U.S. patent application number 14/019007 was filed with the patent office on 2014-03-06 for tumor lysate loaded particles.
This patent application is currently assigned to Orbis Health Solutions LLC. The applicant listed for this patent is Orbis Health Solutions LLC. Invention is credited to Thomas E. Wagner.
Application Number | 20140065173 14/019007 |
Document ID | / |
Family ID | 50187915 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140065173 |
Kind Code |
A1 |
Wagner; Thomas E. |
March 6, 2014 |
TUMOR LYSATE LOADED PARTICLES
Abstract
Dendritic cells containing tumor lysate loaded particles are
prepared. The dendritic cells present tumor antigens to elicit the
Major Histocompatibility Complex class I pathway and can be used as
a vaccine to treat cancer, including ocular melanoma.
Inventors: |
Wagner; Thomas E.;
(Greenville, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Orbis Health Solutions LLC |
Greenville |
SC |
US |
|
|
Assignee: |
Orbis Health Solutions LLC
Greenville
SC
|
Family ID: |
50187915 |
Appl. No.: |
14/019007 |
Filed: |
September 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61697498 |
Sep 6, 2012 |
|
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|
Current U.S.
Class: |
424/184.1 ;
435/325; 435/408 |
Current CPC
Class: |
A61K 2039/5154 20130101;
A61K 39/0011 20130101; A61K 35/15 20130101; A61K 2039/572 20130101;
C12N 5/0639 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/184.1 ;
435/325; 435/408 |
International
Class: |
C12N 5/0784 20060101
C12N005/0784 |
Claims
1. An isolated dendritic cell comprising a phagocytosed component
consisting essentially of (i) a particle and (ii) a tumor lysate
loaded within the particle.
2. The dendritic cell of claim 1, wherein the tumor lysate is
present in an amount from about 200 .mu.g to about 500 .mu.g.
3. The dendritic cell of claim 1, wherein the tumor lysate is
present in an amount of about 200 .mu.g.
4. The dendritic cell of claim 1, wherein 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.
5. The dendritic cell of claim 1, wherein the particle is a yeast
cell wall particle.
6. The dendritic cell of claim 1, wherein the dendritic cell is an
immature cell that has been isolated for no more than 8 days.
7. A vaccine comprising the isolated dendritic cell of claim 1.
8. 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.
9. The method of claim 8, further comprising (a) resuspending the
tumor lysate loaded particle in solution and (b) freeze-drying the
resuspended solution before step (iii).
10. The method of claim 8, wherein the tumor lysate is produced by
freezing and thawing the tumor.
11. The method of claim 10, further comprising repeating the
freezing and thawing steps.
12. The method of claim 11, further comprising cryopreserving the
tumor lysate at -150.degree. C.
13. The method of claim 8, wherein 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.
14. The method of claim 8, wherein 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.
15. A method for treating cancer, comprising administering a
vaccine comprising the isolated dendritic cell of claim 1 to a
patient in need thereof.
16. The method of claim 15, wherein 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.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
application No. 61/697,498, filed Sep. 6, 2012.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
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 .mu.g to about 500 .mu.g. In specific embodiments, the
tumor lysate is present in an amount of about 200 .mu.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.degree.
C.
[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] 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
[0015] FIG. 1 depicts a process for producing dendritic cells.
[0016] FIG. 2 depicts a process for producing tumor lysate.
[0017] FIG. 3 depicts a process for producing yeast cell wall
particles.
[0018] FIG. 4 depicts a process for loading tumor lysate into yeast
cell wall particles.
[0019] FIG. 5 depicts a process for producing tumor lysate particle
loaded dendritic cells.
[0020] FIG. 6 is a graph comparing the effect of tumor lysate
particle loaded dendritic cells versus antigen pulsing of dendritic
cells on B3Z cells.
[0021] FIG. 7A shows the lungs of control mice afflicted with B16F0
murine melanoma. FIG. 7B shows the lungs of mice afflicted with
Bl6F0 murine melanoma but treated with tumor lysate particle loaded
dendritic cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] Reference is made herein to various methodologies known to
those of ordinary skill in the art. Publications and other
materials setting forth such known methodologies to which reference
is made are incorporated herein by reference in their entirety as
though set forth in full.
