1 RMFPNAPYL 22 Survivin ELTLGEFLKL 23
AFP GVALQTMKQ 24 ELF2M ETVSEQSNV 25 proteinase 3 and VLQELNVTV 26
its peptide PR1 neutrophil elastase VLQELNVTV 27 MAGE EADPTGHSY 28
MART AAGIGILTV 29 tyrosinase RHRPLQEVYPEANAPIGHNRE 30 GP100
WNRQLYPEWTEAQRLD 31 NY-Eso-1 VLLKEFTVSG 32 Herceptin KIFGSLAFL 33
carcino-embryonic HLFGYSWYK 34 antigen (CEA) PSA FLTPKKLQCV 35
[0251] Specific non-limiting examples are angioimmunoblastic
lymphoma or nodular lymphocyte predominant Hodgkin lymphoma.
Angioimmunoblastic lymphoma (AIL) is an aggressive (rapidly
progressing) type of T-cell non-Hodgkin lymphoma marked by enlarged
lymph nodes and hypergammaglobulinemia (increased antibodies in the
blood). Other symptoms may include a skin rash, fever, weight loss,
positive Coomb's test or night sweats. This malignancy usually
occurs in adults. Patients are usually aged 40-90 years (median
around 65) and are more often male. As AIL progresses,
hepatosplenomegaly, hemolytic anemia, and polyclonal
hypergammaglobulinemia may develop. The skin is involved in
approximately 40-50% of patients.
[0252] Nodular lymphocyte predominant Hodgkin lymphoma is a B cell
neoplasm that appears to be derived from germinal center B cells
with mutated, non-functional immunoglobulin genes. Similar to
angioimmunoblastic lymphoma, neoplastic cells are associated with a
meshwork of follicular dendritic cells. PD-1 expression is seen in
T cells closely associated with neoplastic CD20+ cell in nodular
lymphocyte predominant Hodgkin lymphoma, in a pattern similar to
that seen for CD57+ T cells. CD57 has been identified as another
marker of germinal center-associated T cells, along with CXCR5,
findings which support the conclusion that the neoplastic cells in
nodular lymphocyte predominant Hodgkin lymphoma have a close
association with germinal center-associated T cells.
[0253] Expression of a tumor antigen of interest can be determined
at the protein or nucleic acid level using any method known in the
art. For example, Northern hybridization analysis using probes
which specifically recognize one or more of these sequences can be
used to determine gene expression. Alternatively, expression is
measured using reverse-transcription-based PCR assays, such as
using primers specific for the differentially expressed sequence of
genes. Expression is also determined at the protein level, such as
by measuring the levels of peptides encoded by the gene products
described herein, or activities thereof. Such methods are well
known in the art and include, for example immunoassays based on
antibodies to proteins encoded by the genes. Any biological
material can be used for the detection/quantification of the
protein or the activity.
[0254] In one example, the subject has been previously diagnosed as
having cancer. In additional examples, the subject has undergone
prior treatment for the cancer. However, in some examples, the
subject has not been previously diagnosed as having the cancer.
Diagnosis of a solid tumor can be made through the identification
of a mass on an examination, although it may also be through other
means such as a radiological diagnosis, or ultrasound. Treatment of
cancer can include surgery, or can include the use of
chemotherapeutic agents such as docetaxel, vinorelbine gemcitabine,
capecitabine or combinations of cyclophosphamide, methotrexate, and
fluorouracil; cyclophosphamide, doxorubicin, and fluorouracil;
doxorubicin and cyclophosphamide; doxorubicin and cyclophosphamide
with paclitaxel; doxorubicin followed by CMF (Cyclophosphamide,
epirubicin and fluorouracil). In addition, treatment can include
the use of radiation.
[0255] In several examples, a therapeutically effective amount a
PD-1 antagonist is administered to the subject. A therapeutically
effective amount of a tumor antigen, or a nucleic acid encoding the
antigen, is also administered to the subject. The administration
can be concurrent or can be sequential.
[0256] For the treatment of a subject with a persistent infection
(such as a chronic infection) or a tumor, a therapeutically
effective amount of a PD-1 antagonist is administered to the
subject of interest. In one example, a therapeutically effective
amount of a PD-1 antagonist is a biologically active dose, such as
a dose that will induce an increase in CD8+ T cell cytotoxic
activity the increase in the immune response specific to the
infectious agent. Desirably, the PD-1 antagonist has the ability to
reduce the expression or activity of PD-1 in antigen specific
immune cells (e.g., T cells such as CD8+ T cells) by at least 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 100%
below untreated control levels. The levels or activity of PD-1 in
immune cells is measured by any method known in the art, including,
for example, Western blot analysis, immunohistochemistry, ELISA,
and Northern Blot analysis. Alternatively, the biological activity
of PD-1 is measured by assessing binding of PD-1 to PD-L1, PD-L2,
or both. The biological activity of PD-1 is determined according to
its ability to increase CD8+ T cell cytotoxicity including, for
example, cytokine production, clearance of the infectious agent,
and proliferation of antigen specific CD8+ T cells. Preferably, the
agent that reduces the expression or activity of PD-1 can increase
the immune response specific to the infectious agent or the tumor
by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
more than 100% above untreated control levels. The agent of the
present invention is therefore any agent having any one or more of
these activities. Although the agent is preferably expressed in
CD8+ T cells, it is understood that any cell that can influence the
immune response to persistent infections is also amenable to the
methods of the invention and include, for example, B cells.
[0257] Optionally, the subject is administered one or more
additional therapeutic agents. Additional therapeutic agents
include, for example, antiviral compounds (e.g., vidarabine,
acyclovir, gancyclovir, valgancyclovir, nucleoside-analog reverse
transcriptase inhibitor (NRTI) (e.g., AZT (Zidovudine), ddl
(Didanosine), ddC (Zalcitabine), d4T (Stavudine), or 3TC
(Lamivudine)), non-nucleoside reverse transcriptase inhibitor
(NNRTI) (e.g., (nevirapine or delavirdine), protease inhibitor
(saquinavir, ritonavir, indinavir, or nelfinavir), ribavirin, or
interferon), antibacterial compounds, antifungal compounds,
antiparasitic compounds, anti-inflammatory compounds,
anti-neoplastic agent (chemotherapeutics) or analgesics.
[0258] The additional therapeutic agent is administered prior to,
concomitantly, or subsequent to administration of the PD-1
antagonist. For example, the PD-1 antagonist and the additional
agent are administered in separate formulations within at least 1,
2, 4, 6, 10, 12, 18, or more than 24 hours apart. Optionally, the
additional agent is formulated together with the PD-1 antagonist.
When the additional agent is present in a different composition,
different routes of administration may be used. The agent is
administered at doses known to be effective for such agent for
treating, reducing, or preventing an infection.
[0259] Concentrations of the PD-1 antagonist and the additional
agent depends upon different factors, including means of
administration, target site, physiological state of the mammal, and
other medication administered. Thus treatment dosages may be
titrated to optimize safety and efficacy and is within the skill of
an artisan. Determination of the proper dosage and administration
regime for a particular situation is within the skill of the
art.
[0260] Optionally, the subject is further administered a vaccine
that elicits a protective immune response against the infectious
agent that causes a persistent infection. For example, the subject
receives a vaccine that elicits an immune response against human
immunodeficiency virus (HIV), tuberculosis, influenza, XMRV,
polyoma JC virus, or hepatitis C, amongst others. Exemplary
vaccines are described, for example, in Berzofsky et al. (J. Clin.
Invest. 114:456-462, 2004). If desired, the vaccine is administered
with a prime-booster shot or with adjuvants. The vaccine can also
be a tumor vaccine, such as a therapeutically effective amount of a
tumor antigen. In several embodiments, a therapeutically effective
amount of an antigenic polypeptide, such as a viral or a tumor
antigen, is administered to the subject.
[0261] A therapeutically effective amount of the tumor antigen, or
a nucleic acid encoding the tumor antigen can be administered to
the subject. The polynucleotides include a recombinant DNA which is
incorporated into a vector into an autonomously replicating plasmid
or virus or into the genomic DNA of a prokaryote or eukaryote, or
which exists as a separate molecule (such as a cDNA) independent of
other sequences. The nucleotides be ribonucleotides,
deoxyribonucleotides, or modified forms of either nucleotide. The
term includes single and double forms of DNA.
[0262] A number of viral vectors have been constructed, including
polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol.,
73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol.
Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques,
6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin
et al., 1992, Proc. Nad. Acad. Sci. USA, 89:2581-2584; Rosenfeld et
al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids
Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene
Ther., 1:241-256), vaccinia virus (Mackett et al., 1992,
Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992,
Curr. Top. Microbiol. Immunol., 158:91-123; On et al., 1990, Gene,
89:279-282), herpes viruses including HSV and EBV (Margolskee,
1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al.,
1992, J. Virol., 66:29522965; Fink et al., 1992, Hum. Gene Ther.
3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371;
Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199), Sindbis
viruses (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167;
U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.
Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al.,
1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses
of avian (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754;
Petropouplos et al., 1992, J. Virol., 66:3391-3397), murine
(Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24; Miller et
al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al., 1984, Mol.
Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401-407),
and human origin (Page et al., 1990, J. Virol., 64:5370-5276;
Buchschalcher et al., 1992, J. Virol., 66:2731-2739). Baculovirus
(Autographa californica multinuclear polyhedrosis virus; AcMNPV)
vectors are also known in the art, and may be obtained from
commercial sources (such as PharMingen, San Diego, Calif.; Protein
Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).
[0263] In one embodiment, the polynucleotide encoding a tumor
antigen or a viral antigen is included in a viral vector. Suitable
vectors include retrovirus vectors, orthopox vectors, avipox
vectors, fowlpox vectors, capripox vectors, suipox vectors,
adenoviral vectors, herpes virus vectors, alpha virus vectors,
baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors
and poliovirus vectors. Specific exemplary vectors are poxvirus
vectors such as vaccinia virus, fowlpox virus and a highly
attenuated vaccinia virus (MVA), adenovirus, baculovirus and the
like.
[0264] Pox viruses of use include orthopox, suipox, avipox, and
capripox virus. Orthopox include vaccinia, ectromelia, and raccoon
pox. One example of an orthopox of use is vaccinia. Avipox includes
fowlpox, canary pox and pigeon pox. Capripox include goatpox and
sheeppox. In one example, the suipox is swinepox. Examples of pox
viral vectors for expression as described for example, in U.S. Pat.
No. 6,165,460, which is incorporated herein by reference. Other
viral vectors that can be used include other DNA viruses such as
herpes virus and adenoviruses, and RNA viruses such as retroviruses
and polio.
[0265] In several embodiments, PD-1 antagonists are administered in
an amount sufficient to increase T cell, such as CD8+ T cell,
cytotoxicity. An increase in T-cell cytotoxicity results in an
increased immune response and a reduction in the persistent
infection, or a reduction in a sign or a symptom of a tumor. An
increased immune response can be measured, for example, by an
increase in immune cell proliferation, such as T-cell or B cell
proliferation, an increase in cytokine production, and an increase
in the clearance of an infectious agent or a reduction in tumor
burden. Thus, the method can result in alleviation of one or more
of symptoms associated with the persistent infection or tumor.
Thus, administration of the PD-1 antagonist reduces the persistent
infection, inhibits the growth/size of a tumor, or alleviates one
or more symptoms associated with the persistent infection or tumor
by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as
compared to an untreated subject.
[0266] Treatment is efficacious if the treatment leads to clinical
benefit such as, a reduction of the load of the infectious agent or
a reduction of tumor burden in the subject. When treatment is
applied prophylactically, "efficacious" means that the treatment
retards or prevents an infection from forming, such as for a period
of six months, one year, two years, three years or more. Efficacy
may be determined using any known method for diagnosing or treating
the particular infection or tumor.
[0267] Thus, the methods include administering to a subject a
pharmaceutical composition that includes a therapeutically
effective amount of a PD-1 antagonist. An effective amount of a
therapeutic compound, such as an antibody, can be for example from
about 0.1 mg/kg to about 150 mg/kg. Effective doses vary, as
recognized by those skilled in the art, depending on route of
administration, excipient usage, and coadministration with other
therapeutic treatments including use of other anti-infection agents
or therapeutic agents for treating, preventing or alleviating a
symptom of a particular infection or cancer. A therapeutic regimen
is utilized for a human patient suffering from (or at risk of
developing) an infection or cancer, using standard methods.
[0268] The PD-1 antagonist is administered to such an individual
using methods known in the art. Any PD-1 antagonist can be
utilized, such as those disclosed herein. In addition, more than
one PD-1 antagonist can be utilized. A PD-1 antagonist can be
administered locally or systemically. For example, the PD-1
antagonist is administered orally, rectally, nasally, topically
parenterally, subcutaneously, intraperitoneally, intramuscularly,
and intravenously. The PD-1 antagonist can be administered
prophylactically, or after the detection of an infection or tumor.
The PD-1 antagonist is optionally formulated as a component of a
cocktail of therapeutic drugs to treat infection. Examples of
formulations suitable for parenteral administration include aqueous
solutions of the active agent in an isotonic saline solution, a 5%
glucose solution, or another standard pharmaceutically acceptable
excipient. Standard solubilizing agents such as PVP or
cyclodextrins are also utilized as pharmaceutical excipients for
delivery of the therapeutic compounds.
[0269] The therapeutic compounds described herein are formulated
into compositions for other routes of administration utilizing
conventional methods. For example, PD-1 antagonist is formulated in
a capsule or a tablet for oral administration. Capsules may contain
any standard pharmaceutically acceptable materials such as gelatin
or cellulose. Tablets may be formulated in accordance with
conventional procedures by compressing mixtures of a therapeutic
compound with a solid carrier and a lubricant. Examples of solid
carriers include starch and sugar bentonite. The PD-1 antagonist
can be administered in the form of a hard shell tablet or a capsule
containing a binder, such as lactose or mannitol, a conventional
filler, and a tableting agent. Other formulations include an
ointment, suppository, paste, spray, patch, cream, gel, resorbable
sponge, or foam. Such formulations are produced using methods well
known in the art.
[0270] Additionally, PD-1 antagonists can be administered by
implanting (either directly into an organ (e.g., intestine or
liver) or subcutaneously) a solid or resorbable matrix which slowly
releases the compound into adjacent and surrounding tissues of the
subject. For example, for the treatment of gastrointestinal
infection, the compound may be administered systemically (e.g.,
intravenously, rectally or orally) or locally (e.g., directly into
gastric tissue). Alternatively, a PD-1 antagonist-impregnated wafer
or resorbable sponge is placed in direct contact with gastric
tissue. The PD-1 antagonist is slowly released in vivo by diffusion
of the drug from the wafer and erosion of the polymer matrix. As
another example, infection of the liver (i.e., hepatitis) is
treated by infusing into the liver vasculature a solution
containing the PD-1 antagonist.
[0271] Where the therapeutic compound is a nucleic acid encoding a
PD-1 antagonist, the nucleic acid can be administered in vivo to
promote expression of the encoded protein, by constructing it as
part of an appropriate nucleic acid expression vector and
administering it so that it becomes intracellular (such by use of a
retroviral vector, by direct injection, by use of microparticle
bombardment, by coating with lipids or cell-surface receptors or
transfecting agents, or by administering it in linkage to a
homeobox-like peptide which is known to enter the nucleus (See,
e.g., Joliot, et al., Proc Natl Acad Sci USA 88:1864-1868, 1991),
and the like. Alternatively, a nucleic acid therapeutic is
introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination or remain episomal.
[0272] For local administration of DNA, standard gene therapy
vectors can be used. Such vectors include viral vectors, including
those derived from replication-defective hepatitis viruses (such as
HBV and HCV), retroviruses (see, PCT Publication No. WO 89/07136;
Rosenberg et al., N. Eng. J. Med. 323(9):570-578, 1990, adenovirus
(see, Morsey et al., J. Cell. Biochem., Supp. 17E, 1993),
adeno-associated virus (Kotin et al., Proc. Natl. Acad. Sci. USA
87:2211-2215, 1990), replication defective herpes simplex viruses
(HSV; Lu et al., Abstract, page 66, Abstracts of the Meeting on
Gene Therapy, September 22-26, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1992, and any modified versions of these
vectors. Any other delivery system can be utilized that
accomplishes in vivo transfer of nucleic acids into eukaryotic
cells. For example, the nucleic acids may be packaged into
liposomes, such as cationic liposomes (Lipofectin),
receptor-mediated delivery systems, non-viral nucleic acid-based
vectors, erythrocyte ghosts, or microspheres (such as
microparticles; see, e.g., U.S. Pat. No. 4,789,734; U.S. Pat. No.
4,925,673; U.S. Pat. No. 3,625,214). Naked DNA may also be
administered.
[0273] With regard to nucleic acid inhibitors, a therapeutically
effective amount is an amount which is capable of producing a
medically desirable result, e.g., a decrease of a PD-1 gene product
in a treated animal. Such an amount can be determined by one of
ordinary skill in the art. Dosage for any given patient depends
upon many factors, including the patient's size, body surface area,
age, the particular compound to be administered, sex, time and
route of administration, general health, and other drugs being
administered concurrently. Dosages may vary, but a preferred dosage
for intravenous administration of DNA is approximately 106 to 1022
copies of the DNA molecule.
[0274] Typically, plasmids are administered to a mammal in an
amount of about 1 nanogram to about 5000 micrograms of DNA.
Desirably, compositions contain about 5 nanograms to 1000
micrograms of DNA, 10 nanograms to 800 micrograms of DNA, 0.1
micrograms to 500 micrograms of DNA, 1 microgram to 350 micrograms
of DNA, 25 micrograms to 250 micrograms of DNA, or 100 micrograms
to 200 micrograms of DNA. Alternatively, administration of
recombinant adenoviral vectors encoding the PD-1 antagonist into a
mammal may be administered at a concentration of at least 105, 106,
107, 108, 109, 1010, or 1011 plaque forming unit (pfu).
[0275] In some embodiments, for the treatment of neurological
infections, the PD-1 antagonist can be administered intravenously
or intrathecally (for example, by direct infusion into the
cerebrospinal fluid). For local administration, a
compound-impregnated wafer or resorbable sponge is placed in direct
contact with central nervous system (CNS) tissue. The compound or
mixture of compounds is slowly released in vivo by diffusion of the
drug from the wafer and erosion of the polymer matrix.
Alternatively, the compound is infused into the brain or
cerebrospinal fluid using standard methods. For example, a burr
hole ring with a catheter for use as an injection port is
positioned to engage the skull at a burr hole drilled into the
skull. A fluid reservoir connected to the catheter is accessed by a
needle or stylet inserted through a septum positioned over the top
of the burr hole ring. A catheter assembly (described, for example,
in U.S. Pat. No. 5,954,687) provides a fluid flow path suitable for
the transfer of fluids to or from selected location at, near or
within the brain to allow administration of the drug over a period
of time.
[0276] In additional embodiments, for cardiac infections, the PD-1
antagonist can be delivered, for example, to the cardiac tissue
(such as the myocardium, pericardium, or endocardium) by direct
intracoronary injection through the chest wall or using standard
percutaneous catheter based methods under fluoroscopic guidance.
Thus, the PD-1 antagonist may be directly injected into tissue or
may be infused from a stent or catheter which is inserted into a
bodily lumen. Any variety of coronary catheter or perfusion
catheter may be used to administer the compound. Alternatively, the
PD-1 antagonist is coated or impregnated on a stent that is placed
in a coronary vessel.
[0277] Pulmonary infections can be treated, for example, by
administering the PD-1 antagonist by inhalation. The compounds are
delivered in the form of an aerosol spray from a pressured
container or dispenser which contains a suitable propellant, such
as a gas such as carbon dioxide or a nebulizer.
[0278] One in the art will understand that the patients treated can
have been subjected to the same tests to diagnose a persistently
infected subject or may have been identified, without examination,
as one at high risk due to the presence of one or more risk factors
(such as exposure to infectious agent, exposure to infected
subject, genetic predisposition, or having a pathological condition
predisposing to secondary infections). Reduction of persistent
infection symptoms or damage may also include, but are not limited
to, alleviation of symptoms, diminishment of extent of disease,
stabilization (not worsening) state of disease, delay or slowing of
disease progression, and amelioration or palliation of the disease
state. Treatment can occur at home with close supervision by the
health care provider, or can occur in a health care facility.
[0279] Methods for measuring the immune response following
treatment using the methods disclosed herein are well known in the
art. The activity of T cells may be assessed, for example, by
assays that detect cytokine production, assays measuring T cell
proliferation, assays that measure the clearance of the microbial
agent, and assays that measure CD8+ T cell cytotoxicity. These
assays are described, for example, in U.S. Pat. No. 6,808,710 and
U.S. Patent Application Publication Nos. 20040137577, 20030232323,
20030166531, 20030064380, 20030044768, 20030039653, 20020164600,
20020160000, 20020110836, 20020107363, and 20020106730, all of
which are hereby incorporated by reference. The measurement of a B
cell response, such as a memory B cell response, is described
below.
[0280] Optionally, the ability of a PD-1 antagonist to increase
CD8+ T cell cytotoxicity is assessed by assays that measure the
proliferation of CD8+ T cells (for example, thymidine
incorporation, BrdU assays, and staining with cell cycle markers
(for example, Ki67 and CFSE), described, for example, by Dong et
al. (Nature 5:1365-1369, 1999). In one example, T-cell
proliferation is monitored by culturing the purified T-cells
expressing PD-1 with a PD-1 antagonist, a primary activation signal
as described above, and .sup.3H-thymidine. The level of T-cell
proliferation is determined by measuring thymidine
incorporation.
[0281] CD8+ T cell cytotoxicity also can be assessed by lysis
assays (such as .sup.51Cr release assays or assays detecting the
release of perforin or granzyme), assays that detect caspase
activation, or assays that measure the clearance of the microbial
agent from the infected subject. For example, the viral load in a
biological sample from the infected subject (e.g., serum, spleen,
liver, lung, or the tissue to which the virus is tropic) may be
measured before and after treatment.
[0282] The production of cytokines such as IFN.gamma., TNF-.alpha.,
and IL-2 may also be measured. For example, purified T-cells are
cultured in the presence of the PD-1 protein antagonist and a
primary activation signal. The level of various cytokines in the
supernatant can be determined by sandwich enzyme-linked
immunosorbent assays or other conventional assays described, for
example, in Dong et al. (Nature 5:1365-1369, 1999).
[0283] If desired, the efficacy of the PD-1 antagonist is assessed
by its ability to induce co-stimulation of T cells. For example, a
method for in vitro T-cell co-stimulation involves providing
purified T-cells that express PD-1 with a first or primary
activation signal by anti-CD3 monoclonal antibody or phorbol ester,
or by antigen in association with class II MHC. The ability of a
candidate compound agent to reduce PD-1 expression or activity and
therefore provide the secondary or co-stimulatory signal necessary
to modulate immune function, to these T-cells can then be assayed
by any one of the several conventional assays well known in the
art.
[0284] The B cell response to the PD-1 antagonist can be assessed
by LCMV specific ELISA, plasma cell ELISPOT, memory B-cell assay,
phenotyping of B cell, and analysis of germinal centers by
immunohistochemistry.
Methods of Treatment: Adoptive Immunotherapy
[0285] Methods are disclosed herein for the treatment of a subject
of interest, such as a subject with a persistent viral infection
(such as a chronic infection) or a tumor. The methods include the
administration of a therapeutically effective amount of cytoxic T
cells specific for an antigen of interest, such as a viral antigen
or a tumor antigen, and a therapeutically effective amount of a
PD-1 antagonist. In some embodiments, the method can also include
measuring memory B cell proliferation in a sample from the subject
(see below). In additional embodiments, the methods include
measuring naive B cells in a sample from the subject. In further
embodiments, the methods also include measuring T cells that
express CD28. In some embodiments, the methods include measuring
neutralizing antibodies. Thus, the disclosed methods include
measuring at least one of neutralizing antibodies, memory B cell
proliferation, naive B cells, and T cells that express CD28. Two,
three or all of these parameters can be measured using the methods
disclosed herein.
[0286] Methods are disclosed herein for increasing the immune
response, such as enhancing the immune system in a subject.
Administration of the purified antigen-specific T cells and PD-1,
as disclosed herein, will increase the ability of a subject to
overcome pathological conditions, such as an infectious disease or
a tumor, by targeting an immune response against a pathogen (such
as a virus or fungus) or neoplasm. Therefore, by purifying and
generating a purified population of selected antigen-specific T
cells from a subject ex vivo and introducing a therapeutic amount
of these cells, the immune response of the recipient subject is
enhanced. The administration of a therapeutically effective amount
of a PD-1 antagonist also enhances the immune response of the
recipient.
[0287] Methods of inducing an immune response to an antigen of
interest in a recipient are provided herein. The recipient can be
any subject of interest, including a subject with a chronic
infection, such as a viral or fungal infection, or a subject with a
tumor. These infections are described above.
[0288] Infections in immune deficient people are a common problem
in allograft stem cell recipients and in permanently
immunosuppressed organ transplant recipients. The resulting T cell
deficiency infections in these subjects are usually from
reactivation of viruses already present in the recipient. For
example, once acquired, most herpes group viruses (such as CMV,
EBV, VZV, HSV) are dormant, and kept suppressed by T cells.
However, when patients are immunosuppressed by conditioning
regimens, dormant viruses can be reactivated. For example, CMV
reactivation, Epstein Barr virus (EBV) reactivation which causes a
tumor in B cells (EBV lymphoproliferative disease), and BK virus
reactivation which causes hemorrhagic cystitis, can occur following
immunosuppression. In addition, HIV infection and congenital immune
deficiency are other examples of T cell immune deficiency. These
viral infections and reactivations can be an issue in
immunosuppressed subjects.
[0289] In several embodiments, an immune response against a tumor
is provided to the recipient of a bone marrow transplant.
Anti-tumor immunity can be provided to a subject by administration
of antigen-specific T cells that recognize a tumor-antigen. Such
administration to a recipient will enhance the recipient's immune
response to the tumor by providing T cells that are targeted to,
recognize, and immunoreact with a tumor antigen of interest.
[0290] In one example, the method includes isolating from the donor
a population of donor cells including T cells (such as peripheral
blood mononuclear cells) and contacting a population of donor cells
comprising T cells with a population of antigen presenting cells
(APCs) from the donor that are presenting an antigen of interest,
optionally in the presence of PD-1, thereby producing a population
of donor cells comprising activated donor CD4.sup.+ and/or
CD8.sup.+ T cells depleted for alloreactive T cells that recognize
an antigen of interest. A therapeutically effective amount of the
population of donor activated CD4+ and/or CD8+ cells into the
recipient, thereby producing an immune response to the antigen of
interest in the recipient. Administration of the purified
antigen-specific T cells can increase the ability of a subject to
overcome pathological conditions, such as an infectious disease or
a tumor, by targeting an immune response against a pathogen (such
as a virus or fungus) or neoplasm. Thus, an immune response is
produced in the recipient against the antigen of interest.
[0291] In several embodiments the method also includes
administering a therapeutically effective amount of a PD-1
antagonist to the subject. The administration of PD-1 antagonists
is described in detail above.
[0292] Any antigenic peptide (such as an immunogenic fragment) from
an antigen of interest can be used to generate a population of T
cells specific for that antigen of interest. Numerous such
antigenic peptides are known in the art, such as viral and tumor
antigens (see, for example, Tables 1-3). This disclosure is not
limited to using specific antigen peptides. Particular examples of
antigenic peptides from antigens of interest, include, but are not
limited to, those antigens that are viral, fungal, and tumor
antigens, such as those shown in Tables 1-3. Additional antigenic
peptides are known in the art (for example see Novellino et al.,
Cancer Immunol. Immunother. 54(3):187-207, 2005, and Chen et al.,
Cytotherapy, 4:41-8, 2002, both herein incorporated by
reference).
[0293] Although Tables 1 and 3 disclose particular fragments of
full-length antigens of interest, one skilled in the art will
recognize that other fragments or the full-length protein can also
be used in the methods disclosed herein. In one example, an antigen
of interest is an "immunogenic fragment" of a full-length antigen
sequence. An "immunogenic fragment" refers to a portion of a
protein which, when presented by a cell in the context of a
molecule of the MHC, can in a T-cell activation assay, activate a
T-cell against a cell expressing the protein. Typically, such
fragments that bind to MHC class I molecules are 8 to 12 contiguous
amino acids of a full length antigen, although longer fragments may
of course also be used. In some examples, the immunogenic fragment
is one that can specifically bind to an MHC molecule on the surface
of an APC, without further processing of the epitope sequence. In
particular examples, the immunogenic fragment is 8-50 contiguous
amino acids from a full-length antigen sequence, such as 8-20 amino
acids, 8-15 amino acids, 8-12 amino acids, 8-10 amino acids, or 8,
9, 10, 11, 12, 13, 14, 15 or 20 contiguous amino acids from a
full-length antigen sequence. In some examples, APCs are incubated
with the immunogenic fragment under conditions sufficient for the
immunogenic fragment to specifically bind to MHC molecules on the
APC surface, without the need for intracellular processing.
[0294] In one example, an antigen includes a peptide from the
antigen of interest with an amino acid sequence bearing a binding
motif for an HLA molecule of the subject. These motifs are well
known in the art. For example, HLA-A2 is a common allele in the
human population. The binding motif for this molecule includes
peptides with 9 or 10 amino acids having leucine or methionine in
the second position and valine or leucine in the last positions
(see examples above). Peptides that include these motifs can be
prepared by any method known in the art (such as recombinantly,
chemically, etc.). With knowledge of an amino acid sequence of an
antigen of interest, immunogenic fragment sequences predicted to
bind to an MHC can be determined using publicly available programs.
