Note: Descriptions are shown in the official language in which they were submitted.
CA 02874065 2014-12-04
METHODS AND COMPOSITIONS FOR TREATING BREAST CANCER WITH
DENDRITIC CELL VACCINES
BACKGROUND OF THE INVENTION
A. Field of the Invention
[0001] The invention generally relates to the field of medicine. More
particularly, it
concerns the use of dendritic cell vaccines in immunotherapy of breast cancer.
In certain aspects,
the dendritic cells display cyclin B1 and WT-1 peptide epitopes and are
administered to breast
cancer patients.
B. Background
[0002] Women with breast cancer who are treated with preoperative
chemotherapy have
the same survival as those who receive adjuvant therapy; however, pathologic
complete response
(pCR) after preoperative chemotherapy is a predictor of improved outcomes
(Fisher, et al., 1997;
Rastrogi, et al., 2008). Those treated with preoperative therapy who achieve a
pCR or near pCR
have significantly better distant relapse-free survival than those with
extensive residual disease
independent of pathologic subtype.
[0003] Women with triple-negative breast cancer (TNBC) have an increased
pCR rate as
compared to women with non-TNBC, and those with pCR have a 90% disease-free
survival
(Liedtke, et al., 2008; Von Minckwitz, et al., 2011). However, women with TNBC
who do not
achieve a pCR (i.e., those that have residual disease after neoadjuvant
chemotherapy) have an
increased risk of recurrence, decreased overall survival, and post-recurrence
survival as
compared to women with non-TNBC who do not achieve a pCR. The risk of
recurrence and
death is time-dependent and significantly higher for women with TNBC in the
first 3 years of
follow-up, versus women with non-TNBC (Liedtke, et al., 2008).
[0004] These patients have a great unmet medical need as there is no known
effective
therapy which can improve outcome. Therefore, a high priority for clinical
research in patients
with locally advanced TNBC is to increase the pathologic complete response
(pCR) rate in breast
and axilla following preoperative therapy. Patients with T3 and T4 cancers and
with clinically
- I -
CA 02874065 2014-12-04
N1/N2 axillary disease are at highest risk of not achieving a pCR with
standard therapy, and of
developing metastatic disease.
SUMMARY OF THE INVENTION
[0005] Methods and compositions are provided concerning a breast cancer
immunotherapy that can improve outcomes for breast cancer patients.
Vaccination of a subject
with dendritic cells ("DCs") loaded with cyclin B1 and WT-1 peptide antigens
can induce both
therapeutic T cell immunity by activating effector T cells and protective T
cell immunity by
creating tumor-specific memory T cells that can control tumor relapse.
Treatment with DC
vaccines can be administered in combination with standard chemotherapy,
radiation treatment,
and surgery in order to improve outcomes for those treatments. DC vaccines can
also be
administered in combination with the IL-1R antagonist anakinra, which
decreases the chronic
inflammation that is associated with many solid tumors and that promotes
cancer cell survival
and metastasis.
[0006] In some embodiments, there is disclosed a pharmaceutical
composition
comprising: isolated, active dendritic cells displaying cyclin B1 peptide
epitopes; isolated, active
dendritic cells displaying WT-1 peptide epitopes; and/or isolated, active
dendritic cells
displaying both cyclin B1 peptide epitopes and WT-1 peptide epitopes; or any
combination
thereof. In some embodiments, the composition can comprise isolated, active
dendritic cells
wherein each dendritic cell may display both cyclin B1 peptide epitopes and WT-
1 peptide
epitopes. In some embodiments, the composition can comprise a mixture of
isolated, active
dendritic cells, some of which display cyclin B1 peptide epitopes and not WT-1
peptide epitopes
and some of which display WT-1 peptide epitopes and not cyclin B1 peptide
epitopes. In some
embodiments, the isolated, active dendritic cells in the composition display
only cyclin B1
peptide epitopes or only WT-1 peptide epitopes. In some embodiments, the
cyclin B1 peptide
epitopes comprise sequences corresponding to SEQ ID NO:1 and/or SEQ ID NO:2 or
fragments
thereof. In some embodiments, the isolated, active dendritic cells displaying
cyclin B1 peptide
epitopes have been incubated with cyclin B1 peptide antigens comprising SEQ ID
NO:1 and/or
SEQ ID NO:2. In some embodiments, the WT-1 peptide epitopes comprise sequences
corresponding to one or more of SEQ ID NOs: 3 ¨ 8 or fragments thereof. In
some embodiments,
the isolated, active dendritic cells displaying WT-1 peptide epitopes have
been incubated with
- 2 -
CA 02874065 2014-12-04
WT-1 peptide antigens comprising one or more of SEQ ID NOs:3 ¨ 8. In some
embodiments, the
isolated, active dendritic cells displaying cyclin B1 and the isolated, active
dendritic cells
displaying WT-1 peptide epitopes have been activated by incubation with
lipopolysaccharide,
CD40 ligand, and CL075. In certain embodiments, the WT-1 peptide comprises or
consists of the
amino sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6,
SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, dendritic cells are exposed
to 1, 2, 3, 4,
5, 6, 7, or all 8 peptide sequences.
[0007] "Peptide epitopes" as used herein includes polypeptides displayed
on the surface
of dendritic cells in complex with MHC class II molecules. Peptide epitopes
can be derived from
peptide antigens with which dendritic cells have been incubated. Dendritic
cells can take up the
peptide antigens and process the peptide antigens for display on the cell
surface, a process
referred to herein as "antigen loading." Dendritic cells incubated with
antigens and displaying
peptide epitopes on their surface are in some instances referred to herein as
"antigen-loaded
dendritic cells." The "peptide epitopes" referred to herein are at least 9
amino acids in length.
[0008] "Isolated dendritic cells" as used herein refers to dendritic cells
that are found
outside the body, separate from the environment in which dendritic cells are
usually found in the
body.
[0009] In some embodiments, isolated dendritic cells in the compositions
and methods
described herein are derived from monocytes that have been isolated from a
subject's blood.
Monocytes can be isolated from a subject's blood by any process known to those
of skill in the
art. As an example, monocytes can be isolated by a process that begins with
removing white
blood cells from a subject's blood stream by apheresis, which can result in a
blood composition
enriched for white blood cells with minimal presence of red blood cells.
Monocytes can be
further isolated or enriched from the apheresis product by elutriation, which
results in a
composition enriched for monocytes. The elutriation product may contain at
least or at most 70,
75, 80, 85, 90, 95% or more monocytes as a percentage of total cells present
in the composition
(or any range derivable therein). Thus, as used herein, "isolated monocytes"
can include
compositions that include some proportion of other types of cells and
"isolating monocytes" can
refer to a process that results in enrichment of monocytes and not necessarily
complete
purification of monocytes.
-3 -
CA 02874065 2014-12-04
[0010] In some embodiments of the methods described herein, dendritic
cells are derived
from monocytes by incubating the monocytes with IFNa and GM-CSF. This process
is also
referred to as differentiation. The dendritic cells that are formed by
incubating monocytes with
IFNa and GM-CSF are in some instances referred to herein as "IFN-DCs."
[0011] Also disclosed is a pharmaceutical composition for treating breast
cancer in a
subject comprising isolated, active dendritic cells that (i) are derived from
monocytes isolated
from the subject's blood and differentiated into dendritic cells in vitro and
(ii) display cyclin B1
peptide epitopes, WT-1 peptide epitopes, or both cyclin B1 peptide epitopes
and WT-1 peptide
epitopes. In some embodiments, the isolated, active dendritic cells display WT-
1 peptide
epitopes. In some embodiments, the isolated, active IFN-dendritic cells
display both cyclin B1
peptide epitopes and WT-1 peptide epitopes. In some embodiments, the cyclin B1
peptide
epitopes comprise sequences corresponding to SEQ ID NO:1 and/or SEQ ID NO:2 or
fragments
thereof. In some embodiments, the isolated, active dendritic cells have been
incubated with
cyclin B1 peptide antigens comprising SEQ ID NO:1 and/or SEQ ID NO:2. In some
embodiments, the WT-1 peptide epitopes comprise sequences corresponding to one
or more of
SEQ ID NOs:3 ¨ 8 or fragments thereof. In some embodiments, the isolated,
active dendritic
cells have been incubated with WT-1 peptide antigens comprising one or more of
SEQ ID NOs:3
¨ 8. In some embodiments, the isolated, active dendritic cells have been
derived from monocytes
isolated from the subject by incubating the monocytes with IFNa and GM-CSF. In
certain
embodiments, one or more peptide antigens may be excluded in the embodiment.
[0012] Also disclosed is a pharmaceutical composition for treating breast
cancer in a
subject made by a method comprising: isolating monocytes from the subject's
blood;
differentiating the isolated monocytes into dendritic cells; incubating the
dendritic cells with one
or more isolated cyclin B1 peptide antigens and/or WT-1 peptide antigens; and
activating the
dendritic cells. In some embodiments, the dendritic cells are incubated with
one or more isolated
WT-1 peptide antigens. In some embodiments, the dendritic cells are incubated
with isolated
cyclin B1 and WT-1 peptide antigens. In some embodiments, the one or more
isolated cyclin B1
peptide antigens comprise SEQ ID NO:1 and/or SEQ ID NO:2. In some embodiments,
the one or
more isolated cyclin B1 peptide antigens comprise SEQ ID NO:1 and SEQ ID NO:2.
In some
embodiments, the one or more isolated WT-1 peptide antigens comprise one or
more of SEQ ID
- 4 -
CA 02874065 2014-12-04
NOs:3 ¨ 8. In some embodiments, the one or more isolated WT-1 peptide antigens
comprise
SEQ ID NOs:3 ¨ 8. In some embodiments, the step of differentiating the
isolated monocytes is
performed by incubating the isolated monocytes with IFNa and GM-CSF. In some
embodiments,
the step of activating the dendritic cells is performed by incubating the
dendritic cells with
lipopolysaccharide, CD40 ligand, and CL075 before, during, or after the time
that the dendritic
cells are incubated with one or more isolated cyclin B1 peptide antigens
and/or WT-1 peptide
antigens. In certain embodiments, one or more peptide antigens may be excluded
in the
embodiment.
[0013] Also disclosed is a method of treating breast cancer in a subject
comprising
administering to the subject any of the pharmaceutical compositions described
herein. In some
embodiments, the subject has triple negative breast cancer. In some
embodiments, the subject has
been diagnosed with triple negative breast cancer. In some embodiments, the
subject has
previously been administered a chemotherapeutic agent. In some embodiments,
the
chemotherapy did not result in a pathologic complete response in the subject.
In some
embodiments, the breast cancer is resistant to chemotherapy. In some
embodiments, the breast
cancer has been determined to be resistant to chemotherapy. In some
embodiments, the method
further comprises administering to the subject an IL-1 receptor (IL-1R)
antagonist in
combination with the composition of any one of claims I to 26. In some
embodiments, the IL-1R
antagonist is anakinra. In some embodiments, the method further comprises
administering to the
subject one or more chemotherapeutic agents in combination with the
composition of any one of
claims 1 to 26. In some embodiments, the one or more chemotherapeutic agents
comprise one or
more of doxorubicin, cyclophosphamide, Adriamycin, Cytoxan, and paclitaxel. In
some
embodiments, the method further comprises treating the breast cancer by
performing surgery
and/or radiation. It is specifically contemplated that one or more
chemotherapeutic agents or
surgery or radiation may be excluded in certain embodiments.
[0014] Methods of treating breast cancer described herein can include
methods that result
in decreased growth of cancer cells, inhibition of growth of cancer cells,
killing of cancer cells,
and/or shrinking of cancer tumors. The methods described herein can also be
used to reduce the
metastagenicity of breast cancer, to enhance the effectiveness of other cancer
treatments such as
chemotherapy, surgery, or radiation, or to reduce the likelihood of recurrence
of a breast cancer.
- 5 -
CA 02874065 2014-12-04
[0015] In the methods described herein, administration of pharmaceutical
compositions
comprising antigen-loaded dendritic cells can be performed in conjuction or in
combination with
surgery, radiation, chemotherapy, or other breast cancer treatments.
Administration of antigen-
loaded DCs can be performed before, during, or after the time that the other
treatments are
administered. The other cancer treatments administered in conjuction with
pharmaceutical
compositions comprising antigen-loaded dendritic cells can be any treatment
known by those of
skill in the art.
[0016] Also disclosed herein is a method of making active, antigen-loaded
dendritic cells
for treating breast cancer in a subject comprising: isolating monocytes from
the subject's blood;
differentiating the isolated monocytes into dendritic cells; incubating the
dendritic cells with one
or more isolated cyclin B1 peptide antigens and/or WT-1 peptide antigens; and
activating the
dendritic cells. In some embodiments, the dendritic cells are incubated with
isolated WT-1
peptide antigens. In some embodiments, the dendritic cells are incubated with
one or more
isolated cyclin B1 peptide antigens and one or more WT-1 peptide antigens. In
some
embodiments, the one or more isolated cyclin B1 peptide antigens comprise SEQ
ID NO:1
and/or SEQ ID NO:2. In some embodiments, the one or more isolated cyclin Bl
peptide antigens
comprise SEQ ID NO:1 and SEQ ID NO:2. In some embodiments, the one or more
isolated WT-
1 peptide antigens comprise one or more of SEQ ID NOs:3 ¨ 8. In some
embodiments, the one or
more isolated WT-1 peptide antigens comprise SEQ ID NOs:3 ¨ 8. In some
embodiments, the
step of differentiating the isolated monocytes is performed by incubating the
isolated monocytes
with IFNot and GM-CSF. In some embodiments, the step of activating the
dendritic cells is
performed by incubating the dendritic cells with lipopolysaccharide, CD40
ligand, and CL075
before, during, or after the time that the dendritic cells are incubated with
one or more isolated
cyclin 81 peptide antigens and/or WT-1 peptide antigens. The step of
activating the dendritic
cells can also be performed by incubating the dendritic cells with a
composition comprising
lipopolysaccharide, CD40 ligand, and/or CL075. In certain embodiments, one or
more peptide
antigens may be excluded in the embodiment.
[0017] Also disclosed is a method of treating breast cancer in a subject
comprising:
isolating monocytes from the subject's blood; differentiating the monocytes to
form dendritic
cells; incubating the dendritic cells with an antigenic composition comprising
(i) one or more
isolated cyclin B1 peptide antigens and/or WT-1 peptide antigens and (ii) one
or more dendritic
- 6 -
CA 02874065 2014-12-04
cell activating agents to form activated, antigen-loaded dendritic cells; and
administering to the
subject a first pharmaceutical composition comprising the activated, antigen-
loaded dendritic
cells. In some instances, the one or more cyclin B1 peptide antigens and/or
one or more WT-1
peptide antigens are added to a composition comprising the dendritic cells
before the dendritic
cell activating agents are added. In other instances, one or more peptide
antigens can be added to
the dendritic cells at the same time as or after the dendritic cell activating
agents. "Dendritic cell
activating agents" include agents that enhance the ability of dendritic cells
to stimulate an
immune response when administered to a subject. In some embodiments, the
method further
comprises obtaining blood from the subject. In some embodiments, the antigenic
composition
comprises one or more isolated WT-1 peptide antigens. In some embodiments, the
antigenic
composition comprises one or more isolated cyclin B1 and WT-1 peptide
antigens. In some
embodiments, the one or more isolated cyclin B1 peptide antigens comprise SEQ
ID NO:1
and/or SEQ ID NO:2. In some embodiments, the one or more isolated cyclin B1
peptide antigens
comprise SEQ ID NO:1 and SEQ ID NO:2. In some embodiments, the one or more
isolated WT-
1 peptide antigens comprise one or more of SEQ ID NOs:3 ¨ 8. In some
embodiments, the one or
more isolated WT-1 peptide antigens comprise SEQ ID NOs:3 ¨ 8. In some
embodiments, the
step of differentiating the isolated monocytes is performed by incubating the
isolated monocytes
with IFNa and GM-CSF. In some embodiments, the one or more dendritic cell
activating agents
comprise lipopolysaccharide, CD40 ligand, and/or CL075. In some embodiments,
the one or
more dendritic cell activating agents comprise lipopolysaccharide, CD40
ligand, and CL075. In
some embodiments, the subject has triple negative breast cancer. In some
embodiments, the
subject has been diagnosed with triple negative breast cancer. In some
embodiments, the subject
has previously been administered chemotherapy. In some embodiments, the
chemotherapy did
not result in a pathologic complete response in the subject. In some
embodiments, the breast
cancer is resistant to chemotherapy. In some embodiments, the breast cancer
has been
determined to be resistant to chemotherapy. In some embodiments, the method
further comprises
administering to the subject an IL-1R antagonist. In some embodiments, the IL-
1R antagonist is
anakinra. In some embodiments, the first pharmaceutical composition is
administered
intratumorally, subcutaneously, or intraveinously. In some embodiments, the
first pharmaceutical
composition is administered to the subject in multiple doses. In some
embodiments, each dose of
the first pharmaceutical composition comprises between about 0.5 x 106 and 15
x 106 viable cells
- 7 -
CA 02874065 2014-12-04
of the active, antigen-loaded dendritic cells. In some embodiments, each dose
of the first
pharmaceutical composition comprises at least about 15 x 106 viable cells of
the active, antigen-
loaded dendritic cells. In some embodiments, the concentration of the active,
antigen-loaded
dendritic cells in the first pharmaceutical composition is between about 1 x
106 and 15 x 106
viable cells/ml. In some embodiments, is approximately or is at least or at
most about 15 x 106
viable cells/ml. In some embodiments, the first pharmaceutical composition is
administered to
the subject in combination with one or more chemotherapeutic agents. In some
embodiments, the
one or more chemotherapeutic agents comprise one or more of doxorubicin,
cyclophosphamide,
and paclitaxel. In some embodiments, the first pharmaceutical composition is
administered to the
subject intratumorally. In some embodiments, the first pharmaceutical
composition is
administered to the subject in multiple doses. In some embodiments, each dose
of the first
pharmaceutical composition comprises approximately 0.6 x 106 viable cells of
the active,
antigen-loaded dendritic cells. In some embodiments, the concentration of the
active, antigen-
loaded dendritic cells in the first pharmaceutical composition is about 3 x
106 viable cells/ml. In
some embodiments, the method further comprises administering to the subject a
second
pharmaceutical composition comprising the active, antigen-loaded dendritic
cells. In some
embodiments, the second pharmaceutical composition is administered
subcutaneously. In some
embodiments, the second pharmaceutical composition is administered to the
subject in multiple
doses. In some embodiments, each dose of the second pharmaceutical composition
comprises
approximately 15 x 106 viable cells of the active, antigen-loaded dendritic
cells. In some
embodiments, the concentration of the active, antigen-loaded dendritic cells
in the second
pharmaceutical composition is approximately or at least about 15 x 106 viable
cells/ml. In some
embodiments, the second pharmaceutical composition is administered to the
subject in
combination with one or more chemotherapeutic agents. In some embodiments, the
one or more
chemotherapeutic agents comprise one or more of doxorubicin, cyclophosphamide,
and
paclitaxel. In some embodiments, the method further comprises surgically
removing breast
cancer tissue and/or administering radiation treatment.
[0018]
Also disclosed is a method of treating cancer in a subject comprising:
isolating
monocytes from the subject's blood; differentiating the monocytes into
dendritic cells by
incubating the monocytes with IFI\la and GM-CSF; incubating the dendritic
cells with an
antigenic composition comprising one or more isolated cyclin B1 peptide
antigens and/or WT-1
- 8 -
CA 02874065 2014-12-04
peptide antigens, lipopolysaccharide, CD40 ligand, and CL075 to form
activated, antigen-loaded
dendritic cells; and administering to the subject a pharmaceutical composition
comprising the
activated, antigen-loaded dendritic cells in combination with one or more
chemotherapeutic
agents and an antagonist of IL-1R.
[0019] Also disclosed is a method of thawing a frozen dendritic cell
vaccine for
administration to a subject comprising: thawing frozen dendritic cells by
suspending the
dendritic cells in Lactated Ringer's solution; washing the dendritic cells
with Lactated Ringer's
solution; and suspending the dendritic cells in Lactated Ringer's solution at
a concentration of
approximately or at least about 15 x 106 viable cells/ml. In some embodiments,
the frozen
dendritic cells are active, antigen-loaded dendritic cells. In some
embodiments, the active-
antigen-loaded dendritic cells display cyclin B1 and/or WT-1 peptide epitopes,
which may be
derived from SEQ ID NOs:1 ¨ 8. In certain embodiments, one or more peptide
epitopes may be
excluded in an embodiment.
[0020] Also disclosed is a method of treating cancer in a subject
comprising
administering to the subject a chemotherapeutic agent and administering to the
subject a
therapeutic agent that blocks IL-1(3 and/or blocks the IL-1 inflammation
pathway. In some
embodiments, the agent is an antibody that specifically binds IL-1 receptor or
IL-1[3. It may be a
monoclonal or humanized or chimeric antibody. Alternatively, it may be a
single-chain antibody.
In further embodiments, the agent is a peptide or polypeptide. In some
embodiments, the
therapeutic agent that blocks IL-113 and/or blocks the IL-1 inflammation
pathway is anakinra. In
some embodiments, the therapeutic agent that blocks IL-1f3 and/or blocks the
IL-1 inflammation
pathway is rilonacept or canakinumab. In some embodiments, the
chemotherapeutic agent is Nab
paclitaxel, eribulin, capecitabine, or vinorelbine. In some embodiments,
anakinra is administered
to the subject before the first time the chemotherapeutic agent is
administered to the subject. In
some embodiments, the anakinra may be administered to a subject who has
previously been
administered chemotherapy but is not currently undergoing chemotherapy at the
time the
administration of anakinra begins. In such embodiments, anakinra
administration may begin
before the beginning of another chemotherapy treatment regimen. In some
embodiments,
anakinra is administered to the subject daily for 14 days before the first
time the
chemotherapeutic agent is administered to the subject. In some embodiments,
anakinra is
administered to the subject daily during the time in which the
chemotherapeutic agent is
- 9 -
CA 02874065 2014-12-04
administered to the subject. In some embodiments, each dose of anakinra is 100
mg or about 100
mg. In some embodiments, each dose of anakinra is between about 50 and 150 mg,
between
about 75 and 125 mg, or between about 90 and 110 mg.
[0021] In some embodiments, it is contemplated that whole cells may be
excluded as the
source of antigen and that, accordingly, the antigenic composition with which
dendritic cells are
incubated may exclude whole cells. Thus, in some embodiments, the only source
of cyclin B1
and/or WT-1 peptide for loading dendritic cells is isolated polypeptides.
[0022] It is contemplated that other blockers of IL-113 and/or the IL-1
inflammation
pathway, including but not limited to rilonacept and canakinumab, may be
administered instead
of or in addition to anakinra in any of the embodiments described herein in
which anakinra is
administered.
[0023] The compositions and methods described herein may include dendritic
cells that
display positive control peptide epitopes derived from one or more of the
peptide antigens in the
CEF protein library (SEQ ID NOs:9 ¨ 40) or that have been incubated with the
peptides of SEQ
ID NOs:9 ¨ 40. In certain embodiments, one or more peptides may be excluded in
an
embodiment.
[0024] The terms "a" and "an" are defined as one or more unless this
disclosure
explicitly requires otherwise.
[0025] The term "substantially" is defined as being largely but not
necessarily wholly
what is specified (and include wholly what is specified) as understood by one
of ordinary skill in
the art. In any disclosed embodiment, the term "substantially" may be
substituted with "within
[a percentage] of' what is specified, where the percentage includes 0.1, 1, 5,
and 10 percent.
[0026] The terms "comprise" (and any form of comprise, such as "comprises"
and
"comprising"), "have" (and any form of have, such as "has" and "having"),
"include" (and any
form of include, such as "includes" and "including") and "contain" (and any
form of contain,
such as "contains" and "containing") are open-ended linking verbs. As a
result, the methods and
systems of the present invention that "comprises," "has," "includes" or
"contains" one or more
elements possesses those one or more elements, but is not limited to
possessing only those one or
more elements. Likewise, an element of a method or system of the present
invention that
- 10 -
CA 02874065 2014-12-04
"comprises," "has," "includes" or "contains" one or more features possesses
those one or more
features, but is not limited to possessing only those one or more features.
[0027] The feature or features of one embodiment may be applied to other
embodiments,
even though not described or illustrated, unless expressly prohibited by this
disclosure or the
nature of the embodiments.
[0028] Any method or system of the present invention can consist of or
consist
essentially of¨rather than comprise/include/contain/have¨any of the described
elements and/or
features and/or steps. Thus, in any of the claims, the term "consisting of' or
"consisting
essentially of' can be substituted for any of the open-ended linking verbs
recited above, in order
to change the scope of a given claim from what it would otherwise be using the
open-ended
linking verb.
[0029] Details associated with the embodiments described above and others
are presented
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The following drawings illustrate by way of example and not
limitation. In all
figures, IL-la indicates IL-la, and IL-lb indicates IL-113.
[0031] FIG. 1 ¨ Study design. This exploratory pilot safety, open label
trial will evaluate
the combination of preoperative chemotherapy and Dendritic Cell (DC)
vaccinations in 2 groups
of patients with LA TNBC.
[0032] FIG. 2 ¨ DC vaccination schedule. Schedule of administration of DC
vaccines in
combination with preoperative chemotherapy, surgery, and radiation.
[0033] FIG. 3 ¨ Overview of the BIIR-BrcaVax-001 DC vaccine manufacturing
process.
[0034] FIG. 4A-4E. A) HS-578t or MDA-MB231 cells were treated with medium
alone,
ng/ml of IL-113, IL-la, TNF-a, or IL-6 for the indicated time course
individually. Cells were
harvested and measured for TSLP mRNA level by quantitative real-time PCR.
Values were
normalized to internal control GAPDH. Bars show the mean SEM for triplicate
wells from a
representative experiment. *** p<0.0001, **p<0.01, *p<0.05. B) Luminex
analysis of TSLP in
supernatants of MDA-MB231 cells as indicated after 48h of culture in media (M)
alone, or in the
- 11 -
CA 02874065 2014-12-04
presence of different doses of IL-6 plus sIL6r, TNF-a, IL-lb, or IL-la, or PMA
and ionomycin.
Values are plotted as mean SEM from triplicate experiments. C) Luminex
analysis of TSLP in
supernatants of MDA-MB231 cells after different time points of culture with M
alone, or in
presence of 10 ng/ml IL-1[3, or IL-113 together with anti-IL-1P neutralizing
or non-neutralizing
antibody. Values are plotted as mean SEM from triplicates. D) MDA-MB231 cells
were
cultured in chamber well in presence of different dose of IL- 1f3 as
indicated, or M alone for 24
hours. Cells were fixed in situ, and TSLP was stained with anti-TSLP antibody.
Nuclear was
counter-stained with DAPI. Bar: 60 um. See also FIG. 9. E) levels of TSLP and
IL-10 were
determined by luminex in supernatants of breast tumor fragments post
PMA/ionomycin
stimulation. Levels of TSLP were plotted in contrast to IL-1P from the same
patient. Analysis
used nonparametric spearman correlation to determine the level of correlation
between two
cytokines.
[0035] FIG. 5A-5D. A) level of IL-113 is determined by luminex in
supernatants of breast
tumor fragments (T) or macroscopic uninvolved surrounding tissue (ST) post
PMA/ionomycin
stimulation; B-D) Frozen tissue sections from patients were analyzed by
immunofluorescence
staining. B) Primary tumor from patient was stained with anti-TSLP(green),
anti-IL-113 (red),
anti-cytokeratin-19 (blue) antibodies. Bar: 20 um. C) Primary tumor sections
from different
patients (P235, P255, P256, P345, P357, from above to bottom) were stained for
immune
infiltrates markers, including CD1 lc, HLA-DR, CD14, CD163, and CD68, together
with anti-IL-
O (red) antibody. Bar: 90um. D) Metastatic tumor of surgery removed auxiliary
lymph node
tissue was stained for cytokeratin-19 (red) for cancer cells, TSLP (green), 1L-
113 (red), and
CD11 c (green). Bar: 180 um (top), 90 um (bottom).
[0036] FIG. 6A-6F. A) Cancer cells (MDA-MB231 or HS-578t), co-cultured
with blood
monocytes, monocyte-derived DCs, mDCs or monocyte-derived macrophages in
regular wells or
transwell to separate two types of cells in culture for 48 hours. Supernatants
were harvested to
determine IL-1p level by Luminex. Values are plotted as mean SEM from
triplicate
experiments. B) mDCs were co-cultured with MDA-MB231 cells, HS-578t cells or
HS-Bst cells
for 16 hours. Intracellular IL-1f3 level was measured by FACS. Gated on viable
myeloid cells.
The percentages of IL-1(3 positive DCs were plotted. Each dot represents one
experiment. C)
Surface activated form of TGF-01 was stained using anti-TGF-P 1 antibody and
acquired by
FACS. D) MDA-MB231 cells were co-cultured with mDCs for 48 hours, in presence
of different
- 12 -
CA 02874065 2014-12-04
dose of TGF-PR kinase inhibitor or anti-TGF-13 neutralizing antibody, DMSO, or
isotype control
respectively. IL-1I3 level in the sups was detected by Luminex. Values are
plotted as mean SEM.
E) MDA-MB231 cells and mDCs were co-cultured for 16 hours, in presence or
absence of TGF-
13R kinase inhibitor and or anti-TGF-P neutralizing antibody. Intracellular
staining with anti-IL-
113 antibody was done and acquired by FACS. Gated on viable mDCs. The
percentages of IL-1J3
positive CD11c cells were plotted. Dot represents each experiment. F) MDA-
MB231 cells and
mDCs were co-cultured in presence or absence of TGF-PR kinase inhibitor and or
anti-TGF-P
neutralizing antibody for different time periods as indicated. Cells were
harvested and IL-0
mRNA level was detected using quantative RT-PCR. Values were normalized to
GAPDH. Bars
show the mean SEM for triplicate wells from a representative experiment. ***
p<0.0001,
**p<0.01, *p(0.05. n.s means no significance. See also FIG. 12.
[0037] FIG. 7A-7F. A-B) MDA-MB231 and mDCs were co-cultured in chamber
wells
for 18 hours, in presence of caspasel inhibitor or DMSO. Cells were fixed in
the well and
stained for A) pro-IL-10 (red), HLA-DR (green), and DAPI (blue). Right panel:
summary data of
the proportion of proIL-1f3+DR+cells. B) mature IL-0 (red), HLA-DR (green),
and DAPI
(blue). Right panel: summary data of the proportion of mature IL-113+DR+cells.
Bar: 90um. C)
monocytes were treated with MDA-MB231 culture sups for 16 hours in presence of
200 nm
TAK1 inhibitor or DMSO. Cells were harvested and activated caspase-1 and CD11c
were
stained. Right panel: Summary data of the percentage of activated caspase-1 in
DCs. D) mDCs
were co-cultured with MDA-MB231 cells for different time periods as indicated;
pTAK1 and
total TAK1 was detected by specific staining and analyzed on FACS. E) mDCs
were co-
cultured with MDA-MB231 cells in presence or absence of anti-TGF-P
neutralizing antibody
plus TGF-13R kinase inhibitor (TGF-P blocking) for 60 min, pTAK1 was detected
by specific
staining and analyzed on FACS. F) MDA-MB231 cells were co-cultured with mDCs
for 48
hours in presence of different doses of TAK1 inhibitor or DMSO. IL-113 levels
were detected by
Luminex in the sups after 48 hours of co-culture.
[0038] FIG. 8A-8C. Breast cancer cells was injected subcutaneously in
irradiated
NOD/SCID132-/- mice. Autologous DCs plus CD4+ T cells and CD8+ T cells were co-
injected
intratumorally. Mice were treated with anti-TGF-13 neutralizing antibody on
D3,6,9, with
Anakinra daily since D3, with anti-TSLPR neutralizing antibody on D3,6,9, or
with isotype and
PBS as control. A) Combined data for kinetics of tumor growth from multiple
experiments was
- 13 -
CA 02874065 2014-12-04
shown. Number of mice in each group was indicated. B) cytokine concentration
as measured by
Luminex in PBS group vs. Anakinra group vs. a-TGFf3 neutralizing antibody
group, as
determined by Luminex in supernatants of Day16 harvested mice breast tumor
fragments were
stimulated for 16 hours with PMA and ionomycin. C) IL-1f3 concentration in TGF-
P blocking
group vs. isotype control group, as determined by Luminex in supernatants of
Day 16 harvested
mice breast tumor fragments stimulated for 16 hours with PMA and ionomycin.
See also FIG.
15.
[0039] FIG. 9A-9C. A-B) Luminex analysis of TSLP in supernatants of breast
cancer
cell lines as indicated after 48h of culture in media (M) alone, or in the
presence of different
doses of cytokines. Values are plotted as mean SEM from triplicate
experiments. A) MDA-
MB231 cells were cultured, while in B) HS-578T cells were cultured for TSLP
induction
experiment. C) TSLPR+/IL-7Ra+ Baf3 cells were seeded in 96-well plate. Serial
dilution was
done to IL-1P-treated MDA-MB231 culture sups, IL-1P-treated HS-578t culture
sups, tumor
sups without IL-1f3 treatment, or IL-1f3. The conditioned sups with different
dilution were used to
treat Baf3 cells. The proliferative values were measured based on MTT assay.
[0040] FIG. 10A-10C. A) levels of IL-18, IL-25, IL-33, GM-CSF, TSLP is
determined
by luminex or ELISA in supernatants of breast tumor fragments post
PMA/ionomycin
stimulation; B) level of IL-1Ra is determined by luminex in supernatants of
breast tumor
fragments (T) or macroscopic uninvolved surrounding tissue (ST) post
PMA/ionomycin
stimulation; C) level of IL-la is determined by luminex in supernatants of
breast tumor
fragments (T) or macroscopic uninvolved surrounding tissue (ST) post
PMA/ionomycin
stimulation.
