U.S. patent application number 10/742622 was filed with the patent office on 2004-11-11 for generation and isolation of antigen-specific t cells.
This patent application is currently assigned to XCYTE Therapies, Inc.. Invention is credited to Bonyhadi, Mark, Kalamasz, Dale.
Application Number | 20040224402 10/742622 |
Document ID | / |
Family ID | 33476667 |
Filed Date | 2004-11-11 |
United States Patent
Application |
20040224402 |
Kind Code |
A1 |
Bonyhadi, Mark ; et
al. |
November 11, 2004 |
Generation and isolation of antigen-specific T cells
Abstract
The present invention relates generally to methods for
generating, expanding, and isolating antigen-specific T cells.
Compositions of antigen-specific T cells activated and expanded by
the methods herein are further provided.
Inventors: |
Bonyhadi, Mark; (Issaquah,
WA) ; Kalamasz, Dale; (Redmond, WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
XCYTE Therapies, Inc.
Seattle
WA
|
Family ID: |
33476667 |
Appl. No.: |
10/742622 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60469122 |
May 8, 2003 |
|
|
|
Current U.S.
Class: |
435/372 |
Current CPC
Class: |
A61P 37/02 20180101;
A61K 2039/5158 20130101; A61K 2039/515 20130101; A61P 35/00
20180101; A61P 7/00 20180101; A61P 1/16 20180101; A61P 1/00
20180101; A61P 25/00 20180101; A61P 31/06 20180101; A61K 2035/124
20130101; A61P 15/00 20180101; A61P 11/00 20180101; C12N 2501/515
20130101; C12N 5/0636 20130101; A61K 2035/122 20130101; A61P 33/00
20180101; A61P 1/18 20180101; A61P 17/00 20180101; A61P 31/12
20180101; A61P 31/10 20180101; A61P 13/12 20180101; A61P 37/04
20180101; A61P 31/04 20180101; A61P 31/00 20180101; C12N 2501/599
20130101; C12N 2501/58 20130101; A61P 35/02 20180101 |
Class at
Publication: |
435/372 |
International
Class: |
C12N 005/08 |
Claims
1. A method for expanding a population of antigen-specific T cells
comprising: contacting a population of cells wherein at least a
portion thereof comprises antigen-specific T cells, with a surface,
wherein said surface has attached thereto a first agent and a
second agent, wherein said first agent ligates a CD3/TCR complex on
said T cells and said second agent ligates an accessory molecule on
said T cells, and wherein said ligation by said first and second
agent of said T cells induces proliferation of antigen-specific T
cells and wherein said surface is present at a ratio of surface to
T cells of 1:2 or less.
2. The method according to claim 1 wherein said surface is selected
from the group consisting of paramagnetic beads, lipids, and cell
surfaces.
3. The method according to claim 2 wherein said surface comprises
paramagnetic beads.
4. The method according to claim 3 wherein said beads comprise
beads conjugated to an antibody.
5. The method of claim 1 wherein said surface is present in a ratio
of surface to T cells of about 1:2.5.
6. The method of claim 1 wherein said surface is present in a ratio
of surface to T cells of about 1:5.
7. The method of claim 1 wherein said surface is present in a ratio
of surface to T cells of about 1:10.
8. The method of claim 1 wherein said surface is present in a ratio
of surface to T cells of about 1:25.
9. The method of claim 1 wherein said surface is present in a ratio
of surface to T cells of about 1:50.
10. The method of claim 1 wherein said surface is present in a
ratio of surface to T cells of about 1:100.
11. A method for generating and/or enriching antigen-specific T
cells comprising: (a) exposing a first population of cells wherein
at least a portion thereof comprises antigen presenting cells to a
surface wherein said surface has antigen attached thereto, such
that said surface with antigen attached thereto is ingested by said
APC; (b) exposing a second population of cells wherein at least a
portion thereof comprises T cells to the population of cells in
part (a); thereby generating and/or enriching antigen-specific T
cells.
12. The method according to claim 11 wherein said APC are in direct
contact with said antigen-specific T cells.
13. The method according to claim 12 wherein said APC in direct
contact with said antigen-specific T cells are isolated by exposing
said APC to a magnetic field.
14. The method according to claim 13 wherein said antigen-specific
T cells are expanded according to the following method: (a)
exposing said T cells to an anti-CD3 antibody which is immobilized
on a surface; and (b) stimulating an accessory molecule on the
surface of the T cells with an anti-CD28 antibody, wherein said
anti-CD28 antibody is immobilized on the same surface as the
anti-CD3 antibody; thereby inducing expansion of said
antigen-specific T cells.
15. The method according to claim 14, further comprising exposing
said T cells to IL-15.
16. The method according to claim 14, further comprising exposing
said T cells to a natural ligand for CD137.
17. The method according to claim 14, further comprising exposing
said T cells to an anti-CD137 antibody.
18. The method according to claim 14, further comprising exposing
said T cells to an anti-NKG2D antibody or a natural ligand for
NKG2D.
19-20. (Canceled)
21. The method according to claim 11 wherein said antigen is
selected from the group consisting of protein, glycoprotein,
peptides, antibody/antigen complexes, whole tumor or virus-infected
cells, fixed tumor or virus-infected cells, heat-killed tumor or
virus-infected cells, tumor lysate, non-soluble cell debris,
apoptotic bodies, necrotic cells, whole tumor cells from a tumor or
a cell line that have been treated such that they are unable to
continue dividing, allogeneic cells that have been treated such
that they are unable to continue dividing, irradiated tumor cells,
irradiated allogeneic cells, natural or synthetic complex
carbohydrates, lipoproteins, lipopolysaccharides, transformed cells
or cell line, transfected cells or cell line, transduced cells or
cell line, and virally infected cells or cell line.
22. The method according to claim 11 wherein said antigen is
attached to said surface by an antibody/ligand interaction.
23. The method according to claim 22 wherein said antibody/ligand
interaction comprises an interaction between an antibody/ligand
pair selected from the group consisting of anti-MART-1
antibody/MART-1 antigen, anti-WT-1 antibody/WT-1, anti-PR1 antibody
/PR1, anti-PR3 antibody /PR3, anti-tyrosinase antibody/tyrosinase
antigen, anti-MAGE-1 antibody/MAGE-1 antigen, anti-MUC-1
antibody/MUC-1 antigen, anti-.alpha.-fetoprotein
antibody/.alpha.-fetoprotein antigen, anti-Her2Neu
antibody/Her2Neu, anti-HIV gp120 antibody/HIV gp120, anti-influenza
HA antibody/influenza HA, anti-CMV pp65/CMV pp65, anti-hepatitis C
antibody/hepatitis C proteins, anti-EBV EBNA 3B antibody/EBV EBNA
3B antigen, and anti-human Ig heavy and lignt chains/Ig from a
myeloma cancer patient, and anti-human Ig heavy and lignt chains/Ig
from a CLL cancer patient.
24. The method according to claim 11 wherein said antigen is
chemically attached to said surface.
25. The method according to claim 11 wherein the attachment of said
antigen to said surface comprises a biotin-avidin interaction.
26. The method according to claim 11 wherein said population of
cells wherein at least a portion thereof comprises APC is derived
from a source selected from the group consisting of leukapheresis
product, peripheral blood, lymph node, tonsil, thymus, tissue
biopsy, tumor, spleen, bone marrow, cord blood, CD34.sup.+ cells,
monocytes, and adherent cells.
27-29. (Canceled)
30. A population of antigen-specific T cells generated according to
the method of any one of claims 1.
31. A composition comprising the antigen-specific T cells according
to claim 30 and a pharmaceutically acceptable excipient.
32. A method for stimulating an immune response in a mammal
comprising, administering to the mammal the composition of claim
31.
33. A method for reducing the presence of cancer cells in a mammal
comprising, exposing the cells to the composition of claim 31.
34. The method of claim 33 wherein the cancer cells are from a
cancer selected from the group consisting of melanoma,
non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma,
sarcoma, glioma, thymoma, breast cancer, prostate cancer,
colo-rectal cancer, kidney cancer, renal cell carcinoma, pancreatic
cancer, esophageal cancer, brain cancer, lung cancer, ovarian
cancer, cervical cancer, multiple myeloma, hepatoma, acute
lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),
chronic myelogenous leukemia (CML), and chronic lymphocytic
leukemia (CLL).
35. A method for inhibiting the development of a cancer in a
mammal, comprising administering to the mammal the composition of
claim 31.
36. The method of claim 35 wherein the cancer cells are from a
cancer selected from the group consisting of melanoma,
non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma,
sarcoma, glioma, thymoma, breast cancer, prostate cancer,
colo-rectal cancer, kidney cancer, renal cell carcinoma, pancreatic
cancer, esophageal cancer, brain cancer, lung cancer, ovarian
cancer, cervical cancer, multiple myeloma, hepatoma, acute
lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),
chronic myelogenous leukemia (CML), and chronic lymphocytic
leukemia (CLL).
37. A method for ameliorating an immune response dysfunction in a
mammal comprising administering to the mammal the composition of
claim 31.
38. A method for reducing the presence of an infectious organism in
a mammal comprising, administering to the mammal the composition of
claim 31.
39. The method of claim 38 wherein said organism is selected from
the group consisting of a virus, a single-stranded RNA virus, a
single-stranded DNA virus, a double-stranded DNA virus, Human
Immunodeficiency Virus (HIV), Hepatitis A, B, or C virus, Herpes
Simplex Virus (HSV), Human Papilloma Virus (HPV), Cytomegalovirus
(CMV), Epstein-Barr virus (EBV), a parasite, a bacterium, M
tuberculosis, Pneumocystis carinii, Candida, Aspergillus.
40. A method for inhibiting the development of an infectious
disease in a mammal, comprising administering to the mammal the
composition of claim 31.
41. The method of claim 40 wherein said organism is selected from
the group consisting of a virus, a single-stranded RNA virus, a
single-stranded DNA virus, a double-stranded DNA virus, Human
Immunodeficiency Virus (HIV), Hepatitis A, B, or C virus, Herpes
Simplex Virus (HSV), Human Papilloma Virus (HPV), Cytomegalovirus
(CMV), Epstein-Barr virus (EBV), a parasite, a bacterium, M
tuberculosis, Pneumocystis carinii, Candida, Aspergillus.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Technical Field
[0002] The present invention relates generally to methods for
generating, isolating, and expanding antigen-specific T cells. The
present invention also relates to compositions of antigen-specific
T cells.
[0003] 2. Description of Related Art
[0004] The identification of antigens recognized by T cells in a
variety of cancers and infectious diseases has contributed
significantly to the interest in the use of antigen-specific
immunotherapy for the treatment of malignancies and infectious
diseases. Adoptive therapy using antigen-specific T cells
represents a conceptually attractive strategy by providing a means
to manipulate the specificity, phenotype and magnitude of the
intended immune response. Methods to routinely and reproducibly
expand antigen-specific T cell clones for use in clinical trials of
adoptive therapy would be desirable. Current technologies for
generating therapeutic doses of antigen-specific T cells remain
limited and could be improved by simplifying the manufacturing
process while maintaining or perhaps improving the function of the
infused T cells.
[0005] The various techniques available for expanding human T-cells
have relied primarily on the use of accessory cells (primarily
antigen presenting cells (APC)) and/or exogenous growth factors,
such as interleukin-2 (IL-2). IL-2 has been used together with an
anti-CD3 antibody to stimulate T-cell proliferation, predominantly
expanding the CD8.sup.+ subpopulation of T-cells. Both APC signals
are thought to be required for optimal T-cell activation,
expansion, and long-term survival of the T-cells upon re-infusion.
The requirement for MHC-matched APCs as accessory cells presents a
significant problem for long-term culture systems because APCs are
relatively short-lived. Therefore, in a long-term culture system,
APCs must be continually obtained from a source and replenished.
The necessity for a renewable supply of accessory cells is
problematic for treatment of immunodeficiencies in which accessory
cells are affected. In addition, when treating viral infection, if
accessory cells carry the virus, the cells may contaminate the
entire T-cell population during long-term culture.
[0006] Further, similar syStems require vaccination with antigen
(e.g. tumor/viral antigen), pulsing of antigen-presenting cells
with antigens followed by infusion of cells. Expansion of
antigen-specific T cells to generate large numbers of
antigen-specific T cells often requires labor intensive and
expensive cloning, and/or multiple rounds of activation/expansion
to achieve therapeutically relevant T cell numbers.
[0007] Therefore, there is a need in the art for improved methods
to routinely and reproducibly expand antigen-specific T cell clones
for use in clinical trials of adoptive therapy and for a simplified
manufacturing process that maintains or even improves the function
of the antigen-specific T cells.
[0008] The present invention provides methods to generate an
increased number of highly responsive antigen-specific T cells that
have surface receptor and cytokine production characteristics that
are more more desirable than other expansion methods. The instant
invention does not require knowledge of a particular antigen
(although known antigens can be used in the context of this
invention) and provides for a single, or double, round of expansion
to achieve a therapeutically relevant dose of antigen-specific T
cells, both of the CD4 and CD8 lineage (and either may be selected
if desired).
SUMMARY OF THE INVENTION
[0009] Generally, the present invention relates to methods for
activating, stimulating and isolating antigen-specific T cells. The
present invention also relates to compositions of antigen-specific
T cells and methods of their use in the treatment and prevention of
cancer, infectious diseases, autoimmune diseases, immune
disfunction related to aging, or any other disease state where
antigen-specific T cells are desired for treatment.
[0010] In one aspect of the present invention, a method for
expanding a population of antigen-specific T cells is provided,
comprising contacting a population of cells wherein at least a
portion thereof comprises antigen-specific T cells, with a surface,
wherein said surface has attached thereto a first agent and a
second agent, wherein said first agent ligates a CD3/TCR complex on
said T cells and said second agent ligates an accessory molecule on
said T cells, and wherein said ligation by said first and second
agent of said T cells induces proliferation of antigen-specific T
cells and wherein said surface is present in a ratio of surface to
T cells of 1:2 or less. In certain embodiments the ratio of surface
to T cells is between about 1:1 and about 1:50 and any ratio
therebetween. In certain embodiments the ratio of surface to T
cells is from about 1:2, 1:2.5, 1:5, 1:10, 1:25, 1:50, 1:75, 1:100,
or lower. In one embodiment, the surface includes but is not
limited to paramagnetic beads, lipids, and cell surfaces. In
certain embodiments, the surface comprises paramagnetic beads
conjugated to one or more antibodies. In certain embodiments, the
surface can have 1, 2, 3, 4, or more antibodies or natural ligands
conjugated thereto.
[0011] Another aspect of the present invention provides a method
for generating antigen-specific T cells comprising exposing a first
population of cells wherein at least a portion thereof comprises
antigen presenting cells (APC) to a surface wherein said surface
has antigen attached thereto, such that said surface with antigen
attached thereto is ingested by said APC; exposing a second
population of cells wherein at least a portion thereof comprises T
cells to the population of cells in part (a); thereby generating
antigen-specific T cells. Antigen may be attached or coupled to, or
integrated into a surface by a variety of methods known and
available in the art and described herein. In one embodiment, the
antigen is crosslinked to said surface. In a further embodiment,
the attachment to said surface is by covalent or noncovalent,
electrostatic, or hydrophobic and may be accomplished by a variety
of attachment means, including for example, chemical, mechanical,
enzymatic, electrostatic, or other means whereby the antigen(s) is
capable of stimulating the cells. For example, the antibody to an
antigen first may be attached to a surface, or avidin or
streptavidin may be attached to the surface for binding to a
biotinylated antigen. The antibody to the ligand may be attached to
the surface via an anti-idiotype antibody. Another example includes
using protein A or protein G, or other non-specific antibody
binding molecules, attached to surfaces to bind an antibody.
