Note: Descriptions are shown in the official language in which they were submitted.
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MANUFACTURING MULTI-DOSE INJECTION READY DENDRITIC CELL VACCINES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part application of Serial No.
PCT/US] 5/41022 filed July 17, 2015 which in turn claims priority and benefit
from U.S.
Provisional Application Serial No. 62/025,673, filed July 17, 2014, U.S.
Provisional
Application Serial No. 62/165,445, filed May 22, 2015, U.S. Provisional
Application
Serial No. 62/025,685, filed July 17, 2014, and U.S. Provisional Application
Serial No.
62/028,774, flied July 24, 2014, the contents of each of which are
incotporated by
reference herein in their entireties.
ACKNOWLEDGMENT
The present invention was developed in part with government support
under grant number ROI CA096997 awarded by the National Institutes of Ilealth.
The
government has certain rights in this invention.
BACKGROUND
Dendrific cells (DCs) are white blood cells that acquire protein antigens
from microbes or even cancerous cells and show, or "present" these antigens to
T cells.
The T cells, thus activated by the DCs, then initiate systemic immune
responses to
challenge the threat Traditional vaccines against microbes contain additiv'es
known as
"adjuvants" that by a number of possible means enhance DC activity within the
vaccinated individual and amplify vaccine-induced immune responses. 'fhe
requirements
of vaccines against cancer, however, present a number of unique problems. For
example,
traditional adjuvants do not provide the proper signals to Des that allow them
to initiate
optimal immunity against cancer. Also, the tumors themselves produce an
environment
that can affect the proper activation of DC's.
A popular solution to this problem is to extract DCs from cancer patients,
load them with tumor allkeTIS in vitro, and then supply unique activation
signals to the
cells before re-administering them to the body. This ensures proper DC
activation
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removed from the influence of the tumor environment_ When returned to the
body, the
DCs can then interact with Teas and initiate powerful anti-tumor immunity.
Whereas
the use of extra-corporealized'DCs has solved many efficacy issues, it has
historically
come at the price of practical limitations. For example, since the DC vaccines
are
comprised of living cells, a special cell processing and vaccine production
facility has
been required at the physical location of the medical center administering the
therapy.
This is an expensive and inefficient way to deliver the therapy because every
institution
administering such treatment would have to build and maintain their own
special-use
Management of breast cancer currently relies on a combination of early
diagnosis and aggressive treatment, which can include one or more treatments
such as
surgery, radiation therapy, chemotherapy, and hormone therapy. Herceptin
(trastuzumab)
was developed as a targeted therapy for HER2fErb-B2 positive breast cancer
cells, often
used in conjunction with other therapies, including the mitotic inhibitor
paclitaxel (sold
under the trademark Taxol).
The efficacy of Herceptin as a monotherapy is estimated to be. less than.
30%; combinatorial treatment with microtubtile stabilizing drugs such as
paclitaxe-t.
increases efficacy to approximately 60% (Burris et al., 2000, Semin Oncol 27:
19-23).
Treatment with Herceptin results in accumulation of the Cdk inhibitor p27 and
20. subsequent GUS cell.cycle arrest, and paclita.xel stalls the entry Of
mitosis which canlead
to cell death: In spite of great promise, however, high doses- of Herceptin or
paclitaxel
result in undesirable side effects. Further, the cancer often develops
resistance to
Herceptin and/or paclitaxel.
Therefore, there remains an unmet need for compositions andefficient.
methods for producing maximal therapeutic DC vaccines and for new methods of
treating
cancer using :Herceptin. Accordingly, there is a need in the art to have
additional
immtmotherapeutic approaches for treating or preventing breast cancer and
other
Malignancies. The present embodiments fulfill this need.
BRIEF DESCRIPTION OF THE DRAWINGS
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=
The followMg detailed description of preferred embodiments will be better
understood when read in conjunction with the appended drawings. For the
purpose of
illustrating the embodiments, there are shown in the drawings embodiments
which are
presently preferred. It should be understood, however, that the embodiments
are not
limited to the precise arrangements and instrumentalities of the embodiments
shown in
the drawings.
Figure us a chart showing the viability and yield of post-thawed
ciyopreserved DC1. Recovery of cells was on average 89% and viability was 95%
when
cells were directly thawed and counted.
Figure 2 is a chart showing that here was no significant difference in the
viability (p-,4807), and recovery (p-.1220) of the cells.
Figure 3 is a chart showing that both populations had similar initial (7
hours post LPS addition) 1L-12 p70 secretion (v.0768). The populations
continued to
exhibit comparable secretion levels of IL-12 p70 over a 30 hour observation
period with
no significant differences between the populations.
Figure 4 is a chart showing that there was no significant difference
between populations and production of IL-1p (p=0.7690), IL-In (v0.0841),
Raines
(p-0.902), MDC (V0.1514), 1L-8 (v0,7844); (p-
.2673), IP-10 (v0.7366), IL-
6 (p-0.24), TINF-u (p---4).8972); 1L-5 (p-0.0735),1L-15(p-0.8878), IL-10 (p-
.1937),
Nup-ip (p-A8.9217),
Figure 5 is a chart showing production of1FN-f-froiri cryopreserved and
non-cryopreserved DCs.
Figures 6A-6D show Th Icytokines TNF-a and IFN-y synergize to induce
senescence in breast cancer cells and the doses required are in an inverse
correlation with
the LIER2 expression. Figure 6A shows results of studies in which SK-BR-3
breast
cancer cells were incubated with 10 ngtml TNF-a and 100 Ii/m1IFN-7 for 5 days,
cultured for 2 more passages in absence of crokines and then stained for SA-p-
uaiactosidase (SA-0-gal) expression (senescence marker) and compared to
untreated
control cells. Only paired cytokines induced senescence. Top panel shows
photographs
of representative data horn I of 3 independent experiments. Bottom
pariel.shows a
histogram of densitometfic analysis. Data are presented as '.'4) of SA-0-gal-
positive cells
3
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and presented as mean + S.D. (n=3). ). P- values were caleulated using a
paired Students
t-test. Statistical significance was determined at *P < 0.05. Figure 613 shows
photographs
of western blot analysis in which cell lysates of the cells described in
Figure 6A were
analyzed for p15INKb and p 1 6INK4a expression. Vinaulin was used as loading
control.
Figure 6C shows results of studies in which 1-471) breast cancer cells were
untreated (1)
or incubated with the following concentrations of TNF-a and INF-y; 10 nglad
and 100
U ml (2), 50:tigimi and 5001J:Iinl (3), 75 rigirtil and 750 U ml (4), and 100
tiginil and.
1000 .1.11m1 (5) for 5 days and i::ultured tot 2 more :passages in absence
:.of cytokines. The
cells were then stained for SA--gal and compared to control untreated cells or
those
treated with 8 u114. etoposide as a positive control (6). Top panel shows
photographs of
representative data from 1 of 3 independent experiments. Bottom panel shows a
histogram of densitometric analysis of the 6 studies. Data are presented as %
of SA-0-
gal-positive cells and presented as mean + S.D. (n=3). P-values were
calculated using a
paired Student's t-test. Statistical significance was determined at *P <0.05.
**P < 0.01,
***I' < 0.001. Figure 6D is a histogram showing remits of studies in which
combination
treatment with nil cytokines IFINI-1 and INF-ct resulted in greater senescence
in SK-13R-
3 (10 rig/nil TNF-a + 100 U.fml IFN,y) and1-47D (100 nglird NF-ci + 1000 -
Ultril IFN-
y) cells, compared with untreated controls; NIDA-MB-231 cells (200 nglinl TN.F-
a +
2000 1.1/ml :IFN-y) remained largely unaffected by dual 1EN-7 + TNF-a.
treatment. Data
are presented as % of SA-0-gal-positive cells and presented as mean + S.D.
(n=3). P-
values were calculated using a paired Student's t-test. Statistical
significance was
determined at **,P < 0.01, ***P < 0,001.
Figures 7A-7B show HER2 induces senescence and apoptpsi lvIDA-
MB-231 breast cancer cells, Figure 7A, left panel, is a histogram showing
results of
densitometric analysis in which SA-0-gal staining was performed in Mak-NI13-
231 cells
transfected with wt IHER2 (pcDNAHER2) or with empty vector (peDNA3) which were
treated with the listed concentrations of TNF-a and IFN-y for 5 days and
cultured for 2
more passages in absence of cytokines. Inset above the histogram are
photographs of
western blot analysis in which MDA-MB-231 cells transfected with pcDNAHER2 or
pcDNA3 and probedwith HER2 specific antibody were analyzed for confirmation
of.
expression or lack thereof VinCtiiin was used as loading control. Data are
4
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presented a % of SA-I3-ga1-positiv'e cells and presented as mean + S.D, P-
values
were calculated using a paired Student's t-test. Statistical significance was
determined at
**''`F <o= 0= Figure 7A, right panel, shows photographs of representative data
from I
of 3 independent experiments. Figure 713 shows photographs of western blot
analysis of
the cell lysates of the cells described in Figure 7A for expression of p15INKb
and
cleaved caspase-3. Vinciilin was used as loading control.
Figures $,-sp show combined HER2 and HER blockage expression
enhances h Icytokines TNE-a.-and lFN.y senescenceindoction and apoptoSisin 5K-
I3R-3 breast cancer cells. Figure 8A shows results of studies in. which SA-13-
gal staining
was pertbrmed in SK-BR-3 cells transfected with non,target siRNA (siRNA mr.),
HER2
siRNA, HER3 siRNA or a combination of HER2 and HER3 siRNA, and then treated
with the concentrations listed of TNF-a and IFN-y for 5 days and cultured for
2 more
passages in absence of cytokines. Left panel shows a histogram of den
sitometric
analysis. Data are presented as % of SA-0-gal-positive cells and presented as
mean +
- SD. (r3), P-values were calculated using a paired .Student's mest.
Statistical
significance as de.terthined at ***14 < 0.001. Inset shows photographs of
western blot
analysis of SK-BR-3 cells transfected with NT, HER2, or HER3 siRNA probed with
HER and HER3 specific antibodies. Vinculin was used as loading control.
Similar
results were observed in 3 independent experiments. Right panel shows
photographs of
representative data from 1 of 3 independent experiments. Figure 8B shows
results of
studies in which lysates of the cells described in Figure 8A were analyzed by
western
blotting for 0151NKb. and cleaved. easpase-3 expression
Figures.9A-9C show combined HER2 inhibition and .HER2-HER3
dimerization inhibition enhances Thl cytokines rtkw.-a. and EFN-li senescence
inditction
and apoptosis in SK-BR-3 breast cancer cells. Figure 9A shows results of SA-13-
gal
staining performed in SK-BR-3 cells which were untreated (1) or treated with
10 ngluil
TNF-a and 100 1.11m1 IFN-7 (2), or with 10 u.g/m1 of trastuzumab (Tan),
pertuzumab
(Per)(3), or with the combination of both treatments (4) for 5 days and
cultured for 2
more passages in absence of the antibodies and the cytokines. Left panel is a
histogram
of densitometric analysis. Data are presented as % of SA--gal-positive cells
and
presented as mean + S.D. (n=3). ). P-values were calculated using a paired
Student's t-
5
CA 02986687 2017-11-21
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test Statistical significance was determined at ***P <0.001. Right panel are
photographs showing representative data from I of 3 independent experiments.
Figure 911
shows photographs of western blot analysis in which cell lysates of the cells
described in
Figure 9A were analyzed for p151NKb or cleaved caspase-3 expression. Vinculin
was
used as loading control. Similar results were observed in 3 independent.
experiments.
Figure 9C shows results of studies of induction of apoptosis of SK-BR-3 cells
untreated
or treated as described above performed by staining for annexin V and Pi and
analyzed
by flow cytometry_ Top panel shows plots of representative data from 1 of 3
independent
experiments. Bottom panel, shows a histogram of densitometric analysis. Data
are
presented as average SEM of annexin V Pr cells from 3 independent
experiments. P-
values were calculated using a paired Student's t-test. Statistical
significance was
determined at **P <0.01.
Figures I0A-1013 show combined treatment with trastuzumab: and
pertuzuniab enhance C1)4:' Thi -mediated Senescence and apoptosis of HE R2-
ovexpressing human breast cancer cells. Figure 10A shows results of studies in
which.
using a transwell system, 0.5x 105 SK-BR-3 cells were co-cultured with 5x I 03
CD4' T-
cells alone (CD4' only), CD4' 1-cells + 05x10 each of HER2 Class :1:1 peptide
(DC H)-
or irrelevant Class B. BRAF or survivin peptides (DC B. or DC S)-pulsed type I
polarized
mature DCs, and CD4' T-cells + HER2 (iDC 11)-pulsed immature DCs arx: H), with
or
without 10 rig nil of trastuzumab (Tztn).andpertuzumah(Per) for 5 days: The
cells were
then cultured for 2 more passages in absence of the blocking antibodies' and
the immune
system cells and then stained for SA-0-gal expression and compared to
untreated control
cells. Top panel is a histogram of densitometric analysis. Data are presented
as
SA-13-gal-positive cells and presented as mean + S.D. (n=3). P-values were
calculated
using a paired Students t-test. Statistical significance was determined at
***P <0.000L
Bottom panel shows photographs of representative data from I of 3 independent
experiments. Figure 108 shows photographs of western blot analysis of cell
lysates of
SK-BR-3 cells co-cultured as indicated and analyzed for p15INK4b and cleaved
caspace-
3 expression. Increased pl 5INK4b and cleaved caspase-3 expression is seen
that
suggests induced senescence and apoptosis of SK-BR-3 cells, respectively when
co-
cultured with the DC H/CD4'1.-cells in presence of trastuzumab and pertuzumab,
but not
6
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'front DC B, DC S. and-iDC 11 giNPS. Vint").din as used as iloading:control.
:Results are
representative of 3 independent- experiments. photographs of western blot
analysis in
which cell tysates of the cells described in Figure 9A were analyzed for
p15INKb or
cleaved caspase-3 expression,
Figures 11A-111) show. Thi cytokineS TM -a and IFNI sensitize
trastuzumab and pertuzumab resistant breast cancer cellsita senescence and
apoptosis
induction, figure 11A shows results of SA:1)7gal staining performed in HCC-
1419 and
mwr- t cells respectively untreated (1) or treated with $0 rig ml INS4 and 500
11 nil
IFNI (2), or treated with 10 rig/m1 of trastuzumab (Tzm), pertuzumab (Per)
(3), or
treated with the combination of the same concentrations of trastuzumab,
pertuzumab and
INF-a, IFN-y (4) for 5 days and cultured for 2 more passages in absence of the
antibodies and the cytokines. Top panel is a histogram of densitometric
analysis. Data
are presented as % of SA-0-gal-positive cells and presented as mean + S.D.
P-
values were calculated using a paired Student's t-test. Statistical
significance was
determined at **P <0.01. Bottom panel, top layer, shows photographs of
representative
data from I of 3 independent experiments in HC-1419 cells. Bottom panel,
bottom
layer, shows photographs of representative data from 1 of 3 independent
experiments in
JIMT- I cells, Figure 1111 shows results of cell lysates of the cells
described in Figure
11A which were analyzed by western blotting for pi 5INKb or cleaved caspase-3
expression in HC-1419 (left panel) and .11MT-1 (right panel). Vinculin was
used as
loading control. Similar results were observed in 3 independent experiments.
Figure 12 shows 1FN-yRa and TNF-RI are expressed in Similar levels in
breast cell lines independently from their -HER2 level. IFN-yRet, INF-RI and
HER2
expression in immortalized MCF-I OA mammary epithelial cells and breast cancer
cell
lines (SK-BR-3, BT-474, MCF-7, T-47D and MDA-MB-231) as determined by Western
blot. Vinculin was used as loading control. Similar results were observed in 3
independent experiments.
Figures 13A-13B show combined HER2 and FIER3 blockage expression
enhances Tb 1cytokines INF-a and IFNI senescence and appptosis induction in MC
F-7
breast cancer cells. Figure 13A shows'results of studies in which SA43-gal
staining Was
performed in MICF-7 cells transfected with non-target .s.iRNA (siRNA NT), HER2
7
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siRNA ora-combination of 11ER2 and HER3 sill:NA, and then treated
with the concentrations listed of INF-a and UN-'y for 5 days and cultured for
2 more
passages in absence of cytokines. Left panel shows a histogram of
densitometrie
analysis. Data are presented as % of SA-fl-gal-positive cells and presented as
mean
SD. (n-3), P-values were calculated using a paired Student's t-test,
Statistical
significance was determined at "*P < 0.001. Inset shows photographs of western
blot
analysis of MCF-7 cells transf.ected with NT. HER2 or HER3 or a combination of
HER2
and HER3 sikNA probed with HER?. and HER3 specific antibodies. Vinculin was
used
as loading control. Similar results were observed in 3 independent experiments
Right
panel shows photographs of representative data from I of 3 independent
experiments.
Figure 1311 shows results of studies in which cell lysates of the cells
described in Figure
13A were analyzed by western blotting for p15INKb or cleaved caspase-3
expression.
***P <0.001. Inset shows photographs of western blot analysis of SK-BR-3 cells
transfected with NT, HERZ, or HER.3 sikNA probed with HER and HER3 specific
antibodies. Vineulin was used as loading control. Similar results were
observed in 3.
independent experiments.
Figures 14A-I 413 show the effect of Th I-elaborated cytokines on SK-BR-
3 senescence and apoptosis. Figure I4.Ashows the results of studies using a
transwell
system, whereby 0.5x105 SK-BR-3 cells were co-cultured with 5x] 05 CD4.' T-
cells alone
(CD4.i. only), CD4' 0.5x105 each of 11ER2 Class fl peptide (DC -H)- or
irrelevant Class II BRAE peptide (DC B)7pulsed type 1:polariZed mature DC :s,
and CD4'"
T-cells HE.R2 (iDC H)- or BRAF (iDC B)-pulsed immature DCs for 5 days. The
cells
were then cultured for 2 more passages in absence of immune system cells and
then
stained .for SA-0-gal expression and compared to untreated control cells,
Compared with
IgG isotype control, senescence induced in SK-BR-3 treated with CD4VDC H is
partially
rescued by neutralizing IFNI and TNF-a with specific antibodies (75.27 %
rescue). Top
panel is a histogram of densitometric analysis. Data are presented as % of SA-
13-gal-
positive cells and presented as mean + S.D. (n3),). P-values were calculated
using a
paired Student's Hest. Bottom panel shows corresponding photographs of
representative
data from 1 of 3- independent experiments. Figure 1411 shows photographs Of
western
blots show tog pi 5INK4h
and cleaved caspase 3-expressions which suggest
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senescence and apoptosis induction of SK-BR-3 cells µvhen co-cultured With
Dell,"CD4*
T-cells compared with DC B, IDC H, and iDC B groups. Compared with IgG isotype
control, senescence and apoptosis induced in SK-BR-3 treated with CD4"/DC H
were
partially rescued by neutralizing IFN-7 and TNF-u specific antibodies.
Vinculin was
used as loading control. Results are representative of 3 experiments.
Figure 15 shows the effect of trastuzumab and pertuzumab on AKT
activation by heregulin in breast cancer cell lines. Serum-starved T-47D, HCC-
1419 and
JIM`17-1 cells were treated with trastuzumab (Tzm) and pertuzumab (Per 10
uglini, 90
min) and then stimulated with (HRG, 20 ng/ml, 5 min). Top panel, shows
representative
data from 1 of 3 independent experiments. Data are expressed as % of the HRG
response
in the absence of trastuzumab and pertuzumab and presented as mean S.D.
Figure 16 shows vaccination procedure_ Patients with biopsy diagnosed
HER2P's DCIS were enrolled in the trial. Patient's monocytes were collected by
leukapheresis and elutriation. The monocytes were rapidly matured into type 1
DC:s, and
the pre-vaccination anti-HER2 CD4 nu immune response was measured. Patients
underwent 4-6 weekly vaccinations (+/- anti-estrogen therapy). Patient's
monocytes
were collected again by a second leukapheresis and elutriation, and the post-
vaccination
anti-HER2 CD4 Thl immune response was measured_ Following vaccination,
patients
underwent surgical resection to cure them of residual disease. The clinical
response was
measured in the surgical specimen and the itrumme response was mugged in the
Sentinel
lymph noties(SLNS") When available.
Figures 17A-17B shows results of SKBR3 and MCF7 breast cancer cell
lines treated with Thl eytokines (IFNy and INFO, a tamoxifen metabolite
(l¨Ifydroxy-
Tamoxifen, "4HT"), or both. SKBR3 (ER) (Figure 17A) increased anti-tumor
activity
in response to Thl cytokine treatment, but not in response to anti-estrogen
treatment or
the combination treatment. 1VICF7 (ER') (Figure 17B) did not increase anti-
tumor
activity in response to either Th I cytokine treatment or anti-estrogen
treatment, but the
combination reaulted in an increase in imetabolicaetiity
Figure 18 shows patient distribution for the combination anti!-estrogen
("AE") therapy and anti-HER2 DC1 vaccination study. HER-2 positivity was
defined as
>5% of cells expression 2+ or 3+ intensity of the HER-2 protein on
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immunohistochemistry. AE therapy (Tamoxifen, LetrOzole, or Anastrozole) as
gh,en
concurrently with DC vaccination.
Figure 19 is a histogram showing pathologic complete response rate
comparing patients by ER status and AE treatment. (ER "-% ER" W/0 AE; ER" w
AE).
Figures 20A-20B shows subsequent breast events of the study patiets
comparing patients by pathologic complete response ("pC1r) (Figure 20A) and ER
status
and AE treatment (Figure 208).
Figures 21A-21C show C1)4+ systemic immune response measured in the
peripheral blood. By each metric of Th I response (responsivity (Figure 21A);
response
repertoire (Figure 218) and cumulative response (Figure 21C)), there was a
significant
increase in the immune response following anti-HER2 DC1 vaccination. However,
the
pre- and post-vaccination immune responses were similar across all three
groups.
Figures 22A-22C show CD4 local regional immune response measured in
patient sentinel lymph nodes. By each metric (responsivity (Figure 22A);
response
repertoire (Figure 228) or cumulative response (Figure 22C)) the post-
vaccination
immune responses were higher in the ER patients who received AE compared to
the
EW'' patients who did not receive AE.
Figure 23 shows CD8' systemic immune response measured in the
peripheral blood. Responsivity of patients with ElVeg status and those with ER
P' status
with and without anti estrogen treatment (ER P"' wi`o AE; ER" wAE) are shown,
Figure 24 shows BRAFwmE-DC1 vaccines overcome vemurafenib
resistance in BRAF-mutant murine melanoma.
