U.S. patent application number 10/900046 was filed with the patent office on 2005-04-21 for compositions and methods for eliminating undesired subpopulations of t cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation.
This patent application is currently assigned to XCYTE Therapies, Inc.. Invention is credited to Berenson, Ronald J., Bonyhadi, Mark, Kalamasz, Dale.
Application Number | 20050084967 10/900046 |
Document ID | / |
Family ID | 46302417 |
Filed Date | 2005-04-21 |
United States Patent
Application |
20050084967 |
Kind Code |
A1 |
Berenson, Ronald J. ; et
al. |
April 21, 2005 |
Compositions and methods for eliminating undesired subpopulations
of T cells in patients with immunological defects related to
autoimmunity and organ or hematopoietic stem cell
transplantation
Abstract
The present invention relates generally to methods for
stimulating T cells, and more particularly, to methods to eliminate
undesired (e.g. autoreactive, alloreactive, pathogenic)
subpopulations of T cells from a mixed population of T cells,
thereby restoring the normal immune repertoire of said T cells. The
present invention also relates to compositions of cells, including
stimulated T cells having restored immune repertoire and uses
thereof.
Inventors: |
Berenson, Ronald J.; (Mercer
Island, WA) ; 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: |
46302417 |
Appl. No.: |
10/900046 |
Filed: |
July 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10900046 |
Jul 27, 2004 |
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10729822 |
Dec 5, 2003 |
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10729822 |
Dec 5, 2003 |
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10603577 |
Jun 24, 2003 |
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60442001 |
Jan 22, 2003 |
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60431212 |
Dec 4, 2002 |
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60393042 |
Jun 28, 2002 |
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Current U.S.
Class: |
435/372 |
Current CPC
Class: |
A61P 37/00 20180101;
Y02A 50/475 20180101; C12N 5/0636 20130101; C12N 5/0087 20130101;
Y02A 50/30 20180101; C12N 2501/48 20130101; C12N 2502/1114
20130101; A61K 35/17 20130101 |
Class at
Publication: |
435/372 |
International
Class: |
C12Q 001/68; C12N
005/08 |
Claims
1. A method for eliminating at least a substantial portion of a
clonal T cell subpopulation from a mixed population of T cells from
an individual, comprising, exposing a population of cells, wherein
at least a portion thereof comprises T cells, to one or more
pro-apoptotic or growth inhibiting compositions wherein said
exposure induces apoptosis or growth inhibition in at least a
substantial portion of at least one clonal T cell population
present in the mixed population of T cells; thereby eliminating at
least a substantial portion of said clonal T cell population from
the mixed population of T cells.
2. The method of claim 1 further comprising expanding the remaining
mixed population of T cells.
3. The method of claim 2 wherein the remaining mixed population of
cells is expanded by exposing the remaining mixed population of
cells to a surface wherein the surface has attached thereto one or
more agents that ligate a cell surface moiety of at least a portion
of the remaining T cells and stimulates said remaining T cells.
4. The method of claim 3, 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
of said T cell.
5. A population of T cells generated according to the method of any
one of claims 1-3.
6. The method of claim 1 wherein the pro-apoptotic or growth
inhibiting composition comprises an autoantigen.
7. The method of claim 6, wherein the autoantigen is selected from
the group consisting of myelin basic protein (MBP), MBP 84-102, MBP
143-168, pancreatic islet cell antigens, collagen, thyroid
antigens, Scl-70, nucleic acid, acetylcholine receptor, S Antigen,
and type II collagen.
8. The method of claim 1 wherein the pro-apoptotic composition
comprises allogeneic or xenogeneic cells.
9. The method of claim 1 wherein said population of cells, wherein
at least a portion thereof comprises T cells, is exposed to one or
more pro-apoptotic compositions in vivo.
10. The method of claim 1 wherein said population of cells, wherein
at least a portion thereof comprises T cells, is exposed to one or
more pro-apoptotic compositions ex vivo.
11. The method of claim 3 wherein the exposure of said cells to
said surface is for a time sufficient to increase
polyclonality.
12. The method of claim 11 wherein the increase comprises a shift
from mono to oligoclonality or to polyclonality of the T cell
population as measured by a V.beta., V.alpha., V.gamma., or
V.delta. spectratype profile of at least one V.beta., V.alpha.,
V.gamma., or V.delta. family gene.
13. A population of T cells generated according to the method of
claim 6 or 11.
14. A method for treating autoimmune disease in a patient
comprising administering to the patient the population of T cells
of claim 13.
15. The method of claim 14 wherein the patient has been treated
with an immunoablative agent prior to administering the population
of T cells of claim 10.
16. The method of claim 15 wherein the immunoablative agent is
selected from the group consisting of campath, anti-CD3 antibodies,
cyclophosphamide, fludarabine, cyclosporine, FK506, mycophenolic
acid, steroids, FR901228, and irradiation.
17. The method of claim 14 wherein the patient has been treated
with a T cell ablative therapy prior to administering the
population of T cells of claim 10.
18. The method of claim 1 wherein the pro-apoptotic or growth
inhibiting composition comprises one or more compositions selected
from the group consisting of, anti-CD3 antibody, anti-CD2 antibody,
anti-CD20 antibody, target antigen, MHC-peptide tetramers or
dimers, Fas ligand, anti-Fas antibody, IL-2, IL-4, TRAIL, rolipram,
doxorubicin, chlorambucil, fludarabine, cyclophosphamide,
azathioprine, methotrexate, cyclosporine, mycophenolate, FK506,
inhibitors of bcl-2, topoisomerase inhibitors, interleukin-1.beta.
converting enzyme (ICE)-binding agents, Shigella IpaB protein,
staurosporine, ultraviolet irradiation, gamma irradiation, tumor
necrosis factor, target antigens nucleic acid molecules, proteins
or peptides, and non-protein or non-polynucleotide compounds.
19. The method of claim 3, wherein at least one agent is an
antibody or an antibody fragment.
20. The method of claim 3, wherein the first agent is an antibody
or a fragment thereof, and the second agent is an antibody or a
fragment thereof.
21. The method of claim 3, wherein the first and the second agents
are different antibodies.
22. The method of claim 3, wherein the first agent is an anti-CD3
antibody, an anti-CD2 antibody, or an antibody fragment of an
anti-CD3 or anti-CD2 antibody.
23. The method of claim 3, wherein the second agent is an anti-CD28
antibody or antibody fragment thereof.
24. The method of claim 3, wherein the first agent is an anti-CD3
antibody and the second agent is an anti-CD28 antibody.
25. A method for eliminating at least a substantial portion of a
clonal T cell subpopulation from a mixed population of T cells from
an individual, comprising, (a) exposing a population of cells
wherein at least a portion thereof comprises T cells to one or more
compositions that sensitize at least a portion of the T cells to
further activation or stimulation, (b) exposing the population of
cells to a surface wherein the surface has attached thereto one or
more agents that ligate a cell surface moiety of at least a portion
of the sensitized T cells and stimulates said sensitized T cells,
wherein the exposure of said sensitized T cells to said surface is
for a time sufficient to induce apoptosis of said sensitized T
cells; thereby eliminating said sensitized T cells from the
population.
26. The method of claim 25 wherein step (b) further comprises
exposing said population of cells to said surface for a time
sufficient to stimulate at least a portion of the remaining T cells
and wherein said at least a portion of the remaining cells
proliferates.
27. The method of claim 25, 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
of said T cell.
28. The method of claim 26 wherein the exposure of said cells to
said surface is for a time sufficient to increase
polyclonality.
29. The method of claim 28 wherein the increase comprises a shift
from mono to oligoclonality or to polyclonality of the T cell
population as measured by a V.beta., V.alpha., V.gamma., or
V.delta. spectratype profile of at least one V.beta., V.alpha.,
V.gamma., or V.delta. family gene.
30. A population of T cells generated according to the method of
claim 28.
31. The method of claim 25 wherein the individual requires a
hematopoietic stem cell transplant.
32. The method of claim 31, wherein the composition that sensitizes
comprises recipient PBMCs that have been treated such that they are
unable to continue dividing and the population of cells comprises
donor T cells.
33. A population of T cells generated according to the method of
claim 32.
34. A method for reducing the risk of, or the severity of, an
adverse GVHD effect in a patient who is undergoing a hematopoietic
stem cell transplant, comprising administering to said patient the
population of T cells according to claim 30 or 33.
35. The method of claim 25 wherein the individual requires an organ
transplant.
36. The method of claim 35 wherein the composition that sensitizes
comprises donor cells that have been treated such that they are
unable to divide and the population of cells comprises recipient T
cells.
37. The method of claim 36 wherein the exposure of said cells to
said surface is for a time sufficient to increase
polyclonality.
38. The method of claim 37 wherein the increase comprises a shift
from mono to oligoclonality or to polyclonality of the T cell
population as measured by a V.beta., V.alpha., V.gamma., or
V.delta. spectratype profile of at least one V.beta., V.alpha.,
V.gamma., or V.delta. family gene.
39. A population of T cells generated according to the method of
claim 36 or 37.
40. A method for reducing the risk of organ rejection in a patient
who is receiving an organ transplant, comprising administering to
the patient the population of T cells of claim 39.
41. The method of claim 40 wherein the patient has been treated
with a T cell ablative therapy prior to administration of the
population of T cells of claim 36.
42. The method of claim 25 wherein the composition that sensitizes
comprises an autoantigen.
43. The method of claim 42, wherein the autoantigen is selected
from the group consisting of myelin basic protein (MBP), MBP
84-102, MBP 143-168, Scl-70, pancreatic islet cell antigens, S
Antigen; and type II collagen.
44. The method of claim 43 wherein the exposure of said cells to
said surface is for a time sufficient to increase
polyclonality.
45. The method of claim 44 wherein the increase comprises a shift
from mono to oligoclonality or to polyclonality of the T cell
population as measured by a V.beta., V.alpha., V.gamma., or
V.delta. spectratype profile of at least one V.beta., V.alpha.,
V.gamma., or V.delta. family gene.
46. A population of T cells generated according to the method of
claim 42.
47. A method for treating autoimmune disease in a patient
comprising administering to the patient the population of T cells
of claim 46.
48. The method of claim 47 wherein the patient has been treated
with a T cell ablative therapy prior to administering the
population of T cells of claim 46.
49. The method of claim 26, wherein at least one agent is an
antibody or an antibody fragment.
50. The method of claim 26, wherein the first agent is an antibody
or a fragment thereof, and the second agent is an antibody or a
fragment thereof.
51. The method of claim 50, wherein the first and the second agents
are different antibodies.
52. The method of claim 26, wherein the first agent is an anti-CD3
antibody, an anti-CD2 antibody, or an antibody fragment of an
anti-CD3 or anti-CD2 antibody.
53. The method of claim 26, wherein the second agent is an
anti-CD28 antibody or antibody fragment thereof.
54. The method of claim 26, wherein the first agent is an anti-CD3
antibody and the second agent is an anti-CD28 antibody.
55-61. (canceled)
62. A method for activating and expanding a population of T cells
by cell surface moiety ligation, comprising: contacting a
population of cells, wherein at least a portion thereof comprises T
cells, with a surface, wherein said surface has attached thereto
one or more agents that ligate a cell surface moiety of at least a
portion of the T cells and stimulates said T cells, wherein said
surface is present at a ratio of said surface to said cells such
that at least a substantial portion of at least one population of
antigen-specific T cells is deleted after about 8 days of
culture.
63. The method of claim 62 wherein said ratio is from about 10:1 to
about 5:1.
64. The method of claim 62 wherein said ratio is about 5:1.
65. The method of claim 62 wherein said ratio is about 10:1.
66. The method of claim 1 further comprising expanding the mixed
population of T cells, comprising, exposing the remaining mixed
population of T cells to the pro-apoptotic composition, wherein
said exposure induces proliferation in the mixed population of T
cells.
67. The method of claim 66 wherein said pro-apoptotic composition
comprises anti-CD3 and anti-CD28 antibodies co-immobilized on a
bead.
68. A method for treating a patient afflicted with an autoimmune
disease comprising: (a) contacting a population of cells from the
patient, wherein at least a portion thereof comprises T cells, with
a surface, wherein said surface has attached thereto one or more
agents that ligate a cell surface moiety of at least a portion of
the T cells and stimulates said T cells, wherein said surface is
present at a ratio of said surface to said cells such that at least
a substantial portion of at least one population of
antigen-specific T cells is deleted after about 8 days of culture;
and (b) administering to the patient an effective amount of T cells
from (a) such that in vivo homeostatic proliferation is inhibited;
thereby treating autoimmune disease.
69. The method of claim 68 wherein said ratio is from about 10:1 to
about 5:1.
70. The method of claim 68 wherein said ratio is about 5:1.
71. The method of claim 68 wherein said ratio is about 10:1.
72. A method for treating a patient afflicted with an autoimmune
disease comprising: (a) contacting a population of cells from the
patient, wherein at least a portion thereof comprises T cells, with
a surface, wherein said surface has attached thereto one or more
agents that ligate a cell surface moiety of at least a portion of
the T cells and stimulates said T cells; (b) administering to the
patient the T cells of (a) at a dose such that homeostatic
proliferation of endogenous T cells is inhibited; thereby treating
autoimmune disease.
73. A method for treating a patient infected with HIV comprising:
(a) contacting a population of cells from the patient, wherein at
least a portion thereof comprises T cells, with a surface, wherein
said surface has attached thereto one or more agents that ligate a
cell surface moiety of at least a portion of the T cells and
stimulates said T cells, wherein said surface is present at a ratio
of said surface to said cells such that at least a substantial
portion of HIV-infected T cells is deleted after about 8 days of
culture; and (b) administering to the patient an effective amount
of T cells from (a) such that in vivo homeostatic proliferation is
inhibited; thereby treating the patient infected with HIV.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to methods for
stimulating T cells to restore normal immune repertoire. The
present disclosure includes methods to eliminate undesired (e.g.
autoreactive, alloreactive, pathogenic) subpopulations of T cells
from a mixed population of T cells, thereby restoring the normal
immune repertoire of said T cells. The present invention also
relates to compositions of cells, including stimulated T cells
having restored immune repertoire and uses thereof.
[0003] 2. Description of the Related Art
[0004] The ability of T cells to recognize the universe of antigens
associated with various cancers or infectious organisms is
conferred by its T cell antigen receptor (TCR), which is made up of
both an .alpha. (alpha) chain and a .beta. (beta) chain or a
.gamma. (gamma) and a .delta. (delta) chain. The proteins which
make up these chains are encoded by DNA, which employs a unique
mechanism for generating the tremendous diversity of the TCR. This
multisubunit immune recognition receptor associates with the CD3
complex and binds to peptides presented by the major
histocompatibility complex (MHC) class I and II proteins on the
surface of antigen-presenting cells (APCs). Binding of TCR to the
antigenic peptide on the APC is the central event in T cell
activation, which occurs at an immunological synapse at the point
of contact between the T cell and the APC.
[0005] To sustain T cell activation, T lymphocytes typically
require a second co-stimulatory signal. Co-stimulation is typically
necessary for a T helper cell to produce sufficient cytokine levels
that induce clonal expansion. Bretscher, Immunol. Today 13:74,
1992; June et al., Immunol. Today 15:321, 1994. The major
co-stimulatory signal occurs when a member of the B7 family ligands
(CD80 (B7.1) or CD86 (B7.2)) on an activated antigen-presenting
cell (APC) binds to CD28 on a T cell.
[0006] Methods of stimulating the expansion of certain subsets of T
cells have the potential to generate a variety of T cell
compositions useful in immunotherapy. Successful immunotherapy can
be aided by increasing the reactivity and quantity of T cells by
efficient stimulation. Furthermore, in the settings of autoimmunity
or transplantation, successful immunotherapy can be aided by the
elimination of unwanted autoreactive or alloreactive cells.
[0007] The various techniques available for expanding human T cells
have relied primarily on the use of accessory cells 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 the APC signals directed towards the TCR/CD3
complex and CD28 on the surface of T cells 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.
[0008] In the absence of exogenous growth factors or accessory
cells, a co-stimulatory signal may be delivered to a T cell
population, for example, by exposing the cells to a CD3 ligand and
a CD28 ligand attached to a solid phase surface, such as a bead.
See C. June, et al. (U.S. Pat. No. 5,858,358); C. June et al. WO
99/953823. While these methods are capable of achieving
therapeutically useful T cell populations, increased robustness and
ease of T cell preparation remain less than ideal.
[0009] Methods previously available in the art have made use of
anti-CD3 and anti CD28 for the expansion of T cells. In addition,
the methods currently available in the art have not focused on
short-term expansion of T cells or obtaining a more robust
population of T cells and the beneficial results thereof. None of
these methods has described using such or similar methods to
eliminate an undesired clonal or oligoclonal T cell population from
a T cell population nor the beneficial results thereof. Moreover,
the methods previously available tend to further skew the clonality
of the T cell population rather than eliminate undesired reactive
clones from a T cell population, and restore a normal immune
repertoire. For maximum in vivo effectiveness, theoretically, an ex
vivo- or in vivo-generated, activated T cell population should be
in a state that can maximally orchestrate an immune response to
cancer, infectious disease, or other disease states. In the setting
of autoimmunity or transplantation, the activated T cell
populations should be in a state to reconstitute a normal T cell
repertoire with a reduced presence or entirely without the presence
of autoreactive or potentially pathogenic alloreactive T cells.
Currently, patients with autoimmune diseases are treated with
long-term immunosuppression to inhibit the autoreactive T cells
that cause disease. When the immunosuppressive agents are stopped,
disease recurs often concomitant with reappearance of disease
causing T cells that re-emerge in these patients. The major problem
in hematopoietic stem cell transplantation is graft-versus-host
disease (GVHD), which is caused by alloreactive T cells present in
the infused hematopoietic stem cell preparation. In organ
transplantation, graft rejection mediated by alloreactive host T
cells is the major problem, usually overcome by long-term
immunosuppression of the transplant recipient.
[0010] The present invention provides methods to generate an
increased number of more highly activated and more pure T cells
that have surface receptor and cytokine production characteristics
that appear more healthy and natural than other expansion methods
and further provides for the diminution or elimination of undesired
autoreactive or alloreactive populations of T cells. The present
invention provides methods for the use of said populations of T
cells in the setting of autoimmune diseases, hematopoietic stem
cell, and organ transplantation, as well as other settings where
reconstitution of an ablated, abrogated, or otherwise dysfunctional
T cell immune system is desired. In addition, the present invention
provides compositions of cell populations of any target cell,
including T cell populations and parameters for producing the same,
as well as providing other related advantages.
[0011] Additionally, it is becoming well recognized that the aging
immune system is characterized by a progressive decline in the
responsiveness to exogenous antigens and tumors in combination with
a paradoxical increase in autoimmunity (C. Weyand et al. Mechanisms
of Ageing and Development 102:131-147, 1998; D. Schmidt et al.
Molecular Medicine 2:608-618, 1996; G. Liuzzo et al. Circulation
100:2135-2139, 1999). These studies have described that aging is
associated with the emergence of a subset of T helper cells that
are characterized by the loss of CD28 expression. CD4.sup.+
CD28.sup.- T cells are long lived, typically undergo clonal
expansion in vivo, and react to auto-antigens in vitro. The loss of
CD28 expression is correlated with a lack of CD40 ligand expression
rendering these CD4.sup.+ T cells incapable of promoting B cell
differentiation and immunoglobulin secretion. Aging-related
accumulation of CD4.sup.+ CD28.sup.- T cells results in an immune
compartment that is skewed towards auto-reactive responses and away
from the generation of high-affinity B cell responses against
exogenous antigens.
BRIEF SUMMARY OF THE INVENTION
[0012] One aspect of the present invention provides for a method
for eliminating at least a substantial portion of a clonal T cell
population from a mixed population of T cells from an individual,
comprising, providing a population of cells wherein at least a
portion thereof comprises T cells; exposing the population of cells
ex vivo to one or more pro-apoptotic compositions wherein said
exposure induces apoptosis in at least a portion of the T cells;
thereby eliminating at least a substantial portion of said clonal T
cells from the mixed population.