For example, an HLA binding motif program on the Internet
(Bioinformatics and Molecular Analysis Section-BIMAS) can be used
to predict epitopes of any tumor-, viral-, or fungal-associated
antigen, using routine methods. Antigens of interest (either
full-length proteins or an immunogenic fragment thereof) then can
be produced and purified using standard techniques. For example,
epitope or full-length antigens of interest can be produced
recombinantly or chemically synthesized by standard methods. A
substantially pure peptide preparation will yield a single major
band on a non-reducing polyacrylamide gel. In other examples, the
antigen of interest includes a crude viral lysate.
[0295] In one example, the antigen of interest is a tumor
associated antigen and the amino acid sequences bearing HLA binding
motifs are those that encode subdominant or cryptic epitopes. Those
epitopes can be identified by a lower comparative binding affinity
for the HLA molecule with respect to other epitopes in the molecule
or compared with other molecules that bind to the HLA molecule.
[0296] Through the study of single amino acid substituted antigen
analogs and the sequencing of endogenously bound, naturally
processed peptides, critical residues that correspond to motifs
required for specific binding to HLA antigen molecules have been
identified (see, for example, Southwood et al., J. Immunol.
160:3363, 1998; Rammensee et al., Immunogenetics 41:178, 1995;
Rammensee et al., J. Curr. Opin. Immunol. 10:478, 1998; Engelhard,
Curr. Opin. Immunol. 6:13, 1994; Sette and Grey, Curr. Opin.
Immunol. 4:79, 1992). Furthermore, x-ray crystallographic analysis
of HLA-peptide complexes has revealed pockets within the peptide
binding cleft of HLA molecules which accommodate, in an
allele-specific mode, residues borne by peptide ligands; these
residues in turn determine the HLA binding capacity of the peptides
in which they are present. (See, for example, Madden, Annu. Rev.
Immunol. 13:587, 1995; Smith et al., Immunity 4:203, 1996; Fremont
et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994;
Jones, Curr. Opin. Immunol. 9:75, 1997; Brown et al., Nature
364:33, 1993.)
[0297] The antigen of interest is selected based on the subject to
be treated. For example, if the subject is in need of increased
antiviral or antifungal immunity one or more target viral or fungal
associated antigens are selected. Exemplary antigens of interest
from viruses include antigens from Epstein bar virus (EBV),
hepatitis C virus (HCV) cytomegalovirus (CMV), herpes simplex virus
(HSV), BK virus, JC virus, and human immunodeficiency virus (HIV)
amongst others. Exemplary antigens of interest from fungi include
antigens from Candida albicans, Cryptococcus, Blastomyces, and
Histoplasma, or other infectious agent. In another example, the
subject is in need of increased anti-tumor immunity. Exemplary
antigens of interest from tumors include WT1, PSA, PRAME. Exemplary
antigens of interest for infectious agents are listed in Table 1.
In some examples, the antigen of interest includes both a viral
antigen and a tumor antigen, both a fungal antigen and a tumor
antigen, or a viral antigen, a fungal antigen, and a tumor
antigen.
[0298] For the treatment of a subject with a tumor, the tumor
antigen of interest is chosen based on the expression of the
protein by the recipient's tumor. For example, if the recipient has
a breast tumor, a breast tumor antigen is selected, and if the
recipient has a prostate tumor, a prostate tumor antigen is
selected, and so forth. Table 2 lists exemplary tumors and
respective tumor associated antigens that can be used to generate
purified antigen-specific T cells that can be administered to a
subject having that particular tumor. However, one skilled in the
art will recognize that the same and other tumors can be treated
using additional tumor antigens.
[0299] In one example, antigen-specific T cells that recognize a
tumor antigen are administered in a therapeutically effective
amount to a subject who has had, or will receive, a stem cell
allograft or autograft, or who has been vaccinated with the tumor
antigen. For example, a therapeutic amount of antigen-specific T
cells can be administered that recognize one or more
tumor-associated antigens, for example at least one of the antigens
of interest listed in Tables 2-3.
[0300] In particular examples where the recipient has a tumor and
has or will receive a stem cell allograft, donor tumor
antigen-specific T cells and a therapeutically effective amount of
a PD-1 antagonist are administered in a therapeutically effective
amount after the stem cell allograft to prevent, decrease, or delay
tumor recurrence, or to treat a malignant relapse. The purified
antigen-specific T cells can be introduced back into the subject
after debulking. In yet another example, the recipient is
vaccinated with the tumor antigen of interest, purified
antigen-specific T cells purified from the recipient and then
re-introduced into the recipient with a therapeutically effective
amount of a PD-1 antagonist to increase the recipient's immune
system against the tumor.
[0301] Administration of a therapeutic amount of tumor
antigen-specific T cells and a therapeutically effective amount of
a PD-1 antagonist can be used prophylactically to prevent
recurrence of the tumor in the recipient, or to treat a relapse of
the tumor. Such antigen-specific T cells can kill cells containing
the tumor-associated antigen or assist other immune cells.
[0302] In a specific example, a recipient has a tumor and has or
will receive a stem cell allograft to reconstitute immunity.
Following bone marrow irradiation or administration of a cytotoxic
drug that has ablated or otherwise compromised bone marrow
function, at least two types of donor antigen-specific T cells are
administered in a therapeutically effective amount;
antigen-specific T cells that specifically recognize a
viral-associated antigen (or a fungal-associated antigen) and
antigen-specific T cells that specifically recognize a
tumor-associated antigen. In addition, a therapeutically effective
amount of a PD-1 antagonist is administered to the subject. Such
administration can be used to induce an anti-tumor effect and an
anti-viral effect (such as an anti-viral effect).
[0303] In order to produce a population of antigen-specific T cells
for administration to a subject of interest, a population of cells
including T cells can be contacted with antigen presenting cells
(APCs), such as dendritic cells or T-APCs, to present the antigen
of interest. In some embodiments, the responder T cells (such as
lymphocytes or PBMCs) are treated with an antagonist of PD-1 and
are added to the APCs presenting one or more antigens of interest,
and incubated under conditions sufficient to allow the interaction
between the APCs presenting antigen and the T cells to produce
antigen-specific T cells. The treatment of the responder T cells
with the PD-1 antagonist can be simultaneously with the contact or
the APCs. The treatment with the PD-1 antagonist can also be
immediately prior to the contact with the APCs.
[0304] Thus, methods are provided herein for producing an enriched
population of antigen-specific T cells. Generally, T-APCs present
antigens to T cells and induce an MHC-restricted response in a
class I (CD8+ T cells) and class II (CD4+ T cells) restricted
fashion. The typical T cell response is activation and
proliferation. Thus, a population is produced that includes T cells
that specifically recognize an antigen of interest. Thus a
therapeutically effective amount of this population of cells can be
administered to a subject to produce an immune response, such as a
subject with a chronic infection or a tumor.
[0305] Generally, the APCs and the T cells are autologous. In
specific, non-limiting examples, the APCs and the responder T cells
are from the same individual. However, the APCs and the responder T
cells can be syngeneic. The APC can be used to present any antigen
to a population of autologous T cells. One of skill in the art will
appreciate that antigenic peptides that bind to MHC class I and II
molecules can be generated ex vivo (for example instead of being
processed from a full-length protein in a cell), and allowed to
interact with (such as bind) MHC I and II molecules on a cell
surface. Generally, APCs present antigen in the context of both MHC
class I and II.
[0306] In one example, the antigen of interest incubated with the
APCs is a fusion protein that includes an amino acid sequence from
the antigen of interest (such as 8-50 contiguous amino acids, for
example 8-15 or 8-12 contiguous amino acids from the antigen of
interest). Thus, a series of MHC binding epitopes can be included
in a single antigenic polypeptide, or a single chain trimer can be
utilized, wherein each trimer has an MHC class I molecule, a b2
microglobulin, and an antigenic peptide of interest (see Nature
2005; vol. 436, page 578). In some examples, only a single antigen
is used, but in other embodiments, more than one antigen is used,
such as at least 2 different antigens, at least 3 different
antigens, at least 4 different antigens, at least 5 different
antigens, at least 10 different antigens, at least 15 different
antigens, at least 20 different antigens, or even at least 50
different antigens.
[0307] In yet other examples, an antigen of interest is a
full-length antigen amino acid sequence (such as a full-length
fungal antigen, tumor antigen, or viral antigen, for example a
viral lysate or full-length cathepsin G). In additional examples,
one or more antigens from any infectious agent can be utilized. In
some examples, the full-length antigen of interest is expressed by
the APC.
[0308] APCs can be produced using methods known to one of skill in
the art (see Melenhorst et al, Cytotherapy 7, supp. 1, 2005;
Melenhorst et al., Blood 106: 671a, 2005; Gagliardi et al., Int.
Immunol. 7: 1741-52, 1995, herein incorporated by reference). In
one example, to produce T-APCs, donor peripheral blood monocytes
are activated using IL-2 and an antibody that specifically binds
CD3 (such as OKT3) for about three or more days, such as about one
to two weeks, such as for about seven to ten days.
[0309] It has been observed that in the presence of presenting
antigen, T cells that recognize the antigen bind to antigen
presenting cells (APCs) presenting an antigen of interest more
strongly than do T cells that are not specific for the antigen (and
are thus not binding in an antigen-specific manner). In a
particular example, antigen-specific T cells are selected by
exposing APCs to a target peptide antigen (such as a target viral
or tumor associated antigen) against which desired T cells are to
be targeted in the presence of a PD-1 antagonist, such that the APC
presents the antigen in association with a major histocompatability
complex (MHC) class I and/or class II. For example, APCs can be
exposed to a sufficient amount of a antigen of interest to
sufficiently occupy MHC molecules on the surface of the APC (for
example, at least 1% of the MHC molecules are occupied, such at
least 5%, at least 7.5% or at least 10%) and stimulate preferential
binding of target T cells in the presence of a PD-1 antagonist to
the APCs presenting the antigen of interest (as compared to APCs
that do not present the antigen of interest). A population of T
cells, such as population that has been primed for the antigen of
interest, is then incubated with the APCs, optionally in the
presences of a PD-1 antagonist, such as an antibody that
specifically binds PD-1, to preferentially activate the cells,
thereby producing a population of cells enriched with the desired T
cells that recognize the antigen of interest.
[0310] T cells, such as those present in a population of PBMCs or
lymphocytes, can be incubated with one or more antigens of
interest, optionally in the presence of a PD-1 antagonist to
generate a T cell population that is primed for the one or more
antigens of interest. T cells can be primed using any method known
in the art. In particular examples, PBMCs or lymphocytes are
incubated in the presence of a purified target peptide antigen,
optionally in the presence of a PD-1 antagonist. In some examples,
the antigen of interest is an antigen of an infectious agent, or a
tumor antigen, such as, but not limited to, one or more of the
antigens of interest listed in the above tables. The antigen of
interest can be in a purified form, such as a chemically
synthesized peptide. In other examples, the antigen of interest is
present in a non-purified form, such as in a crude lysate, for
example a viral lysate.
[0311] The amount of antigen used to prime T cells can be readily
determined using methods known in the art. Generally, if the
antigen is used in a purified form, about 1-10 .mu.g/ml of peptide
is used. When a viral lysate is used, about 0.1-100 .mu.l of
lysate, such as about 75 .mu.l, can be used. When T-APCs are used,
about 4-6 million T-APCs presenting the antigen of interest can be
used for every 40-60 million T cells (or lymphocytes or PBMCs).
[0312] In a specific example, lymphocytes are primed in vitro by
incubating them with soluble antigen or viral lysate for 5-7 days
under conditions that permit priming of T cells. Viable T cells are
recovered, for example by Ficoll-Hypaque centrifugation, thereby
generating primed T cells. If desired, the viable primed T cells
can be primed again one or more times, for example by incubation
with the antigen for another 5-7 days under the same conditions as
those used for the first priming, and viable T cells recovered.
[0313] In another example, lymphocytes are primed in vivo by
inoculating a subject with the antigen, for example in the form of
a vaccine. In this example, T cells obtained from the subject
following immunization are already primed. For example, lymphocytes
or PBMC obtained from a subject are then incubated with APCs in the
presence of a PD-1 antagonist as described herein, without the need
for additional priming.
[0314] The method can further include generating the APCs that
present the antigen of interest. For example, APCs can be incubated
with a sufficient amount of one or more different peptide antigens,
under conditions sufficient for the target peptide(s) to be
presented on the surface of the APCs. This generates a population
of APCs that present the antigen of interest on MHC molecules on
the surface of the APC. The disclosed methods are not limited to
particular methods of presenting the antigen of interest on the
surface of an APC.
[0315] Antigens can also be expressed by the APC either naturally
or due to the insertion of a gene containing the DNA sequence
encoding the target protein (antigen). A nucleic acid encoding the
antigen of interest can be introduced into the T cells as messenger
RNA, or using a vector, such as a mammalian expression vector, or a
viral vector (for example, a adenovirus, poxvirus, or retrovirus
vectors). The polynucleotides encoding an antigen of interest
include a recombinant DNA which is an autonomously replicating
plasmid or virus, or which is incorporated into the genomic DNA of
a eukaryote, or which exists as a separate molecule independent of
other sequences. A nucleic acid encoding an antigen of interest can
also be introduced using electroporation, lipofection, or calcium
phosphate-based transfection.
[0316] A number of viral vectors have been constructed, including
polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol.,
73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol.
Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques,
6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin
et al., 1992, Proc. Nad. Acad. Sci. USA, 89:2581-2584; Rosenfeld et
al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids
Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene
Ther., 1:241-256), vaccinia virus (Mackett et al., 1992,
Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992,
Curr. Top. Microbiol. Immunol., 158:91-123; On et al., 1990, Gene,
89:279-282), herpes viruses including HSV, CMV and EBV (Margolskee,
1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al.,
1992, J. Virol., 66:29522965; Fink et al., 1992, Hum. Gene Ther.
3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371;
Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199), Sindbis
viruses (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167;
U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.
Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al.,
1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses
of avian (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754;
Petropouplos et al., 1992, J. Virol., 66:3391-3397), murine
(Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24; Miller et
al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al., 1984, Mol.
Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401-407),
and human origin (Page et al., 1990, J. Virol., 64:5370-5276;
Buchschalcher et al., 1992, J. Virol., 66:2731-2739). Baculovirus
(Autographa californica multinuclear polyhedrosis virus; AcMNPV)
vectors are also known in the art, and may be obtained from
commercial sources (such as PharMingen, San Diego, Calif.; Protein
Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).
[0317] In one embodiment, the polynucleotide encoding an antigen of
interest is included in a viral vector for transfer into APC.
Suitable vectors include retrovirus vectors, orthopox vectors,
avipox vectors, fowlpox vectors, capripox vectors, suipox vectors,
adenoviral vectors, herpes virus vectors, alpha virus vectors,
baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors
and poliovirus vectors. Specific exemplary vectors are poxvirus
vectors such as vaccinia virus, fowlpox virus and a highly
attenuated vaccinia virus (MVA), adenovirus, baculovirus and the
like.
[0318] Pox viruses of use include orthopox, suipox, avipox, and
capripox virus. Orthopox include vaccinia, ectromelia, and raccoon
pox. One example of an orthopox of use is vaccinia. Avipox includes
fowlpox, canary pox and pigeon pox. Capripox include goatpox and
sheeppox. In one example, the suipox is swinepox. Examples of pox
viral vectors for expression as described for example, in U.S. Pat.
No. 6,165,460, which is incorporated herein by reference. Other
viral vectors that can be used include other DNA viruses such as
herpes virus and adenoviruses, and RNA viruses such as retroviruses
and polio.
[0319] Suitable vectors are disclosed, for example, in U.S. Pat.
No. 6,998,252, which is incorporated herein by reference. In one
example, a recombinant poxvirus, such as a recombinant vaccinia
virus is synthetically modified by insertion of a chimeric gene
containing vaccinia regulatory sequences or DNA sequences
functionally equivalent thereto flanking DNA sequences which to
nature are not contiguous with the flanking vaccinia regulatory DNA
sequences that encode an antigen of interest. The recombinant virus
containing such a chimeric gene is effective at expressing the
antigen. In one example, the vaccine viral vector comprises (A) a
segment comprised of (i) a first DNA sequence encoding an antigen
and (ii) a poxvirus promoter, wherein the poxvirus promoter is
adjacent to and exerts transcriptional control over the DNA
sequence encoding an antigen polypeptide; and, flanking said
segment, (B) DNA from a nonessential region of a poxvirus genome.
The viral vector can encode a selectable marker. In one example,
the poxvirus includes, for example, a thymidine kinase gene (see
U.S. Pat. No. 6,998,252, which is incorporated herein by
reference).
[0320] The population of APCs that present a sufficient density of
the antigen(s) are incubated with T cells (such as lymphocytes or
PBMCs), optionally in the presence of an effective amount of a PD-1
antagonist, under conditions sufficient to allow binding between
the APCs presenting the antigen and the T cells that can
specifically immunoreact with the antigen (antigen-specific T
cells). A sufficient number of APCs expressing a sufficient density
of antigen in combination with MHC to stimulate enhance binding of
a target T cell to the APC are used. In particular examples, at
least 20% of the APCs are presenting the desired antigen on MHC
molecules on the APC surface, such as at least 30% of the APCs, at
least 40% of the APCs, at least 50% of the APCs, or at least 60% of
the APCs. The optimal amount of T cells added can vary depending on
the amount of APCs used. In some examples, a T cell:APC ratio of at
least 6:1 is used, such as at least 8:1, at least 10:1, at least
12:1, at least 15:1, at least 16:1, at least 20:1, or even at least
50:1.
[0321] To increase the number of antigen-specific T cells,
proliferation of the cells can be stimulated, for example by
incubation in the presence of a cytokine, such as IL-2, IL-7, IL-12
and IL-15. The amount of cytokine added is sufficient to stimulate
production and proliferation of T cells, and can be determined
using routine methods. In some examples, the amount of IL-2, IL-7,
IL-12, or IL-15 added is about 0.1-100 IU/mL, such as at least 1
IU/mL, at least 10 IU/mL, or at least 20 IU/mL.
[0322] After a sufficient amount of binding of the antigen specific
T cells to the APCs, T cells that specifically recognize the
antigen of interest are produced. This generates a population of
enriched (such as purified) antigen-specific T cells that are
specific for the antigen of interest. In some examples, the
resulting population of T cells that are specific for the antigen
of interest is at least 30% pure, such as at least 40% pure, or
even at least 50% pure. The purity of the population of antigen
specific T cells can be assessed using methods known to one of
skill in the art.
[0323] In one example, during stimulation of proliferation of
antigen-specific T cells, the cells can be counted to determine the
cell number. When the desired number of cells is achieved, purity
is determined. Purity can be determined, for example, using markers
present on the surface of antigen-specific T cells concomitant with
the assessment of cytokine production upon antigen recognition,
such as interferon (IFN).gamma., tumor necrosis factor
(TNF).alpha., interleukin (IL)-2, IL-10, transforming growth factor
(TGF).beta.1, or IL-4. Generally, antigen-specific T cells are
positive for the CD3 marker, along with the CD4 or CD8 marker, and
IFN-.gamma. (which is specific for activated T cells). For example,
fluorescence activated cell sorting (FACS) can be used to identify
(and sort if desired) populations of cells that are positive for
CD3, CD4 or CD8, and IFN-.gamma. by using differently colored
anti-CD3, anti-CD4, anti-CD8 and anti-IFN-.gamma.. Briefly,
stimulated T antigen-specific cells are incubated in the presence
of anti-CD3, anti-CD4, anti-CD8 and anti-IFN-.gamma. (each having a
different fluorophore attached), for a time sufficient for the
antibody to bind to the cells. After removing unbound antibody,
cells are analyzed by FACS using routine methods. Antigen-specific
T cells are those that are INF-.gamma. positive and CD8 positive or
CD4 positive. In specific examples, the resulting population of
antigenic T cells is at least 30% pure relative to the total
population of CD4+ or CD8+ positive cells, such as at least 40%
pure, at least 50% pure, at least 60% pure, or even at least 70%
pure relative to the total population of CD4 positive or CD8
positive cells.
[0324] In another example, the method further includes determining
the cytotoxicity of the antigen-specific T cells. Methods for
determining cytotoxicity are known in the art, for example a
.sup.51Cr-release assay (for example see Walker et al. Nature
328:345-8, 1987; Qin et al. Acta Pharmacol. Sin. 23(6):534-8, 2002;
all herein incorporated by reference).
[0325] The antigen-specific T cells can be subjected to one or more
rounds of selection to increase the purity of the antigen-specific
T cells. For example, the purified antigen-specific T cells
generated above are again incubated with APCs presenting the
antigen of interest in the presence of a PD-1 antagonist under
conditions sufficient to allow binding between the APCs and the
purified antigen-specific T cells. The resulting antigen-specific T
cells can be stimulated to proliferate, for example with IL-2.
Generally, the resulting antigen-specific T cells that specifically
immunoreact with the antigen of interest are more pure after
successive stimulations with APCs than with only one round of
selection. In one example, the population of purified
antigen-specific T cells produced is at least 90% pure relative to
all CD3+ cells present, such as at least 95% pure or at least 98%
pure. In a particular example, the population of purified
antigen-specific T cells produced is at least 95% pure relative to
all CD4+ cells present, such as at least 98% pure. In another
example, the population of purified antigen-specific T cells
produced is at least 90% pure relative to all CD3+ cells present,
such as at least 93% pure.
[0326] The present disclosure also provides therapeutic
compositions that include the enriched (such as purified)
antigen-specific T cells and a PD-1 antagonist. In particular
examples, the resulting enriched population of antigen-specific T
cells (specific for the antigen of interest) are placed in a
therapeutic dose form for administration to a subject in need of
them. The PD-1 antagonist is also present in a therapeutic dose
form for administration to a subject in need of treatment.
[0327] In one example, the population of purified antigen-specific
T cells produced is at least 30% pure relative to all CD3+ cells
present, such as at least 40% pure, at least 50% pure, at least 80%
pure, or even at least 90% pure. In a particular example, the
population of purified antigen-specific T cells produced is at
least 30% pure relative to all CD3+ cells present, such as at least
40% pure, at least 50% pure, at least 80% pure, at least 90% pure,
at least 95% pure, or even at least 98% pure. In another example,
the population of purified antigen-specific T cells produced is at
least 50% pure relative to all CD3+ cells present, such as at least
60% pure, at least 75% pure, at least 80% pure, at least 90% pure,
or even at least 93% pure. Expanded and selected antigen-specific T
cells can be tested for mycoplasma, sterility, endotoxin and
quality controlled for function and purity prior cryopreservation
or prior to infusion into the recipient.
[0328] A therapeutically effective amount of antigen-specific T
cells is administered to the subject. Specific, non-limiting
examples of a therapeutically effective amount of purified
antigen-specific T cells include purified antigen-specific T cells
administered at a dose of about 1.times.10.sup.5 cells per kilogram
of subject to about 1.times.10.sup.9 cells per kilogram of subject,
such as from about 1.times.10.sup.6 cells per kilogram to about
1.times.10.sup.8 cells per kilogram, such as from about
5.times.10.sup.6 cells per kilogram to about 75.times.10.sup.6
cells per kilogram, such as at about 25.times.10.sup.6 cells per
kilogram, or at about 50.times.10.sup.6 cells per kilogram.
[0329] Purified antigen-specific T cells can be administered in
single or multiple doses as determined by a clinician. For example,
the cells can be administered at intervals of approximately two
weeks depending on the response desired and the response obtained.
In some examples, once the desired response is obtained, no further
antigen-specific T cells are administered. However, if the
recipient displays one or more symptoms associated with infection
or the presence or growth of a tumor, a therapeutically effective
amount of antigen-specific T cells can be administered at that
time. The administration can be local or systemic.
[0330] The purified antigen-specific T cells disclosed herein can
be administered with a pharmaceutically acceptable carrier, such as
saline. The PD-1 antagonist can also be formulated in a
pharmaceutically acceptable carrier, as described above. In some
examples, other therapeutic agents are administered with the
antigen-specific T cells and PD-1 antagonist. Other therapeutic
agents can be administered before, during, or after administration
of the antigen-specific T cells, depending on the desired effect.
Exemplary therapeutic agents include, but are not limited to,
anti-microbial agents, immune stimulants such as interferon-alpha,
chemotherapeutic agents or peptide vaccines of the same antigen
used to stimulate T cells in vitro. In a particular example,
compositions containing purified antigen-specific T cells also
include one or more therapeutic agents.
Methods of Treatment and Evaluation
[0331] It is disclosed herein that administration of a
therapeutically effective amount of PD-1 antagonist affect B cells,
such as by increasing the proliferation of memory B cells. Methods
of treatment are provided herein that include the administration of
a PD-1 antagonist to a subject, as described above. These methods
include measuring B cells, such as but not limited to measuring the
proliferation of memory B cells in the subject. In some examples,
the methods include measuring naive B cells in a sample from the
subject. In some embodiments, the methods include measuring CD28 T
cells and/or measuring neutralizing antibodies to an antigen of
interest. Thus, the methods can include measuring one or more of
memory B cell proliferation, naive B cells, CD28 T cells, and
neutralizing antibodies.
[0332] Methods are also provided herein to treat, and measure the
efficacy of a PD-1 antagonist, in a variety of infections and
cancers. The present disclosure encompasses methods to determine if
treatment methods are effective in any subject of interest. In
these methods, a subject of interest is selected, such as a subject
with a persistent infection or cancer. This subject is administered
a therapeutically effective amount of a PD-1 antagonist. In some
examples, memory B cell proliferation is assessed to determine if
the treatment method was effective, and/or to determine if the dose
of the PD-1 antagonist should be altered. In additional examples,
the methods include measuring naive B cells. In further examples,
the methods include measuring CD28 T cells and/or measuring
neutralizing antibodies to an antigen of interest. Thus, the
methods can include measuring one or more of memory B cell
proliferation, naive B cells, CD28 T cells, and neutralizing
antibodies.
[0333] The subject can be any mammal such as human, a primate,
mouse, rat, dog, cat, cow, horse, and pig. In several examples, the
subject is a primate, such as a human. In additional examples, the
subject is a murine subject, such as a mouse. In several
embodiments, the subject is at risk of developing infection, as
discussed above. A subject at risk of developing infection is a
subject that does not yet have the infection, but can be infected
by the infectious agent of interest. In additional examples, a
subject is selected for treatment that has an infection, such as a
persistent infection. In other embodiments, the subject is at risk
of developing cancer or has cancer, as discussed above. These
subjects can be identified by standard methods suitable by one of
skill in the art, such as a physician. The disclosed methods
include selecting a subject of interest, and administering a PD-1
antagonist, as described above. Memory B cell proliferation is then
assessed. In some examples, the number of naive B cells is also
assessed.
[0334] In some embodiments, the subject has a persistent infection
with a bacteria virus, fungus, or parasite, as described above. A
therapeutically effective amount of a PD-1 antagonist is
administered to treat the subject. Memory B cell proliferation is
then assessed to determine if the treatment method was effective,
and/or to determine if the dose of the PD-1 antagonist should be
altered. Generally, persistent infections, in contrast to acute
infections are not effectively cleared by the induction of a host
immune response. The infectious agent and the immune response reach
equilibrium such that the infected subject remains infectious over
a long period of time without necessarily expressing symptoms.
Persistent infections include for example, latent, chronic and slow
infections. Persistent infection occurs with viruses such as human
T-Cell leukemia viruses, XMRV, polyoma JC virus, Epstein-Barr
virus, cytomegalovirus, herpesviruses, varicella-zoster virus,
measles, papovaviruses, prions, hepatitis viruses, adenoviruses,
parvoviruses and papillomaviruses. Additional persistent infections
are described above. These methods can include measuring naive B
cells, CD28 T cells and/or neutralizing antibodies.
[0335] In further embodiments, the subject has a tumor. A
therapeutically effective amount of a PD-1 antagonist is
administered to the subject to treat the tumor, as described above.
Memory B cell proliferation is then assessed to determine if the
treatment method was effective, and/or to determine if the dose of
the PD-1 antagonist should be altered. In several examples, a
therapeutically effective amount of a tumor antigen, or a
nucleotide encoding the tumor antigen, is also administered to the
subject. The PD-1 antagonist and the tumor antigen, or nucleotide
encoding the tumor antigen, can be administered simultaneously or
sequentially. These methods can include measuring naive B cells,
CD28 T cells and/or neutralizing antibodies.
[0336] In additional embodiments, the subject is administered a
therapeutically effective amount of cytoxic T cells specific for an
antigen of interest, such as a viral antigen or a tumor antigen,
and a therapeutically effective amount of a PD-1 antagonist.
Administration of the purified antigen-specific T cells and PD-1,
as disclosed herein, will increase the ability of a subject to
overcome pathological conditions, such as an infectious disease or
a tumor, by targeting an immune response against a pathogen (such
as a virus or fungus) or neoplasm. Therefore, by purifying and
generating a purified population of selected antigen-specific T
cells from a subject ex vivo and introducing a therapeutic amount
of these cells, the immune response of the recipient subject is
enhanced. The administration of a therapeutically effective amount
of a PD-1 antagonist also enhances the immune response of the
recipient. Memory B cell proliferation is then assessed to
determine if the treatment method was effective, and/or to
determine if the dose of the PD-1 antagonist and/or cytotoxic T
cells should be altered. These methods can include measuring naive
B cells, CD28 expressing (CD28+) T cells and/or neutralizing
antibodies.
[0337] Thus, the methods disclosed herein for determining if a PD-1
antagonist is effective, or for determining the dose of a PD-1
antagonist is effective, can be used in combination with any of the
therapeutic methods (and in any of the subjects) described
above.
