Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
Tumor Lysate Loaded Particles
100011
BACKGROUND OF THE INVENTION
100021 A tumor cell exists in part because it has selected for one or more
mutations that
allows it to partially or completely escape immune surveillance in vivo.
100031 In an attempt to elicit an immune response to a tumor cell, previous
researchers have
used dendritic cells, which are professional antigen-presenting cells, to
present tumor
antigens to the immune system. For example, dendritic cells pulsed with
peptide or tumor
lysate have been used to vaccinate melanoma patients.
[0004] However, simply presenting tumor antigens to the immune system in the
foregoing
manner has not been effective because such antigens were merely endocytosed by
the
dendritic cells and generally presented through the Major Histocompatibility
Complex
(MHC) class II, which elicits only helper T cells and does not provide a
robust immune
response.
[0005] In contrast, presenting tumor antigens via the MHC class I pathway
contributes to a
more robust anti-tumor immunity by activating CD8+ T cells. Previous
researchers have
attempted to present tumor antigens through the MHC class I pathway by using
gene transfer
methods. However, these methods have disadvantages, including (1) a limited
ability to
identify all of the important tumor-specific antigens, (2) a limited ability
to map the genes of
specific tumor antigens, (3) only one or a small number of known tumor antigen
genes can be
introduced into a dendritic cell and (4) the methods are time-consuming and
cumbersome.
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SUMMARY OF THE INVENTION
[0006] Some embodiments relate to an isolated dendritic cell comprising a
phagocytosed
component consisting essentially of (i) a particle and (ii) a tumor lysate
loaded within the
particle. In specific embodiments, the tumor lysate is present in an amount
from about 200
jig to about 500 jig. In specific embodiments, the tumor lysate is present in
an amount of
about 200 g.
[0007] In some embodiments, the tumor lysate is a lysate selected from a
cancer selected
from the group consisting of breast cancer, small cell lung cancer, non-small
cell lung cancer,
glioma, medulloblastoma, neuroblastoma, Wilms tumors, rhabdomyosarcoma,
osteosarcoma,
liver cancer, pancreatic cancer, melanoma, prostate cancer and ocular
melanoma.
[0008] In some embodiments, the particle is a yeast cell wall particle. In
some
embodiments, the dendritic cell is an immature cell that has been isolated for
no more than 8
days.
[0009] Another embodiment relates to a vaccine comprising the foregoing
isolated dendritic
cell.
[0010] Some embodiments relate to a method for producing an isolated dendritic
cell
containing a tumor lysate loaded particle comprising: (i) loading the tumor
lysate into the
particle to produce the tumor lysate loaded particle; (ii) freeze-drying the
tumor lysate loaded
particle; and (iii) incubating the tumor lysate loaded particle with a
dendritic cell, wherein the
incubating causes the dendritic cell to phagocytose the tumor lysate loaded
particle.
[0011] In specific embodiments, the foregoing method further comprises (a)
resuspending
the tumor lysate loaded particle in solution and (b) freeze-drying the
resuspended solution
before step (iii). In specific embodiments, the tumor lysate is produced by
freezing and
thawing the tumor. In specific embodiments, the foregoing method further
comprises
repeating the freezing and thawing steps. In specific embodiments, the
foregoing method
further comprises cryopreserving the tumor lysate at -150 C.
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[0012] In specific embodiments, step (iii) comprises: (a) adding tumor lysate
into a yeast
cell wall particle, (b) incubating the yeast cell wall particle, (c) freeze-
drying the yeast cell
wall particle and (d) washing the yeast cell wall, wherein steps (b)-(c) are
repeated at least
once with a step of adding water into the yeast cell particle before step (b)
is repeated.
[0013] In specific embodiments, step (iii) comprises: (a) contacting the tumor
lysate loaded
particle and the dendritic cell at a ratio of about 100:1, (b) incubating the
tumor lysate loaded
particle with the dendritic cell for 1 to 2 hours and (c) collecting the
dendritic cell and
washing the cell.
[0014] In a preferred embodiment, a small amount of one or more immune
response
enhancing adjuvants is added to the tumor lysis buffer prior to incubating
with a dendritic
cell. The addition of one or more adjuvants increases the effects of the tumor
lysate on the
dendritic cell while dramatically decreasing any systemic effects from such
adjuvants.
Commonly used adjuvants include but are not limited to proteins, peptides,
nucleic acids and
carbohydrates.
[0015] Exemplary adjuvants include but are not limited to monophosphoryl lipid
A, GpC
ologonucleotides (such as GpC DNA), Poly I:C, Poly ICLC, potent MHC II epitope
peptides,
beta glucan, and dendritic cell stimulating cytokines such as IL-12. Suitable
adjuvants are
those molecules known to interact with receptors on dendritic cells in order
to activate
dendritic cells and further stimulate a more robust generation of T cells,
such as CD4+ and
CD8+ T cells.