[0013] The present invention provides a method for eliminating at
least a substantial portion of a clonal T cell subpopulation from a
mixed population of T cells from an individual, comprising,
exposing a population of cells, wherein at least a portion thereof
comprises T cells, to one or more pro-apoptotic or growth
inhibiting compositions wherein said exposure induces apoptosis or
inhibits growth in at least a substantial portion of at least one
clonal T cell population present in the mixed population of T cells
thereby eliminating at least a substantial portion of said clonal T
cell population from the mixed population of T cells. In one
embodiment, the method further comprises expanding the mixed
population of T cells, by exposing the remaining mixed population
of T cells to the pro-apoptotic composition, wherein said exposure
induces proliferation in the mixed population of T cells. In one
particular embodiment, the pro-apoptotic composition comprises
anti-CD3 and anti-CD28 antibodies co-immobilized on a bead. In
certain embodiments, the pro-apoptotic composition used to
eliminate at least a substantial portion of said clonal T cell
population from the mixed population of T cells is the same
composition used to expand the remaining mixed population of T
cells.
[0014] In one embodiment, the method further comprises expanding
the remaining population of cells. In another embodiment, the
method further comprises expanding the remaining population of
cells by exposing the remaining population of cells to a surface
wherein the surface has attached thereto one or more agents that
ligate a cell surface moiety of at least a portion of the remaining
T cells and stimulates said remaining T cells. In a related
embodiment, the 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 of said T cell.
[0015] In one embodiment, the agent attached to the surface is an
antibody or an antibody fragment. In another embodiment, the first
agent is an antibody or a fragment thereof, and the second agent is
an antibody or a fragment thereof. In one embodiment the first and
the second agents are different antibodies. In one particular
embodiment, the first agent is an anti-CD3 antibody; an anti-CD2
antibody, or an antibody fragment of an anti-CD3 or anti-CD2
antibody. In another embodiment, the second agent is an anti-CD28
antibody or antibody fragment thereof. In a further embodiment, the
first agent is an anti-CD3 antibody and the second agent is an
anti-CD28 antibody.
[0016] In another embodiment, the cells are exposed to the surfaces
of the present invention for a time sufficient to increase
polyclonality. In certain embodiments, this increase in
polyclonality comprises a shift from mono to oligoclonality or to
polyclonality of the T cell population as measured by a V.beta.,
V.alpha., V.gamma., or V.delta. spectratype profile of at least one
V.beta., V.alpha., V.gamma., or V.delta. family gene.
[0017] Illustrative pro-apoptotic compositions of the present
invention include but are not limited to anti-CD3 antibody,
anti-CD2 antibody, anti-CD28 antibody, anti-CD20 antibody, target
antigen, MHC-peptide tetramers, Fas ligand, anti-Fas antibody,
IL-2, IL-4, TRAIL, rolipram, doxorubicin, chlorambucil,
fludarabine, cyclophosphamide, azathioprine, methotrexate,
cyclosporine, mycophenolate, FK506, inhibitors of bcl-2,
topoisomerase inhibitors, interleukin-1.beta. converting enzyme
(ICE)-binding agents, Shigella IpaB protein, staurosporine,
ultraviolet irradiation, gamma irradiation, tumor necrosis factor,
target antigens nucleic acid molecules, proteins or peptides, and
non-protein or non-polynucleotide compounds. In certain
embodiments, one or more of these compositions are used at the same
time.
[0018] In certain embodiments of the present invention, the
pro-apoptotic compositions comprises an autoantigen. Illustrative
autoantigens of the present invention include but are not limited
to, myelin basic protein (MBP), MBP 84-102, MBP 143-168, pancreatic
islet cell antigens, collagen, CLIP-170, thyroid antigens, nucleic
acid, acetylcholine receptor, S Antigen, and type II collagen.
[0019] The present invention further provides a population of T
cells generated according to any of the methods described
above.
[0020] The present invention provides a method for eliminating at
least a substantial portion of a clonal T cell subpopulation from a
mixed population of T cells from an individual, comprising,
exposing a population of cells, wherein at least a portion thereof
comprises T cells, to one or growth inhibiting compositions wherein
said exposure inhibits growth in at least a substantial portion of
at least one clonal T cell population present in the mixed
population of T cells; the method further comprises expanding the
mixed population of T cells, by exposing the population of cells
that is not growth inhibited, i.e., the remaining mixed population
of T cells to a surface having attached thereto one or more agents
that bind to a cell surface molecule. In one embodiment said
surface comprises anti-CD3 and anti-CD28 antibodies co-immobilized
on a bead.
[0021] One aspect of the present invention provides for methods for
treating autoimmune disease in a patient comprising administering
to a patient the populations of T cells of the present invention.
In one embodiment the patient has been treated with a
chemotherapeutic agent prior to administering the population of T
cells. Illustrative chemotherapeutic agents of the present
invention include but are not limited to campath, anti-CD3
antibodies, cytoxin, fludarabine, cyclosporine, FK506, mycophenolic
acid, steroids, FR901228, and irradiation. In certain embodiments,
the patient is treated with a T cell ablative therapy prior to
administration of the populations of T cells of the present
invention.
[0022] One aspect of the present invention is a method for
eliminating at least a substantial portion of a clonal T cell
population from a population of T cells from an individual,
comprising, providing a population of cells wherein at least a
portion thereof comprises T cells; exposing the population of cells
to one or more agents that sensitize at least a portion of the T
cells to further activation or stimulation, exposing the population
of cells to a surface wherein the surface has attached thereto one
or more agents that ligate a cell surface moiety of at least a
portion of the sensitized T cells and stimulates said sensitized T
cells, wherein the exposure of said sensitized T cells to said
surface is for a time sufficient to induce apoptosis of said
sensitized T cells; thereby eliminating said sensitized T cells
from the population. In one embodiment, the method further
comprises exposing said population of cells to said surface for a
time sufficient to stimulate at least a portion of the remaining T
cells and wherein said at least a portion of the remaining cells
proliferates. In a further embodiment, the method provides that
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 of said T cell. In one
embodiment, at least one agent is an antibody or an antibody
fragment. In another embodiment, 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 a related embodiment,
the first agent is an anti-CD3 antibody, an anti-CD2 antibody, or
an antibody fragment of an anti-CD3 or anti-CD2 antibody. In yet
another embodiment, 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.
[0023] In a related embodiment, cells are exposed to said surface
for a time sufficient to increase polyclonality. In another
embodiment, the increase in polyclonality comprises a shift from
mono to oligoclonality or to polyclonality of the T cell population
as measured by a V.beta., V.alpha., V.gamma., or V.delta.
spectratype profile of at least one V.beta., V.alpha., V.gamma., or
V.delta. family gene.
[0024] In certain embodiments, the patient requires a hematopoietic
stem cell transplant. In a related embodiment, the composition that
sensitizes recipient PBMCs that have been treated such that they
are unable to continue dividing and the population of cells
comprises donor T cells. The present invention also provides for
populations of T cells generated according to the above methods.
The present invention also provides methods for reducing the risk
of, or the severity of, an adverse GVHD effect in a patient who is
undergoing a hematopoietic stem cell transplant, comprising
administering to said patient the population of T cells according
to the methods described herein.
[0025] In certain embodiments, the patients to receive the cells of
the present invention require an organ transplant. In a related
embodiment the composition that sensitizes comprises irradiated
donor cells and the population of cells comprises recipient T
cells. The present invention also provides for a population of
cells generated according to this method. In one embodiment, these
cells are administered to a patient receiving an organ transplant
to reduce the risk of organ rejection. In a related embodiment, the
organ transplant patient is treated with a T cell ablative therapy
prior to administration of the population of T cells.
[0026] In one aspect of the present invention the composition that
sensitizes comprises an autoantigen. Illustrative autoantigens of
the present invention include but are not limited to myelin basic
protein (MBP), MBP 84-102, MBP 143-168, pancreatic islet cell
antigens, S Antigen, and type II collagen. In one embodiment of the
present invention, a patient with an autoimmune disease is treated
by administration of a population of T cells generated according to
this method. In a related embodiment, the patient is treated with a
T cell ablative therapy prior to administering the population of T
cells.
[0027] The present invention also provides a method for eliminating
a clonal B cell population from a population of B cells from an
individual, comprising, providing a population of cells wherein at
least a portion thereof comprises B cells; exposing the population
of cells to one or more pro-apoptotic compositions wherein said
exposure induces apoptosis in at least a portion of the B cells;
thereby eliminating said portion of B cells from the population. In
one embodiment, the method further comprises exposing the remaining
population of cells to a surface wherein the surface has attached
thereto one or more agents that ligate a cell surface moiety of at
least a portion of the remaining B cells and stimulates said
remaining B cells. In certain embodiments, the pro-apoptotic
composition comprises an autoantigen.
[0028] The present invention also provides for compositions of
B-cells generated according to the above methods.
[0029] In one embodiment of the present invention, a patient with
an autoimmune disease is treated with a composition comprising the
populations of B-cells generated using the methods of the present
invention. In a related embodiment, the patient is treated with a B
cell ablative therapy prior to administering the population of B
cells.
[0030] One aspect of the present invention provides methods for
generating a substantially pure population of T cells from a
population of T cells from an individual, comprising providing a
population of cells wherein at least a portion thereof comprises T
cells: exposing the population of T cells ex vivo to a composition
that preferentially selects and/or stimulates surface CD3.sup.+ and
CD28.sup.+ molecules, thereby generating a substantially pure
population of CD3.sup.+/CD28.sup.+ T cells. In a related embodiment
the population of pure T cells generated is a substantially pure
population of CD4.sup.+/CD3.sup.+/CD28.sup.+ T cells. In a related
embodiment the population of pure T cells is a substantially pure
population of CD8.sup.+/CD3.sup.+/CD28.sup.+ T cells.
[0031] In one aspect of the invention the purity of the
CD3.sup.+/CD28.sup.+ T cells is at least 90% pure. In further
embodiments, the purity of the CD3.sup.+/CD28.sup.+ T cells is 91%,
92%, 93%, 94%, 95%, 96%, 97%, or 98% pure. In another embodiment
the purity of the CD3.sup.+/CD28.sup.+ T cells is at least 99%
pure. In a related embodiment the purity of the
CD3.sup.+/CD28.sup.+ T cells is at least 99.9% pure. Therefore, one
aspect of the present invention is a population of
CD3.sup.+/CD28.sup.+ T cells comprising less than 10% of CD28-
cells. In certain embodiments, the population of
CD3.sup.+/CD28.sup.+ T cells comprises less than 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% contaminating CD28.sup.- T
cells.
[0032] In one embodiment the CD3.sup.+ surface molecule is
stimulated using anti-CD3 antibodies and the CD28.sup.+ surface
molecule is stimulated using anti-CD28 antibodies.
[0033] Therefore, the present invention also provides methods for
the generation of a substantially pure population of CD3.sup.+
CD28.sup.+ T cells, including CD4.sup.+ CD3.sup.+CD28.sup.+ T
cells, and CD8.sup.+ CD3.sup.+CD28.sup.+ T cells. These T cell
populations could then be used in the treatment of people suffering
from autoimmune diseases such as, 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, pemphigus vulgaris (PV),
paraneoplastic pemphigus (PNP), and rheumatic fever.
[0034] The present invention further provides a method for
activating and expanding a population of T cells by cell surface
moiety ligation, comprising providing a population of cells wherein
at least a portion thereof comprises T cells, contacting the
population of cells with a surface, wherein the surface has
attached thereto one or more agents that ligate a cell surface
moiety of at least a portion of the T cells and stimulates said T
cells, wherein said surface is present at a ratio of said surface
to said cells such that at least a substantial portion of at least
one population of antigen-specific T cells is deleted after about 8
days of culture. In one embodiment of the invention, the ratio is
from about 50:1 to about 5:1. In certain embodiments, the ratio is
from about 100:1 to about 2:1. In one embodiment the ratio is at
least about 45:1. In certain embodiments, the ratio is at least
about 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1,
10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. In one particular
embodiment the ratio is about 5:1.
[0035] The present invention provides a method for eliminating at
least a substantial portion of a clonal T cell subpopulation from a
mixed population of T cells from an individual, comprising,
exposing a population of cells, wherein at least a portion thereof
comprises T cells, to one or more pro-apoptotic compositions
wherein said exposure induces apoptosis in at least a substantial
portion of at least one clonal T cell population present in the
mixed population of T cells thereby eliminating at least a
substantial portion of said clonal T cell population from the mixed
population of T cells.
[0036] The present invention further provides methods for improved
transplant efficacy by administration of XCELLERATED.TM. T cells
following high-dose chemotherapy and autologous stem cell
transplantation.
[0037] The present invention also provides a method for treating a
patient afflicted with an autoimmune disease comprising contacting
a population of cells from the patient, wherein at least a portion
thereof comprises T cells, with a surface, wherein said surface has
attached thereto one or more agents that ligate a cell surface
moiety of at least a portion of the T cells and stimulates said T
cells, wherein said surface is present at a ratio of said surface
to said cells such that at least a substantial portion of at least
one population of antigen-specific T cells is deleted after about 8
days of culture; and administering to the patient an effective
amount of T cells from (a) such that in vivo homeostatic
proliferation is inhibited; thereby treating autoimmune disease. In
certain embodiments, the ratio is from about 10:1 to about 5:1.
[0038] The present invention further provides a method for treating
a patient afflicted with an autoimmune disease comprising
contacting a population of cells from the patient, wherein at least
a portion thereof comprises T cells, with a surface, wherein said
surface has attached thereto one or more agents that ligate a cell
surface moiety of at least a portion of the T cells and stimulates
said T cells; administering to the patient the T cells of (a) at a
dose such that homeostatic proliferation of endogenous T cells is
inhibited; thereby treating autoimmune disease.
[0039] The present invention also provides a method for treating a
patient infected with HIV comprising contacting a population of
cells from the patient, wherein at least a portion thereof
comprises T cells, with a surface, wherein said surface has
attached thereto one or more agents that ligate a cell surface
moiety of at least a portion of the T cells and stimulates said T
cells, wherein said surface is present at a ratio of said surface
to said cells such that at least a substantial portion of
HIV-infected T cells is deleted after about 8 days of culture; and
administering to the patient an effective amount of T cells from
(a) such that in vivo homeostatic proliferation is inhibited;
thereby treating the patient infected with HIV.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0040] FIG. 1 is a dot plot showing the presence of CD3+
CD8+HLA-A2CMVpp65 antigen specific T cells in an HLA-A2-positive
donor.
[0041] FIG. 2 is a dot plot showing an increase in CD25 expression
in CMV-activated HLA-A2CMVpp65 antigen-specific T cells.
[0042] FIG. 3 is a dot plot showing the upregulation of CD25 on
restimulated cells, and the deletion of prestimulated
tetramer-positive cells (i.e., CMVpp65-Ag-specific) by the
secondary strong stimulation provided by the 3.times.28 beads. At
day 14 post-primary stimulation, cultures were either left
unstimulated (Panels A1-A4) or were restimulated using the
XCELLERATE.TM. process with 3.times.28 beads for 16 hours (Panels
B1-B4). CD25 is upregulated on restimulated cells (Panel B2), but
tetramer-positive (i.e., CMVpp65-Ag-specific) prestimulated cells
were deleted by the secondary strong stimulation provided by the
3.times.28 beads (Panel B3).
[0043] FIG. 4 is a histogram showing the increase in expression of
key effector molecules, including CD95, on leukemic B-cells
co-cultured with XCELLERATED T cells.TM..
[0044] FIG. 5 is a dot plot showing the induction of apoptosis in
leukemic B-cells co-cultured with XCELLERATED T cells.TM..
[0045] FIG. 6 is a graph showing the disappearance of leukemic
B-cells during the XCELLERATE.TM. process and the concomitant
expansion of T cells.
[0046] FIG. 7 is a graph comparing fold increase of polyclonal T
cells to the fold increase of CMV pp65 A2-tetramer+
(antigen-specific) T cells using varying bead:cell ratios. Solid
bars represent polyclonal T cells. Striped bars represent
CMV-specific T cells.
[0047] FIG. 8 shows the spectratype analysis of T cells from a
rheumatoid arthritis patient pre-XCELLERATE.TM.(left panels) and
post-XCELLERATE.TM. (right panels) (5:1 bead:T cell ratio).
Restoration of healthy T cell repertoire was observed (see in
particular TCRBV 13a and TCRBV 3 panels, pre and post
XCELLERATE.TM.).
[0048] FIG. 9 is a bar graph showing the Th1 phenotype of
XCELLERATED.TM. T cells from patients with scleroderma, Crohn's
Disease, lupus, and rheumatoid arthritis.
[0049] FIG. 10 is a 4 panel dot plot showing the deletion of
islet-specific CD8+ autoreactive T cells in a mouse diabetes model.
Islet-specific T cells were detected using flow cytometry and
MHC-class I tetramer staining.
DETAILED DESCRIPTION OF THE INVENTION
[0050] 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.
[0051] The term "biocompatible", as used herein, refers to the
property of being predominantly non-toxic to living cells.
[0052] 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 up- or down-regulate expression of cell surface molecules
such as receptors or adhesion molecules, or up- or down-regulate
secretion of a molecule, such as downregulation of Tumor Growth
Factor beta (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.
[0053] The term "activation", as used herein, refers to the state
of a cell following sufficient cell surface moiety ligation to
induce a measurable morphological, phenotypic, and/or functional
change. Within the context of T cells, such activation may be 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/or secretion, and up- or down-regulation of
expression of cell surface molecules such as receptors or adhesion
molecules, or up- or down-regulation of secretion of certain
molecules, 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.
[0054] The term "target cell", as used herein, refers to any cell
that is intended to be stimulated by cell surface moiety
ligation.
[0055] An "antibody", as used herein, includes both polyclonal and
monoclonal antibodies (mAb); 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.
[0056] The term "protein", as used herein, includes proteins,
glycoproteins and other cell-derived modified 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.
[0057] 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.
[0058] The term "cell surface moiety" as used herein may refer to a
cell surface receptor, an antigenic determinant, or any other
binding site present on a target cell population.
[0059] The terms "agent that binds a cell surface moiety" and "cell
surface moiety", as used herein, should be viewed as a
complementary/anti-complementary set of molecules that demonstrate
specific binding, generally of relatively high affinity.
[0060] 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 and/or activation.
[0061] "Separation", as used herein, includes any means of
substantially purifying one component from another (e.g., by
filtration, affinity, buoyant density, or magnetic attraction).
[0062] 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.
[0063] "Monoclonality", as used herein, in the context of a
population of T cells, refers to a population of T cells that has a
single specificity as defined by spectratype analysis (a measure of
the TCR V.beta., V.alpha., V.gamma., or V.delta. chain
hypervariable region repertoire). A population of T cells is
considered monoclonal (or mono-specific) when the V.beta.,
V.alpha., V.gamma., and/or V.delta. spectratype profile for a given
TCR V.beta., V.alpha., V.gamma., and/or V.delta. family has a
single predominant peak. Spectratype analysis distinguishes
rearranged variable genes of a particular size, not sequence. Thus,
it is understood that a single peak could represent a population of
T cells expressing any one of a limited number of rearranged TCR
variable genes (V.beta., V.alpha., V.gamma., or V.delta.)
comprising any one of the 4 potential nucleotides (adenine (a),
guanine (g), cytosine (c), or thymine (t)) or a combination of the
4 nucleotides at the junctional region. In certain embodiments of
the present invention, it may be desirable to clone and sequence a
particular band to determine the sequence(s) of the rearranged
variable gene(s) present in the band representing a particular
length.