Alternatively, antigen may be attached to the surface by chemical
means, such as cross-linking to the surface, using commercially
available cross-linking reagents (Pierce, Rockford, Ill.) or other
means. In certain embodiments, antigens are covalently bound to the
surface. Further, in one embodiment, commercially available
tosyl-activated DYNABEADS.TM. or DYNABEADS.TM. with epoxy-surface
reactive groups are incubated with the polypeptide antigen of
interest according to the manufacturer's instructions. Briefly,
such conditions typically involve incubation in a phosphate buffer
from pH 4 to pH 9.5 at temperatures ranging from 4 to 37 degrees
C.
[0012] In one embodiment, the APC are in direct contact with the
antigen-specific T cells. In a further embodiment, the APC that are
in direct contact with said antigen-specific T cells are isolated
by exposing said APC to a magnetic field, wherein said surface
comprises a paramagnetic, magnetic, or magnetizable component. In
another embodiment, the antigen-specific T cells are expanded by
exposing said T cells to a surface wherein said surface has
attached thereto a first agent that ligates a first T cell surface
moiety of a T cell, and the same or a second surface has attached
thereto a second agent that ligates a second moiety of said T cell,
wherein said ligation by the first and second agent induces
proliferation (expansion) of said antigen-specific T cells. In
certain embodiments, at least one agent is an antibody or an
antibody fragment. In other embodiments, the first agent is an
antibody or a fragment thereof, and the second agent is an antibody
or a fragment thereof. In yet another embodiment, the first and the
second agents are different antibodies. In certain embodiments, the
first agent is an anti-CD3 antibody, an anti-CD2 antibody, or an
antibody fragment of an anti-CD3 or anti-CD2 antibody and the
second the second agent is an anti-CD28 antibody or antibody
fragment thereof. In another embodiment, the first agent is an
anti-CD3 antibody and the second agent is an anti-CD28 antibody. In
further embodiments, the anti-CD3 antibody and the anti-CD28
antibody are present at a ratio of about 1:1 to about 1:100. In a
further embodiment, the antigen-specific T cells are expanded by
exposing said antigen-specific T cells to a mitogen, such as
phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and
ionomycin, lipopolysaccharide (LPS), and superantigen.
[0013] In a further embodiment, the antigen of the present
invention includes but is not limited to protein, glycoprotein,
peptides, antibody/antigen complexes, whole tumor or virus-infected
cells, fixed tumor or virus-infected cells, heat-killed tumor or
virus-infected cells, tumor lysate, virus lysate, non-soluble cell
debris, apoptotic bodies, necrotic cells, whole tumor cells from a
tumor or a cell line that have been treated such that they are
unable to continue dividing, allogeneic cells that have been
treated such that they are unable to continue dividing, irradiated
tumor cells, irradiated allogeneic cells, natural or synthetic
complex carbohydrates, lipoproteins, lipopolysaccharides,
transformed cells or cell line, transfected cells or cell line,
transduced cells or cell line, and virally infected cells or cell
line. In certain embodiments, antigen is attached to said surface
by an antibody/ligand interaction. An antibody/ligand interaction
includes but is not limited to an interaction between an
antibody/ligand pair selected from the group consisting of
anti-MART-1 antibody/MART-1 antigen, anti-WT-1 antibody/WT-1,
anti-PR1 antibody /PR1, anti-PR3 antibody /PR3, anti-tyrosinase
antibody/tyrosinase antigen, anti-MAGE-1 antibody/MAGE-1 antigen,
anti-MUC-1 antibody/MUC-1 antigen, anti-.alpha.-fetoprotein
antibody/.alpha.-fetoprotein antigen, anti-Her2Neu
antibody/Her2Neu, anti-HIV gp120 antibody/HIV gp120, anti-influenza
HA antibody/influenza HA, anti-CMV pp65/CMV pp65, anti-hepatitis C
antibody/hepatitis C proteins, anti-EBV EBNA 3B antibody/EBV EBNA
3B antigen, and anti-human Ig heavy and lignt chains/Ig from a
myeloma cancer patient, and anti-human Ig heavy and lignt chains/Ig
from a CLL cancer patient. In certain embodiments, the antigen is
chemically attached to a surface. In one embodiment, the attachment
of said antigen to said surface comprises a biotin-avidin
interaction. In a further embodiment, the population of cells
wherein at least a portion thereof comprises APC is derived from a
source selected from the group consisting of a leukapheresis
product, peripheral blood, lymph node, tonsil, thymus, tissue
biopsy, tumor, spleen, bone marrow, cord blood, CD34.sup.+ cells,
monocytes, and adherent cells.
[0014] Another aspect of the present invention provides a method
for generating and expanding antigen-specific T cells comprising
exposing a first population of cells wherein at least a portion
thereof comprises antigen presenting cells to antigen such that
said antigen is taken up by said APC; exposing a second population
of cells wherein at least a portion thereof comprises T cells to
the population of cells in part (a); thereby generating
antigen-specific T cells; and exposing said antigen-specific T
cells of part (b) to a surface wherein said surface has attached
thereto a first agent that ligates a first T cell surface moiety of
a T cell, and the same or a second surface has attached thereto a
second agent that ligates a second moiety of said T cell, wherein
said ligation by the first and second agent induces proliferation
(expansion) of said antigen-specific T cells. In certain
embodiments, at least one agent is an antibody or an antibody
fragment. In other embodiments, the first agent is an antibody or a
fragment thereof, and the second agent is an antibody or a fragment
thereof. In yet another embodiment, the first and the second agents
are different antibodies. In certain embodiments, the first agent
is an anti-CD3 antibody, an anti-CD2 antibody, or an antibody
fragment of an anti-CD3 or anti-CD2 antibody and the second the
second agent is an anti-CD28 antibody or antibody fragment thereof.
In another embodiment, the first agent is an anti-CD3 antibody and
the second agent is an anti-CD28 antibody. In further embodiments,
the anti-CD3 antibody and the anti-CD28 antibody are present at a
ratio of about 1:1 to about 1:100. In one embodiment said
antigen-specific T cells are isolated by contacting said T cells
with antibodies specific for T cell activation markers. In another
embodiment said antibodies are selected from the group consisting
of anti-CD25, anti-CD54, anti-CD69, anti-CD38, anti-CD45RO,
anti-CD49d, anti-CD40L, anti-CD137, anti-CD62L, and anti-CD134.
[0015] A further aspect of the present invention provides a
population of antigen-specific T cells generated according to any
one of the methods described herein.
[0016] An additional aspect of this invention is a composition
comprising the antigen-specific T cells according to any of the
methods described herein and a pharmaceutically acceptable
excipient.
[0017] A further aspect of the present invention provides methods
for stimulating an immune response in a mammal comprising,
administering to the mammal compositions comprising the
antigen-specific T cells of the present invention.
[0018] An additional aspect of the invention provides for reducing
the presence of cancer cells in a mammal comprising, exposing the
cancer cells to the compositions comprising antigen-specific T
cells. In one embodiment, the cancer cells are from a cancer
selected from the group consisting of melanoma, non-Hodgkin's
lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma,
glioma, thymoma, breast cancer, prostate cancer, colo-rectal
cancer, kidney cancer, renal cell carcinoma, pancreatic cancer,
esophageal cancer, brain cancer, lung cancer, ovarian cancer,
cervical cancer, multiple myeloma, hepatoma, acute lymphoblastic
leukemia (ALL), acute myelogenous leukemia (AML), chronic
myelogenous leukemia (CML), and chronic lymphocytic leukemia
(CLL).
[0019] One aspect of the present invention provides a method for
inhibiting the development of a cancer in a mammal, comprising
administering to the mammal the composition comprising
antigen-specific T cells fo the present invention. In certain
embodiments, the cancer cells are from a cancer selected from the
group consisting of melanoma, non-Hodgkin's lymphoma, Hodgkin's
disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast
cancer, prostate cancer, colo-rectal cancer, kidney cancer, renal
cell carcinoma, pancreatic cancer, esophageal cancer, brain cancer,
lung cancer, ovarian cancer, cervical cancer, multiple myeloma,
hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenous
leukemia (AML), chronic myelogenous leukemia (CML), and chronic
lymphocytic leukemia (CLL).
[0020] A further aspect of the present invention provides a method
for ameliorating an immune response dysfunction in a mammal
comprising administering to the mammal the compositions comprising
antigen-specific T cells generated using any one of the methods
described herein.
[0021] Yet another aspect of the invention provides a method for
reducing the presence of an infectious organism in a mammal
comprising, administering to the mammal a composition comprising
antigen-specific T cells generated using any one of the methods
described herein. Within this context, an infectious organism can
include but is not limited to a virus, a single-stranded RNA virus,
a single-stranded DNA virus, a double-stranded DNA virus, Human
Immunodeficiency Virus (HIV), Hepatitis A, B, or C virus, Herpes
Simplex Virus (HSV), Human Papilloma Virus (HPV), Cytomegalovirus
(CMV), Epstein-Barr virus (EBV), a parasite, a bacterium, M.
tuberculosis, Pneumocystis carinii, Candida, Aspergillus.
[0022] An additional aspect of the present invention provides a
method for inhibiting the development of an infectious disease in a
mammal, comprising administering to the mammal the compositions
comprising antigen-specific T cells generated using any one of the
methods described herein. In this regard an infectious disease can
be caused by an infectious organism including but not limited to a
virus, an RNA virus, a DNA virus, Human Immunodeficiency Virus
(HIV), Hepatitis A, B, or C virus, Herpes Simplex Virus (HSV),
Human Papilloma Virus (HPV), Cytomegalovirus (CMV), Epstein-Barr
virus (EBV), a parasite, a bacterium, M. tuberculosis, Pneumocystis
carinii, Candida, Aspergillus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a photograph showing the tight association of
antigen-specific T cells and bead-loaded antigen presenting cells
(APC) post magnetic separation.
[0024] FIG. 2 is a plot showing upregulation of CD25 in
re-stimulated memory CD8 CMV tetramer+ T cells expanded ex vivo.
Panel A is a negative control from an HLA-A2+, CMV- donor. Panel B
is a negative control showing uncoated bead stimulation from an
HLA-A2+, CMV+ donor. Panel C shows CMV antigen-coated bead
stimulation of cells from an HLA-A2+, CMV+ donor.
[0025] FIG. 3 is a plot showing the effect of varying bead:cell
ratio on expansion or deletion of CMV-specific T cells.
[0026] FIG. 4 panels A and B is a bar graph showing the effect on T
cell expansion of sequential bead addition at varying bead:cell
ratios at varying times during cutlure. Panel A shows a comparison
of total T cell expansion over 15 days, comparing standard static
cuture (beads at day 0 at either 1:2.5 or 1:5 bead to cell ratio)
or additional beads added at day 5, 7, or 9 at 1:10, 1:25, 1:50 or
1:100 bead to cell ratios. Panel B shows CMV-specific T cell
expansion under the same experimental conditions as Panel A.
[0027] FIG. 5 is a graph showing the effect on T cell expansion of
low bead:T cell ratio and sequential addition of beads on Melanoma
gp100(M)-specific T cells.
[0028] FIG. 6 is a graph depicting the fold increase of T-cells
over time following stimulation with anti-CD3 and anti-CD28
co-immobilized beads with varying ratios of anti-CD3:CD28
antibodies attached thereto.
[0029] FIG. 7 is a graph depicting the fold increase of
CMVpp65-specific T-cells over time following stimulation with
anti-CD3 and anti-CD28 co-immobilized beads with varying ratios of
anti-CD3:CD28 antibodies attached thereto.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
that will be used hereinafter.
[0031] The term "biocompatible", as used herein, refers to the
property of being predominantly non-toxic to living cells.
[0032] The term "stimulation", as used herein, refers to a primary
response induced by ligation of a cell surface moiety. For example,
in the context of receptors, such stimulation entails the ligation
of a receptor and a subsequent signal transduction event. With
respect to stimulation of a T-cell, such stimulation refers to the
ligation of a T-cell surface moiety that in one embodiment
subsequently induces a signal transduction event, such as binding
the TCR/CD3 complex. Further, the stimulation event may activate a
cell and upregulate or downregulate expression or secretion of a
molecule, such as downregulation of TGF-.beta.. Thus, ligation of
cell surface moieties, even in the absence of a direct signal
transduction event, may result in the reorganization of
cytoskeletal structures, or in the coalescing of cell surface
moieties, each of which could serve to enhance, modify, or alter
subsequent cell responses.
[0033] The term "activation", as used herein, refers to the state
of a cell following sufficient cell surface moiety ligation to
induce a noticeable biochemical or morphological change. Within the
context of T-cells, such activation, refers to the state of a
T-cell that has been sufficiently stimulated to induce cellular
proliferation. Activation of a T-cell may also induce cytokine
production and performance of regulatory or cytolytic effector
functions. Within the context of other cells, this term infers
either up or down regulation of a particular physico-chemical
process.
[0034] The term "target cell", as used herein, refers to any cell
that is intended to be stimulated by cell surface moiety
ligation.
[0035] An "antibody", as used herein, includes both polyclonal and
monoclonal antibodies; primatized (e.g., humanized); murine;
mouse-human; mouse-primate; and chimeric; and may be an intact
molecule, a fragment thereof (such as scFv, Fv, Fd, Fab, Fab' and
F(ab)'.sub.2 fragments), or multimers or aggregates of intact
molecules and/or fragments; and may occur in nature or be produced,
e.g., by immunization, synthesis or genetic engineering; an
"antibody fragment," as used herein, refers to fragments, derived
from or related to an antibody, which bind antigen and which in
some embodiments may be derivatized to exhibit structural features
that facilitate clearance and uptake, e.g., by the incorporation of
galactose residues. This includes, e.g., F(ab), F(ab)'.sub.2, scFv,
light chain variable region (V.sub.L), heavy chain variable region
(V.sub.H), and combinations thereof.
[0036] The term "protein", as used herein, includes proteins,
polypeptides and peptides; and may be an intact molecule, a
fragment thereof, or multimers or aggregates of intact molecules
and/or fragments; and may occur in nature or be produced, e.g., by
synthesis (including chemical and/or enzymatic) or genetic
engineering.
[0037] The term "agent", "ligand", or "agent that binds a cell
surface moiety", as used herein, refers to a molecule that binds to
a defined population of cells. The agent may bind any cell surface
moiety, such as a receptor, an antigenic determinant, or other
binding site present on the target cell population. The agent may
be a protein, peptide, antibody and antibody fragments thereof,
fusion proteins, synthetic molecule, an organic molecule (e.g., a
small molecule), or the like. Within the specification and in the
context of T-cell stimulation, antibodies are used as a
prototypical example of such an agent.