[0041] FIG. 11A-11B. A) MDA-MB231 cells culture sups were used to treat
different
myeloid cells (monocytes, monocyte-derived dendritic cells, mDCs, and monocyte-
derived
macrophages) as indicated for 48 hours. Supernatant were harvested to
determine IL-113 level by
Luminex. Values are plotted as mean SEM from triplicate experiments. B)
Surface expression
of TGF-PRI, II, and III were detected on monocytes, MDDC, and mDCs,
respectively by FACS.
Y-axis indicates the expression level.
[0042] FIG. 12A-12C. A) MDA-MB231 cells were co-cultured with mDCs for 48
hours,
in presence of different dose of TGF-PR kinase inhibitor or anti-TGF-P
neutralizing antibody,
- 14
CA 02874065 2014-12-04
DMSO, or isotype control respectively. IL-113 level in the sups was detected
by Luminex. Values
are plotted as mean SEM. B) MDA-MB231 cells and mDCs were co-cultured for 16
hours, in
presence or absence of TGF-f3R kinase inhibitor and or anti-TGF-P neutralizing
antibody.
Intracellular staining with anti-IL-1P antibody was done and acquired by FACS.
Gated on viable
mDCs. The percentages of IL-1P positive CD11c cells were plotted. Dot
represents each
experiment. C) MDA-MB231 cells and mDCs were co-cultured in presence or
absence of TGF-
OR kinase inhibitor and or anti-TGF-f3 neutralizing antibody for different
time periods as
indicated. Cells were harvested and IL-1P mRNA level was detected using
quantative RT-PCR.
Values were normalized to GAPDH. Bars show the mean SEM for triplicate wells
from a
representative experiment. *** p<0.0001, **p<0.01, *p<0.05. n.s means no
significance.
[0043] FIG. 13. Cancer cells express CD105 but not CD36 on their surface.
Breast
cancer cells in culture were harvested and surface expression level of CD105
and CD36 were
detected by FACS analysis. anti-CD105, anti-CD36, or matched isotype control
antibodies were
used. Histogram shows relative expression in contrast to isotype staining.
[0044] FIG. 14A-14C. A) monocytes were treated with rhuTGF-P1 (10 ng/ml)
for
different time period as indicated. pTAK1 and total TAK1 was detected by
specific staining and
analyzed on FACS. B) MDA-MB231 cells were co-cultured with monocytes or MDDCs
for 16-
48 hours in presence of TAK1 inhibitor or DMSO. B) IL-1P levels were detected
by Luminex in
the sups after 48 hours of co-culture. C) IL-1P expressing CD1 lc cells after
16 hours co-culture
were quantified by intracellular staining with anti-IL-1P antibody, and
analyzed on FACS.
[0045] FIG. 15A-15D. A) Experimental scheme. B) Frozen tissue sections
from tumor-
bearing xenograft were analyzed by immunofluorescence staining. Primary tumor
was stained
with anti-TSLP(green), anti-IL-1p (red), antibodies, and DAPI (blue). Bar: 90
um. C) 3
representative tumors harvested from each treatment group were shown. D) on
D16, single cell
suspensions were generated for intracellular cytokine expression analysis by
FACS. Gate was
based on viable CD4+ T cells. Dot plot shows IL-13 vs. IFN-y.
[0046] FIG. 16A-16B. A-B) levels of cytokines were determined by luminex
or ELISA
in supernatants of breast tumor fragments post PMA/ionomycin stimulation; A)
levels of IL-13
were plotted against TSLP from the same patient. B) Levels of IL-18, IL-25, IL-
33, GM-CSF,
respectively were plotted against the level of IL-13 from the same patient.
Analysis was
- 15 -
CA 02874065 2014-12-04
performed using nonparametric spearman correlation to determine the level of
correlation
between two cytokines. Nonparametric t test was used.
[0047] FIG. 17A-17B. A-B) levels of IL-la, IL-1[3, and IL-13 were
determined by
luminex or ELISA in supernatants of breast tumor fragments post PMA/ionomycin
stimulation;
A) levels of IL-la and IL-113 were plotted against IL-13 from the same
patient. B) IL-113
concentrations were plotted based on patients' clinical stage information.
Numbers on the x-axis
indicate the number of tissue samples from different patients tested. IL-1 a
concentrations were
plotted based on patients' clinical stage information. Numbers on the x-axis
indicate the number
of tissue samples from different patients tested. Nonparametric t test was
used. See also FIG. 16.
DETAILED DESCRIPTION OF THE INVENTION
[0048] Various features and advantageous details are explained more fully
with reference
to the non-limiting embodiments that are illustrated in the accompanying
drawings and detailed
in the following description. It should be understood, however, that the
detailed description and
the specific examples, while indicating embodiments of the invention, are
given by way of
illustration only, and not by way of limitation. Various substitutions,
modifications, additions,
and/or rearrangements will become apparent to those of ordinary skill in the
art from this
disclosure.
[0049] In the following description, numerous specific details are given
to provide a
thorough understanding of the disclosed embodiments. One of ordinary skill in
the relevant art
will recognize, however, that the invention may be practiced without one or
more of the specific
details, or with other methods, components, materials, and so forth. In other
instances, well-
known structures, materials, or operations are not shown or described in
detail to avoid obscuring
aspects of the invention.
A. Breast Cancer and Immunotherapy
[0050] Immunotherapy is an attractive strategy for overcoming chemotherapy
resistance
in TNBC patients and some preliminary studies have been carried out (Park, et
al., 2008;
Knutson, et al., 2001; Anderson, 2009; Disis & Schiffman, 2001). Briefly,
recent studies have
shown that human breast cancers can be immunogenic, and that enhancing the
immune effector
function already present may augment the cytotoxic effects of standard
therapies (Anderson,
- 16 -
CA 02874065 2014-12-04
2009; Disis & Schiffman, 2001). In one preclinical study, IGFBP-2 was found at
elevated levels
in breast cancer patients' sera, and an IGFBP-2¨specific T-cell response
inhibited tumor growth
in a breast cancer mouse model (Park, et al., 2008). In a phase I clinical
trial, 19 patients with
HER2-overexpressing breast cancer were vaccinated with HER2 peptide-specific T-
cells,
resulting in the generation of both CD4 and CD8 T-cell immunity. The resulting
peptide-specific
T-cells recognized endogenous HER2 protein and the immunity was maintained for
a median of
12 months after the last vaccination (Knutson, et al, 2001). More recently,
breast cancer tissues
from a phase III clinical trial were analyzed for lymphocytic infiltrate, and
the results
demonstrated that increased infiltration of lymphocytes in tumor and stroma
was associated with
an overall good prognosis in ER-negative/HER2-negative breast cancer patients.
These findings
were consistent regardless of the chemotherapy type administered,
demonstrating that greater
lymphocytic infiltration was a prognostic factor for ER-negative/HER2-negative
breast cancer
(Loi, et al., 2013).
[0051] The presence of naturally occurring immunity against a broad range
of tumor-
associated antigens including HER-2/neu, MUC1, cyclin B1 and surviv in has now
been
documented in patients with breast cancer (Finn, 2003). However, the natural
immune response
to the cancer co-exists with the cancer, and is therefore not protective,
either because of tumor
escape, for example, through clonal evolution, or because it might have been
generated in and/or
elicited an inappropriate immunosuppressive microenvironment.
[0052] There are numerous strategies under investigation aimed at
enhancing a patient's
immunologic resistance to cancer. Among these are 1) non-specific activation
of the immune
system with microbial components or cytokines; 2) antigen-specific adoptive
immunotherapy
with antibodies and/or T cells; and 3) antigen-specific active immunotherapy
(vaccination). The
major limitation of antibodies is that target proteins must be expressed on
the cell surface
whereas targets for T cells can be intracellular proteins whose peptides are
presented on the cell
surface in complexes with MHC molecules (Townsend, et al., 1985). The
identification of
defined tumor antigens in humans (Doon, et al., 1994; Rosenberg, 1997)
prompted the
development of adoptive T cell therapy. Yet, vaccination remains the most
attractive strategy
because of its expected inducement of both therapeutic T cell immunity
(effector T cells) and
protective T cell immunity (tumor-specific memory T cells that can control
tumor relapse) (Finn,
2003; Pardoll, 1998; Gilboa, 1999).
- 17 -
CA 02874065 2014-12-04
[0053] Several clinical studies have now demonstrated that immunity
against tumor
antigens can be enhanced in cancer patients by vaccination with ex vivo-
generated tumor
antigen-loaded dendritic cells (DCs). This strategy capitalizes on the unique
capacity of DCs to
prime lymphocytes and to regulate and maintain immune responses. Whereas a
number of
antigen-presenting cells can activate memory T cells, only DCs can prime naive
T cells. This
feature is essential to successful vaccination as it might allow generation of
a "new" immune
response, possibly not compromised by the cancer (Palucka & Banchereau, 2012).
B. Therapeutic Dendritic Cell Vaccines
[0054] Treatment methods described herein involve administering antigen-
loaded
dendritic cells to a subject. Multiple phase I/II clinical trials testing ex
vivo-generated DC
vaccines in patients with stage IV melanoma, HIV, and more recently pancreatic
cancer, have
been performed. It has been found that some patients can experience durable
tumor regressions
as well as prolonged survival (reviewed in Banchereau & Palucka, 2005). A DC
vaccine
optimized for CD8+ T cell responses, i.e., GM-CSF/IFN-a-generated DCs
activated with TLR
ligands and CD4OL, has also been developed. A closed system for vaccine
generation has been
developed, as has a frozen vaccine that has been successfully administered in
multicenter clinical
trials in patients with melanoma and in patients with HIV.
[0055] The dendritic cells used in the vaccines are generated from
autologous monocytes
by culturing in the presence of GM-CSF and IFN-a (IFN-DC), which demonstrate
powerful
priming functions in vitro. The in vivo activity IFN-DC has been tested in
phase I/II clinical
trials in patients with stage IV melanoma and in patients with HIV. Early
results show the
induction of immune and clinical responses as well as a good
safety/tolerability profile.
[0056] In some embodiments, the dendritic cell vaccines comprise dendritic
cells
displaying a variety of antigens, including a mixture of tumor antigens and
control antigens. The
tumor antigens consist of eight immunogenic long-peptides: Cyclin B1207-242
(36mer Peptide),
Cyclin B1285334 (50mer Peptide),WT161_114 (54mer Peptide), WT1115-174 (60mer
Peptide),
WTI 175-222 (48mer Peptide), WT1223-280 (58mer Peptide), WT1281-334 (54mer
Peptide) and
WT1 367-421 (55mer Peptide). The control antigens are the cytotoxic T-
lymphocyte (CTL)-CEF-
Class I Peptide Library Pool comprised of 32 peptides, each corresponding to a
defined HLA
class I restricted T-cell epitope from Cytomegalovirus, Epstein-Barr virus and
Influenza virus.
- 18 -
CA 02874065 2014-12-04
[0057] The dendritic cells can be activated at the same time that they are
incubated with
peptide antigens. Antigen loading and dendritic cell activation can also be
performed in
subsequent steps. Activation can be accomplished by incubation of the
dendritic cells with
lipopolysaccharide (LPS), CD40 ligand (CD4O-L), and the TLR7/8 agonist CL075.
Research
results demonstrate that the combination of LPS (TLR4 ligand), CD4OL and CL075
(TLR 7/8
agonist) is superior to poly I:C (TLR3) and/or a mixture of inflammatory
cytokines in priming
IFN-DC to secrete IL-12p40, IL-12p70 and IL-23, which are potent T-cell
signals.
LPS/CD4OL/CL075-activated IFN-DC induce potent antigen-specific CD8+T-ce11
responses in
vitro.
2. Synopsis¨parameters for manufacture of BHR-BrcaVax-001 DC vaccine
[0058] The following parameters have been selected for the manufacture of
the BIIR-
BrcaVax-001 DC vaccine product intended for treating breast cancer patients
enrolled in the
proposed Phase I/II clinical trial.
[0059] Source of Monocytes: Elutriation enriched from the patient's PBMC
collected by
apheresis.
[0060] Cell Culture: DC generated by culturing the monocytes in serum-free
media
supplemented with GM-CSF/IFN-a for 3 days.
[0061] Antigen Loading: Three different sets of peptide antigens listed
below will be
loaded onto the DC in the BIIR-BrcaVax-001 vaccine product. These include:
Cyclin B1 36mer
and 50mer antigen peptides, six long-peptides derived from the WT-1 tumor
antigen, and CEF
which is a mixture of viral antigens as a vaccine positive control.
Cyclin B1 peptide antigens
= Cyclin B1207-242 (36mer Peptide) NH2-DWLVQV QMKFRL LQETMY MTVSII
DRFMQN NCVPKK-COOH (SEQ ID NO:1)
= Cyclin B1285-334 (50mer Peptide) NH2-MEMKIL RALNFG LGRPLP LHFLRR ASKIGE
VDVEQH TLAKYL MELTML DY-COOH (SEQ ID NO:2)
WT-1 peptide antigens
= WT161-114 (54mer Peptide) NH2-ASGSEP QQMGSD VRDLNA LLPAVP SLGGGG
GCALPV SGAAQW APVLDF APPGAS-COOH (SEQ ID NO:3)
- 19 -
CA 02874065 2014-12-04
= WT1115-174 (60mer Peptide) NH2-AYGSLG GPAPPP APPPPP PPPPHS FIKQEP
SWGGAE PHEEQC LSAFTV HFSGQF TGTAGA-COOH (SEQ ID NO:4)
= WT1175-222 (48mer Peptide) NH2-CRYGPF GPPPPS QASSGQ ARMFPN APYLPS
CLESQP AIRNQG YSTVTF-COOH (SEQ ID NO:5)
= WT1223-280 (58mer Peptide) NH2-DGTPSY GHTPSH HAAQFP NHSFKH EDPMGQ
QGSLGE QQYSVP PPVYGC HTPTDS CTGS-COOH (SEQ ID NO:6)
= WT1281-334 (54mer Peptide) NH2-QALLLR TPYSSD NLYQMT SQLECM TWNQMN
LGATLK GVAAGS SSSVKW TEGQSN-COOH (SEQ ID NO:7)
= WT1367-421 (55mer Peptide) NH2-DVRRVP GVAPTL VRSASE TSEKRP FMCAYP
GCNKRY FKLSHL QMHSRK HTGEKPY-COOH (SEQ ID NO:8)
CEF control peptide antigens
= The CTL-CEF-Class I Peptide Library Pool contains 32 peptides, each
corresponding to a
defined HLA class I restricted T-cell epitope from Cytomegalovirus, Epstein-
Barr virus
and Influenza virus. The CEF antigen peptides are manufactured by Bio-
Synthesis, Inc.,
Lewisville, TX, to standards consistent with Phase 0 Guidelines, Catalog
Number: 13686.
The CTL-CEF-Class I Peptide Library Pool contains 32 peptides, each
corresponding to a
defined HLA class I restricted T-cell epitope from Cytomegalovirus, Epstein-
Barr virus
and Influenza virus. The lyophilized peptide pool is reconstituted with DMSO
to 10 mM.
The reconstituted peptide pool is aliquoted in cryovials, frozen and stored at
-80 C. To
manufacture DC vaccine batches an aliquot of the peptide is diluted to 2 mM
with sterile
water prior to use, i.e., 50 [IL to 250 }AL. The amino acid sequences of the
CEF peptide
antigens are listed in Table 1.
- 20 -
CA 02874065 2014-12-04
Table 1. CEF Peptide Antigens
HLA Allele Virus Peptide Sequence SEQ ID
A1 Influenza A NH2-VSDGGPNLY-000H SEQ ID NO:9
Influenza A NH2-CTELKLSDY-COOH SEQ ID NO:10
Influenza M NH2-GILGFVFTL-COOH SEQ ID NO:11
A2 Influenza A NH2-FMYSDFHFI-COOH SEQ ID NO:12
EBV LMP2A NH2-CLGGLLTMV-COOH SEQ ID NO:13
EBV BMLF1 NH2-GLCTLVAML-COOH SEQ ID NO:14
A0201 HCMV pp65 NH2-NLVPMVATV-COOH SEQ ID NO:15
Influenza NP NH2-KTGGPIYKR-COOH SEQ ID NO:16
Influenza NP NH2-RVLSFIKGTK-COOH SEQ ID NO:17
AA68 Influenza A NH2-ILRGSVAHK-COOH SEQ ID NO:18
EBV NH2-RVRAYTYSK-COOH SEQ ID NO:19
EBV NH2-RLRAEAQVK-000H SEQ ID NO:20
A3, A11,
Influenza M NH2-SIIPSGPLK-COOH SEQ ID NO:21
A60B1
EBV EBNA SEQ ID NO:22
NH2-AVFDRKSDAK-COOH
4NP
A11
EBV NH2-IVTDFSVIK-COOH SEQ ID NO:23
EBV NH2-ATIGTAMYK-000H SEQ ID NO:24
A24 EBV RTA NH2-DYCNVLNKEF-COOH SEQ ID NO:25
B7 Influenza NP NH2-LPFDKTTVM-COOH SEQ ID NO:26
EBV NH2-RPPIFIRRL-COOH SEQ ID NO:27
Influenza NP NH2-ELRSRYWAI-COOH SEQ ID NO:28
EBV BZLF-1 NH2-RAKFKQLL-COOH SEQ ID NO:29
B8
EBV EBNA 3A NH2-FLRGRAYGL-COOH SEQ ID NO:30
EBV EBNA 3 NH2-QAKWRLQTL-COOH SEQ ID NO:31
B18 HCMV NH2-SDEEEAIVAYTL-COOH SEQ ID NO:32
Influenza NP NH2-SRYWAIRTR-COOH SEQ ID NO:33
B27 Influenza M NH2-ASCMGLIY-COOH SEQ ID NO:34
EBV EBNA 3C NH2-RRIYDLIEL-COOH SEQ ID NO:35
B35 EBV EBNA3A NH2-YPLHEQHGM-COOH SEQ ID NO:36
CMV pp65 NH2-IPSINVHHY-000H SEQ ID NO:37
B44 EBV NH2-EENLLDFVRF-COOH SEQ ID NO:38
HCMV NH2-EFFWDANDIY-COOH SEQ ID NO:39
B0702 HCMV NH2-TPRVTGGGAM-COOH SEQ ID NO:40
[0062] Activation: The antigen-loaded DC will be activated for
approximately 24 hours
with a combination of LPS, CD40 and CL075.
[0063] Clinical Rationale: The safety and potential efficacy of LPS and
LPS/CD4OL
activated DC vaccines has been established. Based on the activity of CL075 it
is anticipated that
-21 -
CA 02874065 2014-12-04
DC vaccines activated with the agent will elicit stronger antigen-specific
CD8+ T-cell responses
in humans.
C. Intratumoral Injection
[0064] Treatment methods described herein involve administering antigen-
loaded
dendritic cells to a subject. The dendritic cells can be administered by
various routes, including
but not limited to subcutaneous, intratumoral, and intravenous administration
routes.
[0065] Delivering active, antigen-loaded dendritic cells directly into
tumor tissue can be
particularly effective. Although it is generally believed that cytotoxic
antineoplastic agents
mediate their therapeutic effects in a cancer cell-autonomous fashion, recent
results indicate that
at least some chemotherapeutics inhibit tumor growth also indirectly, via the
immune system.
Indeed, it has been shown that a variety of transplantable or chemically
induced primary mouse
cancers respond more efficiently to anthracyclines when they develop in hosts
carrying an intact
immune system (reviewed in Kroemer, et al., 2013). Tumors evolving in
immunodeficient mice
fail to show a reduction in growth after anthracycline treatment in conditions
in which the same
tumors growing in immunocompetent mice do exhibit a significant inflection in
their
progression. Accordingly, clinical studies indicate that anthracycline-killed
tumor cells are
particularly efficient in stimulating a therapeutic immune response in cancer
patients.
Anthracycline-based neoadjuvant therapy of breast cancer patients is more
effective when the
tumor is infiltrated by T cells before chemotherapy is initiated as well as if
chemotherapy causes
a significant influx of CD8+ T cells into the tumor bed and/or reduces the
presence of
immunosuppressive T regulatory (Treg) cells (reviewed in Kroemer, et al.,
2013). The reason
why anthracyclines provoke this complex anticancer immune response has only
been partially
elucidated. In contrast to many other cytotoxic chemotherapeutics,
anthracyclines stimulate
immunogenic cell death that is characterized by a compendium of subtle
biochemical changes in
the plasma membrane surface and in the microenvironment of dying cancer cells.
These changes
include the pre-apoptotic exposure of calreticulin on the plasma membrane
surface (to facilitate
the engulfment of portions of the dying cells by antigen-presenting cells,
APC) and the post-
apoptotic exodus of high mobility group B1 (HMGB1) from the nucleus (to engage
with TLR4
receptors and to stimulate antigen presentation) (reviewed in Kroemer, et al.,
2013). Moreover,
ATP release by autophagy-competent dying tumor cells (positive for LC3-II) is
essential for the
- 22 -
CA 02874065 2014-12-04
induction of an anticancer immune response, both by stimulating the
recruitment of
inflammatory cells (CD11b I,yC61."g" CD11c1' CD86+) into the tumor bed and by
ligating
P2RX7 receptors on dendritic cells, hence facilitating the activation of the
NLRP3
inflammasome and the consequent secretion of IL-1(3 by APC (reviewed in
Kroemer, et al.,
2013).
[0066] How chemotherapy-induced cell death leads to efficient antigen
presentation to T
cells has remained an open conundrum. It has been shown in mice that
intratumoral
CD11c+CD11b+Ly6Chigh cells, which shared some characteristics of inflammatory
dendritic cells
(DC) and contained granulomonocytic precursors, were crucial for the induction
of anticancer
immunity post-chemotherapy (Ma, et al., 2013). First, ATP released by dying
tumor cells is
essential for the recruitment of myeloid cells into tumor beds and for the
local differentiation of
inflammatory DC. Second, manipulations aimed at avoiding the intratumoral
accumulation of
these CD11c+CD11b+Ly6ChIgh cells, such as local overexpression of the ATP-
degrading enzyme
CD39, pharmacological blockade of purinergic receptors, or neutralization of
CD11b, abolished
the immune-dependent inhibition of tumor growth by anthracyclines. Third,
CD11c+CD11b+Ly6ChIgh were efficient in capturing and presenting tumor cell
antigen to T cells
and protected mice upon their adoptive transfer against challenge with cancer
cells. Altogether,
the results identify a population of tumor-infiltrating leukocytes as therapy-
relevant antigen-
presenting cells.
[0067] The two immunogenic cell death markers HMGB1 and LC3-II were
evaluated on
paraffin-embedded BC specimens in a test (50 early BC treated with adjuvant
anthracyclines that
relapse at 3 years paired with 50 cases that were disease-free at 10 years)
and a validation cohort
on 150 Her2 negative early BC treated with adjuvant anthracyclines.
Preliminary data suggest
that LC-3-II staining was negative in the vast majority of cases of early
breast cancers (>70%).
These "autophagy deficient" cancers are also less infiltrated with CD8+ T
cells but contained
more CD68+ cells and had a greater chance of recurrence following adjuvant
chemotherapy. A
larger across Europe validation study is ongoing.
[0068] Blocking ectoATPases (CD39) restored the recruitment of DC in
tumors and the
efficacy of chemotherapy in autophagy deficient murine cancers (Michaud, et
al., 2011).
However, anti-CD39 Ab are not available for use in the human at this time. We
propose to
- 23 -
CA 02874065 2014-12-04
substitute the functional DCs via adoptive transfer of ex vivo generated
autologous mature DCs
injected locally into LA 'TNBCs at 48 hours post-systemic anthracyclines.
Whereas we will not
stratify in this early phase of DC vaccine assessment, all samples will be
tested for LC3-II
staining (and others such as CD68, CD8, Foxp3, phosphoSTAT6).
[0069] Another objective for intra-tumoral vaccination is the possibility
to enhance the
access of DCs to draining lymph nodes. Indeed, recent studies suggest that the
route of DC
injection might determine the homing of elicited T cells. Indeed, for mucosal
cancer vaccines,
the homing to and retention of CD8+ T cells in the mucosa are critical for
efficacy (Sandoval, et
al., 2013). In this context, the growth of orthotopic head and neck or lung
cancers can be
inhibited by a cancer vaccine provided that it is administered by the
intranasal mucosal route, but
not the intramuscular route. This is explained by the induction through
intranasal vaccination of
mucosal CD8+ T cells expressing the mucosal integrin CD49a, the expression of
which is
essential for the efficacy of cancer vaccines.
D. Cyclin B1 Antigen
[0070] In some embodiments, the dendritic cell vaccines disclosed herein
are loaded with
cyclin B1 peptide antigens. Cyclin B1 is also known as CCNB1, CCNB, CCNB 1, G2
mitotic
specific cyclin B1, and G2/mitotic-specific cyclin-B1. The cyclin B1 peptide
antigens can
comprise the full length cyclin B1 sequence. The cyclin B1 peptide antigens
can also comprise
shorter immunogenic peptide fragments. Upon loading with cyclin B1 peptide
antigens, the
dendritic cells process the peptides into smaller fragments and present them
on the cell surface in
complex with MHC class II molecules. Antigen-loaded dendritic cells can then
be activated and
administered to a patient to induce an immune response.
[0071] Transcriptional profiling of triple negative breast cancers
demonstrates a very
strong proliferation signature (Schneider, et al., 2008; Sorlie, et al., 2001)
including enhanced
transcription of cyclin B1. Cytoplasmic accumulation of cyclin B1 has been
identified as an early
event in breast cancer development (Kao, et al., 2001) Furthermore, cyclin B1
genes are among
the transcripts analyzed in the 21-gene assay Oncotype Dx, the first
clinically validated
multigene assay that quantifies the likelihood of breast cancer recurrence
(Strayer, et al., 2010).
- 24 -
CA 02874065 2014-12-04
[0072] Cyclin B1 is a regulatory protein that is an essential component of
the mitotic cell
cycle. The natural peak of cyclin B1 occurs between the G2-M phases of the
cell cycle, and is
reduced to near zero afterwards. However, in cancer cells, this protein is
over-expressed during
all phases of the cell cycle. Additionally, cyclin B1 is found in normal cells
in the nucleus,
whereas in cancer cells it is found in the cytoplasm (Egloff, et al., 2006).
Several studies have
shown that inactivation of the tumor suppressor gene p53, which occurs in all
triple negative
breast cancers, directly contributes to the aberrant regulation of cyclin B1
in tumor cells (Yu, et
al., 2002). Cyclin B1 has been found to be over-expressed in multiple forms of
cancer, including
breast cancer, and in most cancer cell lines (Egloff, et al., 2006). While
studies involving the
immunogenicity of cyclin B1 are limited, there are some indications that it is
an important
antigen to pursue (Yu, et al., 2002). Cyclin B1-specific antibodies are found
in the blood of
patients with many cancer types, at both the premalignant and established
phases (Suzuki, et al.,
2005). Cyclin B1-specific T cells can be also found in healthy volunteers
(Neidhardt-Berard, et
al., 2004). Both antibodies and T cells against cyclin B1 protect from cancer
in mouse models
(Neidhardt-Berard, et al., 2004). Because cyclin B1 is necessary for cancer
cell division, loss of
the antigen is an unlikely means of tumor escape.
[0073] It has been shown that DCs loaded with killed breast cancer cells
expressing
cyclin B1 induce differentiation of cyclin B1-specific T cells, and that these
T cells are able to
kill breast cancer tumors in vitro (Neidhard-Berard, et al., 2004; Saito, et
al., 2006). In
preliminary studies preparatory to the clinical trial proposed herein, it was
also found that
patients with various breast cancer subtypes can display a cyclin B1-specific
memory T cell
repertoire in their blood. These observations further support the targeting of
this antigen for
breast cancer immunotherapy. In the study described herein, LA TNBC patients
will be
immunized with cyclin B1 peptide-loaded DC vaccines, along with standard
preoperative
chemotherapy.
[0074] In some embodiments of the methods described herein, the following
cyclin B1
peptides will be incubated with DCs under conditions that cause cyclin B1
peptide epitopes to be
displayed on the surface of DCs:
=Cyclin B1207_242 (36mer Peptide) NH2-DWLVQV QMKFRL LQETMY MTVS11
DRFMQN NCVPKK-COOH (SEQ ID NO:1)
- 25 -
CA 02874065 2014-12-04
=Cyclin B 1285-334 (5 Mel' Peptide) NH2-MEMKIL RALNFG LGRPLP LHFLRR
ASKIGE VDVEQH TLAKYL MELTML DY-COOH (SEQ ID NO:2)
E. Wilms Tumor Antigen (WT-1)
[0075] In some embodiments, the dendritic cell vaccines disclosed herein
are loaded with
WT-1 peptide antigens. The WT-1 peptide antigens can comprise the full-length
WT-1 sequence.
The WT-1 peptide antigens can also comprise shorter immunogenic peptide
fragments. Upon
loading with WT-1 peptide antigens, the dendritic cells process the peptides
into smaller
fragments and present them on the cell surface in complex with MHC class II
molecules.
Antigen-loaded dendritic cells can then be activated and administered to a
patient to induce an
immune response.
[0076] The zinc finger transcription factor WT-1 is expressed at 10-1000x
fold higher
levels in leukemic cells compared to normal CD34+ cells, and the magnitude of
expression
correlates with clinical aggressiveness of acute myeloid leukemia (AML),
myelodysplastic
syndromes (MDS), and acute lymphoid leukemia (ALL) (Chapuis, et al., 2013).
Although
essential during embryogenesis, WT-1 expression after birth is limited to low
levels
predominantly in kidney podocytes and CD34+ hematopoietic stem cells (HSC). WT-
1-specific
CD8+ T lymphocytes can distinguish over-expressing targets from normal cells
and have been
demonstrated to inhibit the growth of and to lyse leukemic but not normal
CD34+ cells. Recent
whole genome and transcriptome sequencing analysis of metastatic tumor tissue
obtained from
14 TNBC patients, has delineated the wide array of somatic genomic alterations
in these
advanced tumors. Genes mutated in multiple tumors included TP53, LRP1B, HERC1,
CDH5,
RBI, and NFL WT-1 was among the genes that contained focal structural
mutations as were
CTNNA1, PTEN, FBXW7, BRCA2, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP.
Furthermore, WT-1 was found to be overexpressed on RNA sequencing in all 14
samples (Craig,
et al., 2013). Furthermore, the analysis of public microarray datasets of 266
early breast cancer
patients showed that WT-1 mRNA expression was correlated with higher
histological grades,
ER-negative and basal-like and ERBB2 molecular breast cancer subtypes (Qi, et
al., 2012).
Disease-free survival analysis showed worse prognosis the WT-1 high expression
group, and
WT-1 was found to be an independent prognostic indicator in multivariate
analysis. Finally, WT-
- 26 -
CA 02874065 2014-12-04
1 promotes proliferation and oncogenicity, and loss of expression is
disadvantageous for the
tumor, making outgrowth of antigen-loss variants less likely.
[0077] In some embodiments of the methods described herein, the following
WT-1
peptides will be incubated with DCs under conditions that cause WT-1 peptide
epitopes to be
displayed on the surface of DCs:
= WT161-114 (54mer Peptide) NH2-ASGSEP QQMGSD VRDLNA LLPAVP SLGGGG
GCALPV SGAAQW APVLDF APPGAS-COOH (SEQ ID NO:3)
= WT1115-174 (60mer Peptide) NH2-AYGSLG GPAPPP APPPPP PPPPHS FIKQEP
SWGGAE PHEEQC LSAFTV HFSGQF TGTAGA-COOH (SEQ ID NO:4)
= WT1175-222 (48mer Peptide) NH2-CRYGPF GPPPPS QASSGQ ARMFPN APYLPS
CLESQP AIRNQG YSTVTF-COOH (SEQ ID NO:5)
= WT1223-280 (58mer Peptide) NH2-DGTPSY GHTPSH HAAQFP NHSFKH EDPMGQ
QGSLGE QQYSVP PPVYGC HTPTDS CTGS-COOH (SEQ ID NO:6)
= WT128I-334 (54mer Peptide) NH2-QALLLR TPYSSD NLYQMT SQLECM TWNQMN
LGATLK GVAAGS SSSVKW TEGQSN-COOH (SEQ ID NO:7)
= WT1 367-421 (55mer Peptide) NH2-DVRRVP GVAPTL VRSASE TSEKRP FMCAYP
GCNKRY FKLSHL QMHSRK HTGEKPY-COOH (SEQ ID NO:8)
F. Cancer Chemotherapy
[0078] In some embodiments, treatment methods described herein involve
administration
of antigen-loaded dendritic cells in combination with chemotherapy.
Administration of antigen-
loaded dendritic cells can be performed before, during, or after chemotherapy
to help improve
outcomes for subjects with cancer. Embodiments described herein also involve
administering
chemotherapy in combination with anakinra without administration of dendritic
cell vaccines.
[0079] Adjuvant chemotherapy can substantially reduce the risk of breast
cancer
recurrence and death in high-risk patients (Early Breast Cancer Trialists'
Collaborative Group,
1998), and there are many chemotherapy regimens with established efficacy and
safety data. The
value of chemotherapy is established from the data from individual randomized
trials and from
the Early Breast Cancer Trialists' Collaborative Group's (EBCTCG) l5-year meta-
analyses of
randomized chemotherapy trials (Early Break Cancer Trialists' Collaborative
Group's, 2005).