[0064] "Oligoclonality", as used herein, in the context of a
population of T cells, refers to a population of T cells that has
multiple, but narrow antigen specificity. This can be defined by
spectratype analysis (a measure of the TCR V.beta., V.alpha.,
V.gamma., or V.delta.) chain hypervariable region repertoire). A
population of T cells is considered oligoclonal when the V.beta.
spectratype profile for a given TCR V.beta., V.alpha., V.gamma., or
V.delta. family has between about 2 and about 4 predominant peaks.
This can also be defined by generation and characterization of
antigen-specific clones to an antigen of interest.
[0065] "Polyclonality", as used herein, in the context of a
population of T cells, refers to a population of T cells that has
multiple and broad antigen specificity. This can be by spectratype
analysis (a measure of the TCR V.beta., V.alpha., V.gamma., or
V.delta. chain hypervariable region repertoire). A population of T
cells is considered polyclonal when the V.beta.spectratype profile
for a given TCR V.beta., V.alpha., V.gamma., or V.delta. family has
multiple peaks, typically 5 or more predominant peaks and in most
cases with Gaussian distribution. Polyclonality can also be defined
by generation and characterization of antigen-specific clones to an
antigen of interest.
[0066] "Restoring or increasing the polyclonality", as used herein
refers to a shift from a monoclonal profile to an oligoclonal
profile or to a polyclonal profile, or from an oligoclonal profile
to a polyclonal profile, in expressed TCR V.beta., V.alpha.,
V.gamma., and/or V.delta. genes in a population of T cells, as
measured by spectratype analysis or by similar analysis such as
flow cytometry or sequence analysis. The shift from a monoclonal
V.beta., V.alpha., V.gamma., and/or V.delta. expression profile in
a population of T cells to an oligoclonal profile or to a
polyclonal profile is generally seen in at least one TCR V.beta.,
V.alpha., V.gamma., and/or V.delta. family. In one embodiment of
the present invention, this shift is observed in 2, 3, 4, or 5
V.beta.families. In certain embodiments of the present invention, a
shift is observed in 6, 7, 8, 9, or 10 V.beta.families. In a
further embodiment of the present invention, a shift is observed in
from 11, 12, 13, or 14 V.beta. families. In a further embodiment of
the present invention, a shift is observed in from 15 to 20 V.beta.
families. In a further embodiment of the present invention, a shift
is observed in 20 to 24 V.beta. families. In another embodiment, a
shift is seen in all V.beta. families. The functional significance
of restoring or increasing the polyclonality of a population of T
cells is that the immune potential, or the ability to respond to a
full breadth of antigens, of the population of T cells is restored
or increased. In certain aspects of the present invention, some T
cells within a population may not have their TCRs engaged by the
methods set forth herein (e.g., T cells with downregulated TCR
expression). However, by being in close proximity to T cells
activated by the methods described herein, and the factors secreted
by them, these T cells may in turn upregulate their TCR expression
thereby resulting in a further increase in the polyclonality of the
population of T cells. Restoration or increase in polyclonality can
also be measured by determining the breadth of response to a
particular antigen of interest, for example by measuring the number
of different epitopes recognized by antigen-specific cells. This
can be carried out using standard techniques for generating and
cloning antigen-specific T cells in vitro.
[0067] The term "clonal T cell population" as used herein, refers
to a T cell population that has a given range of specificities
against a given target antigen. This can be measured by any number
of assays known in the art, for example by generating and measuring
the breadth of specificities (i.e. number of different
specificities) of antigen-specific clones in a given population. A
clonal T cell population can also be defined by having either
monoclonal or oligoclonal specificity as defined by spectratype
analysis (a measure of the TCR V.beta., V.alpha., V.gamma., or
V.delta. chain hypervariable region repertoire).
[0068] The term "animal" or "mammal" as used herein, encompasses
all mammals, including humans. Preferably, the animal of the
present invention is a human subject.
[0069] The term "exposing" as used herein, refers to bringing into
the state or condition of immediate proximity or direct
contact.
[0070] The term "proliferation" as used herein, means to grow or
multiply by producing new cells.
[0071] "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.
[0072] "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.
[0073] "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.
[0074] "Particles" or "surface" as used herein, may include a
colloidal particle, a microsphere, nanoparticle, a bead, or the
like. A surface may be any surface capable of having a ligand bound
thereto or integrated into, including cell surfaces (for example
K562 cells), and that is biocompatible, that is, substantially
non-toxic to the target cells to be stimulated. 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., New York; PURABEADS.TM., Prometic
Biosciences, magnetic beads from Immunicon, Huntingdon Valley, Pa.,
microspheres from Bangs Laboratories, Inc., Fishers, Ind.).
[0075] "Paramagnetic particles" as used herein, refer to particles,
as defined above, that localize in response to a magnetic
field.
[0076] A "pro-apoptotic composition" "apoptotic compositions" or
"inducer of apoptosis", as used herein refers to any composition or
stimulus that increases the apoptotic activity of a cell either
when administered alone or in conjunction with other pro-apoptotic
compositions. The pro-apoptotic compositions used in the methods of
the present invention preferably induce apoptosis in activated T
cells, NKT, NK or B-cells. In certain embodiments, a pro-apoptotic
composition of the present invention will induce apoptosis without
further activation/stimulation. Illustrative examples of such
compositions or stimuli include, but are not limited to,
deprivation of a growth factor, oxidizing conditions, heat stress,
serum starvation, phorbol myristate acetate (PMA) and ionomycin,
superantigens (e.g. SEA, SEB, and the like) various antibodies,
such as anti-CD2, anti-CD3, anti-CD28, anti-CD20, anti-Fas
antibody, or any combination thereof, MHC-peptide tetramers or
dimers, Fas ligand, IL-2, IL-4, TRAIL, rolipram, doxorubicin,
chlorambucil, fludarabine, corticosteroids, glucocorticoids,
cyclosporine, cyclophosphamide, FK506, azathioprine, methotrexate,
mycophenolate, annexin, caspases, inhibitors of bcl-2,
topoisomerase inhibitors, interleukin-1.beta. converting enzyme
(ICE)-binding agents, Shigella IpaB protein, staurosporine,
ultraviolet irradiation, gamma irradiation, radiation, tumor
necrosis factor, various histone deacetylase inhibitors, and others
well known in the art. In certain embodiments, the pro-apoptotic
compositions comprises a surface, such as a magnetic bead, having
attached thereto one or more agents that binds a cell surface
moiety. In this regard, the agent can be any agent as described
herein. In one embodiment, the surface has attached thereto at
least anti-CD3 antibodies. In another embodiment, the surface has
attached thereto anti-CD3 and anti-CD28 antibodies. In addition, a
stimulator of apoptosis can be a polypeptide that is capable of
increasing or inducing the apoptotic activity of a cell. Such
polypeptides include those that directly regulate the apoptotic
pathway such as Bax, Bad, Bcl-xS, Bak, Bik, and active caspases as
well as those that indirectly regulate the pathway. In certain
embodiments, the pro-apoptotic composition comprises activated T
cells, such as XCELLERATED T cells.TM. (such as those described in
U.S. patent application Ser. No. 10/133,236), in particular for
inducing apoptosis in populations of B-cells. Other illustrative
pro-apoptotic compositions include, but are not limited to,
irradiated cells (e.g. donor or recipient (allogeneic) cells),
target antigens (e.g. defined autoimmune target antigens for
example, in multiple sclerosis, the target antigen identified as
myelin basic protein (MBP) MBP 84-102, or MBP 143-168; pancreatic
islet cell antigens; in uveitis, the S Antigen; or in rheumatoid
arthritis, type II or other types of collagen; in Grave's disease,
thyroid receptor; in Myasthena gravis, acetylcholine receptor),
cytoplasmic linker protein-170 (CLIP-170), nucleic acid molecules,
proteins or peptides, and non-protein or non-polynucleotide
compounds.
[0077] A "composition that sensitizes cells to further activation
or stimulation" or "sensitizing composition" as used herein is any
composition which sensitizes cells to subsequent
activation/stimulation. Upon subsequent activation/stimulation,
sensitized cells undergo apoptosis. Sensitizing compositions of the
present invention also sensitize cells to the effects of
pro-apoptotic compositions. Illustrative compositions that
sensitize cells to further activation, stimulation, or the effects
of pro-apoptotic compositions include cells that have been treated
such that they are unable to continue dividing, for example by
irradiation, (e.g. donor or recipient (allogeneic) cells),
superantigens (e.g. SEA, SEB, and the like), target antigens (e.g.
defined autoimmune target antigens for example, in multiple
sclerosis, the target antigen identified as myelin basic protein
(MBP) MBP 84-102, or MBP 143-168; pancreatic islet cell antigens;
in uveitis, the S Antigen; or in rheumatoid arthritis, type II or
other types of collagen; in Grave's disease, thyroid receptor; in
Myasthena gravis, acetylcholine receptor, nucleic acid molecules,
proteins or peptides, and non-protein or non-polynucleotide
compounds), protein, glycoprotein, peptides, antibody/antigen
complexes, cell lysate, non-soluble cell debris, apoptotic bodies,
necrotic cells, whole cells from a cell line that have been treated
such that they are unable to continue dividing, natural or
synthetic complex carbohydrates, lipoproteins, transformed cells or
cell line, transfected cells or cell line, or transduced cells or
cell line, or any combination thereof.
[0078] Apoptosis, for purposes of the present invention, is defined
as programmed cell death. Apoptosis is a programmed cell death
which is a widespread phenomenon that plays a crucial role in the
myriad of physiological and pathological processes. Apoptosis
occurs in embryogenesis, metamorphosis, endocrine-dependent tissue
atrophy, normal tissue turnover, and death of immune thymocytes
(induced through their antigen-receptor complex or by
glucocorticoids) (Itoh et al., Cell 66:233, 1991). During
maturation of T cells in the thymus, T cells that recognize
self-antigens are destroyed through the apoptotic process, whereas
others are positively selected. The possibility that some T cells
recognizing certain self epitopes (e.g., inefficiently processed
and presented antigenic determinants of a given self protein)
escape this elimination process and subsequently play a role in
autoimmune diseases has been suggested (Gammon et al., Immunology
Today 12:193, 1991). Necrosis is an accidental cell death which is
the cell's response to a variety of harmful conditions and toxic
substances. Apoptosis, morphologically distinct from necrosis, is a
spontaneous form of cell death that occurs in many different
tissues under various conditions. Apoptosis occurs in two stages.
The cell undergoes nuclear and cytoplasmic condensation, and may
eventually break into a number of membrane-bound fragments
containing structurally intact apoptotic bodies, which are
phagocytosed by neighboring cells and rapidly degraded.
Alternatively, cells entering the apoptotic pathway may be
phagocytosed prior to degeneration into membrane bound bodies.
Apoptosis is observed in many different tissues, healthy and
neoplastic, adult and embryonic. Death occurs spontaneously, or is
induced by physiological or noxious agents. Apoptosis is a basic
physiological process that plays a major role in the regulation of
cell populations.
[0079] Methods for measuring apoptosis are well known in the art.
Apoptosis can be determined by methods such as, for example, DNA
ladder, electron or light microscopy, flow cytometry, and different
commercially available kits for the determination of apoptosis.
[0080] As used herein, a "growth inhibiting composition" is any
substance that inhibits growth in cells, or otherwise renders cells
dysfunctional and unable to divide either when administered alone
or in conjunction with other compositions of the present invention.
The growth-inhibiting compositions used in the methods of the
present invention preferably inhibit growth in activated T cells,
NKT, NK or B-cells. Illustrative examples of such compositions or
stimuli include, but are not limited to, but are not limited to,
deprivation of a growth factor, oxidizing conditions, heat stress,
serum starvation, phorbol myristate acetate (PMA) and ionomycin,
superantigens (e.g. SEA, SEB, and the like) various antibodies,
such as anti-CD2, anti-CD3, anti-CD28, anti-CD20, anti-Fas
antibody, or any combination thereof, MHC-peptide tetramers or
dimers, Fas ligand, IL-2, IL-4, TRAIL, rolipram, doxorubicin,
chlorambucil, fludarabine, corticosteroids, glucocorticoids,
cyclosporine, cyclophosphamide, FK506, azathioprine, methotrexate,
mycophenolate, annex in, caspases, inhibitors of bcl-2,
topoisomerase inhibitors, interleukin-1.beta. converting enzyme
(ICE)-binding agents, Shigella IpaB protein, staurosporine,
ultraviolet irradiation, gamma irradiation, radiation, tumor
necrosis factor, various histone deacetylase inhibitors, and others
well known in the art. In certain embodiments, the growth
inhibiting compositions comprises a surface, such as a magnetic
bead, having attached thereto one or more agents that binds a cell
surface moiety. In this regard, the agent can be any agent as
described herein. In one embodiment, the surface has attached
thereto at least anti-CD3 antibodies. In another embodiment, the
surface has attached thereto anti-CD3 and anti-CD28 antibodies. In
addition, a growth inhibiting composition can comprise a
polypeptide that is capable of inhibiting growth of a cell. Such
polypeptides include those peptides such as Bax, Bad, Bcl-xS, Bak,
Bik, and active caspases. Other illustrative growth inhibiting
compositions include, but are not limited to, irradiated cells
(e.g. donor or recipient (allogeneic) cells), target antigens (e.g.
defined autoimmune target antigens for example, in multiple
sclerosis, the target antigen identified as myelin basic protein
(MBP) MBP 84-102, or MBP 143-168; pancreatic islet cell antigens;
in uveitis, the S Antigen; or in rheumatoid arthritis, type II or
other types of collagen; in Grave's disease, thyroid receptor; in
Myasthena gravis, acetylcholine receptor), cytoplasmic linker
protein-170 (CLIP-170), nucleic acid molecules, proteins or
peptides, and non-protein or non-polynucleotide compounds.
[0081] As used herein, a "substantially pure" population of
CD3.sup.+/CD28.sup.+ T cells is a population of cells that is
comprised of at least about 90% CD3.sup.+/CD28.sup.+ T cells. In
certain aspects of the invention a "substantially pure" population
of CD3+/CD28+ T cells is a population of cells that is comprised of
at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%
CD3.sup.+/CD28.sup.+ T cells, preferably at least about 99%, and
even more preferably about 99.9% or more.
[0082] Sources of Mixed Population of Cells
[0083] In one embodiment, cells to be exposed to the pro-apoptotic
or growth inhibiting compositions and/or sensitizing compositions
are from the circulating blood of an individual and are obtained
from one or more units of blood or from an 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.
Prior to exposure to a sensitizing composition and subsequent
activation and/or stimulation, a source of T cells is obtained from
a subject. The term "subject" is intended to include living
organisms in which an immune response can be elicited (e.g.,
mammals). Examples of subjects include humans, dogs, cats, mice,
rats, and transgenic species thereof. 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. In certain
embodiments of the present invention, T cells can be obtained from
a unit of blood collected from a subject using any number of
techniques known to the skilled artisan, such as ficoll separation.
In one preferred embodiment, 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 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, calcium (Ca)-free, magnesium (Mg)-free PBS.
Alternatively, the undesirable components of the apheresis sample
may be removed and the cells directly resuspended in culture
media.
[0084] In another embodiment, T cells are isolated from peripheral
blood lymphocytes by lysing or removing the red blood cells and
depleting the monocytes, for example, 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, in one preferred embodiment, T cells are
isolated by incubation with anti-CD3/anti-CD28 (i.e.,
3.times.28)-conjugated beads, such as DYNABEADS.RTM. M-450 CD3/CD28
T, for a time period sufficient for positive selection of the
desired T cells. In one embodiment, the time period is about 30
minutes. In a further embodiment, the time period ranges from 30
minutes to 36 hours or longer and all integer values there between.
In a further embodiment, the time period is at least 1, 2, 3, 4, 5,
or 6 hours. In yet another preferred embodiment, the time period is
10 to 24 hours. In one preferred embodiment, the incubation time
period is 24 hours. For isolation of T cells from patients with
leukemia, use of longer incubation times, such as 24 hours, can
increase cell yield. Longer incubation times may be used to isolate
T cells in any situation where there are few T cells as compared to
other cell types, such in isolating tumor infiltrating lymphocytes
(TIL) from tumor tissue or from immunocompromised individuals.
Further, use of longer incubation times can increase the efficiency
of capture of CD8+ T cells. 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.
[0085] An additional aspect of the present invention provides a T
cell population or composition that has been depleted or enriched
for populations of cells expressing a variety of markers, such as
CD62L, CD45RA or CD45RO, cytokines (e.g. IL-2, IFN-.gamma., IL-4,
IL-10), cytokine receptors (e.g. CD25), perforin, adhesion
molecules (e.g. VLA-1, VLA-2, VLA-4, LPAM-1, LFA-1), and/or homing
molecules (e.g. L-Selectin), prior to sensitization, stimulation
and expansion. In one embodiment, cells expressing any of these
markers are depleted or positively selected by antibodies or other
ligands/binding agents directed to the marker. One of ordinary
skill in the art would readily be able to identify a variety of
particular methodologies for depleting or positively selecting for
a sample of cells expressing a desired marker.
[0086] Monocyte populations (i.e., CD14.sup.+ cells) may be
depleted from blood preparations prior to ex vivo expansion by a
variety of methodologies, including anti-CD14 coated beads or
columns, or utilization of the phagocytotic activity of these cells
to facilitate removal or through adherence to plastic. Accordingly,
in one embodiment, the invention uses paramagnetic particles of a
size sufficient to be engulfed by phagocytotic monocytes. In
certain embodiments, the paramagnetic particles are commercially
available beads, 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 aspect, other non-specific
cells are removed by coating the paramagnetic particles with
"irrelevant" proteins (e.g., serum proteins or antibodies).
Irrelevant proteins and antibodies include those proteins and
antibodies or fragments thereof that do not specifically target the
T cells to be expanded. In certain embodiments the irrelevant beads
include beads coated with sheep anti-mouse antibodies, goat
anti-mouse antibodies, and human serum albumin.
[0087] In brief such depletion of monocytes is performed by
preincubating PBMC that have been isolated from whole blood using
Ficoll, or apheresed peripheral blood with one or more varieties of
irrelevant or non-antibody coupled paramagnetic particles at any
amount that allows for removal of monocytes (approximately a 20:1
bead:cell ratio)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. Preincubation can also be done
at temperatures as low as 3-4 degrees C. 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 requisite
depletion can be monitored by a variety of methodologies known to
those of ordinary skill in the art, including flow cytometric
analysis of CD14 positive cells, before and after said
depletion.
[0088] T cells for exposure to pro-apoptotic and/or sensitizing
compositions and subsequent stimulation may also be frozen 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 a final concentration of
10% DMSO and 4% human serum albumin, or other suitable cell
freezing media, the cells then are 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.
[0089] Elimination of Undesired Subpopulations of Cells from a
Mixed Population of Cells
[0090] Direct Exposure to Pro-Apoptotic Compositions
[0091] The present invention provides for methods to eliminate at
least a portion of undesired clonal populations of cells, typically
T cells, B cells, NKT, or NK cells, from a population of immune
cells. The present invention further provides for compositions
comprising populations of cells that no longer contain undesired
cells, or have a significantly reduced number of undesired cells,
and uses thereof.
[0092] Undesired populations of cells can be eliminated or reduced
by a statistically significant amount directly through the exposure
of said cells to a pro-apoptotic composition. Exposure to the
pro-apoptotic composition can take place in vivo or in vitro.
Without being bound by theory, the previously activated cells are
thought to be more sensitive to apoptotic compositions than nave or
unactivated cells. Therefore, exposure to apoptotic compositions
either in vivo or in vitro, using doses and conditions that induce
apoptosis, will selectively kill highly activated cells such as
unwanted autoreactive cells in a patient. In preferred embodiments
of the present invention, the autoreactive cells to be eliminated
comprise T cells, NKT, NK, or B cells.