[0338] In some embodiments, memory B cells are measured. An
increase in the proliferation of memory B cells from the a
biological sample as compared to a control indicates that the dose
of the PD-1 antagonist is of use treating the subject, and wherein
an absence of a significant alteration in the proliferation of
memory B cells as compared to the control indicates that the dose
of the PD-1 antagonist is not of use to treat the subject.
[0339] In additional embodiments, the methods include detecting
neutralizing antibodies in a biological sample from the subject,
wherein an increase in neutralizing antibodies as compared to a
control indicates that the dose of the PD-1 antagonist is of use
treating the subject, and wherein an absence of a significant
alteration in neutralizing antibodies as compared to the control
indicates that the dose of the PD-1 antagonist is not of use to
treat the subject. In further embodiments, the methods include
detecting CD28 expressing (CD28+) T cells in a biological sample
from the subject, wherein an increase in CD28+ T cells as compared
to a control indicates that the that a dose of the PD-1 antagonist
is of use treating the subject, and wherein an absence of a
significant alteration in CD28+ T cells as compared to the control
indicates that the dose of the PD-1 antagonist is not of use to
treat the subject. These measurements can be performed in addition
to measuring memory B cells, but can also be performed in the
absence of measuring memory B cells.
[0340] Additional methods are disclosed herein to determine whether
a particular PD-1 antagonist, or a particular dose of a PD-1
antagonist, is effective for treating a subject. These methods
include measuring the proliferation of memory B cells, such as in a
sample from the subject. These methods can also include measuring
naive B cells in a sample from the subject. For example, the
expression of CD27, CD20 and CD21 can be evaluated (see below). In
some examples, the measurement of memory B cells and/or naive B
cells occurs after a sufficient period of time for the PD-1
antagonist to decrease PD-1 activity in the subject.
[0341] The methods can also be used to evaluate the dose of a PD-1
antagonist that is therapeutically effective for a subject. For
example, the methods disclosed herein can be used to determine if
the dose administered to a subject of interest can be lowered and
still be effective. The methods disclosed herein also can be used
to determine if the dose administered to a subject is too low, and
thus must be increased to be therapeutically effective.
[0342] In some embodiments, a first dose of a PD-1 antagonist is
administered to the subject. An increase in proliferating memory B
cells, as compared to a control, indicates that this dose is
effective. In some cases, it can be advantageous to decrease the
amount of an agent administered to a subject, such as to decrease
side effects. Thus, if the first dose increases the proliferation
of memory B cells, a second lower dose of the PD-1 antagonist can
be administered to the subject, and a second sample including B
cells can be obtained. An increase in the proliferation of memory B
cells from the second sample as compared to a control indicates
that the second dose of the PD-1 antagonist is of use treating the
subject, and thus determines that the lower dose will be
therapeutically effective for treating the subject. An absence of a
significant alteration in the proliferation of memory B cells in
the second sample as compared to the control indicates that the
second dose of the PD-1 antagonist is not therapeutically effective
to treat the subject. The method can be repeated to determine the
lowest therapeutically effective dose for a subject of
interest.
[0343] In additional embodiments, a first dose of a PD-1 antagonist
is administered to the subject. A lack of an increase in the
proliferation of memory B cells, as compared to a control,
indicates that this dose is not therapeutically effective for
treating the subject. If the first dose did not increase the
proliferation of memory B cells, a second higher dose can be
administered to the subject, and a second sample including B cells
can be obtained. An increase in the proliferation of memory B cells
from the second sample as compared to a control indicates that the
second higher dose of the PD-1 antagonist is of use treating the
subject, and thus determines that the higher dose will be
therapeutically effective for treating the subject. An absence of a
significant alteration in the proliferation of memory B cells in
the second sample as compared to the control indicates that the
second dose of the PD-1 antagonist is not therapeutically effective
to treat the subject, and thus that a third higher dose is
required. Thus, the method can be repeated to determine a
therapeutically effective dose for a subject of interest.
[0344] The methods disclosed can also be used to determine if a
particular PD-1 antagonist is therapeutically effective for
treating a subject, and thus should be continued, or if the
particular PD-1 antagonist is not effective for treating a subject,
and thus that a different PD-1 antagonist should be utilized to
treat the subject. These methods include administering a particular
PD-1 antagonist to the subject, and assessing the proliferation of
memory B cells in the sample from the subject. An increase in the
proliferation of memory B cells in the sample as compared to a
control indicates that the particular PD-1 antagonist is of use
treating the subject. An absence of a significant alteration in the
proliferation of memory B cells in the sample as compared to the
control indicates that the particular PD-1 antagonist is not
therapeutically effective to treat the subject, and that a
different PD-1 antagonist or other therapeutic agent should be
administered to the subject.
[0345] Thus, the efficacy of a specific PD-1 antagonist can be
monitored, or the effective dose of a PD-1 antagonist can be
determined, using the methods disclosed herein. Generally, an
increase in proliferation of memory B cells from a sample from a
subject administered a PD-1 antagonist, as compared to a control,
indicates that the PD-1 antagonist is therapeutically effective for
a subject, and/or indicates that the dose is sufficient for
treating the subject.
[0346] Generally, measuring the proliferation of memory B cells
includes obtaining a sample that includes B cells from a subject,
and determining the presence or number of proliferating memory B
cells in the sample. In some examples, the sample is a biopsy
sample, a blood sample, or a sample of peripheral blood mononuclear
cells. The sample can be purified, for example to separate B cells,
such as memory B cells and/or naive B cells. In some embodiments,
the methods include measuring the quantity of proliferating memory
B cells and/or the quantity of naive B cells in a sample from a
subject administered a PD-1 antagonist of interest. In some
examples, the quantity of proliferating memory B cells and/or the
quantity of naive B cells is compared to a control. With regard to
proliferating memory B cells, the control can be a previously
determined standard value, or the quantity of proliferating memory
B cells from a subject not administered the PD-1 antagonist, or the
quantity of proliferating memory B cells from a subject
administered a control substance, such as vehicle alone. Similarly,
with regard to naive B cells, the control can be a previously
determined standard value, or the quantity of naive B cells from a
subject not administered the PD-1 antagonist, or the quantity of
naive B cells from a subject administered a control substance, such
as vehicle alone, or the quantity of naive B cells in a subject,
respectively.
[0347] In some examples, memory B cells are identified that express
CD27, such as those cells that express CD20 and CD27, but do not
express CD21 (CD20.sup.+CD27.sup.+CD21.sup.-) compared to naive B
cells, which express CD20 and CD21, but do not express CD27
(CD20.sup.+CD27.sup.-CD21+). Memory B cells and naive B cells can
be isolated and/or detected using antibodies that specifically bind
CD20, CD21 and CD27. In some embodiments, memory B cells express
CD27 (CD27.sup.+). In some examples, memory B cells are identified
as CD27.sup.+CD21.sup.- B cells, such as
CD20.sup.hi/CD21.sup.-/CD27.sup.+ (activated memory).
[0348] Methods for isolating and detecting B cells are known in the
art, and exemplary protocols are provided herein. Methods also are
known in the art to measure the proliferation of memory B cells
and/or to measure naive B cells. These methods generally involve
the use of molecular and/or biochemical techniques and not simple
visual observation. In some examples, fluorescence activated cell
sorting (FACS) is utilized. FACS can be used to sort (isolate)
cells such as immature B cells or differentiated plasma cells or
memory cells, by contacting the cells with an appropriately labeled
antibody. In one embodiment, several antibodies (such as antibodies
that bind CD27, CD20, CD21, CD45R, CD40, CD19, and/or IgM) and FACS
sorting can be used to produce substantially purified populations
of immature B cells, plasma cells and or memory B cells.
[0349] Methods are also known for measuring CD28 T cells in a
sample from a subject. These methods generally involve the use of
molecular and/or biochemical techniques and not simple visual
observation. In some examples, fluorescence activated cell sorting
(FACS) is utilized. FACS can be used to sort (isolate) cells such
as immature B cells or differentiated plasma cells or memory cells,
by contacting the cells with an appropriately labeled antibody. In
one embodiment, several antibodies (such as antibodies that bind
CD3, CD4, CD8 and CD28) and FACS sorting can be used to produce
substantially purified populations of CD28+ T cells. Methods for
the detection of neutralizing antibodies are also known in the art.
These assays include obtaining a biological sample and detecting
the binding of antibodies to an antigen of interest, as well as
specific neutralization assays, such as for a virus, for example
HIV.
[0350] FACS employs a plurality of color channels, low angle and
obtuse light-scattering detection channels, and impedance channels,
among other more sophisticated levels of detection, to separate or
sort cells. Any FACS technique can be employed as long as it is not
detrimental to the viability of the desired cells. (For exemplary
methods of FACS see U.S. Pat. No. 5,061,620).
[0351] However, other techniques of differing efficacy can be
employed to purify and isolate desired populations of cells. The
separation techniques employed should maximize the retention of
viability of the fraction of the cells to be collected. The
particular technique employed will, of course, depend upon the
efficiency of separation, cytotoxicity of the method, the ease and
speed of separation, and what equipment and/or technical skill is
required.
[0352] Separation procedures include magnetic separation, using
antibody-coated magnetic beads, affinity chromatography, cytotoxic
agents, either joined to a monoclonal antibody or used in
conjunction with complement, and "panning," which utilizes a
monoclonal antibody attached to a solid matrix, or another
convenient technique. Antibodies attached to magnetic beads and
other solid matrices, such as agarose beads, polystyrene beads,
hollow fiber membranes and plastic petri dishes, allow for direct
separation. Cells that are bound by the antibody can be removed
from the cell suspension by simply physically separating the solid
support from the cell suspension. The exact conditions and duration
of incubation of the cells with the solid phase-linked antibodies
will depend upon several factors specific to the system employed.
The selection of appropriate conditions, however, is well within
the skill in the art.
[0353] The unbound cells then can be eluted or washed away with
physiologic buffer after sufficient time has been allowed for the
cells expressing a marker of interest (e.g., CD45R or CD27) to bind
to the solid-phase linked antibodies. The bound cells are then
separated from the solid phase by any appropriate method, depending
mainly upon the nature of the solid phase and the antibody
employed.
[0354] Antibodies can be conjugated to biotin, which then can be
removed with avidin or streptavidin bound to a support, or
fluorochromes, which can be used with a fluorescence activated cell
sorter (FACS), to enable cell separation.
[0355] For example, cells expressing CD45R and/or CD27 are
initially separated from other cells by the cell-surface expression
of CD45R or CD27. In one specific, non-limiting example,
CD45R.sup.+ or CD27+ cells are positively selected by magnetic bead
separation, wherein magnetic beads are coated with CD45 or CD27
reactive monoclonal antibody. The CD45R.sup.+ or CD27+ cells are
then removed from the magnetic beads.
[0356] Release of the CD45R.sup.+ cells or CD27.sup.+ cells from
the magnetic beads can effected by culture release or other
methods. Purity of the isolated CD45R.sup.+ cells or CD27.sup.+
cells is then checked, such as with a FACSCAN.RTM. flow cytometer
(Becton Dickinson, San Jose, Calif.), if so desired. In one
embodiment, further purification steps are performed, such as FACS
sorting the population of cells released from the magnetic beads.
In one example, this sorting can be performed to detect expression
of MHC class II, IgM, CD19, and CD40, in order to detect or isolate
immature B cells. In another example, mature B cells can be
isolated and/or detected on the basis of expression of IgD and/or
CD21, in addition to MHC class II, IgM, CD14, and CD40.
[0357] Methods for analyzing B cell proliferation, such as the
assessment of the proliferation of memory B cells are known in the
art. For example, membrane dye dilution approaches can be utilized
which include ex vivo chemical labeling of cells of interest with
fluorescent dyes. Labeling with tritiated nucleoside analogues
(commonly .sup.3H-thymidine deoxyribonucleoside, .sup.3H-TdR) or
bromodeoxyuridine (BrdU) can be utilized. FACS analysis is
available for the measurement of BrdU incorporation. Surrogate
markers of proliferation such as DNA content and cell
cycle-associated proteins, can also be used.
[0358] In one example, measurement of Ki67 or PCNA can be utilized.
Ki67 antigen is the prototypic cell cycle related nuclear protein
that is expressed by proliferating cells in all phases of the
active cell cycle (G1, S, G2 and M phase). It is absent in resting
(G0) cells. Ki67 antibodies are useful in establishing
proliferation. Ki67 antibodies can be used to quantify
proliferating cells among and resting cells (Ki67 index). Ki67 is
routinely used as a marker of cell cycling and proliferation;
antibodies to Ki67 are commercially available, such as from
ABCAM.RTM., and methods are available to use these antibodies in
immunohistochemical and FACS analyses.
[0359] Other methods can be used to detect those cells that are in
the active cell cycle at the time of sampling. Proliferation of
lymphocytes, such as memory B cells, can also be measured by using
methods that utilize stable isotopes to label DNA in biological
samples including cells. DNA is uniformly and highly labeled via
the de novo synthesis pathway. The stable isotope labels used, e.g.
.sup.2H-glucose or heavy water (2H.sub.2O or H.sub.2.sup.18O), are
non-toxic to animals and humans, and generally regarded as safe by
the US Food and Drug Administration (FDA) (see U.S. Patent
Application Publication No. 2009/0155179). The measurement of
stable isotope label incorporation into lymphocyte DNA comprises
the following steps: (i) extraction of DNA or its release from
chromatin without further isolation, hydrolysis of DNA to
deoxyribonucleotides, (ii) selective release of deoxyribose from
purine deoxyribonucleotides, (iii) derivatization of purine
deoxyribose to a volatile derivative (e.g., pentane tetraacetate,
pentafluorobenzyl tetraacetyl derivative, or another suitable
derivative) suitable for analysis by gas chromatography/mass
spectrometry (GC/MS), (iv) GC/MS analysis of said derivative, (v)
analysis of the pattern of mass isotopomer abundance of said
derivative, and (vi) calculation from said pattern of an excess
enrichment value that is a measure of stable isotope incorporation.
Specific embodiments of each of these methods have been taught (see
U.S. Pat. No. 5,910,40).
In Vitro Assay
[0360] Methods are disclosed herein for selecting a PD-1
antagonist. These methods include determining if an agent of
interest is a PD-1 antagonist. Thus, the methods include screening
a number of agent to determine if they function as PD-1
antagoinists. This can be a library of compounds, small molecules
or antibodies, and the assay can be conducted in a high-throughput
format.
[0361] The methods also include determining if a specific PD-1
antagonist will be of use to treat a specific individual of
interest. Thus, these disclosed methods can be used for
"personalized medicine" wherein the population of cells is from a
specific individual of interest, and a number of potential PD-1
antagonist are tested to determine the PD-1 antagonist most suited
for treating that particular individual.
[0362] The methods include contacting an isolated population of
cells comprising memory B cells with an agent in vitro. In some
embodiments, the population of cells is peripheral blood
mononuclear cells or purified memory B cells, such as activated or
resting memory B cells. In one example, the population of cells is
a memory B cell line.
[0363] The methods can include detecting the proliferation of
memory B cells and/or detecting the differentiation of memory B
cells into antibody secreting cells.
[0364] In several embodiments, the methods include assays to detect
IgM, IgG and antibody-producing B cells. The assay can be an
ELISPOT assay. ELISPOT assays employ a technique very similar to
the sandwich enzyme-linked immunosorbent assay (ELISA) technique.
Either a monoclonal or polyclonal capture antibody is coated
aseptically onto a PVDF (polyvinylidene fluoride)-backed
microplate. These antibodies are chosen for their specificity for
the analyte in question. The plate is blocked, usually with a serum
protein that is non-reactive with any of the antibodies in the
assay. After this, cells of interest are plated out at varying
densities, along with antigen or mitogen, and then placed in a
humidified 37.degree. C. CO.sub.2 incubator for a specified period
of time.
[0365] Cytokine (or other cell product of interest, such as IgM or
IgG antibodies) secreted by activated cells is captured locally by
the coated antibody on the high surface area PVDF membrane. After
washing the wells to remove cells, debris, and media components, a
biotinylated polyclonal antibody specific for the chosen analyte is
added to the wells. This antibody is reactive with a distinct
epitope of the target and thus is employed to detect the captured
producted of interest. Following a wash to remove any unbound
biotinylated antibody, the detected product is then visualized,
such as using an avidin-enzyme, and a precipitating substrate for
the enzyme. The colored end product (a spot, usually colored)
typically represents an individual product-producing cell. The
spots can be counted manually (such as with a dissecting
microscope) or using an automated reader to capture the microwell
images and to analyze spot number and size.
[0366] The proliferation of memory B cells can also be assessed.
Suitable assays are disclosed herein (see above). Methods for
analyzing B cell proliferation, such as the assessment of the
proliferation of memory B cells are known in the art. For example,
membrane dye dilution approaches (commonly .sup.3H-thymidine
deoxyribonucleoside, .sup.3H-TdR) or bromodeoxyuridine (BrdU) can
be utilized. FACS analysis is available for the measurement of BrdU
incorporation. Surrogate markers of proliferation such as DNA
content and cell cycle-associated proteins, can also be used. In
one example, measurement of Ki67 or PCNA can be utilized. Other
methods can be used to detect those cells that are in the active
cell cycle at the time of sampling. Proliferation of lymphocytes,
such as memory B cells, can also be measured by using methods that
utilize stable isotopes to label DNA in biological samples
including cells. A exemplary, non-limited protocol for one assay of
use is provided in the Examples section below.
[0367] Generally, an increase of the proliferation of memory B
cells and/or an increase in the differentiation of memory B cells
into antibody secreting cells and/or in increase in antibody
production indicates that the agent is a PD-1 antagonist. The
increase of the proliferation of memory B cells and/or an increase
in the differentiation of memory B cells into antibody secreting
cells and/or in increase in antibody production can indicate that a
specific PD-1 antagonist will be of use in treating a subject.
[0368] The disclosure is illustrated by the following non-limiting
Examples.
Examples
Example 1: Inhibition of the PD-1 Pathway in Chronically-Infected
Mice Using Anti-PD-L1 Antibodies
[0369] Mice infected with various strains of the lymphocytic
choriomeningitis virus (LCMV) were used to study the effect of
chronic viral infection on CD8+ T cell function. The LCMV Armstrong
strain causes an acute infection that is cleared within 8 days,
leaving behind a long-lived population of highly functional,
resting memory CD8+ T cells. The LCMV C1-13 strain, in contrast,
establishes a persistent infection in the host, characterized by a
viremia that lasts up to 3 months. The virus remains in some
tissues indefinitely and antigen specific CD8+ T cells become
functionally impaired. DbNP396-404 CD8+ T cells are physically
deleted, while DbGP33-41 and DbGP276-286 CD8+ T cells persist but
lose the ability to proliferate or secrete anti-viral cytokines,
such as IFN-.gamma. and TNF-.alpha..
[0370] C57BL/6 mice were purchased from the National Cancer
Institute (Frederick, Md.). Mice were infected intravenously (i.v.)
with 2.times.10.sup.6 pfu of LCMV-C1-13. CD4 depletions were
performed by injecting 500 .mu.g of GK1.5 in PBS the day of
infection and the day following the infection. LCMV immune mice are
generated by infecting mice i.p. with 2.times.105 pfu LCMV
Armstrong.
[0371] Gene array analysis was performed on FACS-purified naive
DbGP33-41 specific P14 transgenic CD8+ T cells, DbGP33-41 specific
memory CD8+ T cells derived from LCMV Armstrong immune mice, and
DbGP33-41 specific or DbGP276-286 specific CD8+ T cells derived
from CD4+ depleted LCMV C1-13 infected mice. RNA isolation and gene
array analysis were performed as described in Kaech et al., (Cell
111:837-51, 2002). PD-1 mRNA was highly expressed in exhausted CD8+
T cells relative to memory CD8+ T cells (FIG. 1A). Furthermore,
PD-1 was expressed on the surface of CD8+ T cells in LCMV C1-13
infected mice, but was not present on the surface of CD8+ T cells
after clearance of LCMV Armstrong (FIG. 1B). Chronically infected
mice also expressed higher levels of one of the ligands of PD-1,
PD-L1, on most lymphocytes and APC compared to uninfected mice.
Thus, viral antigen persistence and CD8+ T cell exhaustion are
concomitant with an induction in PD-1 expression.
[0372] To test the hypothesis that blocking the PD-1/PD-L1 pathway
may restore T cell function and enhance viral control during
chronic LCMV infection, the PD-1/PD-L1 co-inhibitory pathway was
disrupted during chronic LCMV infection using .alpha.PD-L1 blocking
antibodies. A blocking monoclonal antibody against PD-L1 was
administered intraperitoneally (i.p.) every third day to mice
infected with LCMV C1-13 (200 .mu.g of rat anti-mouse PD-L1 IgG2b
monoclonal antibodies (clone 10F.5C5 or 10F.9G2)) from day 23 to
day 37 post-infection. At day 37, there was approximately 2.5 fold
more DbNP396-404 specific CD8+ T cells and 3 fold more DbGP33-41
specific CD8+ T cells in treated mice relative to the untreated
controls (FIG. 2A). The induction in proliferation was specific to
CD8+ T cells since the number of CD4+ T cells in the spleen were
approximately the same in both treated mice and untreated mice
(.about.6.times.104 IAbGP61-80 of CD4+ T cells per spleen).
[0373] In addition to an increase in CD8+ T cell proliferation, the
inhibition of PD-1 signaling also resulted in an increased
production of anti-viral cytokines in virus-specific CD8+ T cells.
The production of IFN-.gamma. and TNF-.alpha. by CD8+ T cells to
eight different CTL epitopes was determined. The combined response
was 2.3 fold higher in treated mice as compared to untreated mice
(FIGS. 2B and 2C). A 2-fold increase in the frequency of
TNF-.alpha. producing cells was also observed following treatment
(FIG. 2D). Viral clearance was also accelerated as the virus was
cleared from the serum, spleen, and liver of treated mice. Reduced
viral titers were observed in the lung and kidney (.about.10 fold)
by day 37 post-infection (14 days following initiation of
treatment) in treated mice. Untreated mice, however, displayed
significant levels of virus in all these tissues (FIG. 2E). Viral
titers in serum and tissue homogenates were determined using Vero
cells, as described in Ahmed et al. (J. Virol. 51:34-41, 1984). The
results showing that a PD-1 antagonist increases CD8+ T cell
proliferation and viral clearance therefore indicate that the
inhibition of PD-1 signaling restores CD8+ T cell function.
Furthermore, inhibition of PD-1 signaling also enhanced B cell
responses as the number of LCMV specific antibody secreting cells
in the spleen was also increased (>10-fold) following
treatment.
[0374] CD4+ T cells play a key role in the generation and
maintenance of CD8+ T cell responses. In this regard, CD8+ T cells
primed in the absence of CD4+ T cell (so-called "helpless" CD8+ T
cells) are incapable of mounting normal immune responses.
Furthermore, chronic LCMV infection is more severe in the absence
of CD4+ T cells. Accordingly, helpless T cells generated during
LCMV-C1-13 infection display an even more profound functional
impairment than T cells generated in the presence of CD4+ T cells.
DbNP396-404 specific CD8+ T cells are deleted to undetectable
levels, and DbGP33-41 and DbGP276-286 CD8+ T cells completely lose
the ability to secrete IFN-.gamma. and TNF-.alpha..
[0375] CD4+ T cells were depleted at the time of LCMV-C1-13
infection and mice were treated with anti-PD-L1 antibodies
treatment from day 46 to day 60 post-infection. LCMV-specific CD4+
T cells were not detectable by intracellular IFN-.gamma. staining
before or after treatment. Following treatment, treated mice had
approximately 7 fold more DbGP276-286 CD8+ T cells and 4 fold more
DbGP33-41 CD8+ T cells in their spleen than untreated control mice
(FIG. 3A). The number of virus-specific CD8+ T cells in the spleen
was also increased (FIG. 3B). This increase in virus-specific CD8+
T cells in treated mice was attributed to an increase in
proliferation, as detected by BrdU incorporation. 43% of
DbGP276-286 CD8+ T cells incorporated intermediate levels of BrdU
and 2% incorporated high levels of BrdU in untreated mice, while
50% DbGP276-286 CD8+ T cells incorporated intermediate levels of
BrdU and 37% incorporated high levels of BrdU in treated mice. BrdU
analysis was performed by introducing 1 mg/ml BrdU in the drinking
water during treatment and staining was performed according to the
manufacturer's protocol (BD Biosciences, San Diego, Calif.).
Moreover, treated mice contained a higher percentage of CD8+ T
cells expressing the cell cycle-associated protein Ki67 (60% versus
19% in untreated mice, FIG. 3C). Response to treatment in CD8+ T
cells in the PBMC was restricted to mice having high levels of CD8+
T cell expansion.
[0376] PD-1 inhibition also increased anti-viral cytokine
production in helpless, exhausted virus-specific CD8+ T cells.
Following treatment, the number of DbGP33-41 and DbGP276-286 CD8+ T
cells that produce IFN-.gamma. was markedly increased (FIG. 4A),
though higher numbers of DbNP396-404, KbNP205-212, DbNP166-175, and
DbGP92-101 specific CD8+ T cells were also detected in treated mice
(FIG. 4A). 50% of DbGP276-286 specific CD8+ T cells from treated
mice can produce IFN-.gamma. compared to the 20% of DbGP276-286
specific CD8+ T cells in control untreated mice. (FIG. 4B). Levels
of IFN-.gamma. and TNF-.alpha. produced by DbGP276-286 specific
CD8+ T cells from treated mice, however, were lower than fully
functional DbGP276-286 specific memory cells (FIG. 4C).
[0377] PD-1 inhibition also increased the lytic activity of
helpless, exhausted virus-specific CD8+ T cells. Ex vivo lytic
activity of virus-specific CD8+ T cells was detected following
treatment, using a .sup.51Cr release assay (Wherry et al., 2003. J.
Virol. 77:4911-27). Viral titers were reduced by approximately 3
fold in the spleen, 4 fold in the liver, 2 fold in the lung, and 2
fold in serum after 2 weeks of treatment relative to untreated mice
(FIG. 4E).
[0378] These results therefore demonstrate that blocking the PD-1
pathway breaks CTL peripheral tolerance to a chronic viral
infection, and that exhausted CD8+ T cells deprived of CD4+ T cell
help are not irreversibly inactivated.
Example 2: Administration of Anti-Viral Vaccine and PD-1
Antagonist
[0379] One approach for boosting T cell responses during a
persistent infection is therapeutic vaccination. The rationale for
this approach is that endogenous antigens may not be presented in
an optimal or immunogenic manner during chronic viral infection and
that providing antigen in the form of a vaccine may provide a more
effective stimulus for virus-specific T and B cells. Using the
chronic LCMV model, mice were administered a recombinant vaccinia
virus expressing the LCMV GP33 epitope as a therapeutic vaccine
(VVGP33), which resulted in a modest enhancement of CD8+ T cell
responses in some chronically infected mice. Four out of the nine
chronically infected mice that received the therapeutic vaccine
showed a positive response while none of the control mice had a
significant increase in the immune response against GP33. When this
therapeutic vaccination was combined with a PD-L inhibitor, LCMV
specific T cell responses were boosted to a greater level than
compared to either treatment alone and the effect of combined
treatment was more than additive.
Example 3: Inhibition of the PD-1 Pathway in Chronically-Infected
Mice Using PD-1 RNAi
[0380] RNA interference (RNAi) is capable of silencing gene
expression in mammalian cells. Long double stranded RNAS (dsRNAs)
are introduced into cells and are next processed into smaller,
silencing RNAs (siRNAs) that target specific mRNA molecules or a
small group of mRNAs. This technology is particularly useful in
situations where antibodies are not functional. For example, RNAi
may be employed in a situation in which unique splice variants
produce soluble forms of PD-1 and CTLA-4.
[0381] PD-1 silencer RNAs are inserted into a commercially
available siRNA expression vector, such as pSilencer.TM. expression
vectors or adenoviral vectors (Ambion, Austin, Tex.). These vectors
are then contacted with target exhausted T cells in vivo or ex vivo
(see Example 4 below).
Example 4: Ex Vivo Rejuvenation of Exhausted T Cells
[0382] Virus-specific exhausted CD8+ T cells are isolated from
LCMV-C1-13 chronically infected mice using magnetic beads or
density centrifugation. Transfected CD8+ T cells are contacted with
a monoclonal antibody that targets PD-L1, PD-L2 or PD-1. As
described in Example 1, inhibition of the PD-1 pathway results in
the rejuvenation of the CD8+ T cells. Accordingly, there is an
increase in CD8+ T cell proliferation and cytokine production, for
example. These rejuvenated CD8+ T cells are reintroduced into the
infected mice and viral load is measured as described in Example
1.
Example 5: In Vitro Screening of Novel CD8+ T Cell Rejuvenator
Compounds
[0383] Compounds that modulate the PD-1 pathway can be identified
in in vivo and ex vivo screening assays based on their ability to
reverse CD8+ T cell exhaustion resulting from chronic viral
infection.
[0384] Exhausted CD8+ T cells are derived from mice chronically
infected with LCMV-C1-13 and next contacted with a test compound.
The amount of anti-viral cytokines (for example, IFN-.gamma. or
TNF-.alpha.) released from the contacted T cell is measured, for
example, by ELISA or other quantitative method, and compared to the
amount, if any, of the anti-viral cytokine released from the
exhausted T cell not contacted with the test compound. An increase
in the amount of anti-viral cytokine released by treated cells
relative to such amount in untreated cells identifies the compound
as a PD-1 antagonist, useful to modulate T cell activity.