DETAILED :DESCRIPTION
The present embodiments provide compositions arid methods for
producing an FDA-approved injectable multi-dose antigen pulsed dendrific cell
vaccine
for the personalized treatment and prevention of cancer or other disorders. In
one
entbodiment, the embodiments provide cotnpositiODS and methods for producing
an
FDA-approved injectable multi-dose antigen pulsed type I polarized dendritic
cell
vaccine (DC1).
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In one embodiment, there is provided a method to ciyopreservedendritic
cells in multiple-dose aliquots that are in an antigen-loaded, pre-activated
state that is
"syringe-ready", i.e. suitable for immediate injection into the patient
without the
necessity of any further cell processing that would require (e.g., by FDA
mandate)
additional facilities and quality control/assurance steps.
In one embodiment, there is provided a method to efficiently produce
injectable multi-dose antigen pulsed dendritic cell vaccine, preferably
injectable multi-
dose antigen pulsed type I polarized dendritic cell vaccine that exhibit
maximal efficacy.
In one embodiment, an FDA-approved injectable multi-dose antigen
pulsed dendritic cell vaccine is produced by collecting DCs in a single
patient
leukapheresis. Preferably, the leukapheresis and production of the dendritic
cell vaccine
is performed at a first location whereby the first location can be a
centralized vaccine
production facility where the DCs are manipulated to create an activated.
antigen-loaded
DC vaecine comprised of an initial immunizing dose and multiple "booster doses
thereof An advantage of the present embodiments is that all FDA mandated
quality
control/quality assurance steps would be performed at the central facility,
and after
completion and release, all vaccine doses are cryopreserved and shipped to
remote
medical centers for serial administration to the patient. In one embodiment.,
the FDA-
approved injectable multi-dose antigen pulsed dendritic cell vaccine of the
embodiments
dOes not requirement any Mandated quality .control/quality assurance steps at
the
administration site
In another aspect, the present embodiments are based on the discovery that
an effective therapy to treat cancer includes changing the immune response in
the tumor
so that the immune cells in the tumor site are more effective in attacking the
tumor cells.
hi some instances, the effective therapy includes improving the migration and
activity of
immune cells in the tumor site. Accordingly, the embodiments provide
compositions and
methods of using a dendritic cell vaccine in combination with a composition
that inhibits
one or moie Of KR-2 and. HER,3 trastwaLtriab, pertigOW), and the like) asia
treatment regimen to treat cancer_ In one entodiment,. the treatment regimen
comprises
the use of dendritic cell vaccines, an inhibitor of HER-2, arid a chemokine
modulator.
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In one embodiment there is provided compositions and methods for using
a dendritic cell vaccine in combination with blockage crone or more of HER-2
and
HER-3 as a treatment regimen to treat cancer. En another embodiment, there is
provided
compositions and methods of using a dendritic cell vaccine in combination with
blockage
of HER-2 and HER-3 with the addition of TNE-ct and IFN-y. In another
embodiment
there is provided compositions and methods of blocking one or more of HER-2
and HER-
3 with the addition of TNF-a. and .IFN-y as a treatment regimen to treat
cancer.
In one embodiment the treatment regimen of the embodiments comprise a
combination therapy of inducing an anti-oncodriver Thl immune response (e.g.,
INF-rx.
and IFN-y) and oncodriver blockade for one or more of HER-2 and HER-3
In one embodiment, the treatment regimen of the embodiments can be
used to treat cancer and therefore can be considered as a type of anti-cancer
therapy. In
another embodiment, the treatment regimen of the embodiments can be used in
combination with another anti-cancer therapy including but is not limited to
surgery,
chemotherapy, radiation therapy (e.g. X ray), gene therapy, immunotherapy,
hormone
therapy, viral therapy, DNA. therapy, RNA therapy, protein therapy, cellular
therapy,
nanotherapy, and the like.
En one embodiment, the treatment regimen of the embodiements is used
prior to receiving the other anti-cancer therapy. In another embodiment, the
treatment
regimen of the embodiments is used concurrently with receiving the other anti-
cancer
therapy_ In another embodiment, the treatment regimen of the embodiments is
used after
receiving the other anti-cancer therapy,
in another embodiment concurrent neoadjuvmut anti-estrogen therapy and
anti-HER2 DC1 vaccination increases the immune response in the local sentinel
lymph
nodes and the rate of pathological complete response in HER21"VERI" DCIS
patients.
Definitions
Unless defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in the
art to
which these embodiments belong. Although any methods and materials similar or
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tClUiVaittit to those described herein can be used in the ptoc.tice ot testing
of the present
embodiments, the preferred methods and materials are described.
Generally, the nomenclature used herein and the laboratory procedures in
cell culture, molecular genetics, organic chemistry, and nucleic acid
chemistry and
hybridization are those well-known and commonly employed in the art.
Standard techniques are used for nucleic acid and peptide synthesis. The
techniques and procedures are generally performed according to conventional
methods in
the art and various general references (e,g,. Sambrook and Russell, 2012,
Molecular
Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor,
NY,
and Ausubel et al., 2012, Current .Protocols in Molecular Biology, John Wiley
& Sons,
NY), which are provided throughout this document.
The nomenclature used herein and the laboratory procedures used in
analytical chemistry and organic syntheses described below are those well-
known and
cc:Muni:m-1y -employed in the att. Standard techniques or modifications
thereof are used for
chemical syntheses and chemical analyses.
As used herein, each of the following terms has the Meaning- associated
with it in this section.
The articles "a" and an arc used berein.to irefer to one or to more than
one (i.e., to at least one) of the grammatical object of the article, By way
of example an
element" Means one element Or more than one element,
"About" as used herein when referring to a measurable value, such as an
amount, a temporal duration, and the like, is meant to encompass variations of
20%, or
.-110%, or I.5%, or - 1%, or +0.1% from the specified value, as such
variations are
appropriate to perform the disclosed methods_
The term "abnormal" when used in the context of organisms, tissues, cells
or components thereof, refers to those organisms, tissues, cells or components
thereof
that differ in at least one observable or detectable characteristic (e.g.,
age, treatment, time
ad* etc.) from those OrganisinS, tissues, cells or components thereof that
.display the
"normal" (expected) respective characteristic. Characteristics which are
normal or
expected for one cell or tissue type, might be .abnormal for a different cell
or tissue type.
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Tho term 'antigen" or µat.)," as used herein is-defined:as a molecule that
provokes an immune response. This immune response may involve either antibody
production, or the activation of specific immunologically-competent cells, or
both. The
skilled artisan will understand that any macromolecule,- including virtually
all proteins or
peptides, can serve as an antigen. Furthermore, antigens can be derived from
recombinant
or genomic DNA. A skilled artisan will understand that any DNA, which
comprises a
nucleotide sequences or a partial nucleotide sequence encoding a protein that
elicits an
immune response therefore encodes an "antigen" as that term is used herein.
Furthermore, one skilled in the art will understand that an antigen need not
be encoded
solely by a full length nucleotide sequence of a gene. It is readily apparent
that the
present embodiments include, but are not limited to, the use of partial
nucleotide
sequences of more than one gene and that these nucleotide sequences are
arranged in
various combinations to elicit the desired immune response. Moreover, a
skilled artisan
will understand that an antigen need not be encoded by a "gene" at all. It is
readily
apparent that an antigen can be generated synthesized or can be derived from a
biological
sample. Such a biological sample can include, but is not limited to a tissue
sample, a
tumor sample, a cell or a biological fluid.
"An antigen presenting celr (APC) is :a cell that.arecapable.of aCtivating
T cells, and includes, but is not limited to, monocytesimacrophages. B cells
and denctritic
cells (PCs): =
'Antigen-loaded APC." or.an "antigen-pulsed APC" includes an APC,
which has been exposed to an antigen and activated by the antigen. For
example, an APC
may become Ag-loaded in vitro, e.g., during culture in the presence of an
antigen. The
.APC may also be loaded in vivo by exposure to an antigen. An "antigen-loaded
APC" is
traditionally prepared in one of two ways: ( 1) small peptide fragments, known
as
antigenic peptides, are "pulsed" directly onto the outside of the APCs; or (2)
the APC is
incubated with whole proteins or protein particles which are then ingested by
the APC.
These proteins are digested into small peptide .fragments by the APC and are
eventually
transported to and presented on the APC surface. In addition, the antigen-
loaded APC can
also be generated by introducing a wiyinicleotide encoding an antigen into the
cell.
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"Ann-HER2 response is the immune response specifically againstHER2
protein.
"Apoptosis" is the process of programmed cell death. Caspase-3 is a
frequently activated death protease.
The term "autoimmune disease" as used herein is defined as a disorder
that results from an autoimmune response. An atitthrIMUTIC disease is the
result of an
Inappropriate and excessive response to a self-antigen. Examples of autoimmune
diseases
include but are not limited to, Addision's disease, alopecia amata, ankylosing
spondylitis,
autoimmune hepatitis, autoimmune pm-otitis, Crohn's disease., diabetes (Type
I),
dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves'
disease,
Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus
erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris,
psoriasis,
rheumatic fever, rheumatoid arthritis, sarcoidosis, sclerodenna, Sjogren's
syndrome,
spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious
anemia,
ulcerative colitis, among others.
As used herein, the term "autologous" is meant to refer to any material
derived from the same individual to which it is later to be re-introduced into
the
individual.
The term "B cell" as used herein is defined as a cell derived from the bone
marrow and/or spleen. B cells can develop into plasma. cells which produce
antibodies_
The term "cancer" as used herein is defined as a hypetproliferation of cells
whose unique trait--loss of normal control--results in unregulated growth,
lack of
differentiation, local tissue invasion, and/or metastasis, Examples include
but are not
limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer,
skin cancer,
pancreatic cancer, colorectal cancer, renal cancer and lung cancer.
"CD4 Thl cells," "Thl "CD4+ T-
helper type !cells," "CD4+ T
cells," and the like are defined as a subtype of T-helper cells that express
the surface
protein CD4 and produce high levels of the cytokine &e also,
"T-helper cells."
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"Cumulative response" -means the combined immune response of a patient
group:expressed as the total sum ofteactive spots (spot-forming cells "SFC"
per 10 cells
from IFN-T:ELISPOT analysis) frorri all 6 MEC class II binding peptides from a
given
patient group.
The term "cryopreserved" or "clyopresmation7 as used herein refers to
cells that have been resuspended in a freezing medium and frozen at a
temperature of
around -70 C or lower,
"DC vaccination," -DC immunization," '"Del immunization," and the.
like refer to a strategy using autologous dendritic cells to harness the
immune system to
recognize specific molecules and mount specific responses against them.
The ternO'dendritie cell" (DC) is an antigen presenting cell existing in
vivo, in vitro, ex -Vivo, or in a host or subject, or which can be derived
from a
hematopoietic stem cell or a monocyte. Death-itic cells and their precursors
can be
isolated from a variety of lymphoid organs, e.g., spleen, lymph nodes, as well
as from
bone marrow and peripheral blood. The DC has a characteristic morphology with
thin
sheets (lamellipodia) extending in multiple directions away from the dendritic
cell body.
Typically, dendritic cells express high levels of MBE and costimulatory
(e.g.,137-1 and
B7,.2) molecules. Dendritic cellsean induce antigen specific differentiation
of T cells in
vitro, and are able. to .initiate primary T cell responses in vitro and in
vivo.
As used herein, an "activated 'DC" is a DC that has been exposed to a Toll-
like receptor ationist The activated DC may or may not be loaded with an
antigen.
The term "mature -DC" as used herein, is defined as a dendritic cell that
expresses molecules, including high levels ofMliC class Ti, CD80 (137.1) and
CD86
(B7.2). In contrast, immature dendritic cells express low levels of MI-1C
class It, CD80
(117.1) and CD86 (137.2) molecules, yet can still take up an antigen. "Mature
DC" also
refers to an antigen presenting:cell existing in vivo, in vitro, ex vi.vp, or
in a host or
subject that is DC1-polatized.(i.e., folly capable. of promoting cell-mediated
immapity).
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A `4disease " is a state .of health at animal *herein the animal cannot
maintain homeostasis, and-wherein if the disease is not ameliorated then the
animal's
health continues to deteriorate.
A "disorder" in an animal is a state of health in which the animal is able to
maintain homeostasis, but in .which the animal s-state-of health is less
favorable than it
wonld be in the absence of the disorder: Left untreated, a disorder does not
rie,cessatily
cause a further decrease in the animal's state of health.
A disease or disorder is "alleviated" if the severity or frequency of at least
one sign or symptom of the disease or disorder experienced by a patient is
reduced.
"Effective amount" or "therapeutically effective amount" are used
interchangeably herein, and refer to an amount of a. compound, formulation,
material, or
composition, as described herein effective to achieve a particular biological
result. Such
results may include, but are not limited to, the inhibition of virus infection
as determined
by any means suitable in the art.
As used herein "endogenous" refers to any material from or produced
inside an organism, cell, tissue or system.
As used herein, the term "exogenous" refers to any material introduced
from or produced outside an organism, cell, tissue or system.
"Estrogen receptor (ER) positive" cancer is cancer which tests positive for
expression of ER. Conversely, "ER negative" cancer tests negative for such
expressiOn.
Analysis of ER status can be performed by any method known in the art
The term "freezing medium" as used herein refers to any medium mixed
with a cell sample in preparation for freezing, such that at least some of
cells within the
cell sample can be recovered and remain viable after thawing.
"HER2" is a member of the human epidermal growth factor receptor
("EGER") family. HER2 is overexpressed in approximately 20-25% of human breast
cancer and is expressed in many other cancers
A "HER.reeeptor is a receptor poteio tyrOsitie kuase1.Vhich belongs to
the HER receptor family and includes EGER (EibBl, HERO, HER2 (ErbB2), HER3
(Erb133) and HERA (Erb134) receptors. The HER receptor will generally comprise
an
extracellular domain, which may bind an HER ligand andfor dimerize with
another HER
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receptor mole-edit ;, a lipophilie transmenthrane domain; a
conservedintracellular tyrosine
kinase domain; and a carboxyl-terminal signaling domain harboring several
tyrosine
residues which can be phosphorylated. The HER receptor may be a "native
sequence"
HER receptor or an "amino acid sequence variant" thereof Preferably the HER
receptor
is a native sequence human HER receptor.
The "HER pathway" refers to the signaling network mediated by the HER
receptor family.
"HER activation" refers to activation, or phosphorylation, of any one or
more HER receptors. Generally, HER activation results in signal transduction
(e.g. that
caused by an intracellular kinase domain of a HER receptor phosphorylating
tyrosine
residues in the HER receptor or a substrate polypeptide). HER activation may
be
mediated by HER ligand binding to a HER dimer comprising the HER receptor of
interest. HER ligand binding to a HER dimer may activate a kinase domain of
one or
more of the HER receptors in the dimer and thereby results in phosphorylation
of tyrosine
residues in one or more of the HER receptors andlor phosphorylation of
tyrosine residues
in additional substrate polypeptides(s), such as Akt or MAPK intracellular
kinases.
"HER2 binding peptides," "HER2 MHC: class IT binding peptides,"
"binding peptides," "HER2 peptides," "immunogenic MHC class II binding
peptides,"
"antigen binding peptides," "HER2 epitopes," "reactive peptides," and the like
as used
herein refer to MFIC Class II peptides derived from Or: based on the sequence
of the
IIER2inen protein, a target found on approximately 20-25% of all human breast
cancers
and their equivalents. HER2 extracellular domain "ECD" refers to a domain of
HER2
that is outside of a cell, either anchored to a cell membrane, or in
circulation, including
fragments thereof HER2 intracellular domain "IC!)" refers to a domain of the
HER2ineu protein within the cytoplasm of a cell. According to a preferred
embodiment
HER2 epitopes or otherwise binding peptides comprise 6 HER2 binding peptides
which
include 3 HER2 ECD peptides and 3 HER2 [CD peptides.
Preferred HER2 ECD peptides comprise:
Peptide 42-56: EILDML,RHEYQGCQVNT (SEQ NO; 1);
30. Peptide 98-114: .RERIVRGTQLFEDNYAL (SEQ. ID NO: 2);-and
Peptide 328.-345: TQRCEKCSKPCARVCYGL (SEQ ID NO; 3);:.
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Preferred HERZ ICD peptides comprise:
Peptide 776-790: GVOSPYVSRLLGICL (SEQ ID NO: 4);
Peptide 927-941: PAREIPDLLEKGERL (SEQ ID NO: 5); and
Peptide 1166-1180: TLERPKTLSPGKNGV (SEQ JO NO: 6).
:In embodiments where patients are FILA-A2P"9fbave A2.I blood type WIC class I
peptides or epitopes comprise:
Peptide 369-377:KIFGSLAFL (SEQ ID NO:7); and
Peptide 689-697:RLLQETELV (SEQ ID NO:)
"11ER2P"' is the classification or molecular subtype of a type of breast
cancer as well as numerous other types of cancer. 1{EIt2 positivity is
currently defined
by gene amplification by FISH (fluorescent in situ hybridization) assay and 2+
or 3+ on
intensity of pathological staining.
is defined by the lack of tame amplification by FISH and can
encompass a range of pathologic staining from 0 to 2+ in most cases_
The term "hyperproliferative disease" is defined as a disease that results
from a byperproliferation of cells. Exemplary hyperproliferative diseases
include, but are
not limited to, cancer or autoinmiune diseases. Other hyperproliferative
diseases may
include vascular occlusion, restenosis, atherosclerosis, or inflammatory bowel
disease,
for example.
The term "inhibit," as used herein, means to suppress or block an activity
or function, for example about ten percept relative to a control value.
Preferably, the.
activity is suppressed or blocked by 50% compared to a control value, more
preferably by
75%, and even More preferably by 95%. Inhibit," as used herein, also means to
reduce a
molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein's
expression,
stability, function or activity by a measurable amount or to prevent entirely_
:Inhibitors are
compounds that, e.g., bind to, partially or totally block stimulation,
decrease, prevent,
delay activation, inactivate, desensitize, or down regulate a protein, a gene,
and an
mRNA stability, expression, function and activity, e.g.,antagonists.
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As used herein, an "instructional material" includes a publication, a
recording, a. diagram, or any other medium of expression which can be used. to
communicate the usefulness of the compositions and methods of the embodiments.
The
instructional material of the kit of the embodiments may, for example, be
affixed to a
container, which contains the nucteicacid, peptide, and/or composition of the
embodiments Or be shipped together with a container which contains the nucleic
acid,
peptide, and/or composition. Alternatively, the instructional material may be
shipped
separately from the container with the intention that the instructional
material and the
compound be used cooperatively by the recipient.
"Isolated" means altered or removed from the natural state. For example, a
nucleic acid or a peptide naturally present in a living animal is not
"isolated," but. the
same nucleic acid or peptide partially or completely separated from the
coexisting
materials of its natural state is "isolated." An isolated nucleic acid or
protein can exist in
substantially purified form, or can exist in a non-native environment such as,
for
example, a host cell.
"Metrics" of CD4 Thl- responses (or-Thl responses) are defined
for each subject group analyzed for anti-HER2 CD4' Thl immune response: (a)
overall
anti41ER2 responsivity (expressed as percent of subjects responding to ?1
reactive
peptide); (b) response repertoire (expressed as mean number of reactive
peptides (n)
recognized by each subject grow); and (c) curtmlativ-e response (expressed as
total Sum
of -reactive spots (spot-fOrtning "S.FC" per 10' cells from IFNI, ELISPOT
analysis)
from 6 MHC Class II binding peptides from each subject group.
By the term "modulating," as used herein, is meant mediating, a detectable
increase or decrease in the level of a response in a subject compared with the
level of a
response in the subject in the absence of a treatment or compound, and/or
compared with
the level of a response in an otherwise identical but untreated subject. The
term
encompasses perturbing and/or affecting a native signal or response thereby
mediating a
beneficial therapeutic response in a subjeCt, preferably, a huntan.
"Neoadjimun therapy" for breast cancer as used herein refers to treatment
given before primary therapy (i.e., surgery). "Adjuvant. therapy" is treatment
given after
primary therapy to increase the chance of long-term survival.
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As used herein, a "populationr includes reference to an isolated culture
comprising a homogenous, a substantially homogenous, or a heterogeneous
culture of
cells. Generally, a "population" may also be regarded as an "isolated" culture
of cells.
As used herein, a "recombinant cell" is a host cell that comprises a
recombinant polynucleotide.
"Responsivity" or "anti-HER2 responsivity" are used interchangeably
herein to mean the percentage of subjects responding to at least 1 of 6
binding peptides.
"Response repertoire" is defined as the mean number ("n") of reactive
peptides recognized by each subject group.
"Sample" or "biological sample" as used herein means a biological
material from a subject, including but is not limited to organ, tissue,
exosome, blood,
plasma, saliva, urine and other body fluid. A sample can be any source of
material
obtained from a subject.
"Senescence" refers to cells no longer capable of dividing but which are
still alive and metabolically active. Hallmarks of senescent cells include an
essentially
irreversible growth arrest, and expression of SA4-gal, P15INK
4B and pl6INK4a,
"Signal I" as used herein generally referS to the first.biochemicatsignal
passed from an activated DC to a T cell. Signal.] is provided by an antigen
expressed. at
the surface of the. DC and is sensed by the T cell through the T cell
receptor%
Signal. 2" as used herein generally refers to the second signal provided by
-DCs to T cells. Signal 2 is provided by "costimulatoty" molecules on the
activated DC,
usually CD80 and/or CD86 (although there are other co-stimulatory molecules
known),
and is sensed by the I cell through the surface receptor.CD28.
"Signal 3" as used herein generally refers to the signal generated from
soluble proteins (usually cytokines) produced by the activated DC, These are
sensed.
through receptors on the I lymphocyte. The 3rd signal instructs the T cell as
to which
phenotypical or functional features they Should ae4uire to best deal with-the
current
threat.
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-BY the term "specifically binds," as used herein, is meant a molecule, such
as an antibody, which recognizes and binds to another molecule or feature, but
does not
substantially recognize or bind other molecules or features in a sample.
The terms "subject," "patient," "individual," and the like are used
interchangeably herein, and refer to any animal, or cells thereof whether in
viim or in
situ, amenable to the methods described herein. in certain non-limiting
embodiments, the
patient, subject or individual is a human.
"T/C" is defined as trastuzumab and chemotherapy. Tins -refers to patients
that receive both trastuzumab and chemotherapy before/after surgery for breast
cancer.
The term "T cell" as used herein is defined as a thymus-derived cell that
participates in a variety of cell-mediated immune reactions.