[0016] In one embodiment, the amount of one or more immune response enhancing
adjuvants is at least about 5 jig, at least about 10 jig, at least about 15
jig, at least about 20
jig, at least about 25 g, at least about 30 jig, at least about 35 jig, at
least about 40 jig, at
least about 45 jig, at least about 50 g, at least about 60 jig, at least
about 70 gg, at least
about 80 jig, at least about 80 g, at least about 90 jig, or at least about
100 jig. In one
embodiment, the amount of adjuvant represents between 1-10% of the total
lysate amount.
The amount of adjuvant is sufficient to stimulate receptors, such as the toll-
like receptor, on
the dendritic cell.
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[0017] Some embodiments relate to a method for treating cancer, comprising
administering
a vaccine comprising the foregoing isolated dendritic cell. In specific
embodiments, the
cancer is selected from the group consisting of breast cancer, small cell lung
cancer, non-
small cell lung cancer, glioma, medulloblastoma, neuroblastoma, Wilms tumors,
rhabdomyosarcoma, osteosarcoma, liver cancer, pancreatic cancer, melanoma,
prostate
cancer and ocular melanoma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 depicts a process for producing dendritic cells.
[0019] FIG. 2 depicts a process for producing tumor lysate.
[0020] FIG. 3 depicts a process for producing yeast cell wall particles.
[0021] FIG. 4 depicts a process for loading tumor lysate into yeast cell wall
particles.
[0022] FIG. 5 depicts a process for producing tumor lysate particle loaded
dendritic cells.
[0023] FIG. 6 is a graph comparing the effect of tumor lysate particle loaded
dendritic cells
versus antigen pulsing of dcndritic cells on B3Z cells.
[0024] FIG. 7A shows the lungs of control mice afflicted with B16F0 murinc
melanoma.
FIG. 7B shows the lungs of mice afflicted with B16F0 murine melanoma but
treated with
tumor lysate particle loaded dendritic cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] Reference is made herein to various methodologies known to those of
ordinary skill
in the art.
[0026] As used herein, the singular forms "a," "an," and "the" designate both
the singular
and the plural, unless expressly stated to designate the singular only.
Likewise, singular
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forms of terms designate both the singular and plural, unless expressly stated
to designate the
singular only. For example, "vaccine" means "vaccine" or "vaccines."
[0027] The term "about" in connection with numerical values and ranges means
that the
number comprehended is not limited to the exact number set forth herein, and
is intended to
refer to ranges substantially within the quoted range while not departing from
the scope of the
invention. As used herein, "about" will be understood by persons of ordinary
skill in the art
and will vary to some extent on the context in which it is used.
[0028] As used herein "subject" or "patient" denotes any animal in need of
treatment with a
vaccine. For example, a subject may be suffering from or at risk of developing
a condition
that can be treated or prevented with a vaccine. As used herein "subject" or
"patient"
includes humans.
[0029] As used herein, the phrases "therapeutically effective amount" and
"therapeutic
level" mean that vaccine dosage or plasma concentration in a subject,
respectively, that
provides the specific response for which the vaccine is administered in a
subject in need of
such treatment. For convenience only, exemplary dosages, vaccine delivery
amounts,
therapeutically effective amounts and therapeutic levels are provided below
with reference to
adult human subject. Those skilled in the art can adjust such amounts in
accordance with
standard practices as needed to treat a specific subject and/or
condition/disease.
[0030] Tumor Lysate
[0031] As described herein, "tumor lysate" refers to a tumor that has been
lysed. Tumor
lysis can occur under a number of conditions, including repeated freezing and
thawing of the
tumor, physical breakage of the tumor by homogenizing, contact with a hyper-
or hypo-tonic
solution and contact with one or more non-ionic detergents. The tumor lysate
is not cross-
linked during the lysing process. In another embodiment, the tumor lysate is
produced by
mincing, grinding or mashing the tumor, or otherwise pulverizing the tumor
using any known
technique in the art. In another embodiment, the tumor lysate is produced by
mincing,
grinding, mashing or pulverizing the tumor in the presence of phosphate buffer
solution
(PBS), such as 1X PBS.
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[0032] In specific embodiments, the tumor lysate is produced from a solid
tumor weighing
a minimum of 200 to 500 lug.
[0033] In another embodiment, the tumor lysate is produced by mincing,
grinding, mashing
or pulverizing the tumor followed by repeated freezing and thawing. In
specific
embodiments, the minced tumor is frozen and thawed multiple times. In specific
embodiments, the minced tumor is frozen and thawed at least 1, 2, 3 or 4
times. In some
embodiments, the freezing is performed in liquid nitrogen, and can be
performed for 20
minutes. In specific embodiments, the thawing is performed at room
temperature. In another
embodiment, the tumor lysate is stored at a temperature of about -135 C or
below after the
freezing and thawing process. In specific embodiments, the tumor lysate is
store at a
temperature of -150 C or below after the freezing and thawing process.
