U.S. patent application number 17/195236 was filed with the patent office on 2021-07-01 for method for inducing antitumor immunity using sindbis viral vectors and tumor associated antigens.
The applicant listed for this patent is New York University. Invention is credited to Tomer Granot, Daniel Meruelo, Yoshihide Yamanashi.
Application Number | 20210196839 17/195236 |
Document ID | / |
Family ID | 1000005449465 |
Filed Date | 2021-07-01 |
United States Patent
Application |
20210196839 |
Kind Code |
A1 |
Meruelo; Daniel ; et
al. |
July 1, 2021 |
Method For Inducing Antitumor Immunity Using Sindbis Viral Vectors
And Tumor Associated Antigens
Abstract
The subject application is directed to a method for treating a
mammal harboring a tumor comprising identifying a tumor associated
antigen (TAA) expressed by the tumor and parenterally administering
to the mammal a therapeutically effective amount of a Sindbis viral
vector carrying a gene encoding the TAA to the mammal sufficient to
elicit an immune response directed against the tumor, and thereby
treating the tumor.
Inventors: |
Meruelo; Daniel;
(Scarborough, NY) ; Granot; Tomer; (Brooklyn,
NY) ; Yamanashi; Yoshihide; (Brooklyn, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New York University |
New York |
NY |
US |
|
|
Family ID: |
1000005449465 |
Appl. No.: |
17/195236 |
Filed: |
March 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15996271 |
Jun 1, 2018 |
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17195236 |
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14478783 |
Sep 5, 2014 |
10010628 |
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15996271 |
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61874685 |
Sep 6, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/001182 20180801;
A61K 39/001188 20180801; A61K 2039/572 20130101; A61K 48/0058
20130101; A61K 39/0011 20130101; C12N 2770/36143 20130101; A61K
2039/5256 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 39/00 20060101 A61K039/00 |
Goverment Interests
[0002] The United States Government has certain rights to this
invention by virtue of funding received from the U.S. Public Health
grants CA100687 from the National Cancer Institute, National
Institutes of Health and Departments of Health and Human Services.
Claims
1.-22. (canceled)
23. A method of treating a mammal having a tumor, the method
comprising administering by intravenous or intraperitoneal
injection to a site of the mammal harboring said tumor an effective
amount of a replication defective Sindbis viral vector comprising a
gene encoding New York-ESO-1 (NY-ESO-1), wherein the Sindbis viral
vector does not infect the tumor directly, and wherein the tumor is
treated by NY-ESO-1 specific T cell response in the lymph nodes
that drain the site of administration of the Sindbis viral
vector.
24. The method of claim 23, wherein the NY-ESO-1 specific T cell
response in the lymph nodes is followed by generation and influx of
effector and memory NY-ESO-1 specific CD8+ T cells in the tumor,
and wherein effector and memory NY-ESO-1 specific CD8+ T cells
display cytotoxicity against the NY-ESO-1 positive and negative
tumor cells.
25. The method of claim 23, wherein the said Sindbis viral vector
introduces the NY-ESO-1 into the lymph nodes prior to activation of
a NY-ESO-1 specific immune response by antigen presenting cells
(APCs) in the mammal.
26. The method of claim 23, wherein the immune response induced in
the lymph nodes of the subject further induces an adaptive
anti-tumor immune response comprising activation of
NY-ESO-1-specific CD4+ T cells, NK cells, macrophages, monocytes,
dendritic cells and neutrophils in the mammal.
27. The method of claim 23, wherein the subject is human.
28. The method of claim 23, wherein the tumor is selected from the
group consisting of subcutaneous, intraperitoneal, and lung cancer
tumor.
29. The method of claim 23, wherein the tumor is a solid tumor.
30. The method of claim 23, wherein the solid tumor is ovarian
cancer tumor.
31. The method of claim 23, wherein the Sindbis viral vector is
administered in combination with chemotherapy.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a non-provisional application of U.S.
Provisional Application No. 61/874,685 filed Sep. 6, 2013, which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] Oncolytic viruses (OV) are viruses that specifically target
and replicate in tumor cells [1]. Owing to their selectivity and
oncolytic properties, OVs have generated considerable interest as
an alternative or adjunct to conventional cancer therapies [2].
However, a major limitation of OV therapy is inadequate replication
and propagation at the tumor site [3, 4]. Moreover, for safety
reasons, many OVs are designed to be replication deficient in order
to prevent them from spreading to healthy tissues, further limiting
their oncolytic potential [5].
[0004] One possible solution to this problem is to supplement
direct viral oncolysis with a bystander effect, in which tumor
cells not directly infected by the OV will also be destroyed. This
can be achieved, for example, by inserting a therapeutic or
cytotoxic gene into the OV genome for delivery to the tumor site
[6, 7]. Endowed with natural immunogenicity, some OVs are capable
of effective stimulation of the immune system, raising the
possibility of using OVs to induce an immunological anti-cancer
bystander effect [8]. This idea gained further impetus with the
identification [9, 10] and recent prioritization [11] of a variety
of clinically relevant tumor associated antigens (TAA), which can
be delivered by the OV (OV/TAA) to the tumor site [12]. In their
natural state, TAAs are often poorly immunogenic [13]. However, by
redirecting the anti-viral immune response towards the TAA, an
immunogenic OV/TAA could potentially break this immunological
tolerance. A major goal of OV research should therefore be the
development of safe and effective OV/TAA agents. Sindbis virus
(SV), an alphavirus with a positive single-stranded RNA genome
[14], represents one of a select number of viruses that have
demonstrated exceptional potential both as an OV [15, 16] and as a
viral vaccine [17]. It has been previously shown that replication
deficient SV vectors target and inhibit the growth of xenograft,
syngeneic and spontaneous tumors in mice [16, 18].
[0005] Recently, it has also been found that SV induces the
activation of natural killer (NK) cells and macrophages in
tumor-bearing mice [19]. In addition, SV vectors expressing
immune-modulating genes such as interleukin 12 (IL-12) have an
enhanced antitumor [16] and immunostimulatory [19] effect.
Nevertheless, these approaches have not generally led to complete
tumor remission [19]. Moreover, some tumor cells may not be
efficiently targeted by SV [20], underscoring the need to develop
new ways of enhancing SV anti-cancer therapy.
[0006] Previously, it was hypothesized that the unique
characteristics of SV vectors, which make them effective oncolytic
agents and gene delivery systems (e.g. the ability to disseminate
through the bloodstream [15] and deliver high levels of
heterologous proteins [21]) could also be useful for efficient TAA
delivery. Moreover, the SV life cycle, which is characterized by
the absence of a DNA phase, rendering the vectors safer, also
involves the production of high levels of double stranded RNA
(dsRNA), a potent immunological `danger signal` [22], and the
subsequent activation of the type I interferon pathway [23]. The
combination of safety, immunogenicity, efficient dissemination, and
high TAA expression make SV/TAA an attractive OV/TAA candidate.
Therefore, what is needed in the art are methods for treating
mammals suffering from tumors using SV/TAA, thereby taking
advantage of all of the above-mentioned benefits.
SUMMARY OF THE INVENTION
[0007] Disclosed herein, the BALB/c CT26 colon carcinoma tumor
model was used to investigate the use of SV as an OV/TAA agent. It
was found that unlike other tumor models tested, CT26 cells are not
targeted by SV in vivo. Nevertheless, SV vectors carrying .beta.
galactosidase (SV/LacZ) had a remarkable therapeutic effect in mice
bearing LacZ-expressing CT26 tumors. Using the in vivo imaging
system (IVIS) for sensitive in vivo detection of luciferase
activity [24], the mediastinal lymph nodes (MLN) were identified as
a site of early transient heterologous protein expression after
intraperitoneal (i.p) injection of SV vectors carrying the firefly
luciferase gene (SV/Fluc). TAA delivery into the MLN marked the
starting point of a potent immune response that culminated in the
generation of effector and memory CD8.sup.+ T cells displaying
robust cytotoxicity against LacZ positive and negative tumor cells.
This latter phenomenon, known as epitope spreading, has recently
been suggested to be an important component of effective cancer
immunotherapy in patients [25].
[0008] In one aspect, the present invention provides a method for
treating a mammal harboring a tumor comprising the steps of
identifying a tumor associated antigen (TAA) expressed by the
tumor, and parenterally administering to the mammal a
therapeutically effective amount of a Sindbis viral vector carrying
a gene encoding the TAA to the mammal sufficient to elicit an
immune response directed against the tumor, and thereby treating
the tumor.