[0038] The terms "agent that binds a cell surface moiety" and "cell
surface moiety", as used herein, are used in the context of a
ligand/anti-ligand pair. Accordingly, these molecules should be
viewed as a complementary/anti-complementary set of molecules that
demonstrate specific binding, generally of relatively high affinity
(an affinity constant, K.sub.a, of about 10.sup.6 M.sup.-1 or
tighter).
[0039] "Antigen-presenting cell (APC)", as used herein, refers to
those cells that normally initiate the responses of nave and/or
memory T cells to antigen. In this regard, APC refers to any cell
capable of antigen presentation. APCs include, but are not limited
to, dendritic cells, monocytes, macrophages, and B cells. An APC
may express high levels of MHC class II, ICAM-1 and B7-2.
[0040] A "co-stimulatory signal", as used herein, refers to a
signal, which in combination with a primary signal, such as TCR/CD3
ligation, leads to T-cell proliferation.
[0041] A "ligand/anti-ligand pair", as used herein, refers to a
complementary/anti-complementary set of molecules that demonstrate
specific binding, generally of relatively high affinity (an
affinity constant, K.sub.a, of at least about 10.sup.6 M.sup.-1,).
The skilled artisan would understand that this affinity is
illustrative only and that affinity constants of the
ligand/anti-ligand pairs useful in the context of the present
invention might be lower or in some cases higher. For example, in
the case of biotin/streptavidin, the streptavidin on-rate is
comparable to that of monomeric avidin while its off-rate is seven
times lower. The dissociation constant was determined to be
1.3.times.10(-8)M. Exemplary ligand/anti-ligand pairs
enzyme/inhibitor, hapten/antibody, lectin/carbohydrate,
ligand/receptor, and biotin/avidin or streptavidin. Within the
context of the present invention specification receptors and other
cell surface moieties are anti-ligands, while agents (e.g.,
antibodies and antibody fragments) reactive therewith are
considered ligands.
[0042] "Separation", as used herein, includes any means of
substantially purifying one component from another (e.g., by
filtration, magnetic attraction, etc.).
[0043] "Quiescent", as used herein, refers to a cell state wherein
the cell is not actively proliferating.
[0044] A "surface", as used herein, refers to any surface capable
of having an agent attached thereto and includes, without
limitation, metals, glass, plastics, co-polymers, colloids, lipids,
cell surfaces, and the like. Essentially any surface that is
capable of retaining an agent bound or attached thereto. A
prototypical example of a surface used herein, is a particle such
as a bead. As such, the terms "surface" and "particle" are used
herein interchangeably.
[0045] "Immune response or responsiveness" as used herein, refers
to activation of cells of the immune system, including but not
limited to, T-cells, such that a particular effector function(s) of
a particular cell is induced. Effector functions may include, but
are not limited to, proliferation, secretion of cytokines,
secretion of antibodies, expression of regulatory and/or adhesion
molecules, and the ability to induce cytolysis.
[0046] "Stimulating an immune response" as used herein, refers to
any stimulation such that activation and induction of effector
functions of cells of the immune system are achieved.
[0047] "Immune response dysfunction" as used herein, refers to the
inappropriate activation and/or proliferation, or lack thereof, of
cells of the immune system, and/or the inappropriate secretion, or
lack thereof, of cytokines, and/or the inappropriate or inadequate
induction of other effector functions of cells of the immune
system, such as expression of regulatory, adhesion, and/or homing
receptors, and the induction of cytolysis.
[0048] The terms "preventing" or "inhibiting" the development of a
cancer or cancer cells" as used herein, refers to the occurrence of
the cancer being prevented or the onset of the cancer being
delayed.
[0049] The term "treating" or "reducing the presence of a cancer or
cancer cells" as used herein, means that the cancer growth is
inhibited, which is reflected by, e.g., tumor volume or numbers of
malignant cells. Tumor volume may be determined by various known
procedures, e.g., obtaining two dimensional measurements with a
dial caliper.
[0050] "Preventing or inhibiting the development of an infectious
disease" as used herein, means the occurrence of the infectious
disease is prevented or the onset of the infectious disease is
delayed, or the spread of an existing infection is reversed.
[0051] "Ameliorate" as used herein, is defined as: to make better;
improve (The American Heritage College Dictionary, 3.sup.rd
Edition, Houghton Mifflin Company, 2000).
[0052] "Particles" as used herein, may include a colloidal
particle, a microsphere, nanoparticle, a bead, or the like. In the
various embodiments, commercially available surfaces, such as beads
or other particles, are useful (e.g., Miltenyi Particles, Miltenyi
Biotec, Germany; Sepharose beads, Pharmacia Fine Chemicals, Sweden;
DYNABEADS.TM., Dynal Inc., Oslo, Norway; PURABEADS.TM., Prometic
Biosciences, magnetic beads from Immunicon, Huntingdon Valley, Pa.,
microspheres from Bangs Laboratories, Inc., Fishers, Ind.).
[0053] "Paramagnetic particles" as used herein, refer to particles,
as defined above, that localize in response to a magnetic
field.
[0054] "Antigen" as used herein, refers to any molecule 1) capable
of being specifically recognized, either in its entirety or
fragments thereof, and bound by the "idotypic" portion
(antigen-binding region) of a mAb or its derviative; 2) containing
peptide sequences which can be bound by MHC and then, in the
context of MHC presentation, can specifically engage its cognate T
cell antigen receptor.
[0055] To "load" an APC with antigen, as used herein, refers to
exposing an APC to antigen or antigenic peptide for a period of
time sufficient for the APC to uptake, process, and present the
antigen, bound by MHC molecules, to T cells. In some cases, the
antigen, especially peptide, can be bound by MHC molecules and
presented to T cells without being taken up and processed by the
APC.
[0056] The term "animal" or "mammal" as used herein, encompasses
all mammals, including humans. Preferably, the animal of the
present invention is a human subject.
[0057] The term "exposing" as used herein, refers to bringing into
the state or condition of immediate proximity or direct
contact.
[0058] The term "lysate" as used herein, refers to the supernatant
and non-soluble cell debris resulting from lysis of cells. A
skilled artisan will recognize that any number of lysis buffers
known in the art may be used (see for example Current Protocols in
Immunology, John Wiley & Sons, New York. N.Y.). Cell lysis may
also be carried out by freeze-thaw procedures or other means (e.g.
sonication, etc.).
[0059] The term "apoptotic body" as used herein, is defined as the
smaller, intact, membrane-bound fragments that result from
apoptotic cells.
[0060] The term "proliferation" as used herein, means to grow or
multiply by producing new cells.
[0061] The term "infectious disease" as used herein, refers to any
disease that is caused by an infectious organism. Infectious
organisms may comprise viruses, (e.g., RNA viruses, DNA viruses,
human immunodeficiency virus (HIV), hepatitis A, B, and C virus,
herpes simplex virus (HSV), cytomegalovirus (CMV) Epstein-Barr
virus (EBV), human papilloma virus (HPV)), parasites (e.g.,
protozoan and metazoan pathogens such as Plasmodia species,
Leishmania species, Schistosoma species, Trypanosoma species),
bacteria (e.g., Mycobacteria, in particular, M. tuberculosis,
Salmonella, Streptococci, E. coli, Staphylococci), fungi (e.g.,
Candida species, Aspergillus species), Pneumocystis carinii, and
prions (known prions infect animals to cause scrapie, a
transmissible, degenerative disease of the nervous system of sheep
and goats, as well as bovine spongiform encephalopathy (BSE), or
"mad cow disease", and feline spongiform encephalopathy of cats.
Four prion diseases known to affect humans are (1) kuru, (2)
Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-Scheinker
Disease (GSS), and (4) fatal familial insomnia (FFI)). As used
herein "prion" includes all forms of prions causing all or any of
these diseases or others in any animals used--and in particular in
humans and domesticated farm animals.
[0062] Sources of T Cells
[0063] T cells can be obtained from a number of sources, including
peripheral blood mononuclear cells, bone marrow, thymus, tissue
biopsy, tumor, lymph node tissue, gut associated lymphoid tissue,
mucosa associated lymphoid tissue, spleen tissue, or any other
lymphoid tissue, and tumors. T cells can be obtained from T cell
lines and from autologous or allogeneic sources. T cells may also
be obtained from a xenogeneic source, for example, from mouse, rat,
non-human primate, and pig.
[0064] Preferably, cells from the circulating blood of an
individual are obtained by apheresis or leukapheresis. The
apheresis product typically contains lymphocytes, including T
cells, monocytes, granulocytes, B cells, other nucleated white
blood cells, red blood cells, and platelets. In one embodiment, the
cells collected by apheresis or leukapheresis may be washed to
remove the plasma fraction and to place the cells in an appropriate
buffer or media for subsequent processing steps. In one embodiment
of the invention, the cells are washed with phosphate buffered
saline (PBS). In an alternative embodiment, the wash solution lacks
calcium and may lack magnesium or may lack many if not all divalent
cations. As those of ordinary skill in the art would readily
appreciate a washing step may be accomplished by methods known to
those in the art, such as by using a semi-automated "flow-through"
centrifuge (for example, the Cobe 2991 cell processor, Baxter)
according to the manufacturer's instructions. After washing, the
cells may be resuspended in a variety of biocompatible buffers,
such as, for example, Ca.sup.++/Mg.sup.++ free PBS. Alternatively,
the undesirable components of the apheresis sample may be removed
and the cells directly resuspended in culture media.
[0065] In another embodiment, T cells are isolated from peripheral
blood lymphocytes by lysing the red blood cells and by
centrifugation through a PERCOLL.TM. gradient. A specific
subpopulation of T cells, such as CD28.sup.+, CD4.sup.+, CD8.sup.+,
CD45RA.sup.+, and CD45RO.sup.+T cells, can be further isolated by
positive or negative selection techniques. For example, CD3.sup.+,
CD28.sup.+ T cells can be positively selected using CD3/CD28
conjugated magnetic beads (e.g., DYNABEADS.RTM. M-450 CD3/CD28 T
Cell Expander). In one aspect of the present invention, enrichment
of a T cell population by negative selection can be accomplished
with a combination of antibodies directed to surface markers unique
to the negatively selected cells. A preferred method is cell
sorting and/or selection via negative magnetic immunoadherence or
flow cytometry that uses a cocktail of monoclonal antibodies
directed to cell surface markers present on the cells negatively
selected. For example, to enrich for CD4.sup.+cells by negative
selection, a monoclonal antibody cocktail typically includes
antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
[0066] Another method for preparing T cells for stimulation is to
freeze the cells after the washing step, which does not require the
monocyte-removal step. Wishing not to be bound by theory, the
freeze and subsequent thaw step provides a more uniform product by
removing granulocytes and, to some extent, monocytes in the cell
population. After the washing step that removes plasma and
platelets, the cells may be suspended in a freezing solution. While
many freezing solutions and parameters are known in the art and
will be useful in this context, one method involves using PBS
containing 20% DMSO and 8% human serum albumin (HSA), or other
suitable cell freezing media. This is then diluted 1:1 with media
so that the final concentration of DMSO and HSA are 10% and 4%,
respectively. The cells are then frozen to -80.degree. C. at a rate
of 1.degree. per minute and stored in the vapor phase of a liquid
nitrogen storage tank.
[0067] Sources of Antigen-Presenting Cells (APC)
[0068] The source of antigen-presenting cell (APC) is typically a
tissue source comprising APC or APC precursors that are capable of
proliferating and maturing in vitro into professional APC (pAPC)
when loaded with antigen and/or treated with the necessary
cytokines or factors. "Professional APC" (pAPC) or
"antigen-presenting cell" (APC), as used herein, refers to those
cells that normally initiate the responses of nave and/or memory T
cells to antigen. Professional APCs include, but are not limited
to, DC, macrophages, and B cells. pAPC may express high levels of
MHC class II, ICAM-1 and B7-2. In one aspect, APC precursor cells
are capable of proliferating and maturing in vitro into dendritic
cells (DC). While many tissue sources may be used, typical tissue
sources comprise spleen, thymus, tissue biopsy, tumor, afferent
lymph, lymph nodes, bone marrow, apheresis or leukapheresis
product, and/or peripheral blood. In certain embodiments, apheresis
product, bone marrow and peripheral blood are preferred sources.
Fetal tissue, fetal or umbilical cord blood, which is also rich in
growth factors may also be used as a source of blood for obtaining
APC and/or precursor APC. Exemplary precursor cells may be, but are
not limited to, embryonic stem cells, CD34.sup.+ cells, monocyte
progenitors, monocytes, and pre-B cells.
[0069] Further, according to one aspect of the present invention,
APC may be derived from precursor cells comprising monocytes or
CD34.sup.+ cells.
[0070] In one aspect of the present invention, the source of APC
and/or precursor APC is an apheresis or leukapheresis product.
Cells are collected using apheresis procedures known in the art.
See, for example, Bishop et al., Blood, vol. 83, No. 2, pp. 610-616
(1994). Briefly, cells are collected using conventional devices,
for example, a Haemonetics Model V50 apheresis device (Haemonetics,
Braintree, Mass.). Apheresis product typically contains
lymphocytes, including T cells, monocytes, granulocytes, B cells,
other nucleated white blood cells, red blood cells, and platelets.
In one embodiment, the cells collected by apheresis may be washed
to remove the plasma fraction and to place the cells in an
appropriate buffer or media for subsequent processing steps. In
another embodiment of the invention, the cells are washed with
phosphate buffered saline (PBS). In an alternative embodiment, the
wash solution lacks calcium and may lack magnesium or may lack many
if not all divalent cations. As those of ordinary skill in the art
would readily appreciate a washing step may be accomplished by
methods known to those in the art, such as by using a
semi-automated "flow-through" centrifuge (for example, the Cobe
2991 cell processor, Gambro BCT, Lakewood, Colo.) according to the
manufacturer's instructions. After washing, the cells may be
resuspended in a variety of biocompatible buffers, such as, for
example, Ca-free, Mg-free PBS. Alternatively, the undesirable
components of the apheresis sample may be removed and the cells
directly resuspended in culture media.
[0071] When blood is used as a source of APC, blood leukocytes may
be obtained using conventional methods that maintain their
viability. According to one aspect of the invention, blood is
diluted into medium (preferably RPMI) that may or may not contain
heparin (about 100 U/ml) or other suitable anticoagulant. The
volume of blood to medium is about 1 to 1. Cells are concentrated
by centrifugation of the blood in medium at about 1000 rpm (150 g)
at 4.degree. C. Platelets and red blood cells are depleted by
resuspending the cells in any number of solutions known in the art
that will lyse erythrocytes, for example ammonium chloride. For
example, the mixture may be medium and ammonium chloride (at a
final concentration of about 0.839 percent) at about 1:1 by volume.
Cells may be concentrated by centrifugation and washed in the
desired solution until a population of leukocytes, substantially
free of platelets and red blood cells, is obtained, typically about
two times. Any isotonic solution commonly used in tissue culture
may be used as the medium for separating blood leukocytes from
platelets and red blood cells. Examples of such isotonic solutions
are phosphate buffered saline, Hanks balanced salt solution, or
complete growth media including for example RPMI 1640, DMEM, MEM,
HAMS F-12, X-Vivo 15, or X-Vivo 20. APC and/or APC precursor cells
may also purified by elutriation, using, for example, a Beckman
J6ME centrifuge equipped with a J5.0 rotor and a 40 ml elutriation
chamber.