Example 6: In Vivo Screening of Novel CD8+ T Cell Rejuvenator
Compounds
[0385] Exhausted CD8+ T cells are derived from mice chronically
infected with LCMV-C1-13. A test compound is administered
intravenously to the infected mice. The amount of anti-viral
cytokines (such as IFN-.gamma. or TNF-.alpha.) that is released
into the serum of treated and untreated mice is measured, for
example, by ELISA or other quantitative method, and compared. An
increase in the amount of anti-viral cytokine found in the serum in
treated mice relative to such amount in untreated mice identifies
the test compound as a PD-1 antagonist. Alternatively, the viral
titer (e.g., serum viral titer) can be determined prior and
subsequent to treatment of the test compound.
Example 7: Chimpanzees as a Model for Immunotherapy of Persistent
HCV Infection
[0386] Chimpanzees provide a model of HCV persistence in humans.
Defects in T cell immunity leading to life-long virus persistence
both include a deficit in HCV-specific CD4+ T helper cells and
impaired or altered CD8+ T effector cell activity. Persistently
infected chimpanzees are treated with antibodies against CTLA-4,
PD-1, or a combination of the two. The efficacy of blockade of the
inhibitory pathways, combined with vaccination using recombinant
structural and non-structural HCV proteins, and whether such
strategies can enhance the frequency and longevity of
virus-specific memory T cells are determined. The defect in T cell
immunity is exclusively HCV-specific in persistently infected
humans and chimpanzees. The blood and liver of infected chimpanzees
are examined for expression of CTLA-4, PD-1, BTLA and their ligands
and for the presence of Treg cells. Antiviral activity may then be
restored by delivering to chimpanzees' humanized monoclonal
antibodies that block signaling through these molecules.
[0387] Persistently infected chimpanzees are treated with humanized
.alpha.CTLA-4 antibodies (MDX-010, Medarex) or (PD-1 antibodies.
The initial dose of MDX-010 is 0.3 mg/kg followed 2 weeks later by
1.0 mg/kg and then 3, 10, 30 mg/kg at three week intervals. After
treatment with antibodies to co-inhibitory molecules, the humoral
and cellular immune responses as well as the HCV RNA load will be
determined. Samples are collected at weeks 1, 2, 3, 5, and 8, and
then at monthly intervals. Samples include: 1) serum for analysis
of transaminases, autoantibodies, neutralizing antibodies to HCV,
and cytokine responses, 2) plasma for viral load and genome
evolution, 3) PBMC for in vitro measures of immunity,
costimulatory/inhibitory receptor expression and function, 4) fresh
(unfixed) liver for isolation of intrahepatic lymphocytes and RNA,
and 5) fixed (formalin/paraffin embedded) liver for histology and
immunohistochemical analysis. Regional lymph nodes are also
collected at 2 or 3 time points to assess expression of
co-inhibitory molecules and splice variants by immunohistochemistry
and molecular techniques.
[0388] To determine if vaccination with HCV antigens potentiates
the therapeutic effect of antibodies to PD-1, chimpanzees are
treated as follows: 1) intramuscular immunization with recombinant
envelope glycoproteins E1 and E2 (in MF59 adjuvant) and other
proteins (core plus NS 3, 4, and 5 formulated with ISCOMS) at weeks
0, 4, and 24; 2) intramuscular immunization with the vaccine used
in 1) but co-administered with .alpha.CTLA-4 antibodies (30 mg of
each/Kg body weight, intravenously at weeks 0, 4, and 24 when
vaccine is given); 3) identical to 2) except that .alpha.PD-1 (or
BTLA) antibodies are substituted for the CTLA-4 antibodies; 4)
identical to Groups 2 and 3 except that a combination of CTLA-4 and
PD-1 (or BTLA) antibodies are used in addition to the vaccine.
HCV-specific T and B cell responses are monitored at monthly
intervals after immunization for a period of 1 year.
[0389] Markers examined on HCV-tetramer+ and total T cells in this
analysis include markers of differentiation (e.g. CD45RA/RO, CD62L,
CCR7, and CD27), activation (e.g. CD25, CD69, CD38, and HLA-DR),
survival/proliferation (e.g. bcl-2 and Ki67), cytotoxic potential
(e.g. granzymes and perforin), and cytokine receptors (CD122 and
CD127). An interesting correlation exists between pre-therapy
levels of the chemokine IP-10 and response to PEG
IFN-.gamma./ribavirin. IP-10 levels are measured to investigate a
potential correlation between negative regulatory pathways or
HCV-specific T cell responses and IP-10 levels. Expression of
inhibitory receptors and ligands on PBMC are performed by flow
cytometry.
Example 8: PD-1 Immunostaining in Reactive Lymphoid Tissue
[0390] Case material was obtained from the Brigham & Women's
Hospital, Boston, Mass., in accordance with institutional policies.
All diagnoses were based on the histologic and immunophenotypic
features described in the World Health Organization Lymphoma
Classification system (Jaffe E S, et al. 2001) and in all cases
diagnostic material was reviewed by a hematopathologist.
[0391] Immunostaining for PD-1 was performed on formalin-fixed
paraffin embedded tissue sections following microwave antigen
retrieval in 10 mM citrate buffer, pH 6.0 with a previously
described anti-human PD-1 monoclonal antibody (2H7; 5), using a
standard indirect avidin-biotin horseradish peroxidase method and
diaminobenzidine color development, as previously described (Jones
D, et al. 1999; Dorfman D M, et al. 2003). Cases were regarded as
immunoreactive for PD-1 if at least 25% of neoplastic cells
exhibited positive staining. PD-1 staining was compared with that
of mouse IgG isotype control antibody diluted to identical protein
concentration for all cases studied, to confirm staining
specificity.
[0392] Monoclonal antibody 2H7 for PD-1 was used to stain
formalin-fixed, paraffin-embedded specimens of reactive lymphoid
tissue, thymus, and a range of cases of B cell and T cell
lymphoproliferative disorders. In specimens of tonsil exhibiting
reactive changes, including follicular hyperplasia, a subset of
predominantly small lymphocytes in the germinal centers exhibited
cytoplasmic staining for PD-1, with infrequent PD-1-positive cells
seen in the interfollicular T cell zones. The PD-1 staining pattern
in germinal centers was virtually identical to that seen with an
antibody to CD3, a pan-T cell marker, whereas an antibody to CD20,
a pan-B cell marker, stained the vast majority of germinal center B
cells. Similar results were seen in histologic sections of reactive
lymph node and spleen. No PD-1 staining was observed in adult
thymus.
Example 9: PD-1 Immunostaining in Paraffin Embedded Tissue Sections
of B Cell and T Cell Lymphoproliferative Disorders
[0393] A range of B cell and T cell lymphoproliferative disorders
for PD-1 expression were studied; the results are summarized in
Table 4. Forty-two cases of B cell lymphoproliferative disorders
were examined for PD-1 expression, including representative cases
of precursor B lymphoblastic leukemia/lymphoblastic lymphoma, as
well as a range of lymphoproliferative disorders of mature B cells,
including a number of B cell non-Hodgkin lymphomas of follicular
origin, including 6 cases of follicular lymphoma and 7 cases of
Burkitt lymphoma. None of the B cell lymphoproliferative disorders
showed staining for PD-1. In some cases, non-neoplastic reactive
lymphoid tissue was present, and showed a PD-1 staining pattern as
seen in tonsil and other reactive lymphoid tissue noted above.
[0394] Similarly, in 25 cases of Hodgkin lymphoma, including 11
cases of classical Hodgkin lymphoma and 14 case of lymphocyte
predominant Hodgkin lymphoma, the neoplastic cells did not exhibit
staining for PD-1. Interestingly, in all 14 cases of lymphocyte
predominant Hodgkin lymphoma, the T cells surrounding neoplastic
CD20-positive L&H cells were immunoreactive for PD-1, similar
to the staining pattern noted for CD57+ T cells in lymphocyte
predominant Hodgkin lymphoma. These PD-1-positive cells were a
subset of the total CD3+ T cell population present.
[0395] A range of T cell lymphoproliferative disorders were studied
for expression of PD-1; the results are summarized in Table 4.
Cases of precursor T cell lymphoblastic leukemia/lymphoblastic
lymphoma, a neoplasm of immature T cells of immature T cells, were
negative for PD-1, as were neoplasms of peripheral, post-thymic T
cells, including cases of T cell prolymphocytic leukemia,
peripheral T cell lymphoma, unspecified, anaplastic large cell
lymphoma, and adult T cell leukemia/lymphoma. In contrast, all 19
cases of angioimmunoblastic lymphoma contained foci of
PD-1-positive cells that were also immunoreactive for pan-T cell
markers such as CD3. PD-1-positive cells were consistently found at
foci of expanded CD21+ follicular dendritic cells (FDC) networks, a
characteristic feature of angioimmunoblastic lymphoma.
TABLE-US-00011 TABLE 4 PD-1 immunostaining in lymphoproliferative
disorders PD-1 immunostaining B cell LPDs 0/42* B-LL/LL 0/3 CLL 0/4
MCL 0/4 FL 0/6 MZL 0/3 HCL 0/3 DLBCL 0/6 BL 0/7 LPL 0/3 MM 0/3
Hodgkin lymphoma 0/25 Classical 0/11 Nodular lymphocyte predominant
0/14** T cell LPDs 18/55 T-LL/LL 0/5 T-PLL 0/3 AIL 19/19 PTCL,
unspecified 0/14 ALCL 0/12 ATLL 0/3 Abbreviations:
B-LL/LL--precursor B cell lymphoblastic lymphoma/lymphoblastic
leukemia; CLL--chronic lymphocytic leukemia; MCL--mantle cell
lymphoma; FL--follicular lymphoma; MZL--marginal zone lymphoma;
HCL--hairy cell leukemia; DLBCL--diffuse large B cell lymphoma;
BL--Burkitt lymphoma; LPL--lymphoplasmacytic lymphoma; MM--multiple
myeloma; T-LL/L--precursor T lymphoblastic leukemia/lymphoblastic
lymphoma; T-PLL--T cell prolymphocytic leukemia;
AIL--angioimmunoblastic lymphoma; PTCL--peripheral T cell lymphoma,
unspecified; ALCL--anaplastic large cell lymphoma; ATLL--adult T
cell leukemia/lymphoma. *number of immunoreactive cases/total
number of cases **PD-1-positive cells form rosettes around
neoplastic L&H cells in 14/14 cases
Example 10: General Methods for Studying PD-1 Expression on
HIV-Specific Human CD8+ T Cells
[0396] The following methods were used to perform the experiments
detailed in Examples 11-14.
[0397] Subjects:
[0398] Study participants with chronic clade C HIV-1 infection were
recruited from outpatient clinics at McCord Hospital, Durban, South
Africa, and St. Mary's Hospital, Mariannhill, South Africa.
Peripheral blood was obtained from 65 subjects in this cohort, all
of whom were antiretroviral therapy naive at the time of analysis.
Subjects were selected for inclusion based on their expressed HLA
alleles matching the ten class I tetramers that were constructed
(see below). The median viral load of the cohort was 42,800 HIV-1
RNA copies/ml plasma (range 163-750,000), and the median absolute
CD4 count was 362 (range 129-1179). Information regarding duration
of infection was not available. All subjects gave written informed
consent for the study, which was approved by local institutional
review boards.
[0399] Construction of PD-1 and PD-L1 Antibodies:
[0400] Monoclonal antibodies to human PD-L (29E.2A3, mouse IgG2b)
and PD-1 (EH12, mouse IgG) were prepared as previously described
and have been shown to block the PD-1:PD-L1 interaction.
[0401] MHC Class I Tetramers:
[0402] Ten HIV MHC Class I tetramers, synthesized as previously
described (Altman J D, et al. 1996), were used for this study:
A*0205 GL9 (p24, GAFDLSFFL; SEQ ID NO: 1), A*3002 KIY9 (Integrase,
KIQNFRVYY; SEQ ID NO:2), B*0801 DI8 (p24, DIYKRWII; SEQ ID NO:3),
B*0801 FL8 (Nef, FLKEKGGL; SEQ ID NO:4), B*4201 RM9 (Nef,
RPQVPLRPM; SEQ ID NO:5), B*4201 TL9 (p24, TPQDLNTML; SEQ ID NO:6),
B*4201 TL10 (Nef, TPGPGVRYPL; SEQ ID NO:7), B*4201 YL9 (RT,
YPGIKVKQL; SEQ ID NO:8), B*8101 TL9 (p24, TPQDLNTML; SEQ ID NO:9),
and Cw0304 YL9 (p24, YVDRFFKTL; SEQ ID NO:10).
[0403] HLA Class I Tetramer Staining and Phenotypic Analysis:
[0404] Freshly isolated peripheral blood mononuclear cells (PBMC,
0.5 million) were stained with tetramer for 20 minutes at
37.degree. C. The cells were then washed once with phosphate
buffered saline (PBS), pelleted, and stained directly with
fluorescein isothiosyanate (FITC)-conjugated anti-CD8 (Becton
Dickinson), phycoerythrin-conjugated anti-PD-1 (clone EH12), and
ViaProbe (Becton Dickinson). Cells were incubated for 20 minutes at
room temperature, washed once in PBS, and resuspended in 200 .mu.l
PBS with 1% paraformaldehyde and acquired on a
fluorescence-activated cell sorter (FACSCalibur.TM., Becton
Dickinson). A minimum of 100,000 events were acquired on the
FACSCalibur.TM..
[0405] CFSE Proliferation Assays:
[0406] One million freshly isolated PBMC were washed twice in PBS,
pelleted, and resuspended in 1 ml of 0.5 M carboxy-fluorescein
diacetate, succinimidyl ester (CFSE, Molecular Probes) for 7
minutes at 37.degree. C. The cells were washed twice in PBS,
resuspended in 1 ml RO1 medium (RPMI 1640 supplemented with
glutiathione, penicillin, streptomycin, and 10% fetal calf serum
[FCS]), and plated into one well of a 24-well plate. Initial
studies revealed that a final concentration of 0.2 .mu.g/ml peptide
yielded optimal proliferative responses, therefore this was the
final peptide concentration in the well used for each assay.
Negative control wells consisted of PBMC in medium alone, or PBMC
in medium with purified anti-PD-L1 (10 .mu.g/ml), and positive
control wells were stimulated with 10 .mu.g/ml of
phytohemagluttinin (PHA). Following 6-day incubation in a
37.degree. C. incubator, the cells were washed with 2 ml PBS and
stained with PE-conjugated MHC Class I tetramers, ViaProbe (Becton
Dickinson), and anti-CD8-APC antibodies. Cells were acquired on a
FACSCalibur and analyzed by CellQuest.RTM. software (Becton
Dickinson). Cells were gated on ViaProbe-CD8+ lymphocytes. The fold
increase in tetramer+ cells was calculated by dividing the
percentage of CD8+ tetramer+ cells in the presence of peptide by
the percentage of CD8+ tetramer+ cells in the absence of peptide
stimulation.
[0407] Statistical Analysis:
[0408] Spearman correlation, Mann-Whitney test, and paired t-test
analyses were performed using GraphPad Prism Version 4.0a. All
tests were 2-tailed and p values of p<0.05 were considered
significant.
Example 11: PD-1 Expression on HIV-Specific CD8+ T Cells
[0409] A panel of 10 MHC Class I tetramers specific for dominant
HIV-1 clade C virus CD8+ T cell epitopes was synthesized, based on
prevalent HLA alleles and frequently targeted epitopes in Gag, Nef,
Integrase, and RT allowing direct visualization of surface PD-1
expression on these cells. High resolution HLA typing was performed
on the entire cohort, and a subset of 65 antiretroviral therapy
naive persons was selected for study based on expression of
relevant HLA alleles. A total of 120 individual epitopes were
examined, and representative ex vivo staining of PD-1 on HIV
tetramer+ cells is shown in FIG. 5A. PD-1 expression was readily
apparent on these tetramer+ cells, and was significantly higher
than in the total CD8 T cell population from the same individuals
(p<0.0001); in turn, PD-1 expression on both tetramer+ CD8+ T
cells and the total CD8+ T cell population was significantly higher
than in HIV-seronegative controls (FIG. 5B). For eight of the ten
tetramers tested at least one person was identified in whom the
level of expression on antigen-specific CD8+ cells was 100% (FIG.
5C). PBMC from 3 to 25 individuals were stained for each HIV
tetramer response, with median PD-1 expression levels ranging from
68% to 94% of tetramer+ cells (FIG. 5C). These findings were
further confirmed by analysis of the mean fluorescence intensity
(MFI) of PD-1 on both tetramer+ cells and the total CD8+ T cell
population (FIG. 5B, C).
[0410] It was next determined whether there was evidence for
epitope-specific differences in terms of PD-1 expression levels in
persons with multiple detectable responses. Of the 65 persons
examined, 16 individuals had between 3 and 5 tetramer positive
responses each. PD-1 expression was nearly identical and
approaching 100% for each response analyzed for three of the
sixteen subjects; however, the other 13 individuals displayed
different patterns of PD-1 expression depending on the epitope
(FIG. 5D). These data indicate that PD-1 expression may be
differentially expressed on contemporaneous epitope-specific CD8+ T
cells from a single person, perhaps consistent with recent data
indicating epitope-specific differences in antiviral efficacy
(Tsomides T J, et al. 1994; Yang O, et al. 1996; Loffredo J T, et
al. 2005).
Example 12: The Relationship Between PD-1 Expression and HIV
Disease Progression
[0411] The relationship was determined between PD-1 expression on
HIV-specific CD8+ T cells and plasma viral load and CD4+ cell
counts, both of which are predictors of HIV disease progression.
Consistent with previous studies, the relationship between the
number of tetramer positive cells and viral load or CD4+ cell count
failed to show any significant correlation (FIG. 6A, B). In
contrast, there were significant positive correlations with viral
load and both the percentage and MFI of PD-1 expression on HIV
tetramer positive cells (p=0.0013 and p<0.0001, respectively;
FIG. 6A). There were also inverse correlations between CD4 count
and both the percentage and MFI of PD-1 on HIV tetramer positive
cells (p=0.0046 and p=0.0150, respectively; FIG. 6B). Since the
tetramers tested likely represent only a fraction of the
HIV-specific CD8+ T cell population in these subjects, the
relationship between PD-1 expression on all CD8+ cells and these
parameters was also examined. There were significant positive
correlations between viral load and both the percentage and MFI of
PD-1 expression on the total CD8+ T cell population (p=0.0021 and
p<0.0001, respectively; FIG. 6C), and inverse correlations were
also observed between CD4+ cell count and both the percentage and
MFI of PD-1 expression on the total CD8+ T cell population
(p=0.0049 and p=0.0006, respectively; FIG. 6D). In this same group,
PD-1 expression on CMV-specific CD8+ T cells was tested in 5
subjects, and significantly less PD-1 was expressed on these cells
compared to HIV-specific CD8 T cells (median 23% CMV tetramer+
PD-1+, p=0.0036), and was not different than bulk CD8+ T cells in
these same individuals, indicating that high PD-1 expression is not
a uniform feature of all virus-specific CD8+ T cells. These data
suggest increasing amounts of antigen in chronic HIV infection
result in increased expression of PD-1 on CD8+ T cells, and are
consistent with murine data in chronic LCMV infection, in which
PD-1 expression is associated with functional exhaustion of CD8+ T
cells (Barber D L, et al. 2005). Moreover, they provide the first
clear association, in a large study including analysis of multiple
epitopes, between HIV-specific CD8+ T cells and either viral load
or CD4 count.
Example 13: The Relationship Between PD-1 Expression and CD8 T Cell
Memory Status and Function
[0412] PD-1 expression was next analyzed in the context of a number
of additional phenotypic markers associated with CD8+ T cell memory
status and function, including CD27, CD28, CD45RA, CD57, CD62L,
CD127, CCR7, perforin, granzyme B, and Ki67 (FIG. 7).
Representative stainings for these markers on B*4201 TL9 tetramer+
cells from one individual are shown in FIG. 7A, and aggregate data
for 13 subjects are shown in FIG. 7B. These studies were limited to
those tetramer responses that were greater than 95% PD-1 positive,
as multiparameter flow cytometry of greater than 4 colors was not
available in KwaZulu Natal. The HIV tetramer+ PD-1+ cells express
high levels of CD27 and granzyme B, very low levels of CD28, CCR7,
and intracellular Ki67, low levels of CD45RA and perforin, and
intermediate levels of CD57 and CD62L (FIG. 7B). These data
indicate that HIV-specific PD-1+ T cells display an
effector/effector memory phenotype, and are consistent with
previous reports of skewed maturation of HIV-specific CD8+ T cells.
In addition, virus sequencing was performed to determine whether
these cells were driving immune escape. Of 45 of these
tetramer-positive responses evaluated, the viral epitopes in only 5
were different from the South African clade C consensus sequence,
indicating these cells exert little selection pressure in vivo.
[0413] Previous experiments in mice using the LCMV model showed
that in vivo blockade of PD-1/PD-L1 interaction by infusion of
anti-PD-L1 blocking antibody results in enhanced functionality of
LCMV-specific CD8+ T cells as measured by cytokine production,
killing capacity, proliferative capacity, and, most strikingly,
reduction in viral load. Short-term (12-hour) in vitro
antigen-specific stimulation of freshly isolated PBMC from 15 HIV+
subjects, in the presence or absence of 1 .quadrature. .mu.g/ml
purified anti-PD-L1 antibody, failed to increase IFN-.gamma.,
TNF-.alpha., or IL-2 production.
Example 14: Effect of Blockading the PD-1/PD-L1 Pathway on
Proliferation of HIV-Specific CD8+ T Cells
[0414] Because HIV-specific CD8+ T cells also exhibit impaired
proliferative capacity (2004), it was determined whether blockade
of the PD-1/PD-L1 could enhance this function in vitro.
Representative data from a B*4201-positive individual are shown in
FIG. 8A. Incubation of freshly isolated CFSE-labeled PBMC with
medium alone, or medium with anti-PD-L1 antibody, resulted in
maintenance of a population of B*4201-TL9-specific CD8+ T cells
(1.2% of CD8+ T cells) that remained CFSEhi after six days in
culture. Simulation of CFSE-labeled PBMC for 6 days with TL9
peptide alone resulted in a 4.8-fold expansion of CFSElo B*4201 TL9
tetramer+ cells, whereas stimulation of CFSE-labeled PBMC with TL9
peptide in the presence of anti-PD-L1 blocking antibody further
enhanced proliferation of TL9-specific cells, resulting in a
10.3-fold increase in tetramer+ cells. CFSE proliferation assays
were performed on 28 samples in the presence and absence of
purified anti-human PD-L1 blocking antibody. A significant increase
in the proliferation of HIV-specific CD8+ T cells was observed in
the presence of peptide plus anti-PD-L1 blocking antibody as
compared to the amount of proliferation following stimulation with
peptide alone (FIG. 8B; p=0.0006, paired t-test). The fold increase
of tetramer+ cells in the presence of anti-PD-L1 blocking antibody
varied by individual and by epitope within a given individual (FIG.
8C), again suggesting epitope-specific differences in the degree of
functional exhaustion of these responses.
Example 15: Therapeutic Vaccination in Conjunction with Blocking
PD-1 Inhibitory Pathway Synergistically Improves the Immune Control
of Chronic Viral Infection: A Concept Study of Combinatorial
Therapeutic Vaccine
[0415] The functional impairment of T cells including cytokine
proliferation, cytolysis, and proliferation of antigen-specific T
cells, is a defining characteristic of many chronic infections.
Inactivated T cell immune response is observed during a variety of
different persistent pathogen infections, including HIV, HBV, HCV,
and TB in humans. T cell inactivation during chronic infection
might correlate with the magnitude and persistence of the antigen
burden and originate from disrupted proximal T cell receptor
signals, upregulation of inhibitory proteins or down regulation of
costimulatory proteins, and defects in accessory and cytokine
signals. The defect in exhausted T cells is a primary reason for
the inability of the host to eliminate the persisting pathogen.
During chronic infection, exhausted virus specific CD8 T cells
upregulate two key inhibitory proteins: PD-1 and CTLA-4. An in vivo
blockade of PD-1 increases the number and function of
virus-specific CD8 T cells and results in decreased viral load.
[0416] There are several drawbacks of current vaccination
strategies for chronic viral infections. Specifically, effective
boosting of antiviral CD8 T-cell responses is not observed after
therapeutic vaccination. In addition, a high viral load and the low
proliferative potential of responding T cells during chronic
infection are likely to limit the effectiveness of therapeutic
vaccination. Thus, it is important to develop therapeutic vaccine
strategy to boost effectively the host's endogenous T cell
responses to control chronic infection.
[0417] A well-known chronic infection model induced by LCMV
Clone-13 infection was used to determine the effectiveness of using
a PD-1 antagonist in combination with a therapeutic vaccine. A
vaccinia virus expressing GP33 epitope of LCMV was used as a
therapeutic vaccine to monitor an epitope-specific CD8 T cell
immune response. A therapeutic vaccine was combined with anti-PD-L1
antibody for blocking an inhibitory pathway in order to investigate
the synergist effect regarding a proliferation of antigen-specific
CD8 T cells and a resolution of persisting virus.
[0418] The following methods were used in these experiments:
[0419] Mice and Infections:
[0420] C57BL/6 mice (4- to 6-week-old females) were from The
Jackson Laboratory (Bar Harbor, Me.). Mice were maintained in a
pathogen-free vivarium according to NIH Animal Care guidelines. For
the initiation of chronic infections, mice were infected with
2.times.10.sup.6 PFU of LCMV clone-13 (CL-13) as described
previously. Viral growth and plaque assays to determine viral
titers have been described previously.
[0421] In Vivo Antibody Blockade and Therapeutic Vaccination:
[0422] Two hundred micrograms of rat anti-mouse PD-L1 (10F:9G2)
were administered intraperitoneally every third day from 4 weeks
post-infection with CL-13. At the time point of first treatment of
anti-PD-L1, 2.times.10.sup.6 PFU of recombinant vaccinia virus
expressing the GP33-41 epitope (VV/GP33) as therapeutic vaccine or
wild-type vaccinia virus (VV/WT) as control vaccine were given
intraperitoneally.
[0423] Lymphocyte Isolation:
[0424] Lymphocytes were isolated from tissues and blood as
previously described. Liver and lung were perfused with ice-cold
PBS prior to removal for lymphocyte isolation.
[0425] Flow Cytometry:
[0426] MHC class I peptide tetramers were generated and used as
previously described. All antibodies were obtained from BD
Pharmingen except for granzyme B (Caltag), Bcl-2 (R&D Systems),
and CD127 (eBioscience). All surface and intracellular cytokine
staining was performed as described (Barber et al., Nature 439:682,
2006). To detect degranulation, splenocytes were stimulated for 5 h
in the presence of brefeldin, monensin, anti-CD107a-FITC, and
anti-CD107b-FITC.
[0427] Confocal Microscopy:
[0428] Spleens were removed from mice and frozen in OCT
(TissueTek). From these blocks, 10-20 mm cryostat sections were cut
and fixed in ice-cold acetone for 10 minutes. For
immunofluorescence, sections were stained with the following
antibodies: ER-TR7 to detect reticular cells (Biogenesis, Kingston,
N.H.) and polyclonal anti-LCMV guinea-pig serum. Stains were
visualized with Alexa Fluor-488 goat anti-rat and Alexa Fluor-568
goat anti-guinea-pig Ig (Molecular Probes) and analyzed by confocal
microscopy (Leica Microsystems AG, Germany). Images were prepared
using ImageJ (National Institutes of Health) and Photoshop (Adobe
Systems Inc.).
[0429] The results demonstrated that a combination of therapeutic
vaccine and anti-PD-L1 antibody displays a synergistic effect on
proliferation of antigen-specific CD8 T cells and resolution of
persisting virus. Therapeutic vaccine could boost effectively a
functionally restored CD8 T cell population by blockade of
PD-1/PD-L1 inhibitory pathway. Enhanced proliferation of
antigen-specific CD8 T cells and accelerated viral control were
systematically achieved by combinatorial therapeutic vaccination
(FIGS. 9A-9D and FIG. 10A-10D). Combinatorial therapeutic vaccine
guides to a dramatic increase of functionally active CD8 T cells
(FIG. 11A-D). In addition, therapeutic vaccine using vector
expressing specific epitope during blockade of PD-1/PD-L1 pathway
enhances a proliferation of CD8 T cell specific to epitope encoded
in vector (FIGS. 9 and 11). The increased expression level of CD127
seen on antigen-specific CD8 T cells in the group treated with the
combinatorial vaccine reflects the generation of a long-term memory
T cell responses, while decreased expression levels of PD-1 and
Granzyme B correlate to resolution of persisting virus (FIGS.
12A-12B).
[0430] There was a synergistic effect of therapeutic vaccine
combined with PD-L1 blockade on restoration of function in
`helpless` exhausted CD8 T cells (see (FIG. 13A-13E). Mice were
depleted of CD4 T cells and then infected with LCMV clone-13. Some
mice were vaccinated with wild-type vaccinia virus (VV/WT) or LCMV
GP33-41 epitope-expressing vaccinia virus (VV/GP33) at 7-wk
post-infection. At the same time, the mice were treated 5 times
every three days with .alpha.PD-L1 or its isotype. Two weeks after
initial treatment of antibodies, mice were sacrificed for analysis.
The results are shown in FIG. 13A. The frequency of GP33 specific
CD8 T cells was also examined (FIG. 13B). Splenocytes were
stimulated with GP33 peptide in the presence of .alpha.CD107a/b
antibodies and then co-stained for IFN-.gamma.. The shown plots are
gated on CD8-T cells (FIG. 13C). The percentage of
IFN-.gamma..sup.+ cells after stimulation with GP33 peptide per
cells positive for Db-restricted GP33-41 tetramer was also
determined (FIG. 13D), as was the viral titer ((FIG. 13E). The
results demonstrate the synergistic effect of a vaccine combined
with PD-1 blockade.
