U.S. patent application number 14/343053 was filed with the patent office on 2014-07-31 for anti third party central memory t cells, methods of producing same and use of same in transplantation and disease treatment.
This patent application is currently assigned to Yeda Research and Development Co. Ltd.. The applicant listed for this patent is Ran Afik, Esther Bachar-Lustig, Yaki Eidelstein, Assaf Lask, Eran Ophir, Noga Or-Geva, Yair Reisner. Invention is credited to Ran Afik, Esther Bachar-Lustig, Yaki Eidelstein, Assaf Lask, Eran Ophir, Noga Or-Geva, Yair Reisner.
Application Number | 20140212398 14/343053 |
Document ID | / |
Family ID | 47003163 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140212398 |
Kind Code |
A1 |
Reisner; Yair ; et
al. |
July 31, 2014 |
ANTI THIRD PARTY CENTRAL MEMORY T CELLS, METHODS OF PRODUCING SAME
AND USE OF SAME IN TRANSPLANTATION AND DISEASE TREATMENT
Abstract
A method of generating an isolated population of cells
comprising anti-third party cells having a central memory
T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing
cells and/or endowed with anti-disease activity, and capable of
homing to the lymph nodes following transplantation is disclosed.
The method comprising: (a) contacting peripheral blood mononuclear
cells (PBMC) with a third party antigen or antigens in the presence
of IL-21 so as to allow enrichment of antigen reactive cells; and
(b) culturing the cells resulting from step (a) in the presence of
IL-21, IL-15 and IL-7 in an antigen free environment so as to allow
proliferation of cells comprising the central memory T-lymphocyte
(Tcm) phenotype.
Inventors: |
Reisner; Yair; (Old Jaffa,
IL) ; Eidelstein; Yaki; (Rehovot, IL) ; Ophir;
Eran; (Rehovot, IL) ; Lask; Assaf; (Rehovot,
IL) ; Afik; Ran; (Rehovot, IL) ; Or-Geva;
Noga; (Rehovot, IL) ; Bachar-Lustig; Esther;
(Rehovot, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Reisner; Yair
Eidelstein; Yaki
Ophir; Eran
Lask; Assaf
Afik; Ran
Or-Geva; Noga
Bachar-Lustig; Esther |
Old Jaffa
Rehovot
Rehovot
Rehovot
Rehovot
Rehovot
Rehovot |
|
IL
IL
IL
IL
IL
IL
IL |
|
|
Assignee: |
Yeda Research and Development Co.
Ltd.
Rehovot
IL
|
Family ID: |
47003163 |
Appl. No.: |
14/343053 |
Filed: |
September 6, 2012 |
PCT Filed: |
September 6, 2012 |
PCT NO: |
PCT/IL2012/050354 |
371 Date: |
March 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61532172 |
Sep 8, 2011 |
|
|
|
Current U.S.
Class: |
424/93.71 ;
435/325 |
Current CPC
Class: |
A61K 35/28 20130101;
A61P 31/12 20180101; C12N 2502/1121 20130101; A61K 35/39 20130101;
A61K 2035/122 20130101; A61K 35/22 20130101; A61K 2039/5158
20130101; C12N 5/0637 20130101; A61P 35/00 20180101; C12N 5/0636
20130101; A61K 2035/124 20130101; A61P 37/02 20180101; A61K 35/17
20130101; C12N 2501/2321 20130101; A61K 35/34 20130101; A61P 35/02
20180101; A61K 35/26 20130101; A61K 35/36 20130101; A61K 35/38
20130101; A61K 39/001 20130101; C12N 2501/2315 20130101; C12N
2501/2307 20130101; A61K 35/42 20130101; A61K 35/407 20130101 |
Class at
Publication: |
424/93.71 ;
435/325 |
International
Class: |
A61K 35/14 20060101
A61K035/14; A61K 35/28 20060101 A61K035/28; A61K 35/407 20060101
A61K035/407; A61K 35/39 20060101 A61K035/39; A61K 35/26 20060101
A61K035/26; A61K 35/34 20060101 A61K035/34; A61K 35/42 20060101
A61K035/42; A61K 35/36 20060101 A61K035/36; A61K 35/38 20060101
A61K035/38; C12N 5/0783 20060101 C12N005/0783; A61K 35/22 20060101
A61K035/22 |
Claims
1. A method of generating an isolated population of cells
comprising anti-third party cells having a central memory
T-lymphocyte (Tcm) phenotype, said cells being tolerance-inducing
cells and/or endowed with anti-disease activity, and capable of
homing to the lymph nodes following transplantation, the method
comprising: (a) contacting peripheral blood mononuclear cells
(PBMC) with a third party antigen or antigens in the presence of
IL-21 so as to allow enrichment of antigen reactive cells; and (b)
culturing said cells resulting from step (a) in the presence of
IL-21, IL-15 and IL-7 in an antigen free environment so as to allow
proliferation of cells comprising said central memory T-lymphocyte
(Tcm) phenotype, thereby generating the isolated population of
cells.
2. The method of claim 1, further comprising: depleting
non-adherent cells from said PBMC prior to step (a); depleting CD4+
and/or CD56+ cells from said PBMC prior to step (a); and/or
selecting CD45RA+ and/or CD45RO- cells from said PBMC prior to step
(a).
3-5. (canceled)
6. The method of claim 1, further comprising culturing said cells
resulting from step (a) with a third party antigen or antigens in
the presence of IL-21, IL-15 and IL-7 following step (a) and prior
to step (b).
7-10. (canceled)
11. A method of generating an isolated population of cells
comprising anti-third party cells having a central memory
T-lymphocyte (Tcm) phenotype, said cells being tolerance-inducing
cells and/or endowed with graft-versus-leukemia (GVL) activity, and
capable of homing to the lymph nodes following transplantation, the
method comprising: (a) treating non-adherent peripheral blood
mononuclear cells (PBMC) with an agent capable of depleting CD4+
and/or CD56+ cells so as to obtain CD8+ T cells; (b) contacting
said CD8+ T cells with third party dendritic cells in the presence
of IL-21 for 12 hours to 5 days so as to allow enrichment of
antigen reactive cells; (c) culturing said cells resulting from
step (b) with said third party dendritic cells in the presence of
IL-21, IL-15 and IL-7 for 12 hours to 3 days; and (d) culturing
said cells resulting from step (c) in the presence of IL-21, IL-15
and IL-7 in an antigen free environment for 5-20 days so as to
allow proliferation of cells comprising said central memory
T-lymphocyte (Tcm) phenotype, thereby generating the isolated
population of cells.
12. A method of generating an isolated population of cells
comprising anti-third party cells having a central memory
T-lymphocyte (Tcm) phenotype, said cells being endowed with
anti-disease activity, and capable of homing to the lymph nodes
following transplantation, the method comprising: (a) treating
non-adherent peripheral blood mononuclear cells (PBMC) with an
agent capable of depleting CD4+ and/or CD56+ cells so as to obtain
CD8+ T cells; (b) contacting said CD8+ T cells with non-syngeneic
dendritic cells in the presence of IL-21 for 12 hours to 5 days so
as to allow enrichment of antigen reactive cells; (c) culturing
said cells resulting from step (b) with said non-syngeneic
dendritic cells in the presence of IL-21, IL-15 and IL-7 for 12
hours to 3 days; and (d) culturing said cells resulting from step
(c) in the presence of IL-21, IL-15 and IL-7 in an antigen free
environment for 5-20 days so as to allow proliferation of cells
comprising said central memory T-lymphocyte (Tcm) phenotype,
thereby generating the isolated population of cells.
13. The method of claim 12, further comprising selecting CD45RA+
and/or CD45RO- cells from said PBMC following step (a) and prior to
step (b).
14-16. (canceled)
17. The method of claim 1, wherein said contacting in said presence
of IL-21 is effected for 12 hours to 5 days.
18-19. (canceled)
20. The method of claim 1, further comprising: selecting for
activated cells following step (a) and prior to step (b); or
depleting alloreactive cells following step (b).
21. The method of claim 11, further comprising: selecting CD45RA+
and/or CD45RO- cells from said PBMC following step (a) and prior to
step (b); or selecting for activated cells following step (b) and
prior to step (c); or depleting alloreactive cells following step
(d).
22-24. (canceled)
25. The method of claim 1, wherein said culturing in said presence
of IL-21, IL-15 and IL-7 in said antigen free environment is
effected for 5-20 days.
26-32. (canceled)
33. The method of claim 1, wherein said anti-third party cells
having a T central memory phenotype comprises a CD3.sup.+,
CD8.sup.+, CD62L.sup.+, CD45RA.sup.-, CD45RO.sup.+ signature.
34. The method of claim 33, wherein at least 50% of the isolated
population of cells are CD3+CD8+ cells of which at least 50% have
said signature.
35. An isolated population of cells comprising anti-third party
cells having a central memory T-lymphocyte (Tcm) phenotype, wherein
at least 50% of the isolated population of cells are CD3+CD8+ cells
of which at least 50% comprise a CD3.sup.+, CD8.sup.+, CD62L.sup.+,
CD45RA.sup.-, CD45RO.sup.+ signature, and further wherein said
cells are tolerance-inducing cells and/or endowed with anti-disease
activity, and capable of homing to the lymph nodes following
transplantation.
36. An isolated population of cells comprising anti-third party
cells having a central memory T-lymphocyte (Tcm) phenotype, said
cells being tolerance-inducing cells and/or endowed with
anti-disease activity, and capable of homing to the lymph nodes
following transplantation, generated according to the method of
claim 1.
37. A method of treating a disease in a subject in need thereof,
wherein the disease is selected from the group consisting of a
malignant disease, a viral disease and an autoimmune disease, the
method comprising administering to the subject a therapeutically
effective amount of the isolated population of cells of claim 36,
thereby treating the subject.
38-40. (canceled)
41. A method of treating a subject in need of a cell or tissue
transplantation, the method comprising: (a) transplanting a cell or
tissue transplant into the subject; and (b) administering to the
subject a therapeutically effective amount of the isolated
population of cells of claim 36, thereby treating the subject.
42-44. (canceled)
45. The method of claim 41, wherein said cell or tissue transplant
comprises immature hematopoietic cells.