[0034] The tumor lysate can be prepared from any solid tumor including, but
not limited to
carcinomas, and sarcomas. In some embodiments, the solid tumors are from
tumors relating
to breast cancer, small cell lung cancer, non-small cell lung cancer, glioma,
medulloblastoma,
neuroblastoma, Wilms tumors, rhabdomyosarcoma, osteosarcoma, liver cancer,
pancreatic
cancer, melanoma, prostate cancer and ocular melanoma.
[0035] A number of immune response enhancing agents can be added to the tumor
lysis
buffer as adjuvants to boost immune response such that when the tumor lysate
is incubated
with a dendritic cell, the adjuvants exhibit an increased effect on the
dendritic cell while
dramatically decreasing any systemic effects from such adjuvants.
[0036] It is within the purview of one of ordinary skill in the art to select
one or more
suitable adjuvants for this invention. For instance, monophosphoryl lipid A,
GpC
oligonucleotides, Poly I:C, Poly ICLC, potent MHC II epitope peptides, and
dendritic cell
stimulating cytokines such as IL-12, 1L-2, and GM-CSF arc good adjuvant
candidates of this
invention.
[0037] Particle
[0038] As described herein, "particle" refers to any hollow and porous
structure that can
contain tumor lysate therein and also allow the lysate to exit the structure.
In some
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embodiments, the size of the particle is about 0.5 to about 5 gm, which
approximates the size
of bacterium to allow the particle to be ingested by monocytes, such as
dendritic cells. In
specific embodiments, the size of the particle is about 0.5 to about 1 pm. In
specific
embodiments, the size of the particle is about 0.5 to about 2.5 ium. In some
embodiments, the
particle can be any particle with a glycan network, so long as the particle is
about 0.5 to about
pm in size.
[0039] In some embodiments, the particle is a bead vector. The bead vector is
not limited
by shape or material, but can be any shape, size or material that allows the
bead vector to be
phagocytosed by monocytes, including dendritic cells.
[0040] In another embodiment, the particle is a yeast cell wall particle
("YCWP"). The
YCWP is prepared from yeast cell wall such that the particle is porous and can
contain lysate
therein. In one embodiment, the YCWP is prepared from Saccharomyces
cerevisiae. In
another embodiment, the YCWP is a zymosan particle. In another embodiment, the
YCWP
approximates the size of microbial structures that cells of the mononuclear
phagocyte system
and other phagocytic cells typically ingests. In specific embodiments, the
YCWP is about 1-
5 pm.
[0041] In one embodiment, the YCWP is prepared by (a) suspending yeast to
produce a
suspension, (b) incubating the suspension, (c) centrifuging the suspension and
removing the
supernatant and (d) recovering the resulting YCWP. In another embodiment,
steps (a)-(d) are
repeated at least 1, 2, 3 or 4 times.
[0042] In another embodiment, the YCWP is prepared by (a) suspending yeast in
a solution
to produce a first suspension, (b) incubating the first suspension, (c)
centrifuging the first
suspension and removing the supernatant, (d) suspending the resulting pellet
to produce a
second suspension, (e) incubating the second suspension, (f) centrifuging the
second
suspension and removing the supernatant and (g) washing the resulting pellet
to recover the
YCWP. In another embodiment, the YCWP is sterilized.
[0043] In specific embodiments, the yeast is suspended in NaOH, including 1M
NaOH. In
specific embodiments, the first suspension is incubated at about 80 C for
about 1 hour or for
1 hour. In specific embodiments, the centrifuging is performed at about 2000
times gravity
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for about 10 minutes, or at 2000 times gravity for 10 minutes. In specific
embodiments, the
pellet is suspended in water, including water at about pH 4.5 or at pH 4.5. In
specific
embodiments, the second suspension is incubated at about 55 C for about 1
hour or at 55 C
for 1 hour. In specific embodiments, the pellet is washed in water at least 1,
2, 3 or 4 times.
In specific embodiments, the pellet is washed once.
[0044] In another embodiment, the YCWP is sterilized using isopropanol and/or
acetone
following washing of the pellet. In specific embodiments, other known alcohols
arc
appropriate. In specific embodiments, the YCWP is allowed to fully dry after
sterilization.
In another embodiment, the YCWP is resuspended after being allowed to dry. In
specific
embodiments, the YCWP is resuspended in PBS, such as 1X PBS. In another
embodiment,
the YCWP is allowed to dry and then frozen before the tumor lysate is loaded
into the
YCWP, in order to place it in storage before use. In specific embodiments, the
YCWP is
freeze dried and store at about 4 C or lower. In specific embodiments, the
YCWP is freeze
dried and store at 4 C.
[0045] Tumor Lysate Loaded Particle
[0046] The particle is loaded with tumor lysate. In one embodiment, the tumor
lysate is
loaded into the particle by incubating the lysate and a suspension of
particles together and
allowing the lysate to penetrate into the hollow insides of the particles.