The meta-analyses established that anthracycline-containing therapies, such as
doxorubicin and
- 27 -
CA 02874065 2014-12-04
cyclophosphamide (AC) and docetaxel, doxorubicin, and cyclophosphamide (TAC),
offer
superior efficacy, reducing the risk of recurrence by 11% and the risk of
death by 16% compared
with cyclophosphamide, methotrexate, and fluorouracil (CMF) combinations
(Early Break
Cancer Trialists' Collaborative Group's, 2005).
[0080] Significant improvements in disease-free survival (DFS) were
reported with
adjuvant dose-dense chemotherapy in women with node-positive breast cancer in
the Phase III
CALGB 9741 study of 2005 women. Citron et al., 2003 showed that when the
taxane, paclitaxel
(Taxol) (T), was given sequentially following standard chemotherapy,
doxorubicin (A) and
cyclophosphamide (C), in an every two-weekly dose-dense regimen, the rate of
recurrence was
significantly reduced by 26% (P=0.010) and the rate of death was reduced by
31% (P=0.014),
compared to standard every 3-week administration, with an acceptable toxicity
profile.
[0081] In a 2005 report of the findings of NSABP B-28, the addition of a
taxane,
adjuvant paclitaxel, to AC resulted in significant improvement in DFS. NSABP B-
28 was
conducted to determine whether 4 cycles of adjuvant T after 4 cycles of
adjuvant AC (AC--4T)
would increase the DFS and OS compared with 4 cycles of AC alone in patients
with resected
operable, node-positive breast cancer (Mamounas, et al., 2005). Patients
(N=3060) were
randomly assigned to the 2 groups. The addition of AC---T significantly
reduced the hazard for
developing a DFS event by 17% (relative risk [RR], 0.83; 95% CI, 0.72 to 0.95;
P=0.006). Five-
year DFS was 76% 2% for patients randomly assigned to AC--T compared with 72%
+2% for
those randomly assigned to AC. Improvement in OS was small and not
statistically significant
(RR, 0.93; 95% CI, 0.78 to 1.12; P=0.46). Five-year OS was 85% +2% for both
groups. Toxicity
with the AC---T regimen was found to be acceptable in the adjuvant setting.
[0082] Thus, the combination of AC, followed by a taxane such as
paclitaxel (Taxol) is
now widely accepted as an effective adjuvant treatment for early-stage breast
cancer.
[0083] Advances in adjuvant chemotherapy have resulted in improved
outcomes in
patients with ER- breast cancers to a greater extent than for those with ER+
breast cancers
(Berry, et al., 2006). Many of these have been implemented as neoadjuvant
therapy. Standard
AC¨ T given preoperatively to TNBC patients results in pathologic complete
response rates of
30%-40% (Von Minckwitz, et al., 2011; Carey, et al., 2007).
- 28 -
CA 02874065 2014-12-04
[0084] Other conventional cancer therapies and treatments may also be
administered in
combination with the DC vaccines described herein. Cancer treatments that may
be administered
may include surgery and/or radiation. Conventional cancer therapies may also
include one or
more chemotherapeutics, including but not limited to cisplatin (CDDP),
carboplatin,
procarbazine, mechlorethamine, camptothecin, ifosfamide, melphalan,
chlorambucil, busulfan,
nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP 16),
tamoxifen, raloxifene, estrogen receptor binding agents, gemcitabine,
navelbine, farnesyl-protein
transferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin
and methotrexate, or
any analog or derivative variant of the foregoing.
[0085] Other suitable therapeutic agents may also include, for example,
vinca alkaloids,
agents that disrupt microtubule formation (such as colchicines and its
derivatives), anti-
angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine
kinase targeting agent
(such as tyrosine kinase inhibitors), serine kinase targeting agents,
transitional metal complexes,
proteasome inhibitors, antimetabolites (such as nucleoside analogs),
alkylating agents, platinum-
based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides,
therapeutic
antibodies, retinoids (such as all-trans retinoic acids or a derivatives
thereof); geldanamycin or a
derivative thereof (such as 17-AAG), and other standard chemotherapeutic
agents well
recognized in the art.
[0086] Therapeutic agents administered in the methods described herein may
also include
those that are well known for use against breast cancer. These breast cancer
chemotherapeutics
may include capecitabine, carboplatin, cyclophosphamide (Cytoxan),
daunorubicin, docetaxel
(Taxotere), doxorubicin (Adriamycin), epirubicin (Ellence), fluorouracil (also
called 5-
fluorouracil or 5-FU), gemcitabine, eribulin, ixabepilone, methotrexate,
mitomycin C,
mitoxantrone, paclitaxel (Taxol), thiotepa, vincristine, and vinorelbine.
[0087] In some embodiments, the chemotherapeutic agent is any of (and in
some
embodiments selected from the group consisting of) adriamycin, colchicine,
cyclophosphamide,
actinomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, mitomycin,
methotrexate,
mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU,
cisplatin, etoposide,
interferons, camptothecin and derivatives thereof, phenesterine, taxanes and
derivatives thereof
(e.g., paclitaxel and derivatives thereof, taxotere and derivatives thereof,
and the like), topetecan,
- 29 -
CA 02874065 2014-12-04
vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab-5801,
Irinotecan, HKP,
Ortataxel, gemcitabine, HerceptinO, vinorelbine, DoxiI0, capecitabine,
GleevecO, Alimta0,
AvastinS, Velcade0, Tarceva0, Neulasta0, Lapatinib, STI-571, ZD1839, Iressa0
(gefitinib),
SH268, genistein, CEP2563, SU6668, SU11248, EMD121974, and Sorafenib.
[0088] In some embodiments, the chemotherapeutic agent is a composition
comprising
nanoparticles comprising a thiocolchicine derivative and a carrier protein
(such as albumin).
[0089] In further embodiments a combination of chemotherapeutic agents is
administered
to breast cancer cells. The chemotherapeutic agents may be administered
serially (within
minutes, hours, or days of each other) or in parallel; they also may be
administered to the patient
in a premixed single composition. The composition may or may not contain an
Hsp90 inhibitor.
Combinations of breast cancer therapeutics include, but are not limited to the
following: AT
(Adriamycin and Taxotere), AC + T: (Adriamycin and Cytoxan, with or without
Taxol or
Taxotere), CMF (Cytoxan, methotrexate, and fluorouracil), CEF (Cytoxan,
Ellence, and
fluorouracil), FAC (fluorouracil, Adriamycin, and Cytoxan), CAF (Cytoxan,
Adriamycin, and
fluorouracil) (the FAC and CAF regimens use the same medicines but use
different doses and
frequencies), TAC (Taxotere, Adriamycin, and Cytoxan), and GET (Gemzar,
Ellence, and
Taxol).
[0090] In some embodiments, chemotherapeutic agents can be administered in
combination with anakinra and without administration of dendritic cell
vaccines. In some
embodiments, the chemotherapeutic agent that is administered is combination
with anakinra is
one of Nab paclitaxel, eribulin, capecitabine, or vinorelbine. In some
embodiments, the doses to
be used for chemotherapeutic drug products may be as set forth below:
= Nab paclitaxel: 100 mg/m2 administered IV weekly (Day 1, 8, and 15 every
28
days)
= Eribulin: 1.4 mg/m2 administered IV weekly (Day 1 and 8 every 21 days)
= Capecitabine: physician's choice of utilizing 1000 mg/m2 BID 14 days on,
7
days off OR 1000 mg/m2 BID 7 days on, 7 days off (capecitabine is rounded to
the nearest 500 mg increment).
= Vinorelbine: 25 mg/m2 administered IV weekly (Day 1, 8, and 15 every 28
days).
[0091]
- 30 -
CA 02874065 2014-12-04
G. Decreasing iTH2 Cell-Mediated Cancer Inflammation
[0092] In some embodiments, the treatment methods described herein include
administration of therapeutic agents that block pro-inflammatory pathways that
are responsible
for chronic inflammation in many solid tumors. Such therapeutic agents may
include antagonists
of IL-1R, including but not limited to anakinra. In some embodiments, anakinra
may be used in
combination with chemotherapy and DC vaccines. In some embodiments, anakinra
is used in
combination with chemotherapy alone.
[0093] Solid tumors are often associated with chronic inflammation that
promotes cancer
cell survival and metastasis. Linked closely with this is the significant
presence of macrophages,
educated by type 2 cytokines IL-4 and IL-13. Recent studies have demonstrated
a significant
presence of inflammatory CD4+ T cells (iTH2) cells in breast cancer, which
produce high levels
of IL-13, IL-4, and tumor necrosis factor. These iTH2 cells accelerate breast
cancer development
in xenograft models through production of IL-13, whereas in murine models,
they accelerate
metastases by production of 1L-4. iTH2 cells are driven by OX40L+ tumor
infiltrating myeloid
DCs (mDCs) which are conditioned by thymic stromal lymphopoietin (TSLP)
secreted by
malignant cells and infiltrating stromal cells. TSLP-neutralizing antibodies
block upregulation of
OX4OL by tumor- infiltrating mDCs, and consequently block mDCs' capacity to
generate iTH2
cells and to accelerate tumor development in vivo (Coussens, et al., 2013).
Thus, interference
with the TSLP-OX40L-IL-13 axis will allow modification of cancer-associated
inflammation
and thereby offer a novel therapeutic approach for patients with TNBC.
[0094] Recent studies show that TSLP secretion from breast cancer cells is
regulated by
IL-1I3. Results showed high levels of IL-113 in the breast cancer
microenvironment. IL-1J3 induces
TSLP production from breast cancer cell lines in a dose and contact dependent
manner. Cancer
cells induce IL-113 secretion from DCs and monocytes in a contact-dependent
fashion. This is
mediated by cancer cell-derived TGF-P. Administration of the IL-1R antagonist,
anakinra,
prevents tumor growth in vivo, blocks OX40L+ expression on DCs, and blocks
iTH2 generation
in vivo. Clinically, the Th2 signature in breast cancer (Teschendorff, et al.,
2010; Kristensen, et
al., 2012) is associated with poor outcomes. IL-4 and IL-13 exert pro-tumor
activity through
several pathways including: I) the triggering of TGF-P secretion (Terabe, et
al., 2004); 2) the up-
regulation of anti-apoptotic pathways in cancer cells (Zhang, et al., 2008);
and 3) the generation
-31 -
CA 02874065 2014-12-04
of type-2 polarized macrophages that foster tumor growth directly, via
secretion of growth
factors, and indirectly via inhibitory effects on CD8+ T cell function
(DeNardo, et al., 2011).
Indeed, CD8+ T cells are essential for tumor rejection through the generation
of cytotoxic
effectors. The presence of CD8+ T cells in primary tumors is associated with
the long-term
survival of patients with colorectal and breast cancer (DeNardo, et al., 2011;
Galon, et al., 2006).
Thus, iTH2 cells have a broad and profound impact on tumor microenvironment
and tumor
development.
[0095] Thus blockade of IL-1f3 represents a novel approach to breast
cancer
immunotherapy. In some embodiments of the methods described herein, a
therapeutic agent that
blocks the IL-1 inflammatory pathway is administered to a breast cancer
patient in combination
with chemotherapy and/or dendritic cell vaccines. In some embodiments, such
therapeutic agents
include but are not limited to anakinra, rilonacept, and canakinumab.
(Jurrmann et al., 2009).
Rilonacept is a recombinant IL-1 receptor-IG fusion protein and is generally
administered in a
loading dose of 320 mg followed by 160 mg weekly doses. Canakinumab is a
humanized anti IL-
1f3 antibody and is generally administered in a dose of 150 mg subcutaneaously
every 8 weeks.
Agents that block the IL-1 inflammation pathway are also described in Symons
et al, 1995,
Petrasek et al., 2012, Economides et al., 2003, and Jurrmann et al., 2005.
Agents that block the
IL-1 inflammation pathway may include modifiers of IL-10 gene transcription,
modifiers of IL-
1f3 gene translation, siRNAs that reduce expression of IL-1f3, and antagonists
of IL-1 receptor.
[0096] Anakinra is a recombinant soluble non-glycosylated homolog of the
human
interleukin-1 receptor antagonist (IL-1Ra) that competitively inhibits binding
of IL-la and IL-10
to the receptor type I. Anakinra was approved in 2001 as a treatment for
patients with adult
rheumatoid arthritis whose disease has progressed through one or more disease-
modifying anti-
rheumatic drugs. Anakinra is an effective treatment for systemic onset
juvenile arthritis, an IL-1-
driven disease (Pascual et al., 2005). Resolution of clinical symptoms
including fever, marked
leukocytosis, thrombocytosis, anemia, elevated ESR and arthritis were rapid
and sustained
(Pascual et al., 2005). These results have now been confirmed in randomized
clinical trials. A
pilot safety trial administering anakinra in combination with the physician's
choice of nab
paclitaxel, capecitabine, eribulin, and vinorelbine was recently opened in
patients with metastatic
breast cancer at Baylor Sammons Cancer Center. This study will evaluate the
effects of anakinra
on the IL-1-driven immunologic effects on patients' T cell subsets and on a
peripheral blood IL-
- 32 -
CA 02874065 2014-12-04
1 signature signifying adverse IL-1-mediated immunologic effects that has been
developed at
BIIR. Anakinra has a favorable safety profile; the most common adverse
reaction is an injection
site reaction.
H. Combination Therapies
[0097] In some embodiments, the treatment methods described herein include
two or
more therapeutic agents administered in combination. For example, antigen-
loaded dendritic
cells may be administered in combination with chemotherapy, anakinra, or both.
Administration
of DC vaccines may also be combined with radiation and/or surgery. Combination
therapy may
involve administering different therapeutic agents or treatments at the same
time or within a
period of time wherein separate administration of the therapeutic agents or
treatments produces a
desired therapeutic benefit. This may be achieved by administering a single
pharmacological
formulation that includes two or more therapeutic agents, or by administering
two or more
distinct compositions or formulations, wherein one composition includes one
therapeutic agent
and the other includes another.
[0098] The therapeutic agents and treatments disclosed herein may precede,
be co-
current with and/or follow another treatment or agent by intervals ranging
from minutes to
weeks. In embodiments where agents are applied separately to a cell, tissue or
organism, one
would generally ensure that a significant period of time did not expire
between the time of each
delivery, such that the therapeutic agents would still be able to exert an
advantageously
combined effect on the cell, tissue or organism. For example, in such
instances, it is
contemplated that one may contact the cell, tissue or organism with two,
three, four or more
agents or treatments substantially simultaneously (i.e., within less than
about a minute). In other
aspects, one or more therapeutic agents or treatments may be administered or
provided within I
minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes,
2 hours, 3 hours,
4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12
hours, 13 hours, 14
hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours,
22 hours, 22 hours,
23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30
hours, 31 hours, 32
hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours,
40 hours, 41 hours,
42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 1 day, 2
days, 3 days, 4
days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13
days, 14 days, 15 days,
- 33 -
CA 02874065 2014-12-04
16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3
weeks, 4 weeks, 5
weeks, 6 weeks, 7 weeks, or 8 weeks or more, and any range derivable therein,
prior to and/or
after administering another therapeutic agent or treatment.
[0099] Various combination regimens of the therapeutic agents and
treatments may be
employed. Non-limiting examples of such combinations are shown below, wherein
a therapeutic
agent such as a DC vaccine disclosed herein is "A" and a second agent, such as
an anti-cancer
chemotherapeutic, is "B":
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[00100] In some embodiments, more than one course of therapy may be
employed. It is
contemplated that multiple courses may be implemented.
F. EXAMPLES
[00101] The present invention will be described in greater detail by way of
specific
examples. The following examples are offered for illustrative purposes only,
and are not
intended to limit the invention in any manner. Those of skill in the art will
readily recognize a
variety of noncritical parameters which can be changed or modified to yield
essentially the same
results.
EXAMPLE 1: PROPHETIC EXAMPLE
BLOCKING IL-1B TO REDUCE PRO-CANCER INFLAMMATION
[00102] TSLP secretion from breast cancer cells is regulated by IL-1p.
Results showed
high levels of IL-113 in the breast cancer microenvironment. IL-1p induces
TSLP production
from breast cancer cells lines in a dose and contact dependent manner. Cancer
cells induce IL-1J3
secretion from DCs and monocytes in a contact-dependent fashion. This is
mediated by cancer
cell-derived TGF-P. Administration of the IL-1R antagonist, anakinra, prevents
tumor growth in
vivo, blocks OX40L+ expression on DCs, and blocks iTH2 generation in vivo.
Clinically, the
Th2 signature in breast cancer (Teschendorff, et al., 2010; Kristensen, et
al., 2012) is associated
with poor outcomes. IL-4 and IL-13 exert pro-tumor activity through several
pathways
including: 1) the triggering of TGF-P secretion (Terabe & Berzofsky, et al.,
2004); 2) the up-
- 34 -
CA 02874065 2014-12-04
regulation of anti-apoptotic pathways in cancer cells (Zhang, et al., 2008);
and 3) the generation
of type-2 polarized macrophages that foster tumor growth directly, via
secretion of growth
factors, and indirectly via inhibitory effects on CD8+ T cell function
(DeNardo, et al., 2011).
Indeed, CD8+ T cells are essential for tumor rejection through the generation
of cytotoxic
effectors. The presence of CD8+ T cells in primary tumors is associated with
the long-term
survival of patients with colorectal and breast cancer (DeNardo, et al., 2011;
Galon, et al., 2006).
Thus, iTH2 cells have a broad and profound impact on tumor microenvironment
and tumor
development.
[00103] Thus blockade of IL-1(3 represents a novel approach to breast
cancer
immunotherapy. Anakinra is a recombinant soluble non-glycosylated homolog of
the human
interleukin-1 receptor antagonist (IL-1Ra) that competitively inhibits binding
of IL-la and IL-113
to the receptor type I. The study described herein will evaluate the effects
of anakinra on the IL-
1-driven immunologic effects on patients' T cell subsets and on a peripheral
blood IL-1 signature
signifying adverse IL-1-mediated immunologic effects that has been developed
at BIIR.
Anakinra has a favorable safety profile; the most common adverse reaction is
an injection site
reaction.
EXAMPLE 2: PROPHETIC EXAMPLE
CLINICAL TRIAL OF DENDRITIC CELL VACCINE LOADED WITH CYCLIN B1
AND WT-1 ANTIGENS ADMINISTERED IN COMBINATION WITH
CHEMOTHERAPY AND ANAKINRA
1. Study overview and objectives
[00104] The goals of the study are to boost T cell immunity targeted
against breast cancer
utilizing a tumor antigen-loaded DC vaccine, to reverse the immune suppressive
tumor
microenvironment by IL-1 blockade, to enhance chemotherapy effectiveness and
decrease tumor
metastagenicity, and to decrease the recurrence rates of LA TNBC. Patients
with LA TNBC will
be treated with a combination of antigen-loaded DC vaccinations along with
standard
preoperative chemotherapy, to improve TNBC immunogenicity and to increase the
pCR rate
achieved with standard therapy. The trial will consist of 2 patient cohorts.
In the first group,
patients will receive DC vaccinations in combination with preoperative
chemotherapy. In the
second group, IL-1 blockade with anakinra will be added to the preoperative
chemotherapy and
DC vaccine.
- 35 -
CA 02874065 2014-12-04
[00105] The primary objective of this study is to determine the safety and
feasibility of
combining cyclin B1/WT-1/CEF (antigen)-loaded DC vaccination with preoperative
chemotherapy, and to combine DC vaccination with preoperative chemotherapy in
addition to
IL-1 blockade with anakinra in patients with LA TNBC.
[00106] The secondary objectives of this trial are to determine pathologic
complete
response rates, with and without anakinra; disease-free survival; to assess
immune biomarkers of
immunity (antigen-specific CD8+ T cell immunity and TH2 T cells) in breast
cancer biopsy
specimens and blood samples in patients receiving DC vaccinations, with and
without IL-1
blockade with anakinra; and to assess the feasibility of immunizing LA TNBC
patients with
patient-specific tumor antigens.
[00107] This exploratory pilot safety, open label trial will evaluate the
combination of
preoperative chemotherapy and Dendritic Cell (DC) vaccinations in 2 groups of
patients with LA
TNBC. A summary of the study design is shown in FIG.1. The first 10 patients
will be enrolled
to receive DC vaccinations during the 16 weeks of standard preoperative dose-
dense
doxorubicin/cyclophosphamide followed by paclitaxel chemotherapy; the
following 10 patients
will be a staggered enrollment, and will receive DC vaccinations and anakinra
100 mg SC for 7
days, followed by 7 days off, then repeating, during the 16 weeks of
preoperative chemotherapy.
Enrollment in Group 1 will complete before enrollment can begin in Group 2.
For Group 2, there
will be a staggered enrollment, in order to observe the safety of AC/T
chemotherapy, DC
vaccinations, and anakinra. After the first 3 patients have been enrolled in
Group 2, enrollment
will be held for observation of these patients for the 4 months of AC/T
chemotherapy plus
anakinra plus DC vaccine for adverse events, prior to enrolling a second set
of 3 patients.
Observation of these next 3 patients will occur over the 4 months of AC/T,
anakinra, and DC
vaccine for toxicity prior to completing enrollment of the last 4 patients in
Group 2. Study
procedures will be similar in both groups. Patients may only participate in
one group of the
study.
[00108] The screening period is from signature of the informed consent form
to final
eligibility assessments. Eligible patients will undergo apheresis after
registration and entry into
the study. After collection of peripheral blood mononuclear cells, dendritic
cell will be
- 36 -
CA 02874065 2014-12-04
manufactured from the monocyte fraction, aliquoted and frozen. Patients will
be given a total of
7 DC vaccinations.
[00109] Patients will undergo research biopsies of their breast cancer
prior to the start of
treatment and 1-2 days prior to or on Day 1 of Cycle 3 of AC to analyze the
composition of the
immune microenvironment. Four to 5 core biopsies will be obtained prior to
treatment initiation
for whole exome sequencing and expression analysis and for characterization of
the tumor
immune microenvironment.
[00110] Patients will receive standard preoperative
dose-dense
doxorubicin/cyclophosphamide (4 cycles) followed by paclitaxel (4 cycles;
AC/T)
chemotherapy, administered every 2 weeks for 16 weeks combined with antigen-
loaded DC
vaccinations administered intratumoral (one injection of 0.2 mL at 3 x 106
cells/mL) and
subcutaneous (one injection of lmL at 15 x 106 DCs) on on any one individual
day between
Days 9-12 of Cycles 1 and 3 of dose-dense AC and on any one individual day
between Days 9-
12 of Cycles 1 and 3 of T (4 timepoints). Timing of the vaccinations is based
on data that tumor
cell death associated with doxorubicin treatment increases the generation and
functional
activation of CD8+ T cells required for the antitumor activity of doxorubicin
(Mattarollo, 2011).
Standard pegfilgrastim support will be given on Day 2 of each AC treatment.
[00111] After preoperative treatment, patients will undergo definitive
surgery, generally
with mastectomy, and if available, the residual FFPE breast cancer tissue will
be collected for
assessment of the immune microenvironment and for whole exome sequencing to
identify
cancer-associated mutations in the residual, chemotherapy-refractory cancer.
Patients will be
known to have axillary node positive disease at study entry based on biopsy or
clinical criteria
and will generally undergo level 1/2 axillary dissection at definitive
surgery. However, patients
may undergo SLN biopsy before or after AC/T therapy at the physician's
discretion.
[00112] After definitive surgery and during locoregional radiation therapy
to the breast or
chest wall and regional lymphatics per standard of care, patients will receive
3 boost DC
vaccinations subcutaneously of 1 mL (at 15x106 cells/mL) in the ventral
surface of the upper
arm, with antigen-loaded DCs. The first vaccination booster will occur once
after the surgery and
up to 3 days prior to radiation; the second booster will occur 30 days 3
days after radiation is
completed; and the third booster will occur 90 days 3 days after the 2nd
boost.
- 37 -
CA 02874065 2014-12-04
[00113] Blood samples for immunomonitoring studies will be obtained at
baseline, prior
to each DC vaccination, prior to surgery, prior to radiation, and 2 weeks
after the last DC
vaccination.
2. DC vaccine preparation
A. Manufacturing Process
[00114] Following is the detailed description of the manufacturing process
and equipment
used to prepare the patient's frozen batch of BIIR-BrcaVax-001 autologous DC
vaccine product.
FIG. 3 shows an overview of the manufacturing process.
[00115] Preparation of Monocytes for Initiating the DC Culture. The
following steps
are taken to enrich the peripheral blood monocytes to prepare the patient's
frozen DC vaccine
batch. The first step is the collection of peripheral blood mononuclear cells
(PBMC) from the
patient by apheresis (see below). The second step is to enrich the monocytes
from the patient's
apheresis by elutriation employing the Terumo BCT Elutra . The Elutra is a
semi-automatic,
closed system centrifuge that uses continuous counter-flow elutriation
technology to separate
cells into multiple fractions based on size and density. The elutriation
procedure is outlined in
the Elutra operator's manual. The elutriation process begins by installing a
sterile, disposable
tubing set on the Elutra , and then priming the system with HBSS supplemented
with Human
Serum Albumin. Once the Elutra is primed, the apheresis bag containing the
patient's PBMC
is sterile connected to the disposable tubing set and the elutriation program
is started. A sterile,
heat-activated tubing welder (Terumo) is used to make the cuts and seals to
connect the tubing
used throughout the entire monocyte preparation and DC vaccine manufacture
process to
maintain sterility. The Elutra system automatically collects 5 fractions of
cells based on size
and density. Elutriation Fraction 5 normally contains the enriched monocyte
population. The
Coulter AcT5 automated hematology analyzer is employed to determine the purity
of the
Fraction 5 monocytes before they are used for preparation of the patient's
frozen DC vaccine
batch. Based on the aphereses collected from 41 patients with malignant
melanoma (BB-IND
12919) the expectation is that the median monocyte purity in elutriation
Fraction 5 is 92.4%
(range 41.0% to 96.6%; average std dev of 86.7 13.2%). Other cell types
found in Fraction 5
from this particular group of 41 patients with malignant melanoma included:
neutrophils
(average std dev of 5.3 8.5%), lymphocytes (average std dev of 5.5
6.1%), eosinophils
- 38 -
CA 02874065 2014-12-04
(average std dev of 0.2 0.3%) and basophils (average std dev of 1.3
0.5%). On occasion,
depending on the yield and purity of the patient's cells, the enriched
monocytes are found in
elutriation Fraction 4. In these instances either Fraction 4, Fraction 5 or
the combined Fractions
4 and 5 are used to prepare the patient's frozen DC vaccine batch. Once
determined, the
elutriation bag containing the enriched monocyte population is centrifuged to
pellet the cells.
After centrifugation the supernatant is removed from the bag and the cells are
resuspended in
CellGro DC culture medium. A sample is removed from the cell suspension for
in-process QC
testing to determine the cell count and viability in order to fill the cell
culture bags at the
specified cell density for initiation of the DC culture.
[00116] Method of Monocyte Collection. For the proposed clinical trial the
patient
apheresis is performed at the Baylor University Medical Center (BUMC)
Apheresis Collection
Center, Dallas, TX, using the COBE SPECTRATm system. The settings used for
operation of the
COBE SPECTRATm system used by the BUMC Apheresis Collection Center have been
worked
out in collaboration with GAMBRO the manufacturer of the apheresis system.
Furthermore, the
apheresis procedure conducted at the BUMC Apheresis Collection Center has
provided
consistent product for preparation of DC vaccines manufactured by the BIIR for
conducting
various cancer and infectious disease clinical trials.
[00117] Culture Conditions for DC Vaccine Manufacture. The steps in the DC
vaccine
manufacturing process are outlined below. The first step is to establish the
monocytes in cell
culture to induce differentiation of the DC. To establish the cell culture
(Day 0) elutriation
enriched monocytes are suspended at 1x106 viable cells/mL in CellGrog DC
culture media
supplemented with GM-CSF at 100 ng/mL and IFN-a at 500 IU/mL. The transfer
pack (Baxter
Fenwal, 4R 2041) containing the cell suspension is then sequentially attached
to individual cell
culture bags (AFC, 118AC) and 100 mL of the cell suspension is transferred to
each bag. The
cell culture bags are then placed in a 37 C, 5% CO2 incubator. After 24 hours
of cell culture
(Day 1) the media is replenished with fresh GM-CSF at 100 ng/mL and IFN-a at
500 IU/mL in 5
mL of CellGro DC media per culture bag by using sterile syringes with 19G x 1
IA inch needles
to inject the cytokines and media into the cell culture bags. The second step
is to load the
differentiated DC with tumor and control antigens, that is, CEF control
antigens is a mixture of
infectious virus antigen peptides, WT1 is a mixture of 6 Wilm's Tumor antigen
peptides and
- 39 -
CA 02874065 2014-12-04
Cyclin B1 are two long antigen peptides. The antigen loading is performed
after the cells have
been cultured for approximately 48 hours (Day 2). To load the DC with the
antigen peptides a 1
mL syringe with 19G x 11/2 needle is used to add 1.0 !AL of the peptide
mixture per mL of cell
culture media to achieve 2-3 uM of each of the antigen peptides. CEF antigen
peptides is added
to one of the cell culture bags and WT1 and Cyclin BI1 antigen peptides are
added to half of the
remaining cell culture bags, respectively. The third step is to activate the
antigen loaded DC
with LPS, CD4OL and CL075. On Day 2, immediately after adding the antigen
peptides to the
DC, 1 mL syringes with 19G x 11/2 inch needles are used to add 5 EU/mL of LPS,
100 ng/mL of
CD4OL and 1.0 ug/mL of CL075 to each of the cell culture bags. The DC are
cultured for 24 1
hours with the peptide antigens and LPS/CD4OL/CL075 before they are harvested
for
fill/freezing of the DC vaccine. Prior to DC harvest a 6 mL sample of the cell
culture media is
collected from 6 randomly selected cell culture bags. The samples are pooled
and then submitted
for in-process testing for the possibility of bacterial, fungal and mycoplasma
contamination.
After removing the sample for in-process sterility and mycoplasma testing, the
contents of the
cell culture bags are then transferred into 600 mL transfer packs (Baxter
Fenwal, 4R 2023). The
contents of approximately 4 culture bags is transferred to each transfer pack.
By centrifugation
(770 xg for 10 minutes) the cells are pelleted, combined and transferred to a
single 250 mL
centrifuge tube to be washed in Lactated Ringer's. After the last wash the
cells are transferred to
a 50 mL centrifuge tube and resuspended in 50 mL of Lactated Ringer's. A 1 mL
sample of the
cell suspension is taken for a cell count and cell viability determination.
The cells are then
pelleted and resuspended at 30x106 viable cells/mL of freezing solution (80%
heat-inactivated
autologous serum, 10% Plasma-Lyte A (Baxter, NDC Number: 0338-0221-04)
supplemented
with 5% dextrose, and 10% Dimethyl Sulfoxide (DMSO)). The final step of the
process is to
fill/freeze and then store the frozen DC vaccine for clinical use. The DC
vaccine is filled into 2
mL glass vaccine vials according to SOP VP121 "Manual Cryopreservation of
Dendritic Cell
Vaccines in Glass Vaccine Vials". A 5 mL disposable plastic pipette is used to
transfer 1 mL of
the DC vaccine suspension to each glass vaccine vial. The vials are sealed
with a rubber stopper
and metal flip-off cap. The sealed vaccine vials are then placed in a pre-
cooled controlled rate
freezer with the freezing program initiated as described in SOP EQ143 "Kryo
1020-380
Controlled Rate Freezer". After the cells are frozen the vials of DC vaccine
are placed in a -
80 C freezer and held for at least 4 hours but not more than 24 hours. The
frozen vials are then
- 40 -
CA 02874065 2014-12-04
transferred to the quarantine section of the liquid nitrogen tank in the GMP
freezer room. On the
day that QC release testing is initiated, a vial is randomly selected from the
DC vaccine batch for
QC release testing. Upon completion of the QC testing and QA release the DC
vaccine batch is
moved to the released product storage section of the liquid nitrogen tank.
[00118] Process Timing and Intermediate Storage. The entire manufacture of
a BIIR-
BrcaVax-001 DC vaccine batch is conducted in a single continuous process;
thus, there is not an
intermediate storage step in the manufacturing process. The timing of key
steps of the full
manufacturing campaign to prepare a single DC vaccine batch, which runs
approximately 90
hours, is summarized in FIG. 3. Briefly, on Day 0 the patient's peripheral
blood mononuclear
cells are collected by apheresis, which is transferred to the GMP vaccine
manufacturing facility
for enrichment of the monocyte fraction to initiate the DC culture. On Day 1
after 24 hours in
culture fresh GM-CSF and IFN-a are added to the cell culture bags to replenish
these cytokines
in the cell culture media. On Day 2 after approximately 48 hours of cell
culture the tumor and
control antigen peptides are added to the cell culture to load the DC with the
specified antigen
epitopes. On Day 2, immediately after addition of the antigen peptides, the
LPS, CD4OL and
CL075 are added to the culture to activate the antigen loaded DC. After 24 1
hours of
incubation with the antigens, LPS, CD4OL and CL075 the DC vaccine is harvested
from the cell
culture bags, washed, resuspended in freezing solution, filled into glass
vaccine vials, frozen in a
controlled rate freezer and stored overnight in a -80 C freezer. Following 4-
24 hours storage at -
80 C, the frozen batch of DC vaccine product is transferred to a liquid
nitrogen tank for long-
term storage.