46. The method of claim 41, wherein said cell or tissue transplant
is selected from the group consisting of a liver, a pancreas, a
spleen, a kidney, a heart, a lung, a skin, an intestine and a
lymphoid/hematopoietic tissue or organ.
47. The method of claim 41, wherein said cell or tissue transplant
comprises a co-transplantation of several organs.
48. The method of claim 47, wherein said co-transplantation
comprises transplantation of immature hematopoietic cells and a
solid organ.
49. The method of claim 48, wherein said immature hematopoietic
cells and said solid organ are obtained from the same donor.
50. The method of claim 48, wherein said immature hematopoietic
cells are transplanted prior to, concomitantly with, or following
said transplantation of said solid organ.
51. The method of claim 41, wherein said isolated population of
cells are administered prior to, concomitantly with, or following
said cell or tissue transplant.
52-53. (canceled)
54. The method of claim 41, wherein said cell or tissue transplant
and said isolated population of cells are derived from the same
donor.
55. The method of claim 41, wherein said cell or tissue transplant
is syngeneic with the subject and said isolated population of cells
are non-syngeneic with the subject.
56. The method of claim 41, wherein said cell or tissue transplant
is syngeneic with the subject and said isolated population of cells
are syngeneic with the subject.
57. A method of treating a subject in need of an immature
hematopoietic cell transplantation, the method comprising: (a)
transplanting immature hematopoietic cells into the subject; and
(b) administering to the subject a therapeutically effective amount
of the isolated population of cells of claim 36, thereby treating
the subject.
58. The method of claim 57, wherein said isolated population of
cells are administered prior to, concomitantly with, or following
said immature hematopoietic cells.
59. The method of claim 57, wherein said immature hematopoietic
cells and said isolated population of cells are derived from the
same donor.
60. (canceled)
61. The method of claim 57, wherein said immature hematopoietic
cells and said isolated population of cells are derived from the
subject.
62-63. (canceled)
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] The present invention, in some embodiments thereof, relates
to tolerance inducing and/or graft versus leukemia reactive
anti-third party cells comprising central memory T-lymphocyte
phenotype and, more particularly, but not exclusively, to methods
of generating same and to the use of same in transplantation and in
disease treatment.
[0002] Bone marrow (BM) transplantation offers a curative treatment
for many patients with hematological malignancies and other
hematological disorders. However, the BM graft contains donor T
cells which respond to the host antigens (Ags) and cause
multi-system graft-versus-host disease (GVHD). In the early 80's
bone marrow transplant (BMT), without the deleterious effect of
GVHD, was demonstrated in the haploidentical (three HLA loci
mismatched) settings, in severe combined immunodeficiency (SCID)
patients. The problem of GVHD, which is almost uniformly lethal in
such settings, was completely prevented by T cell depletion.
[0003] However, in leukemia patients, the clinical outcome of T
cell depleted BM was disappointing, as the benefit of GVHD
prevention was offset by a markedly increased rate of graft
rejection. The rejection was shown to be mediated by
radiochemotherapy resistant host derived T cells [Reisner et al.,
Proc Natl Acad Sci USA. (1986) 83:4012-4015]. One way to overcome
this problem is to perform BMT following supra-lethal conditioning
and functional inactivation of host T cells using immunosuppressive
drugs. Nevertheless, this strategy is hampered by opportunistic
infections due to slow immune reconstitution and considerable
toxicities of the immunosuppressants.
[0004] While in high risk leukemia patients such transplant-related
mortality can be acceptable, it would be intolerable if applied to
patients with a long life expectancy. Therefore, the use of reduced
intensity conditioning, with less severe immune ablation, to enable
engraftment of T-depleted BM (TDBM) graft, which is associated with
reduced risk for GVHD, is warranted. The establishment of donor
type chimerism under such reduced conditioning represents a most
desirable goal in transplantation biology, as it is generally
associated with durable tolerance towards cells or tissues from the
original donor. Yet, the marked levels of host immune cells
surviving the mild preparatory regimens, represents a difficult
barrier for the engraftment of donor cells.
[0005] One approach to overcome rejection of allogeneic TDBM made
use of large cell doses. It was first demonstrated in rodent models
that a "megadose" of TDBM transplant can overcome T cell mediated
graft rejection [Lapidot et al., Blood (1989) 73:2025-2032;
Bachar-Lustig et al., Nat. Med. (1995) 1:1268-1273; Uharek et al.,
Blood (1992) 79:1612-1621]. However, a significant increase in the
BM inoculum has been difficult to achieve in humans. To overcome
this problem granulocytes colony stimulating factor (G-CSF), which
facilitates mobilization of hematopoietic stem cells (HSCs, CD34+
cells in humans) from the BM, has been used to increase the yield
of HSCs collected from the blood and T cell depleted HSCs were
supplemented to the conventional TDBM [Aversa et al., N Engl J.
Med. (1998) 339:1186-1193; Aversa et al., J Clin Oncol. (2005)
23:3447-3454; Reisner and Martelli, Immunol Today (1999)
20:343-347; Handgretinger et al., Bone Marrow Transplant. (2001)
27:777-783].
[0006] The CD34 "megadose" transplants raised interesting questions
as to how these cells overcome the barrier presented by host
cytotoxic T-lymphocyte precursors (CTL-p). This question was
answered, in part, by the finding that cells within the CD34
fraction are endowed with potent veto activity [Gur et al., Blood
(2005) 105:2585-2593; Gur et al., Blood (2002) 99:4174-4181;
Rachamim et al., Transplantation (1998) 65:1386-1393]. Other cell
types have also been shown to mediate veto activity including T
lymphocytes (e.g. CD8.sup.+ CTLs), natural killer cells and
dendritic cells. Direct comparison of the veto reactivity of
various cell types revealed that CTLs comprise the strongest veto
effect [Reich-Zeliger et al., J. Immunol. (2004)
173:6654-6659].
[0007] One approach developed to generate veto CTLs without GVH
reactivity was described by Reisner and co-workers, in which CTLs
were stimulated against 3.sup.rd-party stimulators in the absence
of exogenous IL-2. This approach was based on the observation that
only activated CTLp were capable of surviving the IL-2 deprivation
in the primary culture. This method was shown in vitro and in vivo
to deplete GVH reactivity from the anti-3.sup.rd party veto CTLs
[PCT Publication No. WO 2001/049243, Bachar-Lustig et al., Blood.
2003; 102:1943-1950; Aviner et al., Hum Immunol. (2005)
66:644-652]. Introduction of these anti-3.sup.rd party veto CTLs
into a recipient (along with a transplant) prevented graft
rejection without inducing GVHD (PCT Publication No. WO
2001/049243).
[0008] Various approaches have been contemplated for graft
transplantation without graft rejection and/or graft versus host
disease, some are summarized infra.
[0009] PCT Publication No. WO 2007/023491 discloses the use of
tolerogenic cells for reducing or preventing graft rejection of a
non-syngeneic graft in a subject. The tolerogenic cells disclosed
(e.g. CD4.sup.+CD25.sup.+ cells) may be derived from any donor who
is non-syngeneic with both the subject and the graft ("third-party"
tolerogenic cells). The graft (e.g. bone marrow) may be derived
from any graft donor who is allogeneic or xenogeneic with the
subject.
[0010] PCT Publication No. WO 2002/102971 discloses the use of
cultured hematopoietic progenitor cells (HPC) comprising enhanced
veto activity for inducing tolerance to a transplant transplanted
from a donor to a recipient. The tolerogenic cells disclosed
preferably express CD33 and are administered prior to,
concomitantly with or following transplantation of the transplant
(e.g. cell or organ transplant).
[0011] PCT Publication No. WO 2002/043651 discloses the use of a
non-GVHD inducing population of immune effector cells for disease
treatment. In order to arrive at the non-GVHD inducing population
of immune effector cells, a first cell population (e.g.
T-lymphocytes) are co-cultured with a second cell population being
non-syngeneic with the subject and non-syngeneic with the first
cell population (e.g. EBV-infected B-lymphocytes) under conditions
which include IL-2 starvation followed by IL-2 supplementation. The
resultant immune effector cells may be used to treat diseases such
as malignant diseases, viral diseases and autoimmune diseases.
[0012] U.S. Pat. No. 6,759,035 discloses methods of inhibiting
graft rejection and inducing T cell tolerance in a solid organ
transplant recipient. The methods disclosed comprise removing
peripheral blood mononuclear cells (PBMC) from a donor and
recipient, culturing the donor and recipient cells together in the
presence of a compound that induces T cell suppressor activity
(e.g. TGF-.beta., IL-15 and IL-2), and administering the recipient
suppressor T cells to the recipient along with the transplant to
prevent the recipient's T cells from killing donor cells, thereby
inducing tolerance and long term survival of the transplant.
[0013] U.S. Pat. No. 6,803,036 discloses methods for treating donor
cells to ameliorate graft versus host disease in a recipient
patient. The methods disclosed comprise removing PBMCs from a donor
and treating the cells with a suppressive composition (e.g. IL-10,
IL-2, IL-4, IL-15 and TGF-.beta.) for a time sufficient to induce T
cell tolerance. The cells are then introduced to a recipient
patient. The treated cells may be added to donor stem cells prior
to introduction into the patient.
[0014] PCT Publication No. WO 2010/049935 disclosed an isolated
population of cells comprising non-GVHD inducing anti-third party
cells having a central memory T-lymphocyte (Tcm) phenotype, the
cells being tolerance-inducing cells and capable of homing to the
lymph nodes following transplantation.
SUMMARY OF THE INVENTION
[0015] According to an aspect of some embodiments of the present
invention there is provided a method of generating an isolated
population of cells comprising anti-third party cells having a
central memory T-lymphocyte (Tcm) phenotype, the cells being
tolerance-inducing cells and/or endowed with anti-disease activity,
and capable of homing to the lymph nodes following transplantation,
the method comprising: (a) contacting peripheral blood mononuclear
cells (PBMC) with a third party antigen or antigens in the presence
of IL-21 so as to allow enrichment of antigen reactive cells; and
(b) culturing the cells resulting from step (a) in the presence of
IL-21, IL-15 and IL-7 in an antigen free environment so as to allow
proliferation of cells comprising the central memory T-lymphocyte
(Tcm) phenotype, thereby generating the isolated population of
cells.