[0093] Thus, the present invention provides methods for the
elimination of at least a substantial portion of any unwanted
subpopulation of clonal cells (such as T, B, NKT, or NK cells) from
a mixed population of immune cells. For the purposes of the present
invention, a substantial portion means at least 70% of the unwanted
subpopulation of cells. In certain embodiments, a substantial
portion means 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and
higher of the unwanted subpopulation of cells. Elimination of cells
can be measured using any number of techniques known in the art,
including but not limited to flow cytometric analysis using a
variety of antibodies and/or peptide-MHC tetramers and functional
assays such as proliferation and chromium release assays.
[0094] Pro-apoptotic compositions or inducers of apoptosis refers
to any composition or stimulus that increases the apoptotic
activity of a cell either when administered alone or in conjunction
with (in combination with, before or after) other pro-apoptotic
compositions. The pro-apoptotic compositions used in the methods of
the present invention preferably induce apoptosis in activated T
cells, NKT cells, NK cells, and B cells. The amount and conditions
under which the pro-apoptotic compositions induce desired apoptosis
may vary and can be determined by the skilled artisan using routine
optimization. In certain embodiments, a pro-apoptotic composition
of the present invention will induce apoptosis without further
activation/stimulation. Illustrative examples of such agents or
stimuli include, but are not limited to, deprivation of a growth
factor, oxidizing conditions, heat stress, freeze-thaw stress,
serum starvation, various antibodies, such as anti-CD2, anti-CD3,
anti-CD28, anti-CD20, or anti-Fas antibody; MHC-peptide tetramers;
Fas ligand, TRAIL, FR901228 (as described in U.S. Pat. No.
6,403,555), FK506, annexin, caspases, cytokines such as IL-2 or
IL-4, cyclophosphamide, chemotherapeutic agents, V, steroids,
corticosteroids, glucocorticoids, rolipram, doxorubicin,
chlorambucil, fludarabine, inhibitors of bcl-2, topoisomerase
inhibitors, interleukin-1.beta. converting enzyme (ICE)-binding
agents, Shigella IpaB protein, staurosporine, ultraviolet
irradiation, gamma irradiation, radiation, tumor necrosis factor,
histone deacetylase inhibitors, and others well known in the art.
In certain embodiments, the pro-apoptotic composition comprises a
surface, such as a magnetic bead, having attached thereto one or
more agents that binds a cell surface moiety. In this regard, the
agent can be any agent as described herein. In one embodiment, the
surface has attached thereto at least anti-CD3 antibodies. In
another embodiment, the surface has attached thereto anti-CD3 and
anti-CD28 antibodies. In addition, a stimulator of apoptosis can be
a polypeptide that is capable of increasing or inducing the
apoptotic activity of a cell. Such polypeptides include those that
directly regulate the apoptotic pathway such as Bax, Bad, Bcl-xL,
Bak, Bik, and active caspases as well as those that indirectly
regulate the pathway. Other illustrative pro-apoptotic compositions
include, but are not limited to, irradiated cells (e.g. donor or
recipient (allo) cells), target antigens (e.g. defined autoimmune
target antigens such as myelin basic protein (MBP), pancreatic
islet cell antigens, cytoplasmic linker protein-170 (CLIP-170),
Sjogren's syndrome antigen A (SS-A/Ro), Sjogren's syndrome antigen
B (SS-B/La), Sjogren's lupus antigen (SL), scleroderma antigen 70
(Scl-70)) nucleic acid molecules, proteins or peptides, and
non-protein or non-polynucleotide compounds.
[0095] In one aspect of the present invention, one or more
pro-apoptotic compositions is administered to an individual in vivo
in conjunction with a pharmaceutically acceptable excipient. Any
combination of pro-apoptotic compositions may be administered, such
as anti-CD3 antibodies, in conjunction with a cytokine such as IL-2
or IL-4, administration of which is described in patent application
number WO9428926. As the skilled artisan will readily recognize,
tests on any pro-apoptotic composition used in the methods of the
present invention would need to be routinely carried out over a
range of doses to determine: 1) the pharmacokinetic behavior of
these substances; and 2) safety and identification of any untoward
effects 3) optimal doses for effective induction of apoptosis in
cells to be eliminated. This would constitute a Phase I clinical
trial. Thus, the particular pro-apoptotic compositions employed in
the methods described herein would require individual routine
optimization. The pro-apoptotic compositions of the present
invention can be administered topically, parenterally, or by
inhalation. The term "parenteral" includes subcutaneous injections,
intravenous, intramuscular, intracisternal injection, or infusion
techniques. These compositions will typically contain an effective
amount of the pro-apoptotic composition, alone or in combination
with an effective amount of any other active material. Such dosages
and desired drug concentrations contained in the compositions may
vary depending upon many factors, including the intended use,
mammal's body weight and age, and route of administration.
Preliminary doses can be determined according to animal tests, and
the scaling of dosages for human administration can be performed
according to art-accepted practices.
[0096] In one aspect of the present invention, the population of
cells is exposed to one or more pro-apoptotic compositions in
vitro. As the skilled artisan will readily recognize, tests on any
pro-apoptotic composition used in the methods of the present
invention would need to be routinely carried out over a range of
doses to determine: 1) the behavior of these substances; and 2)
safety and identification of any untoward effects 3) optimal doses
for effective induction of apoptosis in cells to be eliminated.
Thus, the particular pro-apoptotic compositions employed in the
methods described herein would require individual routine
optimization. In one particular embodiment, the population of
remaining cells that has been cleared of unwanted reactive
subpopulations of cells can then be administered to the patient
without further stimulation/activation or expansion.
[0097] In one embodiment of the present invention, cells are
exposed to pro-apoptotic compositions multiple times either alone
or in combination with other pro-apoptotic compositions. In certain
aspects of the present invention, it may be preferable to
activate/stimulate and in some cases also expand a mixed population
of cells as described below in the sections entitled
"Stimulation/Activation of Cell Populations" and "Expansion of Cell
Populations" prior to exposure to one or more pro-apoptotic
compositions. In one preferred embodiment, the cells remaining in
the population following exposure to a pro-apoptotic compositions
of the present invention, are activated/stimulated and expanded in
vitro as described below in the sections entitled
"Stimulation/Activation of Cell Populations" and "Expansion of Cell
Populations". In certain embodiments, the pro-apoptotic composition
and the composition used to activate/stimulate and expand are the
same composition. In one particular embodiment, a surface having
attached thereto an agent, as described herein, is used as a
pro-apoptotic composition and further, used to active/stimulate and
expand a mixed population. In this regard, certain clonal cells in
the population are induced to undergo apoptosis while others are
stumulated/activated and proliferate in response to the
composition. In this context, an illustrative composition that can
be used both to induce apoptosis in a subpopulation of T cells and
to stimulate/activate and expand a mixed population of T cells
comprises anti-CD3 and anti-CD28 antibodies co-immobilized on beads
(3.times.28 beads).
[0098] In another embodiment of the present invention, the cells
remaining following exposure to one or more pro-apoptotic
compositions, are further stimulated/activated and expanded in
vivo. In vivo stimulation and expansion of the cells of the present
invention can be carried out using any number of cytokines, such as
IL-2 and IL-4 or other agents described herein that simulate
cells.
[0099] In a further embodiment of the present invention, the cells
remaining following exposure to one or more pro-apoptotic
compositions, are further stimulated/activated and expanded in
vitro using the surfaces and agents bound thereto as described
below in the sections entitled "Stimulation/Activation of Cell
Populations" and "Expansion of T cell Populations". The stimulation
and activation of the remaining cells that have not undergone
apoptosis using the surfaces of the present invention, can increase
polyclonality of said remaining population of T cells as measured
by the breadth of the response of the population to a given
antigen. Restoration or increase in polyclonality can be measured
by determining the breadth of response to a particular antigen of
interest, for example by measuring the number of different epitopes
recognized by antigen-specific cells. This can be carried out using
standard techniques for generating and cloning antigen-specific T
cells in vitro.
[0100] The stimulation and activation using the surfaces of the
present invention of the remaining cells that have not undergone
apoptosis, restores polyclonality to said remaining population of T
cells with respect to expressed TCR genes as indicated by
spectratype analysis. Polyclonality of the T cell compositions of
the present invention are as described in U.S. Patent Application
No. 60/375,733. Spectratype analysis is a method for measuring TCR
V.beta., V.alpha., V.gamma., or V.delta. gene usage by a pool of T
cells and levels of nucleotide insertion during the recombination
process in T cell development (as described in U.S. Pat. No.
5,837,447). Spectratype analysis can be used to measure the breadth
or narrowness of the T cell immune response potential.
Additionally, spectratype analysis can be used to determine if
specific undesired clonal populations of T cells have been removed
from a mixed population of T cells.
[0101] The ability of V, D, and J gene segments to combine together
randomly introduces a large element of combinatorial diversity into
the TCR repertoire. The precise point at which V, D, and J segments
join can vary, giving rise to local amino acid diversity at the
junction. The exact nucleotide position of joining can differ by as
much as 10 residues resulting in deletion of nucleotides from the
ends of the V, D, and J gene segments, thereby producing codon
changes at the junctions of these segments. Diversity is further
increased during the rearrangement process when additional
nucleotides not encoded by either gene segment are added at the
junction between the joined gene segments. (The variability created
by this process is called "N-region diversity.") (Janeway, Travers,
Walport. Immunobiology. Fourth Ed., 98 and 150. Elsevier Science
Ltd/Garland Publishing. 1999).
[0102] The level of diversity for the T cell repertoire can be
measured, in part, by evaluating which TCR V.beta., V.alpha.,
V.gamma., or V.delta. chains are being employed by individual T
cells within a pool of circulating T cells, and by the number of
random nucleotides inserted next to the V.beta. gene at the V-D-J
or V-J gene junctions. In general, when the circulating T cell pool
contains T cells expressing the full range of TCR V.beta.,
V.alpha., V.gamma., or V.delta. chains and when those individual V
region chains are derived from gene recombination events which
utilize the broadest array of inserted nucleotides, the T cell arm
of the immune system will have its greatest potential for
recognizing the universe of potential antigens. When the range of
TCR V region chains expressed by the circulating pool of T cells is
limited or reduced, and when expressed TCRs utilize chains encoded
by recombined genes with limited nucleotide insertions, the breadth
of the immune response potential is correspondingly reduced. The
consequences of this are a reduced ability to respond to the wide
variety of antigens leading to increased risks of infection and
cancer.
[0103] Methods for determining apoptosis are known in the art and
are described, for example, in Current Protocols in Immunology,
John Wiley & Sons, New York, N.Y., or in U.S. Pat. No.
6,312,684. Illustrative assays to measure apoptosis comprise DNA
ladder, electron or light microscopy, flow cytometry, and different
commercially available kits for the determination of apoptosis. In
certain embodiments, cells are observed for morphological changes,
such as chromatin condensation, cell shrinkage, increased
granularity and other indicia of apoptosis known to those of skill
in the art. Chromatin condensation can be detected by standard
methods, such as light microscopy of stained cell preparations.
Cell shrinkage and granularity can be readily detected by measuring
the light scattering properties of the cells (Kerr, et al. supra.,
and Wyllie, et al., supra). Observation of single or double
stranded fragmentation of DNA into oligonucleosomal ladders often
is another indication that apoptosis has been induced (Arend, et
al., Am. J. Pathol, 136:593, 1990; Wyllie, et al., J. Pathol,
142.:67, 1984). Sometimes, however, apoptotic cells do not exhibit
double stranded internucleosomal DNA fragmentation (Collins, et
al., Int. J. Rad. Biol., 62:45 1992; Cohen, et al., Biochem. J.,
286:331 1992); instead, single DNA strand breaks will be observed.
Single-strand breaks can readily be detected using a method of in
situ nick end-labeling of the DNA. This method is described by
Wyllie, et al. (Br. J. Cancer, 67:20, 1993).
[0104] In one embodiment, the cells of the present invention are
exposed to a growth inhibiting composition as described herein. In
certain embodiments, the growth inhibiting compositions of the
present invention inhibit growth in at least a substantial portion
of at least one clonal population of T cells such that when a mixed
population of cells is activated/stimulated and expanded as
described herein, the growth inhibited cells do not expand and are
eventually out-competed by the mixed population of cells. The end
result of this being the effective elimination of the growth
inhibited cells from the mixed population of cells.
[0105] Exposure to Compositions that Sensitize Cells to Further
Stimulation/Activation
[0106] Alternatively, at least a substantial portion of an
undesired population of cells can be eliminated by first
sensitizing the cells to further stimulation/activation and then
further simulating or activating them by exposure to a surface of
the present invention. This additional stimulation/activation
induces apoptosis in the sensitized cells, leading to their
elimination from the population. The sensitizing compositions of
the present invention also sensitize cells to the effects of
pro-apoptotic compositions described above. Thus, the present
invention provides for methods to eliminate at least a substantial
portion of an undesired clonal population of cells, typically T
cells or B cells, from a population of immune cells by exposure to
one or more compositions that sensitize the undesired populations
of cells to further stimulation/activation or to the effects of a
pro-apoptotic composition. The present invention further provides
for compositions comprising populations of cells that no longer
contain undesired cells, and uses thereof.
[0107] In one embodiment, exposure to a composition that sensitizes
to further activation or stimulation occurs naturally in vivo, such
as in the setting of autoimmune diseases. In this regard, auoimmune
cells are exposed to autoantigen in vivo and are thus sensitized.
Upon further stimulation/activation, such as by using the methods
of the present invention using a surface as described herein (e.g.,
a surface having attached thereto one or more agents that ligate a
cell surface moiety, such as anti-CD3 and anti-CD28 antibodies),
these autoimmune cells are induced to undergo apoptosis.
[0108] In one aspect of the present invention, a population of
immune cells is exposed to a composition or compositions that
sensitize to further activation or stimulation, at least a portion
of cells, e.g. previously highly activated T cells or B cells. In a
preferred embodiment, the sensitized cells comprise undesired
autoreactive T or B cells (e.g., in the setting of multiple
sclerosis, such sensitized cells would comprise MBP-specific T
cells that are sensitized in vivo as a results of the aberrant
immune regulation associated with this disease). In a further
embodiment, the sensitized cells comprise alloreactive cells
present in donor hematopoietic stem cell. In yet a further
embodiment, the sensitized cells comprise alloreactive cells from a
potential organ transplant recipient.
[0109] In one embodiment of the present invention, the sensitizing
composition comprises irradiated cells. In a particular embodiment,
the irradiated cells are from a hematopoietic stem cell transplant
recipient and the cells to be sensitized are from the hematopoietic
stem cell transplant donor. In another embodiment, the sensitizing
composition comprises irradiated cells from an organ donor and the
cells to be sensitized are cells from the organ recipient. In
certain embodiments, the cells to be sensitized are cells from an
organ recipient post-transplant. Cells are typically irradiated
with gamma rays in the range of about 3000 to 3600 rads, more
preferably at about 3300 rads. Other irradiated cells that may be
useful in the present invention, such as 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.
Cells may also be treated by other means such as with chemical
agents (e.g., etiposide, mitomycin, and the like).
[0110] Sensitizing compositions of the present invention include
any composition or combination of compositions that sensitizes
immune cells, such as T, NKT, NK, or B cells, to subsequent
stimulation such that subsequent stimulation or activation induces
apoptosis. Sensitizing compositions of the present invention also
include any composition or combination of compositions that
sensitizes immune cells, such as T or B cells, to subsequent
exposure to a pro-apoptotic composition. Sensitizing compositions
of the present invention include but are not limited to antibodies
such as anti-CD2, anti-CD3, anti-FAS; MHC-peptide dimers or
tetramers, cytokines such as IL-2, TRAIL, compounds such as
rolipram, doxorubicin, chlorambucil and fludarabine. Sensitizing
compositions also include FAS-ligand and the natural ligands for
CD2 and CD3. Sensitizing compositions also include inhibitors of
bcl-2, such as those described in U.S. Pat. No. 6,277,844,
topoisomerase inhibitors, such as etoposide, CPT-11 and topotecan,
and others, such as described in U.S. Pat. No. 5,834,012. Other
illustrative sensitizing compositions include interleukin-1.beta.
converting enzyme (ICE)-binding agents that induce apoptosis, such
as Shigella IpaB protein described in U.S. Pat. No. 5,972,899, or
compounds described in U.S. Pat. Nos. 6,350,741, 6,294,546 and
6,329,365.
[0111] Illustrative sensitizing compositions of the present
invention also comprise autoantigens. Autoantigens may be defined
autoimmune target antigens e.g. defined autoimmune target antigens
for example, in multiple sclerosis, the target antigen identified
as myelin basic protein (MBP) MBP 84-102, or MBP 143-168;
pancreatic islet cell antigens; in uveitis, the S Antigen; or in
rheumatoid arthritis, type II or other types of collagen; in SLE,
cytoplasmic linker protein-170 (CLIP-170); Sjogren's syndrome
antigen A (SS-A/Ro), Sjogren's syndrome antigen B (SS-B/La),
Sjogren's lupus antigen (SL); scleroderma antigen 70 (Scl-70); in
Grave's disease, thyroid receptor; in Myasthena gravis,
acetylcholine receptor, nucleic acid molecules, proteins or
peptides, and non-protein or non-polynucleotide compounds.
Autoantigens of the present invention also comprise peptide
mixtures eluted from MHC molecules known to be associated with
autoimmunity, for example, HLA-DQ and -DR molecules that confer
susceptibility to several common autoimmune diseases, such as type
1 diabetes, rheumatoid arthritis and multiple sclerosis, or HLA-B27
molecules known to confer susceptibility to reactive arthritis and
ankylosing spondylitis. Autoantigens of the present invention may
also be synthesized peptides predicted to bind to MHC molecules
associated with autoimmune diseases. It should be noted that
sensitization may occur naturally as a process of autoimmune
disease. As such, the "pre sensitized" autoreactive
(autoantigen-specific) T cells exist in patients and can be
eliminated or otherwise substantially reduced directly through the
XCELLERATE.TM. process as described herein.
[0112] The present invention further provides sensitizing
compositions for selectively eliminating at least a substantial
portion of a population of T cells expressing a specific V.beta.,
V.alpha., V.gamma., or V.delta. gene. For example, antibodies
specific for a particular V.beta., V.alpha., V.gamma., or V.delta.
gene can be used to specifically sensitize the T cells according to
the methods of the present invention. Alternatively, T cells
expressing a particular V.beta., V.alpha., V.gamma., or V.delta.
gene of interest can be negatively selected, thereby eliminating at
least a substantial portion of them from a population.
[0113] In further embodiments of the present invention, one or more
sensitizing compositions are used simultaneously and for times
sufficient to induce the desired sensitization.
[0114] As described above with pro-apoptotic compositions, the
present invention provides for methods wherein compositions that
sensitize cells to further stimulation/activation or the effects of
pro-apoptotic compositions, are administered in vivo or in vitro,
or a combination of the two. As with any medicinal substance, or
biologic, tests on any agents that sensitize cells to further
stimulation/activation to be administered in vivo, such as numerous
pro-apoptotic compounds, antibodies, peptides and proteins used for
immunization would need to be routinely carried out over a range of
doses to determine: 1) the pharmacokinetic behavior of these
substances; 2) their immunogenicity; and 3) safety and
identification of any untoward effects. This would constitute a
Phase I clinical trial. Thus, the particular agents that sensitize
cells to further stimulation/activation employed in the methods of
the present invention (for example, in multiple sclerosis, the
target antigen identified as MBP 84-102, or MBP 143-168; in
uveitis, the S Antigen; or in rheumatoid arthritis, type II
collagen) would require individual routine optimization. The
sensitizing compositions of the present invention can be
administered topically, parenterally, or by inhalation. The term
"parenteral" includes subcutaneous injections, intravenous,
intramuscular, intracisternal injection, or infusion techniques.
These compositions will typically contain an effective amount of
the sensitizing composition, alone or in combination with an
effective amount of any other active material. Such dosages and
desired drug concentrations contained in the compositions may vary
depending upon many factors, including the intended use, mammal's
body weight and age, and route of administration. Preliminary doses
can be determined according to animal tests, and the scaling of
dosages for human administration can be performed according to
art-accepted practices.