[0072] In one embodiment of the present invention, isolation of APC
and/or precursor APC is performed by preincubating ficolled whole
blood or apheresed peripheral blood with one or more varieties of
irrelevant or non-antibody coupled paramagnetic particles (approx.
1 vial of beads or 4.times.10.sup.9 beads to one batch of cells
(typically from about 5.times.10.sup.8 to about 2.times.10.sup.10
cells) for about 30 minutes to 2 hours at 22 to 37 degrees C.,
followed by magnetic removal of cells which have attached to or
engulfed the paramagnetic particles. Such separation can be
performed using standard methods available in the art. For example,
any magnetic separation methodology may be used including a variety
of which are commercially available, (e.g., DYNAL.RTM. Magnetic
Particle Concentrator (DYNAL MPC.RTM.)). Assurance of isolation can
be monitored by a variety of methodologies known to those of
ordinary skill in the art, including flow cytometric analysis of
cells before and after said isolation.
[0073] APC obtained from treatment of the tissue source may be
cultured to form a primary culture in an appropriate culture
container or vessel in an appropriate culture medium. In certain
embodiments, the culture medium is supplemented with one or more
cytokines. According to the present invention, the appropriate
culture container or vessel may be any container with tissue
culture compatible surface. Examples include various bags (e.g.,
Lifecell culture bags), flasks, roller bottles, petri dishes and
multi-well containing plates made for use in tissue culture.
Surfaces treated with a substance, for example collagen or
poly-L-lysine, or antibodies specific for a particular cell type to
promote cell adhesion may also be used provided they allow for the
differential attachment of cells as described below. Surfaces may
be also be chemically treated, for example by ionization. Cells are
plated at an initial cell density from about 10.sup.5 to 10.sup.7
cells/cm.sup.2. In one aspect, cells are plated at 10.sup.6 cell
s/cm.sup.2.
[0074] In one embodiment, the primary cultures from the selected
tissue source are allowed to incubate at about 37.degree. C. under
standard tissue culture conditions of humidity, CO.sub.2, and pH
until a population of cells has adhered to the substrate
sufficiently to allow for the separation of nonadherent cells. Some
immature APC in blood initially are nonadherent to plastic,
particularly immature DC, in contrast to monocytes, so that the
precursors can be separated after overnight culture. Monocytes and
fibroblasts are believed to comprise the majority of adherent cells
and usually adhere to the substrate within about 30 minutes to
about 24 hours. In certain aspects, nonadherent cells are separated
from adherent cells between about 1 to 16 hours. Nonadherent cells
may be separated at about 1 to 2 hours. Any method which does not
dislodge significant quantities of adherent cells may be used to
separate the adherent from nonadherent cells. In certain aspects,
the cells are dislodged by simple shaking or pipetting. Pipetting
is most preferred.
[0075] Adherent cells comprising precursor APC (e.g., monocytes)
isolated according to the methods of the invention are allowed to
incubate at about 37.degree. C. under standard tissue culture
conditions of humidity, CO.sub.2, and pH until a population of
cells has reached an immature APC stage. In certain aspects,
according to the present invention, adherent cells are allowed to
incubate for a period of between 4 hours and 7 days. However, one
of ordinary skill in the art will readily appreciate that
incubation times and conditions may vary. "Immature APC" as used
herein, refers to an intermediate differentiation state of an APC
wherein the APC has the capacity to endocytose or phagocytose
antigen, foreign bodies, necrotic and/or apoptosing tissue and/or
cells. Immature APC may be CD14.sup.- or CD14.sup.+depending on the
origin of the precursor cells. Immature APC may also express CD1a,
CD40, CD86, CD54, and intermediate levels of MHC class II (levels
of marker expression on sample cells can be compared by flow
cytometric analysis to levels of expression on MHC class
II-negative cells and cells known to express high levels of MHC
class II). Immature APC typically do not express CCR7.
[0076] In certain aspects of the present invention, it is not
necessary to separate T cells from APC. For example, in one
embodiment, PBMC comprising APC and T cells can be exposed to
antigen as described herein and the resulting antigen-specific T
cells further expanded as described herein.
[0077] In certain aspects of the present invention, it is not
required that the APCs or the T cells described herein be derived
from an autologous source. Thus, the APC and T cells can be
obtained from a matched or unmatched donor, or from a cell line, a
T cell line, or other cells grown in vitro. Methods for matching
haplotypes are known in the art. Furthermore, the APC and T cells
or supernatant therefrom may be obtained from a xenogeneic source,
for example, mouse, rat, non-human primate, and porcine cells may
be used.
[0078] Sources of Antigen
[0079] According to the present invention, the source of antigen
may be, but is not limited to, protein, including glycoprotein,
peptides (including pools of overlapping peptides), superantigens
(e.g., SEA, SEB, TSST-1) antibody/antigen complexes, tumor lysate,
viral lysate (e.g., CMV lysate and the like), non-soluble cell
debris, apoptotic bodies, necrotic cells, whole cells which are
live, fixed, irradiated, heat-killed or otherwise manipulated,
whole tumor cells from a tumor or a cell line that have been
treated such that they are unable to continue dividing, allogeneic
cells that have been treated such that they are unable to continue
dividing, irradiated tumor cells, irradiated allogeneic cells,
natural or synthetic complex carbohydrates, lipoproteins,
lipopolysaccharides, RNA or a translation product of said RNA, and
DNA or a polypeptide encoded by said DNA. Non-transformed cells are
typically irradiated with gamma rays in the range of about 3000 to
3600 rads, more preferably at about 3300 rads. Lymphoblastoid or
tumor cell lines are typically irradiated with gamma rays in the
range of about 6000 to 10,000 rads, more preferably at about 8000
rads. Necrotic and apoptotic cells may be generated by physical,
chemical, or biological means. Necrotic cells are typically
generated by freeze-thawing, while apoptotic cells are generated
using UV irradiation. UV and gamma irradiation, and freeze-thawing
procedures are well known in the art and are described, for
example, in Current Protocols in Molecular Biology or Current
Protocols in Immunology, John Wiley & Sons, New York. N.Y.
[0080] Antigen source may also comprise non-transformed,
transformed, transfected, or transduced cells or cell lines. Cells
may be transformed, transfected, or transduced using any of a
variety of expression or retroviral vectors known to those of
ordinary skill in the art that may be employed to express
recombinant antigens. Expression may also be achieved in any
appropriate host cell that has been transformed, transfected, or
transduced with an expression or retroviral vector containing a DNA
molecule encoding recombinant antigen(s). Any number of
transfection, transformation, and transduction protocols known to
those in the art may be used, for example those outlined in Current
Protocols in Molecular Biology, John Wiley & Sons, New York.
N.Y., or in numerous kits available commercially (e.g., Invitrogen
Life Technologies, Carlsbad, Calif.). In one embodiment of the
present invention, recombinant vaccinia vectors and cells infected
with said vaccina vectors, may be used as a source of antigen.
Recombinant antigen may include any number of defined tumor
antigens described below.
[0081] According to certain methods of the invention, antigen may
comprise viral antigens such as CMV pp65, HIV pg120, and the like.
In certain embodiments, antigen may comprise defined tumor antigens
such as the melanoma antigen Melan-A (also referred to as melanoma
antigen recognized by T cells or MART-1), melanoma antigen-encoding
genes 1, 2, and 3 (MAGE-1, -2, -3), melanoma GP100,
carcinoembryonic antigen (CEA), the breast cancer angtigen,
Her-2/Neu, serum prostate specific antigen (PSA), Wilm's Tumor
(WT-1), PR1, PR3 (antigens implicated in the graft-versus-leukemia
(GVL) effect in chronic myeloid leukemia), mucin antigens, MUC-1,
-2, -3, -4, B cell lymphoma idotypes, and the like. The skilled
artisan would appreciate that any tumor antigen would be useful in
the context of the present invention.
[0082] Activation of Antigen-Specific T Cells
[0083] One aspect of the present invention stems from the
surprising finding that using different bead:cell ratios can lead
to different outcomes with respect to expansion of antigen-specific
T cells. In particular, bead:cell ratios can be varied to
selectively expand or delete antigen-specific (memory) T cells. In
one embodiment, the particular bead:cell ratio used selectively
expands antigen-specific T cells. Thus, in one embodiment of the
present invention, antigen-specific T cells are activated by direct
contact of a population of cells wherein at least a portion thereof
comprises T cells (e.g., a leukaphersis product from an individual,
blood sample, tumor biopsy, etc.), with a surface, wherein said
surface has attached thereto a first agent that ligates a first T
cell surface moiety of a T cell, and the same or a second surface
has attached thereto a second agent that ligates a second moiety of
said T cell, wherein said ligation by the first and second agent
induces proliferation (expansion) of antigen-specific T cells
present within the population of cells.
[0084] Without being bound by theory, it is thought that the
antigen-specific T cells are sensitized to further stimulation.
Thus, the key appears to be the strength of the T cell activation
signal: selective expansion of memory T cells (antigen-specific T
cells) occurs with "weak" signals while selective deletion of
memory T cells occurs with "strong" signals. The quantity of the
CD3/TCR (and CD28) receptors that are bound by ligands determines
the signal strength. Thus, stimulation with high bead:cell ratios
provides a high concentration of stimulating antibody (i.e.,
"strong signal"), leading to over-stimulation of antigen-specific T
cells, causing them to die, either by apoptosis or other
mechanisms. Using lower bead:cell ratios provides a stimulation
signal to antigen-specific T cells that does not over-stimulate,
but rather induces rapid proliferation of these cells.
[0085] In one embodiment of the present invention, antigen-specific
T cells are activated by culturing T cells isolated as described
herein above, with APC that have been loaded with antigen.
[0086] In another embodiment, suitable APC are plated in culture
dishes and exposed to a source of antigen as described herein, in a
sufficient amount and for a sufficient period of time to allow the
antigen to bind and/or be taken up by the APC. In certain aspects,
antigen is exposed to the APC for a period of time between 24 hours
and 4 days. In one particular embodiment, the antigen is exposed to
the APC for 36, 48, or 72 hours. In a further embodiment, the
antigen is exposed to the APC for 2.5, 3, 3.5, or 4 days. In
certain embodiments, antigen may be exposed to the APC for periods
longer than 4 days, for example 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,
8.5, 9, 9.5, or 10 days. The amount and time necessary to achieve
binding and uptake of the antigen by the APC may differ depending
on the source and type of antigen and may be determined by those of
ordinary skill in the art by immunoassay or binding assay. Other
methods known to those of skill in the art may be used to detect
the presence of antigen in the context of MHC on the APC following
their exposure to antigen.
[0087] In yet an additional embodiment, PBMC (e.g., from blood, a
leukapheris product, etc.) from a subject are cultured directly in
the presence of antigen, as described herein, to load APC with the
antigen and to activate/stimulate antigen-specific T cells present
in the PBMC. In this regard, PBMC may be collected from an
individual, contacted with an antigen of interest, such as a tumor
antigen, or a viral lysate, etc. In this manner, the APC present in
the PBMC are loaded with the antigen, which is then presented to
the T cells present in the sample. In an additional embodiment, the
antigen-specific T cells of the present invention may be stimulated
with peptide-MHC tetramers, see for example Altman, et al., Science
1998 Jun. 19; 280(5371):1821.
[0088] The APC of the present invention may be loaded with antigen
through genetic modification. Genetic modification may comprise RNA
or DNA transfection using any number of techniques known in the
art, for example electroporation (using e.g., the Gene Pulser II,
BioRad, Richmond, Calif.), various cationic lipids,
(LIPOFECTAMINE.TM., Life Technologies, Carlsbad, Calif.), or other
techniques such as calcium phosphate transfection as described in
Current Protocols in Molecular Biology, John Wiley & Sons, New
York. N.Y. For example, 5-50 .mu.g of RNA or DNA in 500 .mu.l of
Opti-MEM can be mixed with a cationic lipid at a concentration of
10 to 100 .mu.g, and incubated at room temperature for 20 to 30
minutes. Other suitable lipids include LIPOFECTIN.TM.,
LIPOFECTAMINE.TM.. The resulting nucleic acid-lipid complex is then
added to 1-3.times.10.sup.6 cells, preferably 2.times.10.sup.6,
antigen-presenting cells in a total volume of approximately 2 ml
(e.g., in Opti-MEM), and incubated at 37.degree. C. for 2 to 4
hours. The APC may also be transduced using viral transduction
methodologies as described below.
[0089] In another embodiment of the present invention, APC are
loaded with antigen attached to, coated on, or otherwise
immobilized on particles, such as beads. In the various
embodiments, commercially available beads or other particles, are
useful, e.g., Miltenyi Particles, Miltenyi Biotec, Germany;
Sepharose beads, Pharmacia Fine Chemicals, Sweden; DYNABEADS.TM.,
Dynal Inc., New York. In certain embodiments, paramagnetic
particles or beads are particularly suitable. Such paramagnetic
beads or particles are commercially available, for example, those
produced by Dynal AS under the trade name Dynabeads.TM.. Exemplary
Dynabeads.TM. in this regard are M-280, M-450, and M-500. In one
embodiment, whole cells which are live, fixed, irradiated,
heat-killed or ohterwise manipulated, are immobilized to ingestable
beads, via for example antibody/ligand specific means or chemical
means. Similarly, tumor cell or virus-infected cell lysates, or
antigen-preparations can be attached or otherwise immobilized to
the beads (which may be paramagnetic or otherwise selectable).
These coated or antigen/cell/lysate-attached beads can be mixed
with human or other animal peripheral blood preparations (or other
compositions containing some percentage of antigen-presenting cells
(particularly those capable of ingesting particles and then
processing and presenting antigens associated with the particles).
Phagocytic cells will ingest the beads/particles, process antigens
associated with the particles, and present them to T cells in the
cell mix. As noted elsewhere herein, only T cells with specificity
for the variety of presented antigens will interact in a positive
manner with the APC. APC containing paramagnetic or otherwise
selectable beads can then be isolated carrying with them
antigen-specific T cells.
[0090] In one particular embodiment, the particles of the present
invention comprise a cell surface, such as described in U.S. patent
application Ser. No. 10/336,224, PCT/US03/00339. In this regard,
antigen can be attached to the cells via antibody/ligand specific
means as described herein or through genetic modification. Any
number of transfection, transformation, and transduction protocols
known to those in the art may be used, for example those outlined
in Current Protocols in Molecular Biology, John Wiley & Sons,
New York. N.Y., or in numerous kits available commercially (e.g.,
Invitrogen Life Technologies, Carlsbad, Calif.). Such techniques
may result in stable transformants or may be transient. One
suitable transfection technique is electroporation, which may be
performed on a variety of cell types, including mammalian cells,
yeast cells and bacteria, using commercially available equipment.
Optimal conditions for electroporation (including voltage,
resistance and pulse length) are experimentally determined for the
particular host cell type, and general guidelines for optimizing
electroporation may be obtained from manufacturers. Other suitable
methods for transfection will depend upon the type of cell used
(e.g., the lithium acetate method for yeast), and will be apparent
to those of ordinary skill in the art. Following transfection,
cells may be maintained in conditions that promote expression of
the polynucleotide within the cell. Appropriate conditions depend
upon the expression system and cell type, and will be apparent to
those skilled in the art.