[0431] These results show that combinations of blocking negative
regulatory pathway and boosting CD8 T cells during chronic
infection can be used in the development of therapeutic vaccines to
improve T cell responses in patients with chronic infections or
malignancies. Therapeutic interventions, such as the use of an
antagonist of PD-1, that boost T-cell responses and lower the viral
load could increase disease-free survival and decrease transmission
of the virus. Effective therapeutic vaccination could be used for
chronic viral infections and persisting bacterial, parasitic
infections. This strategy is also of use for the treatment of
malignancies.
Example 16: Enhancement of T Cell Immunotherapy Through Blockade of
the PD1/PDL1 Pathway
[0432] It is important to develop strategies to treat and eliminate
chronic viral infections such as the Human Immunodeficiency virus
and Hepatitis C. The CDC has recently reported that over one
million American's are living with HIV, exemplifying the need for
more effective therapies. It is important to determine how
inhibitory signaling to lymphocytes can contribute to a pathogen's
ability to persistently evade the host immune response.
[0433] The inhibitory immunoreceptor PD-1 (a member of the B7/CD28
family of costimulatory receptors) and its ligand (PD-L1) have been
shown to be dramatically upregulated during states of chronic
infection with lymphocytic choriomeningitis virus (LCMV).
Additional studies using the LCMV model have demonstrated that
blocking of the PD1/PDL1 pathway significantly augments the
endogenous anti-viral CD8 T cell response during the late phases of
chronic infection when CD8 T cells are exhausted. Exhausted T cells
are functionally compromised and do not mount effective immune
responses upon antigen encounter. However, blockade of the
PD1/PD-L1 pathway appears to reverse exhaustion and restore their
functional capacity. Data suggests that these effects persist well
beyond the immediate period of anti-PDL1 treatment.
[0434] The following experiments were performed in order to (1)
assess the ability of anti-PDL1 to enhance the proliferation and
survival anti-viral CD8 T cells upon adoptive transfer of immune
(memory) splenocytes into congenitally infected (carrier) mice, (2)
to evaluate the functionality of virus-specific, memory CD8 T cells
that have expanded in the presence of PD1/PDL1 blockade, and (3) to
determine the expression of various markers of differentiation in
virus-specific CD8 T cells that have expanded in the presence of
PD1/PDL1 blockade.
[0435] The role of the PD-1 pathway was assessed in a
well-developed model of cyto-immune therapy for chronic viral
infection. The model described herein parallels that of T cell
cyto-immune therapy for tumors in regard to the immunological
barriers the limit the applicability of these therapies (such as
corrupted or suppressed T cell/anti-tumor responses). Mice infected
neonatally or in utero with LCMV do not mount endogenous
LCMV-specific immune responses and go on to have high levels of
infectious LCMV in blood and all tissues throughout their lives.
These animals are congenital carriers and are essentially tolerant
to the pathogen. When splenocytes from an LCMV immune mouse are
adoptively transferred into a congenital carrier the transferred
immune memory cells rapidly undergo expansion and establish a
vigorous immune response against the virus. Approximately 2/3 of
the animals receiving adoptive cyto-immune therapy go on to
completely clear the infection when high doses of splenocytes are
transferred.
[0436] The following materials and methods were used in these
experiments:
[0437] Mice and Infections.
[0438] 4-6 week old female B57BL/6 mice were purchased from the
Jackson Laboratory (Bar Harbor, Me.). Acute infection was initiated
by intraperitoneal injection of 2.times.10.sup.5 PFU LCMV
Armstrong. Congenital carrier mice were bred at Emory University
(Atlanta, Ga.) from colonies derived from neonatally infected mice
(10.sup.4 PFU LCMV clone-13, intracerebral).
[0439] Adoptive Immunotherapy and In Vivo Antibody Blockade.
[0440] 40.times.10.sup.6 whole splenocytes from LCMV immune mice
(day 30-90 post-infection) were isolated and transferred
intravenously into 6-12 week old LCMV carrier mice. 200 micrograms
of rat-anti-mouse PD-L1 (10F.9G2) were administered every 3.sup.rd
day for 15 days following adoptive immunotherapy.
[0441] Flow Cytometry and Tetramer Staining.
[0442] MHC class I tetramers of H-2Db complexed with LCMV
GP.sub.33-41 were generated as previously described. All antibodies
were purchased from BD/Pharmingen (San Diego, Calif.). Peripheral
blood mononuclear cells and splenocytes were isolated and stained
as previously described. Data was acquired using a FACSCalibur.TM.
flow cytometer (BD) and analyzed using FlowJoe software (Tree Star
Inc. Ashland, Oreg.)
[0443] Intracellular Cytokine Staining.
[0444] For intracellular cytokine staining 10.sup.6 splenocytes
were cultured in the presence or absence of the indicated peptide
(0.2 .mu.g/ml) and brefeldin A for 5-6 hours at 37.degree. C.
Following staining for surface markers, cells were permeabilized
and stained for intracellular cytokines using the Cytofix/Cytoperm
preparation (BD/Pharmigen).
[0445] The following results were obtained:
[0446] Anti-PD-L1 Therapy Increases the Number of Virus Specific
CD8 T Cells:
[0447] Peripheral blood mononuclear cells (PBMCs) were isolated
from treated or untreated animals on days 7, 11, 15, 22, and 35.
Cells specific for the D.sup.b GP33 epitope were assessed by
tetramer staining. In two independent experiments it was found that
animals treated with anti-PD-L1 therapy during the first 15 days
following adoptive transfer developed significantly larger numbers
of LCMV specific CD8 T cells when normalized to the number of
D.sup.b GP33 positive cells per million PBMC's (FIG. 14). These
data support the role of the PD-1/PD-L1 pathway in conferring some
degree of proliferative suppression in normal memory T cells.
Moreover these results suggest that therapeutic inhibition of this
pathway could augment the development and maintenance of the
secondary immune response generated following adoptive transfer
into a setting of chronic infection with high antigen load.
[0448] PD-1/PD-L1 Blockade Enhances the Functionality of Antigen
Specific CD8 T Cells:
[0449] Spenocytes were isolated from treated and untreated animals
on day 17 post-adoptive transfer and analyzed for the expression of
inflammatory cytokines (IFN-gamma and TNF alpha) or CD107ab
(lysomal associated membrane protein, LAMP). Across all defined CD8
epitopes, IFN gamma expression was found to be enhanced in animals
receiving anti-PD-L1 blockade compared to untreated animals (FIG.
15a). Additionally, coexpression of IFN gamma and TNF alpha and
CD107ab was also increased following anti-PD-L1 therapy (FIGS.
15B-15E). These findings indicate that adoptively transferred
memory splenocytes expanding in the presence of PD-L1 blockade are
functionally superior, in terms of inflammatory cytokine production
and release of cytolytic granules, as compared to splenocytes from
untreated animals.
Example 17: Murine B Cell Responses During PD-1 Blockade
[0450] The following experiments were performed in order to
determine whether PD-1 blockade enhances B cell responses during
chronic LCMV infection. Both B cell and T cell responses are
critical in controlling chronic LCMV infection, thus improving B
cell responses in chronic LCMV infected mice may help lower viral
load and enhance T cell function.
[0451] The following material and methods were used in these
experiments:
[0452] Mice and Virus:
[0453] Four- to six-week-old female C57Bl/6 mice were purchased
from Jackson Laboratory (Bar Harbor, Me.). Prior to infection,
chronic LCMV mice were depleted of CD4 T cells by administration of
gk1.5 antibody. Previous data demonstrates that administration of
500 ug of gk1.5 days -2 and 0 prior to viral challenge results in
95-99% decrease in the number of CD4 T cells in the spleen and
lymph node with the CD4 T cell numbers slowly recovering over 2 to
4 weeks. Mice received 2.times.10.sup.6 PFU of the Clone-13 strain
of LCMV intravenously on day 0 initiate chronic infection. Titers
of virus were determined by a 6 day plaque assay on Vero cells.
[0454] Detection of ASC by ELISPOT:
[0455] Spleen and bone marrow single cell suspensions were depleted
of red blood cells by 0.84% NH.sub.4CL treatment and resuspended in
RPMI supplemented with 5% FCS. Antibody secreting cells were
detected by plating cells onto nitrocellulose-bottom 96-well
Multiscreen HA filtration plates (Millipore). Plates were
previously coated with 100 ul of 5 ug/ml of goat anti-mouse
IgG+IgM+IgA (Caltag/Invitrogen) overnight at 4.degree. C. Plates
were then washed 3.times. with PBS/0.2% tween followed by 1.times.
with PBS and blocked for 2 hours with RPMI+10% FCS to prevent
non-specific binding. Blocking medium was replaced with 100 ul of
RPMI 5% FCS and 50 ul of 1.times.10.sup.7 cells/ml was plated in
serial three-fold dilutions across the plate. Plates were incubated
for 6 hours at 37.degree. C. and 5% CO.sub.2. Cells were removed
and plates were washed 3.times. with PBS and 3.times. with PBS/0.2%
tween. Wells were then coated with biotinylated goat anti-mouse IgG
(Caltag/Invitrogen) diluted 1/1000 in PBS/0.2% tween/1% FCS and
incubated overnight at 4.degree. C. The secondary antibody was
removed and plates were washed 3.times. with PBS/0.2% tween.
Avidin-D HRP (Vector) diluted 1/1000 in PBS/0.2% tween/1% FCS was
incubated for one hour at RT. Plates were washed 3.times. with
PBS/0.2% tween and 3.times. with PBS and detection was carried out
by adding 100 ml of horseradish peroxidase-H.sub.2O.sub.2 chromogen
substrate. The substrate was prepared by adding 150 ul of a freshly
made AEC solution (10 mg of 3-amino-9-ethylcarbazole (ICN) per ml
dissolved in dimethylformamide(Sigma)) to 10 ml of 0.1 M sodium
acetate buffer pH 4.8), filtering it through a 0.2-mm-pore-size
membrane, and immediately before use adding 150 ml of 3%
H.sub.2O.sub.2. Granular red spots appeared in 3 to 5 minutes, and
the reaction was terminated by thorough rinsing with tap water.
Spots were enumerated with a stereomicroscope equipped with a
vertical white light.
[0456] Determination of Total Bone Marrow Cells:
[0457] For calculation of the total ASC response in bone marrow,
the response was multiplied by the marrow cells of two femurs by a
coefficient of 7.9, since .sup.59Fe distribution studies have shown
that 12.6% of total mouse bone marrow is located in both femurs
combined. No differences have been detected among the ASC
activities of bone marrow cells from the femur, tibia, humorous,
rib, or sternum. Typically, two adult femurs yield
2.0.times.10.sup.7 to 2.5.times.10.sup.7 total bone marrow
cells.
[0458] Flow Cytometry:
[0459] Directly conjugated antibodies were purchased from
Pharmingen (anti-B220, anti-CD4, anti-CD138 anti-CD95, anti-Ki67,
anti-IgD biotinylated), or Vector labs (PNA). Strepavidin-APC was
purchased from Molecular Probes. All staining was carried out at
4.degree. C. in PBS supplemented with 1% FCS and 0.1% sodium azide.
Cells were then fixed in 2% formaldehyde (in PBS) and analyzed on a
FACS Calibur using CellQuest software (BD Biosciences).
[0460] Statistical Analysis:
[0461] Tests were performed using Prism 4.0 (GraphPad, San Diego,
Calif.). Statistics were done using two-tailed, unpaired T test
with 95% confidence bounds.
[0462] Total Numbers of Antibody Secreting Cells in the Spleen is
Enhanced Following In-Vivo PD-1 Blockade:
[0463] Mice infected with LCMV Clone-13 were treated with anti
(.alpha.)PD-L1 approximately 60 days post infection. Mice were
administered 200 ug .alpha.PD-L1 every third day for two weeks. At
day 14 of .alpha.PD-L1 treatment, the mice were sacrificed and the
number of antibody secreting cells in the spleen was measured by
ELISPOT and flow cytometric staining. In three separate
experiments, mice treated with .alpha.PD-L1 showed significantly
increased levels of antibody-secreting cells (ASC) in the spleen
(p=0.011) as compared to untreated mice (FIG. 16a). ASC can be
differentiated from B cells in the spleen by their down-regulation
of the B cell marker B220 and by expression of CD138 (syndecam-1).
In agreement with the ELISPOT results, increased numbers of
B220.sup.low/int CD138+ cells were seen in infected mice treated
with .alpha.PD-L1 (FIG. 16b).
[0464] Treatment of Chronic LCMV Infected Mice with .alpha.PD-L1
does not Lead to Elevated Levels of Bone Marrow ASC.
[0465] It was determined whether antibody secreting cells within
the bone marrow were also enhanced during .alpha.PD-L1 treatment.
The majority of long-lived plasma cells reside within the bone
marrow, and these plasma cells are critical to long-term
maintenance of serum antibody levels. Chronic LCMV infected mice
were treated with .alpha.PD-L1 approximately 60 days post
infection. Day 14 of .alpha.PD-L1 treatment, spleen and bone marrow
ASC levels were measured by ELISPOT. Although there were elevated
numbers of ASC in the spleen two weeks post-treatment, there was no
change in the numbers of ASC in the bone marrow at this time-point
(FIG. 17).
[0466] Co-Treatment of Chronic LCMV Infected Mice with .alpha.PD-L1
and .sup.130.alpha.CTLA-4 Results in Synergistic Increases in
Splenic ASC Levels:
[0467] It was further investigated whether blocking signaling with
of another negative regulatory molecule, CTLA-4, would enhance the
effect seen during the PD-1 blockade. CTLA-4 binding to B7 is
thought to both compete with the positive co-stimulatory molecule
CD28 and/or provide directly antagonizing TCR signals. Mice
infected with LCMV Clone-13 were treated with either treated with
.alpha.PD-L1, .alpha.CTLA-4, both or left untreated, and two weeks
post-treatment the levels of antibody secreting cells were measured
by ELISPOT. Although treatment with .alpha.CTLA-4 showed no impact
on ASC levels, co-treatment of .alpha.PD-L1 with .alpha.CTL-4 led
to a synergistic increase in ASC above that seen with .alpha.PD-L1
treatment alone (FIG. 18).
[0468] Enhanced B Cell and CD4 T Cell Proliferation and Germinal
Center Activity in .alpha.PD-L1 Treated Mice:
[0469] Flow cytometric analysis of spleen populations in chronic
mice treated with .alpha.PD-L1 showed enhanced levels of
proliferation by increased Ki-67 staining in both CD4 T cells and B
cells. B cells within the germinal center reaction can be
identified in the spleen by high levels of PNA and FAS staining.
Following .alpha.PD-L1 treatment, there was a large increase in the
frequency of PNA+FAS+ B cells compared to untreated controls (FIG.
19a-19b).
Example 17: PD-1 Expression on Human T Cells
[0470] CD8 T cells are essential for the control of many chronic
infections. As disclosed herein, these CD8 T cells become exhausted
following chronic antigenic stimulation, which is characterized by
the induction of a hypoproliferative state and loss of the ability
to produce anti-viral cytokines. Exhausted T cells have high
expression of programmed death-1 (PD-1) and, also PD-1 is
upregulated by T cell activation and can be triggered by the PD-1
ligands, PD-L1 and PD-L2. It is disclosed herein that the PD-1
inhibitory pathway is an important mediator of CD8 T cell
exhaustion during a chronic viral infection in mice. Virus specific
CD8 T cells maintained high levels of PD-1 expression in response
to a chronic infection, but not in response to an infection that is
successfully eliminated. Blocking the interaction of PD-1/PD-L1
interaction resulted in enhanced CD8 T cell proliferation,
production of anti-viral cytokines, and a reduction in viral
load.
[0471] It was evaluated whether CD8 T cells specific for chronic
infections in humans express PD-1, and whether PD-1 blockade
enhances CD8 T cells responses. This study (1) determined the
expression pattern of PD-1 on subsets of human peripheral blood
mononuclear cells (PBMC): CD4, CD8, B cell, NK, monocytes, DC; (2)
Determined the phenotype of CD4 and CD8 T cells that express PD-1;
(3) determined PD-1 expression on chronic persistent antigen
[(Epstein-Barr virus (EBV and cytomegalovirus (CMV)] and acute
resolved antigen (influenza and vaccinia)-specific cells; and (4)
determined the effect of blocking PD-1/PD-L1 interaction on the
proliferation of antigen-specific cells.
[0472] The following materials and methods were used in these
studies:
[0473] Blood Samples:
[0474] Peripheral blood samples were obtained from 36 healthy
individuals who were seropositive for EBV, CMV, influenza or
vaccinia viruses. These subjects were selected based on their HLA
allele expression matching HLA class I tetramers specific for EBV,
CMV, influenza or vaccinia virus proteins. PBMC were isolated from
the blood samples over lymphocyte-separation medium (Cellgro,
Herndon, Va.).
[0475] Antibodies, Peptides and Tetramers:
[0476] Phycoerythrin-conjugated anti-human PD-1 (EH12, mouse IgG1)
and unconjugated human PD-L1 (29E.2A3, mouse IgG2b) were obtained.
Directly conjugated antibodies were obtained from Beckman Coulter,
San Diego, Calif. (anti-CD3, CD11a, CD27, CD28, CD38, CD45RA, CD57,
CD62L and granzyme-B), BD Pharmingen, San Diego, Calif. (CD8, CD95,
CD195, HLA-DR, Ki-67 and perforin), and R&D systems,
Minneapolis, Mass. (CCR7). Peptides were made at the peptide
synthesis lab at Emory University, Atlanta, Ga. The plasmid
constructs expressing HLA-A2, -B7 and -B8 were kindly provided by
the NIH Tetramer Core Facility, Atlanta, Ga. and APC-labeled MHC
class I/peptide tetramers carrying CTL epitopes of EBV
(HLA-A2-GLCTLVAML (SEQ ID NO: 36), HLA-B8-RAKFKQLL (SEQ ID NO: 37)
and FLRGRAYGL (SEQ ID NO: 38)), CMV(HLA-A2-NLVPMVATV (SEQ ID NO:
39), HLA-B7-TPRVTGGGAM (SEQ ID NO: 40)), influenza
(HLA-A2-GILGFVFTL (SEQ ID NO: 41)) and vaccinia (HLA-A2-CLTEYILWV
(SEQ ID NO: 42) and KVDDTFYYV (SEQ ID NO: 43)).
[0477] Immunophenotyping and CFSE Proliferation:
[0478] Heparinised human whole blood samples (200 ul) were stained
with antibodies or tetramers and then analyzed (Ibegbu et al., J
Immunol. 174: 6088-6094, 2005) on a FACS Calibur using CellQuest
software or on a LSRII flow cytometer using FACSDiva software (BD
Immunocytometry Systems). For CFSE assays, PBMC
(2.times.10.sup.6/ml) were washed thoroughly and labeled with 3
.mu.M carboxy-fluorescein diacetate, succinimidyl ester (CFSE,
Molecular Probes) at room temperature in dark for 5 min (see, for
example, Weston and Parish, J Immunol Methods 133:87-97, 1990). The
CFSE labeled PBMC were stimulated with either peptide alone (1
g/ml) or peptide with anti-PD-L1 antibody (10 .mu.g/ml). Control
cultures consisted of either PBMC alone, PBMC with anti-PD-L1
antibody or PBMC with an isotype control antibody (IgG2b; 10
.mu.g/ml). Following a 6-day incubation at 37.degree. C., the cells
were washed and stained with tetramer along with anti-CD3 and -CD8
antibodies extracellularly.
[0479] The following results were obtained:
[0480] Expression Pattern of PD-1 on PBMC Subsets:
[0481] PD-1 expression was examined on PBMC subsets in healthy
individuals. It was observed that CD8+ T cells, CD4+ T cells and
monocytes (CD14+) express high levels of PD-1, B cells (CD20+)
express low levels of PD-1 and NK cells (CD56+) and DC (CD11c+) do
not express PD-1.
[0482] PD-1 is Preferentially Expressed Among Effector Memory CD8
and CD4 T Cells:
[0483] CD8 T cells from normal healthy individuals were examined
for co-expression of PD-1 with various phenotypic markers
associated with differentiation state and function (FIG. 20A). In
summary, naive and central memory phenotype CD8 T cells only
expressed low levels of PD-1, whereas CD8 T cells that expressed
various markers associated with effector/effector memory/or
exhausted phenotype also expressed high levels of PD-1 (FIG. 20B).
These data suggested that PD-1 was preferentially expressed among
effector memory CD8 T cells. When the CD4 T cells were examined we
found similar trend (FIG. 20C).
[0484] PD-1 is Upregulated on Persistent Antigen-Specific Memory
CD8 T Cells:
[0485] To evaluate whether CD8 T cells specific for chronic
infections in humans show increased expression of PD-1, PD-1
expression on memory CD8 T cells specific for chronic persistent
viruses (EBV and CMV) was compared with acute virus specific T
cells (influenza and vaccinia) in 36 healthy individuals by
staining with EBV-, CMV-, influenza- and vaccinia virus-specific
tetramers (FIGS. 21A-21B). FIG. 21A shows representative PD-1 GMFI
of EBV, CMV, influenza and vaccinia virus-specific CD8 T cells.
PD-1 expression was found to be increased on EBV-specific CD8 T
cells than influenza (p=0.0335) and vaccinia (p=0.0036)
virus-specific CD8 T cells (FIGS. 21A-21B). Similarly, CMV-specific
CD8 T cells more frequently expressed PD-1 than influenza
(p=0.0431) and vaccinia (p=0.019) (FIGS. 21A-21B). These results
suggest a correlation between PD-1 expression and antigen
experience.
[0486] Anti-PD-L1 Blockade Increases Proliferation of Chronic
Persistent Virus-Specific CD8 T Cells:
[0487] It was assessed whether PD-1 blockade enhances persistent
antigen-specific CD8 T cell responses similar to the results
observed in mice. CFSE labeled cells were stimulated with either
EBV, CMV, influenza or vaccinia virus-specific peptides in the
presence or absence of anti-PD-L1 antibodies. After 6 days, the
percentage of tetramer.sup.+ CFSE.sup.lo cells and CD8+ CFSE.sup.lo
cells was compared between cultures that were stimulated with
peptide alone and cultures that were stimulated with peptide and
subsequently blocked with anti-PD-L1. Representative flow cytometry
plots with proliferation of CMV and EBV-specific CD8 T cells are
shown in FIG. 22A. Aggregated data from CMV (n=5), EBV (n=6),
influenza (n=2) and vaccinia (n=2) seropositive individuals are
shown in FIG. 22B. Blocking PD-1/PD-L1 interaction with anti-PD-L1
antibody resulted in increased proliferation of EBV and
CMV-specific CD8 T cells whereas influenza and vaccinia
virus-specific CD8 T cells did not show proliferation following
blocking with anti-PD-L1. These results show that in the presence
of peptide plus anti-PD-L1 blocking antibody, there is up to
3.5-fold increase in the frequency of EBV or CMV-specific CD8 T
cells compared to stimulation with the peptide alone. It was
assessed whether the proliferation of antigen-specific CD8 T cells
following anti-PD-L1 antibody blockade is related to the PD-1
expression by these cells. The data indicate a positive correlation
between PD-1 expression and proliferation of antigen-specific CD8 T
cells (p=0.0083) (FIG. 22C).
Example 18: Liver Infiltrating Lymphocytes in Chronic Human HCV
Infection Display an Exhausted Phenotype with High PD-1 and Low
CD127
[0488] Expression The experiments described below document that
chronic HCV infection, peripheral HCV-specific T cells express high
levels of PD-1 and that blockade of the PD-1/PD-L1 interaction led
to an enhanced proliferative capacity. Importantly, intrahepatic
HCV-specific T cells not only express high levels of PD-1 but also
decreased IL-7 receptor alpha (CD127), an exhausted phenotype that
was HCV antigen specific and compartmentalized to the liver, the
site of viral replication.
[0489] Currently, no vaccine exists to prevent HCV infection and
the only licensed therapy, alpha interferon (IFN.alpha.), either
alone or in combination with the nucleoside analog ribavirin is
expensive, associated with, at best, only a 50% clearance rate for
the most prevalent genotype (genotype 1) and complicated by
significant side effects. The paucity of efficacious anti-HCV
therapeutic options highlights the need for effective interventions
aimed at augmenting or supplementing the natural immune response
that, alone or in concert with antiviral drug therapy, can prevent
the detrimental consequences of HCV infection.
[0490] Currently, little is known about the expression of PD-1 and
its role in T cell exhaustion in chronic HCV infection,
particularly at the site of active infection, the liver. The
present study was undertaken to better understand the T cell
phenotype in HCV infection by measuring expression of PD-1 on
antigen-specific CD8+ T cells in both the liver and peripheral
blood of patients with chronic HCV infection.
[0491] The following materials and method were used in these
studies:
[0492] Subjects:
[0493] Seventeen patients with chronic HCV infection (HCV antibody
and HCV PCR positive) and negative for HIV by antibody screening
were enrolled in the study. All patients were naive to HCV
anti-viral therapies prior to enrollment. Seven of the fifteen
patients were positive for HLA-A2 by FACS analysis. The patient
characteristics are summarized in Table 5.
TABLE-US-00012 TABLE 5 Patient cohort demographic and clinical data
Baseline Patient HLA- HCV Viral Identification Gender Age A2
Genotype Load (IU/ml) ALT 153 HCV* M 43 + 2b 7,340,000 25 178 HCV*
F 48 + 2 18,330,000 62 179 HCV M 54 - 1a 197,000 197 183 HCV F 56 +
1a 1,170,000 45 190 HCV M 52 - 1a 5,990,000 27 193 HCV M 66 + 1a
16,120,000 30 601 HCV M 60 - 1b 4,690,000 25 602 HCV M 48 - 1a
586,000 80 603 HCV M 58 + 1a 1,820,000 36 604 HCV M 58 - 1a
2,850,000 57 605 HCV F 30 - 1 819,000 57 606 HCV M 50 - 1b 591,000
18 607 HCV M 59 + 3a 343,000 31 608 HCV M 57 - 1b 395,000 16 609
HCV M 55 + 1a 833,000 67 611 HCV M 53 - 1a 1,220,000 88 613 HCV M
59 - 1b 6,160,000 40
[0494] HCV Antibody Testing, Viral Load Determination and
Genotyping:
[0495] HCV antibody testing by ELISA was performed using a kit per
the manufacturer's instructions (Abbott Diagnostics, Abbott Park,
Ill.; Bio-Rad Laboratories, Hercules, Calif.). HCV viral load
quantification was performed using a real-time RT-PCR assay (Roche
Molecular Systems, Alameda Calif.). HCV genotyping was performed
using a real-time RT-PCR assay (Abbott Diagnostics, Abbott Park,
Ill.) and using a line probe assay (LIPA) (Bayer Diagnostics,
Research Triangle Park, N.C.).
[0496] Peripheral Blood Mononuclear Cells:
[0497] EDTA and heparin anticoagulated blood (50-70 ml) was
collected from each patient and either used directly for FACS
staining or for PBMC isolation. PBMCs were isolated using
Ficoll-Paque PLUS density gradient (Amersham, Oslo, Norway), washed
twice in PBS, and either analyzed immediately or cryopreserved in
media containing 90% fetal calf serum (Hyclone) and 10% dimethyl
sulfoxide (Sigma-Aldrich, St. Louis, Mo.).
[0498] Liver Biopsy:
[0499] Liver tissue was obtained by either ultrasound-guided needle
biopsy or via transjugular fluoroscopic technique and immediately
put into RPMI-1640 medium (Gibco) containing 10% fetal calf serum
(Hyclone, Logan, Utah) for immunological assays. Another fragment
was fixed in formalin for histological examination.
[0500] Intrahepatic T Cell Isolation:
[0501] The liver biopsy sample obtained in RPMI-1640 medium (Gibco,
Carlsbad, Calif.) containing 10% fetal calf serum (Hyclone, Logan,
Utah) was washed three times with the same media to remove cell
debris and RBCs. Isolation of liver infiltrating lymphocytes was
performed using an automated, mechanical disaggregation system
(Medimachine, Becton Dickinson, San Jose, Calif.). The sample was
inserted into a 50 m Medicon and inserted into the Medimachine and
run for 15 seconds. Dissagregated cells were removed using a
syringe in the syringe port. The Medicon was rinsed twice with RPMI
medium (Gibco, Carlsbad, Calif.) containing 10% fetal calf serum
(Hyclone, Logan, Utah) to ensure maximum cell recovery. Cells were
used immediately for FACS staining.
[0502] Antibodies, HLA-A2 Tetramers and Flow Cytometry:
[0503] Cells were stained with FITC, PE, PerCP and APC labeled
monoclonal antibodies or tetramers according to the manufacturers'
instructions and flow cytometry performed using FACS Calibur
(Becton Dickinson, San Jose, Calif.). FACS data were analyzed with
FlowJo software (Treestar). The following monoclonal antibodies
from BD Pharmingen (BD Biosciences, San Jose, Calif.) were used:
Anti-CD8 PerCP and anti-CD45RA APC. Anti-CD62L FITC, CD3 FITC and
CD127 PE were obtained from Beckman Coulter (Fullerton, Calif.).
Anti-PD-1 PE conjugated antibody (clone EH12) was generated as
described (Dorfman et al., Am. J. Surg. Pathol. 30:802-810, 2006).
HLA-A2 tetramers were specific for the following CD8+ T cell
epitopes: HCV 1073: CINGVCWTV (SEQ ID NO: 44); HCV-1406: KLVALGINAV
(SEQ ID NO: 45). Flow cytometric collection was performed on a
FACSCaliber.TM. (BD Biosciences, San Jose, Calif.) and analysis
performed using FlowJo software (v8.1.1).