The terms "T-helper cells," "helper I cells," "Th cells," and the
like are used herein with reference to cells indicates a sub-group of
lymphocytes (a type
of white blood cell or leukocyte) including different cell types identifiable
by a skilled
person in the art. hi. particular, T-helper cells are effector T-cells (such
as Th I, Th2 and
Th17) whose primary function is to promote the activation and functions of
other B and T
lymphocytes and/or macrophages. Helper T cells differentiate into two major
subtypes of
cells known as "Thl" or "Type 1" and "Th2" or "Type 2" phenotypes. These Th
cells
secrete cytokines, proteins, or peptides that stimulate or interact with other
leukocytes.
"Thl "CD4.'. Thi "CD4 T;lielper type! "CD4'.I cell" and
the like as
used herein refer to a mature T-cell that has expressed the surface.
glycoprotein C04.
CD4* T-helper cells become activated when they are presented with peptide
antigens by
MEC class II molecules which are expressed on the surface of antigen-
presenting
peptides ("APCs") such as dendritic cells. Upon activation of a CD4j. T helper
cell by
the MHC-antigen complex, it secretes high levels of cytokines such as
interferon-I
("IFN-y"). Such cells are thought to be highly effective against certain
disease-causing
microbes that live inside host cells, and are critical in antitumor response
in human
cancer.
"TM T cell" as used herein refers to a I cell that produces high levels 01'-
the cytokine IFN-y and is thought to be highly effective against certain
disease-causing
microbes that live inside host cells, and cancer as well.
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"1h17 T cell" as used herein refers to .a T cell that produces high levels of
the cytokines IL-17 and 1L-22 and is thought to be highly effective against
disease-
causing microbes that live on mucousal surfaces.
"Therapeutically effective amount" is an amount of a compound of the
embodiments, that when administered to a patient, ameliorates a symptom of the
disease.
The amount of a compound of the embodiments which constitutes a
"therapeutically
effective amount" will vary depending on the compound, the disease state and
its
severity, the age of the patient to be treated, and the like. The
therapeutically effective
amount can be determined routinely by one of ordinary skill in the art having
regard to
his own knowledge and to this disclosure.
The terms "treat," "treating," and "treatment," refer to therapeutic or
preventative measures described herein. The methods of "treatment" employ
administration to a subject, in need of such treatment, a composition of the
present
embodiments, for example, a subject: afflicted a disease or disorder, or a
subject who
ultimately may acquire such a disease or disorder, in order to prevent, cure,
delay, reduce
the severity of, or ameliorate one or more symptoms of the disorder or
recurring disorder,
or in order to prolong the survival of a subject beyond that expected in the
absence of
such treatment.
The term "Toll like receptor", or "TLR" as used herein is defined as a
20. class of Proteins that play a:role in the innate-Mu-mine system. =TLRs
are single-
membrane-spanning, non-catalytic receptors that recognize structurally.
conserved
molecules derived from microbes. TLRs activate immune cell responses upon
.binding to
a ligand.
The term "Toll like receptor agonists)% or "TLR. agonists'as used herein is
defined as a ligand that binds to the TLR to activate immune cell response.
The term "vaccine" as used herein is defined as a material used to provoke
an immune response after administration of the material to an animal,
preferably a
mammal, and more preferably a hunian. Upon introduction into a subject the
vaceine Is
able to provoke an immune response including, but notlimited to, the
production of -
antibodies, cytokines and/or other cellular responses.
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Ranges : throughotnthis disclosure various aspects of the embodiments
can be presented in a range format. It Should be understood that the
description in range
format is merely for convenience and brevity and should not be construed as an
inflexible
limitation on the scope of the embodiments. Accordingly, the description of a
range
should be considered to have specifically disclosed all the possible subranges
as well as
individual numerical values within that range. For example, description of a
range such
as from I to 6 should be considered to have specifically disclosed subranges
such as from
I to 3, from I to 4, from I to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc.,
as well as
individual numbers within that range, for example, 1, 2, 22, 3, 4, 5, 53, and
6. This
applies regardless of the breadth of the range.
Description
The present embodiments include a preparation of DCs. In one
embodiment, the DC preparations are greater than 90% pure. In another
embodiment, the
DC preparations are fully activated. For example, the DCs are activated with a
cytokine
and/or a Toll like receptor ligand, a state which is fully maintained by the
cryopreservation technique of the embodiments. A benefit of the DC preparation
of the
embodiments is that the cells are efficiently cryopreserved from a single
leukapheresis
(patient collection) into an initial vaccine plus multiple "booster" doses
(e.g., 10 or more)
20. that can be thawed as needed at remote treatment locations -without any
specialized cell
processing facilities or further required quality control testing.
As contemplated herein, the present embodiments provide a method for
generating and cryopreseiving DCs with superior functionality in producing
stronger
signals to T cells, and thus resulting in a more potent DC-based vaccine. By
effectively
cryopreserving such cells, samples can be stored and thawed for later use,
thereby
reducing the need for repeated pheresis and elutriation processes during.
vaccine
production. Being able to freeze DCs and then thaw them out later is an
advantage
because it means that ..4 single round . vaccine production can he divided
into small.
parts, frozen away, and then administered one at a time to a patient over the
course of
weeks, months, or years to give "booster" vaccinations that strengthen
immunity,
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In one embodiment, the present embodiments includes an FDA- approved
injectable multi-dose antigen pulsed dendritic cell Vaccine produced by
collecting DCs in
a single patient letikapheresis. The FDA- approved injectable multi-dose
antigen pulsed
dendritic cell vaccine comprises an initial immunizing dose and multiple
"booster" doses.
The FDA- approved injectable multi-dose antigen pulsed dendritic cell vaccine
are
mopreserved and can be shipped to remote medical centers for serial
administration to
the patient with no special requirements at the administration site (e.g., FDA
mandated
QC/QA steps).
The present embodiments also relate to the cryopreservation of these
activated DCs in a manner that retains their potency and functionality in
presenting
antigen as well as their production of various cytokines and chemokines after
thawing,
such that the cryopreserved and subsequently thawed activated DCs are as
clinically
effective as freshly harvested and activated DCs.
The present embodiments also relate to inducing tumor senescence and
apoptosis in a cell by blocking one or more of HER-2 and HER-3 in combination
with
activating anti-HER-2 CD4 Thl cells. Accordingly, the embodiments include a
combination and method for promoting an anti-oncodriver Th I immune response
with an
oncodiiver blockade for HER-2 in order to promote tumor senescent in HER-2
expressing breast cancers. in one embodiment, promoting an anti-oncodriver Thl
29 immune response comprises TNF-a. and IFN-y. In one embodiment, an
oncodriver
blockade for HER-2 includes an composition that blocks HER-2 including but is
not
limited to trastuzumab and pertuzumab.
In one embodiment, there are compositions and methods for the
combination of blocking one or more of HER-2 and HER,3 together with the
addition of
TNF-0( and IFN-y for inducing senescence of HER-2 expressing breast cancer. In
one
embodiment, the INF-cL and IFN-y is secreted from CD4 Thl cells.
In one embodiment, HER2 is required in the mechanism of TNF-ct and
IFN-y inducing senescence and apopwsis in breast cancer cells.
in one embodiment, INF-a and IFINI-7 restores the sensitivity to
trastimonab and :pertnzurnab to breast cancer resistant cells In one
embodiment, the Th
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cytokines,IFN-y and INE-a, revert the resistance to the therapeutic agents
that is
affecting cancer patients widely.
DC-Based Immunotherany
Des are derived from pluripotent monocytes that serve as antigen-
presenting cells (APCs), DCs are ubiquitous in peripheral tissues, \here they
are
prepared to capture antigens. Upon antigen capture, DCs process the antigen
into small
peptides and move towards secondary lymphoid organs. It is within the lymphoid
organs
that DCs present antigen peptides to naive T cells, thereby initiating a
cascade of signals
that polarizes I cell differentiation. Upon exposure, DCs present antigen
molecules
bound to either MHC class I or class 11 binding peptides and activate CD8 or
CD4' T
cells, respectively (Steinman, 1991, Annu. Rev. Immunot 9:271-296; Banchereau
et al.,
1998, Nature392,245-252; Steinman, et al.õ 2007. Nature 449:419-426; Ginhoux
et at,
2007, J. ap. Med. 204:3133-3146; Banerjee et al., 2006, Blood 108:2655-2661;
Sallusto et at, 1999, J. Exp. Med. 189:611-614; Reid et al., 2000, Cum Opin,
Immuno1.12:114-121; Bykovskaia et al., 1999, J. Leukoc. Biol. 66:659-666;
Clark et at,
2000, Microbes infect, 2:257-272),
DCs are responsible for the induction, c.00rdination and revilation of the
adaptive immune response and also serve to orchestrate communication between
effectors of the innate arm and the adaptiAT arm of the immune system. These
features
have made DCs strong candidates for immunotherapy, DCs have a unique capacity
to
sample the environment through macropinocytosis and receptor-mediated
endocytosis
(Gerner et al., 2008,3. immunoll 81:155-164; Stoitzner et al., 2008, Cancer
Immunol.
Immunother 57:1665-1673; Lanzevecchia A., 1996, Curr_ Opin. Innnunol.8:348-
354;
Delamarre et at, 2005, Science, 307(5715):1630-1634).
DCs also require maturation signals to enhance their antigen-presenting
capacity. DCs upreaulate the expression of surface molecules, such as CD80 and
CD86
(also known as second signal molecules) by providing additional maturation
signals, such
as TNF-a, CD401, or calcium signaling agents (Czerniecki et at, 1997,. J.
Immunol.159:3823-3837; IBedrosian et al. 2000, J. Immunother. 23:311-320;
Mailliard
et at, 2004, Cancer Res.64,5934-5937; Brossart et at, 1998, Blood 92:4238-
4247; Jin et
26
CA 02986687 2017-11-21 =
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al 2004, Hum. Irimitmol. 65:91-103). It has been established that 4
mixture of cytokines,
including INF-,a, IL-6 and prostaglandin E2 (PGE2), have the ability- to
mature
DC (Jonuleit, et al., 2000, Arch. Derm. Res, 292:325-332). Des can also be
matured with
calcium ionophore prior to being pulsed with antigen.
:En addition to pathogen-recognition receptors, such as .PKR and MDA.-5
(Ka:lab et al., 2008, J. Immunol. 181:2694-2704; Nallagatla et al., 2008, RNA
Biol.
5(3):140-144), Des also contain a series of receptors, known as Toll-like
receptors
(lilts), that are also capable of sensing danger from pathogens. When these
TLRs are
triggered, a series of activational changes are induced in DCs, which lead to
maturation
and signaling of T cells (Boullart et al. 2008, Cancer Immunol. Immunother.
57(11):1589-1597; Kaisho et al., 2003, Curr. Mol. Med. 3(4)373-385; Pulendran
et al.,
2001, Science 293(5528):253-256; Napo! itani et al., 2005, Nat. Inummol.
6(8):769-776).
Des can activate and extend the various arms of the cell-mediated response,
such as
natural killer .-y-8 T and a-13 T cells and, once activated, Des retain their
immunizing
capacity (Steinman, 1991, Annu. Rev, Immunol. 9:271-296; Banchereau et al.,
1998,
Nature 392:245-252; Reid et al., 2000, CUM 0.pin. Immunol. 12:114-121;
Bykovskaia et
al., 1999, J. Leukoc. Bio1,66:659-666; Clark et al., 2000, Microbes Infect.
2:257-272).
The present embodiments include mature, antigen loaded Des activated
by Toll-like receptor agonists that induce clinically effective immune
responses,
20. preferably \Ohen used earlier in the disease proCeSs. The Des of
thepresemembodiments
produce desirable levels of cytokines and chemokines, and further have the,
capacity to
induce apoptosis of tumor cells.
In one embodiment, there is provided a method of large scale production
of antigen pulsed dendritic cell vaccine. In one embodiment, the method
comprises
rapidly maturing dendritic cells, cryopreserving the dendritic cells, and
thawing the
cryopresemed cells wherein the thawed dendritic cells produce an effective
amount of at
least one cytokine to generate a T cell response.
In one embodiment the maturation of dendritic cells eopriptie contacting
the cells with IFN-gamma and LIPS.
In one embodiment, the thawed cells maintain DC1 phenotype to drive a
ml polarized immune response.
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In One etribodiment, the thawed cells maintain the ability to primarily
sensitize T cells.
Generation of a loaded (pulsed) immune cell
The present embodiments include a cell that has been exposed Or
otherwise "pulsed" with an antigen. For example, an APC, such as a DC, may
become
Ag-loaded in vitro, e.g., by culture ex vivo in the presence of an antigen, OT
in vivo by
exposure to an antigen.
A person skilled in the an would also readily understand that an APC can
be "pulsed" in a manner that exposes the APC to an antigen for a time
sufficient to
promote presentation of that antigen on the surface of the APC. For example,
an APC can
be exposed to an antigen in the form of small peptide fragments, known as
antigenic
peptides, which are "pulsed" directly onto the outside of the APCs (Mehta-
Damani el al.,
1994); or APCs can be incubated with whole proteins or protein particles which
are then
ingested by the APCs. These whole proteins are digested into small peptide
fragments by
the APC and eventually carried to and presented on the APC surface (Cohen et
at, 1994).
Antigen in peptide form may be exposed to the cell by standard "pulsing"
techniques
described herein.
Without wishing to be bound by any particular theory, the antigen in the
20. form of a foreign- or an atitoantigen is processed by the APC of the
embodiments in otclet
to retain .the immunogenic form of The antigen. The immunogenic form of the
antigen
Implies processing of the antigen through fragmentation to produce a form of
the antigen
that can be recognized by and stimulate immune cells, for example I cells.
Preferably,
such a foreign or an autoantigen is a protein which is processed into a
peptide by the
APC. The relevant peptide which is produced by the APC may be extracted and
purified
for use as an immunogenic composition. Peptides processed by the APC may also
be
used to induce tolerance to the proteins processed by the APC.
The antigen-loaded APC, Otherwise known as a "pulsed APC" of the
embodiments, is produced by exposure of the APC to an antigen either in vitro
or in. vivo,
In the case where the APC is pulsed in vitro, the APC can be plated on a
culture dish and
exposed to an antigen in a sufficient amount and for a sufficient period of
time to allow
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the antigen to bind to the APC. The amount and time necessary to achieve
binding -of the
antigen to the APC may be determined by using methods known in the art or
otherwise
disclosed herein. Other methods known to those of skill in the art, for
example
immunoassays or binding assays, may be used to detect the presence of antigen
on the
APC following exposure to the antigen.
In a further embodiment, the APC may be transfected with a vector which
allows for the expression of a specific protein by the APC, The protein which
is
expressed by the APC may then be processed and presented on the cell surface.
The
transfected APC may then be used as an immunogenic composition to produce an
immune response to the protein encoded by the vector.
As discussed elsewhere herein, vectors may be prepared to include a
specific polynucleotide which encodes and expresses a protein to which an
immunogenic
response is desired. Preferably, retroviral vectors are used to infect the
cells. More
preferably, adenoviml vectors are used to infect the cells.
In another embodiment, a vector may be targeted to an APC by modifying
the viral vector to encode a protein or portions thereof that is recognized by
a receptor on
the APC, whereby occupation of the APC receptor by the vector will initiate
endocytosis
of the vector, allowing for processing and presentation of the antigen encoded
by the
nucleic acid of the viral vector. The nucleic acid which is delivered by the
virus may be
20- rtati've to the virus, which When expressed on the APC encodes Vital
proteinawhiehare
then processed and presented on the MIIC receptor of the APC.
As contemplated herein, various methods can be used for transfecting a
polytmeleotide into a host celL The methods include, but are not limited to,
calcium
phosphate precipitation, lipofection, particle bombardment, microinjection,
electroporation, colloidal dispersion systems (i.e. macromolecule complexes,
nanocapsules, microspheres, beads, and lipid-based systems including oil-in-
water
emulsions, micelles, mixed micelles, and liposomes). These methods are
understood in
the aft and are:described in published literature o as to enable one skilled
in the art to
perform these methods.
In another embodiment, a polynucleotide encoding an antigen can be
cloned into an expression vector and the vector can be introduced into an APC
to
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otherwise generate a loaded APC. 'Various types of vectors and methods of
introducing
nucleic acids into a cell are discussed in the available published literature.
For example,
the expression vector can be transferred into a host cell by physical,
chemical or
biological means. See, for example, Sambrook et al. (2001, Molecular Cloning:
A
Laboratory Manual, Cold Spring :Harbor Laboratory, New York), and in Ausubel
et al.
(1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).
It is
readily understood that the introduction of the expression vector comprising a
polynucleotide encoding an antigen yields a pulsed cell.
The present embodiments include various methods for pulsing APCs
including, but not limited to, loading APCs with whole antigen in the form of
a protein,
cDNA or mRNA. However, the embodiments should not be construed to be limited
to the
specific. form of the antigen used for pulsing the APC. Rather, the
embodiments
encompass other methods known in the art for generating an antigen loaded APC.
Preferably, the APC is tranfected with mRNA encoding a defined antigen. mRNA
corresponding to a gene product whose sequence is known can be rapidly
generated in
vitro using appropriate primers and reverse transcriptase-polymerase chain
reaction (RT-
PCR) coupled with transcription reactions. Transfection of an APC with an mRNA
provides an advantage over other antigen-loading techniques for generating a
pulsed
APC. For example, the ability to amplify RNA from a microscopic amount of
tissue, i.e.
tumor tissue, extends the use of the APC for accip4tion to a large number of
patients.
For an antigenic composition to be useful as a-vaccine, the antigenic
composition must induce an immune response to the antigen in a cell, tissue or
mammal
(e.g., a human). As used herein, an "immunological composition" may comprise
an
antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen
(e.g., an
antigen expression vector), or a cell expressing or presenting an antigen or
cellular
component. In particular embodiments the antigenic composition comprises or
encodes
all or part of any antigen described herein, or an immunologically functional
equivalent
thereof In Other embodiments, the antigenic Cornposition is in a mixture that
eomprises
an additional immunostimnlatory agent or nucleic acids encoding such an agent.
Immtmostimulatoty agents include but are not limited to an additional antigen,
an
immunomodulator, an antigen presenting cell or an adjuvant. In other
embodiments, one
CA 02986687 2017-11-21
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or more of the additional agent(s) is covalently bonded to the antigen or an.
immunostimulatory agent, in any combination, In certain embodiments, the
antigenic
composition is conjugated to or comprises an HLA anchor motif amino acids.
A vaccine, as contemplated herein, may vary in its composition of nucleic
acid and/or cellular components. In a non-limiting example, a nucleic encoding
an
antigen might also be formulated with an adjuvant. Of course, it will be
understood that
various compositions described herein may further comprise additional
components. For
example, one or more vaccine components may be comprised in a lipid or
liposome. In
another non-limiting example, a vaccine may comprise one or more adjuvants. A.
vaccine
of the present embodiments, and its various components, may be prepared and/or
administered by any method disclosed herein or as would be known to one of
ordinary
skill in the art, in light of the present disclosure.
It is understood that an antigenic composition oldie present embodiments.
may be made by a method that is well known in the art, includingbutnOt limited
to
chemical synthesis by solid phase synthesis and purification away from the
other
products of the chemical reactions by HPLC, or production by the expression of
a nucleic
acid sequence (e.g., a DNA sequence) encoding a peptide or polypeptide
comprising an
antigen of the present embodiments in an in vitro translation system or in a
living cell. In
addition, an antigenic composition can comprise a cellular component isolated
from a
20. biological sample. The antigenic composition isolated and extensively
dialyzed to
remove. one or more undesired small molecular weight molecules andlor
lyophilized for
more ready formulation into a desired vehicle. It is ftuther understood that
additional
amino acids, mutations, chemical modification and such like, its any, that are
made in a
vaccine component will preferably not substantially interfere with the
antibody
recognition of the epitopic sequence.
A peptide or polypeptide carresponding to one Of more antigenic
determinants of the present embodiments should generally be at least five or
six amino
acid tesidnes in length, and may contain op to about. 10 about 15õ about 20,
about 25,
about 3.0, about 35õ about 40, about 45 or about 50 residues or so. A peptide
sequence
may be synthesized by methods known to those of ordinary skill in the art,
such as, for
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example, peptide synthesis using automated peptide synthesis. machines, such
as those
available from Applied Biosystems, Inc., Foster City, CA (Foster City, CA).
Longer peptides or polypeptides also may be prepared, e.g., by
recombinant means. in certain embodiments, a nucleic acid encoding an
antigenic
tompOsitiOli.andlor a component described herein may be used, for example, to
produce
an antigenic composition. in Om or in viva for the various compositions and
methods of
the present embodiments. For example, in certain embodiments, a nucleic acid
encoding
an antigen is comprised in, for example, a vector in a recombinant cell. The
nucleic acid
may be expressed to produce a peptide or polypeptide comprising an antigenic
sequence.
The peptide or polypeptide may be secreted from the. cell, or comprised as
part of or
within the cell.
In certain embodiments, an immune response may be promoted by
transfecting or inoculating a mammal with a nucleic acid encoding an antigen.
One or
more ells comprised within a. target mammal then expresses the sequences
encoded by
the nucleic acid after administration of the nucleic acid to the mammal. A
vaccine may
also be in the form, for example, of a nucleic acid (e.g., a cDNA or an RNA)
encoding all
or part of the peptide or polypeptide sequence of an antigen. Expression in
viva by the
nucleic acid may be, for example, by a plasmid type vector, a. viral vector,
or a
virallplasmid construct vector.
In another einbodiment, the 'nucleic acid comprises a coding region that
encodes allor.part of the sequencesencoding an appropriate-antigen, or an
immunologically functional equivalent thereof. Of course, the nucleic acid may
comprise
andior encode additional sequences, including hut not limited to those
comprising one or
more immunomodulators or adjuvants.
Antigens
As contemplated herein, the present embodiments may include use of any
antigen. Suitable for loading into an: APc to elicit an immune feVOLISe . In
one
embodiment, tumor antigens may be used. Timor antigens can be divided into two
broad
categories: shared tumor antigens; and unique tumor antigens. Shared antigens
are
expressed by many tumors, while unique tumor antigens can result from
mutations
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induced through physical or chemical careinogens, and are therefore expressed
only by
individual tumors. In certain embodiments, shamd tumor antigens are loaded
into the
DCs of the present embodiments. In other embodiments, unique tumor antigens
are
loaded into the DCs of the present embodiments.
In the context of the present embodiments, "-tumor antigee refer to
antigens that are common to specific hyperproliferative disorders in certain
aspects, the
hyperproliferative disorder antigens of the present embodiments are derived
from
cancers, including but not limited to, primary or metastatic melanoma,
thymoma,
lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkins
lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney
cancer and
adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer,
pancreatic
cancer, and the like.