[0009] In another aspect, the present invention provides a method
for inducing a CD8+ T-cell mediated immune response directed
against a tumor in a mammal comprising the steps of identifying at
least one tumor associated antigen (TAA) expressed by the tumor,
and parenterally administering to a mammal in need of such
treatment an amount of a Sindbis viral vector carrying a gene
encoding the TAA effective to elicit a CD8+ T-cell mediated immune
response directed against the tumor.
[0010] These and other aspects of the present invention will be
apparent to those of ordinary skill in the art in light of the
present description, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1a-1c. SV/LacZ inhibits the growth of LacZ-expressing
CT26.CL25 tumors. (a) 0.5.times.10.sup.6 LacZ-expressing CT26.CL25
(left panel) or LacZ-negative CT26.WT (right panel) cells were
injected s.c. into the right flank of BALB/c mice. Starting on day
9 after tumor inoculation, mice were treated i.p. with SV/LacZ,
control SV/Fluc vectors, or media (Mock). Tumor volume (mm.sup.3)
was measured and plotted (N=3-4). Data are representative of at
least two independent experiments. (b) Kaplan-Meier survival plots
of mice bearing peritoneal CT26.CL25 tumors. 2.5.times.10.sup.4
CT26.CL25 cells were injected i.p., and treatment started on day 4
(N=5). Data for the SV/LacZ and mock groups is representative of 2
independent experiments. (c) Representative IVIS images of SV/LacZ
and control-treated mice bearing lung CT26.CL25.Fluc tumors are
shown (left panel). Relative tumor growth (top right panel) was
determined by normalizing the luminescence to the first image (day
2) for each individual mouse, and survival rates were plotted
(bottom right panel) (N=5-8). Data is representative of 2
independent experiments. Data in (a) and (c) are expressed as
mean.+-.SEM. *p<0.05 **p<0.01. SV, Sindbis viral vector.
[0012] FIGS. 2a-2c. TAA expression and T cell activation occur in
the mediastinal lymph nodes. (a) s.c. CT26.CL25 tumor-bearing mice
(left panel) or tumor-free mice (right panel) were treated i.p.
with SV/Fluc. 3 hours after the 5.sup.th (left panel) or 1.sup.st
(right panel) treatment, bioluminescent images were taken to
monitor Fluc expression from the vector. To determine the source of
the upper body signal, the MLN was extracted and imaged separately
(right panel). Red circles in the left panel indicate the location
of the s.c. tumor in each mouse. (b) Mice bearing lung
CT26.CL25.Fluc tumors were treated with SV/LacZ. 24 hours later,
mediastinal and inguinal lymph nodes were extracted and stained to
determine the percentage of T cells (CD3 positive, MEW class II
negative) in the lymph nodes. Representative plots (left panel),
and their quantification (right panel; n=3) are shown. (c) The
expression of CD69 on CD8.sup.+ T cells extracted from mediastinal
and inguinal lymph nodes of lung tumor-bearing mice 24 hours after
i.p. SV/LacZ injection was analyzed. Representative flow cytometry
plots (left panel), and bar graphs showing the percentage of
CD69-high cells (right panel; n=3) are shown. Data in (b) and (c)
are representative of two independent experiments (the second
experiment was done in mice bearing i.p. tumors), and are expressed
as mean.+-.SEM. *p<0.05 **p<0.01. Fluc, firefly luciferase;
MLN, mediastinal lymph node; ILN, inguinal lymph node; S/L, SV/LacZ
(SV, Sindbis viral vector); TAA, tumor-associated antigen.
[0013] FIGS. 3a and 3b. SV/LacZ induces potent CD8.sup.+ T cell
response. (a) Lung CT26.CL25.Fluc tumor bearing mice were treated
with SV/LacZ or media (Mock). 7 days later, peritoneal cells were
analyzed. Representative flow cytometry plots (left panel), and the
calculated number of CD8.sup.+ T cells (right panel) are shown
(mean.+-.SEM, N=3). (b) CD8.sup.+ T cells from the peritoneum and
the lungs were further analyzed to determine their activation
state, using NKG2D and L-selectin as activation markers.
Representative flow cytometry plots (left panel) and the calculated
percentage of activated (NKG2D high, L-selectin low) cells (right
panel) are shown (mean.+-.SEM, N=3). *p<0.05 **p<0.01. SV,
Sindbis viral vector.
[0014] FIGS. 4a-4d. SV/LacZ induces LacZ-specific CD8.sup.+ T cell
response. (a,b) Splenocytes from CT26.CL25 s.c. tumor-bearing mice
were collected and analyzed 2 weeks after SV/LacZ or Mock treatment
started. Representative tetramer plots (a), and the percentage of
tetramer-positive cells (b) are shown (N=5). (c) Cells from the
peritoneal cavity of i.p. and lung tumor-bearing mice 7 days after
therapy started were collected, stained and analyzed (N=4-5). (d)
Lungs from lung tumor-bearing mice 7 days after treatment started
were analyzed, and the percentage of activated (NKG2D high,
L-selectin low) cells in the subsets of LacZ tetramer positive and
negative CD8.sup.+ T cells in the lungs were analyzed and plotted
as in FIG. 3b (N=3). Data in (b)-(d) are expressed as mean.+-.SEM.
*p<0.05 **p<0.01. SV, Sindbis viral vector.
[0015] FIGS. 5a and 5b. Lymphocytes acquire LacZ-specific
cytotoxicity during SV/LacZ therapy. Lung lymphocytes were
extracted from CT26.CL25.Fluc lung tumor-bearing mice 7 days after
Mock (-), SV/GFP (G) or SV/LacZ (L) treatment started. Extracted
lung lymphocytes were co-cultured with CT26.CL25.Fluc cells
(CT26.CL25) or CT26.WT.Fluc cells (CT26.WT) for 2 days to determine
(a) the cytotoxicity of lung lymphocytes against each tumor cell
population, and (b) IFN-.gamma. secretion from the lung lymphocytes
in response to co-culture with each tumor cell population, as
described in Materials and Methods (data in (a) and (b) are
expressed as mean.+-.SD, N=3). **p<0.01 (significantly different
from Mock). N.D, not detected. SV, Sindbis viral vector.
[0016] FIGS. 6a-6c. CD8.sup.+ T cells are required for the enhanced
therapeutic effect of SV/LacZ. The therapeutic effect of SV/LacZ
was compared between intact and CD8.sup.+ T cell-depleted
(CD8.sup.+ T cell (-)) mice in the (a) s.c., (b) i.p., and (c) lung
tumor models. (a) The size of CT26.CL25 s.c. tumors at indicated
time points was measured and plotted for each group (N=5). (b)
Survival rates in CT26.CL25 i.p. tumor-bearing mice were monitored
and plotted as Kaplan-Meier survival plots. N=8-9 (c) Tumor growth
(left panel) and survival rates (right panel) in CT26.CL25.Fluc
lung tumor-bearing mice were analyzed. Relative tumor growth was
quantified as in FIG. 1c, and survival rates are shown as
Kaplan-Meier survival plots (N=5). Data in (a) and (c) are
expressed as mean.+-.SEM. *P<0.05, **P<0.01. N. S, not
significant; SV, Sindbis viral vector.
[0017] FIGS. 7a-7e. Immunity against endogenous CT26 TAAs develops
during SV/LacZ therapy. (a,b) Splenocytes were extracted from
CT26.CL25.Fluc lung tumor-bearing mice at 7 days after Mock (-),
SV/GFP (G) or SV/LacZ (L) treatment started. Extracted splenocytes
were co-cultured with CT26.CL25.Fluc (CT26.CL25) or CT26.WT.Fluc
(CT26.WT) cells for 2 days to determine (a) the cytotoxicity of the
splenocytes towards each tumor cell population, and (b) IFN-.gamma.
secretion from the splenocytes in response to co-culture with each
tumor cell population, as described in Materials and Methods
(mean.+-.SD, N=3). (c) CT26. WT.Fluc tumor was inoculated i.v. into
naive and CT26.CL25 SV/LacZ-treated tumor-cured mice at more than
60 days after the last SV/LacZ treatment, and tumor growth in the
lung was analyzed at the indicated time points by bioluminescent
imaging. The left panel shows representative IVIS images of 2
independent experiments. The right panel shows the quantification
of tumor bioluminescence at the indicated time points (mean.+-.SEM,
N=8. (d) CT26.WT.Fluc tumors were inoculated i.v. into naive (N)
and SV/LacZ-treated tumor-cured mice (S) at more than 30 days after
the last SV/LacZ treatment. 8 days after tumor inoculation,
splenocytes were extracted from each mouse and incubated with LacZ,
gp70, or control peptides for 3 days. After the incubation, LacZ-
or gp70-specific induction of IFN-.gamma. secretion was analyzed as
described in Materials and Methods (mean.+-.SEM, N=3). (e) The
number of gp70-specific CD8.sup.+ T cells in splenocytes extracted
in (d) was quantified by flow cytometry using gp70 tetramers
(mean.+-.SD, N=3). *p<0.05, **p<0.01 (significantly different
from Mock or Naive). N.D, not detected; SV, Sindbis viral
vector.