[0091] Antigen may be attached to the particles, such as beads, by
antibody/ligand specific means, e.g. through particles, such as
beads, conjugated to an antibody or antibodies. Suitable
antibody/ligand pairs may include, but are not limited to
anti-MART-1 antibody/MART-1 antigen, anti-WT-1 antibody/WT-1,
anti-PR1 antibody /PR1, anti-PR3 antibody/PR3, anti-tyrosinase
antibody/tyrosinase antigen, anti-MAGE-1 antibody/MAGE-1 antigen,
anti-MUC-1 antibody/MUC-1 antigen, anti-.alpha.-fetoprotein
antibody/.alpha.-fetoprotein antigen, anti-Her2Neu
antibody/Her2Neu, anti-HIV gp120 antibody/HIV gp120, anti-influenza
HA antibody/influenza HA, anti-CMV pp65/CMV pp65, anti-hepatitis C
antibody/hepatitis C proteins, anti-EBV EBNA 3B antibody/EBV EBNA
3B antigen, and anti-human Ig heavy and lignt chains/Ig from cancer
patient, such as myeloma or CLL patient. Other protein:protein
binding interactions may be suitable for attaching antigen to
particles, such as beads, for example, receptor/ligand interactions
may be utilized. In certain embodiments, the antigen/protein is
attached to the particles, such as beads by chemical means, e.g.
antigen/protein can be bound through non-covalent association of
the antigen and bead, simply by incubating/contacting the two
together for a time and under conditions sufficient for association
to occur. In yet further embodiments, antigen may be attached to
the particles, such as beads by a biotin/avidin or streptavidin
interaction. In certain embodiments, hydrophobic "naked" beads with
p-toluenesulphonyl (tosyl) reactive groups are used. Proteins are
adsorbed hydrophobically on initial coupling with covalent binding
of primary amine groups (NH.sub.2) and sulphydryl groups (SH)
occurring overnight. Coupling reactions can be performed at neutral
pH however high pH and incubation at 37.degree. C. can promote
covalent binding.
[0092] In certain aspects, T cells isolated from a tissue source
are exposed to antigen-loaded APC described herein for a time
sufficient for T cells specific for a given antigen to be
activated, for example as described in U.S. Pat. No. 5,827,642, or
as described in Riddell, et al., 1990, J. Immunol. Methods,
128:189-201. In one embodiment, T cells are exposed to
antigen-loaded APC for a period of between about several hours to
about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or about 20 days.
[0093] In one embodiment, the T cells are exposed to antigen, or
antigen-loaded APC as described herein in vivo. In this regard,
antigen or antigen-loaded APC may be administered to an individual
in order to stimulate and activate the T cells in vivo. The T cells
may then be expanded either in vivo or ex vivo using the methods as
described herein, such as with anti-CD3/anti-CD28 beads. The
quantity and frequency of administration will be determined by such
factors as the condition of the individual, and the type and
severity of disease, although appropriate dosages may be determined
by clinical trials. In certain embodiments the T cells are exposed
to antigen in vivo in an individual prior to onset of a disease or
prior to treatment with other known therapies. In this regard, the
antigen-specific T cells are generated and then isolated and
expanded and preserved for later use.
[0094] In one embodiment of the present invention, isolation of
antigen-specific T cells in direct contact with APC loaded with
antigen immobilized on particles, such as beads, is performed by
magnetic isolation of cells which have attached to or engulfed
paramagnetic particles. Such separation can be performed using
standard methods available in the art. For example, any magnetic
separation methodology may be used including a variety of which are
commercially available, (e.g., DYNAL.RTM. Magnetic Particle
Concentrator (DYNAL MPC.RTM.), MACS, Miltenyi Biotec, Germany). In
this regard, only T cells with specificity for the variety of
presented antigens will optimally interact in a positive manner
with the APC. APC containing paramagnetic (or otherwise selectable)
beads can then be isolated (via magnet or otherwise) carrying with
them antigen-specific T cells. These antigen-specific T cells can
then be activated/expanded by a variety of means, such as via
XCELLERATE.TM. technologies as described herein and U.S. patent
application Ser. Nos. 10/350,305; 10/187,467; 10/133,236;
09/960,264; 09/794,230; PCT/US01/06139; and PCT/US02/28161.
[0095] In another embodiment of the invention, antigen-specific T
cells are isolated by positive selection. Such isolation can be
carried out on T cells freshly isolated from a subject or on T
cells that have been exposed to antigen or antigen-loaded APC as
described herein. Numerous immunoselection methods known to skilled
artisans may be used. Such techniques are described, for example,
in Current Protocols in Immunology, John Wiley & Sons, New
York. N.Y. Markers that may be useful for the positive selection of
antigen-specific cells include, but are not limited to, CD25, CD54,
CD69, CD38, CD45RO, CD49d, CD40L, CD137, CD62L, and CD134. In one
embodiment, fluorescence activated cell sorting may also be used to
isolate desired antigen-specific T cells. In an additional
embodiment, antigen-specific T cells may be isolated using
peptide-MHC tetramers, see for example Altman, et al., Science 1998
Jun. 19; 280(5371):1821.
[0096] In a further embodiment of the invention, antigen-specific T
cells may be genetically modified. Genetic modification may
comprise RNA or DNA transfection using any number of techniques
known in the art, for example electroporation (using e.g., the Gene
Pulser II, BioRad, Richmond, Calif.), various cationic lipids,
(LIPOFECTAMINE.TM., Life Technologies, Carlsbad, Calif.), or other
techniques such as calcium phosphate transfection as described in
Current Protocols in Molecular Biology, John Wiley & Sons, New
York. N.Y. For example, 5-50 .mu.g of RNA or DNA in 500 .mu.l of
Opti-MEM can be mixed with a cationic lipid at a concentration of
10 to 100 .mu.g, and incubated at room temperature for 20 to 30
minutes. Other suitable lipids include LIPOFECTIN.TM.,
LIPOFECTAMINE.TM.. The resulting nucleic acid-lipid complex is then
added to 1-3.times.10.sup.6 cells, preferably 2.times.10.sup.6,
antigen-presenting cells in a total volume of approximately 2 ml
(e.g., in Opti-MEM), and incubated at 37.degree. C. for 2 to 4
hours. The APC may also be transduced using viral transduction
methodologies as described below.
[0097] The antigen-specific T cells of the present invention may
alternatively be genetically modified using retroviral transduction
technologies. In one aspect of the invention, the retroviral vector
may be an amphotropic retroviral vector, preferably a vector
characterized in that it has a long terminal repeat sequence (LTR),
e.g., a retroviral vector derived from the Moloney murine leukemia
virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine
embryonic stem cell virus (MESV), murine stem cell virus (MSCV),
spleen focus forming virus (SFFV), or adeno-associated virus (AAV).
Most retroviral vectors are derived from murine retroviruses.
Retroviruses adaptable for use in accordance with the present
invention can, however, be derived from any avian or mammalian cell
source. These retroviruses are preferably amphotropic, meaning that
they are capable of infecting host cells of several species,
including humans. In one embodiment, the gene to be expressed
replaces the retroviral gag, pol and/or env sequences. A number of
illustrative retroviral systems have been described (e.g., U.S.
Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)
BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy
1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al.
(1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie
and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
[0098] In one aspect of the present invention, genetically modified
antigen-specific T cells can be isolated by any one of numerous
immunoselection methods known to skilled artisans using antibodies
or other receptors/ligands specific for the protein or proteins
expressed from the transgene. Such techniques are known in the art,
for example, in Current Protocols in Immunology, John Wiley &
Sons, New York. N.Y.
[0099] In one particular embodiment, the antigen-specific T cells
may be genetically modified to express a suicide gene, e.g. the
herpes simplex virus thymidine kinase (HSV-TK) as described in
Bonini, et al., 1997 Science, 276(5319):1719-24, and/or other
surface markers (e.g., truncated nerve growth factor (dNGFR)) for
in vivo tracking and/or control of infused antigen-specific T
cells. In a further embodiment, the antigen-specific T cells may be
genetically modified to express a protein for targeting the T cells
to a particular tissue of interest.
[0100] Those of ordinary skill in the art will readily appreciate
that the cell separation and culture methodologies described
herein, may be carried out in a variety of environments (i.e.,
containers). Examples include various bags (e.g., Lifecell culture
bags), flasks, roller bottles, bioreactors, (e.g., CellCube
(Corning Science Products) or CELL-PHARM, (CD-Medical, Inc. of
Hialeah, Fla.)), petri dishes and multi-well containing plates made
for use in tissue culture, or any container capable of holding
cells, preferably in a sterile environment. In one embodiment of
the present invention a bioreactor is also useful. For example,
several manufacturers currently manufacture devices that can be
used to grow cells and be used in combination with the methods of
the present invention. See for example, Celdyne Corp., Houston,
Tex.; Unisyn Technologies, Hopkinton, Mass.; Synthecon, Inc.
Houston, Tex.; Aastrom Biosciences, Inc. Ann Arbor, Mich.; Wave
Biotech LLC, Bedminster, N.J. Further, patents covering such
bioreactors include U.S. Pat. Nos. 6,096,532; 5,985,653; 5,888,807;
5,190,878.
[0101] Suitable complete growth media for the culture of the APC
and antigen-specific T cells of the present invention include for
example RPMI 1640, DMEM, MEM, .alpha.-MEM, AIM-V, HAMS F-12, X-Vivo
15, or X-Vivo 20. In further embodiments, the media can comprise a
cytokine, such as IL-2, IFN-.gamma., IL-4, GM-CSF, IL-10, IL-12,
TGF.beta., and TNF-.alpha., or a vitamin. In further embodiments,
the medium comprises surfactant, an antibody, plasmanate or a
reducing agent (e.g. N-acetyl-cysteine, 2-mercaptoethanol). The
growth medium for the cells at each step of the method of the
invention should allow for the survival of the APC and/or the
antigen-specific T cells. Any growth medium typically used to
culture cells may be used according to the method of the invention
provided the medium is supplemented with the appropriate cytokines,
serum, antibiotics, vitamins, amino acids or other necessary
additives. According to the present invention, the cytokines may
be, but are not limited to, granulocyte-macrophage
colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), or
IL-13. Other exemplary cytokines and growth factors that may be
added to the growth medium include but are not limited to
interleukin 1.alpha. (IL-1.alpha.) and .beta. (IL-1.beta.), IL-2,
tumor necrosis factor alpha (TNF-.alpha.), interleukin 3 (IL-3),
monocyte colony stimulating factor (M-CSF), granulocyte
colony-stimulating factor (G-CSF), stem cell factor (SCF),
interleukin 6 (IL-6), interleukin 15 (IL-15), and Flt3-ligand.
Preferred media include RPMI 1640, AIM-V, DMEM, MEM, .alpha.-MEM,
F-12, X-Vivo 15, and X-Vivo 20, with added amino acids and
vitamins, either serum-free or supplemented with an appropriate
amount of serum (or plasma) or a defined set of hormones, and an
amount of cytokine(s) sufficient to support the expansion of the
antigen-specific T cells. In one aspect, the preferred media
comprises 1 liter of X-Vivo 15, BioWhittaker; with 50 ml heat
inactivated pooled human serum, 20 ml 1 M Hepes, 10 ml 200 mM
L-glutamine with or without about 100,000 I.U. IL-2. In one aspect,
media may include lipids and/or sources of protein. RPMI 1640
supplemented with 1-5% human AB serum preferred. Mixtures of
cytokines may also be used. Cells may also be adapted to grow in
other sera, such as fetal calf (bovine) serum (FCS/FBS), at other
concentrations of serum, or in serum-free media. For example,
serum-free medium supplemented with hormones is also suitable for
culturing the APC precursors. Media may, but does not necessarily,
contain antibiotics to minimize growth of bacteria in the cultures.
Penicillin, streptomycin or gentamicin or combinations containing
them are preferred. The medium, or a portion of the medium, in
which the cells are cultured should be periodically replenished to
provide fresh nutrients including GM-CSF, IL-4, IL-13, IL-15 and/or
other cytokines.
[0102] Expansion of Antigen-Specific T Cells
[0103] Expansion of the antigen-specific T cells of the present
invention is carried out by cell surface moiety ligation that
re-stimulates the antigen-specific T cells to proliferate. In one
embodiment of the present invention, the antigen-specific T cells
are first isolated by methods described herein following exposure
to antigen loaded APC. In another embodiment of the present
invention, the antigen-specific T cells are expanded directly from
the culture with antigen-loaded APC present without an isolation
step.
[0104] In one particular embodiment, antigen-specific T cells are
activated and expanded using XCELLERATE.TM. processes as described
herein and in U.S. patent application Ser. Nos. 10/350,305;
10/187,467; 10/133,236; 09/960,264; 09/794,230, with no addition of
antigen or antigen-coated particles. In this regard, as noted
further herein, antigen-specific T cells that have been previously
stimulated or activated in vivo (e.g. memory T cells) are expanded
by an agent providing a primary activation signal such as an
anti-CD3 antibody and an agent providing a co-stimulatory signal,
such as an anti-CD28 antibody, with both agents co-immobilized to
the same surface, such as a paramagnetic bead. As further described
herein, see in particular the Examples below, varying the bead:cell
ratios during this expansion phase, in particular using low
bead:cell ratios, favors expansion of antigen-specific T cells. For
example, bead to cell ratios of 1:200, 1:150, 1:125, 1:110, 1:100,
1:75, 1:50, 1:25, 1:20, 1:15, 1:10, 1:5 or 1:2.5 are used to expand
antigen-specific T cells. A particular advantage of this aspect of
the present invention is that it is not necessary to add
antigen.
[0105] Generally, expansion is carried out by re-stimulating a
population of antigen-specific T cells and simultaneously
stimulating an accessory molecule on the surface of the
antigen-specific T cells with a ligand which binds the accessory
molecule, as described for example, in U.S. patent application Ser.
Nos. 10/350,305, 10/187,467, 10/133,236, 09/960,264, 09/794,230,
08/253,694, 08/403,253, 08/435,816, 08/592,711, 09/183,055,
09/350,202, and 09/252,150, and U.S. Pat. Nos. 5,858,358;
6,352,694; and 5,883,223.
[0106] Generally, re-stimulation may be accomplished by cell
surface moiety ligation, such as through the T cell receptor
(TCR)/CD3 complex or the CD2 surface protein. A number of
anti-human CD3 monoclonal antibodies are commercially available,
exemplary are, clone BC3 (XR-CD3; Fred Hutchinson Cancer Research
Center, Seattle, Wash.), OKT3, prepared from hybridoma cells
obtained from the American Type Culture Collection, and monoclonal
antibody G19-4. Similarly, stimulatory forms of anti-CD2 antibodies
are known and available. Stimulation through CD2 with anti-CD2
antibodies is typically accomplished using a combination of at
least two different anti-CD2 antibodies. Stimulatory combinations
of anti-CD2 antibodies that have been described include the
following: the T11.3 antibody in combination with the T11.1 or
T11.2 antibody (Meuer et al., Cell 36:897-906, 1984), and the 9.6
antibody (which recognizes the same epitope as T11.1) in
combination with the 9-1 antibody (Yang et al., J. Immunol.
137:1097-1100, 1986). Other antibodies that bind to the same
epitopes as any of the above-described antibodies can also be used.