Malignant .tumors express a number of proteins that can serve as target
antigens for an inmmtne -attack. These Molecules include, but are not limited
to, tissue-
specific antigens such as MART-1, tyrosinase and OP 100 in melanoma, and
prostatic
acid phosphatase (PAP) and prostate-specific antigen wsi-io in prostate
cancer. Other
target molecules belong to the group of transformation-related molecules, such
as the
oncogene HER-2/NeulErbB-2. Yet another group of target antigens are onco-fetal
antigens, such as carcinoembryonic antigen (CEA). In B cell lymphoma, the
tumor-
specific idiotype immunoglobulin constitutes a Indy tumor-specific
immtmoglobtilin
antigen that is unique to the individual tumor, 13 cell differentiation
antigens, such as
CD! 9, CD20 and CD37, are other candidates for target antigens in B cell
lymphoma.
Some of these antigens (CEA, HER-2, CD 19, CD20, idiotype) have been used as
targets
for passive inunwtotherapy with monoclonal antibodies with limited success.
The tumor antigen and the antigenic cancer epitopes thereof may be
purified and isolated from natural sources such as from primary clinical
isolates, cell lines
and the like. The cancer peptides and their antigenic epitopes may also be
obtained by
chemical syntheSis or by recombinant DNA. tediniquesiknoNVii. in the arts.
Techniques for
chemical synthesis are described in Steward et at (1-969);:-Bodansky et at
(1974
Meienhofer (1983); and Schroder et at. (1965). Furthermore, as described in
Renkvist et
al. (2001), there are numerous antigens known in the art. Although analogs or
artificially
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modified .epitopes:are not specifically described, a skilled artisan
recognizes how to
obtain or generate them by standard means in the art. Other antigens,
identified by
antibodies and as detected by the Serex technology (see Sahin et al. (1997)
and Chen et
al. (2000)), are identified in the database of the Ludwig Institute for Cancer
Research.
In yet another embodiment, the present embodiments may include
microbial antigens for presentation by the APCs. As contemplated herein,
microbial
antigens may be viral, bacterial, or fungal in origin. Examples of infectious
virus include:
Retroviridae (e.g. human immunodeficiency viruses, such as HIV -1 (also
referred to as
HTLAT-Ill, LAY or HILN-III/LAY, or tifY411.; and other isolates, such as H1V-
LP;
Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human
coxsackie
viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause
gastroenteritis);
Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae
(e.g. dengue
viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g.
corortaviruses);
Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae
(e.g. chola
viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles
virus,
respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses);
Bungaviridae
(e.g. Hantaan viruses, bunga viruses, phleboviruses and Naito viruses); Arena
viridae
(hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviruses and
rotaviruses);
Bimaviridae; Hepadnaviridae (Hepatitis B virus);1Parvovirida (parvoviruses);
20. Papovaviridae (Papilloma viruses, polyoma iruses); Adenovitidae (most
adertoviruses);
Iletpesviridae (limes simplex virus (11W) I and 2, vaticella zoster virus,
cytomegalovirus (CMV), herpes virus); Poxviridae (variola viruses, vaccinia
viruses, pox
viruses); and rridoviridae (e.g. African swine fever virus); and unclassified
viruses (e.g.
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).
.ExaMples of infectious baCteria includet Helitobacter pyloriS,.E3orelia
burgdorferi, Legionella pneumophilia, IMycobacteria sps (e :g, M.
tuberculosis, M. aviurn,
M. intracellulare, M. kansasii, M. gordonae), Staphylococcus aureus, Neisseria
gonorrboeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus
pyogenes
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(Group A Streptococcus), Streptococcus agalactiae (Group 13 StreptococcuS),
Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic
Clunpylobacter
sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis,
corynebacterium
diphtheriae, corynebacterium sp., Etysipelothtix rhusiopathiae, Clostridium
perfringens,
Clostridium tetani, Enterobacter aemgenes, Klebsiella pneumoniae, PastureIla
multocida,
Bacteroides sp, Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema
Treponema pertenue, Leptospira, and Actinomyces israeilL
Examples of infectious fungi include: Cryptococcus neofbrmans,
Histoplasma capsulatum, Coccidioides immitis, Blastornyces dermatitidis,
Chlamydia
trachomatis, Candida albicans. Other infectious organisms (i.e., protists)
including:
Plasmodium falciparum and Toxoplasma
Activation of DCs
While traditional DC-based vaccines (that have previously dominated
clinical trials) comprise of matured DCs generated from using a cytokine
cocktail mixture
including combinations of TNIF, 1L-6, PGE2 and 1L-113, which ultimately
stimulate
aseptic inflammation, the present embodiments instead utilize TLR agonists to
mature the
DCs and stimulate production of signal_
According to an aspect of the present embodiments, the stimulation of
DCs. with a combination of TLR ligands leads to the production of increased
amounts of
1L-12. Further, activation of DCs with a combination of TLR agonists can yield
a more
pronounced C1)4 and CDS T-cell response (Waraer et at., 2006, Blood 108:544-
550).
Thus, the DCs of the present embodiments can secrete Th1 driving cytokines,
such as IL-
12, by exposure to these ligands that trigger ThRs. For example, the addition
of
poly(1:C), a TLR3 agonist to EL-1 13, INF-a, and IEN-y, can generate a potent
type-I
polarized DC, characterized by robust levels of IL-12 production (Heifler et
al., 1996,
Eur. Inatutiol. 26 6 S9 -668) In CertainettiboditnOts, antigen can be loaded
into. the
DC prior to TLR agonist exposure. in other-embodiments, antigen can be loaded
into the
DC subsequent to MR agonist exposure.
=
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According to an aspect of the present .emhodinients, the injectable multi-
dose antigen pulsed dendritic cell vaccine is produced by collecting DCs in a
single
patient leukapheresis whereby the cells are activated with biomolecules that
simulate
bacterial infection (e.g., LPS). This unique activation method endows the DCs
with
qualities not found in DCs that are matured with a cytokine cocktail of `INF,
11,6, .PGE2
and IL-1p (the "traditional maturation"), which also simulates aseptic
inflammation
(Lombardi et at.. 200, J. Immunol. 182:3372-3379).
In one embodiment, the DCs of the present embodiments can be activated
with the combination of the TLR4 agonist, bacterial lipopolysa.ccharide (LPS),
the
TLR7/8 agonist, resimiquod (R848) andlor IFN-y (Amati et al., 2006, Curt.
Pharm. Des
12:4247-4254). By activating DCs with a TLR4 agonist and bacterial LPS, DCs
are
generated that are at least virtually identical (in phenotype) to DC1s
generated via
traditional maturation methods. These DCs have a high expression of surface
molecules,
Including CD83, (1)80, CD86 and IlLA-DR. In other embodiments, TI.,R2
agonists, such
as lipotechoic acid (LTA), TLR3 agonists, such as poly( [:C), and/or other
TLR4 agoitists,
such as MPL, may be used. As contemplated herein, any TLR. agonist, or
combination of
TLR agonists, can be used to active DCs, provided such ligands stimulate the
production
of cytokine and chemokine signals by the activated:DC.s. Many other TLR
agonists are
known in the art and can be found in the published literature for use with the
present
embodiments.
Even though there are similarities in phenotype between DCs and
traditionally matured DCs, the DCs of the present embodiments display many
marked
advantages.
Cryopreservation
Mier culture initiation and activation, the cells ate harvested and the
vaccine is cryopreserved. For example, peripheral blood monncytes are obtained
by
lenkapheresis. The cells ate cultured in seratt free Medium with:CiNKSF and IL-
4 for a
period of time -followed by pulsing the cells with a desired.antigen.
Following pulsing the
cells with a desired antigen, the antigen pulsed dendritic cells are incubated
with ITN-7
followed by a TLR agonist (e.g., LPS). The activated antigen pulsed dendritic
cell is
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harvested and cryopreserved in a freezing medium and. stored in liquid
nitrogen. In one
embodiment the freezing medium comprises 55% plasmalyte, 40% human serum
albumin, and 5% DMSO.
The cryopreservation aspect of the embodiments allow for the generation
of an FDA- approved injectable multi-dose antigen pulsed dendritic cell
vaccine. An
advantage of the embodiments is that the multi-dose antigen pulsed dendritic
cells retain
their ability to produce signals critical to T cell function after thawing. As
contemplated
herein, the present embodiments include a variety of cryopreservation
techniques and
cryomedia, as would be understood by those skilled in the art_ For example, in
certain
embodiments, the freezing medium comprises 55% plasmalyte, 40% human serum
albumin, and 5% DMSO. Accordingly, the embodiments provide the ability to
produce
the multi-dose antigen pulsed dendritic cell vaccine of the embodiments at a
centralized
area comprising of an initial immunizing dose and multiple "booster" doses.
Therefore
the multi-dose antigen pulsed dendritic cell vaccine can be shipped to remote
medical
centers for serial administration to the patient with no special FDA quality
control/quality
assurance requirements at the administration site.
In one embodiment, the dendritic cell vaccine of the embodiments is
cryppreserved in aliquots for multiple doses. For example, the cells are
cryopreserved at. a
concentration of 30x1 06 cells/mL. For example, a bag of freezing medium
containing a
20- volumecqual to the cell volume is Prepared. 'Working rapidly, the
freezing medium is
added to the cell bag and the cells are transferred to labeled cryovials. In
one
embodiment, the vials are frozen using a rate controlled freezer. For example,
eryovials
are frozen using an automated rate controlled freezer at PC/min and stored in
vapor
phase nitrogen.
In one embodiment, the vials are frozen using a rate controlled freezer.
The vials are placed in a freezing chamber and liquid nitrogen enters the
chamber through
an electronic solenoid valve. Since vaporization is almost instantaneous,
controlling the
rate at which liquid nitrogen enters the chamber directly controls the rate.at
which heat is
absorbed and removed from the freezing chamber and its Contents.
As contemplated herein, the present embodiments include a variety of
cryopreservation techniques and freezing medium, as would be understood by
those
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skilled Mahe- art For example, in certain enibodiments, the freezing .medium -
for cultured
cells can include about 55% plasmalyte, about 40% human serum albumin, and
about 5%
DMSO. In other embodiments, the cryomedia can be serum-free. In certain
etribodiments,
controlled rate freezing may be used, while other embodiments can include use
of
insulated containers in which vials of cells mixed with freezing medium are
placed in the
freezer, such as at temperatures ranging from about -70ri-C to -80"C, The
present
embodiments provide a method to preserve activated DCs in such a manner so as
to
further facilitate clinical application of such cells, and to reduce the need
for extensive
and repeated pherisis and elutriation steps. As contemplated herein,
cryopreservation
techniques may be used for both small-scale and large-scale batches.
When considering the extensive utility for activated DC, the ability to
provide a steady supply of cryopreserved activated DC represents a significant
advantage
that can facilitate various therapeutic uses of such cells. For example, a
large-scale
culture of activated DC may be cryopreserved in aliquots of the appropriate
size
comprising an initial immunizing, dose and multiple "booster" doses, according
to the
methods of the present embodiments, such that individual doses of cells can
later be used
in any particular immunotherapeutic protocol. In certain embodiments,
activated DCs can
be cryopreserved for 2-24 weeks at temperatures of approximately -70 C or
tower. At
lower temperatures, such as at about -120 C. or lower, activated 'Des can be
20- cryopreserved for at least a year or longer.
In one exemplary embodiment, the Des are suspended in human serum
and approximately 5% DMSO (v/v), Alternatively, other serum types, such as
fetal calf
serum, may be used. The suspended cells can be all quoted into smaller
samples, such as
in 1.8 ml vials, and stored at approximately -70 C or lower. In other
embodiments, the
freezing medium may include about 20% serum and about 10% DMSO, and suspended
cells can be stored at about -180 C. Still further embodiments may include
medium
containing about 55% plasmalyte, and about 5% DMSO. Other exemplary freezing
media
May it:a:Jude about 12%,DM50 and about 2:5-40% aernin.
While the present embodiments as described herein may include specific
concentrations of serum, it should be understood by those skilled in the art
that the exact
amount of serum in the freezing medium may vary, and in some embodiments may
be
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entirely absent but will generally be within the range.ofabout '1% to 30%. Of
Mine, any
concentration of serum that results in a cell viability of around 50% and/or a
cell recovery
of around 50% may be used in any DC composition of the present embodiments, as
well
as with any cryopreservation method as described herein. Preferably, cell
viability and
recovery of at least 60%, more preferably at: least about 70%, oreven 80% is
desired
when recovering cryopreserved cells in the selected freezing medium.
Similarly, while the present embodiments as described herein may include
specific. concentrations of DMSO, those skilled in the art should recognize
that DMSO
may be entirely absent in some embodiments, while in other embodiments,
concentrations from about 5% to as high as about 20% may be used in the
freezing
medium and included within the cryopreservation methods described herein.
Generally,
lower concentrations of DMSO are preferred, such as between about 5% to about
10%.
However, any concentration of DMSO that results after thawing. in cell
viability of at
least 50% and a cell recovery of at least 50%, and preferably a cell viability
and recOyery
of at least 60%, more preferably about 70%, more preferably about 80% and even
more
preferably about 90% and higher, may be used.
While the present embodiments as described herein may include reference
to a rate controlled freezing, it should be understood by those skilled in the
art that
methods of freezing in a rate controlled or non-rate controlled manner can
routinely be
employed.
It should also be understood by those skilled in.the- art that. the various
cryopreservation
media as described herein may either include serum or may be serum free.
Examples of
serum free media can include XVIVO 10, XVIVO 15, XVIVO 20, StemPro, as well as
any commercially available serum free media. When utilizing a serum-free
freezing
medium, the cryopreservation methods of the present embodiments are generally
free of
infectious agents, antibodies and foreign proteins, which may be antigenic,
and any other
foreign molecule that may typically be found in serum-based freezing medium.
(r opt aptigto
loaded, aCtive.DCs can Occur at any point
after activation of cells with a TLR agonist: In one embodiment, the activated
DCs =
are cryopreserved approximately 6-8 hr after exposure to the MR agonist.
Preferably, the
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time point chosen to ci-3,,opreserve the activated cells should be based on
the
maximization of signal production of the cells, partieularlylL-12 production.
The present embodiments provide compositions and methods for
producing large scale dendritic cell vaccines. in one embodiment, the large
scale
production of dendritic cell vaccines allows for the production of an FDA-
approved
injectable multi-dose antigen pulsed dendritic cell vaccine for the
personalized treatment
and prevention of cancer or other disorders. in one embodiment, there are
provided
compositions and methods for producing large scale of antigen pulsed type. .1
polarized
dendritic cell vaccine (DC1).
I0 In one embodiment, the embodiments provide a method to cryopreserve
dendritic cells that are in an antigen-loaded, pre-activated state in a large
scale that is
"syringe-ready", i.e. suitable for immediate injection into the patient
without the
necessity of any fin-01er cell processing that would require (e.g., by FDA
mandate)
additional facilities and quality control/assurance steps.
In one embodiment, the embodiments provide a method to efficiently
produce in a large scale injectable multi-dose antigen pulsed dendritic cell
vaccine,
preferably injectable multi-dose antigen pulsed type I polarized dendritic
cell vaccine that
exhibit maximal efficacy.
Packaging Of Compositions or Kit Components
Suitable containeis for compositions of the embodiments (or kit
components) include vials, syringes (e.g. disposable syringes), etc. These
containers
should be sterile.
Where a composition component is located in a Vial, the vial is .preferably
made of a glass or plastic material. The vial is preferably sterilized before
the
composition is added to it. To avoid problems with latex-sensitive patients,
vials are
preferably sealed with a latex-free stopper, and the absence of latex in all
packaging
Material is preferred. The Vial may *Jude a single dose of vactine, or it may
include
more than one dose (a "multidose" vial) e.g. 10 doses. Preferred vials are
made of
colorless glass.
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A vial can have a cap (e.g, a Luet lock) adapted -sit& that a pre-filled-
syringe can be inserted into the cap, the contents of the syringe can be
expelled into the.
vial, and the contents of the vial can be removed back into the syringe. After
removal of
the syringe from the vial, a needle can then be attached and the composition
can be
administered to a patient. The cap is preferably located inside a seal or
cover, such that
the seal or cover has to be removed before the cap can be accessed. A vial may
have a
cap that permits aseptic removal of its contents, particularly for multidose
vials.
Where a compositionicompOnent is packaged into a syringe, the syringe
may have a needle attached to it. if a needle is not attached, a separate
needle may be
supplied with the syringe for assembly and use. Such a needle may be sheathed.
Safety
needles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge
needles
are typical. Syringes may be provided with peel-off labels on which the lot
number,
influenza season and expiration date of the contents may be printed, to
facilitate record
keeping. The plunger in the ,s,,ringe preferably has a stopper to prevent the
plunger from
being accidentally removed during aspiration. The syringes may have a latex
rubber cap
and/or plunger. Disposable syringes contain a single dose of vaccine. The
syringe will
generally have a tip cap to seal the tip prior to attachment of a needle, and
the tip cap is
preferably made of a butyl rubber_ if the syringe and needle are packaged
separately then
the needle is preferably fitted with a butyl rubber shield.
Containers maybe marked to show a-half-dose volume. e.g. to -facilitate
delivery to children. For instance .a syringe. containinga 0S ml dose may have
a mark
showing a 0.25 ml volume.
Where a glass container (e.g. a syringe or a vial) is used, then it is
preferred to use a container made from a borosilicate glass rather than from a
soda lime
glass.
A kit or composition may be packaged (e.g. in the same box) With a leaflet
including details of the vaccine e,a, instructions for administration, details
of the antigens
within the =vaecine, etc, The instructions may also contain at flings e g to
keep a solution
of adrenaline readily available in case of anaphylactic reaction fallowing
vaccination, etc,
Methods for Treating a Disease
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The present embodiments also encompass 'methods of treatment and/or
prevention of a disease caused by pathogenic microorganisms, autoimmune
disorder
and/or a hyperproliferative disease_
Diseases that may be treated or prevented by use of the present
embodiments include diseases caused by viruses, bacteria, yeast, parasites,
protozoa,
cancer cells and the like, The pharmaceutical composition of the present
embodiments
may be used as a generalized immune enhancer (DC activating composition or
system)
and as such has utility in treating diseases. Exemplary diseases that can be
treated and/or
prevented utilizing the pharmaceutical composition of the present embodiments
include,
but are not limited to infections of viral etiology such as HIV, influenza,
Herpes, viral
hepatitis, Epstein Bar, polio, viral encephalitis, measles, chicken pox,
Papilloma virus
etc.; or infections of bacterial etiology such as pneumonia, tuberculosis,
syphilis, etc.; or
infections of parasitic etiology such as malaria, ttypanosomiasis,
leishmaniasis,
trichomoniasis, amoebiasis, etc.
:Preneoplastic or hyperplastic states that may be treated or prevented using
the pharmaceutical composition of the present embodiments (transduced DCs,
expression
vector, expression construct, etc.) of the present embodiments include but are
not limited
to preneoplastic or hyperplastic states such as colon polyps, Crohn's disease,
ulcerative
colitis, breast lesions and the like,
Cancers that may be treated using the composition :of the present
embochments include, but are not limited to primary or metastatic melanoma,
adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma,
thymoma,
lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma,
Hodgkin's
lymphoma, leukemias, uterine cancer, breast cancer, prostate cancer, ovarian
cancer,
pancreatic cancer, colon cancer, multiple myeloma, neurciblastoma, NPC,
bladder cancer,
cervical cancer and the like.
Other hyperproliferative diseases that may be treated using DC activation
systernOf the pm esenternbodiments include, but ate not littited to rheumatoid
arthritis,
inflammatory bowel disease, osteoattritis,:leionryornas, adenomas, lipomas,
hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre-
neoplastic
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iesions.0116-as .adenomatons hyperplasia'and prostatic intraepithelial
neoplasia),
carcinoma in situ, oral hairy leukoplakia, or psoriasis. .
Autoimmune disorders that may be treated using the composition of the
present embodiments include, but are not limited to, AIDS, Addison's disease,
adult
respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis,
bronchitis,
cholec37stitis, Crohn's disease, ulcerative colitis, atopic dermatitis,
dermatomyositis,
diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,
glomerulonephritis,
gout, Graves disease, hypereosinophilia, irritable bowel syndrome, lupus
erythematosus,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis, pancreatitis, Tpolymyositis, rheumatoid
arthritis, scleroderma,
*gives syndrome, and autoimmune thyroiditis; complications of cancer,
hemodialysis,
and extracorporeal circulation; viral, bacterial, fungal, parasitic,
protozoal, and helminthic
infections; and trauma.
in the method of treatment, the administration of the composition of the
embodiments may be for either "prophylactic" or "therapeutic" purpose. When
provided
prophylactically, the composition of the present embodiments is provided in
advance of
any symptom, although in particular embodiments the vaccine is provided
following the
onset of one or more symptoms to prevent further symptoms from developing or
to
prevent present symptoms from becoming worse. The prophylactic administration
of
composition .serves .to prevent or ameliorate ow subsequent infection or
disease. When
provided therapeutically, the pharmaceutical composition is provided at or
after the onset-
of a symptom of infection or disease. Thus, the present 'embodiments may be
provided
either prior to the anticipated exposure to a disease-causing agent or disease
state or after
the initiation of the infection or disease.
An effective amount of the composition would be the amount that
achieves this selected result of enhancing the immune response, and such an
amount
could be determined as a matter of routine by a person Skilled in the art. For
example, an
'effective amount of for treating an immune system deficiency against .cancer
'or pathogen
could be that amountmecessary to cause activation of the immune system,
resulting in the
development of an antigen specific immune response upon exposure to antigen.
The term
is also synonymous with "sufficient amount."
43
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The effective :amount for any particular application can an depending on
such factors as the disease or condition being treated, the particular
composition being
administered, the size of the subject, and/or the severity of the disease or
condition. One
of ordinary skill in the art can empirically determine the effective amount of
a particular
composition of the present embodiments without necessitating undue
experimentation.
Therapeutic applications
The present embodiments include the generation of an antigen loaded,
activated APC that produces significant levels of cytokirtes and che.mokines
when thawed
from cryopresmation, where the antigen loaded and activated APC is used in
immunotherapy for a mammal, preferably a human. The response to an antigen
presented
by an APC may be measured by monitoring the induction of a cytolytic T-cell
response, a
helper T-cell response, and/or antibody response to the antigen using methods
well
known in the art
The present embodiments include a method of enhancing the immune
response in a mammal comprising the steps of: generating immature Des from
monocytes obtained from a mammal (e.g., a patient); pulsing the immature Des
with a
composition comprising an antigenic composition; activating the antigen loaded
Des
with at least one TLR agonist; cryopreserving the activated, antigen loaded
Des; thawing
the activated, antigen leaded Des and then administering the activated,
antigen loaded
Des to a mammal in need thereof The composition includes at least an antigen,
and may
further be a vaccine for ex viva immunization and/or in viva therapy in a
mammal.