[0018] FIG. 8. Four-step model for the activation of CD8.sup.+ T
cells during SV/TAA therapy. Step 1: i.p. injection of SV/LacZ
results in transient immunogenic expression of LacZ in the
mediastinal lymph nodes (dark blue arrow), followed by the
induction of T cell activation at this site and/or in alternative
locations (light blue arrow). NK cells are also activated against
the tumor cells (brown arrow). Step 2 (red arrow): LacZ-specific
CD8.sup.+ T cell cytotoxicity results in the destruction of tumor
cells and the subsequent release of tumor associated antigens such
as LacZ and gp70. Step 3 (green arrows): Antigen-presenting cells
capture and present these antigens to CD8.sup.+ T cells in the
tumor-draining lymph nodes, resulting in epitope spreading,
including the induction of gp70-specific CD8.sup.+ T cells that can
potentially target LacZ(-) tumor cell escape variants. Step 4
(purple arrows): memory CD8.sup.+ T cells against a variety of
tumor-associated antigens are generated. APC, antigen-presenting
cell; LN, lymph node; MLN, mediastinal lymph node; NK, Natural
killer cell; SV, Sindbis viral vector; TAA, tumor-associated
antigen; Tc, cytotoxic CD8.sup.+ T cell; Tm, memory CD8.sup.+ T
cell.
[0019] FIGS. 9a and 9b. The enhanced therapeutic effect of SV/LacZ
in mice bearing lung tumors is dependent on LacZ expression on the
tumors. (a) Tumor growth was analyzed in CT26.CL25.Fluc or
CT26.WT.Fluc lung tumor-bearing mice at indicated time points. The
left panel shows representative IVIS images of two independent
experiments. The right panel shows the relative tumor growth at
indicated time points. Data are expressed as mean.+-.SEM. (N=4-7).
(b) Survival rates of CT26.CL25.Fluc or CT26.WT.Fluc lung
tumor-bearing mice are shown as Kaplan-Meier survival plots
(N=5-7). *p<0.05, **p<0.01. SV, Sindbis viral vector.
[0020] FIGS. 10a and 10b. SV does not target CT26 tumors in the
lung. (a) Tumor-free or CT26.WT lung tumor-bearing mice were
treated i.p. with SV/Fluc every 2 days. Whole body bioluminescent
images were taken at indicated time points after the first SV/Fluc
treatment. (b) On day 6, whole body images were taken, and then the
indicated organs were extracted and imaged separately. Fluc,
firefly luciferase; SV, Sindbis viral vector.
[0021] FIGS. 11a and 11b. SV/Fluc and SV/GFP induce CD8+ T cell
response. (a) Peritoneal tumor bearing mice were treated with
SV/Fluc (left panel), SV/GFP (right panel), or media (Mock). At
indicated time points, peritoneal cells were analyzed using flow
cytometry, and the calculated number of CD8+ T cells in the
peritoneum is shown (mean.+-.SEM, N=2-3 for each time point). (b)
Representative flow cytometry plots show L-selectin expression on
peritoneal CD8+ T cells from SV/GFP or mock-treated mice 7 days
after treatment started (N=2). Fluc, firefly luciferase; GFP, green
fluorescent protein; SV, Sindbis viral vector.
[0022] FIGS. 12a-12c. SV/TAA induces the activation of effector and
memory LacZ-specific CD8+ T cells. (a) Left panel: LacZ-naive,
tumor-free mice were injected with SV/LacZ or media (Mock). Four
days later, peritoneal cells were extracted and analyzed for the
presence of LacZ-specific CD8+ T cells. Right panel: The activation
level of peritoneal CD8+ T cells from Mock- and SV/LacZ-treated
mice were compared to each other, as well as to the LacZ-specific
CD8+ T cells obtained from the SV/LacZ treated mouse (SV/LacZ
tet+). Activated cells were defined as NKG2D high, L-selectin low
cells. (b) LacZ tetramer analysis from peritoneal CT26.CL25 tumor
bearing mice treated with SV/LacZ, SV/Fluc, or media (Mock) are
shown. (c) Splenocytes from naive or SV/LacZ-treated long-term
surviving mice (SV/LacZ survivor) that bore i.p. CT26.CL25 tumors
were stained with anti-CD127 (memory cell marker) and LacZ specific
tetramers to determine the presence of long-lasting LacZ-specific
memory (CD127+, Tetramer+) cells. Data is representative of two
specimens, taken more than 3 months after the treatment was
stopped. All plots show gated CD8+ T cells. Fluc, firefly
luciferase; LacZ, 3-galactosidase; SV, Sindbis viral vector; tet,
tetramer.
[0023] FIGS. 13a and 13b. NK cells are activated at an early stage
of SV therapy. (a) Percentages of lung CD4+ T cells, CD8+ T cells,
LacZ-specific CD8+ T cells, and NK (CD3-CD122+) cells within the
total lung immune cell (CD45+) population from CT26.CL25.Fluc lung
tumor-bearing mice were analyzed at indicated time points after
Mock or SV/LacZ treatment started. (b) Expression of NKG2D on NK
cells in the lung from CT26.CL25.Fluc lung tumor-bearing mice was
analyzed at indicated time points after mock or SV/LacZ treatment
started. Data are expressed as mean.+-.SEM (N=3). *p<0.05,
**p<0.01. LacZ, .beta.-galactosidase; NK, natural killer cell;
SV, Sindbis viral vector; tet, tetramer.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The term "about" or "approximately" usually means within an
acceptable error range for the type of value and method of
measurement. For example, it can mean within 20%, more preferably
within 10%, and most preferably still within 5% of a given value or
range. Alternatively, especially in biological systems, the term
"about" means within about a log (i.e., an order of magnitude)
preferably within a factor of two of a given value.
[0025] The present invention is based on the following discoveries:
(i) SV represents a potentially powerful therapeutic platform for
the immunogenic delivery of TAAs, (ii) the therapeutic benefit
obtained from SV/TAA does not necessarily require the direct
targeting of tumor cells, (iii) SV/TAA therapy involves transient
early delivery of the TAA to lymph nodes draining the injection
site, in particular the MLN in the case of i.p. SV injection, (iv)
SV/TAA therapy induces a potent TAA-specific CD8+ T cell response,
that is subsequently redirected against tumor cells expressing the
cognate TAA, (v) SV/TAA therapy leads to epitope spreading,
providing a possible solution to the problem of tumor escape by TAA
loss or modification, and (vi) SV/TAA therapy ultimately leads to
long-term survival of tumor-bearing mice, and to the generation of
long-lasting memory CD8+ T cells against multiple TAAs.
[0026] Pursuant to the present invention, Sindbis viral vectors
carrying genes encoding tumor associated antigens (TAAs) are used
to elicit an immune response directed against tumors in mammals.
Oncolytic viruses (OVs) have recently emerged as a promising
strategy for the immunogenic delivery of TAAs to cancer patients.
However, prior to the present invention, safe and effective OV/TAA
therapies have not yet been established. It has been previously
demonstrated that vectors based on Sindbis virus (SV) can target
tumor cells, inhibit tumor growth and activate the innate immune
system in mice. It has now been unexpectedly discovered that
parenterally administered SV vectors carrying a gene encoding a
tumor associated antigen (TAA) generate a dramatically enhanced
therapeutic effect in mice bearing subcutaneous, intraperitoneal,
and lung cancers. Surprisingly, SV/TAA efficacy was not dependent
on tumor cell targeting, but was characterized by the transient
expression of TAAs in lymph nodes draining the injection site.