[0016] According to an aspect of some embodiments of the present
invention there is provided a method of generating an isolated
population of cells comprising anti-third party cells having a
central memory T-lymphocyte (Tcm) phenotype, the cells being
tolerance-inducing cells and/or endowed with graft-versus-leukemia
(GVL) activity, and capable of homing to the lymph nodes following
transplantation, the method comprising: (a) treating non-adherent
peripheral blood mononuclear cells (PBMC) with an agent capable of
depleting CD4+ and/or CD56+ cells so as to obtain CD8+ T cells; (b)
contacting the CD8+ T cells with third party dendritic cells in the
presence of IL-21 for 12 hours to 5 days so as to allow enrichment
of antigen reactive cells; (c) culturing the cells resulting from
step (b) with the third party dendritic cells in the presence of
IL-21, IL-15 and IL-7 for 12 hours to 3 days; and (d) culturing the
cells resulting from step (c) in the presence of IL-21, IL-15 and
IL-7 in an antigen free environment for 5-20 days so as to allow
proliferation of cells comprising the central memory T-lymphocyte
(Tcm) phenotype, thereby generating the isolated population of
cells.
[0017] According to an aspect of some embodiments of the present
invention there is provided a method of generating an isolated
population of cells comprising anti-third party cells having a
central memory T-lymphocyte (Tcm) phenotype, the cells being
endowed with anti-disease activity, and capable of homing to the
lymph nodes following transplantation, the method comprising: (a)
treating non-adherent peripheral blood mononuclear cells (PBMC)
with an agent capable of depleting CD4+ and/or CD56+ cells so as to
obtain CD8+ T cells; (b) contacting the CD8+ T cells with
non-syngeneic dendritic cells in the presence of IL-21 for 12 hours
to 5 days so as to allow enrichment of antigen reactive cells; (c)
culturing the cells resulting from step (b) with the non-syngeneic
dendritic cells in the presence of IL-21, IL-15 and IL-7 for 12
hours to 3 days; and (d) culturing the cells resulting from step
(c) in the presence of IL-21, IL-15 and IL-7 in an antigen free
environment for 5-20 days so as to allow proliferation of cells
comprising the central memory T-lymphocyte (Tcm) phenotype, thereby
generating the isolated population of cells.
[0018] According to an aspect of some embodiments of the present
invention there is provided an isolated population of cells
comprising anti-third party cells having a central memory
T-lymphocyte (Tcm) phenotype, wherein at least 50% of the isolated
population of cells are CD3+CD8+ cells of which at least 50%
comprise a CD3.sup.+, CD8.sup.+, CD62L.sup.+, CD45RA.sup.-,
CD45RO.sup.+ signature, and further wherein the cells are
tolerance-inducing cells and/or endowed with anti-disease activity,
and capable of homing to the lymph nodes following
transplantation.
[0019] According to an aspect of some embodiments of the present
invention there is provided an isolated population of cells
comprising anti-third party cells having a central memory
T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing
cells and/or endowed with anti-disease activity, and capable of
homing to the lymph nodes following transplantation, generated
according to the present methods.
[0020] According to an aspect of some embodiments of the present
invention there is provided a method of treating a disease in a
subject in need thereof, wherein the disease is selected from the
group consisting of a malignant disease, a viral disease and an
autoimmune disease, the method comprising administering to the
subject a therapeutically effective amount of the isolated
population of cells of the present invention, thereby treating the
subject.
[0021] According to an aspect of some embodiments of the present
invention there is provided a method of treating a subject in need
of a cell or tissue transplantation, the method comprising: (a)
transplanting a cell or tissue transplant into the subject; and (b)
administering to the subject a therapeutically effective amount of
the isolated population of cells of the present invention, thereby
treating the subject.
[0022] According to an aspect of some embodiments of the present
invention there is provided a method of treating a subject in need
of an immature hematopoietic cell transplantation, the method
comprising: (a) transplanting immature hematopoietic cells into the
subject; and (b) administering to the subject a therapeutically
effective amount of the isolated population of cells of the present
invention, thereby treating the subject.
[0023] According to some embodiments of the invention, the method
further comprises depleting non-adherent cells from the PBMC prior
to step (a).
[0024] According to some embodiments of the invention, the method
further comprises depleting CD4+ and/or CD56+ cells from the PBMC
prior to step (a).
[0025] According to some embodiments of the invention, the method
further comprises selecting CD45RA+ and/or CD45RO- cells from the
PBMC prior to step (a).
[0026] According to some embodiments of the invention, the PBMC
comprise CD8+ T cells.
[0027] According to some embodiments of the invention, the method
further comprises culturing the cells resulting from step (a) with
a third party antigen or antigens in the presence of IL-21, IL-15
and IL-7 following step (a) and prior to step (b).
[0028] According to some embodiments of the invention, the third
party antigen or antigens comprise dendritic cells.
[0029] According to some embodiments of the invention, the
dendritic cells are irradiated dendritic cells.
[0030] According to some embodiments of the invention, the third
party antigen or antigens is selected from the group consisting of
third party cells, a cell antigen, a viral antigen, a bacterial
antigen, a protein extract, a purified protein and a synthetic
peptide presented by autologous presenting cells, non-autologous
presenting cells or on an artificial vehicle or on artificial
antigen presenting cells.
[0031] According to some embodiments of the invention, the third
party cells are stimulatory cells selected from the group
consisting of cells purified from peripheral blood lymphocytes,
spleen or lymph nodes, cytokine-mobilized PBLs, in vitro expanded
antigen-presenting cells (APC), in vitro expanded dendritic cells
and artificial antigen presenting cells.
[0032] According to some embodiments of the invention, the method
further comprises selecting CD45RA+ and/or CD45RO- cells from the
PBMC following step (a) and prior to step (b).
[0033] According to some embodiments of the invention, the CD8+ T
cells comprise naive CD8+ T cells.
[0034] According to some embodiments of the invention, the
dendritic cells comprise in vitro expanded dendritic cells.
[0035] According to some embodiments of the invention, the
dendritic cells comprise irradiated dendritic cells.
[0036] According to some embodiments of the invention, the
contacting in the presence of IL-21 is effected for 12 hours to 5
days.
[0037] According to some embodiments of the invention, the
contacting in the presence of IL-21 is effected for 2-3 days.
[0038] According to some embodiments of the invention, the
contacting in the presence of IL-21 is effected for 3 days.
[0039] According to some embodiments of the invention, the method
further comprises selecting for activated cells following step (a)
and prior to step (b).
[0040] According to some embodiments of the invention, the method
further comprises selecting for activated cells following step (b)
and prior to step (c).
[0041] According to some embodiments of the invention, the
selecting for activated cells is effected by selection of CD137+
and/or CD25+ cells.
[0042] According to some embodiments of the invention, the
selecting for activated cells is effected 12-72 hours after the
contacting.
[0043] According to some embodiments of the invention, the
culturing with the third party antigen or antigens in the presence
of IL-21, IL-15 and IL-7 is effected for 12 hours to 3 days.
[0044] According to some embodiments of the invention, the presence
of IL-21, IL-15 and IL-7 in the antigen free environment is
effected for 5-20 days.
[0045] According to some embodiments of the invention, the
culturing in the presence of IL-21, IL-15 and IL-7 in the antigen
free environment is effected for 7-11 days.
[0046] According to some embodiments of the invention, the method
further comprises depleting alloreactive cells following step
(b).
[0047] According to some embodiments of the invention, the method
further comprises depleting alloreactive cells following step
(d).
[0048] According to some embodiments of the invention, the
depleting the alloreactive cells is effected by depletion of CD137+
and/or CD25+ cells following contacting the cells comprising the
central memory T-lymphocyte (Tcm) with host antigen presenting
cells (APCs).
[0049] According to some embodiments of the invention, the
peripheral blood mononuclear cells (PBMC) are syngeneic with
respect to a subject.
[0050] According to some embodiments of the invention, the
peripheral blood mononuclear cells (PBMC) are non-syngeneic with
respect to a subject.
[0051] According to some embodiments of the invention, the
non-syngeneic PBMC are xenogeneic or allogeneic with respect to a
subject.
[0052] According to some embodiments of the invention, the
anti-third party cells having a T central memory phenotype
comprises a CD3.sup.+, CD8.sup.+, CD62L.sup.+, CD45RA.sup.-,
CD45RO.sup.+ signature.
[0053] According to some embodiments of the invention, at least 50%
of the isolated population of cells are CD3+CD8+ cells of which at
least 50% have the signature.
[0054] According to some embodiments of the invention, the
malignant disease comprises a leukemia or a lymphoma.
[0055] According to some embodiments of the invention, the isolated
population of cells are syngeneic with the subject.
[0056] According to some embodiments of the invention, the isolated
population of cells are non-syngeneic with the subject.
[0057] According to some embodiments of the invention, the method
further comprises conditioning the subject under sublethal, lethal
or supralethal conditions prior to the transplanting.
[0058] According to some embodiments of the invention, the cell or
tissue transplant is syngeneic with the subject.
[0059] According to some embodiments of the invention, the cell or
tissue transplant is derived from a donor selected from the group
consisting of an HLA identical allogeneic donor, an HLA
non-identical allogeneic donor and a xenogeneic donor.
[0060] According to some embodiments of the invention, the cell or
tissue transplant comprises immature hematopoietic cells.
[0061] According to some embodiments of the invention, the cell or
tissue transplant is selected from the group consisting of a liver,
a pancreas, a spleen, a kidney, a heart, a lung, a skin, an
intestine and a lymphoid/hematopoietic tissue or organ.
[0062] According to some embodiments of the invention, the cell or
tissue transplant comprises a co-transplantation of several
organs.
[0063] According to some embodiments of the invention, the
co-transplantation comprises transplantation of immature
hematopoietic cells and a solid organ.
[0064] According to some embodiments of the invention, the immature
hematopoietic cells and the solid organ or obtained from the same
donor.
[0065] According to some embodiments of the invention, the immature
hematopoietic cells are transplanted prior to, concomitantly with,
or following the transplantation of the solid organ
[0066] According to some embodiments of the invention, the isolated
population of cells are administered prior to, concomitantly with,
or following the cell or tissue transplant.
[0067] According to some embodiments of the invention, the isolated
population of cells are syngeneic with the subject.