Preferably, the mammal is a human.
Ex vim procedures are well known in the -art and are discussed more fay
below. Briefly, cells are isolated from a mammal (preferably a human), The
cells can be
administered to a mammalian recipient to provide a therapeutic benefit. The
mammalian
recipient may be a human and the cells can be autologous with respect to the
recipient.
Alternatively, the cells an be allogeneic, syngentic otxenogeneic with.
respect to the
recipient.
In one embodiment, peripheral blood monocytes are obtained from. a
patient by combined leukapheresis and elutriation. The monocytes can be
cultured in
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SFM with -GM-C SF and 1L-4 overnight. The next dayõ immature DCs can be pulsed
with,
antigen, followed by contacting the DCs with IFN-T and LP& The activatedDes
can
then be suspended in a freezing medium and frozen until ready for use in
immunotherapy.
Cryopreserved DCs can be cultured ex Vii,o wide conditions! effective to =
generate the percent recovery and percent viability of the cells as compared
freshly
activated DCs. DCs generated from cryopreserved samples can show similar
stability as
compared to freshly prepared DCs. Furthermore, comparisons of cryopreserved
mature
DCs with those of freshly prepared DCs can show virtually identical phenotypes
as well
as signal secretion profiles. As contemplated herein, DCs can be preserved at
both small
and large scale for approximately 2 to 24 weeks, in the various freezing media
described
herein, at temperatures of approximately -70 C to -80 C. At temperatures below
about -
120 C, the duration of storage can be extended. indefinitely or at least
beyond 24 weeks
without impacting cell recovery, viability, and functionality of the DCs. For
example, in
certain embodiments, the activated cells can be preserved for at least one
year and still
retain their ability to produce signal after thawing. The present embodiments
provide for
effective recovery and viability profiles upon thawing the cells, and
furthermore the
cryopreservation conditions described herein do not affect the ability of DCs
to retain
their signal profiles as explained herein throughout.
20. In an exemPlaty embodiment, ctyopreservation.may.be Performed after
activation of DCs by re-suspending the cells in a freezing medium comprising
about 55%
plasmalyte, about 40% human serum albumin, and about 5% DMSO. The mixture can
then be aliquoted in 1.8 ml vials and frozen at about -80 C in a cryochamber
overnight.
Vials can then be transferred to liquid nitrogen tanks the following day_
After about 2 to
24 weeks of ctyopreservation, the frozen DCs can be thawed and examined for
their
recovery and viability. Recovery of such DCs can be greater than or equal to
about 70%
with a viability of greater than or equal to about 70%. In other embodiments,
DCs or even
inonocyieS can be cryopreserved prior to cell activation.
Alternatively, the procedure for a vivo expansion ofhematopoietic stem
and progenitor cells is described in U.S. Pat. No. 5,199,942, which is
incorporated herein
by reference, can be applied to the cells of the present embodiments. In
addition to the
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cellular growth factors 'described in U.S. PAt. No. 5,199,942, other factors
such as flt3-Lõ
and c-kit ligand, can be used for culturing and expansion of the cells.
A variety of cell selection techniques are known for identiing and
separating cells from a population of cells. For example, monoclonal
antibodies (or other
specific cell binding proteins) can be used to bind to a marker protein or
surface antigen
protein found on the cells. Several such markers or cell surface antigens are
known in the
art
Vaccine Formulations
The present embodiments further include vaccine formulations suitable for
use in immtmotherapy. In certain embodiments, vaccine formulations are used
for the
prevention and/or treatment: of a disease, such as cancer and infectious
diseases. In one
embodiment, the administration to a patient of a vaccine in accordance with
the present
embodiments .for the prevention and for treatment of caneread take place
before otafter
a surgical procedure to remove the cancer, before or after: a
chemotherapeutic. procedure
for the treatment of cancer, and before or after radiation therapy for the
treatment of
cancer and any combination thereof. In other embodiments, the vaccine
formulations may
be administrated to a patient in conjunction or combination with another
composition or
pharmaceutical product. It should be appreciated that the present embodiments
can also
be used to prevent cancer in individual without cancer, but who might be at
risk of
developing cancer.
The administration of a cancer vaccine prepared in accordance with the
present embodiments, is broadly applicable to the prevention or treatment of
cancer,
=
determined in part by the selection of antigens forming part of the cancer
vaccine.
Cancers that can be suitably treated in accordance with the practices of the
present
embodiments include, without limitation, cancers of the lung, breast, ovary,
cervix, colon,
head and neck, pancreas, prostate, stomach, bladder, kidney, bone, liver,
esophagus,
gastroesophageal, brain, testicle, uterus and the Variousleukernias and
lymphomas:
in oneembodiment, vaccines can be derived front the tumor or cancer
cells to be treated. For example, in the treatment of I nag cancer, the lung
cancer cells
would be. treated as described hereinabove to produce a lung cancer vaccine.
Similarly,
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breast cancer vaccine, colon 'cancer vaccine, pancreas = Cancer vaccine,
stomach canter.
vaccine, bladder cancer vaccine, kidney cancer vaccine and the like, would be
produced
and employed as immunotherapeutc agents in accordance with the practices for
the
prevention and/or treatment of the tumor or cancer cell from which the vaccine
was
produced_
In another embodiment, vaccines could, is stated, also be prepared to treat
various infectious diseases which affect mammals, by collecting the relevant
antigens
shed into a culture medium by the pathogen. As there is heterogenecity in the
type of
immunogenic and protective antigens expressed by different varieties of
organisms
causing the same disease, polyvalent vaccines can be prepared by preparing the
vaccine
from a pool of organisms expressing the different antigens of importance.
In another embodiment, the vaccine can be administered by intranodal
injection into groin nodes, Alternatively, and. depending on the vaccine
target, the vaccine
can be intradertnally of subcutaneously administered to the extreinities, arms
and legs, of
the patients being treated. Although this approach is generally satisfactory
for melanoma
and other cancers, including the prevention or treatment of infectious
diseases, other
routes of administration, such as intramuscularly or into the blood stream may
also be
used.
Additionally, the vaccine can be given together with adjuvants andlor
immuno-modulators to boost the activity of the vaccine and the patierit'S.
response. Such
adjuvants andlor iminuno-modulators are understood by those skilled in the
am:and are
readily described in available published literature.
As contemplated herein, and depending on the type of vaccine being
generated, the production of vaccine can, if desired, be Scaled up by
culturing cells in
bioreactors or fermentors or other such vessels or devices suitable for the
growing of cells
in bulk. In such apparatus, the culture medium would be collected regularly,
frequently or
continuously to recover therefrom any materials or antigens before such
materials or
antigens. are degraded in the culture Medinnt.
If desired, devices or compositions containing the vaccine or antigens
produced and recovered, in accordance with the present embodiments, and
suitable for
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sustained or intermittent release could be, in effect, implanted in the body
or topically
applied thereto for a relatively slow or timed release of such materials into
the body.
Other steps in vaccine preparation can be individualized to satisfy the
requirements of particular vaccines. Such additional steps will be understood
by those
skilled in the art. For example, certain collected antigenic materials may be
concentrated
and in some cases treated with detergent and ultracentrifuged to remove
transplantation
alloantigens.
Combination Therapy
The present embodiments provide an effective therapy to treat cancer
Wherein the therapy includes changing the immune response in the tumor so that
the
immune cells in the tumor site are more effective in attacking the tumor cells
In some
instances.. the effective therapy includes improving the migration and
activity of immune
cells- in the tumor site. In one embodiment, there are provided compositions
and methods
of using a dendritic cell vaccine in combination with an inhibitor of one or
more of HER2
and HER3 as a treatment regimen to treat cancer. In another embodiment., the
treatment
regimen comprises the use of a dendritic cell vaccine, an inhibitor of one or
more of
HER2 and HER3, and a chemokine modulator. In one embodiment, the chemokine
modulator is a chemokine-activating agent. An example of a chemokine-
activating agent
is a .MR8. agonist.
In one embodiment, there amprovided.compositions and methods of using
a dendritic cell vaccine in combination with blockage of HER-2 arid HER-3 as a
treatment regimen to treat cancer. In another embodiment, there are provided
compositions and methods of using a dendritic cell vaccine in combination with
blockage
of HER-2 and HER-3 with NF-a and IFN-y_ In another embodiment, there are
provided
compositions and methods of blocking both of HER-2 and HER-3 with the
addition.
TNF-u. and IFN-7 as a treatment regimen to treat cancer.
In one embodiment, a treatment regimen can be used to treat cancer and
therefore can be considered as a type of anti-cancer therapy. In another
embodiment, a
treatment regimen can be used in the context of a combination therapy with
another anti-
cancer or anti-tumor therapy including but not limited to surgety,
chemotherapy,
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radiation therapy (e4 X lay)õ gene therapy,.immunotherapy, hormone .therapy,
viral
therapy, DNA therapy, RNA therapy, protein therapy, cellular therapy, and
nanotherapy.
In one embodiment, there is provided a treatment regimen in combination
with another cancer medicament for the treatment or prevention of cancer in
subjects.
The other canter medicament is administered in synergistic amounts or in
various
dosages or at various time schedules with the treatment regimen of the
embodiments. The
embodiments also relate to kits and compositions concerning the combination of
treatment regimens of the embodiments alone or in combination with a desired
cancer
medicament.
In one embodiment, a treatment regimen is used prior to receiving another
anti-cancer therapy. In another embodiment, a treatment regimen is used
concurrently
with receiving another anti-cancer therapy. In another embodiment, a treatment
regimen
is used after receiving another anti-cancer therapy.
In some embodiments, the present embodiments provide a method of
treating breast cancer that is negative fbr ER in a subject. in some
embodiments, there is
a method of treating breast cancer that is negative for ER and positive for
HER2 in a
subject. hi some embodiments, the breast cancer is a metastatic breast cancer.
In some
embodiments, the breast cancer is at stage I, stage II, or stage M.
En another embodiment, the treatment regimen may be used in
combination with existing therapeutic agents used totreat -cancer, in order to
evaluate
potential therapeutic efficacy of the trea.tmentregimen of the embodiments in
combination with the antitumor therapeutics described elsewhere herein, these
combinations may be tested for antitumor activity according to methods known
in the art.
in one aspect, the present embodiments contemplate that a treatment
regimen may be used in combination with a therapeutic agent such as an anti-
tumor anent
including but is not limited to a chemotherapeutic agent, an anti-cell
proliferation agent
or any combination thereof.
The embodiments - Should not limited to any particular chemotherapeutic
agent, Rather, any chemotherapeutic agent can be used with the treatment
regimen of the
embodiments. For example, any conventional chemotherapeutic agents of the
following
exemplaty classes are included in the embodiments: alkylating agents;
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nitrosoureas; antimetabolites; antitumor antibioties; plant alkyloids; taxanes
; hormonal
agents; and Miscellaneous agents.
Alkylating agents are so named betatise of their ability to add- alkyl groups
to many electronegative groups under conditions present in cells, thereby
interfering with
DNA replication to prevent cancer cells from reproducing. Most alkylating
agents are cell
cycle non-specific. In specific aspects, they stop tumor growth by cross -
linking guanine
bases in DNA double-helix strands. Non-limiting examples include busulfan,
carboplatin,
chlorambucil, cisplafin, cyclophosphamide, dacarbazine, ifosfamide,
mechlorethamine
hydrochloride, melphalan, procarbazine, thiotepa, and uracil mustard.
Anti-metabolites prevent incorporation of bases into DNA during the
synthesis (S) phase oldie cell cycle, prohibiting normal development and
division. Non
limiting examples of antimetabolites include drugs such as 54luorouracil, 6-
mercaptopurine, capecitabine, cytosine arabinoside, floxuridine, fiudarabine,
gemcitabine, methotrexate, and thioguanine.
There are a variety of antitumor antibiotics that generally prevent cell
division by interfering with enzymes needed for cell division or by altering
the
membranes that surround cells. Included in this class ate the anthracyclines,
such as
doxorubicin, which act to prevent cell division by disrupting the structure of
the DNA
and terminate its function. These agents are cell cycle non-specific. Non-
limiting
examples of antitumor antibiotics include. dactinomycin, datutortibicirt,
doxorubicin,
idartibicin, mitomycin,C, and mitoxantrone.
Plant alkaloids inhibit or stop mitosis or inhibit enzymes that prevent cells
from making proteins needed for cell growth. Frequently used plant alkaloids
include
vinblastine, vincristine, vindesine, and vinorelbine. However, the embodiments
should
not be construed as being limited solely to these plant alkaloids.
The taxanes affect cell structures called microttibules that are important in
cellular functions. In normal cell growih, microtubules are formed when a cell
starts
dividing, but once the cell stops dividing, the tilicrottibulea'are
disassetribled or
destroyed. Taxanes prohibit the microtubules from breakMg down such that the
cancer
cells become so clogged with microtubnles that they cannot grow and divide.
Non-
Limiting exemplary taxanes include paclitaxel and docetaxel.
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Hormonal 'agents and hormone-like drugs are utilized for 4,-ertain types of
cancer, including, for example, leukemia, lymphoma, and multiple myeloma. They
are
often employed with other types of chemotherapy drugs to enhance their
effectiveness_
Sex hormones are used to alter the action or production of female or male
hormones and
are used tosloW the growth 'of breast,' Prostate, and endometrial cancers.
Inhibiting the
production (aromatase inhibitors) or action (tarnoxifen) of these hormones can
often be
used as an adjunct to therapy. Some other tumors are also hormone dependent.
Tamoxifen is a non-limiting example of a hormonal agent that interferes with
the activity
of estrogen, which promotes the growth of breast cancer cells.
Miscellaneous agents include chemotherapeutics such as bleomycin,
hydroxyurea, L-asparaginase, and procarbazine that are also useful in the
embodiments.
An anti-cell proliferation agent can fluffier be defined as an apoptosis-
inducing agent or a cytotoxic agent. The apoptosis-inducing agent may be a
granzyme,
Bc1-2 family member, cytochrome C, a caspase, or a combination thereof
Exemplary
granzymes include granzyme A, granzyme 13, granzyme C, granzyme D, granzyme E,
granzyme F, granzyme G. granzyme H, granzyme I, granzyme J, granzyme K,
granzyme
L. gratrzyme M, granzyme N, or a combination thereof. In other specific
aspects, the Bel-
2 family member is, for example, Bax, Bak, r3c1-Xs, Bad, Bid, Bik, Hrk, Bok,
or a
combination thereof
In additional asPects, the caspase is caspase-1,. easPase2, caspase-3
caspase4, caspase;-5, caspase-0.õ caspase-7, caspase-8. Caspaserg, caspase-IA
caspase-11,
caspase-12, caspase-13, caspase-14, or a combination thereof. In specific
aspects, the
cytotoxie agent is TNF-a, gelonin, Prodigiosin, a ribosome-inhibiting protein
(RIP),
Pseudomonas exotoxin, Clostridium difficile Toxin B, Helicobacter pylori VacA,
Yersinia enterocolitica YopT, .Violacein, diethylenetriaminepentaacetic acid,
imfulwn,
Diptheria. Toxin, mitogillin, ricin, boutlinum toxin, cholera toxin, saporin
6, or a
combination thereof.
In one embodiment -a treatment regimen is used in combination with an
anti-tumor agent wherein the anti-tumor agent is an antitutnotalky'lating
agent, antitumor
antimetabolite, antitumor antibiotics, plant-derived antitumor agent,
antitumor platinum
complex, antitumor campthotecin derivative, antitumor tyrosine kinase
inhibitor,
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monoclonal antibody, intert7eron, biological response modifier, hormonal .anti-
tumor
agent, anti-tamor viral agent, angiogenesis inhibitor, differentiating agent,
:PI3KirriT0RJAKT inhibitor, cell cycle inhibitor, apoptosis inhibitor, lisp 90
inhibitor,
tubulin inhibitor, DNA repair inhibitor, anti-angiogenic agent, receptor
tyrosine kinase
inhibitor, topoisomerase inhibitor, taxane, agent targeting Her-2, hormone
antagonist,
agent targeting a growth factor receptor, or a pharmaceutically acceptable
salt thereof. In
some embodiments, the anti-tumor agent is citabine, capecitabine,
valopicitabine or
gemcitabine. In some embodiments, the anti-tumor agent is selected from the
group
consisting of Avastin, Sutent, Nexavar, Recentin, ABT-869, Axitinib,
Irinotecan,
topotecan, paclitaxel, docetaxel, lapatinib, Herceptin, lapatinib, tamoxifen,
a steroidal
aromatase inhibitor, a non-steroidal aromatase inhibitor, Fulvestrant, an
inhibitor of
epidetmal growth factor receptor (EGFR), Cetuximab, Panitumimab, an inhibitor
of
insulin-like growth factor 1 receptor (IGFI R), and CP-751871,
In one embodiment, the anti-tumor agent is a chemotherapeutic agent.. A
chemotherapeutic agent as used herein is a chemical compound useful in the
treatment of
cancer. Examples of chemotherapeutic agents include alkylating agents such as
thiotepa
and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan,
improsullan
and piposulfan; aziridines such as benzodopa, carboqu one, meturedopa, and
uredopa;
ethylenimines and methylamelamines including altretamine, tfiethylenemelamine,
trietylenephosphoramide, ti iethiylenethiophosphoramitkand
tritnethylolotnelamMe;
acelogenins (especially bullatacin and bullatacinone); delta-9-
tetrahydrocannabinol
(dronabinol, MARINOL); beta,lapachone; lapkhol; colchicines; betulinic acid; a
camptotbecin (including the synthetic analogue topotecan (HYCANITIN), CPT-11
(irinotecan, CAMPTOSAR), acetylcamptothecin, scopolecti.n, and 9-
aminocamptothecin); bryostatin; callystatin; CC-1065 (including its
adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic
acid;
teniposide; cr.,,ptophycins (particularly ctyptophycin 1 and cryptophycin 8);
dolastatin;
dtiocarinycin (including the synthetie analogues, KW.-21S9 andCB1-TM
eleutherobin;
pancratistatin; a sarcodictOrt; spongistatin; nitrogen mustards such as
chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine,
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prednimustine, trofosfamide, mad mustard; ilitMSEtteas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;
antibiotics such as
the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gamma
ii and
calkheamicin omegall (see, e.g., Agnew, Chem Irttl. Ed. Engl., 33:183-186
(1994));
4/let/licit), including d3rnemicin A; an esperamicin; as well as
neocarzinostatin
chromophore and related chromoprotein enediyne antiobiotic chromophores),
aelacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norlevcine, doxorubicin (including ADRIAMYCIN,
morpholino-doxonthicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin,
doxorubicin HCI liposome injection (DOX1L)õ liposomal doxorubicin TLC D-99
(MYOCET), peglylated liposomal doxorubicin (CAELYX), and deoxydoxorubicin),
epirubicin, esorubiein, idaruhicin, marcellomycin, mitomycins such as
mitomycin C,
mycophenolic acid, nogalatnycin, olivomycins, peplomycin, potfiromycin,
puromycin,
quelamyein, rodorubicin, streptonigrin, streptozociuõ tubercidin, ubenimex,
zinostatin,
zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR),
tegafur
(UFTORAL), capecitabine (XELODA), an epothilone, and 5-fluorouracil (5-FU);
folic
acid analogues such as denopterin, methotrexate, pteropterin, trimenexate;
purine analogs
such as fiudarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine
analogs such
as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,
dideoxyuridine,
doxifluridine, enocitabine, floxtuidine; anti-adrenals such as
aminoglutethimide,
mitotane, trilostane; folic acid replenisber such as frolinic acid;
aceglatone;
aldophospharnide glycoside; aminolevolinic acid; eniluracil; antsactine;
bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine;
elliptinium
acetate; etoglucid; gallium nitrate; hydroxyurea; leminan; lonidainine;
maytansinoids
such as maytansine and ansamitocins; rnitoguazone; mitoxantrone; mopidanmol;
nitraerine; pentostatin; pheriamet; pirarubicin; losoxantrone; 2-
ethylhydrazide;
procarbazine; PSK polysaccharide complex (11 l' Products, Eugene, Oreg.);
razoxane; rhizoxin; sizotimn; spirogermanium; tenuazonic acid; triaziquone;
trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A,
roridin A and
anguidine); urethan; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman;
53
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.gaCytoSin; .arabinoside ("ArarC''); thiotepa; taxoid,e g paclitaxel(TAXOL)
albumin-
engineered nanoparticle formulation of paclitaxel (ABRAXANE), and docetaxel
(TAXOTERE); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate;
platinum
agents such as cisplatin, oxaliplatin, and carboplatin; vincas, Which prevent
tubulM
polymerization from forming microtubules, including vinblastine (VELBAN),
vincristine
(ONCOVIN), vindesine (ELDISINE, FILDESIN), and virtorelbine (NAVELBINE);
etoposide (VP-I6); itbsfamide; mitoxantrone; leticovovin; novantrone;
edanexate,
datinornycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;
difluorometlhylomithine (DMF0); retinoids such as retinoic acid, including
bexarotene
(TARGRETIN); bisphosphonates such as clodronate (for example, BONEFOSO or
OSTAC), etidronate (D1DROCAL), NE-58095, zoledronic acidizoledronate (ZOMETA),
alendronate (FOSAMAX), pamidronate (ARENA), tiludronate (SKELID), or
risedronate
(ACTONEL); troxacitabine (a 1,3-dioxolatte nucleoside cytosine analog);
antisense
oligonucleotides, particularly those that inhibit expression of genes in
signaling pathways
implicated in aberrant cell proliferation, such as, for example, PKC-alpha,
Rat*,
and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE.RTM,
vaccine and gene therapy vaccines, for example, ALLOVECTIN vaccine, LEUVECTIN
vaccine, and VAXID vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN);
rniRH
(e.g., ABARELIX); 13AY439006 (sorafenib; Bayer); SU-11248 (Pfizer);
perifosine,
COX-2. inhibitor (e.g., celeeoxib or etoricoXib), proteoSonte
inhibitor:(e,g.,.PS341);-
bortezotnib (VELCADE); CC l-779; tipifamib (R11577); orafenib, ABT510; BU-2
inhibitor such as oblimersen sodium (GENASENSE); pixantrone; EGER inhibitors;
tyrosine kinase inhibitors; and pharmaceutically acceptable salts, acids or
derivatives of
any of the above; as well as combinations of two or more of the above such as
CHOP, an
abbreviation for a combined therapy of cyclophosphamideõ doxorubicin,
vincristine, and
prednisolone, and FOLFOX, an abbreviation for a treatment regimen with
oxaliplatin
(ELOXATIN) combined with 5-FU and leticovovin.