[0115] Ample evidence from the development of vaccines suggests
that either synthetic peptides or recombinant DNA-derived proteins
are effective in eliciting an immune response in humans. These
studies also provide guidance as to the range of doses effective
for immunization (Zajoc, B. A., D. J. West, W. J. McAleer and E. M.
Scolnick, Overview of clinical studies with Hepatitis B vaccine
made by recombinant DNA, J. Infect. 13:(Suppl A)39-45 (1986).
Yamamoto, S., T. Kuroki, K. Kurai and S. Iino, Comparison of
results for phase I studies with recombinant and plasma-derived
hepatitis B vaccines, and controlled study comparing intramuscular
and subcutaneous injections of recombinant hepatitis B vaccine, J.
Infect. 13:(Suppl A)53-60 (1986). Francis, D. P. et al., The
prevention of Hepatitis B with vaccine, Ann. Int. Med. 97:362-366
(1982). Putney et al., Features of HIV envelope and development of
a subunit vaccine, AIDS Vaccine Research and Clinical Trials, S.
Putney and B. Bolognesi, eds. (New York: Dekker) pp. 3-62 (1990).
Steven, V. C. and W. R. Jones, Vaccines to prevent pregnancy, New
Generation Vaccines, G. C. Woodrow and M. M. Levine, eds. (New
York: Dekker) pp. 879-900 (1990). Herrington et al., Safety and
immunogenicity in man of a synthetic peptide malaria vaccine
against Plasmodium Falciparium sporozoites, Nature, 328:257-259
(1987)).
[0116] In one embodiment of the present invention, immunization
(i.e. in vivo sensitization) with an agent that sensitizes cells to
further stimulation/activation or exposure to a pro-apoptotic
composition, is then followed by a waiting period during which the
agent activates the subset of cells bearing reactive receptors,
such as T cells bearing reactive TCRs or B cells expressing
specific antibody receptors, causing them to express cytokine
receptors, such as the IL-2 receptor. For example, this process
will induce IL-2 receptors only on T cells that have been
antigenically-stimulated. Based on studies of both human and mouse
T cells in vitro, between about 12 to about 24 hours after antigen
exposure are required to express significant numbers of IL-2
receptors, and as long as about 72 hours are required to express
optimal numbers of IL-2 receptors on the majority of T cells. Thus,
the waiting period can be as short as about 12 hours or as long as
about 72 hours, and in the case of various disease states, due to
retarded immune responsiveness, this period may be as long as 120
hours, becoming increasingly optimal toward the upper end of this
range.
[0117] In one embodiment of the present invention, IL-2, or other
appropriate cytokines, such as IL-4, are administered to the
patient to induce apoptosis in the activated cells as described
above. Administration of IL-2 to humans has been well-studied in
cancer patients, and various doses have been evaluated (Loize, M.
T., L. W. Frana, S. O. Sharrow, R. J. Robb and S. A. Rosenberg, In
vivo administration of purified human interleukin 2. I. Half-life
and immunologic effects of the Jurkat cell line-derived interleukin
2. J. Immunol. 134:157-166 (1985). Lotze, J. T., Y. L. Malory, S.
E. Ettinghausen, A. A. Rayner, S. O. Sharrow, C. A. Y. Seipp, M. C.
Custer and S. A. Rosenberg, In vivo administration of purified
human interleukin 2. II. Half-life, immunologic effects, and
expansion of peripheral lymphoid cells in vivo with recombinant IL
2. J. Immunol. 135:2865-2875 (1985). Donahue, J. H. and S. A.
Rosenberg, The fate of interleukin-2 after in vivo administration,
J. Immunol. 130:2203-2208 (1983). Belldegrun, A., M. M. Muul and S.
A Rosenberg, Interleukin 2 expanded tumor-infiltrating lymphocytes
in human renal cell cancer: isolation, characterization, and
antitumor activity, Cancer Research 48:206-214 (1988). Rosenberg,
S. A., M. T. Lotze, L. M. Muul, S. Leitman, A. E. Chang, S. E.
Ettinghausen, Y. L. Malory, J. M. Skibber, E. Shiloni, J. T. Vetto,
C. A. Seipp, C. Simpson and C. M. Reichert, Observations on the
systemic administration of autologous lymphokine-activated killer
cells and recombinant interleukin-2 to patients with metastatic
cancer, New Eng. J. Med. 313:1485-1492 (1985).). Data indicate that
IL-2 should be given I.V., either as frequent bolus doses or as a
continuous infusion. Doses that have been previously established
range between about 300 to about 3000 units/kg/hour continuous
infusion, or from 104 to 106 units/kg I.V. bolus.
[0118] In one aspect of the present invention, the population of
cells is exposed to one or more sensitizing compositions in vitro.
As the skilled artisan will readily recognize, tests on any
sensitizing composition used in the methods of the present
invention would need to be routinely carried out over a range of
doses to determine: 1) the pharmacokinetic behavior of these
substances; and 2) safety and identification of any untoward
effects 3) optimal doses for effective induction of apoptosis in
cells to be eliminated. Thus, the particular sensitizing
compositions employed in the methods described herein would require
individual routine optimization.
[0119] In one embodiment of the present invention, cells are
collected from an individual previously treated in vivo with an
agent that sensitizes cells to further stimulation/activation.
Cells are then further stimulated/activated to induce apoptosis and
then expanded in vitro as described below.
[0120] Stimulation of Sensitized Cells to Induce Apoptosis of Cells
to be Eliminated From a Mixed Population of Cells
[0121] In one aspect of the present invention, a population of
immune cells comprising sensitized cells as described above is
further activated or stimulated to induce apoptosis as described
below in the section entitled "Stimulation/Activation of Cell
Populations", thereby eliminating the sensitized cells, such as
autoreactive or alloreactive T- or B-cells, from the mixed
population of cells. At the same time, the desired cells that
remain, e.g., those cells that are not sensitized to undergo
apoptosis, are activated and stimulated to expand, thereby
resulting in a population of activated cells from which at least a
substantial portion of unwanted subpopulations of T (or B cells)
have been eliminated. As mentioned previously,
stimulation/activation as described herein may be carried out on
cells remaining following exposure of a mixed population of cells
directly to pro-apoptotic compositions. Furthermore, the subsequent
stimulation and activation provided by the present invention
restores polyclonality to the population of T cells with respect to
expressed TCR genes as indicated by spectratype analysis.
[0122] In one embodiment of the present invention, sensitized cells
are stimulated/activated as described below multiple times with or
without additional sensitizing composition, as many times as is
necessary to eliminate at least a substantial portion of the
undesired cells. For example in the setting of an autoimmune
disease, the present invention provides for methods to stimulate
cells a second or more times in the presence of antigen (i.e.,
sensitizing composition) after the initial round of
stimulation/activation. Likewise, in the setting of hematopoietic
stem cell transplantation, the present invention provides for
methods to stimulate cells from the hematopoietic stem cell donor a
second or more times, or as many times as necessary to eliminate at
least a substantial portion of the undesired cells, in the presence
of irradiated cells from a hematopoietic stem cell transplant
recipient. In the setting of organ transplantation, the present
invention provides for methods to stimulate cells from the organ
recipient a second or more times, or as many times as is necessary
to eliminate at least a substantial portion of undesired cells, if
necessary in the presence of irradiated cells from the organ donor.
In one embodiment, the methods of the present invention are carried
out on cells from a patient (e.g. host cells) post-transplant in
order to eliminate undesired cells. In certain embodiments, it may
not be necessary to eliminate all of the undesired cells, for
example in the setting of hematopoietic stem cell transplantation
for certain types of cancer, graft versus leukemic cell effect may
be desired.
[0123] In certain aspects of the present invention, it may be
preferable to stimulate/activate and in some cases expand a mixed
population of cells as described below in the sections entitled
"Stimulation/Activation of Cell Populations" and "Expansion of Cell
Populations" prior to exposure to one or more agents that sensitize
cells to further stimulation/activation and subsequent
stimulation.
[0124] In further aspects of the present invention, the cells are
sensitized and then exposed to a pro-apoptotic composition, thereby
eliminating at least a substantial portion of cells that have
become sensitized to the effects of the pro-apoptotic composition.
The cells remaining in the population can then be further
stimulated/activated and expanded as described below.
[0125] In one embodiment, the cells of the present invention are
exposed to a growth inhibiting composition as described herein. In
certain embodiments, the growth inhibiting compositions of the
present invention inhibit growth in at least a substantial portion
of at least one clonal population of T cells such that when a mixed
population of cells is activated/stimulated and expanded as
described herein, the growth inhibited cells do not expand and are
eventually out-competed by the mixed population of cells. The end
result of this being the effective elimination of the growth
inhibited cells from the mixed population of cells.
[0126] Generation of a Substantially Pure CD3.sup.+ CD28.sup.+ T
Cell Population
[0127] The present invention provides methods for the generation of
a substantially pure population of CD3.sup.+ CD28.sup.+ T cells
from a population of immune cells. For the purposes of the present
invention, a population of substantially pure CD3.sup.+ CD28.sup.+
T cells contains less than 10% CD3.sup.+ CD28.sup.- T cells. In
certain embodiments, a population of substantially pure CD3.sup.+
CD28.sup.+ T cells contains less than 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, 0.5%, or 0.1% CD3.sup.+ CD28.sup.- T cells.
[0128] A pure population of CD3.sup.+ CD28.sup.+ T cells can be
generated by magnetic concentration, selection, and stimulating the
mixed population of T cells with a composition capable of
stimulating both CD3 and CD28 molecules on the surface of a T cell.
Selection and stimulation of both the CD3 and CD28 molecules on the
surface of a cell results in the activation and proliferation of
this subset of cells. Conversely, under conditions described
herein, exposure of a CD3.sup.+ CD28.sup.- T cell to a composition
capable of selecting and stimulating both CD3 and CD28 surface
molecules would be insufficient to induce both activation and
expansion of this population of T cells. Further, shortening
incubation time with CD3/CD28 beads as described herein, favors
selection of CD3.sup.+ CD28.sup.+ cells at the expense of CD3.sup.+
CD28.sup.- cells (e.g., a 15 minute selection at 1 r.p.m. at room
temperature, followed by magnetic concentration leaves many or most
CD3.sup.+ CD28.sup.- cells behind.) Thus, in one embodiment, a
short incubation with a surface as described herein followed by a
short magnetic selection is used to preferentially select or enrich
for CD28.sup.+T cells while leaving CD28.sup.- cells behind.
Further the temperature of incubation, the rate of mixing during
the incubation, and the exposure to the magnetic field, can all be
varied to preferentially select for CD28.sup.+cells. In certain
embodiments, antibodies other than anti-CD28 or in conjuction with
anti-CD28 can be used, for example anti-NKG2D antibody.
[0129] Triggering of the TCR by either a specific antigen or by a
molecule capable of stimulating the CD3 surface molecule, for
example an anti-CD3 antibody, is considered insufficient to induce
expansion and lymphokine secretion unless supplemented by
co-stimulatory signals, i.e., the specific stimulation of the CD28
molecule. In fact, in the absence of co-stimulation, these T cells
may acquire a state of non-responsiveness or anergy.
[0130] Thus, the methods of the present invention, e.g., the
stimulation and selection of a mixed population of T cells using a
composition capable of triggering CD3 and simulating CD28, would
result in the generation of a substantially pure population of
CD3.sup.+ CD28.sup.+ T cells.
[0131] In certain embodiments, it may be desirable to use the
CD28.sup.- population of T cells. Without being bound by theory,
this population may contain T cells, such as tumor-specific, or
virus-specific T cells of interest that could be enriched and used
for therapy as described herein, either as is or following further
culture/expansion.
[0132] In one embodiment of the present invention, this population
of substantially pure CD3.sup.+ CD28.sup.+ T cells can be used to
treat acute or chronic GVHD. In other embodiments, a population of
substantially pure CD3.sup.+ CD28.sup.+ T cells can be used to
treat autoimmune diseases, such as rheumatoid arthritis, multiple
sclerosis, insulin dependent diabetes, Addison's disease, celiac
disease, chronic fatigue syndrome, colitis, Crohn's disease,
fibromyalgia, lupus, 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 and rheumatic fever. In a further
embodiment, the cells of the present invention can be used to treat
autoimmunity associated with large granular lymphocyte leukemia
(LGL). A mixed population of immune cells could be removed from a
donor and these cells stimulated with a composition capable of
stimulating CD3 and CD28 molecules. While not wanting to be bound
by theory, it is postulated that this stimulation results in the
specific activation and expansion of CD3.sup.+ CD28.sup.+ T cells,
and result in the anergy of T cells that lack the expression of the
co-stimulatory molecule, CD28. Once the pure population of
CD3.sup.+ CD28.sup.+ T cells has been generated, these cells can
then be infused for the treatment of an autoimmune disease, LGL, or
GVHD.
[0133] Stimulation/Activation of Cell Populations
[0134] The stimulated and activated T cells of the present
invention are generated by cell surface moiety ligation that
induces activation. In certain embodiments, the stimulated and
activated T cells are generated by activating a population of T
cells solely via engagement of the TCR, for example using anti-TCR
antibodies, anti-CD3 antibodies, or natural ligands for the TCR. In
certain embodiments, the stimulated and activated T cells are
generated by activating a population of T cells and stimulating an
accessory molecule on the surface of the T cells with a ligand
which binds the accessory molecule, as described for example, in
U.S. patent application Ser. Nos. 08/253,694, 08/435,816,
08/592,711, 09/183,055, 09/350,202, 09/252,150, 10/133,236,
10/187,467, 10/350,305, published PCT application WO03024989, and
patent numbers 6,352,694, 5,858,358 and 5,883,223. In the context
of sensitized cells described above, activating said sensitized
population of T cells and stimulating an accessory molecule on the
surface of said sensitized T cells with a ligand which binds the
accessory molecule induces apoptosis and subsequent elimination of
the cells.
[0135] Generally, T cell activation of cells may be accomplished by
cell surface moiety ligation, such as stimulating 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. Stimulation may
also be achieved through contact with 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.
[0136] To further activate a population of 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 (C D86) (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).
[0137] Other illustrative accessory molecules on the surface of the
T cells that can be stimulated with a ligand that binds the
accessory molecule in the present invention include, but are not
limited to, NKG2D, CD54, LFA-1, ICOS, and CD40.
[0138] 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, growth
factor, cytokine, chemokine, soluble receptor, steroid, hormone,
mitogen, such as PHA, or other superantigens.
[0139] As described earlier, the subsequent stimulation and
activation of the remaining cells that have not undergone apoptosis
or have not been sensitized to undergo apoptosis, restores
polyclonality to said remaining population of T cells with respect
to expressed TCR genes as indicated by spectratype analysis.
[0140] Expansion of Cell Populations
[0141] Generally, the present invention provides for expansion of
the population of cells that remains following exposure of the
population to a pro-apoptotic compositions or a sensitizing
composition and any subsequent induction of apoptosis in undesired
subpopulations of cells, preferably autoreactive or undesired
alloreactive T cells. In one embodiment of the invention, the
remaining T cells may be stimulated by a single agent. In another
embodiment, remaining T cells are stimulated with two or more
agents, one that induces a primary signal and additional agents
that induce one or more co-stimulatory signals. Ligands useful for
stimulating a single signal or stimulating a primary signal and an
accessory molecule that stimulates a second signal may be used in
soluble form, attached to the surface of a cell, or immobilized on
a surface as described herein. A ligand or agent that is attached
to a surface serves as a "surrogate" antigen presenting cell (APC).
In a preferred embodiment both primary and secondary agents are
co-immobilized on a surface. In one embodiment, the molecule
providing the primary activation signal, such as a CD3 ligand, and
the co-stimulatory molecule, such as a CD28 ligand, are coupled to
the same surface, for example, a particle. Further, as noted
earlier, one, two, or more stimulatory molecules may be used on the
same or differing surfaces.
[0142] The cell population may be stimulated 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. Alternatively, a population of cells can be contacted with
PMA and ionomycin. 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).
[0143] 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 or Sc receptors or an
antibody or other binding agent which will bind to the agents. In a
preferred embodiment, the two agents are immobilized on a spherical
or semi-spherical surface, the prototypic examples being beads or
cells, 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 bead in equivalent molecular
amounts. In one embodiment, a 1:1 ratio of each antibody bound to
the beads for T cell expansion and T cell growth is used. In
certain aspects of the present invention, a ratio of anti
CD3:anti-CD28 antibodies (CD3:CD28) 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 anti-CD3:anti-CD28 (CD3:CD28)
antibody bound to the beads ranges from about 100:1 to 1:100 and
all integer values there between. In certain embodiments, the ratio
of CD3:CD28 is at least about 95:1, 90:1, 85:1, 80:1, 75:1, 70:1,
65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1,
10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. 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 1:200 CD3:CD28
ratio of antibody bound to beads is used. In one particular
embodiment, a 1:150 CD3:CD28 ratio of antibody bound to beads is
used. 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 1:45 CD3:CD28 ratio of antibody
bound to beads is used. In another embodiment, a 1:40 CD3:CD28
ratio of antibody bound to beads is used. In another embodiment, a
1:35 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 another embodiment, a 1:25 CD3:CD28 ratio of antibody
bound to beads is used. In another embodiment, a 1:20 CD3:CD28
ratio of antibody bound to beads is used. In another embodiment, a
1:15 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 1:5 CD3:CD28 ratio of
antibody bound to beads is used. In another embodiment, a 1:4
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.
[0144] Ratios of particles to cells from 1:500 to 500:1 and any
integer values in between may be used to stimulate T cells or other
target cells. As those of ordinary skill in the art can readily
appreciate, the ratio of particle to cells may depend 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 cells to particles ranges from
1:100 to 100:1 and any integer values in-between and in further
embodiments the ratio comprises 1:50 to 50:1 and any integer values
in between. In another embodiment, the ratio of cells to particles
ranges from 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 can vary as noted above, however certain preferred
values include at least 1:150, 1:125, 1:100, 1:75, 1:50, 1:40,
1:30, 1:20, 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, and 15:1, with
one preferred ratio being at least 1:1 particles per T cell. In one
particular embodiment, the preferred ratio of particles to cells is
1:5 or 1:10. In one embodiment, a ratio of particles to cells of
1:1 or less is used.
[0145] 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 1:1 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 to 1:10 (based on cell counts on the day of
addition). In another embodiment, the ratio of particles to cells
is at least about 1:2.5 on the first day and additional particles
are added to the cells on day 5 at about 1:10, 1:25, 1:50 or 1:100,
on day 7 at 1:10, 1:25, 1:50, or 1:100 and on day 9 at 1:10, 1:25,
1:50, or 1:100. In one particular embodiment, the ratio of
particles to cells is 1:1 on the first day of stimulation and
adjusted to 1:5 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 1:5 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. In particular, ratios will vary depending
on particle size and on cell size and type.
[0146] 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. Specifically, high bead:cell
ratios, such as about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1,
40:1, 45:1, 50:1, and higher, induce deletion of antigen-specific T
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. Thus, in this regard, the bead compositions described
herein are functioning as a pro-apoptotic composition. Further, in
this regard, as the skilled artisan would appreciate, in certain
embodiments, the same composition used as a pro-apoptotic
composition (e.g., a surface having attached thereto an agent that
stimulates a cell surface moiety, such as the bead compositions
described herein) is used to expand the remaining mixed population
of cells for use in any variety of immunotherapeutic settings as
described herein. 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. In a further embodiment, the particular bead:cell ratio used
selectively expands antigen-specific T cells. The skilled artisan
would readily appreciate that any ratio can be used as long as the
desired expansion or deletion occurs. 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.
[0147] 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 7 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.