[0023] 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 forms of terms
designate both the singular and plural, unless expressly stated to
designate the singular only. For example, "vaccine" means "vaccine"
or "vaccines."
[0024] 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.
[0025] 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.
[0026] 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.
[0027] Tumor Lysate
[0028] 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 1.times.PBS.
[0029] In specific embodiments, the tumor lysate is produced from a
solid tumor weighing a minimum of 200 to 500 .mu.g.
[0030] 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.degree.
C. or below after the freezing and thawing process. In specific
embodiments, the tumor lysate is store at a temperature of
-150.degree. C. or below after the freezing and thawing
process.
[0031] 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.
[0032] Particle
[0033] 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 embodiments, the
size of the particle is about 0.5 to about 5 .mu.m, 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
.mu.m. In specific embodiments, the size of the particle is about
0.5 to about 2.5 .mu.m. In some embodiments, the particle can be
any particle with a glycan network, so long as the particle is
about 0.5 to about 5 .mu.m in size.
[0034] 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.
[0035] 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 .mu.m.
[0036] 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.
[0037] 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.
[0038] In specific embodiments, the yeast is suspended in NaOH,
including 1M NaOH. In specific embodiments, the first suspension is
incubated at about 80.degree. C. for about 1 hour or for 1 hour. In
specific embodiments, the centrifuging is performed at about 2000
times gravity 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.degree.
C. for about 1 hour or at 55.degree. 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.
[0039] In another embodiment, the YCWP is sterilized using
isopropanol and/or acetone following washing of the pellet. In
specific embodiments, other known alcohols are 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 1.times.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.degree. C. or lower. In specific embodiments, the YCWP is
freeze dried and store at 4.degree. C.
[0040] Tumor Lysate Loaded Particle
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] In specific embodiments using YCWPs, the number of YCWPs is
about 1.times.10.sup.9 and the volume of tumor lysate is about 50
.mu.L (generated from about 200 .mu.g of tumor tissue). In specific
embodiments, the number of YCWPs is 1.times.10.sup.9 and the volume
of tumor lysate is 50 .mu.L (from about 200 .mu.g of tumor tissue).
In specific embodiments, the incubation in step (a) is for about 2
hours at about 4.degree. C. In specific embodiments, the incubation
in step (a) is for 2 hours at 4.degree. 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
.mu.L of water or 50 .mu.L of water. In some embodiments, the
resuspended YCWPs are incubated in step (d) for about 2 hours at
about 4.degree. C. or for 2 hours at 4.degree. C.
[0046] Dendritic Cell
[0047] 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.
[0048] 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.
[0049] 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 500 g for about 5 minutes or is performed at
500 g for 5 minutes. In specific embodiments, a third centrifuging
is performed before step (d) and is performed at about 500 g for
about 10 minutes or is performed at 500 g 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.degree. C. for about 1-2 hours or at 37.degree.
C. for 1-2 hours. In specific embodiments, serum-free DC media is
added to the flask.
[0050] 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/ml) and IL-4 (500
units/ml).
[0051] Tumor Lysate Loaded Particles Phagocytosed in Dendritic
Cells
[0052] 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.
[0053] 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.
[0054] 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.times.10.sup.6 cells per ml or
10.times.10.sup.6 cells per ml. In specific embodiments, the
resuspension is frozen for storage before use.
[0055] Vaccine
[0056] 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 .mu.L of a
10.times.10.sup.6 concentration of dendritic cells containing tumor
lysate loaded particles forms one dose of the vaccine.
[0057] In another embodiment, the dose is administered by diluting
the 200 .mu.L 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 .mu.L 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.
[0058] 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
embodiments, the tumor lysate loaded particles are injected in a
patient at or near (1) a tumor or (2) a lymph node.
[0059] 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.
[0060] 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
II-binding p30 peptide.
Example 1
Preparing Dendritic Cells
[0061] 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 1.times.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 1000 g 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 1.times.PBS were
added and the tubes were inverted to mix the contents. The PBMCs
were then centrifuged at 500 g at room temperature for 5 minutes.
Ten ml of 1.times.PBS were added into each tube and the cells were
resuspended and pooled into 1 tube. The PBMCs were again
centrifuged at 500 g 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.degree. C. for 1 to 2 hours. The non-adherent cells were then
removed by rinsing. Afterwards, 10 ml of 1.times.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.degree. C., 5% CO2 for 2 days. On Day 3,
10 ml of complete DC medium was added into each flask. The cells
were then incubated for another 2 days. On Day 6 or 7, the
resulting immature DCs were ready for use.