[0504] CFSE Labeling and Antibody Blockade:
[0505] 10.times.0.sup.6 PBMCs were washed with PBS and labeled with
3 .mu.M CFSE (Molecular Probes). Cells were adjusted to
1.times.10.sup.6 cells/ml and cultured in the presence of 2
.mu.g/ml of A2-HCV 1073 (CINGVCWTV, SEQ ID NO: 44) peptide. 10 U/ml
of IL-2 were added on day 3 post stimulation. An unstimulated
control was included in each assay. Specific blocking antibodies
(anti-PD-L1; clone #29E and anti-PD-1; clone # EH12 (Dofman et al.,
supra) were added to cell cultures at a concentration of 10
.mu.g/ml at the time of stimulation. Cells were incubated for 6
days, harvested and stained with surface antibodies and tetramers
and analyzed by flow cytometry.
[0506] Statistical Analysis:
[0507] Results were graphed and analyzed using GraphPad Prism (v4).
Comparisons made within the same patient were performed using
paired t tests. Comparisons made between patients were made using
unpaired t tests.
[0508] The following results were obtained:
[0509] PD-1 Expression on HCV Antigen Specific CD8+ T Cells:
[0510] Seventeen patients with HCV infection (all HIV negative)
were studied (Table 1). Fifteen patients underwent both blood and
liver sampling for phenotyping by flow cytometric analysis, and all
were untreated with pharmacologic antiviral therapy prior to study
enrollment. Seven patients in the cohort were HLA-A2 positive and
demonstrated a population of HCV specific CD8+ T cells in the
periphery by HLA tetramer staining (Table 1). These HCV specific
CD8+ T cells were evaluated for PD-1 expression (FIG. 23A). The
level of PD-1 expression on total CD8+ T cells in the peripheral
blood from healthy donors was not significantly different from that
of the total pool of peripheral CD8+ T cells from HCV infected
patients (FIG. 23B). In contrast, the majority of HCV-specific
tetramer positive CD8+ T cells sampled from the peripheral blood
were PD-1 positive (mean 85%, SEM 3.6) (FIG. 23A) with
significantly higher expression than that of the total CD8+ T cell
population (p<0.0001) (FIG. 23B). Expression of differentiation,
co-stimulatory, trafficking and effector function molecules on
antigen specific CD8+ T cells was also investigated. The
HCV-specific tetramer positive cells exhibit a memory phenotype
(high CD1 la, low CD45RA), early differentiation markers (high
CD27, high CD28, intermediate expression of CCR7 and CD62L) and low
levels of mediators of effector function granzyme B and perforin.
Interestingly, these HCV tetramer positive T cells in the
peripheral blood expressed high levels of CD127 (IL-7 receptor a
chain), a phenotypic marker that when expressed at low levels
identifies impaired memory T cell differentiation.
[0511] To determine whether the phenotype of CD8+ T cells was
different in the setting of non-chronic infection, Flu-specific T
cells were examined in five healthy HLA-A2+ donors who were not
infected with HCV. The percentage of peripheral Flu tetramer+ CD8+
T cells that expressed PD-1 was 49% (SEM 14.1) (FIG. 23C). Five of
the seven HLA-A2 positive chronic HCV patients were also identified
by tetramer analysis to have Flu specific CD8+ T cells. The
percentage of Flu-specific T cells expressing PD-1 in these
chronically infected HCV patients was not significantly different
from the same population in healthy donors (FIG. 23C). Importantly,
because five of the seven HLA-A2+ HCV patients also had detectable
Flu specific CD8+ T cells, a comparison could be made, within each
patient, of PD-1 for T cells specific for a non-chronic (Flu) and
chronic (HCV) infection. The difference between Flu-specific and
HCV-specific T cell expression of PD-1 expression was significant
(FIG. 23C). The percentage of HCV specific CD8+ T cells expressing
PD-1 (mean 83%, SEM 6.4) was greater than the percentage of PD-1+
Flu specific CD8+ T cells (49%, SEM 12.3) (p=0.048) (FIG. 23C).
[0512] PD-1 Expression on Human Peripheral Blood and Liver
Infiltrating Lymphocytes:
[0513] Peripheral blood and liver biopsies were analyzed for the
expression of PD-from fifteen patients chronically infected with
HCV. Representative flow cytometric analysis from five patients is
shown in FIG. 24A. Whereas in the peripheral blood, 27% (SEM 3.4)
of CD8+ T cells were PD-1+, the frequency of such cells was
increased two fold (57%, SEM 3.6) in the liver (FIG. 24B). Hence,
the liver is enriched in cells expressing high levels of PD-1.
While naive cells should express high levels of both CD62L and
CD45RA, in the liver the majority of CD8+ T cells were CD62L
low/CD45RA low consistent with a memory phenotype (FIG. 24C).
Analysis specifically of this memory population in both the liver
and the periphery showed that PD-1 expression was elevated in the
liver compared with the periphery (FIG. 24C). These data suggest
that the increase in the percentage of cells expressing PD-1 on the
intrahepatic T cells is not merely due to the absence of the naive
population in this compartment. Rather, there is a preferential
enrichment of PD-1+CD8+ T effector memory (CD62L low/CD45RA low)
cells within the liver compared to the peripheral blood (FIG.
23C).
[0514] CD127 Expression on Human Peripheral Blood and Liver
Infiltrating Lymphocytes:
[0515] IL-7 is required for maintenance of memory CD8+ T cells
(Kaech et al., Nat Immunol 4:1191-8, 2003), and the alpha chain of
its receptor, CD127, is downregulated on antigen specific T cells
in persistent LCMV and gammaherpesvirus infections (see, for
example, Fuller et al., J Immunol 174:5926-30, 2005). This loss of
CD127 during chronic infection correlates with impaired cytokine
production, increased susceptibility to apoptosis, and a reduction
in the ability of memory virus-specific CD8+ T cells to persist in
the host. Accordingly, resolution of acute hepatitis B virus (HBV)
infection correlates with upregulation of CD127 expression and
concomitant loss of PD-1 expression (Boettler et al., J Virol
80:3532-40, 2006). Interestingly, in the chronic HCV patients, only
20% (SEM 4.8) of total peripheral CD8+ T cells were CD127 negative,
but in the hepatic CD8+ T cell infiltrates, this percentage
increased significantly to 58% (SEM 4.4) (FIG. 24D). Hence, the
liver is enriched in cells expressing an exhausted phenotype with
high PD-1 and low CD127 cells predominating. These data suggest
that liver infiltrating CD8+ T cells in chronic HCV patients do not
phenotypically mirror the peripheral CD8+ T cell population. In the
setting of HIV infection where the virus infects T cells and
monocytes in the peripheral blood, low levels of CD127 are
associated with functional or memory T cell defects (Boutboul et
al., Aids 19:1981-6, 2005). In this study, the hepatic
compartmentalization of the cells showing this exhausted phenotype
suggests that the phenotype is intimately tied to the site of
persistent viral replication.
[0516] PD-1 and CD127 Expression on HCV Antigen Specific CD8+ T
Cells in the Liver:
[0517] Two of our HLA-A2 patients in the cohort also had an
identifiable HCV specific population by tetramer staining in the
liver (FIG. 25). Expression of PD-1 and CD127 was directly compared
on HCV specific tetramer positive CD8+ T cells in the liver versus
the periphery of these individuals. HCV specific CD8+ T cells from
the periphery were mostly PD-1 positive (mean 85%, SEM 3.6) and
CD127 positive (mean 84%, SEM 4.0), while the hepatic HCV specific
CD8+ T cells were mostly PD-1 positive (mean 92%) but only rarely
CD127 positive (mean 13%) (FIG. 25). At the site of viral
replication, there appeared to be an expansion of CD127 negative
cells expressing high levels of PD-1. That peripheral antigen
specific CD8+ T cells differentially express CD127 compared with
the intrahepatic compartment could be related to the level or
timing of antigen exposure needed to cause downregulation of CD127.
In LCMV infection of mice, exposure to persistent antigen load with
chronic infection, CD127 was persistently downregulated whereas
short-lived exposure to LCMV antigen using GP33 only temporarily
suppressed CD127 expression and failed to induce T cell exhaustion
(Lang et al., Eur J Immunol 35:738-45, 2005). Dependence on
availability of antigen and time of exposure was also observed to
affect the expression of CD62L and CD127, whereas persistent
antigen led to persistent downregulation of both CD62L and CD127
(Bachmann et al., J Immunol 175:4686-96, 2005). Without being bound
by theory, in chronic HCV infection, the few HCV specific CD8+ T
cells detected in the periphery may not be continuously exposed to
sufficient antigen to maintain low levels of CD127. Thus, the T
cells may "believe" that the virus has been cleared.
[0518] Blockade of PD-1/PD-L1 Leads to Increased Expansion of HCV
Specific Tetramer Positive CD8+ T Cells:
[0519] Evidence from the patient population suggests that blockade
of the PD-1/PD-L1 interaction with anti-PD-L1 or anti-PD-1 antibody
increases the proliferative capacity of HCV-specific T cells (FIG.
26). Addition of blocking antibodies in the presence of IL-2 and
HCV-specific peptide resulted in a four-fold increase in expansion
of the HCV-specific T cells as demonstrated by monitoring the
frequency of carboxyfluorescein succinimidyl ester (CFSE).sup.low
tetramer labeled CD8+ T cells after stimulation with cognate
peptide for 6 days.
[0520] The results show that at the site of infection, the liver,
the frequency of HCV specific CD8+ T cells expressing PD-1 is high.
Second, the majority of HCV specific CD8+ T cells from the
peripheral blood of patients with chronic HCV infection express
high levels of CD127. The phenotype of T cells in chronic HCV
infection was characterized by studying the expression of the PD-1
molecule linked to impaired effector function and T cell
exhaustion. The results show that the majority of HCV specific T
cells in the intrahepatic compartment express PD-1 but lack CD127,
a phenotype consistent with T cell exhaustion. Thus, PD-1
antagonists are of use as therapeutic agents for the treatment of
HCV infection.
Example 19: PD1 Blockade Induces Expansion of SIV-Specific CD8
Cells In Vitro
[0521] Anti-viral CD8 T cells play a critical role in the control
of HIV/SIV infections. A central role for CD8 T cells has been
shown by viral re-emergence during transient in vivo depletions in
SIV-infected macaques. Consistent with this, contemporary vaccine
strategies designed to elicit high frequencies of anti-viral CD8 T
cells have contained pathogenic SHIV and SIV challenges in macaques
(see, for example Barouch et al., Science 290, 486-92 (2000);
Casimiro et al., J Virol 79, 15547-55 (2005).
[0522] Both the function and the frequency of anti-viral CD8 T
cells are crucial for the control of chronic viral infections such
as HIV (Migueles et al. Nat Immunol 3, 1061-8, 2002) and
Lymphocytic choriomeningitis virus (LCMV). Effective anti-viral CD8
T cells possess a number of functional properties including the
ability to produce different cytokines, cytotoxic potential, and
high proliferative potential and low apoptosis. In chronic viral
infections virus-specific CD8 T cells undergo exhaustion that is
associated with the loss of many of these functions (Zajac et al.,
J Exp Med 188, 2205-13, 1998). Similarly, HIV-specific CD8 T cells
from individuals with progressive disease have been shown to be
impaired for their function. These CD8 T cells can produce
cytokines such as IFN-.gamma. but are impaired for the production
of IL-2, a cytokine that is critical for the T cell proliferation
and survival; expression of perforin (Appay et al., J Exp Med 192,
63-75, 2000, a molecule that is critical for cytolytic function;
and proliferative capacity, a property that has been implicated to
be critical for the control of HIV (see, for example, Harari et
al., Blood 103, 966-72, 2004) and SIV. HIV-specific T cells express
high levels of PD-1 and this expression is directly proportional to
the level of viremia. A transient blockade of interaction between
PD-1 and PD-L1 in vitro restores HIV-specific T cell function.
[0523] The expression of PD-1 on SIV-specific CD8 T cells following
infection with a pathogenic SIV239 in macaques was investigated.
The results demonstrate that SIV-specific CD8 T cells express high
levels of PD-1 and blockade of PD-1:PDL-1 pathway in vitro results
in enhanced expansion of these cells. The following results were
obtained:
[0524] Elevated PD-1 Expression on SIV-Specific CD8 T Cells
Following SIV239 Infection:
[0525] The level of PD-1 expression on CD8 T cells from normal
healthy and SIV-infected macaques was investigated to understand
the role of PD-1 expression and its relationship with the control
of SIV-infection. A significant proportion (40-50%) of total CD8 T
cells from normal healthy macaques expressed PD-1 (FIG. 27A). The
PD-1 expression was predominantly restricted to memory cells and
was absent on naive CD8 T cells. A similar PD-1 expression pattern
was also observed for total CD8 T cells from SIVmac239-infected
macaques (FIGS. 27B and C). However, the majority (>95%) of SIV
Gag CM9-specific CD8 T cells were positive for PD-1 expression and
a significant proportion of these cells further up regulated PD-1
expression (MFI of 580) compared to total CD8 T cells (MFI of 220)
(FIG. 27D). Collectively, these results demonstrate that a
significant proportion of memory CD8 T cells from normal and
SIV-infected macaques express PD-1 and the level of PD-1 expression
is further elevated on the SIV-specific CD8 T cells.
[0526] In Vitro Blockade of PD-1 Results in Enhanced Expansion of
SIV-Specific CD8 T Cells:
[0527] To study the effect of PD-1 blockade on the function of
SIV-specific CD8 T cells, proliferation assays were conducted in
the presence and absence of a blocking antibody to human PD-1
molecule that is cross reactive to macaque PD-1. PBMC from Mamu
A*01 positive rhesus macaques that were infected with a pathogenic
simian and human immunodeficiency virus 89.6P (SHIV 89.6P) were
stimulated with P11C peptide (Gag-CM9 epitope) in the absence and
presence of anti-PD-1 blocking Ab for six days. The frequency of
Gag CM-9 tetramer positive cells was evaluated at the end of
stimulation. Unstimulated cells served as negative controls. As can
be seen in FIG. 28A-28B, stimulation with P11C peptide resulted in
an about 4-80 fold increase in the frequency of tetramer positive
cells. In addition, in four out of six macaques tested,
stimulations with P11C peptide in the presence of anti-PD-1
blocking Ab resulted in about 2-4 fold further enhancement in the
frequency of tetramer positive cells over stimulations with P11C
peptide in the absence of blocking antibody.
[0528] These results demonstrate that PD-1 blockade enhances the
proliferative capacity of SIV-specific CD8 T cells in SIV-infected
macaques.
Example 20: Role of PD-L2
[0529] Two PD-1 ligands differ in their expression patterns: PD-L1
is constitutively expressed and upregulated to higher amounts on
both hematopoietic and nonhematopoeitic cells, whereas PD-L2 is
only inducibly expressed on dendritic cells (DCs) and macrophages.
Although some studies for evaluating the role that PD-L2 plays in T
cell activation have demonstrated inhibitory function for PD-L2,
other studies reported that PD-L2 stimulate T cell proliferation
and cytokine production. To delineate the role of PD-L2 on T cell
immune response, the kinetics of PD-L2 expression on different cell
types ex vivo was examined after LCMV Armstrong infection (FIG.
29). In contrast to PD-L1 expression, PD-L2 expression was
expressed limitedly on DC during a very short time (day 1-4
post-infection). This result suggests that PD-L2 expression is
closely related to DC regulation and results in regulation of T
cell activation.
Example 21: PD-1 is Expressed by the Majority of Effector Memory
CD8 T Cells in the Blood of Healthy Humans
[0530] PD-1 expression on CD3+/CD8+ T cells from the blood of
healthy human adults was investigated. In human blood 20-60% of CD8
T cells expressed PD-1. The relationship between T cell
differentiation state and PD-1 expression was examined. CD3+/CD8+ T
cells were delineated into naive, central memory (T.sub.CM),
effector memory (T.sub.EM), and terminally differentiated effector
(T.sub.EMRA) subsets based on patterns of CD45RA and CCR7
expression. PD-1 was not expressed by naive T cells, and by
approximately one third of T.sub.CM and T.sub.EMRA. In contrast,
60% of T.sub.EM expressed PD-1. These data demonstrate that the
majority of T.sub.EM isolated from the blood of healthy human
adults express PD-1.
[0531] Based on these analyses, T cells were subdivided into
multiple populations based on CD45RA and CCR7 expression. An
additional relationship was found between CD45RA expression and
PD-1 expression. Specifically, CCR7-/CD8+ T cells with the lowest
CD45RA expression contained the highest proportion of PD-1+ cells.
In conclusion, PD-1 was predominantly expressed by T.sub.EM, to a
lesser extent by T.sub.EMRA and T.sub.CM, and was not expressed
among naive CD8 T cells. These data illustrate that a large
proportion of T.sub.EM CD8 T cells express PD-1 among healthy human
adults.
[0532] To characterize the properties of PD-1+CD8 T cells further,
the co-expression of PD-1 and several T cell differentiation
markers was examined. The majority of PD-1+CD8 T cells bore markers
associated with antigen experience and effector/effector memory
differentiation. For instance,
CD11a+/CCR7-/CD62L-/CD45RA-/KLRG1+/granzyme B+/perforin+CD8 T cells
were enriched in PD-1 expression. In contrast, naive phenotype
(CD11a-/CCR7+/CD62L+/CD45RA+/KLRG1-) CD8 T cells expressed low
levels of PD-1. Thus, PD-1 was preferentially expressed on
antigen-experienced CD8 T cells with effector/effector memory
qualities.
Example 22: PD-1 is Expressed by the Majority of Effector Memory
CD4 T Cells in Blood of Healthy Humans
[0533] PD-1 expression among CD3+CD4+ T cells was then
investigated. Thirty percent of CD4 T cells expressed PD-1 in the
blood of healthy adults. Similar to CD8 T cells, naive CD4 T cells
expressed little PD-1. While a minority of T.sub.CM CD4 T cells
expressed PD-1, PD-1 expression was preferentially enriched among
T.sub.EMCD4 T cells (50%).
[0534] To further characterize the properties of CD4 T cells that
expressed PD, CD4+/CD3+ T cells were assayed from the blood of
healthy individuals for the co-expression of PD-1 and several T
cell differentiation markers. Similar to CD8 T cells, PD-1
expression was enriched on CD4 T cells with an effector/effector
memory phenotype, including CD62L-, CD95+, CD45RA-, CCR7-, and
CCR5+ cells.
Example 23: PD-1 is More Highly Expressed on CD8 T Cells Specific
for EBV and CMV Infections in Humans
[0535] To test whether PD-1 expression is correlated with viral
antigen persistence, PD-1 expression was compared on EBV, CMV,
influenza, and vaccinia virus specific CD8 T cells. EBV and
CMV-specific CD8 T cells expressed high levels of PD-1. In
contrast, influenza virus specific memory CD8 T cells expressed
intermediate levels of PD-1 and vaccinia virus specific CD8 T cells
express low levels of PD-1. Hence memory CD8 T cells specific for
chronic infections (EBV and CMV) expressed higher levels of PD-1
than acute (influenza and vaccinia) infections. These results show
that CD8 T cells specific for chronic infections (EBV and CMV)
expressed higher levels of PD-1 than acute infections (influenza
and vaccinia viruses). CD8 T cells specific for very common chronic
infections can express high levels of PD-1.
Example 24: Anti-PD-L1 Blockade Increases Proliferation of CD8 T
Cells Specific for EBV and CMV Infections in Humans
[0536] Blockade of the PD-1 inhibitory pathway results in enhanced
clonal expansion of HIV-specific CD8 T cells upon in vitro
stimulation. As CD8 T cells specific for common chronic infections
also express PD-1, it was tested whether blockade of the PD-1/PD-L1
pathway could enhance the proliferation of CD8 T cells specific for
EBV, CMV, and also vaccinia virus (an acute infection resulting in
PD-1 memory CD8 T cells). Lymphocytes were isolated from the blood
of individuals containing CD8 T cells specific for CMV, EBV, or VV
were labeled with CFSE and cultured for 6 days under various
conditions. As expected, incubation of freshly isolated peripheral
blood mononuclear cells (PBMC) with medium alone, or medium with
anti-PD-L1 antibody, did not induce proliferation of virus-specific
CD8 T cells. Stimulation of PBMC for 6 days with virus-derived
peptides resulted in division of tetramer+ CD8 T cells. However,
peptide stimulation of PBMC in the presence of anti-PD-L1 blocking
antibody further enhanced division of EBV and CMV-specific CD8 T
cells, resulting in a greater fold-expansion than peptide alone The
enhanced division induced by anti-PD-L1 blocking antibody varied
among individuals and even among different epitopes within a given
individual. Moreover, PD-1 blockade did not result in enhanced
expansion of vaccinia or influenza specific CD8 T cells. The degree
of enhanced division induced by blocking PD-L1 in culture could be
related to the amount of PD-1 expressed by antigen specific CD8 T
cells prior to stimulation. These data suggest that PD-1 expression
on CD8 T cells specific for chronic infections inhibits their
proliferative capacity upon antigenic stimulation.
Example 25: Sustained PD-L1 Blockade Further Increases
Proliferation of CD8 T Cells Specific for Chronic Infections
[0537] Upon in vitro stimulation, the addition of PD-L1 blocking
antibody led to increased division among CD8 T cells specific for
EBV and CMV. Anti-PD-L1 mAb was added once (day 0), and
proliferation was assessed at the end of the six-day culture
period. In vivo anti-PD-L1 treatment in mice involved multiple
injections of blocking antibody. Furthermore, in these murine
studies, in vivo PD-L1 blockade resulted in a rapid upregulation of
PD-1 expression among CD8 T cells specific for chronic viral
antigen. For these reasons, it was tested whether repeated
additions of anti-PD-L1 to stimulated T cell cultures would further
enhance proliferation. The addition of a-PD-L1 mAb on days 0, 2,
and 4 of culture resulted in an even greater accumulation of EBV
specific CD8 T cells than a single addition of mAb at day 0.
Similar data was observed for CMV specific CD8 T cells. These data
suggest that continued blocking of PD-1 signaling can optimize the
ability to increase the numbers of CD8 T cells specific for chronic
antigens.
Example 26: Additional Methods for Studies Described in Example
27
[0538] Study Group:
[0539] Fourteen Indian rhesus macaques (Macaca mulatta) infected
with SIV were studied. Eight macaques were used for the early
chronic phase and were infected intravenously with 200 TCID50 of
SIV251. Six macaques were used for the late chronic phase, three
were infected with SIV251 intrarectally and three were infected
with SIV239 intravenously. All macaques, except RDb11, were
negative for Mamu B08 and Mamu B17 alleles. RDb11 was positive for
Mamu B17 allele.
[0540] In Vivo Antibody Treatment:
[0541] Macaques were infused with either partially humanized mouse
anti-human PD-1 antibody (clone EH12-1540) (Dofrman et al., Am J
Surg Pathol 30, 802-810 (2006)) or a control antibody (SYNAGIS).
The anti-PD-1 antibody has mouse variable heavy chain domain linked
to human IgG1 (mutated to reduce FcR and complement binding) (Xu et
al., Cell Immunol 200, 16-26 (2000)) and mouse variable light chain
domain linked to human K. The clone EH12 binds to macaque PD-1 and
blocks interactions between PD-1 and its ligands in vitro (Velu et
al., J Virol 81, 5819-5828 (2007). SYNAGIS is a humanized mouse
monoclonal antibody (IgG1.kappa.) specific to F protein of
respiratory syncytial virus (Medimmune). Antibodies were
administered intravenously at 3 mg kg.sup.-1 of body weight on days
0, 3, 7 and 10.
[0542] Immune Responses:
[0543] Peripheral blood mononuclear cells from blood and
lymphocytes from rectal pinch biopsies were isolated as described
previously (Velu et al., J Virol 81, 5819-5828 (2007). Tetramer
staining (Amara et al., Science 292, 69-74 (2001)), intracellular
cytokine production (Kannanganat et al., J Virol 81, 8468-8476
(2007)) and measurements of anti-SIV Env binding antibody (Lai et
al., Virology 369, 153-167 (2007)) were performed as described
previously.
[0544] B Cell Responses:
[0545] A total of 100 .mu.l of blood was surface stained with
antibodies to CD3 (clone SP34-2, BD Biosciences), CD20 (2H7,
e-Biosciences), CD21 (B-ly4, Becton Dickson) CD27 (M-T2712, Becton
Dickson) and PD-1 (clone EH-12), each conjugated to a different
fluorochrome. Cells were lysed and fixed with FACS lysing solution,
and permeabilized using FACS perm (BD Biosciences) according to the
manufacturer's instructions. Cells were then stained for
intracellular Ki67 using an anti-Ki67 antibody conjugated to
phycoerythrin (PE) (clone B56, Becton Dickson). After staining,
cells were washed and acquired using LSRII (BD Biosciences), and
analysed using FLowJo.TM. software.
[0546] Titres of Anti-PD-1 Antibody and Monkey Antibody Response
Against Anti-PD-1 Antibody in Serum:
[0547] To measure the levels of anti-PD-1 antibody, plates were
coated with goat anti-mouse immunoglobulin (pre-absorbed to human
immunoglobulin, Southern Biotech), blocked and incubated with
different dilutions of serum to capture the blocking antibody.
Bound antibody was detected using anti-mouse IgG conjugated to HRP
(pre-absorbed to human immunoglobulin, Southern Biotech). Known
amounts of blocking antibody captured in the same manner were used
to generate a standard curve. To measure the levels of monkey
antibody response against the anti-PD-1 antibody, plates were
coated with anti-PD-1 antibody (5 .mu.g ml.sup.-1), blocked and
incubated with different dilutions of serum to capture the
anti-blocking antibody. Bound antibody was detected using
anti-human .lamda.-chain-specific antibody conjugated to HRP
(Southern Biotech). This detection antibody does not bind to the
blocking antibody because only the constant regions of the heavy
and light chains were humanized and the constant region of light
chain is K. The amount of captured monkey immunoglobulin was
estimated using a standard curve that consisted of known amounts of
purified macaque immunoglobulin that had been captured using
anti-macaque immunoglobulin.
[0548] Quantification of SIV Copy Number:
[0549] SIV copy number was determined using a quantitative
real-time PCR as previously described (Amara et al., Science 292,
69-74 (2001)). All specimens were extracted and amplified in
duplicates, with the mean result reported.
[0550] Amplification and Sequencing of the Tat TL8 Epitope:
[0551] A 350-nucleotide fragment including Tat TL8 epitope was
amplified by limiting dilution RT-PCR. Viral RNA was extracted
using the QIAAMP.TM. Viral RNA mini kit (Qiagen) from plasma. vRNA
was reverse transcribed with the SIVmac239-specific primer Tat-RT3
(5'-TGGGGATAATTTTACACAAGGC-3') and Superscript III (Invitrogen)
using the manufacturer's protocol. The resultant cDNA was diluted
and copy number was determined empirically in our nested PCR
protocol. Limiting dilution, nested PCR was performed at .about.0.2
copies per reaction using the Expand HiFi PCR kit (Roche Applied
Sciences) with the following primers:
[0552] outer primers:
TABLE-US-00013 (SEQ ID NO: 53) Tat-F1
(5'-GATGAATGGGTAGTGGAGGTTCTGG-3') (SEQ ID NO: 54) Tat-R2
(5'-CCCAAGTATCCCTATTCTTGGTTGCAC-3')
54)
[0553] inner primers:
TABLE-US-00014 (SEQ ID NO: 55) Tat-F3 (5'-TGATCCTCGCTTGCTAACTG-3')
(SEQ ID NO: 56) Tat-R3 (5'-AGCAAGATGGCGATAAGCAG-3').
[0554] The first round reactions were cycled using the following
program: 94.degree. C. for 1 min, followed by 10 cycles of
94.degree. C. for 30 s, 55.degree. C. for 30 s, and 68.degree. C.
for 1 min, followed by 25 more cycles identical to the first ten
but for the addition of 5 s to the extension time at every cycle,
followed by a final extension at 68.degree. C. for 7 min. The
second round reactions were cycled using the following programme:
94.degree. C. for 1 min, followed by 35 cycles of 94.degree. C. for
30 s, 53.degree. C. for 30 s, and 68.degree. C. for 1 min, followed
by a final extension at 68.degree. C. for 7 min. After clean-up
with ExoSap-IT (USB Corporation), PCR products were sequenced
directly using the inner primers on an automated sequencer. Contigs
were assembled using Sequencher 4.8 (Gene Codes Corporation).
Amplicons containing nucleotides with double chromatogram peaks
were excluded.
[0555] Statistical Analyses:
[0556] Linear mixed effects models were used to determine
differences in blood chemistry and complete blood count values
between anti-PD-1-antibody-treated and control-antibody-treated
animals. The Bonferroni method was used to adjust P values for
multiple tests. A paired t-test was used for comparison of immune
responses before and after PD-1 blockade. Log-transformed data were
used when the data were not normal, but log-normal. A Wilcoxon
rank-sum test was used to compare the fold reductions in viral
loads between the groups. A Mantel-Haenszel log rank test was used
to compare the survival curves between the groups. Statistical
analyses were performed using S-PLUS 8.0. A two-sided P<0.05 was
considered statistically significant.
Example 27: Proliferation of Memory B Cells Induced by PD-1
Blockade
[0557] Chronic immunodeficiency virus infections are characterized
by dysfunctional cellular and humoral antiviral immune responses.