[0068] According to some embodiments of the invention, the isolated
population of cells are non-syngeneic with the subject.
[0069] According to some embodiments of the invention, the cell or
tissue transplant and the isolated population of cells are derived
from the same donor.
[0070] According to some embodiments of the invention, the cell or
tissue transplant is syngeneic with the subject and the isolated
population of cells are non-syngeneic with the subject.
[0071] According to some embodiments of the invention, the cell or
tissue transplant is syngeneic with the subject and the isolated
population of cells are syngeneic with the subject.
[0072] According to some embodiments of the invention, the isolated
population of cells are administered prior to, concomitantly with,
or following the immature hematopoietic cells.
[0073] According to some embodiments of the invention, the immature
hematopoietic cells and the isolated population of cells are
derived from the same donor.
[0074] According to some embodiments of the invention, the donor is
non-syngeneic with the subject.
[0075] According to some embodiments of the invention, the immature
hematopoietic cells and the isolated population of cells are
derived from the subject.
[0076] According to some embodiments of the invention, the method
further comprises conditioning the subject under sublethal, lethal
or supralethal conditions prior to the transplanting.
[0077] According to some embodiments of the invention, the subject
is a human subject.
[0078] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0079] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0080] In the drawings:
[0081] FIGS. 1A-B are schematic diagrams depicting human autologous
(FIG. 1A) and allogeneic (FIG. 1B) settings. Of note, the two
settings differ from each other in the origin of the bone marrow
(BM) donor (host vs. allogeneic), the responders (host vs.
allogeneic) and stimulators (any allogeneic donor vs. 3.sup.rd
party not cross-reactive with host MHC) that are involved in the
Tcm generation.
[0082] FIGS. 2A-B are schematic diagrams depicting mouse syngeneic
(FIG. 2A) and allogeneic (FIG. 2B) settings. Of note, the two
settings differ from each other in the origin of the BM donor
(syngeneic or F1 vs. allogeneic), the responders (syngeneic or F1
vs. allogeneic) and stimulators (allogeneic vs. 3.sup.rd party)
that are involved in the Tcm generation.
[0083] FIGS. 3A-B are schematic diagrams depicting the autologous
human protocol for generation of Tcm (FIG. 3A) in comparison to the
syngeneic mouse protocol (FIG. 3B).
[0084] FIGS. 4A-C depict the kinetics of anti-3.sup.rd party
central memory generation ("reference control experiments"). Naive
CD8 T cells were stimulated with irradiated allogeneic 3rd party DC
at a ratio of 4:1 in a medium containing IL-21 for 3 days.
Thereafter, the cells received no further activation and were
expanded in medium containing IL-7 and IL-15 until day 12.5. On
days 7.5, 10.5 and 12.5, cells were evaluated for phenotype
(surface marker expression) (FIG. 4A), and percentage of Tcm
(CD62L+CD45RO+) from CD8 T cells using FACS analysis (FIG. 4B), and
for cell numbers by trypan blue exclusion (FIG. 4C). For each time
point data represent average.+-.SE of n independent
experiments.
[0085] FIGS. 5A-C depict a typical experiment demonstrating the
role of priming with allogeneic DC. Of note, IL-21 alone or IL-7
plus IL-15 without DC priming does not induce central memory
phenotype in naive CD8 T cells and poorly supports their expansion.
FIG. 5A illustrates naive CD8 T cells which were stimulated with
irradiated allogeneic 3rd party DC at a ratio of 4:1 in a medium
containing IL-21 for 3 days. Thereafter, the cells received no
further activation and were expanded with IL-7 and IL-15 until day
13 ("reference control group"=d(0-3) IL21+DC d(3-13)IL7+IL15);
FIGS. 5B-C illustrate naive CD8 T cells which were cultured with
IL-21 (FIG. 5B) or with a combination of IL-7 and IL-15 (FIG. 5C)
in the absence of stimulation until day 10 or day 13,
respectively.
[0086] FIGS. 6A-B depict the role of priming with allogeneic DC
demonstrated by the average relative impact on Tcm level and fold
expansion compared to the reference control group. Naive CD8 T
cells were stimulated with irradiated allogeneic 3rd party DC at a
ratio of 4:1 in a medium containing IL-21 for 3 days. Thereafter,
the cells received no further activation and were expanded with
IL-7 and IL-15 until day 13 ("Reference control group"=d(0-3)
IL21+DC d(3-13)IL7+IL15). Alternatively, naive CD8 T cells were
cultured with IL-21 or with combination of IL-7 and IL-15 in the
absence of stimulation until day 13. Cells were evaluated for cell
numbers by trypan blue exclusion (FIG. 6A), and percentage of Tcm
(CD62L+CD45RO+) from CD8 T cells using FACS analysis (FIG. 6B). For
each time point, data represent the average.+-.SE of n independent
experiments.
[0087] FIGS. 7A-C depict a typical experiment demonstrating the
role of IL-21 in the priming and expansion phases of anti-3rd party
Tcm. Of note, removal of IL-21 from the priming phase reduced both
expansion and Tcm induction, while the presence of IL-21 throughout
the culture increased Tcm induction. FIG. 7A illustrates naive CD8
T cells which were stimulated with irradiated allogeneic 3rd party
DC at a ratio of 4:1 in a medium containing IL-21 for 3 days.
Thereafter, the cells received no further activation and were
expanded with IL-7 and IL-15 until day 13 ("reference control
group"=d(0-3) IL21+DC d(3-13)IL7+IL15); FIGS. 7B-C illustrate naive
CD8 T cells which were stimulated with irradiated allogeneic 3rd
party DC at a ratio of 4:1 in the absence of IL-21 for 3 days.
Thereafter the cells received no further activation and were
expanded with IL-7 and IL-15 until day 13 (FIG. 7B), or stimulated
with irradiated allogeneic 3rd party DC at a ratio of 4:1 with
continuous presence of IL-21 in both the priming phase (IL-21
alone) and in the expansion phase (together with IL-7 and IL-15)
(FIG. 7C).
[0088] FIGS. 8A-B depict the requirement for IL-21 for optimal Tcm
yield (average of several independent experiments). Naive CD8 T
cells were treated as above and cultures were evaluated for cell
number by trypan blue exclusion (FIG. 8A), and percentage of Tcm
(CD62L+CD45RO+) from CD8 T cells using FACS analysis (FIG. 8B).
Results of each experiment are shown separately and lines indicate
average results over n experiments.
[0089] FIGS. 9A-B depict the optimal responder/DC ratio for the
induction of Tcm phenotype and robust expansion. 4.times.10.sup.5
naive CD8 T cells were stimulated against irradiated allogeneic 3rd
party DC at increasing numbers in the presence of IL-21 for 3 days.
Thereafter the cells received no further activation and were
expanded with IL-7 and IL-15 until day 13 ("reference control
group"=d(0-3) IL21+100,00 DC d(3-13) IL7+IL15). Cultures were
evaluated for cell numbers by trypan blue exclusion (FIG. 9A), and
percentage of Tcm (CD62L+CD45RO+) from CD8 T cells using FACS
analysis (FIG. 9B). Results of each experiment are shown separately
and Lines indicate average results over n experiments.
[0090] FIG. 10 depicts an evaluation of the effect of different GMP
grade reagents on the enrichment of CD8+ and naive CD8+CD45RA+ T
cells. Donor PBMC were depleted from adherent cells by overnight
incubation in plates specifically designed to remove adherent
myeloid cells (upper panel), and on day 0, non adherent cells were
divided to four test groups, each subjected to a different magnetic
sorting protocols. Cells were evaluated for cell composition and
Tcm phenotype by FACS analysis. The results in the left column
(CD45RO and CD45RA) are gated on CD3+CD8+ cells. The results
represent a typical experiment out of two independent experiments
performed.
[0091] FIG. 11 depicts a typical experiment showing the effect of
different GMP grade reagents used for isolation of CD8 T cells, on
the proportion of CD8+ T cells with a Tcm phenotype, and
contamination with NK and NKT cells, 7 days after stimulation with
3.sup.rd party DCs. Unstimulated cells maintained in culture with
IL-7 alone (upper panel) were used as a reference. The results in
the left most column (CD45RO and CD45RA) and right most column
(CD62L and CD45RO) are gated on CD3+CD8+ cells.
[0092] FIG. 12 depicts a typical experiment showing the effect of
different GMP grade reagents used for isolation of CD8 T cells, on
the proportion of CD8+ T cells with a Tcm phenotype, and
contamination with NK and NKT cells, 14 days after stimulation with
3.sup.rd party DCs. Unstimulated cells maintained in culture with
IL-7 alone (upper panel) were used as a reference. The results in
the left most column (CD45RO and CD45RA) and right most column
(CD62L and CD45RO) are gated on CD3+CD8+ cells.
[0093] FIGS. 13A-B depict the effect of different GMP grade
reagents used for isolation of CD8 T cells on the levels of CD8 T
cells with a Tcm phenotype after 7 days of stimulation against 3rd
party DCs. Average percent of CD3+CD8+ NKT- T cells (FIG. 13A) and
Tcm (FIG. 13B) are shown as percent of the levels attained in the
optimal control group making use of all 4 reagents
(CD4/CD56/CD19/CD45RA).
[0094] FIGS. 14A-C depict the effect of different GMP grade
reagents used for isolation of CD8 T cells on the final yield of
CD8 T cells with a Tcm phenotype after 10 days of stimulation
against 3rd party DCs. Average fold expansion from day 0 at day 10
(FIG. 14A), and average yield after magnetic sorting (FIG. 14B) are
shown as percent of the levels attained in the optimal control
group making use of all four selection reagents
(CD4/CD56/CD19/CD45RA). The yield of Tcm at day 10 (FIG. 14C) was
calculated by multiplication of the yield after magnetic sorting
(at day 0) with the fold expansion from day 0 (at day 10).
[0095] FIG. 15 depicts that changing the source for allogeneic DC
stimulators had only minor effect on the expansion potential of the
Tcm cells. CD8 T cells were enriched from thawed leukapheresis by
depletion of CD4+ and CD56+ cells using the CliniMacs system. The
enriched CD8 T cells were then divided into two test groups, each
stimulated with different irradiated allogeneic 3rd party DC, at a
ratio of 6:1 in a medium containing IL-21 for 3 days in culture
bags. Thereafter, the cells received no further activation and were
expanded in medium containing IL-7, IL-15 and IL-21 until day 11.