[0148] For inducing long-term stimulation of a population of
CD4.sup.+ and/or CD8.sup.+ T cells, it may be necessary to
reactivate and re-stimulate the T cells with a stimulatory agent
such as an anti-CD3 antibody and an anti-CD28 antibody (e.g. B-T3,
XR-CD28 (Diaclone, Besan.cedilla.on, France)) several times to
produce a population of CD4.sup.+ or CD8.sup.+ cells increased in
number from about 10 to about 1,000-fold the original T cell
population. For example, in one embodiment of the present
invention, T cells are stimulated as described for 2-3 times. In
further embodiments, T cells are stimulated as described for 4 or 5
times. Using the present methodology, it is possible to achieve T
cell numbers from about 100 to about 100,000-fold that have
increased polyclonality as compared to prior to stimulation.
Moreover, T cells expanded by the method of the present invention
secrete substantial levels of cytokines (e.g., IL-2, IFN-.gamma.,
IL-4, GM-CSF and TNF-.alpha.) into the culture supernatants. For
example, as compared to stimulation with IL-2, CD4.sup.+ T cells
expanded by use of anti-CD3 and anti-CD28 co-stimulation secrete
high levels of GM-CSF and TNF-.alpha. into the culture medium.
These cytokines can be purified from the culture supernatants or
the supernatants can be used directly for maintaining cells in
culture. Similarly, the T cells expanded by the method of the
present invention together with the culture supernatant and
cytokines can be administered to support the growth of cells in
vivo.
[0149] In one embodiment, T cell stimulation is performed, for
example 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, increased cytokine production, etc.
[0150] In further embodiments of the present invention, the cells,
such as T cells are combined with agent-coated or conjugated 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 or conjugated 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
and/or polarization of the activation signal.
[0151] By way of example, when T cells are the target cell
population, the cell surface moieties may be ligated by allowing
paramagnetic beads to which anti-CD3 and anti-CD28 antibodies are
attached (3.times.28 beads) to contact the T cells prepared. In one
embodiment the cells (for example, 104 to 109 T cells) and beads
(for example, DYNABEADS.RTM. M-450 CD3/CD28 T paramagnetic beads at
a ratio of 1:1) are combined in a buffer, preferably PBS (without
divalent cations such as, calcium and magnesium). Again, those of
ordinary skill in the art can readily appreciate any cell
concentration may be used. For example, the target cell may be very
rare in the sample and comprise only 0.01% of the sample or the
entire sample (i.e. 100%) may comprise the target cell of interest.
Accordingly, any cell number is within the context of the present
invention. In certain embodiments, it may be desirable to
significantly decrease the volume in which particles and cells are
mixed together (i.e., increase the concentration of cells), to
ensure maximum contact of cells and particles. For example, in one
embodiment, a concentration of about 2 billion cells/ml is used. In
another embodiment, greater than 100 million cells/ml is used. In a
further embodiment, a concentration of cells of 10, 15, 20, 25, 30,
35, 40, 45, or 50 million cells/ml is used. In yet another
embodiment, a concentration of cells from 75, 80, 85, 90, 95, or
100 million cells/ml is used. In further embodiments,
concentrations of 125 or 150 million cells/ml can be used. Using
high concentrations can result in increased cell yield, cell
activation, and cell expansion. Further, use of high cell
concentrations allows more efficient capture of cells that may
weakly express target antigens of interest, such as CD28-negative T
cells. Such populations of cells may have therapeutic value and
would be desirable to obtain. For example, using high concentration
of cells allows more efficient selection of CD8+ T cells that
normally have weaker CD28 expression.
[0152] In a related embodiment, it may be desirable to use lower
concentrations of cells. By significantly diluting the mixture of T
cells and particles, interactions between particles and cells is
minimized. This selects for cells that express high amounts of
desired antigens to be bound to the particles. For example, CD4+ T
cells express higher levels of CD28 and are more efficiently
captured and stimulated than CD8+ T cells in dilute concentrations.
In one embodiment, the concentration of cells used is about
5.times.10.sup.6/ml. In other embodiments, the concentration used
can be from about 1.times.10.sup.5/ml to about 1.times.10.sup.6/ml,
and any integer value in between.
[0153] The buffer that the cells are suspended in may be any that
is appropriate for the particular cell type. When utilizing certain
cell types the buffer may contain other components, e.g. 1-5%
serum, necessary to maintain cell integrity during the process. In
another embodiment, the cells and beads may be combined in cell
culture media. The cells and beads may be mixed, for example, by
rotation, agitation or any means for mixing, for a period of time
ranging from one minute to several hours. The container of beads
and cells is then concentrated by a force, such as placing in a
magnetic field. Media and unbound cells are removed and the cells
attached to the beads or other surface are washed, for example, by
pumping via a peristaltic pump, and then resuspended in media
appropriate for cell culture.
[0154] In one embodiment of the present invention, the mixture may
be cultured for 30 minutes to several hours (about 3 hours) to
about 14 days or any hourly or minute 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. As described above,
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) or
interleukin-2 (IL-2). insulin, or any other additives for the
growth of cells known to the skilled artisan. 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).
[0155] In one embodiment of the present invention, bead:cell ratios
can be tailored to obtain a desired T cell phenotype. In one
particular embodiment, bead:cell ratios can be vaired 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. The skilled artisan would readily
appreciate that any ratio can be used as long as the desired
expansion or deletion of antigen-specific T cells occurs.
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.
[0156] In another embodiment, the time of exposure to stimulatory
agents such as anti-CD3/anti-CD28 (i.e., 3.times.28)-coated beads
may be modified or tailored in such a way to obtain a desired T
cell phenotype. Alternatively, a desired population of T cells can
be selected using any number of selection techniques, prior to
stimulation. One may desire a greater population of helper T cells
(T.sub.H), typically CD4.sup.+ as opposed to CD8.sup.+ cytotoxic or
regulatory T cells, because an expansion of T.sub.H cells could
improve or restore overall immune responsiveness. While many
specific immune responses are mediated by CD8.sup.+
antigen-specific T cells, which can directly lyse or kill target
cells, most immune responses require the help of CD4.sup.+ T cells,
which 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.
Increased numbers of CD4.sup.+ T cells can increase the amount of
cell-expressed CD40L introduced into patients, potentially
improving target cell visibility (improved APC function). Similar
effects can be seen by increasing the number of infused cells
expressing GM-CSF, or IL-2, all of which are expressed
predominantly by CD4.sup.+ T cells. 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) which can be generated and
expanded using the methods described herein. Alternatively, in
situations where CD4-help is needed less and increased numbers of
CD8.sup.+ T cells are desirous, the XCELLERATE.TM. 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. or increased cytolysis of a target cell is preferred.
One may also modify time and type of exposure to stimulatory agents
to expand T cells with a desired TCR repertoire, e.g. expressing
desired V.beta. family genes.
[0157] To effectuate isolation of different T cell populations,
exposure times to the particles may be varied. For example, in one
preferred embodiment, T cells are isolated by incubation with
3.times.28 beads, such as DYNABEADS.RTM. M-450, for a time period
sufficient for positive selection of the desired T cells. In one
embodiment, the time period is about 30 minutes. In a further
embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours.
In yet another preferred embodiment, the time period is 10 to 24
hours or more. In one preferred embodiment, the incubation time
period is 24 hours. For isolation of T cells from cancer patients,
use of longer incubation times, such as 24 hours, can increase cell
yield.
[0158] In certain embodiments, stimulation and/or expansion times
may be 10 weeks or less, 8 weeks or less, 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)
or any value between these numbers). In some embodiments in may be
desirable to clone T cells using, for example, limiting dilution or
cell sorting, wherein longer stimulation time may be necessary. 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 more robust and
healthy activated T cells that can continue to proliferate in vivo
and more closely resemble the natural effector T cell pool. As the
availability of T cell help is often the limiting factor in
antibody responses to protein antigens, the ability to selectively
expand or selectively infuse a CD4.sup.+ rich population of T cells
into a subject is extremely beneficial. Further benefits of such
enriched populations are readily apparent in that activated helper
T cells that recognize antigens presented by B lymphocytes deliver
two types of stimuli, physical contact and cytokine production,
that result in the proliferation and differentiation of B
cells.
[0159] In the various embodiments, one of ordinary skill in the art
understands removal of the stimulation signal from the cells is
dependent upon the type of surface used. For example, if
paramagnetic beads are used, then magnetic separation is the
feasible option. Separation techniques are described in detail by
paramagnetic bead manufacturers' instructions (for example, DYNAL
Inc., Oslo, Norway). Furthermore, filtration may be used if the
surface is a bead large enough to be separated from the cells. In
addition, a variety of transfusion filters are commercially
available, including 20 micron and 80 micron transfusion filters
(Baxter). Accordingly, so long as the beads are larger than the
mesh size of the filter, such filtration is highly efficient. In a
related embodiment, the beads may pass through the filter, but
cells may remain, thus allowing separation. In one particular
embodiment, the biocompatible surface used degrades (i.e. is
biodegradable) in culture during the exposure period.
[0160] Although the antibodies used in the methods described herein
can be readily obtained from public sources, such as the American
Type Culture Collection (ATCC), antibodies to T cell accessory
molecules and the CD3 complex can be produced by standard
techniques. Methodologies for generating antibodies for use in the
methods of the invention are well-known in the art and are
discussed in further detail herein.
[0161] Ligand Immobilization on a Surface
[0162] As indicated above, the methods of the present invention
preferably use ligands bound to a surface. The surface may be any
surface capable of having a ligand bound thereto or integrated into
and that is biocompatible, that is, substantially non-toxic to the
target cells to be stimulated. The biocompatible surface may be
biodegradable or non-biodegradable. The surface may be natural or
synthetic, and a synthetic surface may be a polymer. The surface
may comprise collagen, purified proteins, purified peptides,
polysaccharides, glycosaminoglycans, extracellular matrix
compositions, liposomes, or cell surfaces. A polysaccharide may
include for example, cellulose, agarose, dextran, chitosan,
hyaluronic acid, or alginate. Other polymers may include
polyesters, polyethers, polyanhydrides, polyalkylcyanoacryllates,
polyacrylamides, polyorthoesters, polyphosphazenes,
polyvinylacetates, block copolymers, polypropylene,
polytetrafluorethylene (PTFE), or polyurethanes. The polymer may be
lactic acid or a copolymer. A copolymer may comprise lactic acid
and glycolic acid (PLGA). Non-biodegradable surfaces may include
polymers, such as poly(dimethylsiloxane) and poly(ethylene-vinyl
acetate). Biocompatible surfaces include for example, glass (e.g.,
bioglass), collagen, chitin, metal, hydroxyapatite, aluminate,
bioceramic materials, hyaluronic acid polymers, alginate, acrylic
ester polymers, lactic acid polymer, glycolic acid polymer, lactic
acid/glycolic acid polymer, purified proteins, purified peptides,
or extracellular matrix compositions. Other polymers comprising a
surface may include glass, silica, silicon, hydroxyapatite,
hydrogels, collagen, acrolein, polyacrylamide, polypropylene,
polystyrene, nylon, or any number of plastics or synthetic organic
polymers, or the like. The surface may comprise a biological
structure, such as a liposome or cell surface, such as red blood
cells (RBCs). The surface may be in the form of a lipid, a plate,
bag, pellet, fiber, mesh, or particle. A particle 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., New York;
PURABEADS.TM., Prometic Biosciences).
[0163] When beads are used, the bead may be of any size that
effectuates target cell stimulation. In one embodiment, beads are
preferably from about 5 nanometers to about 500 .mu.m in size.
Accordingly, the choice of bead size depends on the particular use
the bead will serve. For example, if the bead is used for monocyte
depletion, a small size is chosen to facilitate monocyte ingestion
(e.g., 1.0 .mu.m and 4.5 .mu.m in diameter or any size that may be
engulfed, such as nanometer sizes); however, when separation of
beads by filtration is desired, bead sizes of no less than 50 .mu.m
are typically used. Further, when using paramagnetic beads, the
beads typically range in size from about 2.8 .mu.m to about 500
.mu.m and more preferably from about 2.8 .mu.m to about 50 .mu.m.
Lastly, one may choose to use super-paramagnetic nanoparticles
which can be as small as about 10.sup.-5 nm. Accordingly, as is
readily apparent from the discussion above, virtually any particle
size may be utilized.
[0164] An agent may be attached, incorporated into, coupled to, or
integrated into a surface by a variety of methods known and
available in the art. The agent may be a natural ligand, a protein
ligand, or a synthetic ligand. The attachment may be 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 a
ligand is capable of stimulating the cells. The attachment of the
agent may be direct or indirect (e.g. tethered). For example, the
antibody to a ligand first may be attached to a surface (direct
attachment), or avidin or streptavidin, or a second antibody that
binds the first, may be attached to the surface for binding to a
biotinylated ligand (indirect attachment). With respect to cell
surfaces, the attachment may be via genetic expression of the agent
using any number of technologies known in the art, such as
transfection or transduction, etc of an expression vector
comprising the coding region of the agent of interest. 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, the ligand 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,
the ligands 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 ligand 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.
[0165] In one aspect, the agent, such as certain ligands may be of
singular origin or multiple origins and may be antibodies or
fragments thereof while in another aspect, when utilizing T cells,
the co-stimulatory ligand is a B7 molecule (e.g., B7-1, B7-2).
These ligands are coupled to the surface by any of the different
attachment means discussed above. The B7 molecule to be coupled to
the surface may be isolated from a cell expressing the
co-stimulatory molecule, or obtained using standard recombinant DNA
technology and expression systems that allow for production and
isolation of the co-stimulatory molecule(s) as described herein.
Fragments, mutants, or variants of a B7 molecule that retain the
capability to trigger a co-stimulatory signal in T cells when
coupled to the surface of a cell can also be used. Furthermore, one
of ordinary skill in the art will recognize that any ligand useful
in the activation and induction of proliferation of a subset of T
cells may also be immobilized on beads or culture vessel surfaces
or any surface. In addition, while covalent binding of the ligand
to the surface is one preferred methodology, adsorption or capture
by a secondary monoclonal antibody may also be used. The amount of
a particular ligand attached to a surface may be readily determined
by flow cytometric analysis if the surface is that of beads or
determined by enzyme-linked immunosorbant assay (ELISA) if the
surface is a tissue culture dish, mesh, fibers, bags, for
example.
[0166] In a particular embodiment, the stimulatory form of a B7
molecule or an anti-CD28 antibody or fragment thereof is attached
to the same solid phase surface as the agent that stimulates the
TCR/CD3 complex, such as an anti-CD3 antibody. In addition to
anti-CD3 antibodies, other antibodies that bind to receptors that
mimic antigen signals may be used. For example, the beads or other
surfaces may be coated with combinations of anti-CD2 antibodies and
a B7 molecule and in particular anti-CD3 antibodies and anti-CD28
antibodies.
[0167] 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 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 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. 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 1:200 CD3:CD28 ratio of antibody bound to beads is
used. In one particular embodiment, a 1:150 CD3:CD28 ratio of
antibody bound to beads is used. 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 1:45 CD3:CD28
ratio of antibody bound to beads is used. In another embodiment, a
1:40 CD3:CD28 ratio of antibody bound to beads is used. In another
embodiment, a 1:35 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 another embodiment, a 1:25 CD3:CD28
ratio of antibody bound to beads is used. In another embodiment, a
1:20 CD3:CD28 ratio of antibody bound to beads is used. In another
embodiment, a 1:15 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 1:5
CD3:CD28 ratio of antibody bound to beads is used. In another
embodiment, a 1:4 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.
[0168] Surface-Associated Agents
[0169] Agents contemplated by the present invention include protein
ligands, natural ligands, and synthetic ligands. Agents that can
bind to cell surface moieties, and under certain conditions, cause
ligation and aggregation that leads to signaling include, but are
not limited to, lectins (for example, phyotohaemagluttinin (PHA),
lentil lectins, concanavalin A), antibodies, antibody fragments,
peptides, polypeptides, glycopeptides, receptors, B cell receptor
and T cell receptor ligands, MHC-peptide dimers or tetramers,
extracellular matrix components, steroids, hormones (for example,
growth hormone, corticosteroids, prostaglandins, tetra-iodo
thyronine), bacterial moieties (such as lipopolysaccharides),
mitogens, superantigens and their derivatives, growth factors,
cytokines, adhesion molecules (such as, L-selectin, LFA-3, CD54,
LFA-1), chemokines, and small molecules. The agents may be isolated
from natural sources such as cells, blood products, and tissues, or
isolated from cells propogated in vitro, prepared recombinantly, by
chemical synthesis, or by other methods known to those with skill
in the art.
[0170] In one aspect of the present invention, when it is desirous
to stimulate T cells, useful agents include ligands that are
capable of binding the CD3/TCR complex, CD2, and/or CD28 and
initiating activation or proliferation, respectively. Accordingly,
the term ligand includes those proteins that are the "natural"
ligand for the cell surface protein, such as a B7 molecule for
CD28, as well as artificial ligands such as antibodies directed to
the cell surface protein. Such antibodies and fragments thereof may
be produced in accordance with conventional techniques, such as
hybridoma methods and recombinant DNA and protein expression
techniques. Useful antibodies and fragments may be derived from any
species, including humans, or may be formed as chimeric proteins,
which employ sequences from more than one species.
[0171] Methods well known in the art may be used to generate
antibodies, polyclonal antisera, or monoclonal antibodies that are
specific for a ligand. Antibodies also may be produced as
genetically engineered immunoglobulins (Ig) or Ig fragments
designed to have desirable properties. For example, by way of
illustration and not limitation, antibodies may include a
recombinant IgG that is a chimeric fusion protein having at least
one variable (V) region domain from a first mammalian species and
at least one constant region domain from a second distinct
mammalian species. Most commonly, a chimeric antibody has murine
variable region sequences and human constant region sequences. Such
a murine/human chimeric immunoglobulin may be "humanized" by
grafting the complementarity determining regions (CDRs), which
confer binding specificity for an antigen, derived from a murine
antibody into human-derived V region framework regions and
human-derived constant regions. Antibodies containing CDRs of
different specificities can also be combined to generate
multi-specific (bi or tri-specific, etc.) antibodies. Fragments of
these molecules may be generated by proteolytic digestion, or
optionally, by proteolytic digestion followed by mild reduction of
disulfide bonds and alkylation, or by recombinant genetic
engineering techniques.
[0172] Antibodies are defined to be "immunospecific" if they
specifically bind the antigen with an affinity constant, K.sub.a,
of greater than or equal to about 10.sup.4 M.sup.-1, preferably of
greater than or equal to about 10.sup.5 M.sup.-1, more preferably
of greater than or equal to about 10.sup.6 M.sup.-1, and still more
preferably of greater than or equal to about 10.sup.7 M.sup.-1.
Affinities of binding partners or antibodies can be readily
determined using conventional techniques, for example, those
described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660,
1949) or by surface plasmon resonance (BIAcore, Biosensor,
Piscataway, N.J.) See, e.g., Wolff et al., Cancer Res.,
53:2560-2565, 1993).
[0173] Antibodies may generally be prepared by any of a variety of
techniques known to those having ordinary skill in the art (See,
e.g., Harlow et al., Antibodies: A Laboratory Manual, 1988, Cold
Spring Harbor Laboratory). In one such technique, an animal is
immunized with the ligand as antigen to generate polyclonal
antisera. Suitable animals include rabbits, sheep, goats, pigs,
cattle, and may include smaller mammalian species, such as, mice,
rats, and hamsters. Antibodies of the present invention may also be
generated as described in U.S. Pat. Nos. 6,150,584, 6,130,364,
6,114,598, 5,833,985, 6,071,517, 5,756,096, 5,736,137, and
5,837,243.
[0174] An immunogen may be comprised of cells expressing the
ligand, purified or partially purified ligand polypeptides or
variants or fragments thereof, or ligand peptides. Ligand peptides
may be generated by proteolytic cleavage or may be chemically
synthesized. Peptides for immunization may be selected by analyzing
the primary, secondary, or tertiary structure of the ligand
according to methods know to those skilled in the art in order to
determine amino acid sequences more likely to generate an antigenic
response in a host animal (See, e.g., Novotny, Mol. Immunol.
28:201-207, 1991; Berzoksky, Science 229:932-40, 1985).