Early T cell activation at this site was followed by a robust
influx of NKG2D expressing antigen-specific cytotoxic CD8.sup.+ T
cells into the tumor site, subsequently leading to the generation
of long-lasting memory T cells. Such cells conferred protection
against re-challenge with TAA-positive as well as--negative tumor
cells. As described herein, by combining in vivo imaging, flow
cytometry, cytotoxicity/cytokine assays, and tetramer analysis, the
relationship between these events has been discerned. As a result,
a model for CD8.sup.+ T cell activation during SV/TAA therapy and a
method to treat mammals suffering from tumors by eliciting an
immune response directed against a tumor is provided.
[0027] SV/TAA can be combined with chemotherapy, as it has been
previously shown that SV and chemotherapy can synergize (e.g. see
U.S. patent application Ser. No. 13/133,680). This includes, but is
not limited to, chemotherapy that stimulates the immune system, or
that inhibits suppressor elements in the immune system, or that
affects tumor cells and makes them more susceptible to T cell (or
other immune cell) cytotoxicity. For example, there are certain
chemotherapies that could facilitate SV/TAA therapy because they
suppress immunosuppressive cells, thereby enhancing SV/TAA
immunostimulation. There have also been reports in the literature
suggesting that chemotherapy enhances tumor cell susceptibility to
T cell mediated cytotoxicity, for example, Ramakrishnan et al.
Journal of Clinical Investigation, 120(11):4141-4154, 2010.
[0028] In the method of the present invention, a patient afflicted
with a tumor is examined to identify a TAA associated with the
tumor. Examples of solid tumors that can be treated according to
the invention include sarcomas and carcinomas such as, but not
limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, synovioma, mesothelioma, Ewing's tumor.
leiomyosarcoma, rhabdomyo sarcoma. Colon carcinoma, pancreatic
cancer, breast cancer, ovarian cancer, prostate cancer, squamolls
cell carcinoma, basal cell carcinoma, epidermoid carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma.
hepatoma. bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma. Wilms'.tumor. cervical cancer, testicular tumor, lung
carcinoma. small cell lung carcinoma. Bladder carcinoma, epithelial
carcinoma, glioma. astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma. hemangioblastoma. acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
neuroglioma. and retinoblastoma.
[0029] Hematologic malignancies can also be treated according to
the invention provided that the specific TAA can be identified.
[0030] Pursuant to the present invention, the tumor and the SV must
express either the same TAA or a similar but not identical TAA that
is immunologically cross-reactive with the TAA expressed by the
SV/TAA. TAAs are well known in the art. For example, Cheevers et
al. (Clin Cancer Res 15: 5323-5337, 2009) disclosed 75
representative TAAs for comparison and ranking, assembling
information on the predefined criteria for the selected antigens,
and ranking the antigens based on the predefined, pre-weighted
criteria. Any TAA expressed by the tumor can be utilized. However,
it is expected that there is wide variability between the efficacy
of different TAAs, with some TAAs potentially inducing much
stronger responses (immunodominant TAAs); exactly which ones are
preferred can be determined using routine investigation well known
to those of ordinary skill in the art.
[0031] The TAA expressed by a patient's tumor can be identified
from a biopsy or from blood tests when a biopsy is not possible.
Serological analysis of expression cDNA libraries (SEREX) has
previously been used to identify human TAAs. Alternative methods
can also be used.
[0032] After the relevant TAA has been identified, a Sindbis viral
vector carrying a gene encoding the TAA is constructed using
techniques well known in the art such as those described in the
Materials and Methods below. The nucleotide sequences encoding the
TAAs are also well known in the art and can be easily obtained from
the literature. For example, the sequence of NY-ESO-1, a testicular
antigen aberrantly expressed in human cancers was published in 1997
(http://www.pnas.org/content/94/5/1914.full, Yao-Tseng
Chen*.dagger..dagger-dbl., Matthew J. Scanlan.dagger., Ugur
Sahin.sctn., Ozlem Tureci.sctn., Ali O. Gure.dagger., Solam
Tsang.dagger., Barbara Williamson.dagger., Elisabeth
Stockert.dagger., Michael Pfreundschuh.sctn., and Lloyd J.
Old.dagger. PNAS 1997.), whereas the Carcinoembryonic antigen
sequence was published in 1987
(http://mcb.asm.org/content/7/9/3221.short Isolation and
characterization of full-length functional cDNA clones for human
carcinoembryonic antigen. N Beauchemin, S Benchimol, D Cournoyer, A
Fuks and C P Stanners, Molecular and Cellular Biology 1987.)
[0033] Any Sindbis viral vector can be used in the present
invention, including replication competent (described, for example,
in U.S. Pat. No. 8,282,916) and replication defective (described,
for example, in U.S. Pat. Nos. 7,303,898, 7,306,792, and
8,093,021). Replication defective vectors are preferred for use in
the present invention in order to prevent infection of healthy
tissues.
[0034] Pursuant to the present invention, a single i.p. injection
of a therapeutically effective amount of SV/TAA sufficient to
infect the cells of the mediastinal lymph nodes (MLN) leads to
their rapid immunogenic delivery to the MLN. Such therapeutically
effective amounts broadly range between about between about 10
million and about 100 billion vector particles. Although in mice a
single i.p. injection of SV/TAA is sufficient to elicit a
detectable CD8+ mediated immune response directed against the
tumor, other regimens may be necessary for achieving a maximal
response. For example, between 1 and about 8 i.p. injections over a
time period of between 1 week and many weeks, with the possibility
of injecting one or more booster injections 1 or more years later,
may be preferably administered for a maximum effect.
[0035] The MLN has previously been shown to drain the peritoneum
[27, 28], and represents an environment in which antigens delivered
by SV vectors (e.g., TAAs) can potentially be processed and
presented to T cells by antigen presenting cells (APC) in the
context of SV viral danger signals such as double stranded (ds) RNA
[22]. One of the main functions of lymph nodes is to facilitate the
induction of an adaptive immune response. Viral danger signals are
components of the virus (or of infected cells) that stimulate the
immune system. Double stranded RNA is such a danger signal because
it is not normally found in cells, and is associated with viral
infections. The MLN provides the location for the induction of a
CD8+ T cell mediated immune response directed against the TAA.
Consistent with this finding, the number of T cells in the MLN
significantly increased 24 hours after SV/TAA treatment using LacZ
as a model antigen.
[0036] It is also possible to use two (or more) different vectors,
including the injection of different vectors carrying different
cytokines at different time points to facilitate the induction and
progression of an enhanced immune response against the TAA or
TAAs.
[0037] In addition to CD8+ T cells, SV/TAA therapy can also
activate additional immune (or non-immune) cells, including (but
not limited to) CD4+ T cells, NK cells, macrophages, monocytes,
dendritic cells, neutrophils, and other cells, as well as the
humoral immune response. Epitope spreading can occur not only in
CD8+ T cells, but also in CD4+ T cells. As can be seen in Example
2, tumor cell targeting is not required for effective SV/TAA
therapy, suggesting that immune cell activation during SV/TAA
therapy may occur far away from the tumor site (in this case the
lungs), e.g. in lymph nodes that drain the SV injection site.
[0038] As shown in Example 3, using flow cytometry, it was
confirmed that a large number of CD8+ T cells influx into the
peritoneum 7 days after the first SV/TAA injection. These
peritoneal CD8+ T cells were activated, as evidenced by the
upregulation of NKG2D [30] and downregulation of lymph node homing
receptor L-selectin [31]). In addition to the robust influx of
activated CD8+ T cells into the peritoneum, a small number of NKG2D
high, L-selectin low CD8+ T cells could also be seen in the lungs
of mice bearing lung CT26.CL25 tumors that were treated with
SV/TAA. It was found that a subset of the LacZ-specific CD8+ T
cells generated during SV/LacZ therapy eventually develop into
memory T cells. Splenocytes from SV/LacZ-treated long-term
surviving mice that bore i.p. CT26.CL25 tumors were analyzed. Using
LacZ tetramers in combination with the memory marker CD127, a
population (roughly 1% of the CD8+ T cell splenocyte population) of
LacZ-specific, CD127+ memory CD8+ T cells in these mice was
identified more than 3 months after the last SV/LacZ injection.
Therefore, treatment pursuant to the present invention led to the
long term maintenance of antitumor activity.