In another embodiment combinatiOns of immunotherapieS :are used to treat
estrogen receptor,positiveiRER2-positive:(ERP9'1HERP') DCIS: breast cancer
patients.
Anti-estrogen therapy such as, for example, tamoxifen is combined with amti-
HER2
dendritic cell vaccination to improve pathologic complete response.
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These methods described herein are by no means all-inclusive, and further
methods to suit the specific application will be apparent to the ordinary
skilled artisan.
Moreover, the effective amount of the compositions can be farther approximated
through
analogy to compounds known to exert the desired effect.
EXPERIMENTAL EXAMPLES
The embodiments are further described in detail by reference to the
following experimental examples. These examples are provided for purposes of
illustration only, and are not intended to be limiting unless otherwise
specified. Thus, the
embodiments should in no way be construed as being limited, to the following
examples,
but rather, should be construed to encompass any and all variations which
become
evident as a result of the teaching provided. herein.
Without further description, it is believed that one of ordinary skill in the
art can, using the preceding description and the following illustrative
examples, make and
utilize the present embodiments and practice the claimed methods. The
following
working examples therefore, specifically point out the preferred embodiments,
and are
not to be construed as limiting in any way the remainder of the disclosure.
20. Example :A:CropreServed, pre-activated, : multi-dose dendritie cell
vaccine;
To be able to produce These large scale vaccines, a process was developed
to produce fully activated DCI vaccines pulsed with tumor antigen whereby the
fully
activated TX.:1 vaccines are cryopreserved as multi-dose syringe ready 6 pack
DC I
vaccines, The DC1 are cryopreserved activated as DC1 as described elsewhere
herein.
For example, they are oyopreserved in 55% Plasmalyte medium with 40% Human
serum
albumin and 5% DMSO. These vaccines have been generated and extensively tested
in
the laboratory and consistently meet quality standards set by the FDA for
administration
tOipatients.
Thematerials.aud methods employed in the experiments and examples
disclosed herein are now described,
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Preparation ofJul hin aid D(.7Jul enOprescowtioti
Freshly elutriated myeloid monocytes were cultured in 6 well microplates
(12x106cells/well). Culture medium consisted of Serum Free Medium ( SFM
invitrogen
Carlsbad CA). The final concentration of added GMCSF was 5Ongtml and of IL4 is
1000
Wail. Cells were cultured overnight at 3T C in 5% CO2. In some batches, the
cells were
pulsed with the adequate peptides after 16-20 hr and cultured for additional 6-
8 hr, after
which 1000LI/m1IFN-T was added. Dendritic cells were matured with TLR agonist
LPS
(TLR 4, lOnginil) or R848 (TLR.8, li.tgim1). The maturation time was at least
about 6hr.
After that, the TLR agonist-activated DCs were ready for cryopreservation Or
immediate
use.
To induce the production of the Thl-polarizing cytokine IL-12, the -DCs
are activated with combinations of the cytokine IFNI, or the. TLR agonists
bacterial LPS
and/or R848. This should induced T cells that produce IFN-y. Alternatively,
the DCs can
be activated with combinations of ATP, bacterial LTA, LPS and prostaglandin E2
(PGE2).. This can cause ILL23., 1L-6 and IL ID to be amplified, leading an
immune
response dontinated by 11 -17 and II,-22-secreting Th17 cells%
Olvpreerivtion of DC
Des were harvested by gentle scraping. MI medium and the cells were
kept at wet ice at all times_ Cells were gently washed by centrifugation at
about 800RPM
for 10 min. Cells (e.g., 10 x106 cells) were cryopreserved in freezing medium
of
plasmalyte 55%, human serum albumin 40% with 5% :DMS0 and stored in liquid
nitrogen,
The results of the experiments presented herein are now described,
Multi-dose DC1 vaccines
Experiments Were performed .to assess the recovery, viability and sterility
Of the cell's., Briefly, .peripheral blood monocytes were.- obtained by
cOrnhined
leukapheresis the counter current elutriation and were cultured in serum free
medium
with GM-CSF and 1L-4 overnight. The next day they were pulsed with HER-2
peptides
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then W.NPt tbiloWed by LPs. The DC.1 were harvested at 40 hours and
eiyopreServed in.
freezing medium of pla,smalyte 55%, human serum albumin 40% with 5% DMSO and
stored in liquid nitrogen. Following I week they were thawed and release
criteria were
obtained including viability, yield, endotoxin testing, and sterility
cultures. All 12
cultures had no bacterial growth and <0.1 EU endotoxin, Figure I demonstrates
the
viability and yield of cryopreserved DC1. As can be seen in Figure 1, recovery
of cells
was OP average.9% and viability waS 95% When cells were direetly thawed and
counted.
These data demonstratethe cryopreservation and viabilityof the Del vaccine is
maintained in Medium containing below the FDA allowable 7.5% DIVISO,
It was observed that cell function was maintained following
crypreservation..1L-12 and Thl chemokines were produced for 36 hours post thaw
from
multi-dose DC1 vaccines. Thawed cells produced high levels of IL-12 from about
6 hours
post thaw through 36 hours. These levels of 1L-12 are comparable to prepared
DC.1
vaccines made from cryopreserved monocytes.
.15 The results presented herein demonstrate that cryopreserved,
preactivated,
multi-dose dendritic cell vaccine sensitized against the HER-2 tumor target
antigen on
breast cancer is therapeutic. However, the embodiments are applicable to a
variety of
additional pathophysiological conditions.
The multi-dose syringe ready pack DC I vaccines can be used in HER-2
non-expressing breast cancer. HER-2 is expressed on approximately 25% of all
breast
Cancers. Breast cancers that do not produce detectable levels of HER -2 may
not be
susceptible to vaccination. To address this limitation, additional. target
proteins can be
added to the vaccine. For example, many breast cancers that do not produce
high levels
of HER-2 instead produce other, related proteins including HER-I and HER,3.
Without
wishing to be bound by any particular theory, it is believed that adding these
other
proteins to the vaccine would allow the targeting of these other breast cancer
phenotypes.
The multi-dose syringe ready pack DC I vaccines can be used in other
cancer types: besides breast cancer. Anticipated target proteins such as HER-
4, HER,=2
and 1-1ER.-3 c,air also be present on other types Of cancer including ovarian,
prostate,
pancreatic, colorectal, gastric, head and neck and non-small cell lung
carcinoma, as well
as other common cancers.
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The multi-dose syringe ready pack .DCI vaccines can be used to treat
chronic infectious diseases, including but not limited to, chronic infections
like HIV or
hepatitis virus C. Here, proteins specific for these viruses would replace the
HER-2 or
other cancer proteins to mobilize the patient's immune response against these
persistent
infections. It is possible that the enhanced immunity would greatly reduce
viral load and
attendant disease symptoms and progression, or could possibly help clear the
infection
entirely.
The multi-dose syringe ready pack Del 'vaccines can-be used to treat
= autoimmune diseases. Diseases like rheumatoid arthritis and Lupus 'occur
when the
immune system mistakenly attacks the body's own normal tissues. Whereas the
current
vaccinetimmunotherapy formulation is designed to initiate and strengthen
immune
responses, without wishing to be bound by any particular theory, it is
believed that in
vitro signals can be provided to the DCs during vaccine production that induce
these cells
to switch off pathological immune responses.
Example 2: Crvopreservation of Activated DC1 Makes Large Scale Dendritic Cell
'Vaccines Feasible in Cancer Therapy
Dentiritic cell-based vaccine therapy is :a promising directed therapy against
a
variety of cancers. While a variety of strategies have been employed to mature
DCs to a
20- phenotype that optimizes sensitization of CD,1-4- and CD8+ I cells
turecognize tumor
epitopes and elicit antitumor immunity, experiments were designed to utilize
a:method
that employs the rapid maturation of monocytes in serum free media (SFM) using
Interferon gamma (IFN-7) and lipopolysaccharide (1,PS), a toil-like receptor
(TI.,R) 4
agonist, resulting in mature DCs capable of polarizing the immune response to
a Thl
type response and eliciting sensitization via an IL-12 dependent mechanism.
Results
demonstrate the potential for this vaccine strategy to be used as an adjunct
therapy in
early breast cancer. Cryopreservation of dendritic cells (DCs) in a matured
state permits
'easier production of accessibility to personalized therapy.
Rapid maturation of dendritic cells
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Both freshly matured :DCs (DC .I s) and IX s ctyopreserved in a mature
State (cryoDCS) and subsequently thawed were compared for viability and
recover as well
as far phenotypic maturity as determined by expression of cell surface markers
by flow
cytometry. Function of dendritic cells was determined by measuring production
of
various cytokines, in particular, interleukin 12p70 (IL-12p70). The ability of
these cells
to stimulate naïve CD4+ and CD8+ tells was also assessed.
There was no significant difference in the viability (p-.4807), and
recovery (p=.1220) (Figure 2).
Both populations had similar initial (7 hours post LPS addition) IL-12 p70
secretion (p=.0768). The populations continued to exhibit comparable secretion
levels of
IL-12 p70 over a 30 hour Observation period with no significant differences
between the
populations. (Figure 3),
There was no significant difference between populations and-production
of IL-lbeta (p-0.7690), IL-1 alpha (p-0.0841), Ratites (r0,902), MDC (r0.1514)
IL-
10 (p---.1937), (p--,2673), IP-10 (p-0.7366), IL-6 (p----0.24), IL-5 (p-
0.0735),
INF-beta (p=0,9422), IL-15(p=0.8878), MIP- I beta (1)=0.9217), INF-alpha (p-
0.8972),
111L-8(p=0,7844). (Figure 4).
Cell surface markers denoting DC maturity between Deis and cryoDcs
exhibit no significant difference in expression. Both populations elicited
nonspecific
alloantigeri CD4 .T cell responses as well as antigcn-specific CD S+I cells
recognition
with a:Thl polarized response. CM, 83, and 86 demonstrate DC maturation,
expression
revealed no significant difference. Functional abilities were induced by CD4+
T cells
from diff7erent donors co-cultured with each population.resulting in a
nonspecific
alloantigen response and CD4+ I cells secreting INF-gamma (DC1s 107.40 ne,/mL
and
cryoDCis 129.23 ng/mL). Antigen specific sensitizations initiated by co-
culturing the
two populations with tumor antigen specific CDS+ I cells elicit comparable
:I:FN.-7
secretions for both groups and result in a TI-I1 polarized response. (Figure
5).
The results: presented herein demonstrate that rapid maturation method Of
DC1 can be mopreserved functionally matured and maintains, phenotype and
function,
thus can be used to manufacture syringe ready DC:1 for use world-wide in
cancer therapy.
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In conjunction ith maintaining the DC 1 phenotype:to-drive a 'fhl
polarized immune response, the results presented. herein demonstrate that the
cryoDCs
maintained the ability to primarily sensitize T cells. This also likely
relates to the
maturation strategy, as the DCIs matured with [FM-gamma and LPS exhibit an
enhanced
ability to primarily sensitize Cl) 4+ T cells compared to cytokine matured
Des.
The present protocol for cry,ropreserved mature DC preparations can be
easily adapted to current good manufacturing practice guidelines thereby
increasing the
availability of emerging therapy.
Example 3: Cvtokines from CD4 T cells and Herceptin make hieh HER-2-expressine
breast cells susceptible to killing by CD8 T cells
It has been demonstrated that that high HER-2-expressing breast cancer
cells down-regulate molecules on the cell surface that make these cancer cells
visible to
C1)8 T cells and allow them to be killed by these immune cells. It has been
shown that
the cytokines interferon-gamma (IFNI) and tumor necrosis factor-alpha (TNIz-a)
produced by CD4 cells, when combined with Herceptin, cause the intermediate
and high
:HER-2-expressing breast cancer cells to increase their Class I molecule
expression. As a
result of this, the CDS T cells are able to better see the breast cancer cells
and kill them or
to produce cytokines to kill them.
20. Trials were designed to assess the therapeutic effects of using
Herceptin in
combination with dendritie cell vaccines (e.g.. DC1 vaccines).
A phase I DCIS vaccine trial combining Herceptin with DC1 vaccines was
designed. For example, a Phase I trial was designed for patients with high
HER-2--
expressing DCIS to receive a DCI vaccine combined with 2 doses of Herceptin at
week 1
and week 4. Without wishing to be bound by any particular theory, it is
believed that this
combination will increase the complete response rate from 30% to greater than
50% in
patients with HER-2-expressing DCIS.
In addition, a Phase Ill DcIS Vaccine trial was designed. FQ:e e*ainple,
vaccine:trial was developed to prevent recurrence of breast cancer in patients
with
estrogen independent (ER"), DCIS. There are three treatment arms in
the study: 1) a standard therapy (surgery and radiation), 2) receiving a DCI
vaccine
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before stirgety, and 3)=teceivina DC:',1 vaccine plus Herceptin before
surgery, Without
wishing to be bound by any particular theory, those patients who have complete
responses to the treatment can avoid radiation after surgery. It is also
believed that this
trial serves demonstrate the prevention of recurrence in patients with DC1S
using a
vaccine.
In addition, a phase I neoadjuvant DC1 vaccine in combination with
Ilerceptin in patients with early invasive FIER-2-P"''' breast cancer was
designed. For
example, a Phase i trial was designed to test whether the combination of
Herceptin and
vaccines along with a chemokine modulator (e.g., a chemokine-activating agent)
can
eliminate small HER-2-expressing invasive breast cancers prior to surgery and
avoid the
need for chemotherapy. Without wishing to be bound by any particular theory.,
it is
believed that this neoadjuv ant (before surgery) strategy, with the
possibility of adding an
immune antibody that takes the brakes off the immune response, may eliminate
the need
for toxic chemotherapies for the treatment of breast cancer and therefore make
immune
therapy the standard of care for this disease, That is, the treatment regimen
disclosed
herein provides a step forward in the quest to eradicate breast cancer using
the natural
immune response, which can be restored with vaccines regimens of the
embodiments.
The regiments discussed herein can drive immune cells into the tumor by
changing the
immune response in the tumor and enable the immune cells to work longer by
taking the
brakes .off the Cells. It is believed that combining DCI Niaccines with
Fiereeptin and also
addingthe ehemokine modulator improves the migration and activity of the
immune cells
within the tumor in the breast.
Without wishing to be bound by any particular theory, it is believed that in
many cancers, if there are large numbers of good immune-fighting cells
present, those
patients do better with any type of therapy_ This means that if one gets
immune cells into
the tumor to tight it before it is removed, the patient likely does better and
responds better
to other therapies, including surgery, chemotherapy, and radiation.
Without wishing to be bound by any partiettlat an effective therapy to
treat cancer would include agents that rapidly change the immune response in
the tumor
prior to surgery to improve outcomes for patients with breast cancer and other
types of
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cancet7rhiS strategy can be applied to a variety of cancer including but is
not limited to
colon cancer, melanoma, lung, brain, pancreas, prostate, esophagus, and the
like.
Experiments were designed to develop immune fluorescence assays to
measure and quantify the types of immune cells that migrate into the breast
after
vaccination, the types of chemokines that are made to bring cells in, and the
molecules
they express to help eliminate cancer cells. These assays allow multiple cell
types to be
visualized at the same time and show where they are located in the tumor
microenvironment. It has been observed that at least in some of the vaccinated
patients,
their tumors produced chemokines to recruit cells into the environment to kill
the tumor
cells,
Example 4: Combination of Blockade of HER-2 and HER-3
The results presented herein demonstrate that the combination .of an
effective anti-1-lER2 C1)4+ Th I response combined with HER2 and-IER3 blockade
is
extremely effective in causing tumor senescence and apoptosis in HER-2
expressing
breast cancers.
It was observed that the combination of blockade of HER-2 and HER-3
together with the addition of combinations of Thl crokines TNE-tt and IFN-7
from CD4
Thl cells causes significant senescence and apoptosis of HER-2 expressing
breast cancer,
This has been verified in several different cells lines of both: high and
intermediate
HER2-expresSing breast cancer cells, The results demonstrate that this
combination can
be potent for prevention and prevention of recurrence.
METHODS-
Cell culture and treatments
Human breast. cancer cell lines SK-BR-3, 13T-474, T-47D,
HCC-
1419 and MDA-MB-231 were obtained from the American Type Culture Collection
(Manassas, VA) and grown in RP1µ41 640 (Life technologies, Grand1Sland, NY)
supplemented With 10% FRS ((ellgro. flerndon, VA). J1MT- I cells were a kind
gift
from Dr, Pravin Kaumaya (Ohio State University, Columbus, OH) and were grown
in
Dulbecco's modified Eagle's medium (MEM) (hvitrogen, Wallin), MA)
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supplemented ;with 10% PBS. Normal immortalized MCF,10 cells _N,i.ere:
obtained from
the Karmanos Cancer Institute (Detroit, ME) and grown in DMEM/FI2 (Invitrogen)
supplemented with 10 mM HEPES, 10 pginil insulin, 20 nginil EGF, 100 ngtml
cholera
toxin, 30 niM sodium bicarbonate, 0.5 gglml hydrocortisone, and 5% fetal horse
serum.
All cells were grown at 37"C in a humidified 5% CO2 incubator.
Three hundred thousand of breast cancer cells were treated for 5 days with
the indicated concentrations of human recombinant TNF-a (R&D Systems,
Minneapolis,
MN) and human recombinant IFN-y (R&D Systems) and then cultured for 2 more
passages in absence of cytokines. Cells were subjected to senescence
associated 0-
.10 g,alactosidase enzyme (SA-fl-gal) detection or Ilysed and subjected to
western blot
analysis for pl5INK4b, pl6INK4a AND CLEAVED CASPACE-3.
In the some cases, the cells were treated with 10 Lig/nil trastuzumab
(HERCEPTINTh) and pertuzumab (PERJETATm) (both, Genentech, San Francisco, CA)
for the indicated times. This treatment was combined with cytokines or with
human
retombinant heregtilin (R&D Systems).
Fitly thousand cancer cells were also cultured in the lower chamber of a
transwell system (BD Biosciences, San Jose, CA) with co-culture of I Ox j5
human CD4'.
T-cells and 105 mature (i.e. type 1 polarized) or immature human dendritic
cells (DC's) in
the upper chamber. DCs and CD4 T-cells were obtained from select trial
subjects
(Sharma et al., 2012 Cancer 118: 4354-4362). Mature and immature DC's were
pulsed
with Class II-derived HER2 or control irrelevant (BRAF and survivine) peptides
(20
pg ml) for 5.days at 37"C, Control wells contained C04' T-cells only In
additionõ
0,5x10' cells Were incubated in the presence of Dc/CD,V coeulture
Supernatants
for 5 days at 37 Cõ In both approaches, cells were then cultured for 2 more
passages in
absence of cytokines and subjected to senescence studies (SA-0-gal activity at
pH 6 and
p15INK4b and pl6INK4a western blot) or apoptosis studies (cleaved caspase-3
western
blot). The following antibodies were added to cells 60 min before incubation
with the co-
caine of DC and CD4' T-cells to neutralize Thl-elaborated cytokines:
polyclimal goat
IgG anti-human TNF-a. (0.06 jig/nil per 0.75 ngiml TNF-a) and liFN-y (0,3
uglml per 5
ngitril IFN-t), and. goat IgG isotype :as the corresponding negative control
(all from R&D
Systems).
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Plasmid transfections
MDA-MB-231 cells were transiently transfected for 48 h with 2 pg of the
wt HER2 expression vector (pcDNAHER2). As a control, cells were transfected
with 2
pg of the empty vector (pcDNA3). Both vectors were kindly provided by Dr_ Mark
Greene (University of Pennsylvania, Philadelphia, PA). The cells were
transfected in
complete medium without antibiotics with Ttubofect (Thermo Scientific,
Waltham, MA),
Transfection efficiency was evaluated by western blot 48 h after transfection.
Forty eight
hours later, transfected cells were transferred to complete culture medium
containing 0.4
mglinl G418 (Life Technologies). After 15 days of culture, colonies resistant
to G418
were selected by limiting dilution. Transfection efficiency was evaluated by
western blot.
RNA interference (RNAi) transfections
Small interfering RNA (siRNA) SMART Pool: ON TARGET Plus HER2
siRNA, HER3 siRNA and SMART Pool: ON-TARGETplus Non-targeting Pool were
purchased front GE Dharmacon-(Lafayette, CO). The following target sequences
were
used: HER2: UGGAAGAGAUCACAGGLIUA (SEQ ID NO: 9),
GACIACCCGCLICiAACAAUAC (SEQ ID NO: 10), GGAGGAAUGCCGAGLIACUG
(SEQ ID NO: 11), GCUCAUCGCUCACAACCAA (SEQ ID NO: 12); HER3:
GCGAUGCLIGAGAACCAALIA (SEQ ID NO: 13), AGAIJUGUGCLICACGGGACA
(SEQ 1D NO: 14), GCAGUGGAUUCGAGAAGUG (SEQ ID NO: 15),
UCCRICAUGULIGAACUAUA (SEQ ID NO: 16); Non-targeting:
LIGGULWACAUGLICGACUAA (SEQ ID NO: 17), UGGUULIACAUGULIGUGUGA
(SEQ ID NO: 18), UCKILTULJACAUGULTUUCUGA (SEQ ID NO: 19),
UGGLIUUACAUGLIUIJUCCLIA (SEQ ID NO: 20). Three hundred thousand cells were
transfected with siRNA sequences (25 nM) using RNA I Max Lipofectarnine (Life
Technologies) in serum free medium, and afier 1 h the medium was supplemented
with
10% FBS. Sixteen hours later, cells were subjected to 48 h of serum starvation
followed
by various designated treatments and western blot to check expression levels.