On days 5, 7, 9 and 11 of culture cell numbers was determined by
trypan blue exclusion.
[0096] FIGS. 16A-B depict that changing the source for allogeneic
DC stimulators had only minor effect on the cell composition. CD8 T
cells were enriched from thawed leukapheresis by depletion of CD4+
and CD56+ cells using the CliniMacs system for large scale
isolation. The enriched CD8 T cells were then divided into two test
groups, each stimulated with different irradiated allogeneic 3rd
party DC (FIGS. 16A and 16B, respectively), at a ratio of 6:1 in a
medium containing IL-21 for 3 days in culture bags. Thereafter, the
cells received no further activation and were expanded in medium
containing IL-7, IL-15 and IL-21 until day 11. On days 0, 5, 9 and
12 of culture cells were evaluated for cell composition by FACS
analysis. All the results are gated from lymphogate and live gate
(7AAD-).
[0097] FIGS. 17A-B depict that changing the source for allogeneic
DC stimulators had only minor effect on the cell composition. CD8 T
cells were enriched from thawed leukapheresis by depletion of CD4+
and CD56+ Cells using the CliniMacs system. The enriched CD8 T
cells were then divided into two test groups, each stimulated with
different irradiated allogeneic 3rd party DC, at a ratio of 6:1 in
a medium containing IL-21 for 3 days in culture bags. Thereafter,
the cells received no further activation and were expanded in
medium containing IL-7, IL-15 and IL-21 until day 11. On days 0, 5,
9 and 12 of culture cells were evaluated for Tcm phenotype
(CD45RO+CD62L+) composition by FACS analysis. All the results are
gated from lymphogate and live gate (7AAD-) and CD8 T cell
(CD3+CD8+CD56-CD16-).
[0098] FIG. 17C depicts the percent apoptotic cells after 22 hours
of mixed lymphocyte reaction (MLR) with B-cell lymphoma and plasma
cell leukemia cell lines. CalceinAM pre-labeled Daudi, H.My2 C1R
HLA A2 K66A mutant or L363 cell lines were incubated for 22 hours
with or without 5-fold excess of anti-3.sup.rd party Tcm. Annexin
V+ cells were determined by FACS. Data is shown as mean.+-.SD of
pentaplicate cultures. ***p<0.001 values indicate statistically
significant changes compared to samples cultured in the absence of
Tcm.
[0099] FIG. 18 depicts a typical experiment showing enrichment for
CD8 T cells, at day 14 before graft versus leukemia (GVL) assay, by
extensively depleting non CD8 T cells (i.e., CD4+ T cells,
.gamma./.delta. T cells, B cells, NK cells, dendritic cells,
monocytes, granulocytes, and erythroid cells) using magnetic bead
sorting.
[0100] FIGS. 19A-D depict H.My C1R ("Neo") and H.My C1R HLA A2 K66A
mutant transfectant ("K66A") B-cell lymphoblastoid cell lines which
were labeled with CalceinAM, a vital dye that is released upon cell
death and then incubated for 22 hours with or without anti-3.sup.rd
party Tcm cells at a 1 to 5 ratio in favor of anti-3.sup.rd party
Tcm cells. After 22 hours, cells were recovered and analyzed for
survival by measuring the number of surviving Calcein+ stained
cells, and for apoptosis by AnnexinV+ cells from the calcein+
population by FACS. FIGS. 19A-B and FIGS. 19C-D represent two
independent experiments, respectively; FIGS. 19A and 19C show
killing while FIGS. 19B and 19D show apoptosis.
[0101] The percentage of B lymphoblast line cells killing was
calculated by the following formula:
( 1 - The number of live B lymphoblast line cells in the assessed
well The number of live B lymphoblast line cells in the control
well ) .times. 100 ##EQU00001##
[0102] Negative values signify that the B-cell lymphoblastoid cell
lines proliferated in the presence of Tcm.
[0103] The percentage of B lymphoblast line cells undergoing
specific apoptosis was calculated by the following formula: =(%
Calcein+AnnexinV+B lymphoblast line cells in the assessed well)-(%
Calcein+AnnexinV+B lymphoblast line cells in the control well).
[0104] FIG. 20 depicts the effect of different GMP grade reagents
used for isolation of CD8 T cells on levels of K66A killing. H.My
C1R HLA A2 K66A mutant transfectant cell lines were incubated for
22 hours with or without anti-3rd party Tcm cells at a 1 to 5 ratio
in favor of anti-3rd party Tcm cells. After 22 hours, cells were
recovered and analyzed for survival by measuring the number of
surviving Calcein+ stained cells a by FACS (mean of two independent
experiments). Average percent of killing of H.My C1R HLA A2 K66A
mutant cells shown as percent of the levels attained by the optimal
control group isolated making use of all four reagents
(CD4/CD56/CD19/CD45RA).
[0105] FIGS. 21-22 are schematic illustrations depicting protocols
for generation of Tcm for autologous (FIG. 21) and allogeneic (FIG.
22) transplantation.
[0106] FIGS. 23A-D depict a typical experiment demonstrating the
role of timing of addition of cytokines on the induction of Tcm
phenotype in CD8 T cells stimulated by allogeneic 3.sup.rd party
monocyte-derived mature DC. FIG. 23A illustrates naive CD8 T cells
which were stimulated with irradiated allogeneic 3rd party DC at a
ratio of 4:1 in a medium containing IL-21 for 3 days. Thereafter
the cells received no further activation and were expanded with
IL-7 and IL-15 until day 13 ("reference control group"=d(0-3)
IL21+DC d(3-13)IL7+IL15); FIG. 23B illustrates naive CD8 T cells
which were stimulated with irradiated allogeneic 3rd party DC at a
ratio of 4:1 in the presence of IL-21 for 7 days. Thereafter the
cells received no further activation and were expanded with IL-7
and IL-15 until day 13; FIG. 23C illustrates CD8 T cells which were
stimulated with irradiated allogeneic 3rd party DC at a ratio of
4:1 with continuous presence of IL-21 in both the priming phase
(IL21 alone) and in the expansion phase (together with IL-15); FIG.
23D illustrates CD8 T cells which were stimulated with irradiated
allogeneic 3rd party DC at a ratio of 4:1 with cytokine deprivation
for 7 days. Thereafter, the cells received no further activation
and were expanded with IL-15 alone until day 13.
[0107] FIGS. 24A-B depict the role of timing of addition of
cytokines in the human allogeneic model; summary of experiments.
Naive CD8 T cells were stimulated with irradiated allogeneic 3rd
party DC at a ratio of 4:1 in a medium containing IL-21 for 3 days.
Thereafter the cells received no further activation and were
expanded with IL-7 and IL-15 until day 13 ("reference control
group"=d(0-3) IL21.fwdarw.d(3-13)IL7+IL15). The other groups were
treated as indicated under the graphs. Cultures were evaluated for
cell numbers by trypan blue exclusion (FIG. 24A), and percentage of
Tcm (CD62L+CD45RO+) from CD8 T cells using FACS analysis (FIG.
24B). For each time point data represents average.+-.SE of the
indicated number (n) of independent experiments.
[0108] FIG. 25 depicts enrichment of anti-3.sup.rd party specific
CD8 T cell by positive selection of CD137+ cells. Naive CD8 T cells
were stimulated with irradiated allogeneic 3rd party DC (at a ratio
of 5.7:1) in the presence of IL-21. After 14 hours of activation,
CD137+ cells were positively selected by magnetic sorting. The
expression of CD137 on CD8 T cells was evaluated by FACS.
[0109] FIG. 26 depicts that enrichment of anti-3.sup.rd party
specific CD8 T cell by positive selection of CD137+ cells does not
reduce acquisition of Tcm phenotype. Naive CD8 T cells were
stimulated with irradiated allogeneic 3rd party DC at a ratio of
4:1 in the presence of IL-21 for 3 days. Thereafter, the cells
received no further activation and were expanded with IL-7 and
IL-15 until day 10 ("Reference control group"). Alternatively,
naive CD8 T cells were stimulated with irradiated allogeneic 3rd
party DC at a ratio of 5.7:1 in the presence of IL-21. After 14
hours of activation, CD137+ cells were positively selected by
magnetic sorting. CD137+ cells were then re-stimulated with
irradiated allogeneic 3rd party DC at a ratio of 4:1 in the
presence of IL-21 until day 3. Thereafter the cells were expanded
with IL-7 and IL-15 until day 10. Cells were evaluated for
percentage of Tcm (CD62L+CD45RO+) from CD8 T cells by FACS
analysis.
[0110] FIG. 27 depicts a comparison of proliferation kinetics.
Naive CD8 T cells were stimulated with irradiated allogeneic 3rd
party DC at a ratio of 4:1 in the presence of IL-21 for 3 days. The
cells received no further activation thereafter and were expanded
with IL-7 and IL-15 until day 14 ("Reference control group").
Alternatively, naive CD8 T cells were stimulated with irradiated
allogeneic 3rd party DC at a ratio of 5.7:1 in the presence of
IL-21. After 14 hours of activation, CD137+ cells were positively
selected by magnetic sorting. CD137+ cells were then re-stimulated
with irradiated allogeneic 3rd party DC in at a ratio of 4:1 in the
presence of IL-21 until day 3. Thereafter, the cells were expanded
with IL-7 and IL-15 until day 10. On day 10, cells were divided
into two test groups. In the first group cells continued to be
expanded with IL-7 and IL-15 until day 14 ("Anti 3.sup.rd CD137+")
while cells in the second test group were activated with irradiated
host PBMC in the presence of IL-7 and IL-15 (at a ratio of 1 to 2).
After 24 hours, CD137+ cells were depleted by magnetic sorting. The
CD137 depleted cells were re-plated with IL-7 and IL-15 and
cultured until day 14 ("Anti 3.sup.rd CD137+ and Anti host
CD137-"). On the indicated days, cells were counted by trypan blue
exclusion.