[0039] Use of the methods of the present invention causes epitope
spreading. One of the limitations of prior art cancer vaccine
strategies has been the inherent heterogeneity and genomic
instability of tumor cell populations, coupled with the selective
pressure induced by the treatment, leading to tumor evasion by loss
or modification of the TAA used in the vaccine [38, 39]. In this
context, an important aspect of the present invention is the
induction of epitope spreading, i.e. the expansion of the
anti-tumor T cell response to incorporate novel TAAs that are
endogenous to the tumor, but not delivered by the vector [32]
during SV/TAA therapy. Clinical trials are increasingly
incorporating the analysis of epitope spreading [40], and in some
cases a positive correlation between the induction of epitope
spreading and therapeutic efficacy has been shown [25]. As shown in
Example 7, SV/TAA therapy against CT26.CL25 tumors caused epitope
spreading, which led to the development of immunity against other
unrelated antigen(s) expressed on the CT26 tumors.
[0040] In an alternative embodiment of the present invention, dual
expression SV vectors that carry and deliver genes encoding TAAs in
conjunction with genes encoding appropriate immune stimulating
cytokines to create optimal conditions in the lymph node for T cell
stimulation are employed. Such immune stimulating cytokines
include, without limitation, IL-12 (disclosed in
http://www.jimmunol.org/content/146/9/3074.short Cloning of cDNA
for natural killer cell stimulatory factor, a heterodimeric
cytokine with multiple biologic effects on T and natural killer
cells. S F Wolf, P A Temple, M Kobayashi, D Young, M Dicig, L Lowe,
R Dzialo, L Fitz, C Ferenz and R M Hewick the Journal of
Immunology), and CCL17
(http://www.jbc.org/content/271/35/21514.short Molecular Cloning of
a Novel T Cell-directed CC Chemokine Expressed in Thymus by Signal
Sequence Trap Using Epstein-Barr Virus Vector*-Toshio
Imai.dagger-dbl., Tetsuya Yoshida, Masataka Baba, Miyuki Nishimura,
Mayumi Kakizaki and Osamu Yoshie. The Journal of biological
Chemistry).
[0041] Additional immune stimulating cytokines include, but are not
limited to: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17. Additional
cytokines include IL-18-IL-36. In addition to CCL17, other
chemokines can also be used, including, but not limited to,
CCL1-CCL27 and other CC chemokines, CXCL1-CXCL13 and other CXC
chemokines, C chemokines, and CX.sub.3C chemkines. Cytokine or
chemokine receptors and soluble receptors can also be used.
Additional immune modulators that can be used include TGF-.beta.
and TNF.alpha.. In addition, different combinations of the
above-mentioned (or alternative) cytokines can be used.
[0042] Moreover, because MLN TAA expression is both transient and
re-inducible (unpublished results), different cytokines can be
delivered at different stages of SV/TAA therapy to further tailor
the anti-tumor immune response. For example, SV/IL12 can be
delivered in the early stages of SV/TAA therapy in order to
stimulate a Th1 cytotoxic T cell response, and SV/CCL17 can be
delivered later on, in order to enhance the cross-priming of
additional TAAs, thereby increasing epitope spreading.
[0043] It has been previously demonstrated that SV vectors carrying
the IL-12 gene have an enhanced therapeutic effect in tumor-bearing
mice [16], and promote the IFN-.gamma.-dependent activation of M1
type macrophages [19]. However, the effects of IL-12 delivery to
the MLN have not specifically been investigated before the present
invention.
[0044] In another alternative embodiment, SV vectors are used to
target and/or to deliver payloads to mediastinal masses such as
those derived from certain neurogenic tumors [37]. Since tumors
often metastasize to the lymph nodes (including the mediastinal
lymph nodes), and SV can naturally target certain lymph nodes
(including the mediastinal lymph nodes), SV can be used to deliver
antigens, cytokines, or other payloads directly to the site of
tumor growth.
[0045] Multiple TAAs can also be used either by using one Sindbis
vector expressing multiple TAAs, or by using multiple Sindbis
vectors expressing different TAAs. In addition, the route of
administration is parenteral, including, but not limited to,
intravenous, intraperitoneal, subcutaneous, intramuscular,
intranasal, intraorbital, intranodular, and intratumoral
injections.
[0046] The model for the method is presented below:
[0047] Step 1: i.p. injection of SV/TAA results in transient
immunogenic expression of TAA in the mediastinal lymph nodes,
followed by the induction of T cell activation at this site and/or
in alternative locations; NK cells are also activated against the
tumor cells. Step 2 TAA-specific CD8+ T cell cytotoxicity results
in the destruction of tumor cells and the subsequent release of
tumor associated antigens. Step 3: Antigen-presenting cells capture
and present these antigens to CD8+ T cells in the tumor-draining
lymph nodes, resulting in epitope spreading, including the
induction of TAA-specific CD8+ T cells that can potentially target
TAA(-) tumor cell escape variants. Step 4: memory CD8+ T cells
against a variety of tumor-associated antigens are generated.
[0048] The present invention is described further below in working
examples which are intended to further describe the present
invention without limiting the scope thereof.
[0049] Materials and Methods
[0050] Cell Lines. Baby hamster kidney (BHK), CT26.WT, and
LacZ-expressing CT26.CL25 cells were obtained from the American
Type Culture Collection. Firefly luciferase (Fluc)-expressing CT26
cells (CT26.WT.Fluc and CT26.CL25.Fluc) for noninvasive
bioluminescent imaging were generated by stable transfection of a
Fluc-expressing plasmid into CT26.WT and CT26.CL25 cells. The
Fluc-expressing plasmid was constructed by introducing a SV40
promoter sequence into the multi-cloning site of pGL4.20 vector
(Promega, Wis.).
[0051] Cell Culture. BHK cells were maintained in minimum essential
a-modified media (a-MEM) (Mediatech, VA) with 10% fetal bovine
serum (FBS) (Atlanta Biologicals, Norcross, Ga.). CT26.WT,
CT26.CL25, CT26.WT.Fluc, and CT26.CL25.Fluc cells were maintained
in Dulbecco modified essential media (DMEM) containing 4.5 g/L
glucose (Mediatech) supplemented with 10% FBS. All basal media were
supplemented with 100 mg/mL of penicillin-streptomycin (Mediatech)
and 0.5 mg/mL of amphotericin B (Mediatech). For culturing
CT26.CL25 and CT26.CL25.Fluc cells, 0.4 mg/ml of G418 sulfate
(Mediatech) was added to the basal media. For culturing
CT26.WT.Fluc and CT26.CL25.Fluc cells, 5 mg/ml of puromycin
(Sigma-Aldrich, MO) was added to the basal media.
[0052] SV/TAA Production. SV/LacZ was used as an immunogenic SV/TAA
agent, and SV/Fluc and SV/GFP were used as control vectors. SV/Fluc
was also used for imaging experiments (see below). Vectors were
produced as previously described [16]. Briefly, plasmids carrying
the replicon (SinRep5-LacZ, SinRep5-GFP or SinRep5-Fluc) or DHBB
helper RNAs (SinRep5-tBB) were linearized with Xhol (for
SinRep5-LacZ, SinRep5-GFP and SinRep5-tBB) or Pad (for
SinRep5-Fluc). In vitro transcription was performed using the
mMessage mMachine RNA transcription kit (Ambion, Tex.). Helper and
replicon RNAs were then electroporated into BHK cells and incubated
at 37.degree. C. in .quadrature.-MEM supplemented with 10% FBS.
After 12 hours, the media was replaced with OPTI-MEM I (Invitrogen,
CA) supplemented with CaCl.sub.2) (100 .quadrature.g/mL) and cells
were incubated at 37.degree. C. After 24 hours, the supernatant was
collected, centrifuged to remove cellular debris, and frozen at
-80.degree. C. Titers of the vectors were determined as previously
described [15].
[0053] Mice and Tumor Inoculation. 4-8-week-old female BALB/c mice
were purchased from Taconic (Germantown, N.Y.). For the s.c. tumor
model, 0.5.times.10.sup.6 or 1.times.10.sup.6 CT26.WT or CT26.CL25
cells in 0.2 mL PBS were injected s.c. into the right flank of each
mouse. For the i.p. tumor model, 2.5.times.10.sup.4 or
5.times.10.sup.4 CT26.CL25 cells in 0.2 mL PBS were injected i.p.
into each mouse. For the lung tumor model, 0.3.times.10.sup.6
CT26.WT.Fluc or CT26.CL25.Fluc cells in 0.2 ml PBS were injected
i.v. into each mouse.