[0175] Preparation of the immunogen may include covalent coupling
of the ligand polypeptide or variant or fragment thereof, or
peptide to another immunogenic protein, such as, keyhole limpet
hemocyanin or bovine serum albumin. In addition, the peptide,
polypeptide, or cells may be emulsified in an adjuvant (See Harlow
et al., Antibodies: A Laboratory Manual, 1988 Cold Spring Harbor
Laboratory). In general, after the first injection, animals receive
one or more booster immunizations according to a preferable
schedule for the animal species. The immune response may be
monitored by periodically bleeding the animal, separating the sera,
and analyzing the sera in an immunoassay, such as an Ouchterlony
assay, to assess the specific antibody titer. Once an antibody
titer is established, the animals may be bled periodically to
accumulate the polyclonal antisera. Polyclonal antibodies that bind
specifically to the ligand polypeptide or peptide may then be
purified from such antisera, for example, by affinity
chromatography using protein A or using the ligand polypeptide or
peptide coupled to a suitable solid support.
[0176] Monoclonal antibodies that specifically bind ligand
polypeptides or fragments or variants thereof may be prepared, for
example, using the technique of Kohler and Milstein (Nature,
256:495-497, 1975; Eur. J. Immunol. 6:511-519, 1976) and
improvements thereto. Hybridomas, which are immortal eucaryotic
cell lines, may be generated that produce antibodies having the
desired specificity to a ligand polypeptide or variant or fragment
thereof. An animal--for example, a rat, hamster, or preferably
mouse--is immunized with the ligand immunogen prepared as described
above. Lymphoid cells, most commonly, spleen cells, obtained from
an immunized animal may be immortalized by fusion with a
drug-sensitized myeloma cell fusion partner, preferably one that is
syngeneic with the immunized animal. The spleen cells and myeloma
cells may be combined for a few minutes with a membrane
fusion-promoting agent, such as polyethylene glycol or a nonionic
detergent, and then plated at low density on a selective medium
that supports the growth of hybridoma cells, but not myeloma cells.
A preferred selection media is HAT (hypoxanthine, aminopterin,
thymidine). After a sufficient time, usually about 1 to 2 weeks,
colonies of cells are observed. Single colonies are isolated, and
antibodies produced by the cells may be tested for binding activity
to the ligand polypeptide or variant or fragment thereof.
Hybridomas producing antibody with high affinity and specificity
for the ligand antigen are preferred. Hybridomas that produce
monoclonal antibodies that specifically bind to a ligand
polypeptide or variant or fragment thereof are contemplated by the
present invention.
[0177] Monoclonal antibodies may be isolated from the supernatants
of hybridoma cultures. An alternative method for production of a
murine monoclonal antibody is to inject the hybridoma cells into
the peritoneal cavity of a syngeneic mouse. The mouse produces
ascites fluid containing the monoclonal antibody. Contaminants may
be removed from the antibody by conventional techniques, such as
chromatography, gel filtration, precipitation, or extraction.
[0178] Human monoclonal antibodies may be generated by any number
of techniques. Methods include but are not limited to, Epstein Barr
Virus (EBV) transformation of human peripheral blood cells (see,
U.S. Pat. No. 4,464,456), in vitro immunization of human B cells
(see, e.g., Boerner et al., J. Immunol. 147:86-95, 1991), fusion of
spleen cells from immunized transgenic mice carrying human
immunoglobulin genes and fusion of spleen cells from immunized
transgenic mice carrying immunoglobulin genes inserted by yeast
artificial chromosome (YAC) (see, e.g., U.S. Pat. No. 5,877,397;
Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58, 1997;
Jakobovits et al., Ann. N.Y. Acad. Sci. 764:525-35, 1995), or
isolation from human immunoglobulin V region phage libraries.
[0179] Chimeric antibodies and humanized antibodies for use in the
present invention may be generated. A chimeric antibody has at
least one constant region domain derived from a first mammalian
species and at least one variable region domain derived from a
second distinct mammalian species (See, e.g., Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-55, 1984). Most commonly, a
chimeric antibody may be constructed by cloning the polynucleotide
sequences that encode at least one variable region domain derived
from a non-human monoclonal antibody, such as the variable region
derived from a murine, rat, or hamster monoclonal antibody, into a
vector containing sequences that encode at least one human constant
region. (See, e.g., Shin et al., Methods Enzymol. 178:459-76, 1989;
Walls et al., Nucleic Acids Res. 21:2921-29, 1993). The human
constant region chosen may depend upon the effector functions
desired for the particular antibody. Another method known in the
art for generating chimeric antibodies is homologous recombination
(U.S. Pat. No. 5,482,856). Preferably, the vectors will be
transfected into eukaryotic cells for stable expression of the
chimeric antibody.
[0180] A non-human/human chimeric antibody may be further
genetically engineered to create a "humanized" antibody. Such an
antibody has a plurality of CDRs derived from an immunoglobulin of
a non-human mammalian species, at least one human variable
framework region, and at least one human immunoglobulin constant
region. Humanization may yield an antibody that has decreased
binding affinity when compared with the non-human monoclonal
antibody or the chimeric antibody. Those having skill in the art,
therefore, use one or more strategies to design humanized
antibodies.
[0181] Within certain embodiments, the use of antigen-binding
fragments of antibodies may be preferred. Such fragments include
Fab fragments or F(ab').sub.2 fragments, which may be prepared by
proteolytic digestion with papain or pepsin, respectively. The
antigen binding fragments may be separated from the Fc fragments by
affinity chromatography, for example, using immobilized protein A
or immobilized ligand polypeptide or a variant or a fragment
thereof. An alternative method to generate Fab fragments includes
mild reduction of F(ab').sub.2 fragments followed by alkylation
(See, e.g., Weir, Handbook of Experimental Immunology, 1986,
Blackwell Scientific, Boston).
[0182] Non-human, human, or humanized heavy chain and light chain
variable regions of any of the above described Ig molecules may be
constructed as single chain Fv (sFv) fragments (single chain
antibodies). See, e.g., Bird et al., Science 242:423-426, 1988;
Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988.
Multi-functional fusion proteins may be generated by linking
polynucleotide sequences encoding an sFv in-frame with
polynucleotide sequences encoding various effector proteins. These
methods are known in the art, and are disclosed, for example, in
EP-B1-0318554, U.S. Pat. No. 5,132,405, U.S. Pat. No. 5,091,513,
and U.S. Pat. No. 5,476,786.
[0183] An additional method for selecting antibodies that
specifically bind to a ligand polypeptide or variant or fragment
thereof is by phage display (See, e.g., Winter et al., Annul. Rev.
Immunol. 12:433-55, 1994; Burton et al., Adv. Immunol. 57:191-280,
1994). Human or murine immunoglobulin variable region gene
combinatorial libraries may be created in phage vectors that can be
screened to select Ig fragments (Fab, Fv, sFv, or multimers
thereof) that bind specifically to a ligand polypeptide or variant
or fragment thereof (See, e.g., U.S. Pat. No. 5,223,409; Huse et
al., Science 246:1275-81, 1989; Kang et al., Proc. Natl. Acad. Sci.
USA 88:4363-66, 1991; Hoogenboom et al., J. Molec. Biol.
227:381-388, 1992; Schlebusch et al., Hybridoma 16:47-52, 1997 and
references cited therein).
[0184] Methods of Use
[0185] Monoclonal and oligoclonal T cell populations are associated
with most autoimmune diseases and are often correlated with disease
activity. Further, broad T cell repertoire is restored when
patients achieve disease remission. The present invention relates
generally to methods for stimulating T cells, and more
particularly, to methods to eliminate undesired (e.g. autoreactive,
alloreactive, pathogenic) subpopulations of T cells from a mixed
population of T cells, thereby restoring the normal immune
repertoire of the T cells. Thus, the present invention provides
compositions of cells, including stimulated T cells having restored
immune repertoire and uses thereof.
[0186] Generally, the compositions and methodologies described
herein can be used to to eliminate at least a portion of undesired
clonal populations of cells, typically T cells, B cells, NKT, or NK
cells, from a population of immune cells. The present invention
further provides for compositions comprising populations of cells
that no longer contain undesired cells, or have a significantly
reduced number of undesired cells, and uses thereof. The
compositions and methods of the present invention are also used to
selectively expand a population of cells that have been deleted for
undesired clonal populations for use in the treatment of immune
defects associated with hematopoietic stem cell transplantation
(including allotransplantation and autotransplantation from sources
that include blood, cord blood, and bone marrow), organ
transplantation (e.g., acute or chronic GVHD), and autoimmune
diseases, including autoimmune disease caused by cancers such as
large granular lymphocyte (LGL) leukemia, chronic lymphocytic
leukemia (CLL) or by common variable immunodeficiency. As a result,
a population of cells, in the case of T cells, that express TCRs
that are polyclonal with respect to antigen reactivity, but
essentially homogeneous with respect to either CD4.sup.+ or
CD8.sup.+, can be produced that have been cleared of any undesired
subpopulations of cells, such as autoreactive cells or alloreactive
cells. With respect to B cells, a populations of cells can be
produced that has been cleared of any undesired subpopulations of B
cells producing autoreactive antibodies. In addition, the method
allows for the expansion of the resulting population of T- or
B-cells in numbers sufficient to reconstitute an individual's total
CD4.sup.+ or CD8.sup.+ T cell population or B cell population (the
population of lymphocytes in an individual is approximately
5.times.10.sup.11 cells). The resulting cell population can also be
genetically transduced using a variety of techniques known to the
skilled artisan and used for immunotherapy.
[0187] In one embodiment, the T or B cell compositions of the
present invention may be used in the context of hematopoietic stem
cell transplantation. The major problem in hematopoietic stem cell
transplantation is graft-versus-host disease (GVHD), which is
caused by alloreactive T cells present in the infused hematopoietic
stem cell preparation. Thus, the present invention may be used to
remove alloreative T cells and to expand the remaining T cell
population for infusion into the patient. The cell compositions of
the present invention can be used alone or in conjunction with
other therapies.
[0188] In one embodiment, the T or B cell compositions of the
present invention may be used in the context of any autoimmune
disease. Illustrative autoimmune diseases include, but are not
limited to, systemic lupus erythematosus (SLE), multiple sclerosis
(MS), rheumatoid arthritis, progressive systemic sclerosis,
Sjogren's syndrome, multiple sclerosis, polymyositis,
dermatomyositis, uveitis, arthritis, psoriatic arthritis, reactive
arthritis, Type I insulin-dependent diabetes, Hashimoto's
thyroiditis, Grave's thyroiditis, myasthenia gravis, autoimmune
myocarditis, vasculitis, aplastic anemia, autoimmune hemolytic
anemia, myelodysplastic syndrome, Evan's syndrome, stiff person
syndrome, atopic dermatitis, psoriasis, Behchet's syndrome, Crohn's
disease, biliary cirrhosis, inflammatory bowel disease, ulcerative
colitis, Goodpasture's syndrome, Wegener's granulomatosis,
paroxysmal nocturnal hemaglobinuria, myelodysplastic syndrome,
allergic disorders such as hay fever, extrinsic asthma, or insect
bite and sting allergies, and food and drug allergies.
[0189] Further uses of the T and B cell compositions of the present
invention may include the treatment and/or prophylaxis of:
inflammatory and hyperproliferative skin diseases and cutaneous
manifestations of immunologically mediated illnesses, such as,
seborrhoeis dermatitis, angioedemas, erythemas, acne, and Alopecia
greata; various eye diseases (autoimmune and otherwise); allergic
reactions, such as pollen allergies, reversible obstructive airway
disease, which includes condition such as asthma (for example,
bronchial asthma, allergic asthma, intrinsic asthma, extrinsic
asthma and dust asthma), particularly chronic or inveterate asthma
(for example, late asthma and airway hyper-responsiveness),
bronchitis, allergic rhinitis, and the like; inflammation of mucous
and blood vessels.
[0190] As noted above, the T and B cell compositions of the present
invention may be used in the treatment of immune defects associated
with organ transplantation, e.g., host versus graft disease.
Treatment of immune defects associated with any organ
transplantation is contemplated herein. For example, the methods
and cells of the present invention can be used in the treatment of
immune defects associated with kidney, heart, lung, and liver
transplantation.
[0191] In certain embodiments of the present invention, the cells
of the present invention are administered to a patient following
treatment with an agent such as chemotherapy, radiation,
immunosuppressive agents, such as cyclosporine, azathioprine,
methotrexate, mycophenolate, and FK506, antibodies, or other
immunoablative agents such as CAMPATH, anti-CD3 antibodies,
cyclophosphamide, fludarabine, cyclosporine, 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)).
[0192] In a further embodiment, the cells are administered to a
patient whose immune system has been rendered essentially to a
naive state through treatment with one or more agents as described
herein, followed by an organ transplant in conjunction with an
immunosuppressive regimen. Immunosuppresive regimens useful in this
context include but are not limited to, anti-CD3 antibodies,
anti-CD25 antibodies, ATG, thymoglobulin, campath, fludarabine,
cyclophosphamide, FK506, mycophenolate, cyclosporine, CTLA-4 IG,
anti-CD40 antibody, destruxin, radiation therapy, and the like. In
this regard, many immunosuppressive regimens have been shown to be
effective in animal transplant models (e.g., mouse models) but have
not been successful in the clinic. Without being bound by theory,
one of the major reasons for this decrepancy is thought to be the
presence of antigen-specific memory T cells that cross react with
donor alloantigens in the transplanted organ. Lymphoablation in
vivo followed by infusion of activated T cells grown under
conditions that favor deletion of antigen-specific memory T cells
as described herein while preserving nave T cells may provide
conditions similar to the pathogen-free mice that accept organs
under a variety of immunosuppressive regimens. This would enable
wider and more successful use of immunosuppressive drugs.
Additionally, infusion of the T cells as described herein in this
setting would lead to a decrease in the use and amount of toxic
immunosuppresive drugs, including the number of drugs administered
and the length of time that patients are on these drugs. This in
turn would lead to increased efficacy (fewer organ rejections) and
decreased toxicity. Further, this would also allow the use of organ
transplantation in settings such as severe mismatching and could
allow successful transplants in xenogeneic settings.
[0193] In a further embodiment, the cell compositions of the
present invention are administered to a patient with autoimmune
disease 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 to a patient with autoimmune disease 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).
[0194] In a further aspect of the present invention, at least a
substantial portion of autoreactive cells from a patient are
eliminated in vitro using the methods of the present invention then
further stimulated and expanded and administered to the patient. In
a related embodiment, at least a substantial portion of
autoreactive cells from a patient are eliminated in vitro using the
methods of the present invention then administered to the patient
and expanded in vivo. It is envisioned as one aspect that the
compositions of the present invention can be used in conjunction
with other therapies available in the art for treatment of
autoimmune disease.
[0195] In one embodiment, T cells can be stimulated and expanded as
described herein to induce or enhance responsiveness in an
individual who is immunocompromised as a result of treatment
associated with hematopoietic stem cell transplantation. The
present invention provides methods for reducing the risk of, or the
severity of, an adverse GVHD effect in a patient who is undergoing
a hematopoietic stem cell transplant, comprising administering to
the patient a population of T cells of the present invention. In
one particular embodiment, at least a substantial portion of
alloreactive cells present in the donor hematopoietic stem cells
are eliminated by the methods of the present invention. In a
further embodiment, the T cell compositions of the present
invention are administered to a patient undergoing a hematopoietic
stem cell transplantation following treatment with chemotherapy
agents. In a further embodiment, at least a substantial portion of
alloreactive cells from the donor marrow are eliminated in vitro
using the methods of the present invention then further stimulated
and expanded and then administered to the patient. In a further
embodiment, at least a substantial portion of alloreactive cells
from the donor marrow are eliminated in vitro using the methods of
the present invention then administered to the patient and expanded
in vivo. It is envisioned as one aspect that the compositions of
the present invention can be used in conjunction with other
therapies available in the art for use in hematopoietic stem cell
transplantation, such as administration of G-CSF, IL-2, IL-11,
IL-7, IL-12, and antiviral treatments.
[0196] In one embodiment, T cells can be stimulated and expanded as
described herein to induce or enhance responsiveness in an
individual who is immunocompromised as a result of treatment
associated with organ transplantation, including but not limited
to, kidney, heart, lung, and liver transplantation. In one
particular embodiment, at least a substantial portion of
alloreactive cells present in the recipient are eliminated by the
methods of the present invention. Thus, the present invention
provides methods for reducing the risk of, or the severity of,
organ rejection. In a further embodiment, the T cell compositions
of the present invention are administered to a patient undergoing
an organ transplant following treatment with chemotherapy agents.
In a further embodiment, at least a substantial portion of
alloreactive cells from the transplant recipient are eliminated in
vitro using the methods of the present invention then further
stimulated and expanded and then administered to the patient. It is
envisioned as one aspect that the compositions of the present
invention can be used in conjunction with other therapies available
known in the art for use in organ transplantation.
[0197] Another embodiment of the invention, provides a method for
selectively expanding a population of T.sub.H1 cells from a
population of CD4.sup.+ T cells. In this method, CD4.sup.+ T cells
are co-stimulated with an anti-CD28 antibody, such as the
monoclonal antibody 9.3, inducing secretion of T.sub.H1-specific
cytokines, including IFN-.gamma., resulting in enrichment of
T.sub.H1 cells over T.sub.H2 cells. As described further herein,
XCELLERATED.TM. T cells from patients with autoimmune disease
demonstrate a T.sub.H1-type phenotype (see FIG. 9). Accordingly,
the XCELLERATE.TM. process can be used to expand T cells of a
desired phenotype, including TH1-type phenotype.
[0198] The present invention further provides a method for
selectively expanding a population of T.sub.H2 cells from a
population of CD4.sup.+ T cells. In this method, CD4.sup.+ T cells
are co-stimulated with an anti-CD28 antibody, such as the
monoclonal antibody B-T3, XR-CD28, inducing secretion of
T.sub.H2-specific cytokines, resulting in enrichment of T.sub.H2
cells over T.sub.H1 cells (see for example, Fowler, et al. Blood
1994 Nov. 15;84(10):3540-9; Cohen, et al., Ciba Found Symp
1994;187:179-93).
[0199] The present invention further provides methods for using the
instant cell compositions in conjunction with regulatory T cells.
Without being bound by theory, regulatory T cells may provide help
in suppressing aberrant immune responses or otherwise regulating
immune cells of the present invention. Regulatory T cells can be
generated and expanded using the methods as described herein. The
regulatory T cells can be antigen-specific and/or polyclonal.
Regulatory T cells can also 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.
[0200] The present invention further provides a method for
selectively expanding a population of T cells expressing a specific
V.beta., V.alpha., V.gamma., or V.delta. gene. For example, in this
method, T cells expressing a particular V.beta., V.alpha.,
V.gamma., or V.delta. gene are positively or negatively selected
and then further expanded/stimulated according to the methods of
the present invention. Alternatively, stimulated and expanded T
cells expressing a particular V.beta., V.alpha., V.gamma., or
V.delta. gene of interest can be positively or negatively selected
and further stimulated and expanded.
[0201] One aspect of the present invention is to administer
activated and expanded T cells that proliferate and grow rapidly in
vivo. Without being bound by theory, the infused T cells may
suppress in vivo homeostatic T cell proliferation and prevent
unwanted T cells from proliferating in vivo, for example cancer
cells, autoreactive T cells, alloreactive T cells, HIV infected T
cells, and the like (see King, et al., 2004, Cell 117:265-277).
Accordingly, the compositions described herein comprising T cells
that have been cultured so as to delete at least a substantial
portion of unwanted cells can be infused into a patient. In one
embodiment, the cells are infused at a dose such that the cells
then prevent homeostatic proliferation and therefore, prevent
unwanted cells from regenerating in vivo. The frequency of
administration of the cells of the present invention 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. The precise amount of
the compositions comprising cells of the present invention to be
administered can be determined by a physician with consideration of
individual differences in age, weight, disease severity and
condition of the patient and any other factors relevant to
treatment of the patient.