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SA-0-nal activity at pH 6
Cells were washed twice in PBS, fixed in 3% formaldehyde, and washed
again in PBS. The cells were incubated overnight at 37 C (without CO2) with
freshly
prepared senescence associated acidic 0-galactosidase (SA4I-gal) staining
solution from
Millipore (Billerica, MA) per manufacturer's instructions. The percentage of
SA-0-gal-
positive (blue) cells in each sample was determined after scoring 300 cells
using a bright-
field microscope (Evos Corer Bethel, WA/40X/ 2048 x 1536, 3.2 untlpixel; 3.1
MP
COLOR/ Captured images: Color TIFF, PNG, JPG or BMP-2048 x 1536 pixels)
Western blot analysis
Lysates were prepared from MCF-10Aõ SK-BR-3, and MCF-7, T-47 D or
MDA-MB-231. cells. Cells were lysed in a buffer containing 50 mM Iris (pH
7.4), 150
mM NaC1, II mM EDTA, 1 mIVI ECM., 10% glycerol, 70% Tergitol, 0.1% SDS, 1 mM
Mg2C1 and protease inhibitor cocktail Sigma-Aldrich (St. Louis, MO); Lysates
were
centrifuged at 12,000xg for 15 min at 4 C. Proteins were solubitized in sample
buffer
(Life Technologies) and subjected to SDS-P,NGE. Proteins were electrohlotted
onto
PVDF. Membranes were imunmoblotted with the following antibodies: pl5INK4b (K-
18), p16INK4a (50.1), IFN-yRa (C-20), HER3 (C-17) all from Santa Cruz
Biotechnology
(Santa Cmz, CA); Vinculin (V9131) from Sigma-Aldrich; HER2 (29D8), cleaved
caspase-3 (Aspl 75) and TNF-R1 (C25C1) and phospho-Akt (Ser473) from Cell
Signaling Technologies (Danvers, MA). After washing; membranes were incubated
with
I-MP-conjugated secondary antibody (Bio-16d, Hercules, CA). Bands were
visualized
and quantified by using the enhanced chemilumineseence (ECL) western blot
detection
system and the Image Reader LAS-1000 Lite version 1.0 software (Fuji).
Quantification
of western blots was performed using Imagei software,
Flow cytometrie analysis of cell death
SK-BR-3 cells were untreated, treated with IFN-y (100 Ulm!) and TNF-a
(lOnglin1), treated with trastuzumah (I Oug/m1) and pertuzumab (10ggim1), or
treated
with a combination of IFN-y, TNF-a, trastuzturiab, and pertuzumab, for 24
hours, After
incubation, apoptosis induction was determined using F1TC-Annexin V apoptosis
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'detection kit (BI) biostiences) according to manufactureeS instructions
Briefly,
untreated and treated SKBR3 cells were collected, washed with PBS and
resuspended in
Annexin V binding buffer at a concentration of 1x106 cellthnl. 100p1 of cell
suspension
was incubated with 5p1 P1 and 5p1FITC-annexin V for 15 min at room temperature
in the
dark. After incubation, 150platmexin V binding buffer was added and apoptosis
induction was analyzed using a BD Accuri C6 flow cytometry (BD Biosciences)
and data
was analyzed with CFlow Plus software. UV-irradiated cells were used as
positive
controls.
Tumorigenesis studies
For xenograft experiments, SK-BR-3 (2 x 106 cells mouse in 200 pi PBS)
were injected into the flanks of six-week old female oathymic- (nude) Mice
(Foxn0,
Harlam Laboratories, 5 mice/group). When the tumors were palpable, the animals
were
treated s.c. with trastuzumab and pertuzumab (30 ugikg) and then injected s.c.
twice a
week with brINF-a. and brfFN-y (10 ng/kg). Tumor formation was monitored by
palpation and tumor volume in trim3 was determined with a. caliper twice a
week: widtb2
x length/2. All animal experiments were carried out in compliance with the
institutions
guidelines.
Statistical analysis
Unpaired Student's t-test (two-tailed) analysis was performed using .GraphPad
Prism (GraphPad Software, La Jolla, CAõ USA). A P value of 0.05 or less was
considered
significant. ""P <0,05, **P <0.01, "V <0.001.
RESULTS.
Thl cytokines TNF-a and 1FN-y synergize to induce senescence in breast cancer
cells
SK-BR-3 cells were incubated with human recombinant tumor necrosis
factor alpha (TNF-a) and interferon gamma (IFN-y) alone or combined for 5 days
at
37 C to study if the elaborated crokines produced by the immune system cells
could
induce a specific senescence response in tumor cells. The cells were then
cultured for 2
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more paSS4OS and subjected to senescence studies.. The combination of both
cytokines
resulted in senescence induction of SK-8R-3 cells, evidenced by increased
senescence
associated acidic flialactosidase (SA-11-gal) staining (Figure 6A) and higher
expression
of the senescence-associated markers pl5INK4b and pl 6INK4a (Figure 68)
compared to
the control untreated cells or each cytokine alone detected by western blot.
Similar results
were obtained in 8T-474, MCF-7 and T-471) breast cancer cell lines (data not
shown).
Thus, T-47D cells were treated with several Concentrations of TINFet-, foarn
10 to 100
and IFNI, from 100 to 1000 or in
combination. It was observed that the
induction of the senescence phenotype was dose dependent (Figure 6C), Similar
results
were found in 5K-BR-3, 81-474 and MCF-7 cells.
Inverse correlation between the HER2 expression level in breast cancer cells
and the Tit I
cytokines TINF-a and IFN-1 doses required to induce senescence
Experiments were designed to determine whether the HER2 expression
level plays a role in the senescence induced by the treatment with INF7a, and
IFNI for 5
days at 37 C'followed by 2 more passages without thetytpkines.in SK BR-3
BT..474
MCF-7, 1-471) and MD.A-MB-231 breast cancer cells. In fact, the amount of SA43-
gal
positive cells was augmented in the high HER2 expressing cell lines (SK-13R-3,
Figure
6D and BT-474, data not shown), with lower doses of TNF-a. and IFN-y compared
to the
intermediate HER2 expressing cell lines (T-47D, Figure 6D and MCF-7, data not
shown)
with higher cytokines concentrations. However even highest concentration of
TNF-u. and
IFN-1 could not induce senescence inthe low 1IER2-expressing cell line MDA-M13-
231
(Figure 6D), These results clearly evidence a correlation between the TNT-a.
and IFN-y
inducing senescence and the HER2 expression level..
1IER2 is .required for Thl cvtokines TNF-u. and IFN-'y tuediated_senescence
and
apoptosis in MDA-MB-231 breast cancer cells
Only when MDA-MB-231 cells were stably transfected with a wild type
HER2 plasmid (pcDNAHER2) and treated with high concentrations of TNF-a (200
ng/m1) and IFNI (2000 Li/m1.) for 5 days at 37C followed by 2 more passages in
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absence of cytokines, there was a strong increase in-SA43-0 positive cells
(Figure 7A)
and in p1SINK4b expression (Figure 713). 'Notably, since it was observed that
not only
there -was a relatively higher number of blue senescent cells when HER2-MDA-
M13-231
cells were cultured with highest concentrations of TNF-a and IFN-y, but also
there was a
significantly lower amount of cells, the experiment was repeated and the cells
were
subjected to western blot to detect active easpase-3 to study apoptosis
(Figure 7B). The
treatment of cellstranstected with .the control empty vector (pcDNA3) with
increasing
concentrations Of TNF-ct, front 75 to 200 nglinl, and IFNI, from 750 to 2000
in
combination in the same conditions described above, had no effect on
senescence
induction assessed by SA-gal staining (Figure 7A) or p15INK4b and cleaved
caspase 3
expressions (Figure 78). This finding reinforces that 'FIER2 is required in
the mechanism
of TNF-a and IFNI inducing senescence and apoptosis in breast cancer cells.
Cytokine receptors are expressed in similar levels in breast cell lines
-15. The high HER2-expmssing cell lines SK-.13R4 and BT-474, the
intermediate MCF-Tand T-47D. and the low -HER2-expressing.MDA-MB-231 breast.
cancer cell lines, like the low IHER2 normal immortalized MCF-10 breast cell
line
showed similar IFN-y and TNF-u receptor expression by western blot analysis
(Figure
12). This result demonstrates that the expression level of these two cytokine
receptors is
independent of the HER2 expression level. It is in accordance with reports
that describe
the action of these cytokines in the different phases of the normal breast.
TNF-a has been
'invOlvedin pro1 iferation,. development and branching mot phogenesis of the
normal
mammary Wand (Lee et al., 2000 Endocrinology 141: 3764-3773). The receptor
TNFRI.
expression mediates TNF-a-induced proliferation of mammary epithelial cells,
and
TNFR2 activation induces casein accumulation (Varela et al., 1996
Endocrinology 137:
4915-4924). Similarly, the active form of:117Ni, interacts with its receptor
expressed on
the surface of almost all normal cells (Ealick et al., 1991 Science 252: 698-
702; Farrar et
al., 199$ Annu. Rev. Immunol. 11:571-611).
=
30. Combined HER2 and HER3 blockage expression enhances Th I cytokines TNF-
aand
IFN-y senescence induction in breast cancer cells.
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The previow results showing the enhancement of senescent and apoptosis
phenotypes in high and intermediate HER2-expressing cell lines treated with
the
combination of cytokines led to studying the effect of knocking down HER2 and
HER3
with siRNA. The therapeutic benefit of blocking HER2/HER3 signaling in breast
cancer
has been demonstrated by several studies (Lee-El oeftich et al., 2008 Cancer
Res.
14:5878-87; Berghoff et al., 2014 Breast 14: S0960-9776). Although the
combined
treatment of TNF-a and IFN-7 in HER3-depleted cells did not significantly
enhance the
number of senescent or apoptosis cells, the double knock down with HER2 and
HER3
siRNA strongly increased the SA4-gal staining (Figure 8A) and p15INK4b and
active
caspace-3 expression level (Figure 813) in SK-BR-3 cells. At the same time, it
was
observed that a higher apoptosis induction of the cells treated with the
double knock
down and the cytokines by western blot of active caspase-3 (Figure 8A).
Similar results
were found in MCF-7 cells (Figure 13), BT-474 and T47D cells.
Combined HER2 inhibition and HER2-HER3 dimerization inhibition enhances Thi
cytokines TNE-rx and IFN-7 senescenee induction and aptiptosis. in SK-BR,-3
breast
cancer cells
Trastuzumab and pertuzumab are antibodies that have been widely used in
the clinic to treat HER2-positive breast cancer. To study the Th1 cytokines,-
induced
senescence and apoptosis in a translational approach, experiments were
designed to
pretreat SK-BR-3 cells with trastuzumab and pertuzumab and then, the cells
were
additionally treated with TNE-a.and IFN-y for 5 days at 37'C 'followed by 2
more
passages withOut.the cytokines ,and antibodies. It Was observed that the
amount of blue
cells was highly increased in the cells that received the complete treatment
compared to
the cells treated with cytokines only, as measured by SA-11-gal staining
(Figure 9A) and
p15INK4b increased expression by western blot (Figure 913). interestingly, the
double
treatment also had a significant effect on the induction of apoptosis, both by
increased
active caspase 3 expression by western blot (Figure 9B) and by increased
amount of
anexin v and propidium iodide positive cells by flow cytometry analysis
(Figure 9C),
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C1)4-' Thl-mediated senescence and apoptosis of HER2-ovexpressirig.human
breast
cancer cells
Experiments were designed to co,culture HER2 Class H peptide-primed
CD4 T-cells with SK-BR-3 breast cancer cellS;using a tranSwell culture System
to
Confirm that :the eywk*s produced by the immune system cells in vivo could
also
induce.aSpeeific senescence and apoptotic response in tumor cells. The cells
were co-
cultured for 5 days at 37"C and then cultured for 2 more passages in complete
medium
without immune system cells. The co-culture resulted in senescence and
apoptosis of SK-
BR-3 cells, evidenced by increased amount of SA-13-gal staining (Figure 10A)
and
increased expression of p 15INK4b and cleaved caspase 3 (Figure 108) detected
by
western blot CD4' T-cells primed either with immature dendritic cells (DCs) or
mature
DCs plus irrelevant Class H (BRAF or survivine) peptides were not able to
induce either
senescence, or apoptosis of SK-BR-3 cell (Figure 10).
The senescence and apoptosis observed was highly augmented in SK-BR-
3 when the cells were co-cultured with HER2 Class H peptide-primed CD4' I-
cells in
presence of trastuzumab and pertuzumab in the transwell. This result was
clearly
evidenced by increase SA-Ii-gal staining (Figure 10A) and p15INK4b and cleaved
caspase 3 expression levels (Figure 108).
As another approach, SK-BR-3 cells were co-cultured with the supernatant
from CD4' T cell-mature DC co-culture and a similar specific senescence
response was
observed. The Thi -elaborated cytrikines fEN-y and TNF-a obtained from CD4.' T
cell-
mature DC co-culture supernatants were previously confirmed using ELBA. By
both
experimental approaches, SK-BR,3 senescence and apoptosis..could be partially
rescued
by neutralizing IFN-y and TNF-a. blocking antibodies (Figure 14).
It was also observed that the effect was dose-dependent as increasing
number of immune system cells induced higher SA-11-gal staining, pl 5INK4b and
cleaved caspase 3 expression levels in SK-BR-3 cells by both approaches.
Thl cytokines TNF-ct and 1FN-y sensitize trastuzumab and pertuzumab resistant
breast
cancer cells to senescence and apeptosiS induction
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to continue unraveling the mechanism by which the I hi cytokines could
induce senescence and apoptosis, it is believed that TNF-a and IFN-y could
restore the
sensitivity to trastuzumab and pertuzumab to breast cancer resistant cells. It
was observed
that the treatment with trastuzumab and pertuzumab could not prevent the
activation of
AKT in two resistant cell lines HCC-1419 (O'Brien et al., 2010 Mol Cancer
Ther.
6:1489-502) and JIMT-I (O'Brien et al., 2010 !viol Cancer Ther. 6:1489-502;
Tanner et
al, 2004 Mc! Cancer Ther. 12:1585-92) contrary to T-47D sensitive cells
(Figure 16).
The treatment with INF-0. and IFN-y induced senescence and apoptosis in
a dose dependent manner in HCC-1419 and JIMT-1 cells. When the cells were
treated
with trastuzumab and pertuzumab, senescence and apoptosis could not be
evidenced even
at higher doses (data not shown). However, the double treatment with cytokines
and
antibodies induced senescence by SA-11-gal assay (Figure 11A) and increase
expression
of pl5INK4b (Figure I 1B) in HCC-1419 and HMI- I cells. Moreover, the
combination
of cytokines and antibodies effectively induced cell death (Figure 11B) in HCC-
I419 and
JIMI-1 cells. This result demonstrates that the Thl cytokines IFN-y and INF-a
could
revert the resistance to the therapeutic agents that is affecting cancer
patients widely.
DISCUSSION
Herein it has been demonstrated that INF-a and IFN-y induce senescence and
apoptosis in breast cancer cells in a dose dependent manner. Also revealed is
an inverse
correlation between the HER2 expression level in breast cancer cells and the
doses of
TNF-a and IFN-y required to induce senescence in those cells. It has also been
shown
that cytokine receptors are expressed in similar levels M all the breast cell
lines tested,
implicating that this is not the cause of the differential response.
Furthermore, only when
MDA-MB-231 cells (low HE-R2 level) were stably transfected with a wild type
11ER2
plasmid, high doses ant cytokines were able to induce senescence and
apoptosis.
Thus, it seems that HER2 signaling is required to induce senescence and
apoptosis by thl
cytokines, because in cells that lack HER2 or express very low levels is not
possible to
induce senescence or apoptosis even with high doses of cytokines. However, in
cells that
express hid or intermediate levels of HER2, knocking down the gene induces
senescence and apoptosis through a phenomenon called oncog,ene addiction. It
has
additionally been shown that combining 1{ER2 and HER3 siRNA enhances even more
of
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,the senescence and apoptoSis induced by TNF-et and II N-I in breast cancer
cells, taking
a step ahead of HER3 over counting for the lack of HER2 signaling.
Example 5: Anti-Estrogen Therapy and Anti-HER2 Dendritic Cell Vaccination
Improves
Pathologic Complete Response in ER/HER2 P' DOS
The antigen-presenting capacity of dendritic cells ("DCs") has led to
enthusiasm for their nse in anti-tumor vaCtination. The present group designed
a HER2
peptide-pulsed autologous DC vaccine uniquely engineered to promote anti-HER2
Thl
Sensitization and attraction. See United States Ser. No. 14/658,095, filed
Match 13,
2015; United States Ser. No. 14/985,303, filed December 30, 2015, the
disclosures of
which are incorporated by reference herein in their entireties; Dana, i., et
al.,
Oneohnmunology 4:8 e1022301 (2015) D01:10. 1080/2162402 X.2015. 1022301, and
Dana, I., et al., Breast Cancer Res. 17(1):71 (2015). The feasibility, the
safety, and the
preliminary clinical results following the neoadjuvant use of the anti-HER2
vaccine in
patients with HER2 f"-'s DCIS (1iIER2P ductal carcinoma in situ) has been
reported.
Complete tumor regression (pathological complete response ("pCR")) was induced
in
19% of patients; however, these results were concentrated in patients with
hormone
independent (ER) DOS, suggesting that patients with ERP'sDCIS were less
responsive
to the anti-HER2 DC vaccine than are those patients with hormone independent
DCIS
(Sharma, Aõ et al., Cancer 118(17):4354-62 (2012)). The extensive
bidirectional
crosstalk between the HER2 and ER signaling pathways leads to enhanced
cancettell
proliferation and survival (Arnim. G., et al., Endocrine Reviews 29(2):217-233
(2008)
and Prat, A., et al,, Nat. Qin. Proc. Oncol. 5(9):531-542 (2008)). Combined
targeting of
both of these two receptors may prevent them front continuing to activate each
other. It
is hypothesized that adding anti-estrogen ("AE") therapy to anti-HER2 DC
vaccination
treatment would improve the response in patients with HER2P'-'/ERP"' Dos. In
the
studies reported herein comparisons were made between the clinical and the
immune
responses in anti-HER2 DC vaccinated patients with ER :nn DCIS ("ER), ER1" DOS
patients Who received the anti-HER2 DC- vaccine alone ("ERPN,ithotit At"), and
ER.P"'
DM patients who received both the anti-HER2 DC vaccine and concurrent anti-
estrogen
therapy ("ER P"' with AE").
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T he results .presented. herein-demonstrate that COACUITtat Tie0adjUVant anti-
estrogen therapy and anti-HER2 DC1 vaccination increases the immune response
in the
local sentinel lymph nodes and the rate of pathologic complete response in
HER2106/ERN DC1S. These results further support individualized, targeted,
combination
therapy. -
METHODS
Methods Summary: Eighty-one patients with HERn's DCIS received-a
neoadjuvant anti-HER2 DC vaccine. Clinical response was measured in the
resected
surgical specimen. Immune response anti-HER2 CD4 Thl response - was measured
in the peripheral blood pre- and post- vaccination and in the sentinel lymph
nodes post-
vaccination. Clinical and immune responses were compared between ER""e
patients who
underwent anti-HER2 vaccination alone, ER`" patients who underwent anti-HER2
vaccination alone, and ER"' patients that received anti-estrogen therapy
concurrently
with anti-HER2 vaccination.
The methods carried out are set forth below in detail,
xperiMents
Breast cancer cell.lines., SK13R3 (HER2P"' 3+1 ER"g) and ME. F7 (HERVg 2+1
ER), were treated with Th I cytokines (IFNy and TNFa)õ a tamoxifen metabolite
(4-
hydroxytamoxifen, -41-1r), or both for 72 hours. Metabolic activity was
measured via
Alamar Blue assay.
Trial Design
After approval by the Institutional Review Board of the University of
Pennsylvania, two neoadjuvant clinical trials of a HER2 peptide pulsed DC1
vaccine
(NCT001 070211 and NCT02061332) were conducted. The primary objectives were to
evaluate the feasibility, safety, and efficacy of DC1 vaccination. The
secondary objective
was to assess clinical and immune responses. .Preliminary results of these
trials showed
that the vaccine is safe, well tolerated, and induces decline or eradication
of HER-2
exPression (Sharma ,.A., et al., Cancer I 18(l7):4354-62 (2012) ("Sharma, et
al.")).
Further review of the preliminary results showed that vaccination was more
effective in
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hormone independent (ER) patients [20, 21]. Based On the preclinical data and
the
preliminary results of the clinical trial, an addendum was approved to treat
subsequent
ERP 3 patients with hormone dependent ( ERP's) DCIS with concurrent AE
therapy.
Patient Selection
Female patients older than 18 years of age with biopsy-proven HER2r"DOS and
an ECOG Performance Status Score of 0 or I were eligible for the trial. Al!
tissue
specimens were reviewed by a single pathologist for eligibility; HER-2
positivity was
defined as >5% of cells expression 2+ or 3+ intensity of the 1-[ER-2 protein.
Women of
childbearing age were required to have -a negative serum pregnancy test and
were
required to use a medically acceptable form of birth control. Women with
cardiac
dysfunction, HIV, HepC, coagulopathies, or a pre-existing medical illness or
medications
which might interfere with the study were excluded from the trial. Women who
had
received definitive treatment or whose DCIS was eliminated by excisional
biopsy at
diagnosis were not eligible for the trial. Eighty-one women were enrolled in
the trial and
completed the vaccination treatment. All 81 patients underwent surgical
resection with
pathologic examination of the resected specimen. The immune responses were
measured
in patients in the second trial (NCT02061332) - 53 of 54 patients had pre and
post-
vaccination CD4+ immune responseS measured in the peripheral blood; 40
patients had
post vaccination CD4+ immune responses measured in the sentinel lymph nodes;
and 22
HLA-A21' patients had pre- and post-vaccination CD8+ immune responses measured
in
the peripheral blood.
Vaccination Procedure
Vaccine preparation and delivery have been desctibed in detail previously.
See,
for example, Sharma, et al., Czemiecki, BJ., et al., Cancer Res. 67(4):1842-52
(2007)
(Czerniecki, et al.), and Koski, G.K., et al., i. .Thnnunother. 35(1) 54-56
(2012). The
vaccination procedure is shown in Figure 16. Briefly, monocytic dendtitic cell
precursors
were obtained from patients via tandem leukapheresisicountercurrent
centrifugal
elutriation. Monocytes were cultured at 37 C in serum free medium (SFM)
ativitrogen,
CarlSbad, CA) with gralnliOtyle-maa0phage Coiony-stimulating factor (GM-CS 1)
(Amgen, 'Newbury Park, CA) and IL-4. The following day, the cells were pulsed
With six
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HER,2,derived majorhistocompatibility complex (MEW) class H binding peptides
(American Peptide Corporation, Sunnyvale, CA) - three extracellular domain
(ECD)
peptides (peptide 42-56: FILDMLRFILYQGCQVV (SEQ ID NO: I); peptide 98-114:
RLRIVRGTQLFEDNYAL (SEQ ID NO: 2); and peptide 328-345:
TQRCEKCSKPCARVCYCIL (SEQ NO: 3)) and three intracellular domain (1CD)
peptides (peptide 776-790: GVGSPYAISRLLGICL (SEQ ID NO: 4);. peptide 927-941:
PARE1PDLLEKGERL (SEQ ID NO: 5); and peptide 1166-1180
TILERPKTLSPGKNGV (SEQ ID NO: 6))_ After 8-12 hours, 10001,11mL of:MN-gamma
(Inteimuneõ Brisbane, CA) was added, and 6 hours before harvest, 10mgina: of
clinical
grade LPS (gift from Dr Anthony Suffredini at the National Institute of Health
(NM))
was added to complete the rapid maturation to a type-I dendritic cell (DC 1).