[0062] FIG. 1 provides an overview of the generation of dendritic
cells.
Example 2
Preparing Tumor Lysate
[0063] A tumor sample was obtained from a patient. After separating
fat and necrotic tissue away from the tumor tissue, the tissue was
weighed and 1.times.PBS added (50 .mu.L of PBS per 200 .mu.g of
tissue) and the tumor was minced thoroughly with scalpels in
1.times.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 .ltoreq.-135.degree. C. in preparation for vaccine
preparation.
[0064] FIG. 2 provides an overview of the tumor cell lysate
processing.
Example 3
Preparing YCWP
[0065] YCWPs were prepared from Fleishmans Baker's Yeast. Briefly,
10 g of Fleishmans Baker's yeast was suspended in 100 ml of 1 M
NaOH and heated to 80.degree. C. for one hour. The undissolved
yeast cell walls were recovered by centrifugation at 2000.times.g
for 10 minutes. The recovered yeast cell walls were then
resuspended in 100 ml of water with the pH adjusted to 4.5 with HCl
and incubated at 55.degree. 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.times.10.sup.9 particles and freeze dried for use in
manufacturing the vaccine.
[0066] FIG. 3 provides an overview of the yeast cell wall particles
processing.
Example 4
Loading Tumor Lysate into YCWP
[0067] A suspension of fully anhydrous YCWPs (1.times.10.sup.9) was
placed in contact with 50 .mu.L of tumor lysate in PBS (from 200
.mu.gs of tumor tissue) over a period of 2 hours at 4.degree. 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 .mu.L of water was added to
the loaded YCWPs, incubated for another 2 hours at 4.degree. 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.
[0068] FIG. 4 provides an overview of the YCWPs loading
procedure.
Example 5
Preparing Dendritic Cells Containing Tumor Lysate Loaded
Particles
[0069] After co-incubation for 1 hour with loaded YCWPs, dendritic
cells were collected, washed twice with 1.times.PBS, pelleted by
centrifugation and resuspended in CryoStor.TM. CS-10 freezing
medium at a final concentration of 10.times.10.sup.6 cells/ml. The
cell suspension was aliquoted into several 200 .mu.l aliquots,
frozen, and stored at .ltoreq.-135.degree. C.
[0070] FIG. 5 provides an overview of the vaccine formulation and
filling procedure.
Example 6
Dendritic Cells Containing Tumor Lysate Loaded Particles Versus
Antigen Pulsed Dendritic Cells (DCs)
[0071] B3Z cells are a T cell hybridoma expressing a T-cell
receptor that specifically recognizes the OVA.sub.(257-264)
(SIINFEKL) epitope in the context of H-2K.sup.b and carry a
beta-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.
[0072] Upon activation by interaction with MHC class I molecules on
dendritic cells presenting ovalbumin epitopes, the B3Z cells were
engineered to respond by expressing .beta.-galactosidase.
.beta.-Galactosidase catalyzes a breakdown of X-gal
(5-bromo-4-chloro-indolyl-.beta.-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 I presentation of the ovalbumin epitope. The
results of this experiment, shown in FIG. 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.
Example 7
In Vitro Data
[0073] 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. B16F0 murine melanoma cells were obtained
(ATCC(CRL-6322)) and cultured using standard tissue culture
techniques. The dendritic cells were loaded with YCWPs containing
B16F0 tumor lysate (around 2.times.10.sup.-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.times.10.sup.6 B16F0 melanoma cells in 0.4 ml 1.times.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 2.times.10.sup.6 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.
[0074] 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 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.
TABLE-US-00001 TABLE 1 Number of Metastases in Control and Treated
Mice Number of Metastases Group (including tumors at non-lung
sites) Control: A >50 B >50 C >50 D >50 Treated: A 0 B
0 C 0 D 0
[0075] Moreover, FIG. 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 FIG.
7B shows the lungs of the four treated mice.
Example 8
In Vivo Procedure
[0076] 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
(CryoStor.TM. CS-10, BioLife Solutinos, Inc.). Before injection,
the dendritic 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.
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