Additional antibodies, or combinations of antibodies, can be
prepared and identified by standard techniques. Re-stimulation may
also be achieved through contact with antigen, peptide, protein,
peptide-MHC tetramers (see Altman, et al Science 1996 Oct. 4;
274(5284):94-6), superantigens (e.g., Staphylococcus enterotoxin A
(SEA), Staphylococcus enterotoxin B (SEB), Toxic Shock Syndrome
Toxin 1 (TSST-1)), endotoxin, or through a variety of mitogens,
including but not limited to, phytohemagglutinin (PHA), phorbol
myristate acetate (PMA) and ionomycin, lipopolysaccharide (LPS), T
cell mitogen, and IL-2.
[0107] The antigen-specific cell population may be stimulated or
restimulated as described herein, such as by contact with an
anti-CD3 antibody or an anti-CD2 antibody immobilized on a surface,
or by contact with a protein kinase C activator (e.g., bryostatin)
in conjunction with a calcium ionophore. For co-stimulation of an
accessory molecule on the surface of the T-cells, a ligand that
binds the accessory molecule is used. For example, a population of
CD4.sup.+cells can be contacted with an anti-CD3 antibody and an
anti-CD28 antibody, under conditions appropriate for stimulating
proliferation of the T-cells. Similarly, to stimulate proliferation
of CD8.sup.+T-cells, an anti-CD3 antibody and the anti-CD28
antibody B-T3, XR-CD28 (Diaclone, Besan.cedilla.on, France) can be
used as can other methods commonly known in the art (Berg et al.,
Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.
190(9):1319-1328, 1999; Garland et al., J. Immunol Meth.
227(1-2):53-63, 1999).
[0108] To further re-stimulate a population of antigen-specific T
cells, a co-stimulatory or accessory molecule on the surface of the
T cells, such as CD28, is stimulated with a ligand that binds the
accessory molecule. Accordingly, one of ordinary skill in the art
will recognize that any agent, including an anti-CD28 antibody or
fragment thereof capable of cross-linking the CD28 molecule, or a
natural ligand for CD28 can be used to stimulate T cells. Exemplary
anti-CD28 antibodies or fragments thereof useful in the context of
the present invention include monoclonal antibody 9.3 (IgG2.sub.a)
(Bristol-Myers Squibb, Princeton, N.J.), monoclonal antibody KOLT-2
(IgG1), 15E8 (IgG1), 248.23.2 (IgM), clone B-T3 (XR-CD28; Diaclone,
Besan.cedilla.on, France) and EX5.3D10 (IgG2.sub.a) (ATCC HB11373).
Exemplary natural ligands include the B7 family of proteins, such
as B7-1 (CD80) and B7-2 (CD86) (Freedman et al., J. Immunol.
137:3260-3267, 1987; Freeman et al., J. Immunol. 143:2714-2722,
1989; Freeman et al., J. Exp. Med. 174:625-631, 1991; Freeman et
al., Science 262:909-911, 1993; Azuma et al., Nature 366:76-79,
1993; Freeman et al., J. Exp. Med. 178:2185-2192, 1993).
[0109] In a further embodiment of the invention, activation of a
T-cell population may be enhanced by co-stimulation of other T-cell
integral membrane proteins. For example, binding of the T-cell
integrin LFA-1 to its natural ligand, ICAM-1, may enhance
activation of cells. Another cell surface molecule that may act as
a co-stimulator for T-cells is VCAM-1 (CD106) that binds
very-late-antigen-4 (VLA-4) on T-cells. Ligation of 4-1BB (CD137),
a co-stimulatory receptor expressed on activated T cells, and/or
NKG2D may also be useful in the context of the present invention to
amplify T-cell mediated immunity. It should be noted that more than
one costimulatory molecule as described herein may be stimulated at
a time, and in any combination, such that desired expansion of the
T cells occurs.
[0110] In addition, binding homologues of a natural ligand, whether
native or synthesized by chemical or recombinant techniques, can
also be used in accordance with the present invention. Other agents
may include natural and synthetic ligands. Agents may include, but
are not limited to, other antibodies or fragments thereof, a
peptide, polypeptide, growth factor, cytokine, chemokine,
glycopeptide, soluble receptor, steroid, hormone, mitogen, such as
PHA, or other superantigens.
[0111] The primary stimulatory signal and the co-stimulatory signal
for the T-cell may be provided by different protocols. For example,
the agents providing each signal may be in solution or coupled to a
surface. When coupled to a surface, the agents may be coupled to
the same surface (i.e., in "cis" formation) or to separate surfaces
(i.e., in "trans" formation). Alternatively, one agent may be
coupled to a surface and the other agent in solution. In one
embodiment, the agent providing the co-stimulatory signal is bound
to a cell surface and the agent providing the primary activation
signal is in solution or coupled to a surface. In certain
embodiments, both agents can be in solution. In another embodiment,
the agents may be in soluble form, and then cross-linked to a
surface, such as a cell expressing Fc receptors or an antibody or
other binding agent which will bind to the agents. In a preferred
embodiment, the two agents are immobilized on beads, either on the
same bead, i.e., "cis," or to separate beads, i.e., "trans." By way
of example, the agent providing the primary activation signal is an
anti-CD3 antibody and the agent providing the co-stimulatory signal
is an anti-CD28 antibody; and both agents are co-immobilized to the
same surface, such as a bead, in equivalent molecular amounts. In
one embodiment, a 1:1 ratio of each antibody bound to the beads for
CD4.sup.+ T-cell expansion and T-cell growth is used.
[0112] One aspect of the present invention stems from the
surprising finding that using lower ratios of anti-CD3:anti-CD28
antibodies bound to the beads results in improved expansion of T
cells, including antigen-specific T cells. In certain aspects of
the present invention, a ratio of anti CD3:CD28 antibodies bound to
the beads is used such that an increase in T cell expansion is
observed as compared to the expansion observed using a ratio of
1:1. In one particular embodiment an increase of from about 0.5 to
about 3 fold is observed as compared to the expansion observed
using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28
antibody bound to the beads ranges from 100:1 to 1:100 and all
integer values there between. In one aspect of the present
invention, more anti-CD28 antibody is bound to the particles than
anti-CD3 antibody, i.e. the ratio of CD3:CD28 is less than one. In
certain embodiments of the invention, the ratio of anti CD28
antibody to anti CD3 antibody bound to the beads is greater than
2:1. In one particular embodiment, a 1:100 CD3:CD28 ratio of
antibody bound to beads is used. In another embodiment, a 1:75
CD3:CD28 ratio of antibody bound to beads is used. In a further
embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is
used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody
bound to beads is used. In one preferred embodiment, a 1:10
CD3:CD28 ratio of antibody bound to beads is used. In another
embodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is
used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody
bound to the beads is used.
[0113] Ratios of particles to cells from 1:500 to 500:1 and any
integer values in between may be used to stimulate T-cells. As
those of ordinary skill in the art can readily appreciate, the
ratio of particle to cells may dependant on particle size relative
to the target cell. For example, small sized beads could only bind
a few cells, while larger beads could bind many. In certain
embodiments the ratio of particles to cells ranges from 1:100 to
100:1 and any integer values in-between and in further embodiments
the ratio comprises 1:9 to 9:1 and any integer values in between,
can also be used to stimulate T-cells. The ratio of anti-CD3- and
anti-CD28-coupled particles to T-cells that result in T-cell
stimulation and expansion can vary as noted above, however in
certain embodiments the ratio may be 1:150 or lower. Certain
preferred ratios include 1:150, 1:100, 1:75, 1:50, 1:40, 1:30,
1:25, 1:20, 1:15, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5,
1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, and
20:1 with one preferred ratio being 1:1 particles per T-cell. In
one embodiment, a ratio of particles to cells of 1:1 or less is
used. In one particular embodiment, a preferred particle:cell ratio
is 1:2.5 or 1:5. In further embodiments, the ratio of particles to
cells can be varied depending on the day of stimulation. For
example, in one embodiment, the ratio of particles to cells is from
1:5, 1:2.5, 1:1 to 10:1 on the first day and additional particles
are added to the cells every day or every other day thereafter for
up to 10 days, at final ratios of from 1:1, 1:5, 1:20, 1:25, 1:50,
or 1:100 (based on cell counts on the day of addition). In one
particular embodiment, the ratio of particles to cells is 1:2.5,
1:5, or 1:1 on the first day of stimulation and adjusted to 1:5 on
the third and fifth days of stimulation. In a further embodiment,
the ratio of particles to cells is 1:2.5, 1:5, or 1:1 on the first
day of stimulation and adjusted to 1:10, 1:20, 1:25, 1:50, or 1:100
at day 5, 7, or 9. In another embodiment, particles are added on a
daily or every other day basis to a final ratio of 1:1 on the first
day, and 1:5 on the third and fifth days of stimulation. In another
embodiment, the ratio of particles to cells is 2:1 on the first day
of stimulation and adjusted to 1:10 on the third and fifth days of
stimulation. In another embodiment, particles are added on a daily
or every other day basis to a final ratio of 1:1 on the first day,
and 1:10 on the third and fifth days of stimulation. One of skill
in the art will appreciate that a variety of other ratios may be
suitable for use in the present invention.
[0114] One aspect of the present invention stems from the
surprising finding that using different bead:cell ratios can lead
to different outcomes with respect to expansion of antigen-specific
T cells. In particular, bead:cell ratios can be varied to
selectively expand or delete antigen-specific (memory) T cells. In
one embodiment, the particular bead:cell ratio used selectively
deletes antigen-specific T cells. In a further embodiment, the
particular bead:cell ratio used selectively expands
antigen-specific T cells. For example, bead to cell ratios of
1:100, 1:50, 1:25, 1:5 or 1:2.5 and the like are used to expand
antigen-specific T cells. Low bead:cell ratio can help preserve and
promote expansion of memory (antigen-specific) T cells.
Additionally, when additional beads are added at very low ratios to
cells (1:10, 1:25, 1:50, 1:100) at various days of culture (e.g.
sequential addition at day 5, 7, or 9), one can enhance and even
promote preferential expansion of the memory cells. With either 1:5
or 1:2.5 bead:cell ratio as initial simulus, addition of 1:10,
1:25, and to some extent 1:50 and 1:100 bead:cell raio at days 5
and 7 appear to preserve and enhance further expansion of memory
cells that would otherwise not occur with a single stimulation at
day 0 (see specifically Examples described herein). Therefore, the
compositions and methods described herein can be used to expand
specific populations of T cells, or to delete specific populations
of T cells, for use in any variety of immunotherapeutic settings
described herein.
[0115] It should be noted that the particle:cell ratios described
herein can be used in any combination with the various ratios of
antibodies bound on the beads. For example, beads containing about
1:5 to 1:10 ratio of anti-CD3/anti-CD28 antibodies bound thereto
can be used at a ratio of about 1:5 to 1:10 particles:cell. Or,
beads containing a 1:1 ratio of anti-CD3/anti-CD28 antibodies bound
thereto can be used at a ratio of about 1:5 particles:cell, etc.
Thus, the ratio of anti-CD3:anti-CD28 antibody bound to the beads
ranges from 100:1 to 1:100 and all integer values there between and
such beads can be used at a ratio of particle:cell of anywhere from
about 1:500 to 500:1 and any integer values in between, in any
combination.
[0116] Using certain methodologies it may be advantageous to
maintain long-term stimulation of a population of T-cells following
the initial activation and stimulation, by separating the T-cells
from the stimulus after a period of about 12 to about 14 days. The
rate of T-cell proliferation is monitored periodically (e.g.,
daily) by, for example, examining the size or measuring the volume
of the T-cells, such as with a Coulter Counter. In this regard, a
resting T-cell has a mean diameter of about 6.8 microns, and upon
initial activation and stimulation, in the presence of the
stimulating ligand, the T-cell mean diameter will increase to over
12 microns by day 4 and begin to decrease by about day 6. When the
mean T-cell diameter decreases to approximately 8 microns, the
T-cells may be reactivated and re-stimulated to induce further
proliferation of the T-cells. Alternatively, the rate of T-cell
proliferation and time for T-cell re-stimulation can be monitored
by assaying for the presence of cell surface molecules, such as,
CD154, CD54, CD25, CD137, CD134, which are induced on activated
T-cells.
[0117] In one embodiment, T-cell stimulation is performed with
anti-CD3 and anti-CD28 antibodies co-immobilized on beads
(3.times.28 beads), for a period of time sufficient for the cells
to return to a quiescent state (low or no proliferation)
(approximately 8-14 days after initial stimulation). The
stimulation signal is then removed from the cells and the cells are
washed and infused back into the patient. The cells at the end of
the stimulation phase are rendered "super-inducible" by the methods
of the present invention, as demonstrated by their ability to
respond to antigens and the ability of these cells to demonstrate a
memory-like phenotype, as is evidence by the examples. Accordingly,
upon re-stimulation either exogenously or by an antigen in vivo
after infusion, the activated T-cells demonstrate a robust response
characterized by unique phenotypic properties, such as sustained
CD154 expression and increased cytokine production.
[0118] In further embodiments of the present invention, the cells,
such as T-cells, are combined with agent-coated beads, the beads
and the cells are subsequently separated, and then the cells are
cultured. In an alternative embodiment, prior to culture, the
agent-coated beads and cells are not separated but are cultured
together. In a further embodiment, the beads and cells are first
concentrated by application of a force, resulting in cell surface
moiety ligation, thereby inducing cell stimulation.
[0119] In another embodiment, the time of exposure to stimulatory
agents such as anti-CD3/anti-CD28 (i.e., CD3xCD28)-coated
particles, such as beads, may be modified or tailored to obtain a
desired T-cell phenotype. One may desire a greater population of
helper T-cells (T.sub.H), typically CD4.sup.+ as opposed to
CD8.sup.+ cytotoxic or suppressor T-cells (T.sub.C), because an
expansion of T.sub.H cells could induce desired effector function
(e.g., anti-tumor, anti-viral, anti-bacterial, and the like).
CD4.sup.+ T-cells, express important immune-regulatory molecules,
such as GM-CSF, CD40L, and IL-2, for example. Where CD4-mediated
help is preferred, a method, such as that described herein, which
preserves or enhances the CD4:CD8 ratio could be of significant
benefit. In one aspect of the present invention, it may be
beneficial to increase the number of infused cells expressing
GM-CSF, or IL-2, all of which are expressed predominantly by
CD4.sup.+ T-cells. Alternatively, in situations where CD4-help is
needed less and increased numbers of CD8.sup.+T-cells are desirous,
the T cell activation approaches described herein can also be
utilized, by for example, pre-selecting for CD8.sup.+ cells prior
to stimulation and/or culture. Such situations may exist where
increased levels of IFN-.gamma. is preferred. Further, in other
applications, it may be desirable to utilize a population of
T.sub.H1-type cells versus T.sub.H2-type cells (or vice versa), or
supernatants therefrom. Likewise, it may be desirable in certain
applications to utilize a population of regulatory T cells (e.g.,
Autoimmun Rev. 2002 August;1(4):190-7; Curr Opin Immunol. 2002
December; 14(6):771-8).