For H.LA-
A2P patients, the monocyte pooi was pulsed with WIC class l. binding peptides
369-377
and 689-697.
Four to six weekly injections of 10-20 million HER-2-peptide pulsed Del s were
administered into the breast, the groin lymph nodes, or both the breast and
the groin
lymph nodes.
Patients were monitored for adverse effects for a minimum of 1-2 hours
following
each weekly vaccination. All adverse events were classified by National Cancer
Institute
Common Toxicity Criteria (NCI-CTC version 3.0), were assessed at least weekly
during
vaccination, and were Monitored until their resolution.
Clinical Monitoring
Pathologic response was examined at the time of surgical resection -
lumpectomy
(n 48) or mastectomy (n 33). A pathologic complete response to
immunization was
defined as no residual DCIS or invasive breast cancer at the time of surgical
resection.
Patients were monitored after surgical .resection for the des elOpment Of
subsequent breast
events A subsequent breast event was defined as a lesion ¨ DCIS or invasive
breast
cancer, identified in either the ipsilateral or contra-lateral breast.
Anti-Estrogen Therapy
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ER positive patients were _treated with anti-estrogen therapy concurrent with
four
to six weekly anti-HER2 DC1 vaccinations. A physician investigator decided.
which of
the following anti-estrogen therapies was best suited for each patient:
17amoxifen (4-
hydroxytamoxifen ("4H1") (NOLVADEXTm)); LetrozOle (FEMARATm); Anastrozote
(ARMIDEXTm); Exemestane (AROMASINTm); Raloxifene (EVISTATm); or any other
suitable anti-estrogen agent that blocks or modifies the actions of estrogen.
Immune 'Monitoring
(.7D44.
Systemic. anti-HER2 CD4+ 'f-cell responses were generated from autologous
peripheral blood mononuclear cells (PBMC) pulsed with six HER2-derived major
histocompatibility complex (MHC) class 11 binding peptides peptides. Localized
anti-
HER2 CD4+ T-cell responses were measured in the locoregional sentinel lymph
nodes
(SLN) in 40 patients who underwent SLN biopsy. 1FN-y production was quantified
via
enzyme-linked immunosorbent spot (ELISPOT) assays as previously described in
detail.
(Fracol, M., et aL Aim. Sorg. Oncol. 20(10):3233-9 (2013)) Briefly, PVDF
membrane
plates (Mabtech, Cincinnati, OH) were coated overnight with anti-[FN-y capture
antibody
(ID! K). The following day, after the plates were washed with PBS (Mediatech,
Manassas, VA) and blocked with 10% human serum,I.DMEM, 2x IW PBMCs on SUN
cells. Were plated in each well either unSrimulated, pulsed with HER2-derived
Class li
peptides:(4gg) (42-56, 98-114,.328-345, 776-79k927-941 1166-1180), or pulsed
With
anti-human CD3 and CD28 antibodies (0.5gginiL) (positive control, BD
Pharmingenõ
San Diego, CA), and incubated at 370C+ 5%C.02 for 24-36 hours. After the
plates were
washed with PBS, 10Opt of detection antibody (imgimL; 7 B6-1-biotin) was added
to
each well and the plates were incubated tbr 2 !lours. After the plates were
washed again
with PBS, 100AL of 1:1000 diluted streptavidin-HRP was added to each well and
the
plates were incubated for another hour. TMB substrate solution was added to
reveal spot
fOrMation. Spot forming 'OM (SFC) .viere counted using an automated reader
(ImmtmoSpot CTL).
A positive response to an individual HER2 Class II peptide was defined as a
minimum of 20 SFC/2x105 cells after subtracting the unstimulated background
and at
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least a two-fold increase over the.uristimulated baekground. Three metrics
were used to
quantify the CD4+ Th I response: (I) overall response rate (the proportion of
patients
responding to? 1 peptide), (2) response repertoire (the number of peptides to
which a
patient responds), and (3) cumulative response (the sum of the SFCs across all
6
peptides).
CD84- T-celh
Systemic anti-HER2 CD8+ T-celli responses were measured in twenty two HLA-
.A2im(HLA.2.1) patients . Anti-HER.2 CDS+ T-cell responses were generated by
in vitro
sensitization assays as previously described in detail by Czerniecki, et al...
Briefly, CD8+
T-cells were selected from the cryopreserved 120-140 lymphocyte cell fractions
via
negative selection (StemCell Technologies, Vancouver, BC). Autologous
dendritic cells
were suspended in serum free medium (SFM) (invitrogen, Carlsbad, CA) with GM-
CSF
(long/m1). pulsed with a class] 1-IER2 peptide (lOuglinl) (369-377), and co-
cultured with
the CD8+ T-cells at a ratio of 10:1. IL-2 (30 MIMI) was added on day 1 On day
10, 1-
cells were harvested and were tested against T2 target cells pulsed with
either the Class I
HER.2 peptide or irrelevant controls (p53 and colon cancer peptide). After 24
hours, the
supernatant was harvested and analyzed by enzyme-linked ittuunnosorbent assay
(El. ISA): A. positive response to the HER2 Class I peptide was defined as a
two-fold
increase in CDS+ T-cell IFNy production compared to the irrelevant peptide
controls.
Statistical Methods
Descriptive statistics and univatiate logistic regression was used to evaluate
demographic. and clinical data. P-values <0.05 were considered statistically
significant.
Development of subsequent breast events was compared using Kaplan Meier
analysis.
All analyses were performed with STATA 12.0/IC statistical software (STATA
Corp,
College Station, TX).
RESULTS
Results Summary: Patients with ER DCIS and patients with ER" DCIS who
were treated with anti-estrogen therapy bad a similar rate of pathologic
complete
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response.(31.4% vs-28.6%, p 1.00);.and both rates wete significantly higher
than the
rate of pathologic complete response found in patients with ER P's DCIS that
did not
receive anti-estrogen therapy (4,0%, p 0.035). The anti-HER2 Thl immune
response
measured in the peripheral blood increased significantly following
vaccination, but was
similar across all three groups. In the sentinel lymph nodes, however, the
anti-HER2 TM
immune response was significantly higher in the patients with ERP' DOS Who
were
treated with combination anti4HER2 vaccination and anti-estrogen therapy
compared
with the patients with ERP' DCIS who were treated with anti-HER2 vaccination
alone.
The results are set forth below in detail,
Pre-Clinical Experiments
=
The SKBR3 breast cancer cell line (ER) increased anti-tumor activity in
response to Thl cytokine treatment, but not in response to anti-estrogen
treatment.
Furthermore, adding anti-estrogen treatment to the Thl cytokine treatment had
no effect
on the metabolic activity as shown in Figure 17A,
-Conversely, the MCP breast .cancer cell line (ER!") did nOt increase anti-
tumor
activity in response to either Thl cytokine treatment or .anti-estrogen
treatment; however,
the combination of Thl cytokine treatment and anti-estrogen treatment together
resulted
in an increase in metabolic activity as shown in Figure 17B.
Trial; Patient Selection and Demostraphio
Oldie 81 patients who participated in .the clinical trial, the median age was
55
2$ (interquartile range (IQR) 47-60), median BMI was 25.9 (IQR 22.4-31.0),
and a majority
of patients were post-menopausal (n .r 68; 84,0%) and white (n.-- 65, 80.2%).
All
eligible patients were diagnosed with DOS at the time of biopsy ; however,
a.minority of
patients were found to have early invasive breast cancer (Stage 1) at-the time
of final
surgical resection (n. ¨ 1(, 19.8%). All eligible patients were diagnosed with
2+ In ¨ 28,
34.6%) or 3+ (ll ¨ 53, 65,4%) HER2P" 'DOS. Vaccination was administered into
groin
lymph nodes in 47 patients (58%), into the breast in 18 patients (22.2%), and
into both
the groin lymph nodes and the breast in 16 patients (20%). Surgical resection
was
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completed via lumpectomy in 48 patients (59.3µ,`,10) and via, mastectomy it 33
patients
(40.7%). Of those patients who underwent lumpectomy, 37,5% received post-
operative
radiation therapy.
The vaccine was well tolerated with only grade 1-2 adverse events. The. most
commonly reported adverse events associated with the vaccine were fatigue (n.
41,
50.6%), injection site reaction (n 34, 42.0%), and chills/rigors (n ¨ 26,
32A%). No
patients were unable to complete the trial due to the side effects.
Figure 18 shows that in the overall cohort, 35 patients (43.2%) had ER neg
disease
and 46 patients (56.8%) had ER' " disease. Of those patients with ER1"
disease, 25
patients (543%) received the DC1 vaccine alone and 21 patients (45.7%)
received the
DC1 vaccine and concurrent AE therapy. Demographic and clinical
characteristics of
these treatment groups are summarized in Table 1 below, and did not show any
significant difference between the groups.
Table 1; Demographic and clinical characteristics comparing patients by ER
status and
AE treatment.
Characteristic ER mg ERP s wiOut ERtms wi AE
Univariate
(%) AE (%)
Clot p-valuel
Number of patients $ 432 25 O 9 21(259) i
Median AVeJ101,956 (48-61151145-50)_ _ _54150-57) _
rfiM.Bi*tilignlligilgaiStiVP)pNEVIIARU)MMMMggigagggiligniii.gdidRMIRI
27.1 (23.2- 25.9 (22_6- 23.4 (21.8- 0.19
Median EIMI (IQR) 30.1)
.111100,1011,111.1.1411(131011.111111:40PRISINIORAMEMINSARISEI
tigiggililiiitlifilliellitiggCallag.i2ZIOMOIREE915.0101).E.REEREEntita=
Charison
Comorbidity
Score 27 (77.1) 21 (84.0) , 21 (100.0) 0.07
152 8(22.9) 4(16.0) 0(0.0)
?.3
!*iavr:.E:77:7.7.77.7.7.7.77.77.75717.7,7.!.77:77.77.7..7.7r.T.7.7.7.77.7..!,77
7777:!!.!,:,7R
1111011111:111111111.1111129M9)IIIII17(80)I11111003)1111111140111
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PCT/US2016/021090
2+ 14 (40.0) 3(12.0) 11 (52.4) (1..01
,,,,,,,,,, 22 (88.0) 10 (47.6)
9.11100*Sgeiggiggiaggallillagiogiggiggingligliigiglingnilignigigiggag
Sg Res
111000411111111211410011=111100410111111111#10101111111=1111=111
MildtWAIIII11111110406411111111044411NRAMMENEMIRMEM
DIONEMENEMIEMASEIMEIRd011111101001111111111.11.111111011
,
Lumpectomy 19 (54.3) 13 (52.0) 16
(76.2) 0.18
Mastectomy_ ,16 j45.7)_ 12148.0) 5 (23.81
illtiaattOWNEERNMeggreggiONFINFORROlgireggiORM
EgilliiitOgNESE.:iliiiiingNEERTMESPREM61461Pani.di:41;i4301i4danigg040iNWE':gli
IttntingaiDeangiAMONEEINEMPIENNVIAIRMIERIEMEMEMENN
ER (estrogen receptor), AE (anti-estrogen), IQR (interquartile range), HER2
(human
epidermal growth factor receptor 2).
bold indicates statistical shmificance
'following lumpectomy
Clinical Response Rates to HER2 Vaccination
The clinical response was available for all 81 patients_ Overall, 18 of the 81
immunized patients (22.2%) were found to have no residual disease identified
in the
resected surgical specimen; these patients were considered to have a
pathologic complete
response (pCR). As shown in Figure 19, pathologic, complete response in the
ERneg
group was higher than in the ER I" group. More specifically, pCR in the ER 'e
group (II
= 11, 31_4%) and the ERI" group that received AE therapy (n = 6, 28.6%) were
similar
(p 1.001); however, the rate of pCR. in the ER" group that did not.
receive AE therapy (ti
= 1, 4.0%) was significantly lower than the rate of pCR in the ER"g group (P =
0.01) and
the ER " group that received .AE therapy (p 0.04) (Figure 19).
Pathologic coMplete.responsecorrelates with a decreased 1.7i* of recurrence
See,
for example, Tanioka,. M., et al, . Br. .1. -(7(1/leer 103(30:297.302 (2010)
Subsequent breast
lesions, defined as either DCIS or invasive breast cancer identified in either
breast-,
occurred in 6 vaccinated patients (7.4%). All of the patients who experienced
subsequent
breast events had residual disease identified at the time of surgical.
resection, < pCR
(Figure 20A). Two of the patients had ER' DCIS and four of the patients had
ERP1'
DCIS but did not receive AE therapy. None of the patients who had ER' DCIS and
received: AE therapy have experienced a subsequenthreast..eVent (Figure 20B).
.25 patients enrolled later in the trial were treated with concurrent
vaccination and AE =
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therapy, and, therefore, median follow up was shorter for these patients. Of
course, these
patient S will continue to be monitored.
As shown in Table 1, the rate of lumpectomy was similar across all three
groups;
however, the rate of radiation following lumpectomy was lower in the group of
patients
with ERP" DOS who received AE therapy (18.75%) than in the group of patients
with
ER' "' DOS who did not receive AE therapy (53.8%, p = 0.06). Patients with
ER!' DOS
who received AE therapy have a decreased rate of subsequent breast events
despite the
lower rate of radiation.
Immune Response Rates to HER2 Vaccination
CD4 4- T7I1 Response: Spiemic - Peripheral Blood
The pre-vaccination and post-vaccination immune responses were measured in 53
patients. Overall, responsivity increased significantly from 36.25% pre-
vaccination to
56.25% post-vaccination (p ¨ <0,001), Median response repertoire also
increased
significantly from 1(IQR 0-2) pre-vaccination to 2.9 (IQR 2-4) post-
vaccination (p ¨
<0.001). Finally, median cumulative response increased significantly from 56A
(IQR
23,3-111.7) pm-vaccination to 133.1 (IQR 75.6-240.3) post-vaccination (p
0,002).
Responsivitv: Following vaccination, responsivity increased significantly in
each
group (ER 'eg 58,3% to 87.5%, p < 0,01; ER P"s without AE treatment 50.0% to
75.0%, p <
0.01; ERP' with AE treatment 57.1% to 90.5%, p < 0.01), Pre-vaccination
responsivity
rates were similar across all three groups 58.3%. Eltr"'s without AE
treatment
2$ 50.0%, ER" with AE treatment 57.1%; p = 0,9). Post-vaccination
responsivity rates
were also similar across all three groups (ElVeg 87.5%, ERP' without AB
treatment
75,0%. ER P" with AE treatment 90.5%; p 0.5, Figure 21A).
Response Repertoire: Following vaccination, response repertoire increased in
each. group (ER .1. to 3, p 0.05; ER! " without AE treatment 0 t01.5, p 0.1;
ERP"
with AE treatment 1 to 3, p = 0.01). Pre-vaccination median response
repertoire was
similar across all three groups (EIVeg 1 (1QR 0-2), ERP' without AB treatment
0 (IQR 0-
1.5), ER P"' with AE treatment 1 (IQR 0-2); p 0.5). Post-vaccination median
response
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repertoire :was also. similar across all three groups (ER- 3 (IQR. 2-
4.5)õ.ERP" without AE
treatinent 1.5 (IQR 0.5-3.5), ER P" with AE treatment 3 (IQR 2-5); p = 0.4.
Figure 21B).
Cumulative Response: Following vaccination cumulative response increased in
each group (ER "g 56.3 to 149.7, p <0.01; ER P" without. AE treatment 41.1 to
178.7, p <
0.01; ERP" with AE treatment 58.6 to 100.9, p <0.01). Pre-vaccination median
cumulative response was similar across all three groups (ER" 56.3 (IQR. 26.1-
116.2),
ERP' without AE treatment 41.1 (IQR 9.6-168.6), ER's with AE treatment 58.6
(IQR
24.2-87.9); p = 0.886). Post-vaccination median cumulative response was also
similar
across all three groups (ER's .1493 (IC* 977-246,7), .ER without AEtreatment
178.7
(IQR 64.7-278.3), ER"' with AE treatment 100.9 (T.QR 67,5-174A); p ¨ 0.5,
Figure 21C).
CD4-i- Thi Response: Local Sentinel Lymph Node
The post-vaccination immune responses were measured in 40 patients. Overall,
responsiviti was 80%, median response repertoire was 2 (IQR 1-5), and median
cumulative response was 76.5 (19R 23-197).
Responsivity: Responsivity rates were significantly higher in patients with
HER2P"/MRP"' DOS who received with AE treatment compared to patients with
IIER2P09/ERP"' DCIS who did not receive AE treatment (92.3% vs 43%; p 0.03.
Figure
22A).
Response Repertoire: Median response repertoire was also significantly higher
in
patients with HER2posiERpos DCES who received with AE treatment compared to
patients with HER2pos/ERpos DOS who did not receive AE treatment (4 (IQR 2-6)
vs 0
(IQR. 0-5); p 0.05. Figure 228),
Cumulative Response: Median cumulative response was also significantly higher
in patients with HER2P"./ERP" DCIS who received with AE treatment compared to
patients with FIER2P"8/ERP" DCIS who did not receive AE treatment (102 (IQR 69-
354)
vs 23 (IQR 1-100); p 0.05. Figure 22C).
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CD84. Response: Systemic - Peripheral Blood
The pre-vac.cinationandpost-yaccination CDS+ tmtnune responseS were
measured in 22 IILA-A2+ patients. Overall, tesponsivity increased
significantly from
113% pre-vaccination to 723% post-vaccination (p = <0.0002).
Responsivitv: Following vaccination, responsivity increased in each group
(ER"g
12.5% to 75%, p = 0.04; ER"' without AE treatment 0% to 100%, p < 0.03, ER!'
with
AE treatment 20% to 60%, p =0.1 7. Pre-vaccination responsivity rates were
similar
across all three groups (ER 'g 12.5%, ER'" 'without AE treatment 0%, ER ' with
AE
treatment 20%; p = ). Post-vaccination responsivity rates were also similar
across all
three groups (EIV'eg- 75%, ERix's without AE treatment 100%. ER P"' with AE
treatment
60%; p =. Figure 23).
CONCLUSIONS: The study described herein clearly shows anti-HER2 DC.1
vaccines are clinically effective in ElltnIFIEVb: DCIS.patie.nts. DC1 ,
vaccines are also shown to be safe when combined with anti-estrogen therapy.
It was
shown that concurrent neoadjuvant anti-estrogen therapy and anti-HER2 DC1
vaccination increases the immune response in the local sentinel lymph nodes
and the rate
of pathologic co Piet response in FIER21"/ERPY4DCIS patients The combination
therapy with anti-estrogen may also reduce subsequent Breast Events_ These
results may
offer a personalized approach in DCISItherapy: These results further support
individualized* targeted, combination anti-HER2 DC1 vaccine and anti-estrogen
treatment. One skilled in the art can appreciate that other anti-proliferative
combinations
of anti-HER2 treatments such as Trastuzumab and Pertuzumab, and others, are
possible.
These approaches may limit the need to extensive surgery, eliminate
radiatition therapy,
and reduce long term hormonal treatment.
Example 6:A Novel Dendritic Cell Vaccine Targeting Mutated BEM Overcomes
Vemurafenib Resist= and Synergistically Improves Survival in BRA.F-Mutant
Marine
Melatioina
BRAF inhibitor vemurafenib (PLX) improves survival in :MAP-mutant
(BRAFv6m3-) melanoma, but resistance is common. A BRA.Fv"IE-puised typel-
polarized
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dendritic cell vaccine (BRAF"."-DC1) induces antigen-specific CDr T-cells that
impact murine BRAI"" melanoma. We investigated if combinations of BRAP"'-
DC:1 and PLX elicit a synergistic clinical response.
METHODS
A transplantable BRAFv6mr=PTEN melanoma model was de eloped in the
C57I31/6 background, DC1 were generated from bone marrow precursors using
F1t3, IL-
6, GM-CSF, 1L-4, CpG and LPS, and pulsed with class 1 BRAFw" peptide. In
addition
to untreated and ovalburnin-DC1 controls, BRAF'-DC1 (2x weekly injections) and
PLX were administered alone or in designated combinations to tumor-bearing
mice
(n-10 each). Tumor growth and survival were determined, Induction of BRAF''-
specific CDS T-cell responses from splenocytes were assessed by IFN-y ELISA.
Cytokine mRNA quantification in tumor microenvironments (TME) was performed
by
RTAPCR.
RESULTS
Figure 24 shows mice receiving BRAF'"E-DCI+PLX mithinations, either
initiated concurrently or after BRAP"tx/13-DC I induction, demonstrated
dramatically
delayed tumor growth (P<0.001) and iMpiOVed median survival (86d and 73.5d,
respectively) vs .BRAF'-DC1 (42d), PLX (43.5d), ovalbumin-DC I (28d)õ or
untreated
(24d) cohorts (P<0.00I); 35% were rendered disease-free following BRAF'b-
DCI+PLX therapy, and remained immune to BRAFv6'a tumor rechallenge. BRAFv6"*-
DCI+PLX, compared with individual, treatments induced synergistically improved
systemic CDS' T-cell recognition of BRAFv6("-pulsed antigen-presenting cells
and
BRAFv uE tumor cells (p<0.001) measured by IFN-y release in vitro. In TME.,
13RAF-DC1+FLX generated higher mRNA levels of Thl (IFN-T/TNF-a) and T-cell
homing (CXCL9/CCL5) cytokines, while attenuating PD-L1 expression; CD8' TIL
trafficking was augmented by BRAFv'/".-DC1+PLX.
In conclusion BRAFvml"-DC1 vaccines overcome vernurafenib resistance in
BRAFN.'")E melanoma, and synergistically improve immune and clinical
responses. Such
combinations will find use by those of skill in the art.
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The disclosures of each and every patent, patent application, and
publication cited herein are hereby incorporated herein by reference in their
entirety.
While these embodiments has been disclosed with reference to specific
embodiments, it
is apparent that other embodiments and variations of these embodiments may be
devised
by others skilled in the art without departing from the true spirit and scope
of the
embodiments. The appended claims are intended to be construed to include all
such
embodiments and equivalent variations.