[0047] In another embodiment, after the particle is loaded with tumor lysate,
the
combination is freeze-dried to create an anhydrous tumor lysate within the
particle. By
freeze-drying the particle loaded with tumor lysate, the lysate is trapped
within the particle
and ready to be phagocytosed by a monocyte, such as a dendritic cell. In
specific
embodiments, the freeze-drying is the only mechanism used to trap the lysate
within the
particle. In specific embodiments, the entrapment is not caused by a separate
component
blocking the lysate from exiting the particle, for example, by physical
entrapment,
hydrophobic binding, any other binding. In specific embodiments, the
entrapment is not
caused by crosslinking or otherwise attaching the lysate to the particle
outside of any
attachment that may occur upon freeze-drying.
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[0048] In another embodiment, the particle is resuspended in solution after
the freeze-
drying. In specific embodiments, the solution is water. In specific
embodiments, the particle
is resuspended to allow additional tumor lysate to penetrate the particle and
then the
combination is freeze-dried again. In other embodiments, the combination is
subjected to
multiple freeze-drying and resuspensions. In other embodiments, the tumor
lysate loaded
particle is sterilized in ethanol after the freeze-drying and before use.
[0049] In specific embodiments, the tumor lysate is loaded into the particle
by (a)
incubating the lysate and a suspension of the particles, allowing the lysate
to penetrate into
the hollow insides of the particles and freeze-drying the suspension of
particles loaded with
lysate and (b) optionally resuspending the particles, incubating the
resuspended particles and
freeze drying the resuspended particles and any tumor lysate not already in
the particle.
[0050] In specific embodiments using YCWPs, the number of YCWPs is about 1 x
109 and
the volume of tumor lysate is about 50 iaL (generated from about 200 lag of
tumor tissue). In
specific embodiments, the number of YCWPs is 1 x 109 and the volume of tumor
lysate is 50
(from about 200 jig of tumor tissue). In specific embodiments, the incubation
in step (a)
is for about 2 hours at about 4 C. In specific embodiments, the incubation in
step (a) is for 2
hours at 4 C. In some embodiments, the foregoing suspension is freeze dried
in step (b) over
a period of about 2 hours or over a period of 2 hours. In some embodiments,
the YCWPs in
step (c) are resuspended in water, including about 50 IA of water or 50 IA of
water. In some
embodiments, the resuspended YCWPs are incubated in step (d) for about 2 hours
at about 4
C or for 2 hours at 4 C.
[0051] Dendritic Cell
[0052] As described herein, "dendritic cell" refers to a cell generated from a
peripheral
blood mononuclear cell (-PBMC"). In one embodiment, a dendritic cell is
prepared by (a)
collecting blood, (b) diluting the blood, (c) performing a density gradient
separation of
PBMCs, (d) lysing red blood cells and washing the PBMCs, (e) incubating the
PBMCs, (f)
removing nonadherent cells and (g) culturing adherent cells in media.
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[0053] In some embodiments, the dendritic cell is an immature dendritic cell
that has been
cultured for no more than 5 days. In other embodiments, the dendritic cell has
been cultured
for 6-8 days.
[0054] In specific embodiments, the blood is heparinized. In specific
embodiments, the
density gradient separation at step (c) comprises placing the blood in a
Lymphocyte
Separation Medium and then centrifuging the blood. In specific embodiments,
the
centrifuging is performed at about 1000 times gravity for about 20 minutes or
at 1000 times
gravity for 20 minutes. In specific embodiments, a second centrifuging is
performed before
step (d) and is performed at about 500g for about 5 minutes or is performed at
500g for 5
minutes. In specific embodiments, a third centrifuging is performed before
step (d) and is
performed at about 500g for about 10 minutes or is performed at 500g for 10
minutes. In
specific embodiments, the lysing is performed using an ACK lysing solution,
followed by
incubation, preferably at room temperature for about 5 minutes, and followed
by a
subsequent centrifugation. In specific embodiments, the PBMCs are washed in
RPMI-1640
medium. In specific embodiments, the PBMCs are incubated at step (e) in flasks
at about 37
C for about 1-2 hours or at 37 C for 1-2 hours. In specific embodiments,
serum-free DC
media is added to the flask.
[0055] In some embodiments, one or more cytokines is present in the culture
media,
including, but not limited to, granulocyte macrophage colony stimulating
factor (800
units/m1) and IL-4 (500 units/m1).
[0056] Tumor Lysate Loaded Particles Phagocytosed in Dendritic Cells
[0057] The tumor lysate loaded particle is phagocytosed within a monocyte,
preferably a
dendrite cell. In one embodiment, the tumor lysate loaded particle is
incubated with a
dendritic cell such that the cell phagocytoses the tumor lysate loaded
particle.
[0058] In specific embodiments, the particle is incubated with the dendritic
cell at a ratio of
about 100:1 or at a ratio of 100:1. The incubation can be performed for in
about 1 hour, 1
hour or preferably less than 1 hour.
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[0059] In specific embodiments, the incubated dendritic cell containing the
tumor lysate
particle is collected and washed, for example, at least 1, 2, 3 or 4 times. In
other
embodiments, the dendritic cells are incubated after washing and resuspended
in freezing
medium. In specific embodiments, the resuspension produces a concentration of
about 10 x
106 cells per ml or 10 x 106 cells per ml. In specific embodiments, the
resuspension is frozen
for storage before use.