[0054] Therapeutic Efficacy. In the s.c. tumor model, treatment
started after tumor volume was more than 40 mm.sup.3
(volume=width.times.width.times.length/2). In the i.p. tumor model,
treatment started on day 4 after tumor cell inoculation. In the
lung tumor model, treatment started on day 3 after tumor cell
inoculation. SV/LacZ, SV/GFP or SV/Fluc (.about.10.sup.7 plaque
forming units in 0.5 mL of OPTI-MEM I) and mock treatments (0.5 mL
of OPTI-MEM I supplemented with 100 mg/L CaCl.sub.2)) were
administered i.p. 4 times a week for 2 weeks, for a total of 8
treatments. Therapeutic efficacy was monitored in three ways: tumor
volume (for s.c. tumors, measured with mechanical calipers), tumor
luminescence (for lung tumors), and survival (for i.p. and lung
tumors). Noninvasive bioluminescent imaging was done using the IVIS
Spectrum imaging system (Caliper Life Sciences, Inc., MA), and
tumor growth was quantified using the Living Image 3.0 software
(Caliper Life Sciences) as previously described [16]. Survival was
monitored and recorded daily.
[0055] Bioluminescent Imaging of SV/Fluc. Tumor-bearing and
tumor-free mice were injected with SV/Fluc (.about.10.sup.7 plaque
forming units in 0.5 mL of OPTI-MEM I 0.5 ml) intraperitoneally.
After the treatment, bioluminescence signal was detected by IVIS at
the indicated time points as previously described [16].
[0056] Ex Vivo Cytotoxicity Assay. Lung lymphocytes or splenocytes
from tumor-bearing mice were collected 7 days after SV treatment
started. Lung lymphocytes (1.times.10.sup.5/ml) or splenocytes
(2.times.10.sup.6/ml) were co-cultured with CT26.WT.Fluc cells
(2.times.10.sup.4/ml) or CT26.CL25.Fluc cells (2.times.10.sup.4/ml)
in a 24-well plate for 2 days in 1 ml RPMI 1640 supplemented with
10% FBS. Culture media were then collected for interferon
(IFN)-.gamma. secretion assays, and the remaining cells in each
well were washed twice with PBS. Cells were then lysed with 100
.mu.l of M-PER Mammalian Protein Extraction Reagent (Pierce, Ill.)
per well. Cytotoxicity was assessed based on the viability of the
CT26 cells, which was determined by measuring the luciferase
activity in each well. Luciferase activity was analyzed by adding
100 .mu.l of Steady-Glo reagent (Promega corp., WI) to each cell
lysate, and measuring the luminescence using a GLOMAX portable
luminometer (Promega corp.).
[0057] IFN-.gamma. Secretion Assay. Lung lymphocytes
(1.times.10.sup.5/ml) or splenocytes (2.times.10.sup.6/ml) were
stimulated by CT26 tumor cells (2.times.10.sup.4/ml) or immunogenic
peptides (5 .mu.g/ml) in a 24-well plate in 1 ml RPMI 1640
(Mediatech) supplemented with 10% FBS. The peptides used were the
LacZ peptide TPHPARIGL [43], the gp70 peptide SPSYVYHQF [44], or
the MA peptide LPYLGWLVF as a negative control [45]. After
stimulation, IFN-.gamma. levels in the media were measured using a
mouse IFN-.gamma. Quantikine ELISA kit (R&D systems,
Minneapolis, Minn.). TPHPARIGL and SPSYVYHQF-mediated increase in
IFN-.gamma. secretion was calculated by subtracting the IFN-.gamma.
levels in the control (LPYLGWLVF stimulated) samples from the
IFN-.gamma. levels in the TPHPARIGL and SPSYVYHQF stimulated
samples.
[0058] Flow Cytometry. Anti-mouse antibodies anti-CD8a eFluor.RTM.
450 and eFluor.RTM. 650NC, anti-CD4 PE-Cyanine7, anti-CD69 PE,
anti-CD314 (NKG2D) PE-Cyanine7, anti-CD62L (L-selectin) FITC and
Alexa Fluor.RTM. 700 and anti-CD45 eFluor.RTM. 450 were purchased
from eBioscience (San Diego, Calif.). PE-labeled LacZ tetramers
were obtained from the NYU Vaccine and Cell Therapy Core (New York,
N.Y.), and APC-labeled gp70 tetramers were obtained from the NIH
Tetramer Core Facility (Atlanta, Ga.). For flow cytometry analysis
of lung lymphocytes and splenocytes, mice were euthanized, and
their lungs and spleens were extracted. The extracted lungs were
chopped into small pieces and incubated with a digestion mix
(collagenase I (50 .mu.g/ml), collagenase IV (50 .mu.g/ml),
hyaluronidase V (25 .mu.g/ml) and DNAse I (20 units/ml)) for 30
minutes at 37.degree. C. Extracted spleens and digested lungs were
then mashed through 70-100 .mu.m cell strainers, followed by a
treatment with 1.times.RBC lysis buffer (eBioscience) to eliminate
red blood cells. Peritoneal cells were collected from peritoneal
exudates as previously described [19]. Cells were then stained with
various Abs, washed twice with HBSS (Mediatech) and analyzed using
an LSR II machine (BD biosciences, CA). Data was analyzed using
FlowJo (Tree Star, San Carlos, Calif.).
[0059] CD8.sup.+ T Cell Depletion. CD8.sup.+ T cells were depleted
using anti-CD8 antibody (clone 2.43) (Bio X cell, Lebanon, N.H.).
0.4 mg antibody in 0.2 mL PBS was injected into each mouse,
starting 1 day before the first SV treatment, and then every 2-3
days for 2 weeks. Control mice were injected with PBS.
[0060] Statistics. For flow cytometry, IVIS imaging, ELISA, tumor
growth, and survival experiments, student t tests (2-tailed),
analysis of variance (ANOVA) followed by Dunnett's test, or
Kaplan-Meier log-rank test were done using Prism.RTM. 4 for
Macintosh (GraphPad Software, Inc., La Jolla, Calif.).
Example 1: SV/LacZ Inhibits the Growth of LacZ-Expressing Tumors in
Immunocompetent Mice
[0061] In order to evaluate the use of SV vectors carrying TAAs for
cancer therapy, a LacZ-expressing mouse colon cancer cell line
(CT26.CL25) as a model tumor-TAA system. Initially, SV/TAA
(SV/LacZ) efficacy in mice bearing subcutaneous (s.c.) tumors was
tested. As seen in FIG. 1a, SV/LacZ significantly inhibited the
growth of LacZ-expressing CT26.CL25 tumors, while the control
vector SV/Fluc had no observable therapeutic effect (FIG. 1a, left
panel). On the other hand, both SV/LacZ and SV/Fluc had little
effect on the growth of LacZ-negative CT26.WT tumors (FIG. 1a,
right panel). These results demonstrate that SV/LacZ has a powerful
antigen-dependent therapeutic effect in mice bearing s.c. CT26
tumors.
[0062] In order to investigate SV/LacZ efficacy in a
physiologically relevant model of colon cancer, CT26.CL25 cells
were injected intraperitoneally to mimic peritoneal carcinomatosis
[26]. Therapeutic efficacy in this model was assessed by monitoring
mouse survival. As in the s.c. model, SV/LacZ was found to have a
potent therapeutic effect against these tumors, while the control
vector (SV/Fluc) had only a minor therapeutic effect (FIG. 1b).
Next, the efficacy of SV/LacZ against tumors growing in the lung
was examined. To supplement the survival data in this model,
Fluc-expressing CT26 cell lines (CT26.CL25.Fluc and CT26.WT.Fluc)
were constructed, which can be used to monitor tumor growth
noninvasively using the IVIS imaging system [16]. I.v. injection of
Fluc-expressing CT26.CL25 cells produced lung tumors, and it was
found that SV/LacZ induced complete tumor remission and long-term
survival in this model, while the control vector, SV/GFP, only
slightly delayed tumor growth and did not result in long-term
survival (FIG. 1c). As in the s.c. tumor model, the enhanced
therapeutic effect obtained from SV/LacZ in the lung tumor model
was dependent on the expression of the TAA (LacZ) from both the
vector and the tumor cells, as LacZ-negative CT26.WT tumor growth
was only slightly inhibited by SV/LacZ (FIG. 9). Taken together,
these results demonstrate that SV vectors carrying a TAA induce a
potent therapeutic effect in mice bearing TAA-expressing CT26
tumors, regardless of the site of tumor growth.