[0120] To effectuate isolation of different antigen-specific T-cell
populations, times of cell surface moiety ligation that induces
re-stimulation (activation) may be varied or pulsed. For example
expansion times may be varied to obtain the specific phenotype of
interest and/or different types of stimulatory agents may be used
(e.g., antibodies or fragments thereof, a peptide, polypeptide,
MHC/peptide tetramer, growth factor, cytokine, chemokine,
glycopeptide, soluble receptor, steroid, hormone, mitogen, such as
PHA, or other superantigens). The expression of a variety of
phenotypic markers change over time; therefore, a particular time
point or stimulatory agent may be chosen to obtain a specific
population of T-cells. Accordingly, depending on the cell type to
be stimulated, the stimulation and/or expansion time may be four
weeks or less, 2 weeks or less, 10 days or less, or 8 days or less
(four weeks or less includes all time ranges from 4 weeks down to 1
day (24 hours)). In some embodiments, stimulation and expansion may
be carried out for 6 days or less, 4 days or less, 2 days or less,
and in other embodiments for as little as 24 or less hours, and
preferably 4-6 hours or less (these ranges include any integer
values in between). When stimulation of T-cells is carried out for
shorter periods of time, the population of T-cells may not increase
in number as dramatically, but the population will provide robust
and healthy activated antigen-specific T-cells that can continue to
proliferate in vivo and more closely resemble the natural effector
T-cell pool.
[0121] In one embodiment of the present invention, the mixture may
be cultured for several hours (about 3 hours) to about 14 days or
any hourly integer value in between. In another embodiment, the
mixture may be cultured for 21 days. In one embodiment of the
invention the beads and the T-cells are cultured together for about
eight days. In another embodiment, the beads and T-cells are
cultured together for 2-3 days. Several cycles of stimulation may
also be desired such that culture time of T cells can be 60 days or
more. Conditions appropriate for T-cell culture include an
appropriate media (e.g., Minimal Essential Media or RPMI Media 1640
or, X-vivo 15, (BioWhittaker)) that may contain factors necessary
for proliferation and viability, including serum (e.g., fetal
bovine or human serum), interleukin-2 (IL-2), insulin, IFN-.gamma.,
IL-4, GM-CSF, IL-10, IL-12, TGF.beta., and TNF-.alpha.. or any
other additives for the growth of cells known to the skilled
artisan. Other additives for the growth of cells include, but are
not limited to, surfactant, plasmanate, and reducing agents such as
N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI
1640, AIM-V, DMEM, MEM, .alpha.-MEM, F-12, X-Vivo 15, and X-Vivo
20, with added amino acids and vitamins, either serum-free or
supplemented with an appropriate amount of serum (or plasma) or a
defined set of hormones, and/or an amount of cytokine(s) sufficient
for the growth and expansion of T-cells. Antibiotics, e.g.,
penicillin and streptomycin, are included only in experimental
cultures, not in cultures of cells that are to be infused into a
subject. The target cells are maintained under conditions necessary
to support growth, for example, an appropriate temperature (e.g.,
37.degree. C.) and atmosphere (e.g., air plus 5% CO.sub.2).
[0122] In certain embodiments, it may be desirable to add some
number of feeder cells to augment activation and/or expansion of
antigen-specific cells. Feeder cells can encompass a variety of
cell types, including, irradiated peripheral blood lymphocytes
(autologous or allogeneic) alone or in combination with
EBV-transformed B cell lines (autologous or allogeneic),
immortalized or non-immortalized cell lines of the myelomoncytic
lineage, such as macrophges, dentritic cells, red blood cells,
B-cells, tumor cell lines such as U937, Jurkat, Daudi, MOLT-4, HUT,
CEM, Colo 205, HTB-13, and HTB-70. Feeder cells need not be of
human origin as long as they provide feeder function, e.g. the
ability to facilitate the survival and growth of primary T cells
and ther derived antigen-specific clones.
[0123] Pharmaceutical Compositions
[0124] An additional aspect of the present invention provides a
population or composition of antigen-specific T cells. The present
invention further provides a pharmaceutical composition comprising
antigen-specific T cells and a pharmaceutically acceptable carrier.
Compositions of the present invention may be administered either
alone, or as a pharmaceutical composition in combination with
diluents and/or with other components such as IL-2 or other
cytokines or cell populations. Briefly, pharmaceutical compositions
of the present invention may comprise a target cell population as
described herein, in combination with one or more pharmaceutically
or physiologically acceptable carriers, diluents or excipients.
Such compositions may comprise buffers such as neutral buffered
saline, phosphate buffered saline and the like; carbohydrates such
as glucose, mannose, sucrose or dextrans, mannitol; proteins;
polypeptides or amino acids such as glycine; antioxidants;
chelating agents such as ethylenediaminetetraacetic acid (EDTA) or
glutathione; adjuvants (e.g., aluminum hydroxide); and
preservatives. Compositions of the present invention are, in
certain aspects, formulated for intravenous administration.
[0125] A related embodiment of the present invention further
provides a pharmaceutical composition comprising the
antigen-specific T cells, and a pharmaceutically acceptable
carrier. The pharmaceutically acceptable carrier should be
sterilized by techniques known to those skilled in the art.
[0126] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). The quantity and frequency of administration will
be determined by such factors as the condition of the patient, and
the type and severity of the patient's disease, although
appropriate dosages may be determined by clinical trials.
[0127] The present invention also provides methods for preventing,
inhibiting, or reducing the presence of a cancer or malignant cells
in an animal, which comprise administering to an animal an
anti-cancer effective amount of the subject antigen-specific T
cells.
[0128] The cancers contemplated by the present invention, against
which the immune response is induced, or which is to be prevented,
inhibited, or reduced in presence, may include but are not limited
to melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia,
plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate
cancer, colo-rectal cancer, kidney cancer, renal cell carcinoma,
pancreatic cancer, esophageal cancer, brain cancer, lung cancer,
ovarian cancer, cervical cancer, multiple myeloma, hepatoma, acute
lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),
chronic myelogenous leukemia (CML), chronic lymphocytic leukemia
(CLL), low-grade lymphoma, and other neoplasms known in the
art.
[0129] Alternatively, compositions as described herein can be used
to induce or enhance responsiveness to pathogenic organisms, such
as viruses, (e.g., single stranded RNA viruses, single stranded DNA
viruses, human immunodeficiency virus (HIV), hepatitis A, B, and C
virus, herpes simplex virus (HSV), cytomegalovirus (CMV)
Epstein-Barr virus (EBV), Human Papilloma Virus (HPV)), parasites
(e.g., protozoan and metazoan pathogens such as Plasmodia species,
Leishmania species, Schistosoma species, Trypanosoma species),
bacteria (e.g., Mycobacteria, Salmonella, Streptococci, E. coli,
Staphylococci), fungi (e.g., Candida species, Aspergillus species)
and Pneumocystis carinii.
[0130] In certain embodiments, the methods of the present invention
can be used in conjunction with the generation of T regulatory
cells for specific immunosuppression in the case of inflammatory
disease, autoimmunity, and foreign graft acceptance. Regulatory T
cells can be generated and expanded using the methods of the
present invention. The regulatory T cells can be antigen-specific
and/or polyclonal. Regulatory T cells can be generated using
art-recognized techniques as described for example, in Woo, et al.,
J. Immunol. 2002 May 1;168(9):4272-6; Shevach, E. M., Annu. Rev.
Immunol. 2000, 18:423; Stephens, et al., Eur. J. Immunol. 2001,
31:1247; Salomon, et al, Immunity 2000, 12:431; and Sakaguchi, et
al., Immunol. Rev. 2001, 182:18. Accordingly, T cells of the
present invention can be used for the treatment of autoimmune
diseases such as, but not limited to, rheumatoid arthritis,
multiple sclerosis, insulin dependent diabetes, Addison's disease,
celiac disease, chronic fatigue syndrome, inflammatory bowel
disease, ulcerativecolitis, Crohn's disease, Fibromyalgia, systemic
lupus erythematosus, psoriasis, Sjogren's syndrome,
hyperthyroidism/Graves disease, hypothyroidism/Hashimoto's disease,
Insulin-dependent diabetes (type 1), Myasthenia Gravis,
endometriosis, scleroderma, pernicious anemia, Goodpasture
syndrome, Wegener's disease, glomerulonephritis, aplastic anemia,
paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome,
idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia,
Evan's syndrome, Factor VIII inhibitor syndrome, systemic
vasculitis, dermatomyositis, polymyositis and rheumatic fever.
[0131] The immune response induced in the animal by administering
the subject compositions of the present invention may include
cellular immune responses mediated by cytotoxic T cells, capable of
killing tumor and infected cells, and helper T cell responses.
Humoral immune responses, mediated primarily by helper T cells
capable of activating B cells thus leading to antibody production,
may also be induced. A variety of techniques may be used for
analyzing the type of immune responses induced by the compositions
of the present invention, which are well described in the art;
e.g., Coligan et al. Current Protocols in Immunology, John Wiley
& Sons Inc. (1994).
[0132] When "an immunologically effective amount", "an anti-tumor
effective amount", "an tumor-inhibiting effective amount", or
"therapeutic amount" is indicated, the precise amount of the
compositions of the present invention to be administered can be
determined by a physician with consideration of individual
differences in age, weight, tumor size, extent of infection or
metastasis, and condition of the patient. It can generally be
stated that a pharmaceutical composition comprising the subject
antigen-specific T cells, may be administered at a dosage of
10.sup.4 to 10.sup.7 APC/kg body weight, preferably 10.sup.5 to
10.sup.6 APC/kg body weight, including all integer values within
those ranges. Antigen-specific T cells compositions may also be
administered multiple times at these dosages. The cells can be
administered by using infusion techniques that are commonly known
in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.
319:1676, 1988). The optimal dosage and treatment regime for a
particular patient can readily be determined by one skilled in the
art of medicine by monitoring the patient for signs of disease and
adjusting the treatment accordingly.
[0133] Typically, in adoptive immunotherapy studies,
antigen-specific T cells are administered approximately at
2.times.10.sup.9 to 2.times.10.sup.11 cells to the patient. (See,
e.g., U.S. Pat. No. 5,057,423). In some aspects of the present
invention, particularly in the use of allogeneic or xenogeneic
cells, lower numbers of cells, in the range of 10.sup.6/kilogram
(10.sup.6-10.sup.11 per patient) may be administered. In certain
embodiments, T cells are administered at 1.times.10.sup.5,
1.times.10.sup.6, 1.times.10.sup.7, 1.times.10.sup.8,
2.times.10.sup.8, 2.times.10.sup.9, 1.times.10.sup.10,
2.times.10.sup.10, 1.times.10.sup.11, 5.times.10.sup.11, or
1.times.10.sup.12 cells to the subject. T cell compositions may be
administered multiple times at dosages within these ranges. The
antigen-specific T cells may be autologous or heterologous to the
patient undergoing therapy. If desired, the treatment may also
include administration of mitogens (e.g., PHA) or lymphokines,
cytokines, and/or chemokines (e.g., GM-CSF, IL-4, IL-13, Flt3-L,
RANTES, MIP1.alpha., etc.) as described herein to enhance induction
of the immune response.
[0134] The administration of the subject pharmaceutical
compositions may be carried out in any convenient manner, including
by aerosol inhalation, injection, ingestion, transfusion,
implantation or transplantation. The compositions of the present
invention may be administered to a patient subcutaneously,
intradermally, intramuscularly, by intravenous (i.v.) injection, or
intraperitoneally. In one embodiment, the antigen-specific T cell
compositions of the present invention are administered to a patient
by intradermal or subcutaneous injection. In another embodiment,
the antigen-specific T cell compositions of the present invention
are preferably administered by i.v. injection. The compositions of
antigen-specific T cells may be injected directly into a tumor or
lymph node.
[0135] In yet another embodiment, the pharmaceutical composition
can be delivered in a controlled release system. In one embodiment,
a pump may be used (see Langer, 1990, Science 249:1527-1533; Sefton
1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980;
Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In
another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, 1974, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla.; Controlled Drug Bioavailability, Drug
Product Design and Performance, 1984, Smolen and Ball (eds.),
Wiley, New York; Ranger and Peppas, 1983; J. Macromol. Sci. Rev.
Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190;
During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.
Neurosurg. 71:105). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, thus
requiring only a fraction of the systemic dose (see, e.g., Medical
Applications of Controlled Release, 1984, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla., vol. 2, pp. 115-138).
[0136] The antigen-specific T cell compositions of the present
invention may also be administered using any number of matrices.
Matrices have been utilized for a number of years within the
context of tissue engineering (see, e.g., Principles of Tissue
Engineering (Lanza, Langer, and Chick (eds.)), 1997. The present
invention utilizes such matrices within the novel context of acting
as an artificial lymphoid organ to support, maintain, or modulate
the immune system, typically through modulation of T cells.
Accordingly, the present invention can utilize those matrix
compositions and formulations which have demonstrated utility in
tissue engineering. Accordingly, the type of matrix that may be
used in the compositions, devices and methods of the invention is
virtually limitless and may include both biological and synthetic
matrices. In one particular example, the compositions and devices
set forth by U.S. Pat. Nos. 5,980,889; 5,913,998; 5,902,745;
5,843,069; 5,787,900; or 5,626,561 are utilized. Matrices comprise
features commonly associated with being biocompatible when
administered to a mammalian host. Matrices may be formed from both
natural or synthetic materials. The matrices may be
non-biodegradable in instances where it is desirable to leave
permanent structures or removable structures in the body of an
animal, such as an implant; or biodegradable. The matrices may take
the form of sponges, implants, tubes, telfa pads, fibers, hollow
fibers, lyophilized components, gels, powders, porous compositions,
or nanoparticles. In addition, matrices can be designed to allow
for sustained release seeded cells or produced cytokine or other
active agent. In certain embodiments, the matrix of the present
invention is flexible and elastic, and may be described as a
semisolid scaffold that is permeable to substances such as
inorganic salts, aqueous fluids and dissolved gaseous agents
including oxygen.
[0137] A matrix is used herein as an example of a biocompatible
substance. However, the current invention is not limited to
matrices and thus, wherever the term matrix or matrices appears
these terms should be read to include devices and other substances
which allow for cellular retention or cellular traversal, are
biocompatible, and are capable of allowing traversal of
macromolecules either directly through the substance such that the
substance itself is a semi-permeable membrane or used in
conjunction with a particular semi-permeable substance.
[0138] In certain embodiments of the present invention, the cells
of the present invention are administered to a patient in
conjunction with (e.g. before, simulataneously or following) any
number of relevant treatment modalities, including but not limited
to treatment with agents such as antiviral agents, chemotherapy,
radiation, immunosuppressive agents, such as cyclosporin,
azathioprine, methotrexate, mycophenolate, and FK506, antibodies,
or other immunoablative agents such as CAMPATH, anti-CD3
antibodies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin,
mycophenolic acid, steroids, FR901228, and irradiation. These drugs
inhibit either the calcium dependent phosphatase calcineurin
(cyclosporine and FK506) or inhibit the p70S6 kinase that is
important for growth factor induced signaling (rapamycin). (Liu et
al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321,
1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993; Isoniemi
(supra)). In a further embodiment, the cell compositions of the
present invention are administered to a patient in conjunction with
(e.g. before, simulataneously or following) T-cell ablative therapy
using either chemotherapy agents such as, fludarabine,
external-beam radiation therapy (XRT), cyclophosphamide, or
antibodies such as OKT3 or CAMPATH. In another embodiment, the cell
compositions of the present invention are administered following
B-cell ablative therapy such as agents that react with CD20, e.g.