[00119] Final Harvest. Step 4 of the BIIR-BrcaVax-001 manufacturing process
is harvest
of the DC vaccine from the cell culture bags for fill/freezing. On Day 3,
after approximately 72
hours in cell culture, the antigen-loaded, LPS/CD4OL/CL075 activated DC are
harvested from
the cell culture bags by the following process. Prior to harvesting the cells
6 mL samples are
collected from six of the cell culture bags selected at random for sterility
and mycoplasma
testing. The cells are then collected from the cell culture bags and pooled by
transferring the
contents of the cell culture bags into 600 mL transfer packs, that is, the
contents of <4 cell
culture bags is transferred to each transfer pack. After the cell culture
suspensions are
transferred to the transfer packs, each of the cell culture bags are then
thoroughly washed with 25
- 41 -
CA 02874065 2014-12-04
mL of Lactated Ringer's. The cell culture bags are reconnected to the transfer
packs and the
Lactated Ringer's rinsed cells are transferred to the transfer packs. The
transfer packs are
centrifuged to pellet the cells. The cells are resuspended in Lactated
Ringer's and pelleted by
centrifugation. After the second wash the cell pellet is dispersed and the
cell suspension in each
of the transfer packs is transferred to a single 250 mL centrifuge tube.
Lactated Ringer's is then
added to Q.S. the cell suspension to 250 mL in the centrifuge tube. After
centrifugation the
pelleted cells are then resuspended in 20 mL of Lactated Ringer's and
transferred to a 50 mL
centrifuge tube. The 250 mL tube is rinsed with 20 mL of Lactated Ringer's
that is added to the
50 mL tube and the cell suspension is Q.S. to 50 mL with Lactated Ringer's. A
1 mL in-process
QC sample is taken to determine the cell count and viability. The cells are
pelleted by
centrifugation and then resuspended in heat-inactivated autologous serum at
60x106 viable
cells/mL. At this point the cell suspension is ready for the addition of an
equal volume of 2x
freezing solution to initiate the fill/freeze step of the process.
[00120]
Timing/Methods/Wash Procedures. Cells are washed at several points in the
BIIR-BrcaVax-001 DC vaccine manufacturing process. The basic method is to
pellet the cells
by centrifugation, that is, the cells in either bags or tubes are centrifuged
to pellet the cells and
remove the suspension solution. Following is a brief summary of the points in
the process where
the cells are washed.
1. Washing the elutriation enriched monocytes. The elutriation fraction bag(s)
containing
the enriched monocyte population is connected to a 150 mL transfer pack and
the
monocytes are transferred. An additional transfer pack is used if the volume
of the
elutriation fraction bag exceeds 150 mL. The transfer pack(s) is centrifuged
at 770 xg for
minutes at ambient room temperature to pellet the cells. After centrifugation
the
supernatant is removed from the bag and the cell pellet is dispersed and
transferred to a 2
L transfer pack. The 150 mL transfer pack(s) is rinsed with 25 mL of normal
saline
which is then transferred to the 2 L transfer pack. CellGro0 media is then
added to the
transfer pack to bring the cell concentration to 1x106 viable cells/mL.
2. Washing of the antigen-loaded, LPS/CD4OL/CL075 activated DC at the time of
cell
harvest. The DC cell culture suspension and volume of Lactated Ringer's used
to rinse
the cell culture bags are transferred to 600 mL transfer packs. The transfer
packs are
centrifuged at 770 xg for 10 minutes at ambient room temperature. The
supernatant is
- 42 -
CA 02874065 2014-12-04
removed from the cell pellet by transferring it from the transfer pack to a
sterile
connected empty transfer "waste" pack. The cell pellet is gently dispersed and
approximately 25 mL of Lactated Ringer's is added to each of the transfer
packs. The
cells are pooled into one transfer pack and Lactated Ringer's is added to a
volume of
approximately 500 mL. The transfer packs are centrifuged at 770 xg for 10
minutes at
ambient room temperature. The solution is removed from the cell pellet by
transferring
the supernatant from the transfer pack to a sterile connected empty transfer
"waste" pack.
The Lactated Ringer's wash step is then repeated. The cell pellet in each
transfer pack is
gently dispersed and the cell suspensions transferred to and pooled in a
single 250 mL
centrifuge tube. The cell suspension in the centrifuge tube is Q.S. to 250 mL
with
Lactated Ringer's. The cells are pelleted by centrifugation at 770 xg for 10
minutes at
ambient room temperature. The supernatant is removed, the cell pellet
dispersed and the
cells suspended in 20 mL of Lactated Ringer's. The cell suspension is
transferred to a 50
mL centrifuge tube. The 250 mL tube is rinsed with 20 mL of Lactated Ringer's
that is
transferred to the 50 mL tube. The cell suspension in the 50 mL tube is Q.S.
to 50 mL
with Lactated Ringer's and, after removing a 1 mL sample for in-process QC
testing, the
cells are pelleted by centrifugation at 270 xg for 10 minutes at ambient room
temperature.
During the final wash the in-process QC sample is analyzed to determine the
cell count
and viability. The washed cells are then resuspended in a volume of heat-
inactivated
autologous serum to give a cell concentration of 60x106 viable cells/mL. The
cell
suspension is now ready for addition of the freezing solution and initiation
of the
fill/freeze process.
[001211
Final Formulation. The BIIR-BrcaVax-001 DC vaccine is prepared for injection
into the patient by thawing the requisite number of frozen vials of DC vaccine
and diluting the
contents with USP injection grade sterile Lactated Ringer's (Hospira, NDC
Number: 0409-7953-
02, 250 mL bag for preparing the Vaccine Product for injection; and 0409-7953-
09, I L bag for
use in the DC vaccine manufacturing process) to wash the cells by
centrifugation. The cells are
washed 3 times with Lactated Ringer's. Prior to the third wash a sample is
taken to determine
the cell count and viability. After the third wash the cells are resuspended
in Lactated Ringer's
at a concentration of 15x106 viable cells/mL. The cell suspensions are filled
into a 2 mL sterile
-43 -
CA 02874065 2014-12-04
glass vaccine vial sealed with a serum stopper and metal cap, for delivery to
the clinic.
Therefore, the final formulation is comprised of the DCs suspended in 100%
Lactated Ringer's.
[00122] Extensive QC release testing of the frozen vaccine will include:
a) Cell Count (Recovery) and Viability
b) Evaluation of DC morphology by Giemsa staining of cytospun cells
c) Evaluation of DC phenotype by multiparameter flow cytometry analysis
d) Sterility testing (mycoplasma, gram stain, bacteria/fungus growth, and
endotoxin)
e) Potency testing by phenotype and cytokine secretion.
[00123] QC release testing of the washed DC vaccine for injection will
include:
a) Cell count and viability
b) Sterility testing: gram stain and endotoxin (results available prior to
injection)
c) Sterility testing: bacterial and fungal growth (results available after
injection)
[00124] Excipients. Following is a list of the excipients employed in the
BIIR-BrcaVax-
001 DC vaccine manufacturing process. These excipients, except for the
Lactated Ringer's used
to suspend the DC in the final formulation for injection into the patent, are
not retained in the
final, frozen DC vaccine product or Vaccine Product prepared for injection
because they are
removed by washing the cells.
Elutriation Buffer. The elutriation buffer is prepared by adding 500 mL of 5%
human
serum albumin to a 4 L bag of 1X Hank's Balanced Salt Solution. The components
used
to prepare the elutriation buffer are described below.
Hank's Balanced Salt Solution, (HBSS, BioWhittaker brand, Lonza, Catalog
Number: 08-003A) is a sterile, isotonic solution at pH 7.0 to 7.4 that is
packaged
in 4 L plastic bags and is stored at ambient room temperature.
5% Human Serum Albumin, USP grade (Baxter, NDC Number: 0944-0491-02
or CSL Behring, NDC Number: 0053-7670-32) is a sterile, nonpyrogenic solution
supplied at 500 mL in glass bottles and is stored at ambient room temperature.
Cell Freezing Solution. The final formulation of the DC vaccine freezing
solution is
comprised of 80% heat-inactivated autologous serum, 10% Plasma-Lyte A
supplemented
- 44 -
CA 02874065 2014-12-04
with 5% dextrose, and 10% Dimethyl Sulfoxide (DMSO). The components used to
prepare the freezing solution are described below.
Heat-Inactivated Autologous Serum. Autologous serum is obtained from
peripheral blood drawn from the patient in red top Vacutainer0 tubes prior to
apheresis. The serum is separated from the clotted blood by centrifugation.
The
serum is transferred to a sterile 50 mL tube labeled "Serum" with the
patient's
identification, study number and date. The serum is filter sterilized by
passing it
through Acrodisc 0.2 micron, 37 mm syringe filters for aliquotting into
labeled,
sterile 15 mL tubes. The tubes are placed in a 56 C heat block for 30 minutes
to
heat-inactivate the serum. "Heat-Inactivated" is then added to the tube label
and
the serum is stored refrigerated at 2-8 C until use.
Plasma-Lyte A, (Multiple Electrolytes Injection, Type 1, USP; Baxter, NDC
Number: 0338-0221-04) is a sterile, non-pyrogenic isotonic solution supplied
in a
500 mL VIAFLEX plastic container for intravenous administration and is stored
at ambient room temperature.
70% Dextrose, USP injection grade, (Hospira, NDC Number: 0409-7918-19) is
supplied at 500 mL in partially filled 1 L plastic bags and is stored at
ambient
room temperature.
Dimethyl Sulfoxide, (DMSO, Cryoserv0 brand; NDC Number: 67457-178-10 or
Number: 67457-178-50) is supplied as a sterile, 99% pure solution at 10 or 50
mL, respectively, in flip-top glass vials and is stored at ambient room
temperature.
Lactated Ringer's Injection, USP, (Hospira) is a sterile, nonpryogenic
solution
containing isotonic concentrations of sodium chloride 600 mg, sodium lactate
anhydrous
310 mg, potassium chloride 30 mg and calcium chloride dehydrate 20 mg. May
contain
hydrochloric acid and/or sodium hydroxide for pH adjustment. Lactated Ringer's
is
supplied in either a 250 mL bag (NDC Number: 0409-7953-02) used for
preparation of
the Vaccine Product for injection, or a 1 L bag (NDC Number: 0409-7953-09)
used in the
DC vaccine manufacturing process. The bags are stored at 20 to 25 C (68 to 77
F) and
are protected from freezing.
- 45 -
CA 02874065 2014-12-04
PBS, phosphate buffered saline without Ca++ and Mg ++ at pH 7.2 (GIBCO ,
Catalog
Number: 20012-027) is supplied as a sterile solution in 500 mL plastic bottles
and is
stored at ambient room temperature.
Normal Saline, USP injection grade, 0.9% sodium chloride (Hospira, NDC Number:
0409-7983-03) is supplied as a sterile solution in 500 mL plastic bags and is
stored at
ambient room temperature.
Sterile Water for Injection, USP grade (referred to herein as Sterile Water;
Hospira,
NDC Number: 0409-4887-10) is supplied as a sterile solution in 10 mL flip-top
plastic
vials and is stored at ambient room temperature.
[00125] Cell Density/Concentration in the Final Product. The DC vaccine is
thawed,
washed with Lactated Ringer's and suspended in USP injection grade sterile
Lactated Ringer's at
15x106 viable cells/mL with 1.5 mL of the cell suspension filled into a glass
vaccine vial for use
by the clinic.
[00126] Storage Method Prior to Use. The frozen BIIR-BrcaVax-001 DC vaccine
is
stored at -180 C (liquid nitrogen vapor phase). The thawing and DMSO washout
process is
conducted at ambient room temperature. The cell preparation for injection is
transported to the
bedside at ambient room temperature. The elapsed time from preparation of the
DC vaccine for
injection and vaccination of the patient is approximately 3 hours.
B. In-Process Testing and Criteria.
[00127] In-process testing is conducted at several points in the BIIR-
BrcaVax-001 DC
vaccine manufacturing process. Specifically, to quantify the patient's
monocytes in-process
samples are taken from the apheresis to determine sterility and from the
elutriation fraction to
determine monocyte purity, cell count and cell viability. In-process samples
are taken at the time
of harvest of the DC vaccine for sterility testing, mycoplasma testing, and
determination of cell
count and viability. The various in-process tests are summarized in Table 2
and Table 3 below.
-46-
CA 02874065 2014-12-04
Table 2: In-Process Tests and Specifications
Preparation of the Monocytes
Process
Test Method Result Specification
Step
Performed by BUMC for Apheresis should be
sterile,
Sterility (Gram-Stain and Information Only - Used if Out-of- that is,
gram-stain negative
Apheresis
Microbial Growth) Specification investigation is and free of
bacteria and
required. fungus contamination.
Identity and percentage (purity) of Monocyte purity ?_50%
for
Hematology Analyzer the monocytes in the elutriation Fraction 5;
and 70% for
Elutriation
(Users Manual) fractions. Result used to select Fraction 4
if being combined
the elutriation fraction(s) for culture with Fraction 5.
Cell Count and Viability by Total number and viability of cells
Elutriation Trypan Blue Staining to determine the cell
concentration Report result
(SOP VR109). for cell culture.
Table 3: Further In-Process Tests and Specifications
Manufacture of the DC Vaccine
Process
Test Method Result Specification
Step
Presence/Absence of
Sterility Bacterial and microbes, i.e., bacteria in 14
DC Harvest Fungal Growth day growth cultures and Negative for
bacteria and
fungus
(SOP VR119) fungus in 28 day growth
cultures.
DC Harvest Sterility Gram Stain Presence/Absence of
gram Negative for gram-positive
(SOP VR119) stain positive organisms. organisms
Mycoplasma detection by Presence/Absence of Hoechst
Hoechst Staining and 33342 stained mycoplasma,
DC Harvest Growth Culture (SOP mycoplasma DNA by PCR,
Negative for Mycoplasma
VR120) and PCR (SOP and mycoplasma in 28 day
VR111). growth culture.
Cell Count and Viability by Total number and viability of
Count: 30x106 viable cells per
DC Harvest Trypan Blue Staining cells to determine cell
mL per glass vaccine vial
(SOP VR109). concentration for fill/freezing.
[00128] Monoeyte Identity and Purity. The Coulter AcT5 Hematological
Analyzer is an
automated system used to identify the various peripheral blood cell
populations in the different
elutriation fraction bags. The instrument uses flow impedance, cytochemistry
and light
absorbance to differentiate the blood cell populations. The instrument readout
provides both cell
number and percentage of the various peripheral blood cell populations in the
sample. The
samples are analyzed to identify which elutriation faction contains the
enriched monocyte
- 47 -
CA 02874065 2014-12-04
population (usually Fraction 5) and determine the purity (percentage) of
monocytes in the
elutriation fraction. The printout of the AcT5 results is filed with the
Vaccine Production Record
used for documenting the preparation and QC release of the DC vaccine batch.
[00129] Cell Count and Viability. The total number of cells and
determination of cell
viability is determined by light microscopic examination of the trypan blue
stained cell
suspension loaded onto a hemocytometer. The cell count and viability assay is
performed
according to the method described in SOP VR109 "Cell Count and Viability Using
Trypan Blue
Stain". Briefly, cells are counted on three fields of the hemocytometer. The
number of non-
stained (viable) and trypan blue stained (non-viable) cells are recorded for
each field. The
average number of viable and non-viable cells is calculated from the combined
results of the
three fields. Based on the calculated average, the number of cells per mL of
cell suspension is
established and the percentage of viable cells is determined. The information
is used to make
cell dilutions for setting up cell cultures and filling glass vaccine vials.
In-process samples to
determine cell count and viability are collected from the elutriation fraction
containing the
monocytes to initiation of the DC culture, and the DC harvest.
[00130] Sterility Testing at the Laboratories at Bonfils. A sample is taken
at DC vaccine
harvest and sent to the Laboratories at Bonfils for a gram stain, 14 day
bacterial growth culture,
and 28 day fungal growth culture. Results should be negative; however, the DC
vaccine
products may be released for the first vaccination before the final reports
are received. See the
specific actions to be taken to monitor and if necessary treat the patient
should the frozen,
autologous DC vaccine be shown to be non-sterile after the first vaccination
has been performed
in the sections below for QC release of the final product. Following is a
description of the
sterility testing conducted at the Laboratories at Bonfils.
[00131] Bonfils Laboratories (LABS) is registered with the FDA, FEI
#1000477683, and
conducts sterility testing by USP methodologies. Microbiological testing,
including sterility
testing is performed at LABS following the latest United States Pharmacopeia
(USP) compendia
of methods. LABS also complies with 21 CFR Part 610 General Biological
Products Standards
section 610.12 on Sterility and with the AATB Standards for Tissue Banking,
Section K3.000
regarding Microbiological Testing. LABS is an ISO certified, FDA registered
testing facility
- 48 -
CA 02874065 2014-12-04
that assures that microbiological testing is performed using validated test
methods, validated
equipment and a staff of fully trained microbiologists.
[00132] Microbiological media used for the detection of aerobic, anaerobic
and fungi
organisms are chosen based on requirements in the CFR. Trypticase Soy Broth,
which is the
same as Soybean-Casein Digest Medium, and Fluid Thioglycollate Medium are used
for the
sterility cultures as prescribed. Trypticase Soy Broth is used for aerobic
organisms and for yeast
and mold detection. Fluid Thioglycollate Medium is used for culture of
anaerobes, facultative
anaerobes and for aerobic bacteria.
[00133] LABS purchases TSB and FTM from approved vendors and while the
standard
developed by the Clinical Laboratory Standards Institute does not require QA
testing on these
media if purchased commercially, LABS tests all media for its ability to
promote microbial
growth before it is used in sterility tests. The sterility culture incubation
period is 14 days per the
requirements in the CFR and in the USP. Cultures are read at 3, 4 or 5 days,
at 7 days and a final
reading at 14 days (Bacteria) or 28 days (Fungus). If the media is turbid at
any reading, the
microbiologist performs a gram stain and subculture as appropriate.
[00134] Mycoplasma Testing. A sample of the BIIR-BrcaVax-001 DC vaccine
culture is
taken from the cell culture prior to DC vaccine harvest for mycoplasma
testing. A portion of the
sample is tested in the BIIR QC Laboratory for the presence of mycoplasma DNA
using the PCR
method described in SOP VR111 "Mycoplasma Detection by PCR". The results of
the PCR test
are expected to be negative for the presence of mycoplasma DNA in the DC
culture sample.
Another portion of the sample is sent to Bionique Laboratories for their M700
assay that includes
a 28 day direct culture and an indirect Hoechst stain using indicator cell
lines. Preliminary
results of the direct culture are received on day 7. Any positive result at
any time during the 28
day culture period is reported immediately to the GMP manager, Quality
Assurance (QA) Unit
and Principle Investigator (PI). Final results for the Hoechst stain are
available 5 days after
initiation of the assay. The results should be negative for mycoplasma. The
remainder of the
sample is saved as a "Retain Sample" for future testing if necessary.
[00135] Bionique Testing Laboratoires' FDA registration number is 1318709.
Bionique
Laboratories conducts testing in compliance with cGMP standards outlined in
21CFR parts 210
and 211; that is, "Recommended Procedures for Detection of Mycoplasma
Contamination in
- 49 -
CA 02874065 2014-12-04
Biological Products Produced in Cell Substrates". The purpose of this test is
to determine
whether or not mycoplasmal contaminants are present in cell culture samples or
bioproducts
derived from cell culture substrates. The procedure requires a non-selective
indirect DNA
fluorochrome staining assay to detect non-cultivable mycoplasmas and a direct
culture assay.
The indicator cell/DNA fluorochrome staining procedure requires the
inoculation of the sample
into 2 indicator cell cultures (slide cultures of VERO cells). Two sample (1.0
mL) aliquots are
inoculated into each of two quality controlled slide cultures of mycoplasmal
free VERO cells and
incubated (5% CO2, 95% Air) for 3 to 5 days. Usually on Day 4, each slide
culture is fixed with
Camoy's fixative and stained with Hoechst DNA fluorochrome stain. This assay
is designed to
enhance the level of sensitivity by reducing background and amplifying the
titer of mycoplasmal
contaminants. Appropriate positive control cultures, M hyorhinis and M orale
at <100 CFU,
and a negative control culture are processed with each batch of tests as
specified by the FDA.
The theoretical sensitivity of this assay is approximately 50 CFU. The direct
culture procedure
utilizes Fortified Commercial (FC) broth and agar formulations. A 10 mL sample
aliquot is
inoculated into 50 mL of FC broth supplemented with 20% horse serum. A 0.1 mL
sample is
inoculated onto 4 FC agar plates. The broth culture is subcultured onto like
agar plates on Day 3,
Day 7 and Day 14 post setup as specified per FDA guidelines. The agar plates
are incubated
aerobically and anaerobically (5% CO2, 95% nitrogen). The FC agar plates are
examined
microscopically at 7 day intervals. Two positive controls, M pneumoniae and M.
orale (<100
CFU) and a negative control are processed with each batch of tests as
specified by the FDA. The
theoretical sensitivity of the direct culture assay is approximately 10 CFU.
All media and
supplemental components used for screening purposes are tested for sterility
and for the ability to
support mycoplasma growth. Total testing time is 28 days.
C. Final Product Release Criteria/Specifications and Test Methods
[00136]
Each patient's frozen batch of autologous DC vaccine is tested and released
according to the procedures and specifications outlined in SOP VR151 "Release
Testing for DC
Vaccine Product BIIR-BrcaVax-001". The test results and Certificate of
Analysis for each
frozen batch of autologous DC vaccine product are reviewed and signed-off by
both GMP
Management and the Quality Assurance Unit. The final product release testing
is performed on a
vial of frozen DC vaccine randomly selected from the DC vaccine batch obtained
at the time the
batch is transferred from the -80 C freezer to the quarantine section of the
liquid nitrogen storage
- 50 -
CA 02874065 2014-12-04
tank. The QC test methods and specifications used to assess and release these
final products are
summarized below in Table 4 and Table 5.
Table 4: Test Methods and Specifications for Final Product Release
Frozen Autologous DC Vaccine Products
Sample Test Method Result Specification
Dose:
Total number and viability of Count .50 /0 of the fill
Cell Count and Viability by
cells in the vial after target (Recovery)
Trypan Blue Staining
thawing. Cell Viability ?_50 /0
(SOP VR109)
Identity: Identification of cell with
Giemsa Staining (SOP characteristics of DC Report Result
VR107) morphology.
Identity/Purity: ?_80 /0 HLA-DR+ CD11c+
Percentage of cells with the
Flow Cytometry (SOP cells
specified DC phenotype.
VR137)
Phenotypic Potency: Percentage of cells with the
Portions of the Flow Cytometry (SOP phenotype indicative of
DC ?70% CD80+ Cells
cell suspension VR137) potency.
taken from the Sterility: Presence/Absence of gram-
Gram-Negative
Lactated Ringer's
Gram-Stain (SOP VR119) positive organisms.
diluted thawed
DC vaccine vial. Sterility: Determine the amount of
<0.5 EU/mL
Endotoxin by Endosafe
endotoxin in the product. <5 EU/dose
LAL Test (SOP VR134)
Sterility: Interim and Final Results
Presence/Absence of
Bacterial/Fungal Cultures negative for microbial
bacteria and fungus
(SOP VR119) contamination
Characterization test of the
Functional Potency: amount of IL-23, IL-12p40,
Luminex measurement of and IL-12p70 secreted by
Report Result
DC Cytokine Secretion the DC. Additional
(SOP VR148) cytokines may also be
analyzed.
NOTE: The 14 day bacteria and 28 day fungal and mycoplasma growth culture
results may not be available at the
time of release of the Vaccine Batch Product for preparation of the inoculates
for injection into the patient. Interim
results are monitored during the course to the testing process. Final results
of the bacterial, fungal and mycoplasma
culture are reviewed for final product release by QA (See signature line on
CoA). An action plan (see below) is in
place to respond to a positive result of a sterility test reported post-
release of either the frozen Vaccine Product or
Vaccine Inoculate.
- 51 -
CA 02874065 2014-12-04
Table 5: Test Methods and Specifications for Final Clinical Product Release
(i.e., Inoculate
Filled in the Glass Vaccine Vial).
Washed Autologous DC Vaccine Inoculate
Sample Test Method Result Specification
Dose:T l f vi 15x10b viable cells/mL
ota number oabity
Portions of the Cell Count and Viability by
cells in suspension fiil lled in with 1.5 mL filled into a
cell suspension Trypan Blue Staining the syringe glass
vaccine vial for
after the cells (SOP VR109) . injection.
were thawed and Sterility: Presence/Absence of gram-
Gram-Negative
washed to Gram-Stain (SOP VR119) positive organisms.
prepare the Steril Bacteryiita:
l/Fungal Cultures
inoculate(s). Presence/Absence of Negative for microbial
(SOP VR119) bacteria and fungus contamination
NOTE: The gram-stain, 14 day bacteria and 28 day fungal growth culture results
may not be available at the time of
release of the vaccine inoculates for injection into the patient. Interim
results are monitored during the course to the
testing process. Final results of the gram-stain, bacterial and fungal growth
cultures are reviewed and released by
QA. The following action plan is in place to respond to a positive result of a
sterility test reported post-release of the
vaccine inoculate.
[00137] Cell Count and Viability. The cell count and viability assay is
performed
according to the method described in the In-Process Test Method section above.
[00138] Endotoxin. Endotoxin testing is performed to release each batch of
frozen DC
vaccine product. The amount of endotoxin in the product is determined by an
automated LAL
assay using the Endosafe portable test system. The Endosafe is a rapid,
point-of-use test
system for quantitative LAL test results in approximately 15 minutes. It is
comprised of a test
cartridge along with a hand-held spectrophotometer. The Endosafe endotoxin
test utilizes
existing FDA-licensed LAL formulations. Test cartridges are available to
achieve a level of
sensitivity as low as 0.01 EU/mL. Results of the endotoxin testing will be
available to release
the frozen DC vaccine batches. To release the DC vaccine products endotoxin
levels should be
less than 0.5 EU/mL or 5 EU/dose.
[00139] Sterility Testing at the Laboratories at Bonfils. A sample is taken
from the final
product, i.e., thawed cell suspension from the frozen DC vaccine batches at
the time of QC
release testing and from the DC vaccine inoculate, and sent to the
Laboratories at Bonfils for a
gram stain, 14 day bacterial growth culture, and 28 day fungal growth culture.
Results should be
negative; however, DC vaccine products are released for the first vaccination
before the final
reports are received. The frozen DC vaccine batches may be and the DC vaccine
inoculates are
released for the first vaccination of the patient prior to or on preliminary
growth culture results
- 52 -
CA 02874065 2014-12-04
but the final results are known before subsequent vaccinations. Specified
actions are taken to
monitor and if necessary treat the patient should the frozen, autologous DC
vaccine or inoculate
be shown to be non-sterile after the vaccination has been performed (see the
Notes after Tables 4
and 5 above). Following is a description of the sterility testing conducted at
the Laboratories at
Bonfils.
[00140] Bonfils Laboratories (LABS) is registered with the FDA, FEI
#1000477683, and
conducts sterility testing by USP methodologies. Microbiological testing,
including sterility
testing is performed at LABS following the latest United States Pharmacopeia
(USP) compendia
of methods. LABS also complies with 21 CFR Part 610 General Biological
Products Standards
section 610.12 on Sterility and with the AATB Standards for Tissue Banking,
Section K3.000
regarding Microbiological Testing. LABS is an ISO certified, FDA registered
testing facility
that assures that microbiological testing is performed using validated test
methods, validated
equipment and a staff of fully trained microbiologists.
[00141] Microbiological media used for the detection of aerobic, anaerobic
and fungi
organisms are chosen based on requirements in the CFR. Trypticase Soy Broth,
which is the
same as Soybean-Casein Digest Medium, and Fluid Thioglycollate Medium are used
for the
sterility cultures as prescribed. Trypticase Soy Broth is used for aerobic
organisms and for yeast
and mold detection. Fluid Thioglycollate Medium is used for culture of
anaerobes, facultative
anaerobes and for aerobic bacteria.
[00142] LABS purchases TSB and FTM from approved vendors and while the
standard
developed by the Clinical Laboratory Standards Institute does not require QA
testing on these
media if purchased commercially, LABS tests all media for its ability to
promote microbial
growth before it is used in sterility tests. The sterility culture incubation
period is 14 days per the
requirements in the CFR and in the USP. Cultures are read at 3, 4 or 5 days,
at 7 days and a final
reading at 14 days (Bacteria) or 28 days (Fungus). If the media is turbid at
any reading, the
microbiologist performs a gram stain and subculture as appropriate.
[00143] Giemsa Stain. Evaluation of DC morphology in the DC vaccine is
performed by
Giemsa staining of cytospun cells. Cells should exhibit dendritic cell
morphology. This is
considered a characterization assay, thus, the DC vaccine batches are released
on a report of the
assay results.
- 53 -
CA 02874065 2014-12-04
[00144] Cell Phenotype. The determination of the DC phenotype is performed
by
multiparameter flow cytometric analysis using the BD FACSCantOTM II Flow
Cytometry
System. An antibody panel has been established to determine the phenotype of
the DC vaccine
products. The DC vaccine phenotyping panel consists of the following
fluorescent-labeled
monoclonal antibodies: CD1b/c FITC, CD80 PE, HLA-DR PerCP, CD83 APC, CD14 APC-
H7
and CD11 c Horizon (Pacific Blue). The cells are also stained with anti-CD45
pacific orange
monoclonal antibody, which is used to gate the cells for FACS analysis. A
single tube of cells is
stained with the mixture of phenotyping antibodies and analyzed on the
FACSCanto. The data
are reported as the percentage of cells expressing a particular cell marker or
combination of cell
markers as detected by the fluorescent-labeled monoclonal antibodies in the
phenotyping panel.
For release of each frozen batch of BIIR-BrcaVax-001 DC vaccine product the
following cell
phenotype specifications must be achieved, i.e., for DC identity >80% of the
cells should be
HLA-DR+CD11c+ and for DC phenotypic potency >70% of the cells should be CD80+.
The
results are reported for the other cell phenotype markers analyzed to help
further characterize the
DC vaccine products.
D. Potency ¨ Characterization Assay
[00145] The potency assay for characterization of the BIIR-BrcaVax-001
breast cancer
therapeutic DC vaccine product is currently under development. In addition to
determining the
phenotypic potency of the DC vaccine by flow cytometry (see above) there is a
characterization
assay that will be performed to assess the functional potency of the DC
vaccine product. This
assay will measure by Luminex analysis the secretion of critical cytokines,
i.e., IL-12p40, IL-
12p70 and IL-23, by the DC. The antigen-loaded and activated DC are expected
to secrete
significant levels of these cytokines. In addition to IL-12p40/p70 and IL-23
other cytokines may
also be analyzed.
3. Vaccine administration and vaccine schedule
[00146] Patients will receive preoperative dose-dense dense AC/T
chemotherapy for 16
weeks combined with antigen-pulsed DC vaccinations administered on Day 2 of
Cycle 1 and 3
of dose-dense AC and on Day 3 of Cycle 1 and 3 of T (4 timepoints).
[00147] At each scheduled vaccination during the preoperative phase, the
patient will
receive a total of 2 injections. Each vaccination will consist of:
- 54 -
CA 02874065 2014-12-04
= One intratumoral injection of 0.2 mL (3 x 106 cells/mL)
= One subcutaneous injection of 1 mL (15 x 106 cells/mL) in the ventral
surface of the
upper arm (ipsilateral).
DC vaccinations will be administered to the patient prior to administering
AC/T on the given
day.
[00148] After definitive surgery and during locoregional radiation therapy
to breast or
chest wall and regional lymphatics per standard of care, patients will receive
3 boost DC
vaccinations subcutaneously of 1 mL each (15 x 106 cells/mL) in the ventral
surface of the upper
arm (contralateral).
= The first vaccination booster will occur once after the surgery and prior
to radiation.
= The second booster will occur 30 days 3 days after radiation is
completed.
= The third booster will occur 90 days 3 days after the 2nd boost.
Patients will be monitored post DC infusion for any signs of infusion related
reaction every 15
minutes for 1 hour.
[00149] The DC vaccination schedule is shown in FIG. 2.
4. Anakinra administration
[00150] Anakinra is a recombinant soluble non-glycosylated homolog of the
human
interleukin-1 receptor antagonist (IL-1Ra) that competitively inhibits binding
of IL-1a and IL-113
to the receptor type I. Anakinra differs from native human IL-1Ra in that it
has the addition of a
single methionine residue at its amino terminus. It is produced by recombinant
DNA technology
using an E coli bacterial expression system. Anakinra consists of 153 amino
acids and has a
molecular weight of 17.3 kilodaltons. Anakinra is supplied in single use
prefilled glass syringes
with 27 gauge needles as a sterile, clear, colorless-to-white, preservative-
free solution for daily
subcutaneous (SC) administration. The solution may contain trace amounts of
small, translucent-
to-white amorphous proteinaceous particles. Each prefilled glass syringe
contains: 0.67 mL (100
mg) of anakinra in a solution (pH 6.5) containing sodium citrate (1.29 mg),
sodium chloride
(5.48 mg), disodium EDTA (0.12 mg), and polysorbate 80 (0.70 mg) in Water for
Injection,
USP. Dosing and administration of anakinra will be as follows:
Group 1:
- 55 -
CA 02874065 2014-12-04
= DC vaccine plus preoperative chemotherapy, no treatment with anakinra for
16 weeks: 10
patients
Group 2:
= DC vaccine plus preoperative chemotherapy; anakinra 100 mg 7 days,
followed by 7 days
off, then repeating, subcutaneously for 16 weeks: 10 patients
[00151] Anakinra is self-administered, with the exception of first dose of
anakinra which
will be administered in the clinic. Before any anakinra is administered, each
patient will be
instructed by the Investigator or his/her representative on the proper self-
administration of study
drug and advised to take any missed dose as soon as possible.