Example 2: Mediastinal Lymph Nodes Transiently Express Antigens
Delivered by SV Vectors, and are a Site of Early T Cell Activation
During SV Therapy
[0063] It has been previously shown that SV vectors have oncolytic
potential, and can target certain tumors in vivo [16]. In order to
evaluate the role of tumor cell targeting in the therapeutic effect
observed in the CT26 tumor model, tumor-bearing mice were treated
with SV/Fluc vectors, which can be used to monitor vector
localization in mice [16]. It was found that even after multiple
injections, SV vectors did not target s.c. growing CT26.CL25 tumors
(FIG. 2a, left panel). Similarly, the vectors did not target lung
tumors; instead, SV/Fluc was seen in the peritoneal fat of
tumor-bearing mice 24 hours after the first injection, and in the
liver 5 days later (FIG. 10). This general pattern was not
dependent on the presence of tumor cells, and occurred in
tumor-free mice as well (FIG. 10). These results are consistent
with other studies demonstrating that CT26 cells are not infected
by SV in vitro [20], and suggest that the powerful therapeutic
effect obtained from SV/LacZ is not dependent on tumor cell
targeting.
[0064] Interestingly, by focusing on very early time points after
SV/Fluc injection, it was noticed that a transient Fluc signal can
be seen in the upper body as early as 3 hours after i.p. SV/Fluc
injection (FIG. 2a and FIG. 10). By extracting the mediastinal
lymph nodes (MLN) and imaging them separately, it was determined
that the upper body signal originated from these lymph nodes (FIG.
2a, right panel). Notably, transient Fluc expression in the MLN
occurred in both tumor-bearing and tumor-free mice (FIG. 10).
[0065] The MLN has previously been shown to drain the peritoneum
[27, 28], and represents an environment in which antigens delivered
by SV vectors (such as Fluc, LacZ, or other TAAs) can potentially
be processed and presented to T cells by antigen presenting cells
(APC) in the context of SV viral danger signals such as double
stranded (ds) RNA [22]. The MLN therefore provides a possible
location for the induction of an immune response to SV/TAA.
Consistent with this hypothesis, the number of T cells in the MLN
significantly increased 24 hours after SV/LacZ treatment (FIG. 2b).
As a control lymph node, we used the inguinal lymph nodes (ILN),
which do not directly drain the peritoneal cavity [29], and were
not targeted by i.p. injection of SV/Fluc (FIG. 2a and additional
data not shown). Unlike the MLN, there was no increase in T cells
in the ILN 24 h after SV/LacZ injection (FIG. 2b). In addition to
the apparent influx of T cells into the MLN, the expression of
CD69, which is an early activation marker of T cells, was highly
induced on CD8.sup.+ T cells in the MLN 24 hours after SV/LacZ
treatment (FIG. 2c). In contrast, CD8.sup.+ T cells from the
control ILN were significantly less activated, though a slight
increase in CD69 expression was observed in these cells (FIG. 2c).
Taken together, FIG. 2 demonstrates that tumor cell targeting is
not required for effective SV/LacZ therapy, and suggests that
immune cell activation during SV/LacZ therapy may occur far away
from the tumor site, e.g. in lymph nodes that drain the SV
injection site.
Example 3: SV/LacZ Treatment Induces a Robust Activation of
CD8.sup.+ T Cells
[0066] Because the activation of CD8.sup.+ T cells in lymph nodes
draining the SV injection site was observed, it was anticipated
that activated CD8.sup.+ T cells might subsequently migrate into
the injection site in the peritoneum. Using flow cytometry, it was
confirmed that a large number of CD8.sup.+ T cells influx into the
peritoneum by 7 days after the first SV/LacZ injection (FIG. 3a).
These peritoneal CD8.sup.+ T cells were activated, as evidenced by
their upregulation of NKG2D [30] and downregulation of lymph node
homing receptor L-selectin [31] (FIG. 3b). In addition to the
robust influx of activated CD8.sup.+ T cells into the peritoneum, a
small number of NKG2D high, L-selectin low CD8.sup.+ T cells could
also be seen in the lungs of mice bearing lung CT26.CL25 tumors
that were treated with SV/LacZ (FIG. 3b).
Example 4: SV/LacZ Treatment Induces LacZ-Specific Effector and
Memory CD8.sup.+ T Cells
[0067] The fact that SV therapeutic efficacy depends on the
expression of LacZ from both the vector and the tumor cells (FIG. 1
FIG. 9), in conjunction with the robust activation of CD8.sup.+ T
cells observed during SV/LacZ therapy (FIGS. 2 and 3) suggest that
CD8.sup.+ T cells may be involved in the anti-cancer effect of
SV/LacZ in this model. Nevertheless, CD8.sup.+ T cell activation
also occurred during SV/GFP and SV/Fluc therapy (FIG. 11), even
though these vectors had significantly lower therapeutic efficacy
(FIG. 1). It was hypothesized that what distinguishes SV/LacZ from
the other vectors is its ability to directly stimulate
LacZ-specific CD8.sup.+ T cells that can subsequently target
LacZ-expressing tumors. To demonstrate this concept, SV/LacZ was
injected into a LacZ-naive tumor-free mouse, a robust LacZ-specific
CD8.sup.+ T cell response in the peritoneum 4 days later was
observed (FIG. 12a). An increase in LacZ-specific CD8.sup.+ T cells
was also observed in the spleens of s.c. tumor-bearing mice (FIGS.
4a and b), in the peritoneum of i.p. and lung tumor-bearing mice
(FIG. 4c), and in the lungs of lung tumor-bearing mice (FIG. 4d,
left panel) treated with SV/LacZ. Fewer LacZ-specific CD8.sup.+ T
cells were seen in mice treated with control vectors (FIG. 12b). As
expected, LacZ-specific CD8.sup.+ T cells from SV/LacZ-treated mice
were characterized by an activated (NKG2D high, L-selectin low)
phenotype (FIG. 4d, right panel, and FIG. 12a, right panel). Taken
together, these results demonstrate that SV/LacZ treatment leads to
the potent activation of LacZ-specific CD8.sup.+ T cells, providing
a possible mechanism for the LacZ-dependent efficacy seen in FIG.
1.
[0068] In order to determine if a subset of the LacZ-specific
CD8.sup.+ T cells generated during SV/LacZ therapy eventually
develop into memory T cells, splenocytes from SV/LacZ-treated
long-term surviving mice that bore i.p. CT26.CL25 tumors were
analyzed. Using LacZ tetramers in combination with the memory
marker CD127, a population (roughly 1% of the CD8.sup.+ T cell
splenocyte population) of LacZ-specific, CD127.sup.+ memory
CD8.sup.+ T cells in these mice was identified, more than 3 months
after the last SV/LacZ injection. Control splenocytes from naive
mice had only background levels of this population (under 0.1%)
(FIG. 12c).
Example 5: SV/LacZ Treatment Induces Lymphocyte Cytotoxicity
Against CT26.CL25 Tumor Cells
[0069] As shown in FIG. 4d, LacZ-specific CD8.sup.+ T cells in the
lungs of lung tumor-bearing mice treated with SV/LacZ appeared to
be activated. In order to investigate the function of these cells,
an ex vivo cytotoxicity assay was performed using lung lymphocytes
obtained from lung tumor (CT26.CL25)-bearing mice receiving SV/LacZ
(or SV/GFP) therapy. As shown in FIG. 5a, the viability of
CT26.CL25 tumor cells was significantly lower when they were
co-cultured with lung lymphocytes from SV/LacZ-treated mice
compared to when they were co-cultured with lymphocytes from mock
or SV/GFP-treated mice. Notably, lung lymphocytes from
SV/LacZ-treated mice did not affect the viability of LacZ-negative
CT26.WT tumor cells, demonstrating the antigen-specific nature of
the immune response in the lung. Consistent with this result, only
lung lymphocytes from SV/LacZ-treated mice that were co-cultured
with LacZ-expressing CT26.CL25 tumor cells showed an increase in
IFN-.gamma. production (FIG. 5b).
Example 6: CD8.sup.+ T Cells are Required for the Antigen-Specific
Enhanced Therapeutic Effect of SV/LacZ
[0070] The results of the cytotoxicity and IFN-.gamma. secretion
assays (FIG. 5) are consistent with the in vivo observation that
SV/LacZ has a significantly stronger therapeutic effect against
CT26.CL25 tumors than control vectors (FIG. 1), and with the
observation that SV/LacZ induces a powerful LacZ-specific CD8.sup.+
T cell response in tumor-bearing mice (FIG. 4). Taken together,
these results strongly suggest the involvement of CD8.sup.+ T cells
in the antigen-specific benefits of SV/TAA therapy. In order to
directly determine the role of CD8.sup.+ T cells in the therapeutic
effects observed, the CD8.sup.+ T cell population in mice bearing
s.c. (FIG. 6a), peritoneal (FIG. 6b), and lung (FIG. 6c) tumors was
depleted, and confirmed that in the absence of CD8.sup.+ T cells,
the therapeutic efficacy of SV/LacZ was greatly reduced in all 3
models.