[0111] FIG. 28 depicts depletion of anti-host specific clones by
depletion of CD137+ cells after activation with irradiated host
PBMC. On day 10 of culture, 9 days after positive selection of
anti-3.sup.rd party specific clones, cells were activated by
irradiated host PBMC (at a 1:2 ratio, in favor of the host PBMC) in
the presence of IL-7 and IL-15. After 24 h, cells were depleted of
CD137+ cells. The expression of CD137 on CD8 T cells was evaluated
by FACS analysis.
[0112] FIG. 29 depicts a two stage magnetic sorting technique,
based on CD137 upregulation after antigen specific activation of
CD8 T cells successfully depletes anti-host clones and increases
the percent of cells specific for 3.sup.rd party antigens. Naive
CD8 T cells were stimulated with irradiated allogeneic 3rd party DC
at a ratio of 4:1 in the presence of IL-21 for 3 days. The cells
received no further activation thereafter and were expanded with
IL-7 and IL-15 until day 14 ("Reference control group").
Alternatively, naive CD8 T cells were stimulated with irradiated
allogeneic 3rd party DC at a ratio of 5.7:1 in the presence of
IL-21. After 14 hours of activation, CD137+ cells were positively
selected by magnetic sorting. CD137+ cells were then re-stimulated
with irradiated allogeneic 3rd party DC in at a ratio of 4:1 in the
presence of IL-21 until day 3. Thereafter, the cells were expanded
with IL-7 and IL-15 until day 10. On day 10, cells were divided
into two test groups. In the first group cells continued to be
expanded with IL-7 and IL-15 until day 14 ("Anti 3.sup.rd CD137+")
while cells in the second test group were activated with irradiated
host PBMC in the presence of IL-7 and IL-15 (at a ratio of 1 to 2).
After 24 h, CD137+ cells were depleted by magnetic sorting. The CD
137 depleted cells were re plated with IL-7 and IL-15 and cultured
until day 14 ("Anti 3.sup.rd CD137+ and Anti host CD137-"). On day
14, anti 3rd party and anti-host alloreactivity was evaluated by
CFSE assay against 3rd party or irradiated host PBMCs. For the CFSE
assay, 1.times.10.sup.6 CFSE+ responders were incubated with or
without 2.times.10.sup.6 irradiated (20 gy) PBMC stimulators for 84
h in the presence of IL-7. After 84 h, cells were recovered and
analyzed for cell division by measuring the number of CFSE low
stained CD8 T cells (CD3+CD8+CD56-) cells by FACS. To obtain
absolute values of cells, samples were suspended in a constant
volume and flow cytometric counts for each sample were obtained
during a constant, predetermined period of time. The number of
specific dividing cells=(Number of dividing cell with APC)-(Number
of dividing cell without APC). Negative values signify that the
number of dividing cells in response to activation with host PBMC
was even lower that the number of dividing cell without any
activation.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0113] The present invention, in some embodiments thereof, relates
to tolerance inducing and/or graft versus leukemia reactive
anti-third party cells comprising central memory T-lymphocyte
phenotype and, more particularly, but not exclusively, to methods
of generating same and to the use of same in transplantation and in
disease treatment.
[0114] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0115] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways. Also, it is to be understood that the
phraseology and terminology employed herein is for the purpose of
description and should not be regarded as limiting.
[0116] While reducing the present invention to practice, the
present inventors have uncovered an improved population of
anti-third party central memory T (Tcm) cells which homes to the
lymph nodes following transplantation and induces tolerance and
anti-disease activity (e.g. graft versus leukemia (GVL) activity)
without inducing a graft versus host (GVH) reaction.
[0117] As is shown hereinbelow and in the Examples section which
follows, the present inventors have provided new methods of
generating Tcm cells for allogeneic and autologous applications. As
shown in FIGS. 1A and 21, autologous Tcm cells, which are endowed
with anti-disease activity (e.g. anti-tumor activity), were
generated by first exposing CD8.sup.+ T cells to allogeneic stimuli
(e.g. dendritic cells) in the presence of IL-21 for 3 days and
subsequently adding IL-15 and IL-7 to the cells with the antigenic
stimuli for another 1-2 days. Next, the resultant cells were
cultured in an antigen free environment in the presence of IL-21,
IL-15 and IL-7 for additional 6-8 days.
[0118] As depicted in FIGS. 1B and 22, allogeneic Tcm cells, which
are tolerance inducing cells and are endowed with GVL activity,
were generated by first exposing CD8.sup.+ T cells to a third party
stimuli (e.g. dendritic cells) in the presence of IL-21 for 3 days.
Approximately 14 hours from the beginning of culture, the activated
cells were selected by positive selection of CD137+, and these
cells were re-cultured with IL-21. Subsequently, IL-15 and IL-7
were added to the IL-21 culture with the antigenic stimuli for
another 1-2 days. Next, the resultant cells were cultured in an
antigen free environment in the presence of IL-21, IL-15 and IL-7
for additional 6-8 days. At the end of culture, the Tcm cells were
depleted of alloreactive cells by depletion of CD137+ cells
following contacting of the Tcm cells with host type antigen
presenting cells (e.g. dendritic cells).
[0119] The cells generated by the present inventors comprised more
than 50% CD3+CD8+ cells of which more than 50% are Tcm cells (i.e.
comprise a CD3.sup.+, CD8.sup.+, CD62L.sup.+, CD45RA.sup.-,
CD45RO.sup.+ signature see e.g. Example 1 of the Examples section
which follows) and comprised TCR independent anti-leukemic activity
(see Example 2).
[0120] Taken together, these results substantiate the use of
anti-third party Tcm cells as graft facilitating cells and for use
in disease treatment in situations in which allogeneic
transplantation is warranted (e.g. hematopoietic stem cell
transplantation or in solid organ transplantation). Furthermore,
these results substantiate the use anti-third party Tcm cells in
disease treatment in situations in which autologous transplantation
is needed, such as for hematological malignancies.
[0121] Thus, according to one aspect of the present invention there
is provided an isolated population of cells comprising non-GVHD
inducing anti-third party cells having a central memory
T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing
cells and capable of homing to the lymph nodes following
transplantation.
[0122] The phrase "isolated population of cells" as used herein
refers to cells which have been isolated from their natural
environment (e.g., the human body).
[0123] The term "non-GVHD" as used herein refers to having
substantially reduced or no graft versus host inducing reactivity.
Thus, the cells of the present invention are generated as to not
significantly cause graft versus host disease (GVHD) as evidenced
by survival, weight and overall appearance of the transplanted
subject 100 days following transplantation.
[0124] As used herein, the term "syngeneic" refers to a cell or
tissue which is derived from an individual who is essentially
genetically identical with the subject. Typically, essentially
fully inbred mammals, mammalian clones, or homozygotic twin mammals
are syngeneic.
[0125] Examples of syngeneic cells or tissues include cells or
tissues derived from the subject (also referred to in the art as
"autologous"), a clone of the subject, or a homozygotic twin of the
subject.
[0126] As used herein, the term "non-syngeneic" refers to a cell or
tissue which is derived from an individual who is allogeneic or
xenogeneic with the subject's lymphocytes.
[0127] As used herein, the term "allogeneic" refers to a cell or
tissue which is derived from a donor who is of the same species as
the subject, but which is substantially non-clonal with the
subject. Typically, outbred, non-zygotic twin mammals of the same
species are allogeneic with each other. It will be appreciated that
an allogeneic donor may be HLA identical or HLA non-identical with
respect to the subject.
[0128] As used herein, the term "xenogeneic" refers to a cell or
tissue which substantially expresses antigens of a different
species relative to the species of a substantial proportion of the
lymphocytes of the subject. Typically, outbred mammals of different
species are xenogeneic with each other.
[0129] The present invention envisages that xenogeneic cells or
tissues are derived from a variety of species such as, but not
limited to, bovines (e.g., cow), equids (e.g., horse), porcines
(e.g. pig), ovids (e.g., goat, sheep), felines (e.g., Felis
domestica), canines (e.g., Canis domestica), rodents (e.g., mouse,
rat, rabbit, guinea pig, gerbil, hamster) or primates (e.g.,
chimpanzee, rhesus monkey, macaque monkey, marmoset).
[0130] Cells or tissues of xenogeneic origin (e.g. porcine origin)
are preferably obtained from a source which is known to be free of
zoonoses, such as porcine endogenous retroviruses. Similarly,
human-derived cells or tissues are preferably obtained from
substantially pathogen-free sources.
[0131] The phrase "anti-third party cells" as used herein refers to
lymphocytes (e.g. T lymphocytes) which are directed (e.g. by T cell
recognition) against a third party antigen or antigens.
[0132] As used herein the phrase "third party antigen or antigens"
refers to a soluble or non-soluble (such as membrane associated)
antigen or antigens which are not present in either the donor or
recipient, as depicted in detail infra.
[0133] For example, third party antigens can be third party cells,
antigens of viruses, such as for example, Epstein-Ban virus (EBV)
or cyto-megalo virus (CMV) or antigens of bacteria, such as
flagellin. Viral or bacterial antigens can be presented by cells
(e.g., cell line) infected therewith or otherwise made to express
viral/bacterial proteins. Autologous or non-autologous antigen
presenting cells can be used to present short synthetic peptides
fused or loaded thereto. Such short peptides may be viral derived
peptides or peptides representing any other antigen.
[0134] Dedicated software can be used to analyze viral or other
sequences to identify immunogenic short peptides, i.e., peptides
presentable in context of class I MHC or class II MHC.
[0135] Third party cells can be either allogeneic or xenogeneic
with respects to the recipient (explained in further detail
hereinbelow). In the case of allogeneic third party cells, such
cells have HLA antigens different from that of the donor but which
are not cross reactive with the recipient HLA antigens, such that
anti-third party cells generated against such cells are not
reactive against a transplant or recipient antigens.
[0136] According to an embodiment of the present invention the
allogeneic or xenogeneic third party cells are stimulatory cells
selected from the group consisting of cells purified from
peripheral blood lymphocytes (PBL), spleen or lymph nodes,
cytokine-mobilized PBLs, in vitro expanded antigen-presenting cells
(APC), in vitro expanded dendritic cells (DC) and artificial
antigen presenting cells
[0137] The artificial APC of the present invention may be
engineered to exhibit autologous MHC with a 3.sup.rd party peptide
or a 3.sup.rd party MHC without being pulsed with an exogenous
peptide. Thus, according to one embodiment, the artificial APC
comprises K562 tumor cells transfected with a third party MHC
determinant and a co-stimulatory molecule [as previously described
e.g. Suhoski M M et al., Mol. Ther. (2007) 15(5): 981-8], or
fibroblasts transfected with same.