[00152] Each patient registered in Group 2 only will be instructed to self-
inject
subcutaneously one pre-filled syringe of anakinra (100 mg) for 7 days,
followed by 7 days of
rest, then repeating, at about the same time every day during the 16 weeks of
preoperative
chemotherapy. Anakinra must be stored in a refrigerator and warmed to room
temperature for
60-90 minutes prior to injection. This dose was the recommended dose for the
treatment of
patients with rheumatoid arthritis. Higher doses did not result in a higher
response.
5. Apheresis
[00153] The use of apheresis for the collection of human blood mononuclear
cells is
commonly done in the practice of hematology and oncology. For venous access,
patients will
either undergo venipuncture of the antecubital veins in both arms or a central
venous catheter
will be inserted in those patients whose venous access is insufficient to
undergo apheresis by
venipuncture.
[00154] After the mononuclear cells are collected from the patients, and
received by the
cGMP Facility at BIIR they will undergo further processing for generation of
the DC vaccine.
Specifically the monocytes will be separated from other mononuclear cells
using a closed
elutriation system ELUTRA (Gambro).
6. Efficacy assessments: evaluation of pathologic response
[00155] Pathologic response to therapy is the primary endpoint of the study
protocol.
Patients will undergo surgical resection of residual breast and axillary
malignant tissue after
protocol-directed treatment. The pathologic specimen will be graded according
to the tumor
- 56 -
CA 02874065 2014-12-04
regression grading schema called the Residual Cancer Burden (RCB) (Symmans, et
al., 2007).
The following parameters are required from pathologic examination in order to
calculate RCB
after neoadjuvant treatment:
1. The largest two dimensions (mms) of the residual tumor bed in the breast
(largest
tumor bed if multicentric disease)
2. Submission of the entire largest cross-sectional area of the residual tumor
bed for
histologic mapping, with specific identification of those slides in the
pathology report
(e.g., "the largest cross-sectional area of primary tumor bed was submitted in
cassettes
A5 - A9")
= If the residual tumor is large (i.e. largest diameter > 5 cm), then at
least 5
representative cassettes from the largest cross-sectional area are sufficient,
but
should be identified in the original pathology report (e.g. "representative
sections
from the largest cross-sectional area of primary tumor bed were submitted in
cassettes A5 - A9")
3. Histologic assessment of the percentage of the tumor bed area that contains
carcinoma
(all carcinoma, i.e. invasive and in situ), select one of the following:
o 0%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
= To assess cellularity it is helpful to scan across the sections of tumor
bed and
then estimate the average cellularity from the different microscopic fields.
= When estimating percentage cancer cellularity in any microscopic field,
compare the involved area with obvious standards, e.g. more or less than half,
one
quarter, one fifth, one tenth, one twentieth, etc.
= Expect there to be variable cellularity within the cross section of any
tumor bed,
but estimate the overall cellularity from the average of the estimates in
different
microscopic fields of the tumor bed.
= e.g. if cellularity in different fields of the tumor bed were estimated
as 20%,
10%, 20%, 0%, 20%, 30%, then an average estimate of overall cellularity would
be 20%.
4. Histologic estimate of the percentage of the carcinoma in the tumor bed
that is in situ,
select one of the following:
o 0%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%
- 57 -
CA 02874065 2014-12-04
5. The number of positive (metastatic) lymph nodes
6. The largest diameter (mm) of the largest nodal metastasis
[00156] A pathologic complete response (pCR) is defined as NO pathologic
evidence of
invasive disease in the breast or axillary lymph nodes.
[00157] The presence or absence of a pCR will be assessed separately for
the tumor and
the lymph nodes. For patients who do not achieve a pCR, the size of the
residual cancer in the
tumor, on pathologic exam, will be documented in the as well as the number of
positive lymph
nodes. Patients will have their pathologic response scored using the RCB
scale.
7. Efficacy assessments: statistical methods
[00158] Immunologic studies will explore immune responses in the peripheral
blood and
the primary breast cancer tissues. Peripheral blood lymphocytes at each pre-
and post-
vaccination time-points will be analyzed by flow cytometry for immune
phenotyping and T cell
subtype quantification according to standardized protocols. Breast cancer
tissue analyses from
the baseline biopsy and from the residual tissue obtained at definitive
surgery will include:
qualitative assessment of immune cell subsets such as T effectors, Tregs, NK
cells, dendritic
cells, macrophage subsets, B cells and expression of immune checkpoint targets
such as PD-1
and PD-L1, and iTH2 cells. Blood and breast cancer tissue samples will be
analyzed by
transcriptional profiling for changes over time including in the BIIR-
described transcriptional IL-
1 signature. Quantitative and avidity evaluation of tumor-infiltrating and
peripheral blood cyclin
B1 and patient-specific mutation, antigen-specific T cells will be carried out
for patients who
received anakinra versus patients who did not receive anakinra. Findings will
be correlated with
clinical endpoints.
[00159] Statistical analysis of immunologic studies.. Continuous variables
will be
summarized with means or medians and standard deviations. Dichotomous and
categorical
variables will be summarized using counts and proportions with exact 95%
confidence intervals.
These summaries will be computed for each patient both pre- and post-
administration of each
DC vaccination. Plots will be used to show the changes in immune response over
time both for
each individual and for patients who received anakinra versus patients who did
not receive
anakinra. For each vaccination, comparisons in the pre- and 14-day post-
vaccine responses will
- 58 -
CA 02874065 2014-12-04
be compared using paired t-tests (or Wilcoxon signed rank tests, if
appropriate) for continuous
variables. McNemar's test will be used to identify significant changes in the
percentage of
individuals with a dichotomous characteristic pre- and post-vaccine.
Associations between
immune parameters will be explored graphically (e.g. scatter plots, box plots)
and numerically
(e.g., correlations, x2 tests). The relationships between the immune
parameters and clinical
outcomes (pCR rates and DFS) will be assessed using a variety of statistical
techniques.
Univariate and multivariate modeling will be used to quantify the associations
between immune
correlates, administration of anakinra versus no administration of anakinra,
and clinical
outcomes. In the case of a time-to-event clinical outcome (ie, DFS), the Cox
proportional
hazards model will be used. For binary and continuous outcomes, logistic and
linear regression
will be used. Kaplan-Meier techniques will be used to quantify time-t- event
outcomes (DFS)
and Cox proportional hazards models will be used to assess risk factors and
compare subgroups
of interest.
EXAMPLE 3
OPTIMIZATION OF THE FROZEN DC VACCINE WASHOUT PROTOCOL
[00160] Purpose: This study was conducted to optimize the procedure to
washout the
frozen Dendritic Cells (DC) from the freezing solution for resuspension of the
DC vaccine
product for injection.
1. Background
[00161] During the testing of the control batches for breast cancer
therapeutic DC vaccine
product, BIIR-BrcaVax-001, it was discovered that the washout procedure was
not optimal. The
viability of the washed out DC was lower than anticipated (See the first two
tables below). This
report summarizes the work done to improve cell viability and recovery during
the washout
procedure as outlined in SOP VP143 "Preparing Frozen DC Vaccines for
Injection".
[00162] Briefly, the original washout procedure is outlined below;
1. Thaw two vials of the frozen DC vaccine batch in normal saline and
transfer the
contents to sterile vacutainer tubes.
2. Centrifuge the cells in the vacutainer tubes at 270 xg for 8 minutes.
3. Wash the cells a total of 3 times in normal saline.
- 59 -
CA 02874065 2014-12-04
4. Prior to spinning down the cells in the third wash step take a sample for a
cell
count and viability determination, as well as assessment of sterility by the
endotoxin LAL assay and In-house gram stain assay.
5. After the third wash is completed, resuspend the cells in normal saline at
14x106
viable cells/mL and transfer 1 mL into a 3 mL syringe for subcutaneous
injection
of the patient.
6. Dilute the remaining cells in the vacutainer tube to 2x106 viable cells/mL
and
transfer 0.5 mL into a 1 mL syringe for intratumoral injection of the patient.
7. Send 2 mL of the remaining cell suspension to Bonfils for full sterility
testing,
i.e., gram stain, 14 day bacterial growth and 28 day fungal growth.
[00163] Following are the results of the testing of the first two BrcaVax-
001 control
batches. Presented in Table 6 and Table 7 below are the cell viability,
number, and sterility of
the DC vaccines at the time of the final washout step, and the stability of
the DC vaccine when
filled into the syringes at the cell concentrations planned for subcutaneous
and intratumoral
injection into the patient.
Table 6 Results of BrcaVax-001 control batch washout per following the
procedure
outlined in SOP VP143
BrcaVax-001 Pre-Wash 3t QC Testing
Control In-House
Batch* Viab Conc/mL LAL Gram Bonfils
CB1
60% 2.5x106/mL Negative Negative Negative
V#7 & V#10
CB2
73% 3.5x106/mL Negative Negative Negative
V#7 & V#22
*Two control batches, CB1 and CB2, were processed and analyzed. The vial
number (V#) processed
from each batch is listed.
tThe percentage of viable cells (Viab) and cell concentration (Conc/mL) in the
sample taken prior to the
third cell wash step are presented for both control batches.
- 60
CA 02874065 2014-12-04
Table 7. Stability of the BrcaVax-001 control batch DC vaccines in syringes:
Control 30 min/syringe t 1 HR/syringe 2 HR/Syringe
3 HR/Syringe
Batch* Viab Conc/mL Viab Conc/mL Viab Conc/mL
Viab Conc/mL
CB1
14x106/mL 27% 6.0x106/mL 46% 9.2x106/mL 21% 3.8x106/mL NT NT
CBI
2x106/mL 21% 0.7x106/mL 45% 1.1x106/mL 16% 0.6x106/mL NT NT
CB2
14x106/mL 56% 9.4x106/mL 65% 10.6x106/mL 62% 9.4x106/mL 55% 6.9x106/mL
CB2
2x106/mL 49% 1.2x106/mL 57% 1.4x106/mL 43% 0.9x106/mL 46% 0.9x106/mL
Note: Large clumps of dead cells were observed in all the syringes analyzed.
*BrcaVax-001 control batches CB1 and CB2 were filled into either a 3 mL
syringe at 14x106 viable
cells/mL or a 1 mL syringe at 2x106 viable cells/mL according to SOP VP143.
f The DC vaccine filled syringes were held at ambient room temperature and
sampled for analysis of cell
viability (Viab) and concentration (Conc/mL) at the specific time points (min
= minutes, and HR = hours).
NT indicates that the sample was not tested.
[00164] Conclusion: When following the original washout and resuspension
procedure,
the viability of the washed and resuspended DC is below the acceptable level
for clinical use.
Additionally, the DC vaccine is unstable in the syringes based on the low cell
viability and
concentrations observed at the different stability time-points.
2. Experiments
[00165] The following experiments were performed to determine what
conditions were
needed to optimize the viability of the washed out cells and secondly how to
incorporate those
conditions into a procedure appropriate for sterile washout and resuspension
of patient's DC
vaccine cells for injection.
[00166] Experiment 1. The first parameter changed from the VP143 procedure
was to
substitute conical tubes for vacutainer tubes for washing the cells and filter
the cell suspension
through a 40 micron cell filter to remove clumps prior to the cell count and
filling into the
syringes. The results of this procedure are presented in Table 8 and Table 9
below for first the
cell viability and concentration in the sample taken prior to the third wash
step and second for
the stability of the DC vaccine suspension in the syringes. In this
experiment, only a single
control batch (CBI) was tested.
- 61 -
CA 02874065 2014-12-04
Table 8
BrcaVax-001 Pre-Wash 3
Control Batch Viab Conc/mL
CB1
68% 2.5x106/mL
V#5 & V#I2
Table 9
Control 30 min/syringe 1 HR/syringe
Batch Viab Conc/mL Viab Conc/mL
CBI 53% 12.4x106/mL 41% 9.6x106/mL
14x106/mL
CBI 38% 1.0x106/mL 41% 1.0x106/mL
2x106/mL
Note: Large clumps of dead cells were observed in all the syringes analyzed.
[00167] Experiment 2. The second parameter changed from the VP143 procedure
was to
substitute conical tubes for vacutainer tubes for washing the cells and to
wash the cells in normal
saline containing 50% Heat Inactivated Human AB serum (HIABS) (volume/volume
of the
formula mix). The results of this procedure are presented in Table 10and Table
11 below for
first the cell viability and concentration in the sample taken prior to the
third wash step and
second for the stability of the DC vaccine suspension in the syringes. In this
experiment, only a
single control batch (CB3) was tested.
Table 10
BrcaVax-001 Pre-Wash 3 QC Testing
In-House
Control Batch Viab Conc/mL LAL Gram Bonfils
CB3 79% 6.1x106/mL Negative Negative Negative
V#3 & V#10
Table 11
Control 30 min/syringe 1 HR/syringe 2 HR/Syringe
3 HR/Syringe
Batch Viab Conc/mL Viab Conc/mL Viab Conc/mL
Viab Conc/mL
CB3
14x106/mL 80% 8.1x106/mL 72% 6.8x106/mL 78% 3.8x106/mL 85% 1.6x106/mL
CB3
2x106/mL 78% 0.8x106/mL 73% 0.5x106/mL 62% 0.4x106/mL 44% 0.8x106/mL
Note: Large clumps of cells were observed in all the syringes analyzed.
- 62 -
CA 02874065 2014-12-04
[00168] Conclusion: Adding HIABS to the saline greatly improved the cell
viability after
wash, resuspension and stability of the DC vaccine in syringes over 3 hours.
The viability of the
cells was more stable but there was a significant loss in cell numbers in the
syringes over time.
The cells were less stable in the syringe at the lower concentration of
2x106/mL and there was no
reduction in cell clumping in the syringes.
[00169] Experiment 3. The next modification of the procedure was to wash
the cells in
vacutainer tubes comparing normal saline versus normal saline + 10% HIABS as
the wash
solution. The centrifugal force to wash the cells was reduced from 270 xg to
110 xg. The results
of this procedure are presented in Table 12 and Table 13 below for first the
cell viability and
concentration in the sample taken prior to the third wash step and second for
the stability of the
DC vaccine suspension in the syringes. In this experiment, only a single
control batch (CB3)
was tested.
Table 12
Pre-Wash 3 Normal Saline
Pre-Wash 3 Normal Saline + 10% HIABS
BrcaVax-001 V#16 & V#18 V#11 &V#20
Control Batch Viab Conc/mL Viab
Conc/mL
CB3 74% 2.7x106/mL 89%
4.3x106/mL
Table 13
Control 1 HR/syringe 2 HR/Syringe 3 HR/Syringe
Batch Viab Conc/mL Viab Conc/mL Viab Conc/mL
CB3 NaCI
14x106/mL 62% 11.3x106/mL 36% 7.8x106/mL NT NT
CB3 NaCI
2x106/mL 49% 1.5x106/mL 22% 0.4x106/mL NT NT
CB3 NaCI +
10% HIABS
14x106/mL 87% 6.4x106/mL 82% 5.1x106/mL 88% 7.0x106/mL
CB3 NaCI +
10% HIABS
2x106/mL 86% 0.7x106/mL 75% 0.6x106/mL 79% 0.4x106/mL
Note: Small clumps of dead cells observed at 1 HR and larger clumps at 2 HR in
all the syringes
analyzed.
[00170] Conclusion: The cell viability was higher and more stable in
preparations made
with normal saline + 10% HIABS and there were fewer cell clumps, but there was
still a loss of
- 63 -
CA 02874065 2014-12-04
cells over time. As seen in previous experiments, the viability of the cells
washed in normal
saline alone was unacceptable.
[00171] Experiment 4. When the vacutainer washout procedure was originally
developed,
it was determined that lower centrifugation speed of 110 xg was the best
compromise between
recovery and increased viability. The speed was increased to 270 xg to
maximize cell recovery,
as viability was not expected to be an issue. Since cell numbers were
comparable between the
two speeds and cell viability was higher at lower speed, it was decided to
continue all further
experiments with centrifugation speeds set lower at 110 xg.
[00172] The next modification to the procedure that was tested was to wash
the cells in
conical tubes with normal saline + 10% HIABS, but after the second wash, the
cells were
incubated at ambient room temperature for 1 HR. The cells were then filtered
through a 40
micron cell filter to remove cell clumps before counting the cells prior to
the third wash step.
The results of this procedure are presented in the Table 14, Table 15, and
Table 16 below for first
the cell viability and concentration in the sample taken prior to the third
wash step and second
for the stability of the DC vaccine suspension in the syringes. In this
experiment, only a single
control batch (CB2) was tested.
Table 14
BrcaVax-001 Filtered/Pre-Wash 3 Normal Saline + 10% HIABS
Control Batch Viab Conc/mL
CB2
V#11 & V#18 89% 3.8x106/mL
Table 15
Control 0 HR/syringe 1 HR/Syringe 2 HR/Syringe 3
HR/Syringe
Batch Viab Conc/mL Viab Conc/mL Viab Conc/mL
Viab Conc/mL
CB2 NaCI +
10% HIABS
14x106/mL 90% 12.6x106/mL 88% 10.9x106/mL 90% *21.1x106/mL 91% "1.1x106/mL
CB2 NaCI +
10% HIABS
2x106/mL 89% 1.9x106/mL 81% 1.5x106/mL 86% 1.3x106/mL 87% 1.8x106/mL
*Note; at 2 HR half of the 14x106/mL syringe was sampled and a large clump
came out with the
sample. After the 3 HR time point sample was taken and analyzed the 2 HR and 3
HR samples
were combined, mixed and recounted as presented in the table below.
- 64 -
CA 02874065 2014-12-04
Table 16
2 HR and 3 HR samples combined/14x106/mL Syringe
Viab Conc/mL
92% 10.9x106/mL
[00173] Conclusion: By this procedure the cell viability was higher and
more stable over
time compared to the previous methods; however, there a significant cell loss,
based on lower
cell concentration levels, over time for cell suspensions held in the
syringes.
[00174] Experiment 5. Since it was unlikely that HIABS was an acceptable
formulation
component for preparing the vaccine product for injection into patients, the
next experiment was
conducted where the HIABS was replaced with Heat Inactivated Autologous Serum
(HIAS). In
this experiment the cells were washed in conical tubes with normal saline + 2%
HIAS. After the
cells were washed and resuspended, they were filled into glass vaccine vials,
instead of a
syringes, at concentrations of 14x106 and 2x106 viable cells/mL. The results
of this procedure
are presented in Table 17 and Table 18 below for first the cell viability and
concentration in the
sample taken prior to the third wash step and second the stability of the DC
vaccine suspensions
in the glass vaccine vials. In this experiment, only a single control batch
(CB2) was tested.
Table 17
BrcaVax-001 Pre-Wash 3 Normal Saline +
2% Autologous Serum
Control Batch Viab Conc/mL
CB2
79% 5.1x106/mL
V#9 & V#20
Table 18
Control 0 HR/vial 1 HR/Vial 2 HR/Vial 3 HR/Vial
Batch Viab Conc/mL Viab Conc/mL Viab
Conc/mL Viab Conc/mL
CB2 NaCI +
2% HIAS
14x106/mL 75% 10.8x106/mL 75% 8.8x106/mL 62% 5.9x106/mL 45% 3.7x106/mL
CB2 NaCI +
2% HIAS
2x106/mL 75% 1.4x106/mL 51% 1.0x106/mL 62%
1.0x106/mL 43% 0.3 x106/mL
[00175] Conclusion: The cell viability after washing and resuspension in
normal saline +
2% HIAS was acceptable, but the cells were not stable beyond 1 hour in the
glass vaccine vial.
When suspended in normal saline + 2% HIAS and filled in glass vaccine vials
the viability and
- 65 -
CA 02874065 2014-12-04
cell concentration decreased over time. The cells at the lower concentration
of 2x106/mL were
less stable than the more concentrated sample of 14x106/mL.
[00176] Experiment 6. The previous experiment was repeated using a higher
concentration of HIAS in the normal saline. In this experiment the cells were
washed in conical
tubes in normal saline + 5% HIAS. The cell suspensions, at the two different
cell concentrations,
were filled into glass vaccine vials for stability assessment. The results of
this procedure are
presented in Table 19 and Table 20 below for first the cell viability and
concentration in the
sample taken prior to the third wash step and second for the stability of the
DC vaccine
suspension in the glass vaccine vials. In this experiment only a single
control batch (CB2) was
tested.
Table 19
BrcaVax-001 Pre-Wash 3 Normal Saline + 5% Autologous Serum
Control Batch Viab Conc/mL
CB2
V#10 & v#23 73% 3.5x106/mL
Table 20
Control 0 HR/vial 1 HR/Vial 2 HRNial 3 HRNial
Batch Viab Conc/mL Viab Conc/mL Viab Conc/mL
Viab Conc/mL
CB2 NaCI +
5% HIAS 77% 14.4x106/mL 73% 11.4x106/mL 70% 8.6x106/mL 56%
4.6x106/mL
14x106/mL
CB2 NaCI +
5% HIAS 72% 1.9x106/mL 64% 2.0x106/mL 73%
1.4x106/mL 65% 0.6x106/mL
2x106/mL
Note: No cell clumps were observed in the cell suspensions in glass vaccine
vials.
[00177] Conclusion: Under these conditions the cells were slightly more
stable in normal
saline containing the higher concentration of HIAS compared to that in the
previous experiment.
However, based on the loss of cell number over time this process does not
appear to give
adequate stability to the DC vaccine product.
[00178] Experiment 7. The experiment was repeated, again increasing the
concentration
of HIAS in the normal saline used to wash and resuspend the cells in the DC
vaccine. In this
experiment the cells were washed in conical tubes with normal saline + 10%
HIAS. The two
concentrations of cell suspension were filled into glass vaccine vials for
stability assessment.
The results of this procedure are presented in Tables 21 and 22 below for
first the cell viability
- 66 -
CA 02874065 2014-12-04
and concentration in the sample taken prior to the third wash step and second
for the stability of
the DC vaccine suspensions in the glass vaccine vials. In this experiment only
a single control
batch (CB4) was tested.
Table 21
BrcaVax-001 Pre-Wash 3 Normal Saline + 10% HIABS
Control Batch Viab Conc/mL
CB4
V#5 & v#6 78% 4.6x106/mL
Table 22
Control 0 HR/vial 1 HRNial 2 HRNial 3 HRNial
Batch Viab Conc/mL Viab Conc/mL Viab Conc/mL
Viab Conc/mL
CB4 NaCI +
10% HIAS
14x106/mL 71% 8.5x106/mL 70% 5.9x106/mL 74% 4.3x106/mL 77% 4.0x106/mL
CB4 NaCI +
10% HIAS
2x106/mL 73% 1.3x106/mL 70% 0.8x106/mL 74% 0.8x106/mL 65% 0.8x106/mL
Note: No cell clumps were observed in the cell suspensions in glass vaccine
vials.
[00179] Conclusion: This change did not improve DC vaccine stability,
based on cell
viability and concentration, beyond that observed in the previous experiment.
While both heat
inactivated AB serum and autologous serum improved viability of the DC vaccine
above that
achieved with normal saline alone, after discussion it was decided that adding
serum to the
formulation of the DC vaccine product is not a viable option for the clinical
setting. An
alternative solution to using serum was thus investigated. In the next set of
experiments,
different formulation options were evaluated for washing and resuspending the
DC vaccines for
injection. It should be noted that in the rest of the experiments presented in
this report all the cell
wash steps were performed in vacutainer tubes.
[00180] Experiment 8. In this experiment the cells were washed with either
normal saline
+ 10% HIAS or Plasma-Lyte, which is an injectable electrolyte replacement.
Following the
wash and resuspension process the cells, at two different concentrations, were
filled into glass
vaccine vials for stability assessment. The results of this procedure are
presented in Table 23
and Table 24 below for first the cell viability and concentration in the
sample taken prior to the
third wash step and second for the stability of the DC vaccine suspension in
the glass vaccine
vials. In this experiment only a single control batch (CB2) was tested.
- 67 -
CA 02874065 2014-12-04
Table 23
Pre-Wash 3 PlasmaLyte
Pre-Wash 3 Normal Saline + 10% HIABS
BrcaVax-001 V#14 V#24
Control Batch Viab Conc/mL Viab
Conc/mL
CB2 54% 0.9x106/mL 84% 2.7x106/mL
Table 24
Control OHR/vial 1HRNial 2HRNial 3HRA/ial
Batch Viab Conc/mL Viab Conc/mL Viab
Conc/mL Viab Conc/mL
CB2 NaCI +
10% HIAS
14x106/mL 71% 5.0x106/mL 70% 2.8x106/mL 72% 1.7x106/mL 71% 0.9x106/mL
CB2 NaCI +
10% HIAS
2x106/mL 64%
1.2x106/mL 74% 0.8x106/mL = 67% 0.6x106/mL 68% 0.3x106/mL
CB2
Plasma-Lyte
14x106/mL 45% 10.8x106/mL NT NT NT NT NT NT
CB2
Plasma-Lyte
2x106/mL 36% 1.3x106/mL NT NT NT NT NT NT
Note: No cell clumps were observed in the cell suspensions in glass vaccine
vials.
[00181] Conclusion: The results of this experiment clearly demonstrate
that the cells were
more stable, based on cell viability, when washed and resuspended in normal
saline +10% HIAS
compared to Plasma-Lyte. Clearly, with regard to cell viability, cell number
and stability it is
unacceptable to use Plasma-Lyte in this process. At this point in the study,
due to the limited
availability of autologous serum for the current control batches, future
experiments did not use
serum to compare to other formulation alternatives for the washout and
resuspension solution.
[00182] Experiment 9. In this experiment the cells were washed with normal
saline + 1%
Dextrose. Cell Viability and cell numbers were compared after 1, 2 and 3
washes. Stability
analysis was conducted on cell suspensions, at two different concentrations,
filled into glass
vaccine vials. The results of this procedure are presented in Table 25 and
Table 26 below for
first the cell viability and concentration in the samples taken prior to each
of the wash steps and
second for the stability of the DC vaccine suspension in glass vaccine vials.
In this experiment
only a single control batch (CB2) was tested.
- 68 -
CA 02874065 2014-12-04
Table 25
BrcaVax-001 Normal Saline + 1% Dextrose
Control Batch Viab Conc/mL
CB2 V#13 82% 3.1x106/mL
Pre¨Wash 1
CB2 V#3 62% 1.3x106/mL
Pre¨Wash 2
CB2 V#17
54% 1.3x106/mL
Pre¨Wash 3
Table 26
Control 15 Min/vial 30 Min /Vial 45
MinNial 1 HR/Vial
Batch Viab Conc/mL Viab Conc/mL Viab Conc/mL
Viab Conc/mL
1 wash
14x106/mL 71% 9.2x106/m L 77% 7.2x106/m L 60%
7.6x106/mL 64% 6.2x106/mL
1 wash
70% 1.1x106/mL 54% 0.8x106/mL 61% 0.6x106/mL 60% 0.9x106/mL
2x106/mL
2 washses 61% 11.2x106/mL 58% 9.8x106/mL 51%
8.4x106/mL 45% 7.7x106/mL
14x10 /nnL
2 washes
45% 0.8x106/mL 48% 1.1 x106/mL 29%
0.6x106/mL 19% 0.4x106/mL
2x106/mL
3 washes
14x106/mL 68% 10.0x106/mL 52% 9.2x106/mL 68% 11.8x106/mL 60% 11.7x106/mL
3 washes 56% 1.1x106/mL 49% 1.0x106/mL 62%
1.3x106/mL 51% 0.8x106/nnL
2x106/mL
Note: No cell clumps were observed in the cell suspensions in glass vaccine
vials.
[00183] Conclusion: For cells washed and suspended in normal saline + 1%
dextrose the
cell viability and cell numbers were acceptable prior to the first wash but
cell viability and
numbers declined significantly with each additional wash step. A single wash
will not be
acceptable because a minimum of two washes are required to wash and combine 2
frozen DC
vaccine vials for processing the final vaccine product for injection.
Therefore, all future
experiments were conducted with a minimum of two washes.
[00184] Experiment 10: This experiment was a repeat of the process
followed in
experiment 9 above. It was done to assess a 10-fold higher concentration of
dextrose with two
washes using 2 frozen vaccine vials. The cells were washed with normal saline
+ 10% Dextrose.
Stability testing was conducted on cell suspensions filled into glass vaccine
vials. The results of
- 69
CA 02874065 2014-12-04
this procedure are presented in Table 27 and Table 28 below for first the cell
viability and
concentration in the sample taken prior to the second wash step and second for
the stability of the
DC vaccine suspensions, at two concentrations, in glass vaccine vials. In this
experiment only a
single control batch (CB2) was tested.
Table 27
BrcaVax-001 Pre-Wash 2 Normal Saline + 10% Dextrose
Control Batch Viab Conc/mL
CB2
V#4 & v#6 57% 2.4x106/mL
Table 28
Control 15 Min/vial 30 Min/Vial
Batch Viab Conc/mL Viab Conc/mL
CB2 NaCI +
10%
Dextrose
14x106/mL 51% 11.6x106/mL 44% 7.1x106/mL
CB2 NaCI +
10%
Dextrose
2x106/mL 42% 0.7x106/mL 35% 0.5x106/mL
Note: No cell clumps were observed in the cell suspensions in glass vaccine
vials.
[00185] Conclusion: Based on the low cell viability normal saline plus 10%
Dextrose is
considered to be unacceptable for formulating the DC vaccine product for
injection.
[00186] Experiment 11. In this experiment, the cells were washed with
normal saline +
5% Dextrose + 1% amino acid solution (AA). Results are in Table 29 below.
Because the cell
viability was so low after the first wash (see results in table below) it was
decided to not pursue
the DC vaccine stability assessment using this formulation.
Table 29
BrcaVax-001 Pre-Wash 2 Normal Saline + 5% Dextrose + 1% AA
Control Batch Viab Conc/mL
CB2 24% 2.1x106/mL
V#2 & v#16
[00187] Conclusion: The wash solution consisting of normal saline/5%
dextrose/1% AA
was considered unacceptable for processing the DC vaccine for injection. It
should be noted;
- 70 -
CA 02874065 2014-12-04
however, that the failure of this formulation could be due to the low pH of
the solution, since the
pH of the NaCI + 5% Dex + 1% amino acid solution was found to be 4.0-4.5 using
pH test strips.
[00188] Experiment 12. In this experiment, the cells were washed with IX
Phosphate
Buffered Saline (PBS). Three washes were preformed with a cell count performed
prior to the
second and third washes. ln Table 30 below the cell viability and number is
presented for
sample analyzed prior to the initiation of the second and third wash steps.
Stability testing of the
DC vaccine was not performed using PBS to resuspend the cells.
Table 30
BrcaVax-001 1X PBS
Control Batch Viab Conc/mL
CB2 V#5
70% 1.9x106/mL
Pre-Wash 2
CB2 V#5
50% 1.5x106/mL
Pre-Wash 3
[00189] Conclusion: 1X PBS offers no additional benefit over normal saline
as the wash
buffer for preparing the DC vaccine for injection.
[00190] Experiment 13. In the next experiment fresh DC vaccine prepared
after harvest
of the cells from the culture bags was suspended at three different
concentrations in normal
saline and filled into syringes for stability testing (see results in Table 31
below). This
experiment was conducted with a single control batch of DC vaccine (CB6).
Table 31
Control 1 HR/syringe 2 HR/syringe
Batch 6 Viab Conc/mL Viab Conc/mL
5x106/mL 81% 5.4x106/mL 91% 4.0x106/mL
10x106/mL 93% 15.5x106/mL 89% 9.3x106/mL
15x106/mL 89% 16.3x106/mL 89% 14.6x106/mL
[00191] Experiment 39. Experiment 13 was repeated using Lactated Ringer's
to wash and
resuspend the cells for stability testing. Cell suspensions, prepared with
cells from control batch
7, were filled into a glass vaccine vials for stability testing (see results
in Table 32 below).
- 71 -
CA 02874065 2014-12-04
Table 32
Control 1 HRNial 2 HRNial 64 HRNial
Batch 7 Viab Conc/mL Viab Conc/mL Viab Conc/mL
Sample 1 93% 3.8x106/mL 89% 2.7x106/mL NT NT
Sample 2 94% 4.3x106/mL 92% 4.2x106/mL NT NT
Sample 3 96% 5.2x106/mL 91% 3.8x106/mL 88% 2.4 x106/mL
[00192] Experiment 15. In this experiment, a batch of fresh DC vaccine was
prepared
with cells harvested in either normal saline or Lactated Ringer's. Samples of
the fresh DC
vaccine preparations were removed and filled in glass vaccine vials for
stability assessment (see
Table 33 below). The remainder of the batch was frozen in glass vaccine vials
per the standard
protocol.
Table 33
Control 0 HRNial 2 HRNial 3 HR/Vial 4 HRNial
19.5 HR/Vial
Batch 8
Fresh Viab Conc/mL Viab Conc/mL Viab Conc/mL
DC Viab Conc/mL Viab Conc/mL
Harvest
Normal 91% 4.2x106/mL 83% 2.6x106/mL 77% 2.6x106/mL 75% 1.9x106/mL 27%
0.8x106/mL
Saline
Harvest
Ringers 90% 1.8x106/mL 84% 1.1x106/mL 82% 1.1x106/mL 74% 0.9x106/mL 81%
1.1x106/mL
Note; Initial cell concentrations of the cell suspensions were different in
the two formulation samples as
shown at time zero in the table.
[00193] Conclusion: The cells harvested in the Lactated Ringer's solution
are more stable
than those processed in normal saline. There is much less cell loss over
time in Lactated
Ringer's suspensions compared to that in normal saline and after 19 hours the
cells suspended in
Lactated Ringer's were still very viable compared to those in normal saline
which were mostly
dead.