[0060] Vaccine
[0061] In one embodiment, the dendritic cell containing a tumor lysate loaded
particle is
used as a vaccine to prevent and/or treat a disease, including cancer. The
disease to be
treated is not particularly limiting, but depends on the particular tumor
lysate loaded into the
particle. For example, a vaccine using tumor lysate from a breast cancer tumor
is used to
treat breast cancer. In another embodiment, a patient's own tumor cells are
used to create the
vaccine. For example, the vaccine can be produced using tumor lysate from a
tumor
associated with breast cancer and then administered to the breast cancer
patient from which
the tumor was extracted. In another embodiment, about 200 ht of a 10 x 106
concentration
of dendritic cells containing tumor lysate loaded particles forms one dose of
the vaccine.
[0062] In another embodiment, the dose is administered by diluting the 200 ht
aliquot to a
final volume of 1 ml before administering the dose to a patient. In specific
embodiments, the
aliquot is diluted with sterile saline containing 5% human serum albumin. In
specific
embodiments, the 200 iut aliquot will need to be thawed before dilution. In
such a scenario,
the length of time between thawing and administration of the dose to a patient
will be no
longer than 2 hours. In some embodiments, the diluted aliquot is administered
in a 3 cc
syringe. In some embodiments, a syringe needle no smaller than 23 gauge is
used.
[0063] In another embodiment, a patient is administered at least 1, 2, 3 or 4
doses. In
specific embodiments, a patient is re-vaccinated once every 4 weeks. In
specific
embodiments, about 1-2 million dendritic cells containing tumor lysate loaded
particles will
be administered at each vaccination. In another embodiment, the dendritic
cells containing
tumor lysate loaded particles are administered to a patient by injection. In
specific
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embodiments, the tumor lysate loaded particles are injected in a patient at or
near (1) a tumor
or (2) a lymph node.
[0064] In some embodiments, the vaccine is not administered with any other
immunosuppressive treatment, such as steroids or chemotherapy. The vaccine can
be
administered using any technique, including intravenous injection and
inhalation.
[0065] The vaccine can also contain biological adjuvants, including but not
limited to
nucleic acids such as GpC oligonucleotides, proteins or peptide epitopes such
as the tetanus
toxoid MHC class 11-binding p30 peptide.
[0066] EXAMPLE 1: Preparing Dendritic Cells
[0067] Dendritic cells were generated from a patient's PBMCs. PBMCs were
collected
from the patient by a blood draw of 200 ml following standard operating
procedures. The
blood was then transferred to 250 ml centrifuge tubes and diluted 1:1 with 1X
PBS. Then, 35
ml of the diluted blood was layered over 15 ml of room temperature Lymphocyte
Separation
Medium (LSM; Mediatech) in 50 ml tubes and centrifuged at 1000g for 20 minutes
at room
temperature. The PBMC layers were removed by pipetting from the LSM gradients
and
placed into clean 50 ml centrifuge tubes. Four volumes of 1X PBS were added
and the tubes
were inverted to mix the contents. The PBMCs were then centrifuged at 500g at
room
temperature for 5 minutes. Ten ml of 1X PBS were added into each tube and the
cells were
resuspended and pooled into 1 tube. The PBMCs were again centrifuged at 500g
at room
temperature for 10 minutes, resuspended in 20 to 40 ml of ACK lysing solution
(Cambrex)
and incubated at room temperature for 5 minutes. The cells were then
centrifuged again at
1500 rpm for 5 minutes. The PBMCs were resuspended in 30 ml RPMI-1640 medium
(Mediatech). The cells were then transferred into 2-4 T75 flasks. The flasks
were incubated
at 37 C for 1 to 2 hours. The non-adherent cells were then removed by
rinsing. Afterwards,
ml of 1X PBS were added into each flask, the flask swirled, and the PBS
removed.
Afterwards, 10 ml of complete DC media (serum-free DC Medium + 800 U/ml GM-CSF
+
1000 U/ml IL-4) was added to each flask. The flasks were then incubated at 37
C, 5% CO2
for 2 days. On Day 3, 10 ml of complete DC medium was added into each flask.
The cells
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were then incubated for another 2 days. On Day 6 or 7, the resulting immature
DCs were
ready for use.
[0068] Figure 1 provides an overview of the generation of dendritic cells.
[0069] EXAMPLE 2: Preparing Tumor Lysate
[0070] A tumor sample was obtained from a patient. After separating fat and
necrotic
tissue away from the tumor tissue, the tissue was weighed and 1X PBS added (50
IA of PBS
per 200)tg of tissue) and the tumor was minced thoroughly with scalpels in 1X
PBS. The
tumor cells were then subjected to 4 cycles of freeze and thaw. The freezing
was performed
in liquid nitrogen for 20 minutes and the thawing was performed at room
temperature.
Prepared tumor lysate was quantified by a spectrophotometer. An aliquot was
taken for
quality control testing. The remainder was stored at < -135 C in preparation
for vaccine
preparation. Small amounts of adjuvant can optionally be added after the
freeze thaw cycles.