[0138] Third party antigens can be presented on the cellular, viral
or bacterial surfaces or derived and/or purified therefrom.
Additionally, a viral or bacterial antigen can be displayed on an
infected cell and a cellular antigen can be displayed on an
artificial vehicle such as a liposome or an artificial antigen
presenting cell (e.g. leukemic or fibroblast cell line transfected
with the third party antigen or antigens).
[0139] The third party antigen may further comprise a synthetic
peptide presented by autologous presenting cells, non-autologous
presenting cells or on an artificial vehicle or on artificial
antigen presenting cells.
[0140] In addition, third party antigens can, for example, be
proteins extracted or purified from a variety of sources. An
example of a purified protein which can serve as a third party
antigen according to the present invention is ovalbumin. Other
examples are envisaged.
[0141] Utilizing cells, viruses, bacteria, virally infected,
bacteria infected, viral peptides or bacteria peptides presenting
cells as third party antigens is particularly advantageous since
such third party antigens include a diverse array of antigenic
determinants and as such direct the formation of anti-third party
cells of a diverse population, which may further serve in faster
reconstitution of T-cells in cases where such reconstitution is
required, e.g., following lethal or sublethal irradiation or
chemotherapy procedure.
[0142] Furthermore, when anti-third party cells are directed
against third party antigens, the cells are endowed with
anti-disease activity. The term "anti-disease activity" refers to
the activity (e.g. killing capability) of the Tcm cells against a
diseased cell (e.g. cancer cell, such as graft versus leukemia,
GVL, activity). This activity is typically due to TCR independent
killing mediated by LFA1-I/CAM1 binding [Arditti et al., Blood
(2005) 105(8):3365-71. Epub 2004 Jul. 6].
[0143] According to one embodiment, the third party cells comprise
dendritic cells.
[0144] According to one embodiment, the third party cells comprise
mature dendritic cells.
[0145] Methods of generating third party dendritic cells, which may
be used as stimulatory cells for inducing Tcm cells, are well known
in the art. Thus, as a non-limiting example, peripheral blood
mononuclear cells (PBMC) may be obtained from a third party
non-syngeneic cell donor [e.g. in case the Tcm cells are syngeneic,
e.g. autologous, the dendritic cells (DCs) may be non-syngeneic,
e.g. allogeneic, with respect to the subject; whereas if the Tcm
cells are non-syngeneic, e.g. allogeneic, the DCs are selected from
a donor being non-syngeneic, e.g. allogeneic, and HLA mismatched
with both the subject and the Tcm cells]. Monocytes may then be
isolated by plastic adherence and cultured (e.g. in cell culture
plates) using DC cell medium (e.g. Cellgro DC medium) supplemented
with human serum (e.g. 1% human serum), penicillin/streptomycin and
GM-CSF (800 IU/ml) and IL-4 (20 ng/ml) (available from e.g.
Peprotech, Hamburg, Germany). After about 48 h of culture, DC
medium may be added comprising GM-CSF (1600 IU/ml) and IL-4 (20
ng/ml). About 24 h later, non-adherent cells may be harvested, and
large cells (mostly immature DC) may be resuspended in fresh medium
containing GM-CSF (800 IU/ml), IL-4 (20 ng/ml), LPS (e.g. from E.
coli 055:B5 at 10 ng/ml) and IFN.gamma. 100 IU/ml) (available from
e.g. Peprotech, Hamburg, Germany), plated and incubated overnight.
The next day, non-adherent cells may be discarded, and adherent DCs
may be gently removed using e.g. cold PBS/1% HS after incubation on
ice for 20 minutes, thereby obtaining large cells consisting of
mature DC.
[0146] According to one embodiment, the third party cells comprise
irradiated dendritic cells.
[0147] Thus, according to one embodiment, the DCs are irradiated
with about 5-10 Gy, about 10-20 Gy, about 20-30 Gy, about 20-40 Gy,
about 20-50 Gy, about 10-50 Gy. According to a specific embodiment,
the DCs are irradiated with about 10-50 Gy (e.g. Gy).
[0148] According to some embodiments, the anti-third party cells of
the present invention comprise a central memory T-lymphocyte (Tcm)
phenotype.
[0149] The phrase "central memory T-lymphocyte (Tcm) phenotype" as
used herein refers to a subset of T cytotoxic cells which home to
the lymph nodes. Cells having the Tcm phenotype, in humans,
typically comprise a CD3+/CD8+/CD62L+/CD45RO+/CD45RA-signature. It
will be appreciated that Tcm cells may express all of the signature
markers on a single cell or may express only part of the signature
markers on a single cell.
[0150] It will be appreciated that at least 30%, at least 40%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95% or even 100% of the isolated population of cells are CD3+CD8+
cells. According to a specific embodiment, the isolated population
of cells comprise about 70-90% CD3+CD8+ cells.
[0151] It will be appreciated that at least 30%, at least 40%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95% or even 100% of the CD3+CD8+ cells have the Tcm cell signature.
According to a specific embodiment, about 30-80% of the CD3+CD8+
cells have the Tcm cell signature (e.g. 40-50%).
[0152] According to one embodiment, there is provided an isolated
population of cells comprising anti-third party cells having a
central memory T-lymphocyte (Tcm) phenotype, wherein at least 50%
of the isolated population of cells are CD3+CD8+ cells of which at
least 50% comprise a CD3.sup.+, CD8.sup.+, CD62L.sup.+,
CD45RA.sup.-, CD45RO.sup.+ signature, and further wherein the cells
are tolerance-inducing cells and/or endowed with anti-disease
activity (e.g. graft-versus-leukemia (GVL) activity), and capable
of homing to the lymph nodes following transplantation.
[0153] As mentioned, the Tcm cells typically home to the lymph
nodes following transplantation. According to some embodiments the
anti-third party Tcm cells of the present invention may home to any
of the lymph nodes following transplantation, as for example, the
peripheral lymph nodes and mesenteric lymph nodes. The homing
nature of these cells allows them to exert their tolerance effect
in a rapid and efficient manner.
[0154] Thus, the anti-third party Tcm cells of the present
invention are tolerance-inducing cells.
[0155] The phrase "tolerance inducing cells" as used herein refers
to cells which provoke decreased responsiveness of the recipient's
cells (e.g. recipient's T cells) when they come in contact with
same as compared to the responsiveness of the recipient's cells in
the absence of administered tolerance inducing cells. Tolerance
inducing cells include veto cells (i.e. T cells which lead to
apoptosis of host T cells upon contact with same) as was previously
described in PCT Publication Nos. WO 2001/049243 and WO
2002/102971.
[0156] According to some embodiments, the Tcm cells of the present
invention may be non-genetically modified cells or genetically
modified cells (e.g. cells which have been genetically engineered
to express or not express specific genes, markers or peptides or to
secrete or not secrete specific cytokines). Any method known in the
art may be implemented in genetically engineering the cells, such
as by inactivation of the relevant gene/s or by insertion of an
antisense RNA interfering with polypeptide expression (see e.g.
WO/2000/039294, which is hereby incorporated by reference).
[0157] According to some embodiments of the invention there is
provided a method of generating the isolated population of cells,
the method comprising: (a) contacting peripheral blood mononuclear
cells (PBMC) with a third party antigen or antigens in the presence
of IL-21 so as to allow enrichment of antigen reactive cells; and
(b) culturing the cells resulting from step (a) in the presence of
IL-21, IL-15 and IL-7 in an antigen free environment so as to allow
proliferation of cells comprising the central memory T-lymphocyte
(Tcm) phenotype.
[0158] The anti-third party Tcm cells of the present invention are
typically generated by first contacting syngeneic or non-syngeneic
peripheral blood mononuclear cells (PBMC) with a third party
antigen or antigens (such as described above) in a culture
supplemented with IL-21 (otherwise cytokine-free culture i.e.,
without the addition of any additional cytokines). This step is
typically carried out for about 12-24 hours, about 12-36 hours,
about 12-72 hours, 24-48 hours, 24-36 hours, about 24-72 hours,
about 48-72 hours, 1-2 days, 2-3 days, 1-3 days, 2-4 days, 1-5
days, 2-5 days, 2-6 days, 1-7 days, 5-7 days, 2-8 days, 8-10 days
or 1-10 days and allows enrichment of antigen reactive cells.
According to a specific embodiment, contacting of syngeneic or
non-syngeneic PBMC with a third party antigen or antigens (such as
described above) in a culture supplemented with IL-21 (otherwise
cytokine-free culture) is effected for 1-5 days (e.g. 3 days. This
step is typically carried out in the presence of about 0.001-3000
ng/ml, 0.001-1000 ng/ml, 0.01-1000 ng/ml, 0.1-1000 ng/ml, 1-1000
ng/ml, 10-1000 ng/ml, 10-500 ng/ml, 10-300 ng/ml, 10-100 ng/ml,
100-1000 ng/ml, 1-100 ng/ml, 1-50 ng/ml, 1-30 ng/ml, 10-50 ng/ml,
10-30 ng/ml, 10-20 ng/ml, 20-30 ng/ml, 20-50 ng/ml, 30-50 ng/ml,
30-100 ng/ml, 1-10 ng/ml, 0.1-10 ng/ml, 0.1-100 ng/ml, 1 ng/ml, 10
ng/ml-100 ng/ml IL-21. According to a specific embodiment, the
concentration of IL-21 is 10-50 ng/ml (e.g. 30 ng/ml).
[0159] According to a specific embodiment, contacting the syngeneic
or non-syngeneic PBMC with a third party antigen or antigens is
effected in a cytokine free culture (e.g. supplemented with only
IL-21), such a culture condition enables survival and enrichment of
only those cells which undergo stimulation and activation by the
third party antigen or antigens (i.e. of antigen reactive cells) as
these cells secrete cytokines (e.g. IL-2) which enable their
survival (all the rest of the cells die under these culture
conditions).