[00194] Compared to the reprocessed, frozen DC vaccines analyzed above, the
fresh DC
vaccine preparations were more stable, displaying both higher cell viability
and cell numbers
over time. While this is encouraging, it should be cautioned that
manufacturing fresh vaccine
batches for the proposed cancer therapy clinical trials is not feasible.
Therefore, the efforts were
put back into improving the process for preparing the frozen DC vaccines for
injection.
[00195] Experiment 16. In the next experiment the cells were washed with
either normal
saline or Lactated Ringer's. For this experiment USP injectable Lactated
Ringer's was tested as
- 72 -
CA 02874065 2014-12-04
a clinically acceptable formula for thawing, washing and resuspending the DC
vaccines prior to
injection. The cell suspensions were filled in either syringes or glass
vaccine vials for stability
testing. The results of this procedure are presented in Table 34 and Table 35
below for first the
cell viability and concentration in the sample taken prior to the second wash
step and second for
the stability of the DC vaccine suspensions in glass vaccine vials and
syringes. In this
experiment only a single control batch (CB6) was tested.
Table 34
BrcaVax-001 Pre-Wash 2 Normal Saline V#11 Pre-
Wash 2 Lactated Ringer's v#4
Control Batch Viab Conc/mL Viab Conc/mL
CB6 50% 1.6x 106/mL 65% 2.7x 106/mL
Table 35
Control 1 HR 2 HR 3 HR
Batch 6 Viab Conc/mL Viab Conc/mL Viab Conc/mL
NaCI
8x106/mL
Vial 63% 11.9x106/mL 40% 6.0x106/mL 55% 9.3x106/mL
NaCI
8x106/mL
Syringe 71% 10.4x106/mL 63% 11.1x106/mL 61% 9.4x106/mL
Lactated
Ringer's
10x106/mL
Vial 75% 6.7x106/mL 69% 8.7x106/mL 66% 7.7x106/mL
Lactated
Ringer's
10x106/mL
Syringe 75% 8.7x106/mL 50% 3.2x106/mL 67% 7.3x106/mL
[00196] Conclusion: Cells washed and resuspended in Lactated Ringer's had
slightly
higher viability compared to those processed in normal saline. Regardless
of the
wash/suspension media the cell numbers and viability of the cells suspensions
were comparable
between syringes and vials.
[00197] Experiment 17. In a follow up experiment, the cells were washed
and
resuspended with Lacated Ringer's and filled in either a syringe or a glass
vaccine vial for
stability testing. The results of this procedure are presented in Table 36 and
Table 37 below for
first the cell viability and concentration in the sample taken prior to the
second wash step and
- 73 -
CA 02874065 2014-12-04
second for the stability of the DC vaccine suspensions in syringes and glass
vaccine vials. In this
experiment only a single control batch (CB6) was tested.
Table 36
BrcaVax-001 Pre-Wash 2 Lactated Ringer's
Control Batch Viab Conc/mL
CB6
V#2 & v#6 83% 5.6x106/mL
Table 37
Control 1 HR 2 HR 3 HR
Batch 6 Viab Conc/mL Viab Conc/mL Viab Conc/mL
Lactated
Ringer's
8x106/mL
Vial 78% 7.7x106/mL 79% 6.2x106/nnL 62% 4.5x106/mL
Lactated
Ringer's
8x106/mL
Syringe 76% 7.2x106/mL 71% 5.6x106/mL 66% 4.0x106/mL
[00198] Conclusion: In Lactated Ringer's solution, the DC had higher cell
viability and
were more stable. The stability of the cell suspensions in syringes and vials
were comparable.
The benefit of using the glass vaccine vials over the syringes is that the
vial is a more secure way
to transport the cells to the clinic as a syringe has a plunger that could be
bumped or otherwise
depressed and result in a loss of volume in the syringe.
[00199] Experiment 18. In this experiment, cells were washed and
resuspended in either
Lactated Ringer's or normal saline. Stability was conducted on cell
suspensions filled into glass
vaccine vials. The results of this procedure are presented in Table 38 and
Table 39below for first
the cell viability and concentration in the sample taken prior to the second
wash step and second
for the stability of the DC vaccine suspension in the glass vaccine vials. In
this experiment only
a single control batch (CB6) was tested.
Table 38
BrcaVax-001 Pre-Wash 2 Normal Saline V#7 & V#9
Pre-Wash 2 Lactated Ringer's V#1 & V#15
Control Batch Viab Conc/mL Viab
Conc/mL
CB6 80% 4.5x106/mL 81%
6.3x106/mL
- 74 -
CA 02874065 2014-12-04
Table 39
Control 1 HRNial 2 HRNial 3 HR/vial
Batch 6 Viab Conc/mL Viab Conc/mL Viab Conc/mL
NaCI
14x106/mL 65% 12.5x106/mL 56% 9.8x106/mL 50% 8.4x106/m L
NaCI
2x106/mL 56% 1.4x106/mL 42% 1.1x106/mL 49% 1.2x106/mL
Lactated
Ringer's
14x106/mL 78% 8.4x106/mL 80% 9.7x106/mL 74% 10.7x106/mL
Lactated
Ringer's
2x106/mL 78% 1.4x106/mL 73% 1.3x106/mL 72% 1.1x106/mL
[00200] Conclusion: Cell viability and cell number stability in the glass
vaccine vial was
higher for the cell suspension prepared in Lactated Ringer's than in normal
saline.
[00201] It should be noted that when Vial #1 and Vial #15 (that is, vials
thawed for
washing the cells in Lactated Ringer's) were combined and sampled for a cell
count and
viability, a sample was taken for an in-house gram stain assay and a PTS LAL
Assays. Both
assays were negative for microbial contamination.
[00202] Experiment 19. A different control batch of DC vaccine (CB7) was
used to
repeat a portion of experiment 17 above. That is, the cells were washed and
resuspended with
Lactated Ringer's. The cell suspension at two concentrations was filled into
glass vaccine vials
for stability testing. The results of this procedure are presented in Table 40
and Table 41 below
for first the cell viability and concentration in the sample taken prior to
the second wash step and
second for the stability of the DC vaccine suspensions in glass vaccine vials.
In this experiment
only a single control batch (CB7) was tested.
Table 40
BrcaVax-001 Pre-Wash 2 Lactated Ringer's
Control Batch Viab Conc/mL
CB7 68% 3.5x106
V#1 &V#8 /mL
- 75 -
CA 02874065 2014-12-04
Table 41
Control 1 HRNial 2 HR/Vial 3 HR/vial
Batch 7 Viab Conc/mL Viab Conc/mL Viab Conc/mL
Lactated
Ringer's 64% 10.3x106/mL 65% 13.0x106/mL 59% 13.0x106/mL
14x106/mL
Lactated
Ringer's 63% 1.4x106/mL 58% 1.3x106/mL 57% 1.1x106/mL
2x106/mL
[00203] Conclusion: Despite the overall low starting cell viability of this
control batch, its
stability in the glass vaccine vial was encouraging, especially for cells
suspended at the higher
concentration. In this experiment both the cell viability and number were
stable for about 3
hours in the cell suspension prepared at a concentration of 14x106 viable
cells/mL.
[00204] As in experiment 17, when Vial #1 and Vial #8 were thawed, washed
and
combined, and sampled for a cell count and viability, a sample was taken for
an in-house gram
stain assay and a PTS LAL Assays. Both assays were negative for microbial
contamination.
[00205] Experiment 20. The frozen vaccine vials from batch 8 processed in
experiment
15 were thawed and washed in Lactated Ringer's. Two conditions were tested,
with the first
being the frozen vials that were harvested by washing in normal saline and the
second being the
frozen vaccine that was harvested and washed in Lactated Ringer's prior to
freezing in vaccine
vials. After washing and resuspending the cells in Lactated Ringer's the cell
suspension at
15x106 viable cells/mL was filled into glass vaccine vials for stability
testing. The results of this
experiment are presented in Table 42 and Table 43 below for first the cell
viability and
concentration in the sample taken prior to the second wash step and second for
the stability of the
DC vaccine suspension in glass vaccine vials. In this experiment only a single
control batch
(CB8) was tested.
Table 42
Pre-Wash 2 Harvest in Normal Saline Pre-Wash 2 Harvest in Lactated Ringer's
BrcaVax-001 V#20 & V#21 V#2 & V#9
Control Batch Viab Conc/mL Viab
Conc/mL
CB8 83% 4.8x106/mL 90%
2.6x106/mL
- 76 -
CA 02874065 2014-12-04
Table 43
Control 1 HRNial 2 HRNial 3 HRNial 4 HRNial
Batch 8
Washed Viab Conc/mL Viab Conc/mL
DC Viab Conc/mL Viab Conc/mL
Harvest
Normal 73% 15.5x106/mL 74% 7.2x106/mL 65% 6.9x106/mL 50% 4.7x106/mL
Saline
Harvest
Lactated 78% 21.0x106/mL 68% 13.7x106/mL 64% 12.2x106/mL 57% 9.6x106/mL
Ringer's
[00206] Conclusion: Comparable stability of the cell suspensions in
Lactated Ringer's was
observed, regardless of whether the cells were harvested and washed prior to
freezing in normal
saline or Lactated Ringer's. While the cell viability was comparable at the
different stability test
time-points, the cell number appeared to be more stable for cell suspension
that was prepared
with cells that had been harvested in Lactated Ringer's.
[00207] Both conditions were tested for an in-house gram stain assay and a
PTS LAL
Assays. Both samples tested negative for endotoxin by LAL and negative for
organisms in the
in-house gram stain.
[00208] Experiment 21. In the next set of experiments, we used the
conditions that are
believed to be the best way to optimize the current washout procedure in a
clinically appropriate
way to test control batches 1 through 4. The frozen DC vaccines were thawed,
transferred into
vacutainer tubes and washed twice in Lactated Ringer's solution. The cells
were suspended in
Lactated Ringer's at 15x106 viable cells/mL and filled into glass vaccine
vials for stability
testing. The results of this procedure are presented in Table 44 and Table 45
below for first the
cell viability and concentration in the sample taken prior to the second wash
step and second for
the stability of the DC vaccine suspensions in the glass vaccine vials. The
samples taken prior to
the second wash step were also analyzed for the presence of endotoxin and gram-
positive
organisms.
- 77 -
CA 02874065 2014-12-04
Table 44
Pre-Wash 2
BrcaVax-001 Lactated Ringers QC Testing
In-House
Control Batch Viab Conc/mL LAL Gram
CB 1 75% 4.3x106/mL Negative Negative
V3 & V11
CB 2
85% 5.3x106/mL Negative Negative
V1 & V12
V5 &
CB V13 3
80% 3.8x106/mL Negative Negative
CB 4
V4 & V9 70% 5.5x106/mL Negative Negative
Table 45
Ringers 1 HRNial 2 HRNial 3 HRNial 4 HR/Vial
15x106/mL
Vial Viab Conc/mL Viab Conc/mL Viab Conc/mL Viab Conc/mL
CB1
V3 &V11 65% 9x106/mL 76% 10.7x106/mL 78% 11.2x106/mL 62% 7.6x106/mL
CB2
V1 &V12 79% 10.3x106/mL 79% 9.9x106/mL 75%
9.6x106/mL 63% 8.3x106/mL
CB3
V5 & V13 82% 10.5x106/mL 68% 9.5x106/mL 79% 8.4x106/mL 74% 8.8x106/mL
CB4
V4 & V9 77% 10.2x106/mL 77% 10.7x106/mL 77%
9.5x106/mL 69% 9.0x106/mL
[00209] Conclusion: The viability and cell number are acceptable when the
DC vaccine
cells are washed twice in vacutainer tubes with Lactated Ringer's solution.
The viability of the
cells in the glass vaccine vials over time support a 2 HR expiration time
(from the time the cells
are put into a sterile glass vaccine vial at a concentration of 15x106 viable
cells/mL).
[00210] Comparing the results of Experiment 21 in Table 44 and Table 45
with the results
from the original washout protocol in Table 6 and Table 7, it is clear that
washing twice in
vacutainer tubes, at slower speeds with Lactated Ringer's and filling the DC
vaccine at a higher
dose into a sterile glass vaccine vial improves the cell viability and cell
number stability of the
vaccine product.
3. Summary
- 78 -
CA 02874065 2014-12-04
[00211] The experiments done in this study support a number of conclusions,
which
include:
= DC vaccine cell viability and recovery is improved when the vaccine
product is prepared
at higher cell concentrations.
= DC vaccine cell viability and recovery is increased and cell clumping is
decreased when
the cells suspension is filled into glass vaccine vials rather than syringes.
= Glass vaccine vials is a more secure way to transport the cell suspension
of the vaccine
product to the clinic and provides more flexibility in dosing the patient.
= When Lactated Ringer's solution is used to prepare the vaccine product
for injection it
increases the cell viability and stability of the cells over time compared to
normal saline.
[00212] Recommendation: From these conclusions, it was decided to revise
the original
washout and resuspension procedure in the following ways:
= Thaw and wash the Frozen DC vaccines with Lactated Ringer's.
= Prepare the cells at a concentration of 15x106 viable cells/mL in
Lactated Ringer's.
= Fill a glass vaccine vial for use by the clinic to vaccinate patients
enrolled in approved
clinical trials.
EXAMPLE 4: PROPHETIC EXAMPLE
PILOT SAFETY AND BLOOD IMMUNE CELL TRANSCRIPTIONAL PROFILING
STUDY OF ANAKINRA PLUS THE PHYSICIAN'S CHEMOTHERAPY OF CHOICE
IN METASTATIC BREAST CANCER PATIENTS
1. Introduction
A. Background on breast cancer
[00213] Breast cancer is a genetically heterogenous and biologically
diverse disease.
Although the treatment of metastatic breast cancer has improved, the disease
remains incurable.
One of the most active first-line regimens for patients with Her2-negative
metastatic breast
cancer (MBC) is the combination of weekly paclitaxel and bevacizumab. In a
Phase III trial, the
addition of bevacizumab to weekly paclitaxel resulted in a longer progression
free survival (PFS)
(11.8 months vs. 5.9 months) and higher response rate (50% vs. 25%) than
paclitaxel alone
(Miller 2007). However, further improvement in the efficacy of treatment is
necessary.
B. IL-1 and pro-tumor inflammation in breast cancer
[00214] The IL-1 family plays an important role in inflammation and host
defense. Up to
11 members have been identified to date (Smith 2000). IL-la and IL-1f3 are
proinflammatory
- 79 -
CA 02874065 2014-12-04
cytokines. IL-la is primarily bound to the membrane whereas IL-1I3 is secreted
(Dinarello
2005a, Andre 2010). There are 2 transmembrane IL-1 receptors, types I and II.
The type II IL-1
receptor does not signal and is a decoy receptor for IL-113 . IL-1Ra is an
endogenous receptor
antagonist. IL-1Ra is predominantly produced by activated monocytes and
macrophages. The IL-
1Ra does not bind to the type II receptor but rather primarily to the type I
receptor, which is the
signaling receptor.
[00215] Recently, it has been suggested that tumor microenvironment plays a
role in
cancer progression and chemotherapy drug resistance. Cells in the tumor
microenvironment can
stimulate cancer cell growth and invasion (Andre 2010). Solid tumors are often
associated with
aseptic inflammation. There are 2 types of inflammation that have opposing
effects on tumors,
chronic inflammation that promotes cancer cell survival, and metastasis, and
acute inflammation
which triggers cancer cell destruction. Chronic inflammation is often linked
with the presence of
type 2-polarized macrophages (M2), which are induced by Th2 cytokines, IL-4
and IL-13. It is
evident that there is a functional relationship between chronic inflammation
and cancer
(Bhowmick 2004), and it is thought that carcinogenesis may be promoted by the
polarization of
M2 tumor-associated macrophages via cytokines and production of growth
factors. In metastatic
breast cancer, IL-1 has been shown to be up regulated, and patients with IL-1-
producing tumors
have generally poor prognoses (Lewis 2006). IL-1 is known to be a strong
inducer of IL-6
(Linkhart 1991), which in turn leads to M2 macrophage polarity, secretion of
pro-growth factors,
ultimately creating an environment that favors tumor progression (DeNardo
2007).
[00216] Recent studies have demonstrated the presence of inflammatory Th2
cells in
breast cancers, which produce IL-13, IL-4, and TNF (Aspord 2007). These CD4+ T
cells appear
to play a key role in the disease as they accelerate breast tumor development
in a xenograft
model through the production of 1L-13. Breast cancers appear to play a
critical role in
conditioning the infiltrating myeloid DCs (mDCs) to induce such inflammatory
Th2 cells. It has
also been shown that thymic stromal lymphopoietin (TSLP) secreted by cancer
cells plays a role
in mDCs conditioning (Pedroza-Gonzalez 2011). Breast cancer cell lines and
primary tumors
from patients show TSLP protein expression. TSLP-neutralizing antibodies block
the
upregulation of OX4OL by mDCs exposed to tumor supernatant and consequently
block mDCs
capacity to generate inflammatory Th2 cells in vitro (Pedroza-Gonzalez 2011).
- 80 -
CA 02874065 2014-12-04
[00217] Recent studies show crosstalk between breast cancer cells and mDCs
which
triggers high level of IL-113 production, which feeds back on cancer cells to
induce high TSLP
secretion. Higher IL-10 levels correspond with higher clinical stage of the
tumor indicating the
potential prognostic value of IL-1p . When measuring IL-1 alpha in tumors from
patients,
however, the levels of IL-1 beta are substantially higher. Furthermore, no
difference was found
in the levels of IL-1 alpha between tumors and surrounding macroscopically
uninvolved tissue.
The current hypothesis is that IL-1p is produced by myeloid infiltrate whereas
IL-1 alpha is
most likely produced by cancer cells. Studies show that treatment with the IL-
1R antagonist,
anakinra, prevents tumor development in vivo in humanized mice model of breast
cancer.
C. Fatigue caused by IL-1 in cancer patients
[00218] Patients undergoing chemotherapy frequently experience symptoms of
fatigue,
which has been attributed to the increase in proinflammatory cytokines such as
IL-113 (Wood
2006). This symptom can be attributed not only to therapeutic intervention,
such as
chemotherapy, but also inherent to cancer itself (Kurzrock 2001). IL-1 has
been associated with
factors that contribute towards fatigue, such as anemia, weight loss, fever,
and infection
(Kurzrock 2001), and is among several molecules that could be targetable to
reduce cancer-
related fatigue.
D. Background on Anakinra (kineret )
[00219] Anakinra is a recombinant soluble non-glycosylated homolog of human
IL-1Ra
that competitively inhibits binding of IL-la and IL-113 to the receptor type I
(Dinarello 2005b).
Anakinra was approved in 2001 as a treatment for adult rheumatoid arthritis
patients whose
disease has progressed on one or more disease-modifying anti-rheumatic drugs.
The approved
adult dose is 100 mg administered daily as a subcutaneous (SC) injection. With
this dose,
anakinra has a favorable safety profile; the most common adverse reaction is
injection site
reaction. The uncommon serious adverse reactions included an increased
incidence of serious
infections. Anakinra has been used in large clinical trials of adults and
children (Gartlehner
2006). It resulted in improvement and/or resolution of clinical
manifestations, hematological and
biochemical changes in patients with inherited chronic inflammatory diseases
affecting IL-1
production (Hawkins 2004, Hoffman 2004, Goldbach-Mansky 2006). When
administered to
pediatric patients with Systemic Onset Juvenile Idiopathic Arthritis (SOJIA),
anakinra treatment
- 81 -
CA 02874065 2014-12-04
resulted in remarkable clinical and hematological responses in >70% of
patients, and it was
accompanied by a steroid-sparing effect (Pascual 2005). Resolution of clinical
symptoms
including fever, marked leukocytosis, thrombocytosis, anemia, elevated ESR and
arthritis were
rapid and sustained (Pascual 2005). These results have now been confirmed in
randomized
clinical trials.
E. Background on standard of care agents for metastatic disease
[00220] Nanoparticle albumin-bound (nab) paclitaxel, capecitabine,
eribulin, and
vinorelbine are standard, FDA approved effective cytotoxic agents for MBC that
are minimally
myelosuppressive and do not require steroid premedication. All treatments were
approved based
on Phase III clinical trials whose data determined their efficacy in this
setting (Gradishar 2005,
Blum 2007, Cortes 2011, Blum 1999, Jones 1995).
F. Rationale
[00221] In an attempt to reverse the immune suppressive microenvironment
and to
enhance chemotherapy effectiveness, decrease tumor metastagenicity and
decrease IL-1-induced
fatigue, metastatic breast cancer (MBC) patients will be treated with
chemotherapy plus
anakinra. In this pilot safety, single arm, open label trial, we plan to
determine the safety of
anakinra plus the physician's chemotherapy choice (TPC) of nab paclitaxel,
capecitabine,
eribulin, or vinorelbine in patients with MBC and to define an anakinra-
induced anti-IL-1 whole
blood transcriptional profile.
2. Trial Objectives
A. Primary objectives
[00222] The objective of this protocol is to assess the safety profile of
the IL-1 receptor
antagonist, anakinra, plus TPC of nab paclitaxel, capecitabine, eribulin, or
vinorelbine in patients
with MBC.
B. Secondary objectives
[00223] The secondary objectives of this protocol are to determine
investigator-assessed
objective response rate, clinical benefit rate, progression-free survival, and
rates of chemotherapy
- 82 -
CA 02874065 2014-12-04
or cancer-related anemia (HgB<10), and an anakinra-induced anti-IL-1 blood
transcriptional
signatures in patients who undergo IL-1 receptor blockade therapy.
3. Study Design
[00224] This pilot safety, single arm, open label trial will evaluate the
impact of adding
anakinra to TPC of nab paclitaxel, capecitabine, eribulin, or vinorelbine in
the treatment of
patients with HER2-negative MBC. Patients starting chemotherapy at the time of
study
enrollment will undergo a 2-week run-in treatment of anakinra alone, 100 mg SC
daily, followed
by anakinra plus TPC of standard of care (SOC) nab paclitaxel, capecitabine,
eribulin, or
vinorelbine. Patients enrolling in the study who are currently being treated
with nab paclitaxel,
capecitabine, eribulin, or vinorelbine will not undergo the anakinra run-in
treatment. These
patients will begin administration with anakinra on Day 1 of the next
chemotherapy cycle. End
of Study will occur 6 months after the first dose of anakinra is administered,
or at early
withdrawal, whichever occurs first. After 6 months, patients may continue
their SOC treatment
alone until disease progression or intolerable toxicity. The use of
corticosteroids as an antiemetic
is strongly discouraged to be able to evaluate anakinra's effects on patients'
well being, energy
level, and on plasma cytokines and other immunologic biomarkers.
[00225] Whole blood for immunologic biomarkers will be obtained: prior to
treatment on
Day -14 Cycle 0, after the 2-week run-in with anakinra for those patients
starting chemotherapy
at the time of study enrollment, and monthly thereafter after initiation of
anakinra/TPC of nab
paclitaxel, capecitabine, eribulin, or vinorelbine for all patients.
4. Therapeutic Agents
A. Anakinra (Kineret or Equivalent; Kineret 2009)
[00226] Anakinra is a recombinant, nonglycosylated form of the human
interleukin-1
receptor antagonist (IL-1Ra). Anakinra differs from native human IL-1Ra in
that it has the
addition of a single methionine residue at its amino terminus. It is produced
by recombinant
DNA technology using an E coli bacterial expression system.
[00227] Anakinra is indicated for the reduction in signs and symptoms and
slowing the
progression of structural damage in moderately to severely active rheumatoid
arthritis, in patients
18 years of age or older who have failed 1 or more disease modifying
antirheumatic drugs
- 83 -
CA 02874065 2014-12-04
(DMARDs). Anakinra can be used alone or in combination with DMARDs, other than
TNF
blocking agents (see Section 5.1).
[00228] Anakinra consists of 153 amino acids and has a molecular weight of
17.3
kilodaltons. Anakinra is supplied in single use prefilled glass syringes with
27 gauge needles as a
sterile, clear, colorless-to-white, preservative-free solution for daily
subcutaneous (SC)
administration. The solution may contain trace amounts of small, translucent-
to-white
amorphous proteinaceous particles. Each prefilled glass syringe contains: 0.67
mL (100 mg) of
anakinra in a solution (pH 6.5) containing sodium citrate (1.29 mg), sodium
chloride (5.48 mg),
disodium EDTA (0.12 mg), and polysorbate 80 (0.70 mg) in Water for Injection,
USP.
B. Physician's chemotherapy choice of MBC agents
[00229] Selection of TPC cytotoxic agent will be based on what agent would
have been
offered to the patient, should he or she not be participating in the trial.
TPC agents are limited to
nab paclitaxel, eribulin, capecitabine, or vinorelbine, and the use of
corticosteroids as antiemetics
are discouraged in order to assess possible beneficial effects of anakinra on
patients' well being.
[00230] Preparation and administration will be followed per the site's
guidelines and
standard FDA labeling. The doses to be used for TPC drug products are below:
= Nab paclitaxel: 100 mg/m2 administered IV weekly (Day 1, 8, and 15 every
28
days)
= Eribulin: 1.4 mg/m2 administered IV weekly (Day 1 and 8 every 21 days)
= Capecitabine: physician's choice of utilizing 1000 mg/m2 BID 14 days on,
7
days off OR 1000 mg/m2 BID 7 days on, 7 days off (capecitabine is rounded to
the nearest 500 mg increment).
= Vinorelbine: 25 mg/m2 administered IV weekly (Day 1, 8, and 15 every 28
days).
5. Study Treatment Administration
A. Premedications
[00231] Optional pre-medications for nab paclitaxel should be administered
prior to each
dose of nab paclitaxel:
= Diphenhydramine 25-50 mg IV (or equivalent).
= Ranitidine 50 mg IV (or equivalent).
- 84 -
CA 02874065 2014-12-04
B. Treatment Plan
[00232] Patients starting chemotherapy at the time of study enrollment will
receive 100
mg of anakinra administered SC daily during a 2-week run-in period (Days -14
to Day 0).
Following the 2-week run-in, 100 mg of anakinra administered SC daily plus TPC
of SOC nab
paclitaxel, capecitabine, eribulin, or vinorelbine for a maximum of 6 months.
[00233] Patients enrolling in the study who are currently being treated
with nab paclitaxel,
capecitabine, eribulin, or vinorelbine will begin administration of anakinra
on Day 1 of the next
chemotherapy cycle.
[00234] End of Study will occur 6 months after the first dose of anakinra
is administered,
or at early withdrawal, whichever occurs first. After 6 months, patients may
continue their SOC
treatment alone until disease progression or intolerable toxicity. Anakinra
dosing should be
administered at approximately the same time of day every day. Patients will
self-administer
anakinra. The use of corticosteroids as an antiemetic is strongly discouraged
to be able to
evaluate anakinra's effects on patients' well being, energy level, and on
plasma cytokines and
other immunologic biomarkers.
[00235] One cycle of treatment will be determined by TPC drug product
(either 21 or 28
days).
[00236] The treatment schema is shown in Table 46.
Table 46. Treatment schema
Agent Dose Frequency of Cycle length
Route of
administration (days) administration
Daily for a maximum of
Anakinra 100 mg N/A SCa
6 months
Nab paclitaxel 100 mg/m2 Day 1, 8, 15 28 IV
Eribulin 1.4 mg/m2 Day 1 and 8 21 IV
BID, Choice: 14 days on,
Capecitabine b1000 mg/m2 7 days off OR 7 days on, 21 PO
7 days off
Vinorelbine 25 mg/m2 Day 1, 8, 15 28 IV
Anakinra should be administered at approximately the same time of day every
day.
b capecitabine dose is rounded to the nearest 500 mg increment.
- 85 -
CA 02874065 2014-12-04
[00237] The dose levels for TPC drug products will be modified at the
physician's
discretion.
6. Assessment
A. Anakinra Run-in Visit
[00238] Run-in visit (Day -14 Cycle 0) must begin within 7 working days
after the
patient's registration on the study. Only patients starting chemotherapy at
the time of study
enrollment will undergo the 2-week run-in treatment with anakinra. Patients
enrolling in the
study who are currently being treated with nab paclitaxel, capecitabine,
eribulin, or vinorelbine
will not undergo the anakinra run-in treatment.
[00239] The following will be issued:
1. Study coordinator will dispense anakinra (study drug) and instruct patients
on its
proper administration. Patient instruction from the anakinra package insert
may also
be distributed.
2. Whole blood collection (15 mls) for immunologic biomarkers prior to 1st
dose of
anakinra, and at the end of 14 days.
3. First administration of anakinra will be completed in the clinic.
4. Each patient will receive a diary and will be instructed how to fill in.
Patient will also
be instructed to return with the diary at the next visit.
5. Instruct patients to bring used and/or unused syringe dispensing pack for
assessment
of patient compliance.
B. Anakinra plus TPC of MBC agents
[00240] The following evaluations will be performed during therapy with
anakinra and
TPC of nab paclitaxel, eribulin, capecitabine, or vinorelbine (at the
beginning of each cycle,
unless otherwise specified):
1. A brief medical history, to capture events that have occurred since the
last cycle.
Events that were not captured in the baseline complete medical history should
be
recorded on the AE page of the CRF.
2. A brief physical examination, including vital signs and body weight.
3. Assessment of PS on the ECOG scale (Appendix III).
4. Assessment of concomitant medications Day 1 of each cycle.
5. A CBC with differential and platelet count prior to weekly dosing.
6. A CMP prior to weekly dosing for the first 2 cycles, Day 1 of cycles
thereafter (cycle
length is dependent on TPC drug product).
- 86 -
CA 02874065 2014-12-04
7. Tumor response by clinical assessment of the patient's disease (ie, by
physical
examination) must be performed every 4 weeks during therapy.
8. Radiological assessment of tumors (ie, chest X-ray, chest CT, brain CT
or MRI,
pelvic/abdominal CT or MRI, radionuclide bone scan) used to establish
measurable or
non-measurable disease (PET scan) will be performed every 8-9 weeks. The
methods
used for prestudy assessments (CT, MRI, or PET) should be used throughout the
study. If possible, the same equipment should be used each time. Under RECIST
criteria, PET cannot be used to assess measurable disease. Please see RECIST
definition of measurable disease (Section 10.1.1).
9. Assessments of other sites of disease must be performed only to confirm a
CR.
10. Whole blood collection (15 mls) for immunologic biomarkers, prior to
treatment on
Day -14 Cycle 0, after the 2-week run-in with anakinra (only for patients
starting
chemotherapy at the time of enrollment and undergoing the 2-week run-in
treatment
with anakinra), and monthly thereafter after initiation of anakinra/TPC.
Patients who
are currently being treated with nab paclitaxel, capecitabine, eribulin, or
vinorelbine
will only have one whole blood collection (15 mls) for immunologic biomarkers
prior
to the first administration of anakinra, and monthly thereafter after
initiation of
anakinra in combination with chemotherapy.
11. A toxicity assessment must be performed.
12. Patient diary assessment.
C. Early Withdrawal Assessments
[00241] This is a single assessment that will be performed when patient
goes off treatment
because of PD or toxicity that places patients off treatment, or in cases of
physician decision or
where patient withdraws consent. Patients who withdraw consent may not want
any further
assessment; however, they should be encouraged to have these final assessments
done.
[00242] If patient withdraws for any reason during the treatment phase,
patient should be
asked to come to the clinic within 24-48 hours after the last treatment,
particularly for the whole
blood collection. Any delay within this window is not a deviation. The
following evaluation will
be performed at this visit:
1. A brief medical history should be done to capture events that have
occurred since the
last cycle. Events that were not captured in the baseline complete medical
history
should be recorded on the AE page of the CRF.
2. A brief physical examination, including vital signs and body weight.
3. Assessment of PS on the ECOG scale (Appendix III).
4. A CBC with differential and platelet count.
5. A CMP
6. A tumor clinical assessment of the patient's disease (ie, by physical
examination).
- 87 -
CA 02874065 2014-12-04
7. Radiological assessment of tumors (ie, chest X-ray, chest CT, brain CT
or MRI,
pelvic/abdominal CT or MRI, radionuclide bone scan) used to establish
measurable or
non-measurable disease (PET scan). The methods used for prestudy assessments
(CT,
MRI, or PET) should be used throughout the study. If possible, the same
equipment
should be used each time. Under RECIST criteria, PET cannot be used to assess
measurable disease.
8. A toxicity assessment.
9. Patient diary assessment.
10. Whole blood collection (15 mls) for immunologic biomarkers
D. Follow Up Assessments
[00243] Toxicities will be recorded for the first 30 days following the
last dose of anakinra
at a maximum of 6 months. Patients will be followed every 3 months for disease
progression.
Note: Patients who die or withdraw consent are considered off study and no
further information
will be collected.
7. Safety Evaluation¨Adverse Events
[00244] All Grade 3 and 4 adverse events (AEs), Grades 1 and 2 alopecia,
and all grades
of neutropenia will be recorded in the CRF throughout the trial. In addition,
all treatment-related
Grade 1 and 2 laboratory abnormalities, which are deemed "clinically
significant" by the
Treating Physician will be documented in the CRF.
[00245] Adverse events (AEs) will be recorded throughout the trial.
Toxicities and AEs
will be graded using the Common Terminology Criteria for Adverse Events
(CTCAE) Version
4Ø The events, and the relationship of each event to treatment, will be
assessed by the Treating
Physician and recorded on the CRF. Additional information about each event,
such as treatment
required, eventual outcome, and whether or not therapy had to be interrupted
or dosages reduced,
will also be recorded on the CRF. Adverse events will be recorded for up to 30
days following
the last study treatment.
8. Efficacy Assessments
A. Definitions
[00246] Response and progression will be evaluated in this study using the
international
criteria published by the Response Evaluation Criteria in Solid Tumors
(RECIST) Committee
- 88 -
CA 02874065 2014-12-04
V 1.1 (Eisenhauer 2009). Best response will be determined based on the
sequence of disease
status with corresponding best response.