As such, immune modulatory therapies that enhance and/or restore
the function of virus-specific immunity may protect from disease
progression. The safety and immune restoration potential of
blockade of the co-inhibitory receptor programmed cell death 1
(PD-1) during chronic simian immunodeficiency virus (SIV) infection
was investigated in macaques. It was demonstrated that PD-1
blockade using an antibody to PD-1 is well tolerated and results in
rapid expansion of virus-specific CD8 T cells with improved
functional quality. This enhanced T-cell immunity was seen in the
blood and also in the gut, a major reservoir of SIV infection. PD-1
blockade also resulted in proliferation of memory B cells and
increases in SIV envelope-specific antibody. These improved immune
responses were associated with significant reductions in plasma
viral load and also prolonged the survival of SIV-infected
macaques. Blockade was effective during the early (week 10) as well
as late (.about.week 90) phases of chronic infection even under
conditions of severe lymphopenia. These results demonstrate
enhancement of both cellular and humoral immune responses during a
pathogenic immunodeficiency virus infection by blocking a single
inhibitory pathway and identify a novel therapeutic approach for
human immunodeficiency virus/acquired immunodeficiency syndrome,
and demonstrate that monitoring B cell response can be used to
assess the efficacy of therapy.
[0558] Virus-specific T cells show varying degrees of functional
impairment during chronic infections (Wherry et al., Immunity 27,
670-684 (2007); Klenerman et al., Nat Immunol 6, 873-879 (2005)).
Although these T cells retain some antiviral functions, they are
less polyfunctional compared with antiviral T cells seen in acute
infections. This defect in T-cell function greatly contributes to
the inability of the host to eliminate the persisting pathogen. It
is disclosed herein that the exhaustion of virus-specific T cells
is present during persistent LCMV infection of mice Zajac et al., J
Exp Med 188, 2205-2213 (1998); Galimore et al., J Exp Med 187,
1383-1393 (1998)) and in other viral infections, including human
immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis
C virus (HCV) infections in humans (Letvin et al., Nat Med 9,
861-866 (2003); Pantaleo et al., Nat Med 10, 806-810 (2004);
Rehermann et al., Nat Rev Immunol 5, 215-229 (2005)). The
co-inhibitory receptor PD-1 was highly expressed by the exhausted
virus-specific CD8 T cells (Barber et al., Nature 439, 682-687
(2006); Sharpe et al., Nat Immunol 8, 239-245 (2007)). PD-1 is also
upregulated on HIV-1-specific (Petrovas et al., J Exp Med 203,
2281-2292 (2006); Day et al., Nature 443, 350-354 (2006)) and
SIV-specific (Velu et al., J Virol 81, 5819-5828 (2007)). CD8 T
cells and in vitro blockade of PD-1 enhanced cytokine production
and proliferative capacity of these cells. An SIV/macaque model was
used to evaluate the effects of in vivo blockade of PD-1 on the
safety and restoration of virus-specific cellular and humoral
immunity during chronic immunodeficiency virus infections.
[0559] PD-1 blockade was performed using an antibody specific to
human PD-1 that blocks the interaction between macaque PD-1 and its
ligands (PDLs) in vitro (Velu et al., J Virol 81, 5819-5828 (2007).
Blockade was performed during the early (10 weeks) as well as late
(.about.90 weeks) phases of chronic SIV infection. Nine macaques
(five during the early phase and four during the late phase)
received the anti-PD-1 antibody and five macaques (three during the
early phase and two during the late phase) received an isotype
control antibody (Synagis, anti-Rous sarcoma virus (RSV)-specific)
(Malley et al., J Infect Dis 178, 1555-1561 (1998)).
[0560] PD-1 blockade during chronic SIV infection resulted in a
rapid expansion of SIV-specific CD8 T cells in the blood of all
macaques (FIG. 30a, b). The CD8 T-cell responses to two
immunodominant epitopes, Gag CM9 and Tat SL8/TL8 (Allen et al.,
Nature 407, 386-390. (2000)), was studied using major
histocompatibility complex (MHC) I tetrameric complexes in seven of
the anti-PD-1-antibody-treated and three of the
control-antibody-treated macaques that expressed the Mamu A*01
histocompatibility molecule. Most (>98%) of the Gag-CM9
tetramer-specific CD8 T cells expressed PD-1 before blockade. After
PD-1 blockade, the Gag-CM9 tetramer-specific CD8 T cells expanded
rapidly and peaked by 7-21 days. At the peak response, these levels
were about 2.5-11-fold higher than their respective levels on day 0
(P=0.007) and remained elevated until 28-45 days (FIG. 30b).
Similar results were observed with blockade during the early as
well as late phases of chronic SIV infection. A 3-4-fold increase
in the frequency of Gag-specific interferon (IFN)-.gamma.-positive
CD8 T cells was also observed by day 14 after blockade in the two
Mamu A*01-negative animals (RTd11 and RDb11), demonstrating that
PD-1 blockade can enhance the frequency of virus-specific CD8 T
cells that are restricted by non-Mamu A*01 alleles. As expected,
expansion of SIV-specific CD8 T cells was not observed in the
control-antibody-treated macaques (FIG. 30).
[0561] PD-1 blockade was also associated with a significant
increase in the frequency of virus-specific CD8 T cells that were
undergoing active cell division in vivo with improved functional
quality (FIG. 30b). Consistent with the rapid expansion of
SIV-specific CD8 T cells, the frequency of Gag-CM9
tetramer-specific CD8 cells that co-expressed Ki67 (marker for
proliferating cells) also increased as early as by day 7 after
blockade (P=0.01). Similarly, an increase in the frequencies of
Gag-CM9 tetramer-specific CD8 T cells co-expressing perforin and
granzyme B (cytolytic potential; P=0.001 and P=0.03, respectively),
CD28 (co-stimulation potential; P=0.001), CD127 (proliferative
potential; P=0.0003) (Kaech et al., Nat Immunol 4, 1191-1198
(2003)) and CCR7 (lymph-node homing potential; P=0.001) was
observed (Salusto et al, Nature 401, 708-712. (1999)). A transient
1.5-2-fold increase in the frequency of tetramer-negative and
Ki67-positive CD8 T cells after blockade was also observed. This
could be due to expansion of CD8 T cells specific to other epitopes
in Gag as well as other proteins of SIV, and other chronic viral
infections in these animals. No significant enhancement was
observed for these markers in the three control-antibody-treated
macaques.
[0562] Notably, no expansion was observed for Tat-TL8-specific CD8
T cells after blockade. This could be due to viral escape from
recognition by Tat-TL8-specific CD8 T cells, as PD-1 blockade is
known to result in expansion of T cells only when they
simultaneously receive signals through T-cell receptor. To test
this possibility, the viral genomes present in the plasma just
before the initiation of blockade from all three Mamu A*01-positive
macaques that were infected with SIV251 and received the blocking
antibody during the early phase of infection were sequenced.
Indeed, mutations in the viral genome were found corresponding to
the Tat TL8 epitope region. All these mutations either have been
shown or predicted to reduce the binding of Tat SL8/TL8 peptide to
Mamu A*01 MHC molecule and result in escape from recognition by the
Tat-SL8/TL8-specific CD8 T cells (Allen et al., Journal of
Immunology 160, 6062-6071 (1998); Allen et al., Nature 407,
386-390. (2000)). These results suggest that in vivo blockade of
PD-1 may not result in expansion of T cells that are specific to
escape mutants of viral epitopes.
[0563] PD-1 blockade also resulted in expansion of Gag-CM9-specific
CD8 T cells at the colorectal mucosal tissue (gut), a preferential
site of SIV/HIV replication (Pierson et al., Annu Rev Immunol 18,
665-708 (2000)) (FIG. 30c). Expansion was not observed for two of
the seven macaques, although expansion was evident for one of them
in blood. In contrast to blood, the expansion in gut peaked much
later by day 42 and ranged from 2- to 3-fold compared with their
respective day 0 levels (P=0.003). Similar to blood, the Gag-CM9
tetramer-specific cells that co-expressed Ki67 (P=0.01), perforin
(P=0.03), granzyme B (P=0.01) and CD28 (P=0.01) also increased in
the gut after blockade.
[0564] PD-1 blockade also enhanced the functional quality of
anti-viral CD8 T cells and resulted in the generation of
polyfunctional cells capable of co-producing the cytokines
IFN-.gamma., tumour-necrosis factor (TNF)-a and interleukin (IL)-2
(FIG. 31). On the day of initiation of PD-1 blockade during the
late chronic phase of infection, the frequency of Gag-specific
IFN-.gamma.-positive cells was low and they failed to co-express
TNF-.alpha. and IL-2 (FIG. 31a). However, after the blockade, the
frequency of IFN-.gamma.-positive cells increased in all four PD-1
antibody-treated macaques (P=0.03) and they acquired the ability to
co-express TNF-.alpha. and IL-2. The expansion of
IFN-.gamma.-positive cells peaked by 14-21 days and the peak levels
were 2-10-fold higher than the respective day 0 levels. On day 21,
about 16% of the total Gag-specific cells co-expressed all three
cytokines, and about 30% co-expressed IFN-.gamma. and TNF-.alpha.
(FIG. 31b). This is in contrast to <1% of the total Gag-specific
cells co-expressing all three cytokines (P=0.01), and about 14%
co-expressing IFN-.gamma. and TNF-.alpha. on day 0 (P=0.04).
Similar results were also observed after blockade during the early
chronic phase of infection.
[0565] Chronic immunodeficiency virus infections are associated
with B-cell dysfunction (De Milito, Current HIV Research 2, 11-21
(2004); Moir and Faucci, J Allergy Clin Immunol 122, 12-19; quiz
20-11 (2008)) but very little is known about the role of PD-1 in
regulating B-cell function/exhaustion. The B-cell responses after
PD-1 blockade in SIV-infected macaques (FIG. 32) was characterized.
Analysis of PD-1 expression on different B-cell subsets before PD-1
blockade revealed preferential expression of PD-1 by memory B cells
(CD20.sup.+CD27.sup.+CD21.sup.-) compared to naive B cells
(CD20.sup.+CD27.sup.-CD21+; FIG. 32a, P<0.001). In vivo blockade
of PD-1 resulted in a 2-8-fold increase in the titre of
SIV-specific binding antibody by day 28 after blockade (P<0.001;
FIG. 32b).
[0566] The proliferation of memory B cells was studied in
SIV-infected macaques that were treated simultaneously with
anti-PD-1 antibody and anti-retroviral therapy and observed a
significant increase in Ki67.sup.+ (proliferating) memory, but not
naive, B cells as early as day 3 (FIG. 32c). These results
demonstrate the PD-1-PDL pathway's role in regulating B-cell
dysfunction during chronic SIV infection. Neutralization assays
revealed a twofold increase in titres against the easily
neutralizable laboratory-adapted SIV251 and no increase in titres
against hard-to-neutralize wild-type SIV251 or SIV239. In two of
the nine animals treated with anti-PD-1 antibody, only a minimal
(<2-fold) expansion of SIV-specific antibody was observed after
blockade. Notably, the frequency of total memory B cells in these
two animals was lower (.about.40% of total B cells) compared with
the remaining seven animals (60-90% of total B cells) before
blockade, indicating that the level of SIV-specific memory B cells
before blockade can determine the level of expansion of
SIV-specific antibody after blockade.
[0567] PD-1 blockade resulted in significant reductions in plasma
viraemia (P=0.03) and also prolonged the survival of SIV-infected
macaques (P=0.001; FIG. 33). In two of the five macaques treated
with anti-PD-1 antibody during the early chronic phase, viral load
declined by day 10 and persisted at or below this level until day
90 (FIG. 33a). In one macaque viral load declined transiently and
in the remaining two macaques increased transiently and returned to
pre-blockade levels. In contrast to the early chronic phase, all
four macaques treated with the anti-PD-1 antibody during the late
chronic phase showed a transient increase in viraemia by day 7, but
rapidly reduced the virus load by day 21 to levels that were below
their respective day 0 levels (FIG. 33b). However, the viral RNA
levels returned to pre-blockade levels by day 43. As expected, no
significant reductions in the plasma viral loads were observed in
any of the five macaques treated with the control antibody (FIG.
33c). By 21-28 days after blockade, the viral RNA levels in the
anti-PD-1-antibody-treated animals were 2-10-fold lower than their
respective day 0 levels (P=0.03; FIG. 33d). By day 150 after the
blockade, four of the five macaques in the control group were
killed owing to AIDS-related symptoms (for example loss of
appetite, diarrhoea, weight loss), whereas all nine animals in the
anti-PD-1-antibody-treated group had survived (P=0.001; FIG.
33e).
[0568] The observed initial rise in plasma viraemia levels in all
of the late-phase-treated and some of the early-phase-treated
animals could be due to an increase in the frequency of activated
CD4 T cells. The percentage of Ki67-positive total CD4 T cells was
measured, as well as the frequency of SIV Gag-specific
IFN-.gamma.-producing CD4 T cells (preferential targets for virus
replication (Douek et al., Nature 417, 95-98 (2002)) after
blockade. These analyses revealed a transient increase in the
percentage of Ki67-positive CD4 T cells by day 7-14 after blockade
(P=0.002) and this increase was higher in animals treated during
the late phase than early phase of infection (P=0.015). Similarly,
an increase in the frequency of Gag-specific CD4 T cells was also
observed, but only in animals treated during the late phase of
infection. No significant increases were observed for these
activated CD4 T cells in the control-antibody-treated macaques.
These results suggest that the activated CD4 T cells could have
contributed to the observed initial rise in plasma viraemia levels
after blockade.
[0569] Before initiation of PD-1 blockade, the set point viral load
in plasma and total CD4 T cells in blood and gut were similar
between the anti-PD-1-antibody-treated and control-antibody-treated
groups. However, the frequencies of Gag CM9.sup.+ cells and Gag
CM9.sup.+ cells co-expressing perforin, granzyme B or CD28 were not
similar between the two treatment groups before in vivo blockade
(FIG. 30b). This raises the possibility that these differences
could have contributed to the expansion of Gag CM9.sup.+ cells
after PD-1 blockade. To study the influence of the frequency of Gag
CM9.sup.+ cells before blockade on their expansion after blockade,
the anti-PD-1-antibody-treated group were divided into two
subgroups based on the frequency of Gag CM9.sup.+ cells before
initiation of blockade such that one group has similar levels and
the other group has higher levels of Gag CM9.sup.+ cells compared
with the control-antibody-treated group. These subgroups were then
analysed for expansion of CM9.sup.+ cells after blockade. Expansion
of CM9.sup.+ cells was evident in both subgroups of animals after
blockade of PD-1, irrespective of whether they were at low or high
levels before blockade. Similar results were also observed with
subgroup analyses based on the frequency of CM9.sup.+ cells
co-expressing molecules associated with better T-cell function such
as perforin, granzyme B, CCR7, CD127 or CD28. However, there was a
trend towards better expansion of CM9+CD28.sup.+ cells in animals
with higher levels of CM9+CD28.sup.+ cells before blockade,
suggesting that CD28 expression serves as a biomarker for
predicting the outcome of in vivo PD-1 blockade.
[0570] To evaluate the safety of PD-1 blockade, an extensive
analysis of serum proteins, ions, lipids, liver and kidney enzymes,
and complete blood count after blockade. These analyses revealed no
significant changes for all parameters tested between the
anti-PD-1-antibody-treated and control-antibody-treated macaques.
Similarly, the levels of anti-nuclear antibodies (ANA) in serum
(measure of autoimmunity) also did not change significantly after
treatment with anti-PD-1 antibody.
TABLE-US-00015 TABLE 6 Biochemical parameters of blood after the
anti-PD-1 antibody treatment Post SIV infection, Pre Days after
PD-1 blockade* Markers infection* (n = 5) Biochemical profile (n =
8) Day 0 Day 14 Day 56 ALT (U/L) 16.8 .+-. 5.0 27 .+-. 11.6 24.8
.+-. 7.7 27 .+-. 7.9 AST (U/L) 33.1 .+-. 8.2 35.0 .+-. 8.3 29.8
.+-. 3.6 49.0 .+-. 18.5 Alkaline Phosphatase 466 .+-. 135 410 .+-.
367 367 .+-. 78 451 .+-. 89 (U/L) Bilirubin (g/L) 0.2 .+-. 0.1 0.16
.+-. 0.1 0.12 .+-. 0.0 0.2 .+-. 0.2 Creatinine (mg/dL) 0.9 .+-. 0.1
0.7 .+-. 0.1 0.66 .+-. 0.1 0.6 .+-. 0.1 Total protein (g/dL) 7.3
.+-. 0.3 7.0 .+-. 0.3 7.24 .+-. 0.4 6.9 .+-. 0.4 Albumin g/L 4.5
.+-. 0.3 4.28 .+-. 0.2 4.1 .+-. 0.2 4.1 .+-. 0.2 Globulin (g/dL)
2.7 .+-. 0.2 2.72 .+-. 0.3 3.14 .+-. 0.3 2.8 .+-. 0.2
Albumin/Globulin (ratio) 1.7 .+-. 0.2 1.62 .+-. 0.2 1.32 .+-. 0.1
1.4 .+-. 0.1 Glucose (mg/dL) 82 .+-. 16 69 .+-. 8 66 .+-. 9 64 .+-.
8.0 Cholesterol (mg/dL) 161 .+-. 32 149 .+-. 32 145 .+-. 20 140
.+-. 18 Triglyceriods (mg/dL) 58 .+-. 19 64 .+-. 12 60 .+-. 7 73
.+-. 34 Blood Urea Nitrogen 18 .+-. 3 17 .+-. 3 17 .+-. 3 16 .+-. 3
(mg/dL) Blood urea nitrogen- 21 .+-. 3 24 .+-. 5 26 .+-. 5 26 .+-.
5 creatinine (ratio) Lipase (U/L) 21 .+-. 17 20 .+-. 7 21 .+-. 9 23
.+-. 10 Creatinine Phosphokinase 428 .+-. 272 537 .+-. 303 486 .+-.
129 462 .+-. 312** (U/L) Gamma glutamil 74 .+-. 23 66 .+-. 16 58
.+-. 18 71 .+-. 15 transpeptidase (U/L) Calcium (mg/dl) 10 .+-. 0.5
10 .+-. 0.2 10 .+-. 0.5 10 .+-. 0.3 Chloride (mEq/L) 110 .+-. 3 107
.+-. 3 108 .+-. 1 107 .+-. 2 Potassium (mEq/L) 4 .+-. 0.3 4 .+-.
0.2 4 .+-. 0.1 4 .+-. 0.6 Sodium (mEq/L) 150 .+-. 5 149 .+-. 3 149
.+-. 1 147 .+-. 2 Phosphorus (mg/dL) 5 .+-. 0.9 5 .+-. 0.6 5 .+-.
0.8 6 .+-. 0.4 *Values represent mean .+-. standard deviation **Day
91 values were used because of RBC lysis on day 56
TABLE-US-00016 TABLE 7 Complete blood count after the anti-PD-1
antibody treatment Post SIV infection, Days after PD-1 Pre
blockade* infection* (n = 5) Cell type (n = 8) Day 0 Day 14 Day 56
Red blood cells 5.7 .+-. 0.3 5.9 .+-. 0.3 5.4 .+-. 0.4 5.9 .+-. 0.3
(Millions/mm.sup.3) Hematocrit (%) 41 .+-. 1 42 .+-. 1 38 .+-. 2 41
.+-. 2 White blood cells (per .mu.L) 8500 .+-. 2171 9260 .+-. 3685
7500 .+-. 2068 7800 .+-. 1972 Neutrophils (counts/.mu.L) 3685 .+-.
1883 3274 .+-. 2124 2573 .+-. 865 2028 .+-. 1585 Lymphocytes
(counts/.mu.L) 4477 .+-. 1583 4700 .+-. 1791 4235 .+-. 1880 5041
.+-. 1705 Monocytes (counts/.mu.L) 166 .+-. 116 635 .+-. 374 336
.+-. 123 350 .+-. 206 Eosinophils (counts/.mu.L) 161 .+-. 155 591
.+-. 580 277 .+-. 275 342 .+-. 175 Basophils (counts/.mu.L) 10 .+-.
29 29 .+-. 65 78 .+-. 78 37 .+-. 53 Platelets (counts/.mu.L) 341
.+-. 64 275 .+-. 45 364 .+-. 79 241 .+-. 74 *Values represent mean
.+-. standard deviation
[0571] In one macaque, the levels of ANA increased about 3-fold by
day 10 after blockade, but returned to day 0 levels by day 56.
These results demonstrate that anti-PD-1 antibody treatment during
chronic SIV infection results in no observable toxicity. This is
consistent with a recent study that demonstrated the safety of PD-1
blockade in patients with advanced haematological malignancies
(Berger et al., Clin Cancer Res 14, 3044-3051 (2008)).
[0572] The pharmacokinetics of the partially humanized anti-PD-1
antibody in serum after in vivo blockade was studied. The titre of
anti-PD-1 antibody rapidly declined between days 14 and 28 after
blockade and coincided with macaques generating antibody response
against the mouse immunoglobulin variable domains of anti-PD-1
antibody. Hence completely humanized anti-PD-1 antibody may allow
longer periods of treatment that may further enhance the efficacy
of in vivo blockade.
[0573] The results demonstrate that in vivo blockade of PD-1 during
chronic SIV infection is safe and results in rapid expansion and
restoration of SIV-specific polyfunctional CD8 T cells and enhanced
B-cell responses. Expansion was observed with blockade performed
during the early as well as late phases of chronic infection even
under conditions of high levels of persisting viraemia and AIDS.
Expansion was also observed at the colorectal mucosal tissue, a
preferential site of SIV/HIV replication (Pierson et al., Annu Rev
Immunol 18, 665-708 (2000)). Importantly, PD-1 blockade resulted in
a significant reduction of plasma viral load and also prolonged the
survival of SIV-infected macaques. These results are highly
significant considering the failure of blockade of a related
co-inhibitory molecule CTLA-4 to expand virus-specific CD8 T cells
and to reduce plasma viral load in SIV-infected macaques
(Cecchinato et al. J Immunol 180, 5439-5447 (2008)). The
therapeutic benefits of PD-1 blockade could be improved further by
using combination therapy with anti-retrovirals and/or therapeutic
vaccination.
Example 28: Materials and Methods for Example 29
[0574] Animals, SIV Inoculation and Infection Stages:
[0575] Indian rhesus monkeys (Macaca mulatta) and sooty mangabeys
were utilized. SIV infection was performed by intravenous
inoculation, and the animals were grouped by stage of infection
into: -acute (2 weeks post infection, p.i.), early chronic (10-12
weeks p.i) and late chronic (.gtoreq.1.5 years p.i.).
[0576] Viral Load Measurements:
[0577] Plasma viral load was determined by quantitative real-time
PCR as previously described (Amara et al., Science 292:69-74,
2001). All viral RNA specimens were extracted and assayed in
duplicate, with mean results reported and used in the analyses.
[0578] Phenotypic Analysis by Flow Cytometry:
[0579] Surface lymphocyte stainings were performed using 100 .mu.l
whole blood samples using multi-parameter, multi-color analysis.
Lymphocytes were obtained from necropsy tissue. The following
antibodies were used: mouse anti-human antibodies against CD3
(clone SP34-2), CD21 (clone B-Ly4), CD27 (clone M-T2712), CD80
(clone L307.4), CD11c (clone S-HCL-3), all from BD
BIODSCIENCES.RTM.; CD20 (clone 2H7, eBIOSCIENCES.RTM.), CD40 (clone
MAB89, BECKMAN COULTER.RTM.), CD95 (clone DX2, CALTAG.RTM.) and
PD-1 (clone EH-12). Cells were analyzed on a LSRII flow cytometer
and data analyzed with FLOWJO.RTM. software version 8.8.2.
[0580] Concanavalin A ELISA to Measure SIV Env-Specific Antibody
Titers and Avidity:
[0581] Titers of anti-env IgG Ab were measured using envelope
proteins produced in transient transfections of 293T cells with
DNA/89.6 VLP (51). Briefly, 96-well ELISA plates (Costar, Corning
Life Sciences) were coated with 25 .mu.g/ml concanavalin A (Con A)
in 10 mM Hepes buffer and incubated overnight at 4.degree. C.
Plates were washed six times with PBS containing 0.05% Tween-20
(PBS-T), 100 .mu.l of VLP added to each well followed by 1 hour
incubation at room temperature, another wash and blocking for 1
hour at room temperature with 100 .mu.l blocking buffer (PBS-T with
4% whey and 5% dry milk) per well. Plates were washed and test sera
serially diluted in PBS-T/4% whey added to duplicate wells and
incubated for 1 hour at room temperature. For ELISA assays, the
plates were washed 6 times with PBS-T, and bound Ab detected using
horseradish peroxidase-conjugated anti-monkey IgG (Rockland
Immunochemicals) and tetramethyl benzene (TMB) substrate (KPL), and
reactions stopped with 100 .mu.l of 2N H.sub.2SO.sub.4. Each plate
included a standard curve generated using goat anti-monkey IgG
(Rockland Immunochemicals) and rhesus IgG (Accurate chemicals).
Standard curves were fitted and sample concentrations interpolated
as .mu.g of Ab per ml of serum using SOFTMAX.RTM. 2.3 software.
[0582] Avidity of Ab to viral envelope proteins was determined by
measuring resistance of antibody-envelope complexes to elution by
the chaotropic agent NaSCN in a modification of the env Ab ELISA.
Test sera were added to the plates in quadruplicates, in 3-fold
dilutions starting from 1:100. Following binding of test sera in
the ConA env eLISA, one set of duplicates was treated with PBS and
the other set with 1.5M NaSCN for 10 minutes before washing and
detection with horseradish peroxidase-conjugated anti-monkey IgG
and TMB substrate. Reactions were stopped with 100 .mu.l of 2N
H.sub.2SO.sub.4. The avidity index was calculated by dividing the
dilution of the serum that gave an O.D. of 0.5 with NaSCN treatment
by the dilution of serum that gave and O.D. of 0.5 with PBS,
multiplied by 100.
[0583] Neutralization Assay:
[0584] Neutralization was measured as a function of a reduction in
luciferase (luc) reporter gene expression after single rounds of
infection in 5.25.EGFP.Luc.M7 cells (TCLA SIVmac25) and TZM-b1
cells (293T pseudovirus) as previously reported (51, 52). Values
reported represent the serum dilution at which relative
luminescence units (RLUs) were reduced 50% compared to virus
control wells.
[0585] Apoptosis Assays:
[0586] PBMC form 7 SIV-infected macaques were plated in 96-well
round-bottomed tissue culture plates at 2.5.times.10.sup.5
cells/well under four different culture conditions: complete
RPMI-1640 medium only (spontaneous apoptosis), complete RPMI-1640
medium+10 ng/ml soluble His-tagged rhFasL (R&D Systems)
(Fas-mediated apoptosis) & complete RPMI-1640 medium+10 ng/ml
soluble His-tagged rhFasL+10 .mu.g/ml anti-PD-1 blocking Ab. Plates
were incubated for 24 h at 37.degree. C. after which the cells were
stained for CD20, CD27, CD21 and Annexin-V and immediately analyzed
on an LSRII flow cytometer.
[0587] Huh-7.5 cells (53) were transfected with a plasmid
expressing HLA-A2 under the CMV promoter with a Neomycin resistance
gene. Clones were selected and propagated, and then subsequently
transfected with a second plasmid (pCDNA3.1-Zeo) expressing the
full-length INCYTE.RTM. human cDNA PD-L1 (OPEN BIOSYSTEMS.RTM.,
Huntsville, Ala.). A second round of selection and propagation of
clones resistant to both Neomycin and Zeocin was performed.
Verification of expression of HLA-A2 and PD-L1 was performed by
flow cytometry. The Huh-7.5.A2.PD-L1 cells were used to assess
PD-L1-mediated apoptosis of activated memory B cells, with Huh-7.5
cells as control. Both cell lines were seeded onto separate 24-well
plates and incubated at 37.degree. C. a day before the experiment.
B cells were isolated from PBMC using NHP-specific CD20 microbeads
(Miltenyi Biotec) and isolated B cells were added to the cell lines
and plates incubated for 24 h at 37.degree. C. after which the
cells were stained for CD20, CD27, CD21 and Annexin-V and
immediately analyzed on an LSRII flow cytometer.
[0588] In Vitro PBMC Stimulation and Memory B Cell ELISpot
Assays:
[0589] PBMC were stimulated and used in memory B cell ELISpot
assays using modifications of the method described by Crotty et al
(23). Briefly, PBMC were plated in sterile 24-well tissue culture
plates (Costar) at 0.5.times.10.sup.6 cells/well in complete
RPMI-1640 medium containing 3-2 mercaptoethanol under 3 different
culture conditions-medium only (control); mitogen cocktail-pokeweed
mitogen diluted 1:1000, fixed Staphylococcus aureus Cowan strain,
SAC (SIGMA.RTM.) diluted 1:10,000 and 6 .mu.g/ml CpG ODN-2006
(Qiagen-Operon); mitogen cocktail+10 .mu.g/ml anti-PD-1 blocking Ab
(clone 1540-29C9, provided by GF) in triplicates. Cells were
cultured at 37.degree. C. with 5% CO.sub.2 for 6 days.
[0590] On Day 5 of culture, 96-well filter ELISpot plates were
coated with affinity-purified goat anti-monkey IgM and IgG
(Rockland Immunochemicals) at 10 .mu.g/ml, and SIVmac239 gp130 at 1
.mu.g/ml, and incubated overnight at 4.degree. C.
[0591] On Day 6, plates were washed once with PBS-T and three times
with PBS and blocked with RPMI-1640 for 2 h at 37.degree. C.