Example 7: SV/LacZ Therapy Induces Epitope Spreading
[0071] Surprisingly, it was found that, unlike lung lymphocytes,
splenocytes from SV/LacZ-treated tumor-cured mice acquired
cytotoxicity against not only CT26.CL25 cells, but also
LacZ-negative CT26.WT cells (FIG. 7a). Consistently, an increase in
IFN-.gamma. production was observed when these splenocytes were
co-cultured with CT26.WT cells, although the extent of the
production was lower than when they were co-cultured with CT26.CL25
cells (FIG. 7b). Based on these results, it was hypothesized that
SV/LacZ-treated tumor-cured mice might have acquired resistance to
LacZ-negative CT26.WT tumors. To determine if this was the case,
CT26.WT cells were injected i.v. (FIG. 7c) or i.p. (data not shown)
into SV/LacZ-treated tumor-cured mice, and found that the tumors
did not grow. In contrast, tumor growth was readily observed in
control (naive) mice. These results suggest that an immune response
to endogenous CT26 tumor antigens might have developed as a
consequence of SV/LacZ therapy, a concept known alternatively as
epitope spreading, antigen spreading, determinant spreading, or
antigen cascade [32]. To confirm that epitope spreading occurred
during SV/LacZ therapy, gp70 was focused on, which is an endogenous
CT26 TAA. As shown in FIG. 7d, an increase in IFN-.gamma. secretion
from splenocytes taken from SV/LacZ-treated tumor-cured mice was
observed after culturing these cells with either gp70 or LacZ
peptides, whereas, neither peptide induced IFN-.gamma. secretion
from naive splenocytes. These results indicate that splenocytes
from SV/LacZ-treated tumor-cured mice could respond to endogenous
CT26 TAAs such as gp70 in addition to LacZ. Consistent with this
observation, flow cytometry analysis using gp70 tetramers
demonstrated that the number of gp70-specific CD8.sup.+ T cells was
increased in the spleens of SV/LacZ-treated tumor-cured mice
compared with naive mice (FIG. 7e). Taken together, these results
indicate that SV/LacZ therapy against CT26.CL25 tumors induced
epitope spreading, which led to the development of immunity against
other antigen(s) expressed on the CT26 tumors.
[0072] Disclosed herein, a mouse cancer-TAA system was used to
investigate the use of SV vectors carrying TAAs for cancer therapy,
and the following key observations were made: (i) SV represents a
potentially powerful therapeutic platform for the immunogenic
delivery of TAAs, (ii) the therapeutic benefit obtained from SV/TAA
does not necessarily require the direct targeting of tumor cells,
(iii) SV/TAA therapy involves transient early delivery of the TAA
to lymph nodes draining the injection site, in particular the MLN
in the case of i.p. SV injection, (iv) SV/TAA therapy induces a
potent TAA-specific CD8.sup.+ T cell response, that is subsequently
redirected against tumor cells expressing the cognate TAA, (v)
SV/TAA therapy leads to epitope spreading, providing a possible
solution to the problem of tumor escape by TAA loss or
modification, and (vi) SV/TAA therapy ultimately leads to long-term
survival of tumor-bearing mice, and to the generation of
long-lasting memory CD8.sup.+ T cells against multiple TAAs.
[0073] Based on these findings, a four-step model for the
activation of CD8+ T cell mediated anti-tumor immunity during
SV/TAA therapy (induction, cytotoxicity, epitope spreading, and
memory), is provided.
[0074] Over the last few decades, a variety of methods have been
developed for the immunogenic delivery of TAAs, including the
employment of vectors that target Antigen Presenting Cells (APCs)
[33], or are directly injected into lymph nodes [34]. Disclosed
herein, it was demonstrated that a single i.p. injection of SV/TAA
leads to the rapid immunogenic delivery of TAAs to the MLN. TAA
expression in the MLN is transient, and likely would have remained
unnoticed without the use of the sensitive IVIS imaging system.
I.p. injections are frequently used in animal studies, and are
becoming increasingly common in the clinic [35]. Observations of
transient TAA expression and subsequent T cell activation at this
site (FIG. 2) may therefore have broad implications for the
development of cancer immunotherapies. In this context, Hsu et al.
have recently demonstrated that i.p.-injected cytomegalovirus
resulted in the productive infection of CD169.sup.+ macrophages in
the MLN [28]. Consistent with this, depletion of macrophages
substantially reduced the expression of SV-derived heterologous
protein in the MLN (unpublished results). Notably, however, the
induction of anti-TAA CD8.sup.+ T cell immunity was not
significantly inhibited in macrophage-depleted mice that were
treated with SV/LacZ (unpublished results). This discrepancy may be
resolved by the observation that while both macrophages and
dendritic cells (DC) express viral antigens in draining lymph
nodes, only DC efficiently present these antigens to naive
CD8.sup.+ T cells [36]. Another possible explanation is the fact
that additional lymph nodes besides the MLN drain the peritoneal
cavity. Indeed, transient heterologous protein expression was also
observed in the abdominal cavity of SV-treated mice (FIG. 10, top
panel). Further studies are needed, and are underway, to clarify
the role of TAA delivery to the MLN during SV/TAA therapy.
[0075] Besides the activation of T cells in the MLN, there appears
to be a systemic redistribution of CD8.sup.+ T cells early after
SV/TAA injection. Various tissues, including the peritoneum (FIG.
11a) and the lung (FIG. 13), show a reduction in CD8.sup.+ T cells
in the first 1-2 days after SV/TAA injection. The apparent efflux
of T cells from these tissues coincides with their influx into the
MLN (FIG. 2B). It is interesting to note that during this early
phase, lung tumors in SV/TAA-treated mice already appear to shrink
(FIG. 1c). Moreover, this early therapeutic effect was also
observed in mice treated with control vectors that do not express
the TAA (FIG. 1c), in SV-treated mice bearing tumors that do not
express the TAA (FIG. 9), and in SV/TAA-treated mice that were
depleted of CD8.sup.+ T cells (FIG. 6c). One possible explanation
for this is the activation of natural killer (NK) cells by SV. It
has been previously shown that SV therapy induces a robust NK cell
response in tumor-bearing mice [19]. In the CT26 lung model, a
rapid influx of NKG2D-expressing NK cells into the lung was
observed as early as 2 days after SV injection, several days before
the maximum influx of TAA-specific CD8.sup.+ T cells (FIG. 13).
[0076] One of the limitations of cancer vaccine strategies is that
the inherent heterogeneity and genomic instability of tumor cell
populations, coupled with the selective pressure induced by the
treatment, can lead to tumor evasion by loss or modification of the
TAA used in the vaccine [38, 39]. In this context, an interesting
and therapeutically significant observation is the induction of
epitope spreading, i.e. the expansion of the anti-tumor T cell
response to incorporate novel TAAs that are endogenous to the
tumor, but not delivered by the vector [32] during SV/TAA therapy
(FIG. 7). Clinical trials are increasingly incorporating the
analysis of epitope spreading [40], and in some cases a positive
correlation between the induction of epitope spreading and
therapeutic efficacy has been shown [25]. These developments may
signify a paradigm shift in the design of cancer vaccines, whereby
an emphasis would be placed on the induction of a strong
diversified T cell response that could potentially be effective
even against tumors with heterogeneous antigen expression.
[0077] In summary, the present application provides methods for the
use of SV/TAA for cancer therapy, and provides valuable insight
into the mechanisms underlying SV/TAA efficacy. Pursuant to the
present invention, using SV vectors that carry a TAA not only
greatly enhances SV efficacy, but also abrogates the need for tumor
cell targeting--a hitherto prerequisite for effective oncolytic SV
therapy--thereby paving the way for a much broader application of
SV anti-cancer therapy. The current findings, in addition to
previous investigations into the oncolytic potential of SV [15,
16], compliment and expand upon earlier studies on the use of SV
nucleic acid [41] and replicon particle [42] vaccines, and
illustrate the versatility of SV anti-cancer therapy.
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[0123] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will be apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0124] It is further to be understood that all values are
approximate, and are provided for description.
[0125] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes
* * * * *
References