[0071] Figure 2 provides an overview of the tumor cell lysate processing.
[0072] EXAMPLE 3: Preparing YCWP
[0073] YCWPs were prepared from Fleishmans Baker's Yeast or equivalent.
Briefly, 10 g
of Fleishmans Baker's yeast was suspended in 100 ml of 1 M NaOH and heated to
80 C for
one hour. The undissolved yeast cell walls were recovered by centrifugation at
2000 x g for
minutes. The recovered yeast cell walls were then resuspended in 100 ml of
water with
the pH adjusted to 4.5 with HC1 and incubated at 55 C for an additional hour,
and
subsequently recovered by centrifugation. The recovered YCWPs were then washed
with
water once, isopropanol 4 times and finally acetone 2 times. Once the YCWPs
were fully
dried they were resuspended in PBS, counted, aliquoted into groups of 1 X109
particles and
freeze dried for use in manufacturing the vaccine.
[0074] Figure 3 provides an overview of the yeast cell wall particles
processing.
[0075] EXAMPLE 4: Preparing YCWP
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[0076] Three grams of active dry yeast (Fleischmann's or equivalent) were
washed three
times in water by suspending the yeast in 30 mL of sterile water, vortexing,
and centrifuging
at 800-1000 x g for 5 minutes at room temperature. After decanting the
supernatant, the yeast
pellet was resuspended in 50 mL of 1 M NaOH and heated in a 90 C water bath
for 1 hour.
[0077] The yeast suspension was subsequently centrifuged at 800-1000 x g for 5
minutes,
and the pellet was resuspended in 25-30 mL of acid water (pH adjusted to 4.5
with HCI). The
acid water wash step was repeated until the pH of the suspension is < 7Ø
Then the pellet
was resuspended in 30 mL acid water and incubated in a 75 C water bath for 1
hour. The
yeast pellet was recovered by centrifugation at 1000 x g for 5 minutes, and
then washed with
mL of sterile water 3 times, 10 mL of isopropanol 4 times and finally 10 mL of
acetone 2
times. The acetone was carefully removed, and the pellet was spread evenly on
the glass
surface of a beaker, allowed to air dry overnight.
[0078] The dried YWCPs were collected and stored in a vacuum jar at 4 C and
then
washed in 10-15 mL of filtered 70% ethanol 3 times. The YWCPs were briefly
sonicated on
the final wash, and the sonication was repeated if necessary to disperse
clumps. Once the
ethanol was removed, the YWCPs were washed in sterile water. Aliquots of 100
1 of
YWCPs were dispensed into 2.0 mL rounded bottom snap top centrifuge tubes,
placed in
freezer for 1 hour, freeze dried, and stored in a vacuum jar at 4 C for
future use.
[0079] EXAMPLE 5: Loading Tumor Lysate into YCWP
[0080] A suspension of fully anhydrous YCWPs (1 X 109) was placed in contact
with 50
uL of tumor lysate in PBS (from 200 jigs of tumor tissue) over a period of 2
hours at 4 C,
allowing the lysate to penetrate into the hollow insides of the YCWPs to
produce loaded
YCWPs. The suspension was then freeze dried for 2 hours. After freeze drying,
50 iLtI, of
water was added to the loaded YCWPs, incubated for another 2 hours at 4 C and
again
freeze dried to yield YCWPs with dry tumor lysate within their hollow insides.
The loaded
YCWPs were then sterilized by washing in ethanol and maintained in ethanol.
[0081] Figure 4 provides an overview of the YCWPs loading procedure.
[0082] EXAMPLE 6: Loading YCWPs with Tumor Lysate
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[0083] A patient tumor biopsy sample was mixed carefully with 50-100 p1 of
lysis buffer
(PBS) (depending on the amount of the tumor sample), avoiding bubbles during
mixing, and
was then incubated at 4 C for 30 minutes. The mixture was subjected to freeze-
thaw 3 times
in acetone-dry ice bath and 37 C water bath, and centrifuged at 4 C for 10
minutes at
maximum speed. 50 i.t1 of the prepared tumor lysate was added in a sterile 2
mL centrifuge
tube containing 10 mg of dried YCWPs such that the liquid tumor lysate covered
the
YCWPs. The mixture was incubated at 4 C for 2 hours until the liquid tumor
lysate soaked
into the YCWPs.
[0084] The tube was then placed into a -85 C freezer for 30 minutes for a
quick freeze of
the pellet. The tube was placed on freeze drier overnight. 50 111 of sterile
water was added
onto the dried yeast pellet and incubated at 4 C for 2 hours to allow the
liquid to soak into
the pellets.
[0085] The tube was placed into a -85 C freezer for 30 minutes for a quick
freeze of the
pellet. The tube was then placed on freeze drier overnight. The dried
particles were then
resuspended in 1 mL of 70% ethanol and stored at 4 C for future use.