[0202] In another example, blood is drawn into a stand-alone
disposable device directly from the patient that contains a
sensitizing composition and or two or more immobilized antibodies
(e.g., anti-CD3 and anti-CD28) or other components to stimulate
receptors required for T cell activation prior to the cells being
administered to the subject (e.g., immobilized on plastic surfaces
or upon separable microparticles). In one embodiment, the
disposable device may comprise a container (e.g., a plastic bag, or
flask) with appropriate tubing connections suitable for
combining/docking with syringes and sterile docking devices. This
device will contain a solid surface for immobilization of T cell
activation components (e.g., anti-CD3 and anti-CD28 antibodies);
these may be the surfaces of the container itself or an insert and
will typically be a flat surface, an etched flat surface, an
irregular surface, a porous pad, fiber, clinically acceptable/safe
ferro-fluid, beads, etc.). Additionally when using the stand-alone
device, the subject can remain connected to the device, or the
device can be separable from the patient. Further, the device may
be utilized at room temperature or incubated at physiologic
temperature using a portable incubator.
[0203] As devices and methods for collecting and processing blood
and blood products are well known, one of skill in the art would
readily recognize that given the teachings provided herein, that a
variety of devices that fulfill the needs set forth above may be
readily designed or existing devices modified. Accordingly, as such
devices and methods are not limited by the specific embodiments set
forth herein, but would include any device or methodology capable
of maintaining sterility and which maintains blood in a fluid form
in which complement activation is reduced and wherein components
necessary for T cell activation (e.g., anti-CD3 and anti-CD28
antibodies or ligands thereto) may be immobilized or separated from
the blood or blood product prior to administration to the subject.
Further, as those of ordinary skill in the art can readily
appreciate a variety of blood products can be utilized in
conjunction with the devices and methods described herein. For
example, the methods and devices could be used to provide rapid
activation of T cells from cryopreserved whole blood, peripheral
blood mononuclear cells, other cyropreserved blood-derived cells,
or cryopreserved T cell lines upon thaw and prior to subject
administration. In another example, the methods and devices can be
used to boost the activity of a previously ex vivo expanded T cell
product or T cell line prior to administration to the subject, thus
providing a highly activated T cell product. Lastly, as will be
readily appreciated the methods and devices above may be utilized
for autologous or allogeneic cell therapy simultaneously with the
subject and donor.
[0204] The methods of the present invention may also be utilized
with vaccines to enhance reactivity of the antigen and enhance in
vivo effect. In one embodiment, the compositions of the present
invention are administered to a patient in conjunction with a
composition that enhances T cells in vivo, for example, IL-2, IL-4,
IL-7, IL-10, IL-12, and IL-15. Further, given that T cells expanded
by the present invention have a relatively long half-life in the
body, these cells could act as perfect vehicles for gene therapy,
by carrying a desired nucleic acid sequence of interest and
potentially homing to sites of cancer, disease, or infection.
Accordingly, the cells expanded by the present invention may be
delivered to a patient in combination with a vaccine, one or more
cytokines, one or more therapeutic antibodies, etc. Virtually any
therapy that would benefit by a more robust T cell population is
within the context of the methods of use described herein.
[0205] A variety of in vitro and animal models exist for testing
and validating the cell compositions of the present invention and
their applicability to a particular immune system related disease
or indication. Accordingly, one of ordinary skill in the art could
easily choose the appropriate model from those currently existing
in the art. Such models include the use of NOD mice, where IDDM
results from a spontaneous T cell dependent autoimmune destruction
of insulin-producing pancreatic .beta. cells that intensifies with
age (Bottazzo et al., J. Engl. J. Med., 113:353, 1985; Miyazaki et
al., Clin. Exp. Immunol., 60:622, 1985). In NOD mice, a model of
human IDDM, therapeutic strategies that target T cells have been
successful in preventing IDDM (Makino et al., Exp. Anim., 29:1,
1980). These include neonatal thymectomy, administration of
cyclosporine, and infusion of anti-pan T cell, anti-CD4, or
anti-CD25 (IL-2R) monoclonal antibodies (mAbs) (Tarui et al.,
Insulitis and Type I Diabetes Lessons from the NOD Mouse, Academic
Press, Tokyo, p.143, 1986). Other models include, for example,
those typically utilized for autoimmune and inflammatory disease,
such as multiple sclerosis (EAE model), rheumatoid arthritis,
graft-versus-host disease (transplantation models for studying
graft rejection using skin graft, heart transplant, islet of
Langerhans transplants, large and small intestine transplants, and
the like), asthma models, systemic lupus erythematosus (systemic
autoimmunity--lpr or NZBx NZWF.sub.1 models), and the like. (see,
for example, Takakura et al., Exp. Hematol. 27(12):1815-821, 1999;
Hu et al., Immunology 98(3):379-385, 1999; Blyth et al., Am. J.
Respir. Cell Mol. Biol. 14(5):425-438, 1996; Theofilopoulos and
Dixon, Adv. Immunol. 37:269-389, 1985; Eisenberg et al., J.
Immunol. 125:1032-1036, 1980; Bonneville et al., Nature
344:163-165, 1990; Dent et al., Nature 343:714-719, 1990; Todd et
al., Nature 351:542-547, 1991; Watanabe et al., Biochem Genet.
29:325-335, 1991; Morris et al., Clin. Immunol. Immunopathol.
57:263-273, 1990; Takahashi et al., Cell 76:969-976, 1994; Current
Protocols in Immunology, Richard Coico (Ed.), John Wiley &
Sons, Inc., Chapter 15, 1998).
[0206] Collagen-induced arthritis (CIA) is a T cell dependent
animal model of rheumatoid arthritis (RA) (D. E. Trentham et al.,
"Autoimmunity to Type II Collagen: An Experimental Model of
Arthritis," J. Exp. Med. 146: 857-868 (1977)). Within two weeks
after immunization with type II collagen (CII) in IFA, susceptible
rats develop polyarthritis with histologic changes of pannus
formation and bone/cartilage erosion. In addition, humoral and
cellular responses to CII occur in CIA as well as RA (E. Brahn,
"Animal Models of Rheumatoid Arthritis: Clues to Etiology and
Treatment" in Clinical Orthopedics and Related Research (B. Hahn,
ed., Philadelphia, JB Lippincott Company, 1991). Consequently, CIA
is a useful animal model of RA that serves as an in vivo system for
the investigation of potentially new therapeutic interventions as
described in the present invention.
[0207] Pharmaceutical Compositions
[0208] T cell populations 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 EDTA or glutathione;
adjuvants (e.g., aluminum hydroxide); and preservatives.
Compositions of the present invention are preferably formulated for
intravenous administration. The present invention further provides
for pharmaceutical compositions comprising sensitizing compositions
as described herein.
[0209] 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. When "an
immunologically 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, disease
severity and condition of the patient and any other factors
relevant to treatment of the patient. It can generally be stated
that a pharmaceutical composition comprising the subject T or B
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. Cell
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.
[0210] In certain adoptive immunotherapy studies, T cells are
administered approximately at 1.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 or B cells are administered
at 1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.10, 5.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 or B cell compositions may be administered multiple
times at dosages within these ranges. The T or B cells may be
autologous or heterologous (allogeneic or xenogeneic) 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
restoration of the immune response.
[0211] The present invention also provides methods for preventing,
inhibiting, or reducing the severity of autoimmune disease in an
animal, which comprise administering to an animal an effective
amount of the subject activated polyclonal T cells that have been
cleared of undesired subpopulations of autoreactive T cells. The T
cell compositions of the present invention can be administered in
conjunction with T cell ablative therapy and/or other therapies for
the treatment of autoimmune diseases.
[0212] The present invention also provides methods for preventing,
inhibiting, or reducing the severity of graft-versus-host disease
in an animal requiring a hematopoietic stem cell transplant, which
comprise administering to an animal an effective amount of the
subject donor bone marrow that has been cleared of undesired
subpopulations of alloreactive T cells. The compositions of the
present invention can be administered in conjunction with other
therapies for the treatment of immune defects associated with
hematopoietic stem cell transplantation.
[0213] The present invention also provides methods for preventing,
inhibiting, or reducing the severity of host-versus-graft disease
or graft rejection in an animal requiring an organ transplant,
which comprise administering to an animal an effective amount of
the subject T cell compositions that has been cleared of undesired
subpopulations of alloreactive T cells. The compositions of the
present invention can be administered in conjunction with other
therapies for the treatment of immune defects associated with organ
transplantation.
[0214] One aspect of the present invention is to administer
activated and expanded T cells that proliferate and grow rapidly in
vivo. Without being bound by theory, the infused T cells may
suppress in vivo homeostatic T cell proliferation and prevent
unwanted T cells from proliferating in vivo, for example cancer
cells, autoreactive T cells, alloreactive T cells, HIV infected T
cells, and the like (see King, et al., 2004, Cell 117:265-277).
Accordingly, the compositions described herein comprising T cells
that have been cultured so as to delete at least a substantial
portion of unwanted cells can be infused into a patient. The
infused cells then prevent homeostatic proliferation and therefore,
prevent unwanted cells from regenerating in vivo.
[0215] 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,
intratumorally, or intraperitoneally. Preferably, the T cell
compositions of the present invention are administered by i.v.
injection. The compositions of activated T cells may be injected
directly into a tumor or lymph node.
[0216] 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).
[0217] The T cell and/or sensitizing composition 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 and 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,
liposomes, cells, or nanoparticles. In addition, matrices can be
designed to allow for sustained release of 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.
[0218] 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.
[0219] 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.
EXAMPLE 1
Deletion of Antigen-Specific T Cells Following Restimulation with
CD3/CD28 XCELLERATE.TM. Beads
[0220] This example describes the elimination of antigen-specific T
cells from a mixed population of cells by restimulation with anti
CD3/CD28 XCELLERATE.TM. beads (3.times.28 beads). The generation of
XCELLERATED T cells.TM. using the processes described herein is
essentially as described in U.S. patent application Ser. No.
10/133,236.
[0221] Human PBMC were screened for HLA-A2 CMVpp65 positivity by
flow cytometry using HLA-A2 tetramers loaded with CMVpp65 peptide
(HLA-A2-CMVpp65). Approximately 3% of the CD3+ CD8+T cells in the
donor selected expressed TCR specific for HLA-A2-CMVpp65 (FIG.
1).
[0222] PBMC from the donor (donor 2) and control donor (donor 1)
were activated with CMV antigen coated onto paramagnetic beads and
by day 10 of culture, many cells were shown by flow cytometric
analysis to be CD25 (IL-2R) positive, and all of the HLA-A2
CMVpp65+T cells expressed high levels of CD25, indicating
activation (FIG. 2, right panel).
[0223] At day 14 post-primary stimulation, cultures were then
either left unstimulated (FIG. 3, panels A1-A4) or were
restimulated using the XCELLERATE.TM. process with 3.times.28 beads
for 16 hours (FIG. 3, panels B1-B4). As shown in FIG. 3, CD25 is
upregulated on restimulated cells (panel B2), but tetramer-positive
(i.e., CMVpp65-Ag-specific) prestimulated cells were deleted by the
secondary strong stimulation provided by the 3.times.28 beads
(panels B3 and B4), while the other cells were unaffected. Similar
results were observed when cells were attached to beads or
associated with cells attached to beads, magnetically selected and
placed back into culture prior to restimulation with the 3.times.28
beads. In an additional study, the cells were restimulated for an
additional 4 days. Deletion of the tetramer-positive cells was
still observed after 4 additional days in the 3.times.28
restimulated cultures.
[0224] These results demonstrate that activated
CMVpp65-antigen-specific T cells that are restimulated with
3.times.28 beads are eliminated from the population of cells, most
likely through apoptosis.
EXAMPLE 2
Determination of Apoptosis
[0225] This example describes an illustrative assay for measuring
apoptosis.
[0226] DNA Fragmentation Assay: Cells are lysed in 50 .mu.l of
lysis buffer (10 mM EDTA, 50 mM Tris pH 8, 0.5% sodium dodecyl
sulfate, 0.5 mg/ml proteinase K). RNAse A (0.5 mg/ml) is added and
lysates are incubated for 1 hr. at 37.degree. C. Two phenol
extraction (equal volumes) are performed, followed by one
chloroform extraction. DNA is precipitated with two volumes of
ice-cold ethanol and incubated at -80.degree. C. for 1 hr. DNA is
pelleted by centrifugation at 14,000 rpm for 10 minutes at
4.degree. C. Pellets are air-dried for 30 minutes, resuspended in
50 .mu.l of Tris-EDTA pH 8. DNA is electrophoresed in a 1.8%
agarose gel in 1.times.TBE running buffer (0.05 M Tris base, 0.05 M
boric acid, 1 mM disodium EDTA), according to the methods of
Preston, et al., Cancer Res., 1994, 54, 4214-4223.
EXAMPLE 3
Induction of Apoptosis in B-Cells by Coculture with XCELLERATED T
Cells.TM.
[0227] This example describes the deletion of leukemic B-cells in
B-CLL patient samples by co-culture with XCELLERATED T
cells.TM..
[0228] XCELLERATED T cells.TM., generated essentially as described
in U.S. patent application Ser. No. 10/133,236, were co-cultured
with unmanipulated autologous leukemic cells from B-CLL patients.
Cell surface markers for CD54, CD80, CD95 (FAS) and CD86, and
Annexin/PI (apoptosis) were measured at 24 and 48 hours by flow
cytometry. XCELLERATED T cells.TM. were shown to drive up
expression of CD95 (FAS) on leukemic B cells (FIG. 4). After 48
hours of co-culture with day 12 XCELLERATED T cells.TM., autologous
leukemic B cells show increased expression of CD95 and sensitivity
to anti-FAS as measured by flow cytometry (FIG. 5). As shown in
FIG. 5, addition of anti-FAS antibody to co-cultured T:B cells led
to increased apoptosis in the leukemic B-cells. In an additional
study, it was shown that T cells grow whereas leukemic B-cells are
eliminated during the XCELLERATE.TM. process (FIG. 6).
[0229] In summary, XCELLERATED T cells.TM. upregulate important
effector molecules on leukemic B cells, induce functional FAS on
leukemic B-cells, and can drive leukemic B-cells into apoptosis
pathways. Leukemic B cells were virtually undetectable by the end
of the XCELLERATE.TM. process. Therefore, XCELLERATED T cells.TM.
can be used as a sensitizing composition or a pro-apoptotic
composition for the elimination of leukemic B-cells from a mixed
population of cells.
EXAMPLE 4
Varying Bead:Cell Ratios can Selectively Expand or Delete Memory
CD8 T Cells
[0230] 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) tends to induce
death in antigen-specific T cells while a lower bead:cell ratio
(1:1-1:10) 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. Further, this example demonstrates that at
high bead:cell ratios, the beads described herein can be used as
pro-apoptotic compositions.
[0231] 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 T cells.TM. 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 cryopreserved. Cells were then thawed, and placed in
culture @37.degree. C./5% CO.sub.2 for 1 hour to allow monocytes
and other adherent cells to bind to the culture plate. Non-adherent
cells were transferred to new culture plates for stimulation as
follows. 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.TM. 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.TM. 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.
[0232] For the experiment described below, cultures containing
cells for which adherent cells had been removed then have beads
added at bead:T cell ratios as shown in Table 1. The beads used in
this Example comprised the DYNABEADS.RTM. M-450 CD3/CD28 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
[0233] The results summarized in Table 1 and shown graphically in
FIG. 7 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 EBV-specific
CD8.sup.+ T cells and influenza-specific CD8.sup.+ T cells and
CD4.sup.+ T cells (not shown). Without being bound by theory, it is
thought that the antigen-specific T cells are sensitized to further
stimulation. Stimulation with high bead:cell ratios provides a high
concentration of stimulating antibody, leading to over-stimulation
of antigen-specific T cells, causing them to die, either by
apoptosis or other mechanisms. Thus, in this regard, the beads are
functioning as a pro-apoptotic composition. 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. An increase in proliferation is also
observed in the polyclonal population of T cells using lower
bead:cell ratios. In particular, the results indicate that a
bead:cell ratio of 1:1 is optimal for polyclonal T cell
expansion.
[0234] 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. If deletion of antigen-specific T
cells is the desired outcome, a higher bead:cell ratio is
preferable.
EXAMPLE 5
Deletion of Allo-Reactive T Cells Following Restimulation with
CD3/CD28 XCELLERATE.TM. Beads
[0235] This example describes the deletion of allo-reactive T cells
following restimulation with CD3/CD28 XCELLERATE.TM. beads.
[0236] PBMC were stimulated for 3 days with either allogeneic PBMC
or the JY B-lymphoblastoid allogeneic cell line. On day 3, the
allogeneic PBMC- or JY-stimulated PBMC were then cultured with
CD3/CD28 beads using the XCELLERATE.TM. process essentially as
described in U.S. patent application Ser. No. 10/350,305, with and
without 30 minute positive selection with CD3/CD28 beads. Following
the XCELLERATE.TM. process, the cells were then restimulated with
either allogeneic PBMC or JY allogeneic antigen and CD25
up-regulation was measured. Restimulation with allogeneic cells
following the XCELLERATE.TM. process did not lead to upregulation
of CD25 expression (measured using flow cytometric analysis),
indicating that the allo-reactive cells had been deleted. In
particular, positive selection of JY stimulated CD8+ T cells during
the XCELLERATE.TM. process significantly decreased allo-reactivity.
However, the T cells remained competent to respond to irrelevant
antigens in XCELLERATED.TM. cultures as demonstrated by 3rd party
allogeneic PBMC and JY responses (e.g., restimulation of
JY-stimulated culture with allogeneic PBMC or restimulation of
allo-PBMC-stimulated culture with JY).
[0237] Thus, these results show that activated allo-reactive T
cells are deleted by restimulation with CD3/CD28 beads while the
remaining polyclonal T cells can be expanded exponentially for use
in any number of immunotherapeutic applications.
EXAMPLE 6
Restoration of T Cell Repertoire in Patients with Autoimmune
Disease
[0238] T cells from patients with autoimmune disease were expanded
using the XCELLERATE.TM. process and T cell repertoire observed by
spectratype analysis.
[0239] Samples from patients with systemic lupus erythematosus,
rheumatoid arthritis, scleroderma, Crohn's disease, and psoriatic
arthritis were analyzed. Total T cell expansion in these patients
(n=9) using the XCELLERATE.TM. process was similar to that observed
in normal donors (as seen in FIG. 7) using bead:T cell ratios from
1:5 to 5:1. Further study of T cells from patients with rheumatoid
arthritis, psoriatic arthritis, and Crohn's disease using
spectratype analysis showed that a healthy T cell repertoire is
restored following the XCELLERATE.TM. process (5:1 bead:T cell
ratio) (Representative sample from a patient with rheumatoid
arthritis is shown in FIG. 8. Similar results were observed in a
patient with psoriatic arthritis and a Crohn's disease patient (not
shown)). Further analysis showed that XCELLERATED.TM. T cells from
these patients exhibit a Th1 phenotype (see FIG. 9).
[0240] In summary, in autoimmune disease patients, the
XCELLERATE.TM. process can be used to expand T cells more than one
thousand fold, restore a broad T cell repertoire and generate a Th
1-type T cell population.
EXAMPLE 7
High Bead:T Cell Ratio Deletes Autoreactive CD8+ T Cells in a Mouse
Diabetes Model
[0241] In a related experiment, the XCELLERATE.TM. process (5:1
bead:T cell ratio) was used to expand T cells in a mouse diabetes
model. Cells were expanded using the XCELLERATE.TM. process and
further analyzed using an islet-specific MHC Class I tetramer. The
results shown in FIG. 10 demonstrate that the autoreactive CD8+ T
cells were deleted.
[0242] In summary, using a pre-clinical mouse diabetes model, the
XCELLERATE.TM. process eliminated autoreactive CD8+ T cells.
[0243] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0244] 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.
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