[00247] At baseline, tumor lesions/lymph nodes will be categorized
measurable or
nonmeasurable as follows:
L Measurable Disease
[00248] Lesions that can be accurately measured in at least one dimension
(longest
diameter (LD) to be recorded) with a minimum size of:
= 10 mm with spiral CT scan (irrespective of scanner type) and MRI (no less
than
double the slice thickness and a minimum of 10 mm)
= 10 mm caliper measurement by clinical exam (when superficial)
= 20 mm by chest X-ray (if clearly defined and surrounded by aerated lung)
Nonmeasurable Disease
[00249] All other lesions (or sites of disease) are considered
nonmeasurable disease.
Lesions that are considered as truly nonmeasurable include the following:
1. leptomeningeal disease
2. ascites
3. pleural/pericardial effusion
4. inflammatory breast disease
5. lymphangitis cutis/pulmonis
6. abdominal masses that are not confirmed and followed by imaging techniques
Special Considerations Regarding Lesion Measurability
[00250] Bone lesions, cystic lesions, and lesions with prior local
treatment require special
considerations.
1. Bone lesions
Bone scans, PET scans, and plain films are not considered adequate imaging
techniques to measure lesions. However, imaging techniques can be used to
confirm
presence or disappearance of bone lesions.
Lytic bone with identifiable soft tissue components that can be evaluable by
CT or
MRI are considered measurable.
Blastic lesions are nonmeasurable.
To be assigned a status of PR or CR, changes in tumor measurements must be
confirmed by repeat assessments performed at least 4 weeks after the criteria
for
- 89 -
CA 02874065 2014-12-04
response are first met. In the case of SD, follow-up measurements must have
met the
SD criteria at least once after study entry at a minimum interval of 8 weeks.
A patient is considered to have progressed by bone scan if:
a. The first bone scan with >2 new lesions compared to baseline is observed
<12 weeks from randomization and is confirmed by a second bone scan
taken >6 weeks later showing >2 additional new lesions (a total of >4 new
lesions compared to baseline);
b. The first bone scan with >2 new lesions compared to baseline is observed
>12 weeks from randomization and the new lesions are verified on the
next bone scan >6 weeks later (a total of >2 new lesions compared to
baseline).
2. Cystic lesions
Lesions that meet the criteria for radiographically defined simple cysts
should not be
considered malignant.
Cystic metastases are measurable lesions, if they meet the criteria outlined
in Section
10.1.1.
3. Lesions with prior local treatment
Progression of a previously irradiated or locally treated area would be
considered
measurable.
B. Guidelines for Evaluation of Measurable Disease
i. Measurement of Lesions
[00251] All measurements should be taken and recorded in metric notation
using a ruler or
calipers. All baseline evaluations will be performed as closely as possible to
the beginning of
treatment and never more than 4 weeks before the beginning of the treatment.
11. Methods of Assessments
[00252] The same method of assessment and the same technique should be used
to
characterize each identified and reported lesion at baseline, during treatment
and in follow-up.
Imaging-based evaluation is preferred to evaluation by clinical examination
when both methods
have been used to assess the antitumor effect of treatment.
[00253] Clinical Lesions: Clinical lesions will only be considered
measurable when they
are superficial and > 10 mm diameter using calipers (for example, skin
nodules). In the case of
skin lesions, documentation by color photography (including a ruler to
estimate the size of the
lesion) is recommended.
- 90 -
CA 02874065 2014-12-04
[00254] Chest X-rays: Lesions on chest X-ray are acceptable as measurable
lesions when
they are clearly defined and surrounded by aerated lung. However, CT is
preferable.
[00255] CT and MRI: CT is the best available and reproducible method to
measure
lesions selected for response assessment. This guideline has defined
measurability of lesions on
CT scans based on the assumption is that the CT slice thickness is 5 mm or
less. MRI is
acceptable in certain situations such as body scans.
[00256] Ultrasound: Ultrasound is not useful in assessment of lesion size
and should not
be used as a method of measurement.
[00257] Endoscopy, laparoscopy: The utilization of these techniques for
objective tumor
evaluation is not advised. However, they can be useful to confirm complete
pathological
response when biopsies are obtained or to determine relapse in trials where
recurrence following
complete response or surgical resection is an endpoint.
[00258] Tumor Markers: Tumor markers alone cannot be used to assess
response. If
markers are initially above the upper normal limit, they must normalize for a
patient to be
considered in complete clinical response. Because tumor markers are disease
specific,
instructions for their measurement should be incorporated into protocols on a
disease specific
basis. Specific guidelines for CA125 response (in recurrent ovarian cancer)
and PSA response (in
recurrent prostate cancer) have been published The Gynecologic Cancer
Intergroup has
developed CA125 progression criteria which are integrated with objective tumor
assessment for
use in first-line trials in ovarian cancer.
[00259] Cytology, histology: These techniques can be used to differentiate
between PR
and CR in rare cases if required by protocol (for example, residual lesions in
tumor types such as
germ cell tumors, where known residual benign tumors can remain). When
effusions are known
to be a potential adverse effect of treatment (for example, with certain
taxane compounds or
angiogenesis inhibitors), the cytological confirmation of the neoplastic
origin of any effusion that
appears or worsens during treatment can be considered if the measurable tumor
has met criteria
for response or stable disease in order to differentiate between response (or
stable disease) and
progressive disease.
C. Tumor Response Evaluation
- 91 -
CA 02874065 2014-12-04
L Assessment of Overall Tumor Burden and Measurable Disease
[00260] To assess objective response or future progression, it is necessary
to estimate the
overall tumor burden at baseline and use the baseline assessment for
subsequent measurements.
In studies where the primary endpoint is tumor progression, the protocol must
specify if entry is
restricted to those with measurable disease or whether patients having
nonmeasurable disease are
also eligible.
Baseline Documentation of Target and Non-target Lesions
[00261] When more than 1 measurable lesion is present at baseline all
lesions up to a
maximum of 5 lesions total (and a maximum of 2 lesions per organ,
specifically, if 1 or 2 target
organs are involved only 2 or 4 lesions, respectively, will be recorded).
a. Target lesions
[00262] Target lesions should be selected on the basis of their size
(lesions with the LD),
representative of all target organs and their suitability for accurate
repetitive measurements
(either by imaging techniques or clinically).
[00263] A sum of the LD for all target lesions will be calculated and
reported as the
baseline sum longest diameter. The baseline sum of the LD will be used as
reference to further
characterize the objective tumor response of the measurable dimension of the
disease.
[00264] Pathological lymph nodes are defined as measurable and meet
criterion of a short
axis of >15 mm by CT scan.
b. Non-target lesions
[00265] All other lesions (or sites of disease) should be identified as non-
target lesions
and should also be recorded at baseline. Measurements of these lesions are not
required, and
these lesions should be followed as "present" or "absent" or in rare cases
"unequivocal
progression".
[00266] Pathological nodes >10 to <15 mm short axis should be considered
non-target
D. Response Criteria
L Evaluation of Target Lesions
- 92 -
CA 02874065 2014-12-04
[00267] Complete Response (CR): Disappearance of all target lesions.
[00268] Partial Response (PR): At least a 30% decrease in the sum of the LD
of target
lesions taking as reference the baseline sum LD.
[00269] Progression (PD): At least a 20% increase in the sum of LD of
target lesions
taking as reference the smallest sum LD recorded since the treatment started
or the appearance of
1 or more new lesions. In addition to the relative increase of 20%, the sum
must also demonstrate
the absolute increase of at least 5 mm. (the appearance of 1 or more lesion is
also considered
progression).
[00270] Stable Disease (SD): Neither sufficient shrinkage to qualify for PR
nor sufficient
increase to qualify for PD taking as reference the smallest sum LD since the
treatment started.
Special Notes on Assessment of Target Lesions
[00271] Lymph nodes should always have the actual short axis measurement
recorded
even if the nodes regress to below 10 mm on study. For PR<SD and PD, the
actual short axis
measurement of the nodes is to be included on the sum of target lesions.
[00272] Target lesions that become too small to measure: when lesions or
lymph nodes
become so faint on CT scan to measure comfortably, it is important that a
value be recorded on
the CRF. If the radiologist's opinion is that the lesion has disappeared, then
0 mm is reported. If
a definable lymph node is present, but too small to measure, the default is 5
mm.
[00273] Lesions that split or coalesce on treatment: the LD of the
fragmented pieces
should be added together to calculate the lesion sum. If lesions coalesce, the
vector of the LD in
this new instance should be the maximal LD for the 'coalesced lesion'.
Evaluation of Non-target Lesions
[00274] Complete Response: Disappearance of all non-target lesions and
normalization of
tumor marker level(s) if applicable. All lymph nodes must be non-pathological
in size (<10 mm
short axis).
[00275] Non-CR/Non-PD: Persistence of 1 or more non-target lesions and/or
maintenance
of tumor marker level(s) above the normal limits.
- 93 -
CA 02874065 2014-12-04
[00276] Progressive Disease: Appearance of 1 or more new lesions or
unequivocal
progression of existing non-target lesions.
iv. Special Notes on Assessments of Progression of Non-target Disease
[00277] When a patient also has measurable disease: to achieve 'unequivocal
progression'
on the basis of the non-target disease, there must be an overall level of
substantial worsening in
non-target disease such that, even in the presence of SD or PR in target
disease, the overall tumor
burden has increased sufficiently to merit discontinuation of therapy. The
designation of overall
progression based on non-target disease in the face of SD or PR is rare.
[00278] When a patient has only nonmeasurable disease: because worsening in
non-target
disease cannot be easily quantified, a useful test that can be applied when
assessing patients for
unequivocal progression is to consider if the increase in overall disease
burden on the change in
nonmeasurable disease is comparable in magnitude to the increase that would be
required to
declare PD for measurable disease, specifically, an increase in tumor burden
representing an
additional 73% increase in 'volume' (which is equivalent to a 20% increase in
diameter in a
measurable lesion).
v. New Lesions
[00279] The appearance of new malignant lesions denotes disease
progression.
[00280] A lesion identified on a follow-up study (assessed during treatment
or in the
follow-up phase) in a location that was not scanned at baseline is considered
a new lesion and
will indicate disease progression.
[00281] If a new lesion is equivocal (ie, small size) follow-up assessments
will clarify if it
represents new disease. If repeat scans confirm a new lesion, then progression
should be declared
using the date of the initial scan.
[00282] FDG-PET imaging can complement CT imaging in assessment of 'new'
lesions.
= Negative FDG-PET at baseline, with a positive FDG-PET at a later
assessment is a
sign of PD based on a new lesion.
= No FDG-PET at baseline and a positive FDG-PET at a later assessment
= If positive FDG-PET at a later assessment corresponds to a new site by
CT, this is
PD.
- 94
CA 02874065 2014-12-04
= If the positive FDG-PET at a later assessment is not confirmed by CT,
additional
follow-up CT scans are needed to determine if there is truly progression
occurring
(if so, the date of PD is date of initial abnormal FDG-PET scan)
= If a positive FDG-PET at a later assessment corresponds to a pre-existing
site of
disease on CT that is not progressing on the basis of the anatomic image, this
is
not PD.
E. Evaluation of Best Overall Response
[00283] The best overall response is the best response recorded from the
start of the
treatment until disease progression/recurrence (taking as reference for
progressive disease the
smallest measurements recorded since the treatment started), Table 47. The
patient's best
response assignment will depend on the findings of both target and non-target
disease and will
also take into consideration the appearance of new lesions. Furthermore,
depending on the
protocol and study requirements, it may also require both measurement and
confirmation criteria.
Table 47. Evaluation of Best Overall Response
Target Lesions Non-target Lesions New Lesions Overall Response
CR CR No CR
CR Non-CR/non-PD No PR
CR NE No PR
PR Non-PD or NE No PR
SD Non-PD or NE No SD
Not all evaluated Non-PD No NE
PD Any Yes or No PD
Any PD Yes or No PD
Any Any Yes PD
CR¨complete response, PR= partial response, SD¨stable disease, PD¨progressive
disease, and
NE¨nonevaluable
i. Timepoint Response
Assessments of response are done at specified time points throughout the study
for measurable
disease (Table 47). When patients have nonmeasurable (non-target) disease
only, Table 48 is
used.
- 95 -
CA 02874065 2014-12-04
Table 48. Time Point Response: Patients with Non-target Disease Only
Non-target lesions New lesions Overall response
CR No CR
Non-CR/non-PD No Non-CR/non-PDa
Not all evaluated No NE
Unequivocal PD Yes or No PD
Any Yes PD
CR=complete response, PD=progressive disease, and NE=nonevaluable
a 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease
since SD is increasingly used as
endpoint assessment for efficacy in some trials; to assign this category when
no lesions can be measured is
not advised
ii. Missing Assessments and Nonevaluable (NE) Designation
[00284] When no imaging/measurement is done at all at a specified time
point, the patient
is nonevaluable (NE) at that time point.
Best Overall Response: All Time Points
[00285] The best overall response is determined once all data for the
patient is known.
[00286] Best response determination in trials where confirmation of CR or
PR is not
required: Best response in these trials is defined as the best response across
all time points (for
example, a patient who has SD at 1st assessment, PR at 2nd assessment and PD
on last assessment
has a best overall response of PR). When SD is believed to be the best
response, it must also
meet the protocol specific minimum time from baseline.
[00287] Best Response determination in trials where confirmation of CR or
PR is
required: CR or PR may be claimed only if the criteria for each are met at a
subsequent time
point as specified in the protocol (generally 4 weeks later). In this
circumstance, the best overall
response can be interpreted as in Table 49.
- 96 -
CA 02874065 2014-12-04
Table 49. Best Overall Response When Confirmation of CR and PR is Required
Overall response Overall response Best overall response
First time point Subsequent time point
CR CR CR
CR PR SD, PD, or PRa
CR SD SD provided minimum criteria for SD
duration
met, otherwise PD
CR PD SD provided minimum criteria for SD
duration
met, otherwise PD
CR NE SD provided minimum criteria for SD
duration
met, otherwise NE
PR CR PR
PR PR PR
PR SD SD
PR PD SD provided minimum criteria for SD
duration
met, otherwise PD
PR NE SD provided minimum criteria for SD
duration
met, otherwise NE
NE NE NE
CR=complete response, PR=partial response, SD= stable disease, PD=progressive
disease, NE=nonevaluable
a If a CR is truly met at first time point, then any disease seen at
subsequent time point, even disease meeting PR
criteria relative to baseline, makes the disease PD at that point (since
disease must have reappeared after CR). Best
response would depend on whether minimum duration for SD was met. However,
sometimes 'CR' may be claimed
when subsequent scans suggest small lesions were likely still present and in
fact the patient had PR, not CR at the
first time point. Under these circumstances, the original CR should be changed
to PR and the best response is PR.
iv. Special Notes on Response Assessment
[00288] When nodal disease is included in the sum of target lesions and the
nodes
decrease to 'normal' size ((10 mm), they may still have a measurement reported
on scans. This
measurement should be recorded even though the nodes are normal in order not
to overstate
progression should it be based on increase in size of the nodes. As noted
earlier, this means that
patients with CR may not have a total sum of 'zero' on the CRF.
[00289] Where confirmation of response is required, missing assessments
(NE) may
complicate determination of best response. The analysis plan for the trial
must address how
missing data/ assessments will be addressed in the determinations of response
and progression. It
may be reasonable to consider a patient with time point responses of PR-NE-PR
as a confirmed
response.
- 97 -
CA 02874065 2014-12-04
[00290] Patients with a global deterioration of health status requiring
discontinuation of
treatment without objective evidence of disease progression at that time
should be classified as
having "symptomatic deterioration". Every effort should be made to document
the objective
progression, even after discontinuation of treatment.
[00291] In some circumstances, it may be difficult to distinguish residual
disease from
normal tissue. When the evaluation of complete response depends on this
determination, it is
recommended that the residual lesion be investigated (fine needle
aspirate/biopsy) before
confirming the complete response status.
F. Evaluation of Pathologic Response
[00292] Pathologic response to therapy is the primary endpoint of the study
protocol.
Patients will undergo surgical resection after protocol-directed treatment.
The pathologic
specimen will be graded according to the tumor regression grading schema
proposed by ROdel et
al (Rodel 2005). In this categorization schedule, the response to treatment
and the degree of
tumor regression is categorized as follows:
TRG 0: no pathologic evidence of tumor regression
TRG 1: minor tumor regression with < 25% of the pathologic specimen exhibiting
fibrosis/necrosis.
TRG 2: moderate tumor regression with 25-50% of the pathologic specimen
exhibiting
fibrosis/necrosis.
TRG 3: significant tumor regression with > 50% of the pathologic specimen
exhibiting
fibrosis/necrosis.
TRG 4: complete pathologic response to therapy; no histologic evidence of
persistent
malignancy.
[00293] A pathologic complete response (pCR) is defined as NO pathologic
evidence of
invasive disease at the primary site in the breast or axillary lymph nodes.
[00294] The presence or absence of a pCR will be assessed separately for
the tumor and
the lymph nodes. For patients who do not achieve a pCR, the size of the
residual cancer in the
tumor, on pathologic exam, will be documented in the as well as the number of
positive lymph
nodes.
- 98 -
CA 02874065 2014-12-04
G. PET Response
[00295] PET responses will be assessed by comparing initial and post-
treatment scans.
Scans will be graded as either complete response (CR), partial response (PR),
stable disease
(SD), or progressive disease (PD)(Young 1999). These are based on standardized
uptake values
(SUV), calculated as:
Tissue concentration (i1Ci/gm)
SUV = Inj. Dose (ACi)/body weight (gm)
CR = complete resolution of abnormal [I8F]flouoro-2-deoxy-D-glucose (FDG)
uptake
PR = SUV decreases by at least 25% after 1 cycle of chemotherapy or by at
least
25% after more than 1 cycle.
SD = SUV increases by less than 25% or decreases by less than 15%, and no
visible increase in tumor size
PD = SUV increases by more than 25%, or visible increase in tumor size, or new
lesions
H. Frequency of Tumor Re-Evaluation
[00296] Frequency of tumor re-evaluation while on treatment is protocol
specific (see
Section 8) and adapted to the type and schedule of treatment.
[00297] After the end of the treatment during the follow-up phase, the need
for repetitive
tumor evaluations depends on whether the trial has as a goal the response rate
or the time to an
event (progression/death). If 'time to an event' (for example, time to
progression, disease-free
survival, progression-free survival) is the main endpoint of the study, then
routine scheduled re-
evaluation of protocol specified sites of disease is warranted.
I. Confirmatory IVIeasurementfilluration of Response
L Confirmation
[00298] In nonrandomized trials where response is the primary endpoint,
confirmation of
PR and CR is required to ensure responses identified are not the result of
measurement error. In
randomized trials (Phase II or III) or studies where SD or progression are the
primary endpoints,
confirmation of response is not required since it will not add value to the
interpretation of trial
results.
- 99 -
CA 02874065 2014-12-04
[00299] In the case of SD, measurements must have met the SD criteria at
least once after
study entry at a minimum interval, not less than 4 weeks.
Time to Response
[00300] For patients who achieve a major objective response (CR or PR) the
time to
response will be assessed as the date of start of treatment to the date of
response.
Duration of Response
[00301] The duration of overall response is measured from the time
measurement criteria
are first met for CR/PR until the first date that recurrent or progressive
disease is objectively
documented (taking as reference for progressive disease the smallest
measurements recorded on
study).
iv. Duration of Complete Response
[00302] The duration of overall CR is measured from the time measurement
criteria are
first met for CR until the first date that recurrent disease is objectively
documented.
v. Duration of Stable Disease
[00303] Stable disease is measured from the date of registration until the
criteria for
progression are met, taking as reference the smallest sum on study.
9. Statistical Methods
[00304] The secondary objectives of this protocol are to determine
investigator-assessed
objective response rate, clinical benefit rate, progression-free survival, and
rates of chemotherapy
or cancer-related anemia (HgB<10), and an anakinra-induced anti-IL-1 blood
transcriptional
signatures in patients who undergo IL-1 receptor blockade as therapy.
[00305] Objective response rate, clinical benefit rate, and progression-
free survival will be
determined with 95% confidence intervals. Whole blood transcriptional
profiling will be
performed to determine a gene expression signature that is induced by IL-1
receptor blockade by
anakinra. The gene expression signatures from baseline will be compared to
those signatures
obtained after the 2-week run-in treatment with anakinra alone.
[00306] Descriptive statistics of demographic, clinical, technical, flow
cytometry, and
microarray derived variables will be given overall and by appropriate
classifications (ie, time) for
- 100 -
CA 02874065 2014-12-04
whole blood transcriptional profiling. Continuous variables will be described
by their frequency
of observations, mean, median, standard deviation, minimum, and maximum
values. Categorical
variables will be described by their frequency and percentage. Generalized
linear mixed model
analyses with appropriate distributional assumptions and link functions will
be used to assess
change in variables over time.
[00307] Unsupervised analyses for microarray data will include hierarchical
cluster
analysis and principal component analysis. Differential gene expression
analysis will be
conducted using linear mixed models with the Benjamini and Hochberg false
discovery rate
(FDR) of 5% used to account for multiple testing. Goodness of fit and checks
of model
assumptions will be carried out for all regression analyses. To ensure that
data is not over-fit we
will perform leave-one-out-cross-validation (LOOCV).
EXAMPLE 5
MATERIALS AND METHODS¨MYELOID CELL-DERIVED IL-1B PROMOTES
TSLP-TH2 INFLAMMATION TO FOSTER BREAST TUMOR GROWTH
[00308] Cell lines and Reagents. Breast cancer cell lines (MDA-MB231, HS-
578t, HCC-
1806 and MCF-7) and benign counterpart cell line HS-Bst cells were obtained
from ATCC and
cultured in medium (RPMI supplemented with glutamine 2 mM, penicillin 50 U/ml,
streptomycin 50 g/ml, MEM non-essential amino acids 0.1 mM, HEPES buffer 10
mM, sodium
pyruvate 0.1 mM and 10% of fetal calf serum). The following cytokines were
obtained from
R&D: IL-113, IL-la, IL-18, IL-6, Si16R, TNF-a, TSLP, and IL-4. GM-CSF (Leukine
) and anti-
human CD14 antibody (RM052) were obtained from Immunex. Anakinra (Kineret ,
Amgen
Inc.) was purchased through Baylor University Medical Center pharmacy. PMA,
ionomycin,
and TAK1 inhibitor (5z-7-oxozeaenol) were obtained from Sigma-Aldrich (St.
Louis, MO).
TGF-13R kinase inhibitor was obtained from EMD Millipore (Billerica, MA).
Caspase-1 activity
detection kit was obtained from Oncolmmunin Inc (Gaithersburg, MD). The
following
antibodies were obtained from Invitrogen (Grand Island, NY): anti-rabbit IgG
conjugated to
Alexa Fluor 568, anti-mouse IgG2b conjugated to Alexa Fluor 647, goat anti-
mouse IgG2a
conjugated to Alexa Fluor 568. Anti-human IL-10 (Ab9722), cytokeratin-19 (A53-
B/A2), and
TAK1 (phosphor T187) antibodies were obtained from Abcam (Cambridge, MA). The
following antibodies were obtained from BD (Franklin Lakes, NJ): antibodies to
human CD3
(UCHT1), CD4 (SK3), CD8 (SKI), CD11c (B-1y6), CD19 (HIB19), CD56 (B159), IL-13
- 101 -
CA 02874065 2014-12-04
(JES10-5A2), IFN-y (B27), PE-labeled OX4OL (Ik-1), anti-CD11 c antibody (S-HCL-
3), anti-
HLA-DR antibody (L-243). Anti-CD68 antibody (Y1/82A) was obtained from
Biolegend (San
Diego, CA). Anti-CD163 (EDHu-1) antibody was from AbD Serotec. Anti-IL-1(3
neutralizing
(AB34 41.6E6.4A4) and non-neutralizing (AB34 41.1G12.1B11) antibodies, anti-
TSLPR
neutralizing antibody (AB81_85.1F11) were made in- house. Caspase-1 inhibitor
(Z-WEHD-
FMK), anti-TGF-P1 antibody (chicken IgY) and Anti-TGF-p neutralizing antibody
(1D11) were
purchased from R&D (Minneapolis, MN).
[00309]
Cytokine Production and Analysis of Tumor Samples from Patients. Tumor
samples from patients diagnosed with breast carcinoma (in situ, invasive duct,
and/or mucinous
carcinoma of the breast, as well as lobular carcinoma) were obtained from the
Baylor University
Medical Center Tissue Bank (Institutional Review Board no. 005-145). The
combined
histological grading system including nuclear grade, tubule formation and
mitotic rate, and
staging system (according to tumor size, invasive or not, lymphoid node
involvement, and spread
out or not) were applied according to pathologists' report post-surgery. Whole-
tissue fragments
(4 x 4 x 4 mm, 0.02 g, approximately) were placed in culture medium with 50
ng/ml of PMA
and 1 is/ml of lonomycin for 16 hours. TSLP, IL-1P, IL-la, IL-33, IL-25, GM-
CSF, and IL-13
levels were analyzed in the culture supernatant by Luminex (EMD Millipore,
Billerica, MA).
Concentrations of IL-18 and IL-1Ra from tissue cultured supernatants were
determined by means
of enzyme-linked immunosorbent assays (R&D system, Minneapolis, MN) following
manufacture's protocols. Briefly, 96-well ELISA plate (Nunc, Roskilde,
Denmark) was first
coated with10 g/ml of anti-human immunoglobulin capture antibody in 0.05 M
sodium carbonate
solution (pH=9.6) at 4-8 C for overnight. After washing with washing solution
(50 mM Tris,
0.14 M NaC1, 0.05% Tween 20, p1-1=8.0), blocking solution (50 mM Tris, 0.14 M
NaCI, 1%
BSA, pH=8.0) was added to each well and the plate was incubated at room
temperature for one
hour. Then, samples and standards diluted with sample diluent (50 mM Tris,
0.14 M NaC1, 1%
BSA, 0.05% Tween 20, pH=8.0) were added to each well and incubated for one
hour at room
temperature. After incubation, samples were removed and wells were washed five
times with
washing solution. Then, goat anti-human immunoglobulin antibody HRP conjugated
was
transferred into each well for one hour at room temperature. After incubation,
HRP conjugate
was removed and plate was washed five times with washing solution. Then, 100 I
TMB
substrate reagent (BD) was added into each well at room temperature in the
dark. After five
- 102 -
CA 02874065 2014-12-04
minutes of incubation, 100 1 of 1 M H3PO4 was added subsequently to stop the
reaction and the
plate was read at 450 nm with the ELISA reader (Molecular Devices, Sunnyvale,
CA). The
standard curve was generated and the amount of total human immunoglobulin was
further
calculated.
[00310] Isolation of monocytes and culture of monocyte-derived DC and
macrophages. CD14+ cells were positively selected from PBMCs of healthy donors
using
magnetic selection according to the manufacturer's instructions (Miltenyi
Biotec). The purity
was routinely >95%. Macrophages were generated from CD14+ monocytes by
culturing with
100 ng/ml M-CSF (protocol see Martinez et al, 2006). MDDCs were generated from
the
adherent fraction of PBMCs by culturing with 100 ng/ml GM-CSF and 10 ng/ml IL-
4 (R&D
Systems).
[00311] Tumor Factor Preparation. Cell lines were culture in medium (RPMI
supplemented with glutamine 2mM, penicillin 50 U/ml, streptomycin 50 g/ml,
MEM non-
essential amino acids 0.1 mM, HEPES buffer 10 mM, sodium pyruvate 0.1 mM and
10% of fetal
calf serum), and when the cells reached 90% of confluence fresh medium was
added and left the
cells in culture for additional 48 h. Cellular debris was removed by
centrifugation and the
supernatant was collected and stored at -80 C.
[00312] Isolation and Culture of Myeloid Dendritic Cells. DCs were purified
from
buffy coat of blood from healthy donors. Briefly, DCs were enriched from
mononuclear cells by
negative selection using a mixture of antibodies against linage markers for
CD3, CD9, CD14,
CD16, CD19, CD34, CD56, CD66b and glycophorin A (EasySep, human pan-DC pre-
enrichment kit). Cells from negative fraction were immuno-labeled with anti-
human FITC-
labeled linage cocktail (CD3, CD14, CD16, CD19, CD20 and CD56, BD
biosciences); PE-
labeled CD123 (mIgGl, clone 9F5, BD biosciences), APC-eflour780-labeled HLA-DR
(mIgG2b, clone LN3, Sigma-Aldrich) and APC-labeled CD11c (mIgG2b, clone S-HCL-
3, BD
biosciences). DCs (lin-, CD123-, HLA-DR, CD11c+) were sorted in a FACS Aria
cytometer (BD
Bioscience). DCs were seeded at 100 x 103 cells/well in 200 1 of medium (RPMI
supplemented
with glutamine 2 mM, penicillin 50 U/ml, streptomycin 50 g/ml, MEM non-
essential amino
acids 0.1 mM, HEPES buffer 10 mM, sodium pyruvate 0.1 mM and 10 % of human AB
serum).
- 103 -
CA 02874065 2014-12-04
DCs were cultured with medium alone or in the presence of 40% of cancer
conditioned
supernatant, or different reagents. After 18 h DCs were harvested and washed.
[00313] Co-culture and Transwell experiment. Tumor cell lines were culture
in medium
(RPMI supplemented with 2 mM glutamine, 50 U/ml penicillin, 50 g/m1
streptomycin, 0.1 mM
MEM nonessential amino acids, 10 mM Hepes buffer, 0.1 mM sodium pyruvate, and
10% fetal
calf serum), and when the cells reached 90% of confluence fresh medium or
different treatment
(different doses of IL-113, PMA/Ionomycin, 1 Ong/ml IL-la, 2Ong/m1 IL-18,
lOng/m1 TNF-a,
2Ong/m1 IL-6) were added and the cells were left in culture for an additional
24h, 48h, or 72h.
For tumor cells and mDCs coculture experiment, 100k tumor cells were seeded in
24 well plate
to grow overnight, then media was refreshed and 100k mDCs were added to
coculture for
another 24-72h. For the transwell experiment, 24-well-plate with inserts were
used (Corning).
200K tumor cells were seeded in the plate to grow at least overnight, then
200k mDCs were
added into inserts. After 48h of co-culture, supernantants were harvested to
determine IL-113
level by ELISA (Duoset, R&D System).
[00314] Tissue Immunofluorescence Staining. 6-am-frozen sections from
tissues were
fixed with cold acetone for 5 min. The sections were labeled with 10 g/m1 of
anti-IL-113
antibody (rabbit IgG, Abcam), followed by anti-rabbit IgG conjugated to AF568
(invitrogen);
5ug/m1 of anti-CD11c antibody (mouse IgG2b, BD), followed by anti-mouse IgG2b
conjugated
to AF647 (Invitrogen), or 1 Oug/ml of anti-CD68 antibody (mouse IgG2b,
Biolegend).
Cytokeratin-19 was labeled with monoclonal antibody clone A53-BA2 (IgG2a;
Abcam),
followed by Alexa Fluor 568 goat anti-mouse IgG2a (Invitrogen). Finally,
sections were
counterstained for 2 min with 3 laM of the nuclear stain DAPI (in PBS;
Invitrogen). To confirm
specificity of 1L-1[3 staining, primary anti-IL-1l3 antibody was preincubated
with 100 ag of
recombinant human IL-113 (R&D Systems) for 30 min at room temperature before
staining of
tissue sections that previously showed to be IL-113 positive.
[00315] Real-Time Polymerase Chain Reaction. Samples were treated and lysed
with
Buffer RLT, and then stored at -80 C until RNA extraction. Total RNA was
isolated and
purified from each sample by using RNeasy kit and RNase-free DNase (Qiagen)
according to the
manufacturer's instructions. cDNA was generated from total RNA with iScriptTM
cDNA
Synthesis Kit. The resulting cDNA was then used for quantitative gene
expression analysis on a
- 104 -
CA 02874065 2014-12-04
Sequence Detection System 7,500 (Applied Biosystems). The primers used were as
follows:
human (h)TSLP, 5'-TAGCAATCGGCCACATTGCC-3' (SEQ ID NO:41) and 5'-
CTGAGTTTCCGAATAGCCTG-3 (SEQ ID NO:42), and human (h)IL-113, 5'-
TACCTGTCCTGCGTGTTGAA-3' (SEQ ID NO:43) and 5'-
TCTTTGGGTAATTTTGGGATCT-3' (SEQ ID NO:44), human (h) GAPDH, 5'-
AGCCACATCGCTCAGACAC-3' (SEQ ID NO :45) and 5'-GCCCAATACGACCAAATCC-3'
(SEQ ID NO:46), human (h) ABL I , 5'- TGACAGGGGACACCTACACA-3' (SEQ ID NO:47)
and 5' ATACTCCAAATGCCCAGACG-3' (SEQ ID NO:48), human (h) PGKI , 5'-
CTTCCTCCTTAAAACTCCTCTCC-3' (SEQ ID NO:49) and 5'-
CTAAGGTCTCCAACGCTCTTCT-3' (SEQ ID NO:50), human (h) PES1, 5'-
CATCACCCATCAGATTGTCG-3' (SEQ ID NO:51) and 5'-A
GCTGCACCCCAGAGAAGTA-3' (SEQ ID NO:52). Equal amounts of cDNA were used with
the iTaq SYBR Green Supermix with ROX (Bio-Rad) and primer mix according to
the real-time
PCR protocols supplied by the manufacturer. Amplification efficiencies were
validated against
the housekeeping gene, GAPDH, PES1, and ABL1. The data were normalized to
GAPDH
mRNA level. The relative quantification of target gene expression was done by
the comparative
cycle threshold (CT) method. The formula 2--AACT was used for each run
according to the
manufacturer's instructions and published methods for this system.