[0086] EXAMPLE 8: Preparing Dendritic Cells Containing Tumor Lysate Loaded
Particles
[0087] The tumor lysate loaded YCWPs in 70% ethanol suspension was
centrifuged. The
ethanol was removed carefully and replaced with 1 mL of PBS. The loaded YCWPs
were
sonicated. The loaded YCWPs were washed with sterile lx PBS. After final wash,
the
loaded YCWPs were resuspended in PBS to approximately 1 X 108 particles/100
ittl PBS.
[0088] The loaded YCWPs were added to a dendritic cell culture at a ratio of
1:100, and the
culture was returned to 37 C incubator. Subsequently, the following factors
were added to
the culture: 50itig/mL of TNF-a in sterile water was added to the culture at a
ratio of 1:5000
in volume (2 iLtI, per 10 mL of culture); 10 p.g/mL of IL-113 in sterile water
was added to the
culture at a ratio of 1:1000 in volume; 10 tg/mL of IL-6 in sterile water was
added to the
culture at a ratio of 1:1000 in volume; and 1 mg/mL of PGE2 in 100% ethanol
was added to
the culture at a ratio of 1:1000 in volume. After all factors were added and
mixed into the
culture, the culture was incubated overnight.
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[0089] EXAMPLE 9: Harvest of Dendritic Cells, Preparation and Cryopresrvation
of
Vaccine
[0090] The dendritic cell culture prepared according to Example 9 was removed
from the
incubator. The following procedure was performed in a hood under sterile
conditions. 10
mL of media were removed from culture flask. The culture flask was rinsed with
4.0-4.5 mL
of IX PBS and also added to the media.
[0091] 1.5-2.0 mL of CcllStripperTM was added to the culture flask. The
culture flask was
placed in 37 C incubator for 10-20 minutes. About 4 mL of the culture media
were added
back to the flask from the tube to wash and remove cells. The flask was washed
to harvest as
many cells as possible. The cells were counted on hemacytometer or
CellometerTm. The
supernatant was removed after centrifugation.
[0092] Subsequently, the cells were resuspended in CryoStorTM 10 at 5 X 106
cells/mL,
aliquoted into cryovials properly labeled with patient ID Number, date and
cell concentration
at 1.25 X 106 cells/mL per vial (about 250 4). A 250-500 iaL portion was saved
in a
cryovial for sterility testing, and the remaining vials were stored in
Styrofoam containers and
placed under -86 C to step down freeze.
[0093] EXAMPLE 10: Preparation of the Solid Tumor Vaccine for Patient
Administration
[0094] One cryovial of patient's cell was removed from cryostorage and
carefully thawed at
37 C in a water bath. Under sterile conditions, 1 mL of sterile saline for
injection with 5%
human serum albumin (or 1 mL of sterile 1X PBS) was gently added to the
cryovial
containing the cells. After the cells were carefully resuspended, the entire
volume from the
cryovial was drawn and the syringe containing the tumor cells was used for
administration of
the vaccine to a patient.
[0095] EXAMPLE 11: Dendritic Cells Containing Tumor Lysate Loaded Particles
versus
antigen pulsed dendritic cells (DCs)
[0096] B3Z cells are a T cell hybridoma expressing a T-cell receptor that
specifically
recognizes the OVA(257_264) (SI1NFEKL) epitopc in the context of H-2Kb and
carry a beta-
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galactosidase (lacZ) construct driven by a nuclear factor of activated T cells
elements from
the interleukin 2 promoter (X). These B3Z cells were used to evaluate the
effectiveness of
dendritic cells pulsed with ovalbumin versus those loaded with ovalbumin by
way of
ovalbumin loaded YCWPs for antigen presentation, resulting in a CD8+ T cell
response.
[0097] Upon activation by interaction with MHC class I molecules on dendritic
cells
presenting ovalbumin epitopes, the B3Z cells were engineered to respond by
expressing p-
galactosidase. I3-Galactosidase catalyzes a breakdown of X-gal (5-bromo-4-
chloro-indoly1-13-
D-galactopyranoside) to yield 5-bromo-4-chloro-3-hydroxyindole, a blue colored
product.
Spectroscopic measurement of this blue color gives a measure of the effective
MHC class
presentation of the ovalbumin epitope. The results of this experiment, shown
in Figure 6,
demonstrate that loading dendritic cells by ovalbumin loaded YCWPs gave more
than a 100
fold increase in CD8+ T cell response over dendritic cells pulsed with
ovalbumin.
[0098] EXAMPLE 12: In vitro Data
[0099] Dendritic cells were prepared from cells obtained from the bone marrow
of the
femur and tibia of both hind legs of a female C57BL/6J mouse. Bl6F0 murine
melanoma
cells were obtained (ATCC (CRL-6322)) and cultured using standard tissue
culture
techniques. The dendritic cells were loaded with YCWPs containing B 1 6F0
tumor lysate
(around 2x10-15 g/YCWP) at a ratio of 100:1 particles:DC by adding the
particles at day 7 of
a dendritic cell culture for a period of 2 hours. Three days prior to the
preparation of the
dendritic cells containing tumor lysate loaded particles, female C57BL/6J mice
were
challenged with 0.75 x 106 B16F0 melanoma cells in 0.4 ml 1X PBS by
intravenous
injection. Once the dendritic cells containing tumor lysate loaded particles
were prepared,
each mouse in the treatment group was injected intravenously with 2x106 of
dendritic cells
containing tumor lysate loaded particles and this vaccination was repeated for
three weekly
doses. The mice were monitored up to four weeks for pulmonary metastasis.