Cultured PBMC were washed twice, added to the prepared ELISpot
plates and incubated at 37.degree. C. for 6 hours. Plates were then
washed 3.times. with PBS and 3.times. with PBS-T and incubated
overnight at 4.degree. C. with 1 g/ml biotin-conjugated anti-monkey
IgM (for detection of total IgM ASC) or 1 g/ml anti-monkey IgG (for
detection of total IgG and anti-gp130 ASC) diluted in PBS-T/1% FCS.
Plates were then washed 4.times. with PBS-T and incubated at for 1
hour at room temperature with 5 .mu.g/ml HRP-conjugated Avidin D
(Vector laboratories) diluted in PBS-T/1% FCS. Plates were washed
4.times. with and developed using 3-Amino-9-Ethylcarbazole (AEC).
Spots on developed plates were counted using an ELISpot plate
reader. Data are represented as number of spots (ASC) per 10.sup.6
PBMC.
[0592] Statistical Analyses:
[0593] Statistical analyses were performed using GRAPHPAD
PRISM.RTM..
Example 29: Memory B Cells and PD-1 in Progression of a Chronic
Infection
[0594] Four Distinct B Cell Subsets can be Identified in Rhesus
Macaque Peripheral Blood:
[0595] The rhesus macaque B cell compartment was characterized.
Four distinct B cell subsets in peripheral blood of healthy RM:
CD20.sup.int/CD21.sup.+/CD27.sup.- (naive),
CD20.sup.int/CD21.sup.+/CD27.sup.+ (resting memory),
CD20.sup.hi/CD21.sup.-/CD27.sup.+ (activated memory) and
CD20.sup.hi/CD21.sup.-/CD27.sup.- (unconventional or tissue
memory), all with significantly different mean fluorescence
intensity (MFI) of CD20 (P<0.0001). Naive and activated memory B
cells were the majority subsets, making up 37% and 36% of total B
cells respectively, followed by tissue (18%) and resting (9%)
memory B cells. Cells were stained for surface IgM and IgD and it
was found that unlike in humans there were virtually no IgM-only
cells. The naive B cells were evenly split between IgD-only and
IgD.sup.+IgM.sup.+. All three memory subsets were made up of
.about.20% IgD-only cells; the remaining resting memory B cells
were IgD.sup.+IgM.sup.+ (.about.50%) and IgD.sup.-IgM.sup.-
(.about.30%). The activated memory B cells were the most
class-switched subset, with .about.60% of them IgD.sup.-IgM.sup.-
and .about.20% IgD.sup.+IgM.sup.+. The tissue-like memory B cells
on the other hand were mostly IgD.sup.+IgM.sup.+ (.about.70%) with
only .about.10% IgD.sup.-IgM.sup.-). Thus, a novel B cell subset
was identified for rhesus macaques (RM), which unlike the activated
memory B cell subset lacked CD27 expression, but was also
CD21.sup.-. These B cells could be similar to the unique
tissue-like memory B cell subset of cells, whose defining surface
marker is the immuno-regulatory molecule FCRL4 in humans.
[0596] To further characterize the subsets, the expression of the
activation and differentiation markers CD40, CD80, CD95 and CD11c
was assessed. Virtually all naive and resting memory B cells and
>70% of resting memory B cells were CD40.sup.hi, while the
majority (>70%) of activated memory B cells were CD40nt.
Activated memory B cells expressed the most CD80, CD95 and CD11c,
closely followed by resting memory B cells. CD11c was only
expressed on activated and unconventional memory B cells, with
naive B cells expressing negligible amounts of CD80, CD95 and
CD11c.
[0597] SIV Infection Leads to Depletion of Activated Memory B
Cells:
[0598] The intravenous route of SIV infection, which we used in
this study, has been associated with a more rapid course of disease
progression in non-human primates, with up to 30% of animals
inoculated via this route progressing to AIDS within six months of
infection. Animals that developed AIDS-like symptoms or full-blown
AIDS and died by week 24 of infection were classified as rapid
progressors and all the other animals were classified as typical
progressors. One of the first observable changes occurring in the B
cell compartment following HIV and SIV infections is a marked
decrease in numbers of total B cells but it is not clear which
specific B cell subsets are deleted. It was found that as early as
two weeks following SIV infection, peripheral blood total B cells
were severely depleted, regardless of rate of disease progression.
A rebound in numbers of B cells occurred by the twelfth week of
infection in both rapid and typical progressors, but the B cell
numbers remained significantly different from pre-infection levels
(P<0.0001). The memory B cells in general were depleted
following SIV infection with a significant decrease in percentage
and numbers of activated memory B cells. By 12 weeks post
infection, the rapid progressors had lost 82% of their activated
memory B cells, while the typical progressors had lost only 23%. In
contrast to the rapid progressors, the activated memory B cell
proportions returned to pre-infection levels by week 12 of
infection in the typical progressors. This striking contrast in
degree of activated memory depletion between rapid and typical
progressors prompted the investigation of whether the depletion of
activated memory B cells has any significance for disease
progression and SIV pathogenesis.
[0599] Depletion of Activated Memory B Cells is an Early Predictor
of Rapid Disease Progression:
[0600] Set-point viral load (12 weeks post infection) was shown to
be a good predictor of clinical outcome of SIV infection. The
association between rapid disease progression and viral load and
interestingly was analyzed, both rapid and typical progressors had
similar peak (week 2 post infection) viral loads (P=0.8); set-point
viral load in the rapid progressors was however a log greater than
in the typical progressors (P<0.0001). Given that differences
were observed in activated memory B cell proportions as early as 2
weeks post infection, it was hypothesized that depletion of
activated memory B cells could be a much earlier predictor of rapid
disease progression. Blood central memory (CD28.sup.+CD95.sup.+,
T.sub.CM) and gut CD4.sup.+ T cells have also been suggested as
markers of disease progression in SIV infection so comparisons were
preformed of all these markers to evaluate the predictive value of
each one. Two weeks post SIV infection, the rapid progressors had
significantly lower proportions of activated memory B cells
compared to the typical progressors and activated memory B cells
were the only cell subset whose distribution was significantly
different (P<0.001) between rapid and typical progressors. By 12
weeks post infection, the activated memory B cells were even
further depleted in the rapid progressors (P<0.0001), and
significant differences between rapid and typical progressors also
emerged with respect to proportions of T.sub.CM and gut CD4.sup.+ T
cells (P<0.01)(FIG. 3B, bottom panel). To further confirm the
usefulness of 2-week depletion of activated memory B cells as an
early marker of disease progression, correlation analyses were
performed of the set point (week 12 post SIV infection) viral load
versus week 2 and week 12 percentages of activated memory B cells,
gut CD4.sup.+ T cells and T.sub.CM cells. Whereas both week 2 and
week 12 activated memory B cells were inversely correlated with
set-point viral loads, only week 12 gut CD4.sup.+ percentages
correlated with set-point viremia, and T.sub.CM showed no
correlation with set-point viremia at all. The loss of activated
memory B cells is therefore an early predictor of rapid disease
progression in rhesus macaques (RM), with better early predictive
value than peak viral load, T.sub.CM and gut CD4.sup.+ T cells.
[0601] Depletion of Activated Memory B Cells in Rapidly Progressing
SIV Infection Impairs SIV-Specific Humoral Immune Response and
Resistance to Other Non-SIV Infections:
[0602] RM with rapidly progressing SIV infection were shown to have
low antibody responses as a consequence of the acute destruction of
the B cell compartment. Opportunistic infections and non-SIV
related Ags are a significant cause of mortality in SIV-infected
animals. The loss of activated memory B cells could have important
consequences for the humoral immune response of rapidly progressing
animals to SIV and non-SIV Ags. Thus, the serum titers of SIV
env-binding Abs were measured in both rapid and typical
progressors; it was found that of the 9 rapid progressors assayed,
only 2 mounted a modest env Ab response by week 12 and only 1 of
the animals had sustained Ab titers by week 20. The remaining 7
rapid progressors had undetectable SIV env Ab titers through week
20 of infection. The typical progressors on the other hand
developed strong env Ab responses by 12 weeks post infection, with
even higher titers by week 20.
[0603] Bacterial opportunistic infections are a significant cause
of morbidity in SIV-infected animals and the causative agents of
these infections are usually flagellated. Serum Ab titers to
flagellin (FliC isolated from Salmonella typhimurium) were measured
as a means to assess the effect of loss of activated memory B cells
on pre-existing humoral immunity. Despite starting off with
comparable anti-FliC Ab titers, the rapid progressors had
significantly lower (P=0.001) titers by week 20 post infection
compared to the typical progressors in which titers were unchanged
(P=0.9) (FIG. 5C). Clinical infection data was analyzed for both
groups over a 6-month period following initial SIV infection and it
was found that a wide variety of other infections occurred in the
animals following SIV infection. These included bacterial
(Campylobacter, Shigella, enteropathogenic E. coli), parasitic
(Trichomonas, whip worms, Giardia) and yeast (Candida) infections.
Rapid progressors succumbed to these infections as early as 1 month
p.i. and by 3 months p.i.>50% of the rapid progressors were
infected compared to <10% of the typical progressors. This rate
of infection in rapid progressors was sustained throughout the
6-month period.
[0604] In Vitro PD-1 Blockade Decreases Fas-Mediated Apoptosis, and
Ligation of PD-1 Induces Apoptosis of Activated Memory B Cells:
[0605] PD-1 is mainly expressed on memory B cells of rhesus
macaques. Expression of PD-1 was assessed on all B cell subsets in
more detail before and after SIV infection, and it was found that a
higher proportion of all 3 memory B cell subsets expressed higher
amounts (mean fluorescence intensity, MFI) of PD-1 compared to
naive B cells (P<0.001). The activated memory cells not only
expressed the highest amounts of PD-1, but also had the highest
proportion of PD-1.sup.+ cells compared to the other subsets
(P<0.001). Following SIV infection, irrespective of disease
progression status, there was a preferential depletion of
PD-1.sup.+ memory B cells. This raised the possibility that PD-1
may play a role in depletion of activated memory B cells.
[0606] Memory B cells in HIV-infected humans are primed to undergo
both spontaneous and death receptor-induced apoptosis notably
through the Fas-FasL pathway, but there is little information on
what role the Fas-FasL pathway plays in B cell apoptosis during SIV
infection. In order to determine susceptibility of activated memory
B cells to Fas-mediated apoptosis and to identify a possible role
for PD-1 in activated memory B cell depletion, PBMC from 7
SIV-infected animals were cultured with and without sFasL in
combination with PD-1 blockade and analyzed Annexin-V expression on
activated memory B cells was assessed after 24 hours of culture. In
all 6 animals a significant increase in apoptosis was seen with the
addition of sFasL to the cultures, and in 4 animals a decrease in
FasL-mediated apoptosis was observed following PD-1 blockade,
indicating that PD-1 could contribute to apoptosis of activated
memory B cells.
[0607] To further demonstrate the role of PD-1 on apoptosis of
activated memory B cells, the human hepatoma cell line, Huh-7.5
transfected with PD-L1 (Huh-7.5.A2.PD-L1) was used as a source of
ligand for the PD-1-expressing activated memory B cells. Expression
of PD-L1 as verified by flow cytometry showed no PD-L1 expression
in the non-transfected Huh-7.5 cells (control) compared to >90%
PD-L1 expression on the Huh-7.5.A2.PD-L1 cells. There was an
increased rate of apoptosis in the activated memory B cells
cultured in the presence of PD-L1 compared to the control wells, in
5 out of 7 animals tested. In one animal (4) a similar rate of
apoptosis was observed with or without PD-L1, and in the other
animal (3), the rate of spontaneous apoptosis was >30% and
addition of PD-L1 did not significantly alter the apoptosis. Thus
PD-1 signaling during SIV infection plays a role in activated
memory B cell apoptosis.
[0608] Blocking PD-1-PD-L1 interaction was shown to increase the
capacity of HIV-specific CD8.sup.+ T cells to proliferate and
survive. Thus, the effect of in vitro PD-1 blockade on spontaneous
and Fas-mediated activated memory B cell apoptosis. The effect of
in vitro blockade was assessed on the ability of memory B cells
from SIV-infected animals to survive, proliferate in response to
polyclonal stimulation, and differentiate into antibody-secreting
cells (ASC) in a memory B cell ELISPot assay. Blockade resulted in
slightly decreased Fas-mediated apoptosis of activated memory B
cells, but did not have an effect on spontaneous apoptosis. Cells
stimulated in the presence of PD-1 blocking Ab proliferated better
and produced higher numbers of ASC against total IgM and IgG, but
also env-specific spots.
[0609] RM Activated Memory B Cells have Lower Expression of BAFF-R,
which is Decreased Further by SIV Infection:
[0610] B cell activating factor belonging to the TNF family, BAFF
(also known as B-lys) is an important regulator of B cell
homeostasis (21), and CD21.sup.- B cells in cynomolgus macaques
were shown to express lower expression of one of its receptors,
BAFF-R. CD21.sup.low B cells of HIV viremic patients were also
shown to express lower levels of BAFF-R. It was found that
activated and tissue memory B cells expressed the lowest levels of
BAFF-R compared to naive and resting memory B cells. Expression was
further decreased 2 weeks post infection but interestingly was
restored by week 12. Thus low expression of BAFF-R may be a
contributing factor in the depletion of activated memory B
cells.
[0611] In Vitro PD-1 Blockade Increases Memory B Cell Proliferation
and Antibody Production:
[0612] It was investigated whether the presence of PD-1 on memory B
cells would affect their ability to proliferate and differentiate
into antibody secreting cells (ASC). An in vitro elispot assay was
designed to track IgM, IgG and SIV gp130-producing memory B cells
based on assays that have been described. Following polyclonal
stimulation, there was a significant increase in IgM (P<0.05)
and IgG (P<0.01) ASC in both early (12 weeks, n=3) and late
chronic (>1 year, n=2) infection. Cells stimulated in the
presence of .alpha.-PD-1 blocking Ab generally proliferated better
and produced a higher number of spots than cells stimulated without
blocking Ab. gp130-specific ASC were however detectable only in the
late chronic monkeys and as with the IgM and IgG ASCs, polyclonal
stimulation resulted in a significantly higher number of gp130
specific ASCs, and blockade of PD-1 further increased the numbers
of ASCs.
[0613] In Vivo PD-1 Blockade Results in Increased SIV Env Binding
Antibody Titers with Higher Avidity, and Increased Neutralizing Ab
Titers:
[0614] In vivo blockade of PD-1 in rhesus macaques with chronic SIV
infection resulted in increased titers of SIV env binding Abs. The
avidity of env Abs following in vivo PD-1 blockade was measured. It
was found that not only were the titers of the env Abs increased,
but the avidity of the binding Abs were also increased in the
treated animals. This was not the case in the control Ab-treated
animal in which avidity was decreased following treatment.
[0615] Neutralizing activity was also assessed in the PD-1 treated
animals, and it was found that though neutralization against a
primary SIV isolate was not significantly different, neutralization
against a TCLA SIV strain was significantly different in the
treated animals, with 2 of the animals showing 3-6 fold increase in
neutralizing Ab titers.
[0616] Distribution of B Cell Subsets in Sooty Mangabeys:
[0617] Sooty mangabeys, one of the natural hosts of SIV, do not
develop AIDS despite persistent high viral titers comparable to
those of rhesus macaques. This makes SM an interesting `control`
model for studies of pathogenic SIV infection in RM. A cohort of
uninfected (n=8) and SIV-infected (n=10) sooty mangabeys (SM) was
studied. Healthy SM had far fewer circulating total B cells than
healthy RM and unlike in RM, we did not see a decrease in
percentage of circulating total B cells following SIV infection in
SM. Identical B cell subsets were identified in SM, but the
distribution of subsets in SM was very different from that in RM.
Naive B cells constituted the major peripheral blood B cell subset
(>40%), and the majority memory B cell subset was the
tissue-like memory B cells and not activated memory B cells as in
RM. Like RM, <10% of circulating memory B cells in the SM were
resting memory B cells but compared to RM, the percentage of
activated memory B cells was significantly lower in SM. Following
SIV infection, there was no depletion of activated memory B cells;
in fact there was a slight increase in percentage of both resting
and activated memory B cells, although these changes did not reach
statistical significance. PD-1 expression on B cell subsets of SM,
as in the RM, was highest on the activated memory B cells and
unlike in RM, PD-1 expression was equally high on tissue memory B
cells. Another significant difference between RM and SM was that
unlike in RM, the proportions of PD-1 expressing cells went up
following SIV infection in SM.
Example 30: Method of Determining the Efficacy of a PD-1
Antagonist
[0618] The efficacy of a PD-1 antagonist for treating a subject can
be determined by measuring B cells, such as by measuring the
presence of neutralizing antibodies, the proliferation of memory B
cells, naive B cells, and/or by measuring CD28+ T cells. Generally,
a statistically significant increase in neutralizing antibodies,
the proliferation of memory B cells, naive B cells, and/or by
measuring CD28+ T cells indicates that the PD-1 antagonist is
effective for treating the subject. B cells can be measured, for
example, as described in U.S. Pat. No. 7,378,276 and/or U.S. Pat.
No. 6,376,459, both of which are incorporated herein by reference.
PD-1 antagonists include antibodies that specifically bind PD-L1
and PD-L2, see for example, U.S. Pat. No. 7,432,059.
[0619] Determining the efficacy of a PD-1 antagonist involves
obtaining a biological sample from the subject. A biological
sample, such as a blood sample or a sample of peripheral blood
mononuclear cells is taken from a human subject, such as a subject
with a persistent infection. The presence of proliferating memory B
cells, naive B cells and/or CD28+ T cells is measured using a FACS
analysis. The presence of neutralizing antibodies can be measured,
such as by using an ELISA. The proliferating memory B cells, naive
B cells and/or CD28+ cells, and/or the presence of neutralizing
antibodies can be compared to a control, such as the in a sample
from the subject obtained prior to treatment with the PD-1
antagonist. A statistical test is performed. A statistically
significant increase in proliferating memory B cells, and/or
neutralizing antibodies and/or CD28+ T cells in the blood sample
from the subject following administration to the subject in
comparison to the control demonstrates that the PD-1 antagonist is
effective for treating the subject. However, naive B cells are not
affected by the administration of the PD-1 antagonist.
[0620] A number of types of subject are treated and tested. These
subjects include a subject with an HIV infection, a subject with an
xenotropic murine leukemia virus-related virus (XMRV) infection,
and a subject with an polyomavirus JC infection. The PD-1
antagonist can be administered with anti-retroviral therapy, such
as for treating HIV and XMRV. Suitable subject also include those
with tumors, such as a solid tumor or a lymphoma or a leukemia.
These subject can also be administered a chemotherapeutic agent
and/or a tumor antigen.
[0621] It will be apparent that the precise details of the methods
or compositions described may be varied or modified without
departing from the spirit of the described invention. We claim all
such modifications and variations that fall within the scope and
spirit of the claims below.
Sequence CWU 1
1
521288PRTHomo sapiens 1Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val
Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu
Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Phe Pro
Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr
Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp
Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala
Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90
95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile
Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala
Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala
His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln
Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu
Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala
Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu
Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215
220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe
Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser
Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp
Gly His Cys Ser Trp Pro Leu 275 280 285 2 288PRTMus musculus 2Met
Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln 1 5 10
15 Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp
20 25 30 Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu
Gly Ala 35 40 45 Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser
Glu Asp Leu Met 50 55 60 Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn
Gln Thr Glu Lys Gln Ala 65 70 75 80 Ala Phe Cys Asn Gly Leu Ser Gln
Pro Val Gln Asp Ala Arg Phe Gln 85 90 95 Ile Ile Gln Leu Pro Asn
Arg His Asp Phe His Met Asn Ile Leu Asp 100 105 110 Thr Arg Arg Asn
Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 His Pro
Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val 130 135 140
Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro 145
150 155 160 Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met
Ser Ala 165 170 175 Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp
Ala Leu Ala Val 180 185 190 Phe Cys Ser Thr Ser Met Ser Glu Ala Arg
Gly Ala Gly Ser Lys Asp 195 200 205 Asp Thr Leu Lys Glu Glu Pro Ser
Ala Ala Pro Val Pro Ser Val Ala 210 215 220 Tyr Glu Glu Leu Asp Phe
Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro 225 230 235 240 Thr Ala Cys
Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly 245 250 255 Leu
Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln 260 265
270 Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu
275 280 285 3 290PRTHomo sapiens 3Met Arg Ile Phe Ala Val Phe Ile
Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr
Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met
Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60
Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65
70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu
Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp
Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp
Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys
Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu
His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala
Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly
Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185
190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr
195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala
Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn
Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu
Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys
Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr
Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290
4273PRTHomo sapiens 4Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu
Leu Gln Leu His Gln 1 5 10 15 Ile Ala Ala Leu Phe Thr Val Thr Val
Pro Lys Glu Leu Tyr Ile Ile 20 25 30 Glu His Gly Ser Asn Val Thr
Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40 45 His Val Asn Leu Gly
Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn 50 55 60 Asp Thr Ser
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu 65 70 75 80 Pro
Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp 85 90
95 Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr
100 105 110 Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile
Asn Thr 115 120 125 His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu
Leu Thr Cys Gln 130 135 140 Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser
Trp Pro Asn Val Ser Val 145 150 155 160 Pro Ala Asn Thr Ser His Ser
Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170 175 Thr Ser Val Leu Arg
Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys 180 185 190 Val Phe Trp
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp 195 200 205 Leu
Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His 210 215
220 Ile Phe Ile Pro Ser Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val
225 230 235 240 Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser
Ser Lys Asp 245 250 255 Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg
Glu Val Asn Ser Ala 260 265 270 Ile 523PRTHomo sapiens 5Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys 20 614PRTHomo sapiens 6Trp Tyr Gln Gln Lys
Pro Gly Gln Pro Pro Leu Leu Ile Tyr 1 5 10 732PRTHomo sapiens 7Gly
Val Pro Asp Arg Pro Phe Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10
15 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys
20 25 30 811PRTHomo sapiens 8Phe Gly Gln Gly Gln Thr Lys Leu Glu
Ile Lys 1 5 10 930PRTHomo sapiens 9Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gln Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gln Tyr Thr Phe Thr 20 25 30 1014PRTHomo sapiens 10Trp
Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly 1 5 10
1132PRTHomo sapiens 11Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser
Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys Ala Arg 20 25 30 1211PRTHomo sapiens 12Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 1315PRTArtificial
sequenceExemplary antigenic peptide 13Thr Leu Tyr Lys Lys Met Glu
Gln Asp Val Lys Val Ala His Gln 1 5 10 15 1415PRTArtificial
sequenceExemplary antigenic peptide 14Gly Asn Leu Pro Leu Met Arg
Lys Ala Tyr Leu Arg Lys Cys Lys 1 5 10 15 1515PRTArtificial
sequenceExemplary antigenic peptide 15Thr Phe Ser Arg Met Lys Tyr
Asn Ile Cys Met Gly Lys Cys Ile 1 5 10 15 169PRTArtificial
sequenceExemplary antigenic peptide 16Ser Ile Thr Glu Val Glu Cys
Phe Leu 1 5 179PRTArtificial sequenceExemplary antigenic peptide
17Gln Pro Arg Ala Pro Ile Arg Pro Ile 1 5 189PRTArtificial
sequenceExemplary antigenic peptide 18Asn Leu Val Pro Met Val Ala
Thr Val 1 5 1910PRTArtificial sequenceExemplary antigenic peptide
19Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr 1 5 10 209PRTArtificial
sequenceExemplary antigenic peptide 20Gly Ile Leu Gly Phe Val Phe
Thr Leu 1 5 219PRTArtificial sequenceExemplary antigenic peptide
21Leu Tyr Val Asp Ser Leu Phe Phe Leu 1 5 229PRTArtificial
sequenceExemplary antigenic peptide 22Arg Met Phe Pro Asn Ala Pro
Tyr Leu 1 5 2310PRTArtificial sequenceExemplary antigenic peptide
23Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu 1 5 10 249PRTArtificial
sequenceExemplary antigenic peptide 24Gly Val Ala Leu Gln Thr Met
Lys Gln 1 5 259PRTArtificial sequenceExemplary antigenic peptide
25Glu Thr Val Ser Glu Gln Ser Asn Val 1 5 269PRTArtificial
sequenceExemplary antigenic peptide 26Val Leu Gln Glu Leu Asn Val
Thr Val 1 5 279PRTArtificial sequenceExemplary antigenic peptide
27Val Leu Gln Glu Leu Asn Val Thr Val 1 5 289PRTArtificial
sequenceExemplary antigenic peptide 28Glu Ala Asp Pro Thr Gly His
Ser Tyr 1 5 299PRTArtificial sequenceExemplary antigenic peptide
29Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 3021PRTArtificial
sequenceExemplary antigenic peptide 30Arg His Arg Pro Leu Gln Glu
Val Tyr Pro Glu Ala Asn Ala Pro Ile 1 5 10 15 Gly His Asn Arg Glu
20 3116PRTArtificial sequenceExemplary antigenic peptide 31Trp Asn
Arg Gln Leu Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp 1 5 10 15
3210PRTArtificial sequenceExemplary antigenic peptide 32Val Leu Leu
Lys Glu Phe Thr Val Ser Gly 1 5 10 339PRTArtificial
sequenceExemplary antigenic peptide 33Lys Ile Phe Gly Ser Leu Ala
Phe Leu 1 5 349PRTArtificial sequenceExemplary antigenic peptide
34His Leu Phe Gly Tyr Ser Trp Tyr Lys 1 5 3510PRTArtificial
sequenceExemplary antigenic peptide 35Phe Leu Thr Pro Lys Lys Leu
Gln Cys Val 1 5 10 369PRTArtificial sequenceExemplary antigenic
peptide 36Gly Leu Cys Thr Leu Val Ala Met Leu 1 5 378PRTArtificial
sequenceExemplary antigenic peptide 37Arg Ala Lys Phe Lys Gln Leu
Leu 1 5 389PRTArtificial sequenceExemplary antigenic peptide 38Phe
Leu Arg Gly Arg Ala Tyr Gly Leu 1 5 399PRTArtificial
sequenceExemplary antigenic peptide 39Asn Leu Val Pro Met Val Ala
Thr Val 1 5 4010PRTArtificial sequenceExemplary antigenic peptide
40Thr Pro Arg Val Thr Gly Gly Gly Ala Met 1 5 10 419PRTArtificial
sequenceExemplary antigenic peptide 41Gly Ile Leu Gly Phe Val Phe
Thr Leu 1 5 429PRTArtificial sequenceExemplary antigenic peptide
42Cys Leu Thr Glu Tyr Ile Leu Trp Val 1 5 439PRTArtificial
sequenceExemplary antigenic peptide 43Lys Val Asp Asp Thr Phe Tyr
Tyr Val 1 5 449PRTArtificial sequenceExemplary antigenic peptide
44Cys Ile Asn Gly Val Cys Trp Thr Val 1 5 4510PRTArtificial
sequenceExemplary antigenic peptide 45Lys Leu Val Ala Leu Gly Ile
Asn Ala Val 1 5 10 46273PRTHomo sapiens 46Met Ile Phe Leu Leu Leu
Met Leu Ser Leu Glu Leu Gln Leu His Gln 1 5 10 15 Ile Ala Ala Leu
Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile 20 25 30 Glu His
Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40 45
His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn 50
55 60 Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln
Leu 65 70 75 80 Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln
Val Arg Asp 85 90 95 Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly
Val Ala Trp Asp Tyr 100 105 110 Lys Tyr Leu Thr Leu Lys Val Lys Ala
Ser Tyr Arg Lys Ile Asn Thr 115 120 125 His Ile Leu Lys Val Pro Glu
Thr Asp Glu Val Glu Leu Thr Cys Gln 130 135 140 Ala Thr Gly Tyr Pro
Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val 145 150 155 160 Pro Ala
Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170 175
Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys 180
185 190 Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile
Asp 195 200 205 Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp
Leu Leu His 210 215 220 Ile Phe Ile Pro Phe Cys Ile Ile Ala Phe Ile
Phe Ile Ala Thr Val 225 230 235 240 Ile Ala Leu Arg Lys Gln Leu Cys
Gln Lys Leu Tyr Ser Ser Lys Asp 245 250 255 Thr Thr Lys Arg Pro Val
Thr Thr Thr Lys Arg Glu Val Asn Ser Ala 260 265 270 Ile
4724PRTArtificial sequenceExemplary antigenic peptide 47Cys Glu Leu
Asp Asn Ser His Glu Asp Tyr Asn Trp Asn Leu Trp Phe 1 5 10 15 Lys
Trp Cys Ser Gly His Gly Arg 20 4824PRTArtificial sequenceExemplary
antigenic peptide 48Thr Gly His Gly Lys His Phe Tyr Asp Cys Asp Trp
Asp Pro Ser His 1 5 10 15 Gly Asp Tyr Ser Trp Tyr Leu Trp 20
4924PRTArtificial sequenceExemplary antigenic peptide 49Asp Pro Ser
His Gly Asp Tyr Ser Trp Tyr Leu Trp Asp Tyr Leu Cys 1 5 10 15 Gly
Asn Gly His His Pro Tyr Asp 20 5024PRTArtificial sequenceExemplary
antigenic peptide 50Asp Tyr Leu Cys
Gly Asn Gly His His Pro Tyr Asp Cys Glu Leu Asp 1 5 10 15 Asn Ser
His Glu Asp Tyr Ser Trp 20 5124PRTArtificial sequenceExemplary
antigenic peptide 51Asp Pro Tyr Asn Cys Asp Trp Asp Pro Tyr His Glu
Lys Tyr Asp Trp 1 5 10 15 Asp Leu Trp Asn Lys Trp Cys Asn 20
5224PRTArtificial sequenceExemplary antigenic peptide 52Lys Tyr Asp
Trp Asp Leu Trp Asn Lys Trp Cys Asn Lys Asp Pro Tyr 1 5 10 15 Asn
Cys Asp Trp Asp Pro Tyr His 20
* * * * *