[00316]
Tumor-bearing mice and in vivo experiment. NOD/SCID/132m-/- mice (Jackson
ImmunoResearch Laboratories) were sublethally irradiated (12 cGy/g body weight
of 137Cs
irradiation) the day before tumor implantation. Then 10 million Hs-578t breast
cancer cells
harvested from cultures were injected subcutaneously into the flanks. Mice
were then
reconstituted with 1 million monocyte-derived DCs (MDDCs) and autologous T
cells. CD4 and
CD8+ T cells were positively selected from thawed PBMCs using magnetic
selection according
to the manufacturer's instructions (Miltenyi Biotec). The purity was routinely
>90%. 10 million
CD4+ T cells and 10 million CD8+ T cells were transferred at days 3, 6, and 9
after tumor
implantation. MDDCs were generated from the adherent fraction of PBMCs by
culturing with
100 ng/ml GM-CSF and 10 ng/ml 1L-4 (R&D Systems). Anakinra (2mg/kg body
weight) or PBS
were injected daily in peritumor area since day 3 after tumor engraftment.
200ug TSLPR
blocking antibody was given on day 3,6,9. In some experiments, TGF-f3 blocking
antibody was
given on day 3,6,9. Tumor size was monitored every 2-3 d. Tumor volume
(ellipsoid) was
- 105 -
CA 02874065 2014-12-04
calculated as follows: ([short diameter]2 x long diameter)/2. On day 16, the
tumors were
harvested. In some experiments, only one time DCs and total T cells transfer
was done. Cell
suspension was obtained from breast cancer tissue of above mice. Cell
suspensions were
obtained by digestion with 2.5 mg/ml of collagenase D (Roche Diagnostics,
Indianapolis, IN),
and 200 U/ml of DNAse I (Sigma-Aldrich, St. Louis, MO) for 30 minutes at 37 C.
Washed
three times and analyzed with FACS-CantoIl (Becton Dickinson).
[00317] Flow Cytometry Analysis. The anti-human antibodies used were FITC-
labeled
linage cocktail (CD3, CD14, CD16, CD19, CD20 and CD56, BD Biosciences); PE-
labeled
OX4OL (mIgGl, clone Ik- 1, BD biosciences); PE-labeled TSLPR; APC-labeled HLA-
DR
(mIgG2a, clone L243, BD); APC-labeled CD11c (mIgG2b, clone S-HCL-3, BD
Biosciences);
Pacific Orange-Labeled CD45. TGF-P 1 (chiken IgY, R&D), TGF-PRI, TGF-PRII, TGF-
f3RIII
(goat IgG, R&D); FITC-labeled IL-113 ( R&D); PE- labeled 1L-13 (rat IgGl,
clone JES10-5A2
BD biosciences); PECy7-labeled TNF-a (mIgGl, clone mAbll, BD biosciences);
Alexa Flour-
700 labeled IFN-y (mIgGl, clone B27, BD biosciences). For surface staining,
cells were
incubated with the antibodies for 30 minutes at 4 C in the dark, then washed
three times and
fixed with 1% paraformaldehyde to be analyzed in a FACS Canto (Becton
Dickinson). For
intracellular cytokines, cells were stained using BD cytofix/cytoperm
fixation/permeabilization
kit according to the manufacturer directions. For caspase-1 staining, cells
were incubated with
substrates (CaspaLux-E1D2, OncoImmunin, Inc.) for 50 minutes at 37 C and
washed with
washing buffer 2 times, then stained for surface markers for 10 minutes at
room temperature.
[00318] Statistical analysis. All statistics and graphs were done with
Prism software
(GraphPad, La Jolla, CA). Differences in variables between any 2 groups were
analyzed using
the Mann-whitney test or two-tailed t-test. Differences between any 3 or more
groups were
analyzed by analysis of variance (ANOVA).
EXAMPLE 6
RESULTS: MYELOID CELL-DERIVED IL-1B PROMOTESTSLP-TH2
INFLAMMATION TO FOSTER BREAST TUMOR GROWTH
[00319] Human breast cancer tumor microenvironment displays features of
TSLP-driven
Th2 inflammation that promote tumor development. The inventors have discovered
the
underlying molecular mechanisms by which TSLP is regulated. The results set
forth in this
Example show that IL-1P induces TSLP production from breast cancer cells lines
in a dose
- 106 -
CA 02874065 2014-12-04
dependent manner in vitro. Cancer cells induce both transcription and
secretion of IL-1p in
myeloid dendritic cells (mDCs) and monocytes. This is mediated by cancer cell-
derived TGF-13.
Moreover, TAK1 signaling is involved in caspasel activation and TGF-P-
dependent IL-1p
production. Administration of anti-TGF-p neutralizing antibody or IL-1R
antagonist Anakinra
prevents tumor growth in vivo and blocks iTh2 generation in vivo. Moreover,
significantly
higher levels of IL-10 are present in cancer tissue than surrounding tissue.
The level of IL-ip
correlates positively with the level of IL-13 in tumor tissue of patients.
Importantly, IL-113 level
is associated with the stage of the disease. Thus blockade of IL-113
represents a novel approach to
breast cancer immunotherapy.
[00320] IL-
113 induces TSLP production from breast cancer cells. Different pro-
inflammatory cytokines have the potential to induce TSLP from keratinocytes,
both
transcriptionally and translationally. Whether IL-10 could induce TSLP
production from breast
cancer cells was tested. Breast cancer cell lines HS-578t or MDA-MB231 cells
in culture were
treated with medium alone, IL-1[3, IL-1 a, IL-6 or TNF-a for the indicated
time course. Cells
were harvested and TSLP mRNA levels were measured by quantitative real-time
PCR (FIG.
4A). As early as 2 hours, IL-113 and IL-la could induce significant TSLP
transcription in both
cells lines tested (FIG. 4A, ***P<0.0001, "P<0.01). MDA-MB231 cells were
cultured in
media alone, or treated with different cytokines (IL-6 and soluble IL-6r, TNF-
a, IL-1p or IL-la
at different doses as indicated), or PMA and ionomycin for 48 hours (FIG. 4B,
FIG. 9A). TSLP
levels in the culture supernatants were determined by Luminex. IL-1p could
induce TSLP
production in a dose-dependent manner. 10 ng/ml of IL-1J3 gave rise to
significantly highest
level of TSLP production compared to TNF-a, IL-6 and or soluble IL-6r, or IL-
la (P=0.0001;
FIG. 4B and FIG. 9A). IL-1p shows similar effect on another breast cancer cell
line HS-578t
(FIG. 9B). To determine whether IL-1p was responsible for specific TSLP
production from
cancer cells, MDA-MB231 cells were cultured with IL-1p in the presence of anti-
IL-1P
neutralizing antibody, or non-neutralizing antibody for 24-72 hours. The pre-
treatment of IL-1J3
with neutralizing antibody was able to abolish the ability of IL-1J3 to induce
TSLP from cancer
cells at different time point (P=0.001, FIG. 4C).
[00321] To
investigate whether the TSLP induction effect is due to IL-1p enhanced-
TSLP generation in single cells, or possibly caused by IL-1(3's direct effect
on cancer cell
proliferation if any, MDA-MB231 cells were cultured in chamber wells in
presence or absence
- 107 -
CA 02874065 2014-12-04
of different doses of IL-113 (as indicated) for 24 hours, the last 5 hours
Golgi-plug and Golgi-stop
was added to the cell culture in order to allow TSLP accumulation
intracellularly. 10 ng/ml of
IL-113 induces more accumulation of TSLP compared with a lower dose of IL-113
treatment.
Both doses (lng/ml and 1 Ong/m1) are able to significantly induce TSLP
production from single
cell level, compared to in absence of IL- 10 (FIG. 4D). Furthermore, when the
TSLP-dependent
Baf3 cell (TSLPR+/IL-7Ra+) line cells in presence or absence of IL-1P-treated
breast cancer cells
culture supernatants. The Baf3 cells do not proliferate in presence of IL-113
alone, and show very
limited proliferation upon unstimulated-cancer cell culture sups treatment,
while IL-113
stimulated MDA-MB231 cells culture sups or HS-578t cells culture sups enhance
the
proliferation of the Baf3 cells in a dose-dependent manner (FIG. 9C),
demonstrating IL-113-
induced TSLP is bioactive.
[00322]
High IL-113 level in tumor clinically correlates with TSLP level in patients.
TSLP and IL-1P were screened by Luminex in supernatants of human breast tumor
fragments (T)
from patient, stimulated for 16 hours with PMA and ionomycin. In line with the
findings that
IL-113 induced TSLP production from in vitro breast cancer cell lines culture,
the level of IL-113
in patients tissue is correlated with the level of TSLP in a subset of
patients (FIG. 4E; pairs=147 ,
P=0.019 with spearman r=0.19).
[00323] To
determine the expression pattern of IL-1I3 in breast cancer tissue of
patients,
IL-113 and other innate cytokines were also compared between tumor tissue (T)
and surrounding
tissue (ST) from the same patient, stimulated for 16 hours with PMA and
ionomycin. Tumor
tissue express high amount of IL-1 family cytokines (FIG. 10A). In 138
patients, the tumor tissue
displayed significantly higher level of IL-10 than that of matched
macroscopically uninvolved
surrounding tissue (FIG. 5A; ST: mean SEM = 218.9 75.76 pg/ml; T: mean
SEM = 527
127.8 pg/ml. P(0.0001, n=138). Accordingly, tumor tissue displayed higher
levels of IL-1Ra
(FIG. 10B; ST: mean SEM = 1257 332.2 pg/ml; T: mean SEM = 4489.6 979
pg/ml.
P<0.0001, n=55). No difference of IL-la level exists between surrounding
tissue and tumor
tissue (FIG. 10C; ST: mean SEM = 12.3 3.3 pg/ml; T: mean + SEM = 17.2 3.4
pg/ml.
P=0.281, n=62).
[00324]
Infiltrating myeloid cells express IL-113 in patients, both in primary tumor
tissue and lymph node metastasis. Cancer cells can directly express IL-113
transcripts or induce
- 108 -
CA 02874065 2014-12-04
cells within the tumor microenvironment to do so (Portier et al, 1993).
Studies have documented
constitutive IL-1f3 protein production in human and animal cancer cell lines
including sarcomas
and ovarian and transitional cell carcinomas (Dinarello, 1996). To identify
the cells producing
IL-113 in human breast cancer tissue, frozen tissue sections from surgically
removed primary
tumors or axillary lymph node metastasis of breast cancer patients were
analyzed by
immunofluorescence. All 20 primary tumors screened were positive for tissue IL-
1[3 staining,
and IL-1(3 was present in infiltrating cells rather than in tumor cells
visualized by expression of
TSLF' and cytokeratin-19 (Fig. 58 shows one representative staining pattern).
To further
characterize the infiltrating cellular types which co-express IL-113, frozen
sections were stained
with different combinations of myeloid cells markers and anti-IL-113 antibody.
IL-1I3 expressing
cells were mainly HLA-DRhi, CD1 1 c+, and CD14+ cells. These cells could be
dendritic cells
(DCs) and tissue monocytes. IL-113 was also detected in some CD163+, and CD68+
cells (Figure
2C). Metastatic tumor from surgically removed axillary lymph node tissue was
stained for
cytokeratin-19, TSLP, IL-1(3 and CD1 1 c (FIG. 5D). Myeloid cells express IL-
1(3, although
metastatic tissue shows less IL-113+ infiltrates in contrast to primary tumor
tissue.
[00325]
Cancer surface factor(s) induce IL-1I3 production in mDCs. The inventors
observed that IL-113+ infiltrating myeloid cells are always localized near
TSLP-expressing tumor
cells. Therefore, whether cancer derived factors are able to induce IL-1f3
production from
myeloid cells was tested. To do so, blood monocytes, mDCs, monocyte-derived
dendritic cells
(MDDC), and monocyte-derived macrophages were stimulated for 48 hours by
cancer cell-
culture sups, which is known being able to drive Th2 polarization through
activation of mDCs in
vitro (Pedroza et al., 2012). The cancer cell-culture supernatants could
induce IL-113 from
monocytes, but not from MDDC, mDC, or macrophages (FIG. 11A). Next, the
transwell system
was used to investigate whether the in vitro modulation of IL-113 production
by myeloid cells
would rely on direct contact of surface molecules between cancer cells and
myeloid cells. So
monocytes, MDDCs, monocyte-derived macrophages and blood mDCs were co-cultured
with
MDA-MB231 or HS-578t cells, which were separated by the transwell membrane
with a 0.3um
pore diameter. Cancer cells or myeloid cells alone do not produce detectable
IL-1p after 48
hours of culture. Co-culture with cancer cells in direct contact manner
significantly enhanced
IL-113 production in the supernatant of the well where monocytes, MDDC, and
mDCs were
present, but not macrophages (FIG. 6A). Co-culture with cancer cells in
transwells significantly
- 109 -
CA 02874065 2014-12-04
enhanced the IL-1p production by monocytes, MDDC and mDCs, whereas IL-1(3
production
from macrophages remained unaffected (FIG. 6A). These experiments show that
for MDDC and
mDCs, the modulation of IL-1f3 production is totally contact dependent,
whereas for monocytes
the modulation is partially contact-dependent, and could be induced by a
soluble factor derived
from cancer cells.
[00326] Cancer surface bound TGF-I3 is involved in IL-113 production during
cancer
cells co-culture with mDCs. It is well known that TGF-P expression increases
markedly in
human cancers, including breast cancer. TGF-P-related-signal transduction/gene
activation has
been implicated in the oncogenesis of many human cancers. TGF-P has the
potential to induce
IL-113 mRNA in human monocytes (Allen et al., 1990). In various disease models
where IL-10
plays a pathogenic role, TGF-P is also over-expressed and involved (Lee et
al., 2012; Hideaki et
al., 2008). Another very important characteristic of TGF-P is its abundant
surface expression
pattern and restrictive transformation from latent form to active form to
allow function (Gleizes
et al., 1997). To investigate whether cancer cell-derived TGF-13 contributes
to IL-10 production
in mDCs during contact, mDCs were cultured together with breast cancer cells
(MDA-MB231
and HS-578t) or benign counterpart cells (1-1S-Bst, derived from the same
patients as HS-578t
cells). In presence of the benign HS-Bst cells, IL-113 was barely induced from
mDCs (FIG.
6B); while in presence of HS-578t cells or MDA-MB231 cells, IL-1(3 was induced
in mDCs. We
then examined the cancer cells surface expression for the activated from of
TGF-p. Both MDA-
MB231 cells and HS-578t cells express active TGF-p, whereas HS-Bst cells did
not express
activated TGF-P (FIG. 6C). Furthermore, cancer cells express CD105 but not
CD36 on their
surface, indicating the possibility of surface activation of TGF-13 (FIG. 13).
[00327] To further investigate whether TGF-P signaling is involved in both
transcription
and secretion of IL-113 during co-culture, different doses of TGF-I3R kinase
inhibitor, anti-TGF-p
neutralizing antibody, or control (DMSO, or isotype control respectively) were
used to treat the
cells in co-culture. Block TGF-P using neutralizing antibody or receptor I
kinase inhibitor could
affect IL-1[3 production from mDCs (FIG. 6D). A similar effect could also be
observed when
cancer cells were co-cultured with MDDCs (FIG. 12A left), whereas it partially
but significantly
reduced 1L-1p production from monocytes (FIG. 12A right). The less amount of
IL-113 released
post TGF-p blocking (inhibition of receptor signaling and or blocking the
effect of TGF-P
through neutralizing antibody) could also be reflected by less IL-10
expressing DCs or
- 110-
CA 02874065 2014-12-04
monocytes in co-culture (FIG. 6E, and FIG. 12B). TGF-P blocking resulted in
decreased
mRNA level of IL-113 (FIG. 6F,and FIG. 12C). Thus, TGF-f3/TGF-pR signaling is
involved in
IL-1f3 production during cancer cell co-culture with either mDCs or monocytes.
[00328] IL-
1I3 production requires TAK1-dependent caspase-1 activation. In most
cases, caspase-1 is needed for proteolytic cleavage and secretion of mature IL-
1(3. It was tested
whether TGF-p-dependent IL-113 production also required the activation of
caspase-1. MDA-
MB231 cells and mDCs were co-cultured in chamber well in presence of 1 uM of
caspase-1
inhibitor or DMSO as vehicle control. 18 hours later, cells were fixed and
stained with specific
antibody against pro-peptide of IL-1p (FIG. 7A), or mature IL-1(3 (FIG. 7B),
both accompanied
with HLA-DR staining. With treatment of caspase-1 inhibitor, accumulated pro-
peptide of IL-1
could be detected inside HLA-DR+ DCs, while DMSO treatment showed much less
pro-peptide
staining, suggesting pro-peptide has been processed by functional activated
caspase-1 (FIG. 7A).
Four areas were counted for the percentage of prolL-113 cells within HLA-DR+
cells for both
DMSO or caspase-1 inhibitor treated wells. Almost 95% of mDCs show
accumulation of proIL-
113 intracellularly, while only around 50% of mDCs express proIL-1[3 (FIG. 7A
right panel).
Using a similar experimental system, mature IL-113 production in presence or
absence of caspase-
1 inhibitor was also measured. In contrast to prolL-113, less mature IL-10 was
detected with
defective caspase-1 function (Figure 4B).
[00329]
Since caspase-1 activation is also required for IL-1(3 release during co-
culture,
the inventors considered which factor(s) could possibly link TGF-OR signaling
with caspase-1
activation. TAK1 (TGF-f3R-activating protein kinase 1) is known to stimulate
inflammasome-
caspasel activation (Eicke et al, 2013; Gong et al, 2010), and evidence shows
that TAK1 activity
is important for TGF-p-mediated angiogenesis and metastasis of breast tumors
(Safina et al,
2008). Monocytes were treated with cancer-culture sups for 16 hours in the
presence of a TAK1
specific inhibitor or DMSO as control. Caspase-1 activity was measured using
the FACS based
method. Cells were gated based on CD11c+ viable cells. Around 94% of monocytes
express
active form of caspase-1 after exposure to cancer culture sups, while TAK1
inhibition prevented
the activation of caspase-1 even in presence of cancer derived factors (FIG.
7C). Similar
experiments were repeated using multiple cancer cell lines culture sups, and
similar effect was
observed (FIG. 7C).
- 111 -
CA 02874065 2014-12-04
[00330] IL-1p production requires TGF-p-dependent TAK1 activation in DCs
and
monocytes. To measure whether TGF-f3 resulted in direct activation of TAK1,
monocytes were
treated with 10 ng/ml of TGF-131 for 5-90 minutes. Soluble TGF-131 was able to
induce
phosphorylation of TAK1 as early as 15 minutes (FIG. 14A). When mDCs were co-
cultured
with MDA-MB231 cells for 5-90 minutes, phosphorylated (p)TAK1 could only be
detected after
60 minutes (FIG. 7D). To confirm that the phosphorylation of TAK1 is TGF-P
mediated, TGF-13
signaling blocking reagents (TGF-13 neutralizing antibody and TGF-i3R kinase
inhibitor) were
added to the co-culture of cancer cells and mDCs. Blocking TGF-f3 and its
receptor signaling
prevented phosphorylation of TAK1 (FIG. 7E). To examine whether TAK1-signaling
participate
in modulation of IL-1(3 production, a co-culture of cancer cells and myeloid
cells was used.
Cancer cells and monocytes, MDDC, or mDC were co-cultured for 48 hours, and IL-
113 levels
were measured by Luminex. Substantially lower amounts of IL-113 were secreted
in the co-
culture supernatants upon TAK1 inhibition (FIG. 7F, and FIG. 14B). Similarly,
TAK1 inhibition
prevented the expression of IL-113 by mDCs and monocytes (FIG. 14C).
[00331] Blocking TGF-13-IL-113 in vivo prevents tumor growth and Th2
generation.
Blockade of TSLPR using a neutralizing antibody induces tumor regression in a
xenograft mice
model, where the tumor growth is dependent on the TSLP-induced IL-13 (Pedroza
et al., 2011).
Thus, involvement of IL-1(3 in the TSLP-dependent in vivo model was evaluated.
The
availability of IL-1f3 in the xenograft mice model was first evaluated. HS-
578t cells were
injected subcutaneously into the flank of irradiated NOD/SCID/f32m-/- mice.
MDDCs plus
autologous total T cells were injected intratumorally on D3,6, and 9 after
tumor cells injection
(FIG. 15A). When tumors grew to 100-150 mm3 in volume, mice were sacrificed
and tumors
were frozen in OCT for tissue staining. Similar with what observed in patient
primary tumors
(FIG. 5), IL-1(3+ cells also infiltrated in the tumor of xenograft model (FIG.
15B). Moreover, the
IL-1(3+ cells closely localized around the TSLP+ cancer cells, suggesting a
crosstalk between the
IL-1(3+ cells and cancer cells (FIG. 15B). Then we tried to understand whether
TGF-f3 or IL-1(3
blocking could affect tumor growth through affecting the availability of IL-
1(3. Anti-TGF-13
neutralizing antibody, isotype antibody as control, IL-1R antagonist Anakinra,
or anti-TSLPR
neutralizing antibody were each injected together with DC plus T cells (FIG.
15A). DC+T+PBS
shows accelerated tumor growth, whereas with daily Anakinra injection, the
tumor growth curve
slowly declined shortly after an initial growth (FIG. 8A). The tumor volume
was almost 10-fold
- 112 -
CA 02874065 2014-12-04
increased (mean=128 mm3) in the PBS group compared to the Anakinra group
(mean=13 mm3)
by the end of the observation (FIG. 8A, P<0.0001 on day 16). The tumor volumes
were
comparable between anti-TGF-p neutralizing antibody and Anakinra treatment. On
day 16,
tumors were harvested; 3 representative tumors from each group are shown in
FIG. 15C. In
some experiments, small pieces of tumor tissue from mice were cultured for 16
hours in the
presence of PMA and ionomycin. Th2 cytokines were measured by Luminex in the
culture sups
(FIG. 8B). Anakinra treatment or blocking TGF-b resulted in less IL-13
(P<0.0001, P<0.005
respectively), less IL-4 (P<0.0001, P<0.005 respectively), and Anakinra
treatment also resulted
in less TSLP production (P=0.03) in tumor tissue.
[00332] To confirm whether the decreased IL-13 level is due to less
infiltration of IL-13
producing cells, single cell suspensions were prepared from the tumors
harvested from both the
PBS and the Anakinra treatment groups. Intracellular staining for cytokines
including IL-13, IL-
4, TNF-a, and IFN-y were combined with CD4, CD3 surface staining. Cells were
nalyzed by
FACS, and gated on viable CD4+CD3+T cells. The treatment showed no specific
effect on TNF-
a and IFN-y. In the PBS group, about 10.2-46.4% of CD4+CD3 T cells express IL-
13. In the
Anakinra treatment group, the population dropped to 0.39-2.5%, which is
significant (FIG. 15D).
Importantly, as shown in FIG. 8C, anti-TGF-P neutralizing antibody treatment
resulted in
decreased IL-1P (P=0.02, n=9).
[00333] IL-1p is associated with Th2 inflammation in breast cancer tissue
of patients
and shows clinical significance. The level of IL-13 correlated with that of
TSLP in
approximately 50% of samples (FIG. 16A, pairs=148, P<0.0001 with spearman
r=0.55). The
level of IL-13 also positively correlated with the level of IL-113 (FIG. 17A
right, pairs=149,
P<0.0001 with spearman r=0.43) and IL-la (FIG. 17A left, pairs=101, P=0.0001
with spearman
r=0.375). No correlation existed between IL-13 with other cytokines tested
(FIG. 16B),
including IL-1Ra (pairs=45, P=0.11), IL-18 (pairs=52, P=0.77), IL-33
(pairs=51, P=0.94), and
IL-25 (pairs=42, P=0.64).
[00334] To investigate the clinical significance of IL-1P in breast cancer,
patients were
further categorized by clinical stage (0, I, II, III and IV) and grade status.
Since only one patient
was with stage 0, and one patient with stage IV, we compared the differences
between stage 0-1,
II and Patients with stage II or stage III-IV tumors appeared to have
significantly higher
- 113 -
CA 02874065 2014-12-04
levels of 1L-1J3 than those with stage 0-1 (P=0.006, P=0.01, respectively;
FIG. 17B left). IL-la
does not have similar clinical significance as IL-1p (FIG. 17B right). Thus,
IL-113 is clinical
significant in breast cancer tissue, and is associated with the invasiveness
status of the disease.
No significant difference was found in the level of IL-lp among tumor grades
(Data not shown).
[00335]
Hormone receptor (ER, PR) status and HER2 expression of the patients tumor
tissue were determined by pathologist of Baylor University Medical center,
based on IHC
staining. Statistical significance of IL-113 abundance within each group was
determined using one
sample T test. ER-PR+HER2- (n=1) and ER+PR-HER2- (n=7) are excluded from
analysis
because of limited sample number. ER-PR-HER2-group, and ER+PR+Her2+ group
patients tend
to have higher level of tissue IL-1p (Table 50).
Table 50
Hormone status Case n. Estimate Std. Error t value Pr(>
Jti)
ER-PR-HER2- 13 1947.71 565.68 3.443 O.000779***
ER-PR-HER2+ 21 204.57 445.08 0.460 0.646573
ER+PR-HER2+ 11 743.91 614.96 1.210 0.228645
ER+PR+H ER2- 36 440.71 339.93 1.296 0.197172
ER+PR+H ER2+ 45 859.11 304.05 2.826 O.005483**
IL-1J3 level in tumor tissue was determined via Luminex (described in
methods). Hormone
receptor (ER, PR) status and HER2 expression were determined by pathologist of
Baylor
University Medical center, based on IHC staining. ER: estrogen receptor; PR:
progesterone
receptor; HER2: human epidermal growth factor receptor 2. Statistical
significance of IL-10
abundance within each group was determined using one sample T test. Pr value
indicate the
difference between each group with the number O. ** and *** indicates
significant difference
existing.
* * * * * * * * *
[00336]
Although certain embodiments have been described above with a certain degree
of particularity, or with reference to one or more individual embodiments,
those skilled in the art
could make numerous alterations to the disclosed embodiments without departing
from the scope
of this invention. Further, where appropriate, aspects of any of the examples
described above
may be combined with aspects of any of the other examples described to form
further examples
having comparable or different properties and addressing the same or different
problems.
Similarly, it will be understood that the benefits and advantages described
above may relate to
one embodiment or may relate to several embodiments.
- 114-
CA 02874065 2014-12-04
[00337] The
claims are not to be interpreted as including means-plus- or step-plus-
function limitations, unless such a limitation is explicitly recited in a
given claim using the
phrase(s) "means for" or "step for," respectively.
- 115 -
CA 02874065 2014-12-04
REFERENCES
The following references, to the extent that they provide exemplary procedural
or other
details supplementary to those set forth herein, are specifically incorporated
herein by reference.
Anderson, Cancer Invest. 27:361-368, 2009.
Andre, et al., Curr Opin Oncol. 22:547-551, 2010.
Aspord, et al., J Exp Med. 204:1037-47, 2007.
Banchereau & Palucka, Nat Rev Immunol. 5:296-306, 2005.
Berry, et al., JAMA. 295:1658-1667, 2006.
Bhowmick, et al., Nature. 432:332-337, 2004
Blum, et al., Clin Breast Cancer. 7(11):850-856, 2007.
Blum, et al., J Clin Oncol. 17:485-493, 1999.
Boon, et al., Annu Rev Immunol. 12:337-365, 1994.
Carey, et al., Clin Cancer Res. 13:2329, 2007.
Chapuis, et al., Sci Transl Med. 5(174):174ra27, 2013.
Citron, et al., J Clin Oncol. 21:1431-1439, 2003.
Cobb, et al., J Immunological Methods. 365:27-37, 2011.
Cortes, et al., Lancet. 377:914-923, 2011.
Coussens, et al., Science. 33:286-291, 2013.
Craig, et al., Mol Cancer Ther. 12(1):104-16, 2013.
Cytoxan package insert, 2005.
DeNardo, et al., Breast Cancer Res. 9:212-221, 2007.
DeNardo, et al., Cancer Discov. 1:54-67, 2011.
Dinarello, Blood. 87:2095-147, 1996.
Dinarello, Arthritis Rheum. 52:1960-7, 2005b.
Dinarello, J Exp Med. 201:1355-9, 2005a.
Disis & Schiffman, Semin Oncol. 28:12-20, 2001.
Early Breast Cancer Trialists' Collaborative Group, Lancet. 365:1687-1717,
2005.
Early Breast Cancer Trialists' Collaborative Group, N Engl J Med. 319:1681-
1692, 1988.
Economides, et al., Nature Med. 9:47-52, 2003.
Egloff, et al., Cancer Res. 66:6-9, 2006.
Eisenhauer, et al., Eur J Cancer. 45(2):228-247, 2009.
- 116-
CA 02874065 2014-12-04
Finn, et al., Nat Rev Immunol. 3:630-641, 2003.
Fisher, et al., J Clin Oncol. 15:2483-2493, 1997.
Galon, et al., Science. 313:1960-1964, 2006.
Gartlehner, et al., J Rheumatol. 33:2398-408, 2006.
Gilboa, Immunity. 11:263-270, 1999.
Goldbach-Mansky, et al., N Engl J Med. 355:581-92, 2006.
Gradishar, et al., J Clin Oncol. 23(31):7794-7803, 2005.
Hawkins, et al., Arthritis Rheum. 50:607-12, 2004.
Hoffman, et al., Lancet. 364:1779-85, 2004.
J Smith, "Reprogramming T cell specific immune responses to Cyclic B1 in
breast cancer
patients using a TLR 8/7 agonist," Doctoral Dissertation, 2012.
Jones, et al., J Clin Oncol. 13:2567-2574, 1995.
Jurmann, et al., Am Soc Nutritional Sci. 135(8):1859-64, 2005.
Jurmann, et al., Ann NY Acad Sci. 1182:111-123, 2009.
Kao, et al., J Exp Med. 194:1313-1323,2001.
Kineret package insert, December 2009.
Klechevsky, et al., Blood. 116:1685-1697, 2010.
Knutson, et al., J Clin Invest. 107:477-484 2001.
Kristensen, et al., Proc Natl Acad Sci USA. 109:2802-2807, 2012.
Kroemer, et al., Annu Rev Immunol. 31:51-72, 2013.
Kurzrock, Cancer 92(6 Suppl):1684-8, 2001.
Lewis, et al., J Transl Med. 4:48-60, 2006.
Liedtke, et al., J Clin Oncol. 26:1275-1281, 2008.
Linkhart, et al., J Bone Miner Res. 6(12):1285-94, 1991.
Loi, et al., J Clin Oncol. 31:860-867, 2013.
Ma, et al., Immunity. 38(4):729-41, 2013.
Mamounas, et al., J Clin Oncol. 23:3686-3696, 2005.
Mattarollo, et al., Cancer Res. 71:4809-4820, 2011.
Michaud, et al., Science. 334(6062):1573-7, 2011.
Miller, et al., NEJM. 357:2666-76, 2007.
Neidhardt-Berard, et al., Breast Cancer Res. 6:R322-328, 2004.
- 117-
CA 02874065 2014-12-04
Palucka & Banchereau, Nat Rev Cancer. 12:265-277, 2012.
Pardo11, et al., Nat Med. 4:525-531, 1998.
Park, et al., Cancer Res. 68:8400-8409, 2008.
Pascual, et al., J Exp Med. 201:1479-86, 2005.
Pedroza-Gonzalez, et al., J Exp Med. 208(3):479-90, 2011.
Pedroza-Gonzalez, et al., J Exp Med. 208:479-90, 2011.
Petrasek, et al., Am Soc Clin Invest. 122(10):3476-89, 2012.
Portier, et al., Br J Haematol. 85:514-20, 1993.
Qi, et al., Oncol Rep. 28(4):1231-6, 2012
Rastogi, et al., J Clin Oncol. 26:778-785, 2008.
Rodel, et al., J Clin Oncol. 23(34):8688-8696, 2005.
Rosenberg, et al., Immunol Today. 18:175-182, 1997.
Safina, et al., Oncogene. 27(9):1198-207, 2008.
Saito, et al., Breast Cancer Res. 8:R65, 2006.
Sandoval, et al., Sci Transl Med. 5:72ra20, 2013.
Schneider, et al., Clin Cancer Res. 14:8010-8018, 2008.
Smith, et al., J Biol Chem. 275:1169-75, 2000.
Sorlie, et al., Proc Natl Acad Sci U S A. 98:10869-10874, 2001.
Strayer, et al., Breast Cancer Res Treat. 119:551-558, 2010.
Suzuki, et al., Clin Cancer Res. 11:1521-1526, 2005.
Symmans, et al., J Clin Oncol. 25:4414-4422, 2007.
Symons, et al., PNAS. 92(5):1714-18, 1995.
Taxol package insert, 2011.
Terabe & Berzofsky, Curr Opin Immunol. 16:157-162, 2004.
Teschendorff, et al., BMC Cancer. 10:604, 2010.
Townsend, et al., Cell. 42:457-467, 1985.
Von Minckwitz, et al., SABCS. S3-2 abstract, 2011.
Wood, et al., Oncol Nurs Forum. 33(3):535-42, 2006.
Yi, et al., Cancer Research. 72(24):Suppl 3, 2012.
Young, et al., Eur J Cancer. 35(13):1773-1782, 1999.
Yu, et al., Mol Immunol. 38:981-987, 2002.
- 118 -
CA 02874065 2014-12-04
Zhang, et al., Cytokine. 42:39-47, 2008.
- 119-