[0100] At the end of four weeks (when one of the control mice died), the mice
were
sacrificed and any occurrence of metastases was counted. All four control
animals that were
not treated with dendritic cells containing tumor lysate loaded particles had
more than
50 tumors. However, none of the treated animals had measurable metastases.
This data
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indicates that dendritic cells containing tumor lysate loaded particles is
effective in treating
cancer in a proven animal model system. The data are compiled in Table 1.
[0101] Table 1. Number of Metastases in Control and Treated Mice
Number of Metastases
Group
(including tumors at non-lung sites)
Control:
A >50
>50
>50
>50
Treated:
A 0
0
0
0
[0102] Moreover, Figure 7A shows the lungs of three of the control mice (one
mouse died
prior to the end of the experiment and the lungs were not able to be
photographed) in this
experiment and Figure 7B shows the lungs of the four treated mice.
[0103] EXAMPLE 13: In vivo Procedure
[0104] To vaccinate a subject, a dose of 1.25 million dendritic cells
containing tumor lysate
loaded particles can be cryopreserved in 0.2 mL of a serum-free, 10% dimethyl
sulfoxide
freezing medium (CryoStorlm CS-10, BioLife Solutinos, Inc.). Before injection,
the dendritic
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cells can be thawed and diluted to a 1 mL with sterile saline for injection
containing 5%
human serum albumin (Albuminar -25, Aventis Behring). The dilution can then be
transferred to a 3.0 cc syringe for injection and using a needle no smaller
than 23 gauge,
which should be administered within 2 hours of the thawing. The injection can
be
administered subcutaneously into an area of lymph nodes.
[0105] EXAMPLE 14: Isolation of Mononuclear Cells from Whole Peripheral Blood
using
the SepMate-50 System
Procedure:
STEP PROCEDURE/WORK INSTRUCTIONS
1 Add 15mL of lymphocyte separation medium (LSM) to each
SepMate tube by carefully, yet quickly, pipetting it through the
central hole of each tube insert.
2 Pool the whole blood.
3 Dilute the whole blood sample with twice the initial blood
volume of
1xPBS
4 Add 30 mL diluted blood to the Sepmate tubes.
Centrifuge for 10-15 minutes at room temperature.
6 Pour off the top layer containing the enriched PBMC's from
each
Sepmate tube into new centrifuge tubes.
7 Cap tubes and centrifuge 5 minutes.
8 Resuspend pellet in each tube with up to 1.0 mL ACK lysing
buffer
with pipette. Repeat cycles of adding 1X PBS to resuspend pellet and
centrifuge.
9 Decant the supernatant, and aliquot 50 x 106 cells suspended
in 15
mL RPMI media per flask. Place in the CO2 incubator at 37 C for 1-
1.5 hours.
Remove flasks from incubator. Wash pellet with 1X PBS.
11 add 15 mL complete DC Media (containing IL-4, GM-CSF, and
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Gentamycin) and place in the 37 CO2 incubator for 22-24 hours.
12 On Day 2, continue with inoculation of cultures with YWCP's
and
cytokines after the dendritic cells have incubated at 37 C for approx.
22 hours.
[0106] EXAMPLE 15: Generation of Dendritic Cells Combined with Loaded YCWP's
[0107] Following the procedure in Example 14, the following methods are
performed:
I. Addition of YCWP's
STEP PROCEDURE/WORK INSTRUCTIONS
To each dendritic cell culture flask, add a sufficient volume of tumor
lysate infused YCWP's at a ratio of 1:100. (50-200u1) and incubate
for 1-2 hours.
II. Preparation And Addition of Cytokines
STEP PROCEDURE/WORK INSTRUCTIONS
1 Add TNF-a , 113, IL-6 and PGE2 to each culture flask,
[0108] EXAMPLE 16: Harvest of Cells, Preparation and Cryopreservation of
Vaccine
[0109] Following the procedure in Example 15, the following methods are
performed:
Harvest of cells:
STEP PROCEDURE/WORK INSTRUCTIONS
Add 4.0-4.5 mL 1xPBS to each flask containing 10 mL media.
2 Add 1.5 -2.0 mL CellStripperTM to each flask to release
mature cells
and incubate at 37C.
3 Centrifuge and emove supernatant by decanting until 'dry'
pellet
remains.
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II. Preparation of Vaccine and Cryopreservation:
STEP PROCEDURE/WORK INSTRUCTIONS
Resuspend cells in CryoStorTM 10, aliquot and step down freeze.
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