Rituxan. The dosage of the above treatments to be administered to a
patient will vary with the precise nature of the condition being
treated and the recipient of the treatment. The scaling of dosages
for human administration can be performed according to art-accepted
practices. The dose for CAMPATH, for example, will generally be in
the range 1 to about 100 mg for an adult patient, usually
administered daily for a period between 1 and 30 days. The
preferred daily dose is 1 to 10 mg per day although in some
instances larger doses of up to 40 mg per day may be used
(described in U.S. Pat. No. 6,120,766.
[0139] All references referred to within the text are hereby
incorporated by reference in their entirety. Moreover, all
numerical ranges utilized herein explicitly include all integer
values within the range and selection of specific numerical values
within the range is contemplated depending on the particular use.
Further, the following examples are offered by way of illustration,
and not by way of limitation.
EXAMPLES
Example 1
CMV ANTIGEN COATED BEADS ACTIVATE AND FACILITATE ISOLATION OF
ANTIGEN-SPECIFIC T CELLS
[0140] In this experiment, cytomegalovirus (CMV)-coated beads were
used to activate and isolate antigen-specific T cells.
[0141] CMV lysate prepared using standard techniques was mixed at
room temperature for 1-2 hours with Dynabead M-450 while rotating.
Beads were then washed once, and added to PBMC. Within hours, the
beads were phagocytosed in the APC. Within 72 hours, CMVpp65-HLA-A2
tetramers detected CD25-high (activated) T cell specific for CMV
pp65. Magnetic selection of the bead-loaded APC with the associated
antigen-specific T cells was carried out at day 5, thereby
enriching for CMV-specific T cells. As shown in FIG. 1, following
magnetic separation, CMV-specific T cells were still tightly
associated with bead-loaded APC. It should be noted that magnetic
separation can be carried out anywhere from about day 1 to about
day 10.
Example 2
MEMORY CD8 CMV TETRAMER.sup.+ T CELLS EXPANDED EX VIVO UP-REGULATE
CD25 UPON RE-STIMULATION
[0142] In this example, antigen-coated beads were used to activate
CMV-specific CD8.sup.+T cells ex vivo.
[0143] PBMC from CMV pp65 tetramer-positive and tetramer-negative
donors were stimulated with paramagnetic Dynal M-450 beads coated
with CMV lysate. As controls, CMV pp65 tetramer-negative PBMC were
cultured with CMV-lysate coated beads (FIG. 2, panel A), CMV pp65
tetramer-positive PBMC were cultured with "naked" beads (no CMV
antigen) (FIG. 2, panel B). CMV pp65 tetramer-positive PBMC were
cultured with CMV-lysate coated beads (FIG. 2, panel C). Following
stimulation, activation of CMV-specific T cells was measured on Day
10 by CMV pp65 HLA-A2 tetramer stain and CD25 expression as an
indicator of activation. As shown in FIG. 2, up-regulation of CD25
was observed in memory CD8 CMV tetramer+T cells expanded ex vivo
using antigen-coated beads.
[0144] Antigen-coated beads can be used to activate and stimulate
antigen-specific T cells. These antigen-specific T cells can then
be enriched as described in Example 1 and elsewhere herein. These
antigen-specific T cells can be further expanded as described
herein and in U.S. patent application Ser. Nos. 10/350,305,
10/187,467, 10/133,236, 09/960,264, and 09/794,230. The
antigen-specific T cells of the present invention can be used in
any number of immunotherapeutic settings as described herein.
Example 3
VARYING BEAD:CELL RATIOS CAN SELECTIVELY EXPAND OR DELETE MEMORY
CD8 T CELLS
[0145] This example shows that the bead:cell ratio can have a
profound effect on expansion of different populations of T cells.
In particular, a high bead:cell ratio (3:1-10:1, 20:1 and higher)
tends to induce death in antigen-specific T cells while a lower
bead:cell ratio (1:1-1:10, 1:20, 1:30, 1:40, 1:50 or lower) leads
to expansion of antigen-specific T cells. Further, the data
described below show that lower bead:cell ratios lead to improved
cell expansion in polyclonal cell populations as well. Thus, this
example shows that lower bead:cell ratios improve overall cell
expansion.
[0146] Cells were prepared and stimulated using the XCELLERATE
I.TM. process essentially as described in U.S. patent application
Ser. No. 10/187,467 filed Jun. 28, 2002. Briefly, in this process,
the XCELLERATED.TM. T-cells are manufactured from a peripheral
blood mononuclear cell (PBMC) apheresis product. After collection
from the patient at the clinical site, the PBMC apheresis are
washed and then incubated with "uncoated" DYNABEADS.RTM. M-450
Epoxy T. During this time phagocytic cells such as monocytes ingest
the beads. After the incubation, the cells and beads are processed
over a MaxSep Magnetic Separator in order to remove the beads and
any monocytic/phagocytic cells that are attached to the beads.
Following this monocyte-depletion step, a volume containing a total
of 5.times.10.sup.8 CD3.sup.+ T-cells is taken and set-up with
1.5.times.10.sup.9 DYNABEADS.RTM. M-450 CD3/CD28 T to initiate the
XCELLERATE.TM. process (approx. 3:1 beads to T-cells). The mixture
of cells and DYNABEADS.RTM. M-450 CD3/CD28 T are then incubated at
37.degree. C., 5% CO.sub.2 for approximately 8 days to generate
XCELLERATED T-cells for a first infusion. The remaining
monocyte-depleted PBMC are cryopreserved until a second or further
cell product expansion (approximately 21 days later) at which time
they are thawed, washed and then a volume containing a total of
5.times.10.sup.8 CD3.sup.+ T-cells is taken and set-up with
1.5.times.10.sup.9 DYNABEADS.RTM. M-450 CD3/CD28 T to initiate the
XCELLERATE Process for a second infusion. During the incubation
period of .apprxeq.8 days at 37.degree. C., 5% CO.sub.2, the
CD3.sup.+T-cells activate and expand. The anti-CD3 mAb used is BC3
(XR-CD3; Fred Hutchinson Cancer Research Center, Seattle, Wash.),
and the anti-CD28 mAb (B-T3, XR-CD28) is obtained from Diaclone,
Besan.cedilla.on, France.
[0147] For the experiment described below, prior to plating and
culturing, the monocyte depleted cells were mixed by rotation for
30 minutes with varying amounts of beads as summarized below in
Table 1. The beads used in this Example comprised the
DYNABEADS.RTM. M-450 CD3CD28 T with a 1:1 CD3:CD28 antibody ratio
bound on the beads.
1TABLE 1 Varying Bead:Cell Ratios can Selectively Expand or Delete
Memory CD8 T cells Fold Increase Bead:Cell Ratio Polyclonal T cells
CMV Antigen-Specific T cells 10:1 149 0 5:1 294 0 3:1 346 1.4 1:1
562 20.6 1:5 113 53 1:10 79 45.8
[0148] The results summarized in Table 1 and shown graphically in
FIG. 3 demonstrate that antigen-specific T cells can be selectively
deleted by using high bead:cell ratios and expanded using low
bead:cell ratios (similar results were observed with Influenza- and
EBV-specific cells). Without being bound by theory, it is thought
that the antigen-specific T cells are sensitized to further
stimulation. Thus, the key appears to be the strength of the T cell
activation signal: selective expansion of memory T cells
(antigen-specific T cells) occurs with "weak" signals while
selective deletion of memory T cells occurs with "strong" signals.
The quantity of the CD3/TCR (and CD28) receptors that bound by
ligands determines the signal strength. Thus, stimulation with high
bead:cell ratios provides a high concentration of stimulating
antibody (i.e., "strong signal"), leading to over-stimulation of
antigen-specific T cells, causing them to die, either by apoptosis
or other mechanisms. Using lower bead:cell ratios provides a
stimulation signal to antigen-specific T cells that does not
over-stimulate, but rather induces rapid proliferation of these
cells.
[0149] In further experiments, fold increase of antigen-specific
(e.g., CMV tetramer positive cells) was shown to be excellent using
a 1:30 ratio and also using beads bound with anti-4-1 BB
antibody.
[0150] Therefore, in this Example, evidence is provided to support
the use of differing bead:cell ratios depending on the outcome
desired. For expansion of antigen-specific T cells, a lower
bead:cell ratio is preferable.
Example 4
VARYING BEAD:CELL RATIOS AND SEQUENTIAL ADDITION OF BEADS DURING
CULTURE CAN IMPROVE EXPANSION OF MEMORY T CELLS
[0151] This example shows that sequential addition of beads at a
low bead:cell ratio during culture can improve expansion of memory
T cells.
[0152] Cells were prepared and stimulated essentially as described
in Example 3 with the following modifications: as shown in FIG. 4,
panels A and B, cells were cultured either at a starting static
culture with a bead:cell ratio of 1:2.5 or 1:5 OR at 1:2.5 or 1:5
starting ratio with additional beads added at day 5, 7, or 9 at
1:10, 1:25, 1:50 or 1:100 ratios as noted. A comparison of total T
cell expansion over 15 days shows an increase in expansion of cells
when beads are added sequentially over culturing time, in cultures
with both starting bead:cell ratios of 1:2.5 and 1:5. Comparison of
CMV-specific T cell expansion over 15 days also shows an increase
in expansion of antigen-specific cells when beads are added
sequentially during culture (see FIG. 4 panel A and FIG. 4 panel
B). The most dramatic increase in expansion of polyclonal cells and
antigen-specific T cells over static culture was observed in those
cultures where beads were added at day 0 at a ratio of 1:2.5
beads:cells and sequentially added at a 1:10 ratio at day 5.
[0153] In a related experiment, reduced bead:cell ratio and
sequential addition was used to examine expansion of T cells from
patients vaccinated with Melanoma gp100(M). As shown in FIG. 5,
using a reduced bead:T cell ratio of 1:50 and sequential addition
at days 3, 5, 11, 15, and 19, a dramatic increase in expansion was
observed in Melanoma gp100(M)-specific T cells.
Example 5
ASSESSMENT OF CD4+ T MEMORY ("ANTIGEN-EXPERIENCED") T CELLS IN THE
XCELLERATE EXPANSION PROCESS
[0154] This example describes a model system for assessing CD4 T
cell subsets in the Xcellerate.RTM. expansion process.
[0155] Toxic Shock Syndrome Toxin (TSST) is a superantigen that
specifically stimulates CD4+ T cells expressing TCR V.beta.2. PBMC
are composed of between 1-25% V.beta.2 TCR T cells. A CD4.sup.+
V.beta.2 specific cell line is generated by stimulating PBMC with
TSST for 9-14 days until T cells proliferate out of log phase.
These "antigen experienced" V.beta.2 T cells are then mixed back at
varying percentages of the total culture (e.g., 1%, 2%) with a
V.beta.2 depleted nave PBMC culture and stimulated with CD3/CD28
beads at varying bead:cell ratios as described herein in the
Xcellerate.RTM. process.
[0156] The results showed that the presence of TSST expanded CD4+
VP2 TCR T cells does not inhibit total T cell Xcellerate.RTM.
expansion, with total T cell fold increases in the normal range.
Further, confirming other experiments, antigen-specificity was
maintained during expansion and antigen experienced V.beta.2 TCR T
cells expanded well at bead:cell ratios of 1:10 and 1:30.
Example 6
T CELL EXPANSION USING VARYING ANTI-CD3:ANTI-CD28 ANTIBODY
RATIOS
[0157] T cell expansion was evaluated using varying concentrations
of anti-CD3:anti-CD28 antibody ratios on the 3.times.28
DYNABEADS.RTM. M-450. In the experiments described herein, the
process referred to as XCELLERATE II.TM. was used, as described in
U.S. patent application Ser. No. 10/187,467. Briefly, this process
is similar to XCELLERATE I.TM. as described in Example 3 with some
modifications in which no separate monocyte depletion step was
utilized and in certain processes the cells were frozen prior to
initial contact with beads and further concentration and
stimulation were performed. As shown in FIG. 6, surprisingly, about
a 68-fold expansion after 8 days of culture was observed with an
anti-CD3:CD28 ratio of 1:10 antibodies on the beads. A 35-fold
expansion of T cells was seen after 8 days of culture with a
CD3:CD28 ratio of 1:3 on the beads. At a 1:1 ratio, about a 24-fold
expansion was seen. As shown in FIG. 7, similar results were
observed with CMVpp65-specific CD8.sup.+T cells using
anti-CD3:anti-CD28 antibody ratios as low as 1:30.
Example 7
T CELL EXPANSION USING THE XCELLERATE PROCESS AND THE WAVE
BIOREACTOR
[0158] This example describes the T cells expansion using
essentially the Xcellerate II process as described in U.S. Patent
Application Nos. 10/350,305; 10/187,467; 10/133,236; 09/960,264;
09/794,230; PCT/US01/06139; and PCT/US02/28161, followed by seeding
cells into the Wave Bioreactor.
[0159] Day 0 of the Xcellerate Process--On the first day of the
Xcellerate process essentially, the required number of
cryopreserved Cryocte.TM. containers from were removed from the
storage freezer, thawed washed and filtered.
[0160] Day 0--A volume of cells containing approximately
0.5.times.10.sup.9 CD3.sup.+ cells was then mixed with Dynabeads
M-450 CD3/CD28 T at a ratio of 3:1 Dynabeads M-450 CD3/CD28
T:CD3.sup.+ T cells and incubated with rotation. After the
incubation, the CD3.sup.+ T cells were magnetically concentrated
and simultaneously activated. The CD3.sup.+ T cells were then
resuspended in complete medium in a Lifecell Cell Culture Bag. The
bag containing the cells and beads was then placed in a
patient-dedicated incubator (37.degree. C., 5% CO.sub.2).
[0161] On or around Day 3--The CD3.sup.+ cells were
culture-expanded for .apprxeq.3 days at which point the contents of
the single bag are split into 4 new Lifecell bags. The 4 bags were
then returned to the patient-dedicated incubator (37.degree. C., 5%
CO.sub.2).
[0162] On or around Day 5--The CD3.sup.+ cells were
culture-expanded for .apprxeq.2 additional days at which point the
contents of the culture bags were then seeded into a 20 L Wave
Bioreactor containing a 10 L volume of media. The cells were then
cultured at 37.degree. C., 5% CO.sub.2 with the wave motion at 15
rocks/minute and with perfusion at 1 ml/minute.
[0163] Cell counts were determined each day and compared to cells
stimulated and expanded using the static Xcellerate II process.
Expansion was dramatically improved when cells were cultured in The
Wave Bioreactor. Further, cell densities reached as high as
50.times.10.sup.6 cells/ml in The Wave Bioreactor, as compared to a
maximum cell density of 5.times.10.sup.6 observed in the static
Xcellerate II process. A total cell count of about 800 billion was
achieved at day 12 of culture from a starting cell count of about
0.5.times.10.sup.9 cells using The Wave Bioreactor.
[0164] Thus, The Wave Bioreactor provides an unexpected and
dramatic improvement to the expansion process. Furthermore,
hitherto unobserved cell densities and final absolute cell yields
were achieved using The Wave Bioreactor.
[0165] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
* * * * *