[0160] The ratio of third party antigen or antigens (e.g. dendritic
cell) to PBMC is typically about 1:2 to about 1:10 such as about
1:4, about 1:6, about 1:8 or about 1:10. According to a specific
embodiment, the ratio of third party antigen or antigens (e.g.
dendritic cell) to PBMC is about 1:2 to about 1:8 (e.g. 1:4).
[0161] Next, the anti-third party cells are cultured in the
presence of IL-21, IL-15 and IL-7 in an antigen free environment so
as to allow proliferation of cells comprising the Tcm phenotype.
This step is typically carried out for about 12-24 hours, about
12-36 hours, about 12-72 hours, 24-48 hours, 24-36 hours, about
24-72 hours, about 48-72 hours, 1-20 days, 1-15 days, 1-10 days,
1-5 days, 5-20 days, 5-15 days, 5-10 days, 1-2 days, 2-3 days, 1-3
days, 2-4 days, 2-5 days, 2-8 days, 2-10 days, 4-10 days, 4-8 days,
6-8 days, 8-10 days, 7-9 days, 7-11 days, 7-13 days, 7-15 days,
10-12 days, 10-14 days, 12-14 days, 14-16 days, 14-18 days, 16-18
days or 18-20 days. According to a specific embodiment, the
anti-third party cells are cultured in the presence of IL-21, IL-15
and IL-7 in an antigen free environment for about 7-11 days (e.g. 8
days)
[0162] This step is typically carried out in the presence of IL-21
at a concentration of about 0.001-3000 ng/ml, 0.001-1000 ng/ml,
0.01-1000 ng/ml, 0.1-1000 ng/ml, 1-1000 ng/ml, 10-1000 ng/ml,
10-500 ng/ml, 10-300 ng/ml, 10-100 ng/ml, 100-1000 ng/ml, 1-100
ng/ml, 1-50 ng/ml, 1-30 ng/ml, 10-50 ng/ml, 10-30 ng/ml, 10-20
ng/ml, 20-30 ng/ml, 20-50 ng/ml, 30-50 ng/ml, 30-100 ng/ml, 1-10
ng/ml, 0.1-10 ng/ml, 0.1-100 ng/ml, 1 ng/ml-100 ng/ml IL-21.
According to a specific embodiment, the concentration of IL-21 is
10-50 ng/ml (e.g. 30 ng/ml).
[0163] This step is further carried out in the presence of IL-15 at
a concentration of about 0.001-3000 ng/ml, 0.001-1000 ng/ml,
0.01-1000 ng/ml, 0.05-1000 ng/ml, 0.1-1000 ng/ml, 0.5-1000 ng/ml,
0.05-500 ng/ml, 0.5-500 ng/ml, 0.1-100 ng/ml, 0.1-10 ng/ml, 0.5-100
ng/ml, 1-100 ng/ml, 5-100 ng/ml, 1-50 ng/ml, 5-50 ng/ml, 1-10
ng/ml, 5-10 ng/ml, 1-5 ng/ml, 2-3 ng/ml, 2-5 ng/ml, 2-7 ng/ml, 3-5
ng/ml, 3-7 ng/ml, 4-5 ng/ml, 5-6 ng/ml, 5-7 ng/ml, 1-8 ng/ml,
10-100 ng/ml, 10-1000 ng/ml, 100-1000 ng/ml. According to a
specific embodiment the concentration of IL-15 is 1-10 ng/ml (e.g.
5 ng/ml).
[0164] This step is further carried out in the presence of IL-7 at
a concentration of about 0.001-3000 ng/ml, 0.001-1000 ng/ml,
0.01-1000 ng/ml, 0.05-1000 ng/ml, 0.1-1000 ng/ml, 0.5-1000 ng/ml,
0.05-500 ng/ml, 0.5-500 ng/ml, 0.1-100 ng/ml, 0.1-10 ng/ml, 0.5-100
ng/ml, 1-100 ng/ml, 5-100 ng/ml, 1-50 ng/ml, 5-50 ng/ml, 1-10
ng/ml, 5-10 ng/ml, 1-5 ng/ml, 2-3 ng/ml, 2-5 ng/ml, 2-7 ng/ml, 3-5
ng/ml, 3-7 ng/ml, 4-5 ng/ml, 5-6 ng/ml, 5-7 ng/ml, 1-8 ng/ml,
10-100 ng/ml, 10-1000 ng/ml, 100-1000 ng/ml. According to a
specific embodiment the concentration of IL-7 is 1-10 ng/ml (5
ng/ml).
[0165] The present inventors have collected through laborious
experimentation and screening a number of criteria which may be
harnessed towards to improving the proliferation of anti-third
party cells comprising a central memory T-lymphocyte (Tcm)
phenotype being devoid of graft versus host (GVH) reactive cells
and/or being enhanced for anti-disease (e.g. GVL) reactive
cells.
[0166] According to one embodiment, the PBMCs are depleted of
non-adherent cells prior to contacting with a third party antigen
or antigens in the presence of IL-21.
[0167] According to one embodiment, the PBMCs are depleted of CD4+
and/or CD56+ cells prior to contacting with a third party antigen
or antigens in the presence of IL-21.
[0168] According to one embodiment, the PBMCs are selected for
CD45RA+ cells prior to contacting with a third party antigen or
antigens in the presence of IL-21. Depletion of CD4.sup.+ and/or
CD56+ cells may be carried out using any method known in the art,
such as by affinity based purification (e.g. such as by the use of
MACS beads, FACS sorter and/or capture ELISA labeling). Such a step
may be beneficial in order to increase the purity of the CD8.sup.+
cells within the culture (i.e. eliminate other lymphocytes within
the cell culture e.g. T CD4.sup.+ cells or NK cells) or in order to
increase the number of CD8.sup.+ T cells.
[0169] According to one embodiment, the PBMCs comprise non-adherent
cells.
[0170] According to one embodiment, the PBMCs comprise CD8+ T
cells.
[0171] According to one embodiment, the PBMCs comprise naive CD8+ T
cells.
[0172] Selection of naive CD8+ T cells may be effected by selection
of cells expressing CD45RA+ and/or cells expressing CD45RO- and may
be carried out using any method known in the art, such as by
affinity based purification (e.g. such as by the use of MACS beads,
FACS sorter and/or capture ELISA labeling).
[0173] According to one embodiment, the PBMCs comprise CD45RA+
cells.
[0174] An additional step which may be carried out in accordance
with the present teachings include culturing the PBMCs cells with a
third party antigen or antigens in the presence of IL-21, IL-15 and
IL-7 prior to removing the third party antigen or antigens from the
cell culture (i.e. prior to generating an antigen free
environment). This step is typically carried out for about 12-24
hours, about 12-36 hours, about 12-72 hours, 24-48 hours, 24-36
hours, about 24-72 hours, about 48-72 hours, 1-2 days, 2-3 days,
1-3 days, 2-4 days, 1-5 days or 2-5 days, and is effected at the
same doses of IL-21, IL-15 and IL-7 indicated above. According to a
specific embodiment, culturing the PBMCs cells with a third party
antigen or antigens in the presence of IL-21, IL-15 and IL-7 is
carried out for 12 hours to 4 days (e.g. 1-2 days).
[0175] Additionally or alternatively, an additional two step
process which allows selection and isolation of activated cells may
be carried out. Such a selection step aids in removal of potential
host reactive T cells in situations where the PBMCs are
non-syngeneic with respect to the subject (as described in further
detail below).
[0176] Thus, isolating activated cells may be carried out in a two
stage approach. In the first stage activated cells are selected
before culturing the cells in the presence of IL-15 and IL-7. This
first stage is typically carried out after the initial contacting
of the PBMC with a third party antigen or antigens in the presence
of IL-21. This selection process picks only those cells which were
activated by the third party antigen (e.g. express activation
markers as described below) and is typically affected about 12-24
hours, about 24-36 hours, about 12-36 hours, about 36-48 hours,
about 12-48 hours, about 48-60 hours, about 12-60 hours, about
60-72 hours, about 12-72 hours, about 72-84 hours, about 12-84
hours, about 84-96 hours, about 12-96 hours, after the initial
contacting of the PBMC with a third party antigen or antigens.
According to a specific embodiment, the selection process is
effected about 12-24 hours (e.g. 14 hours) after the initial
contacting of the PBMC with a third party antigen or antigens.
[0177] Isolating activated cells may be effected by affinity based
purification (e.g. such as by the use of MACS beads, FACS sorter
and/or capture ELISA labeling) and may be effected towards any
activation markers including cell surface markers such as, but not
limited to, CD69, CD44, CD25, CFSE, CD137 or non-cell surface
markers such as, but not limited to, IFN-.gamma. and IL-2.
Isolating activated cells may also be effected by morphology based
purification (e.g. isolating large cells) using any method known in
the art (e.g. by FACS). Typically, the activated cells are also
selected for expression of CD8.sup.+ cells. Furthermore, any
combination of the above methods may be utilized to efficiently
isolate activated cells.
[0178] According to an embodiment of the present invention,
selecting for activated cells is effected by selection of CD137+
and/or CD25+ cells.
[0179] The second stage of isolation of activated cells is
typically carried out at the end of culturing (i.e. after culturing
in an antigen free environment with IL-21, IL-15 and IL-7). This
stage depletes alloreactive cells by depletion of those cells which
were activated following contacting of the central memory
T-lymphocyte (Tcm) with irradiated host antigen presenting cells
(APCs e.g. dendritic cells). As mentioned above, isolating
activated cells may be effected by affinity based purification
(e.g. such as by the use of MACS beads, FACS sorter and/or capture
ELISA labeling) and may be effected towards any activation markers
including cell surface markers such as, but not limited to, CD69,
CD44, CD25, CFSE, CD137 or non-cell surface markers such as, but
not limited to, IFN-.gamma. and IL-2.
[0180] According to an embodiment of the present invention,
depleting the alloreactive cells is effected by depletion of CD137+
and/or CD25+ cells.
[0181] Following are a number of non-limiting examples of protocols
which can be used according to some embodiments of the
invention.
[0182] According to one embodiment of the invention, there is
provided a method of generating an isolated population of cells
comprising anti-third party cells having a central memory
T-lymphocyte (Tcm) phenotype, the cells being tolerance-inducing
cells and/or endowed with anti-disease activity (e.g.
graft-versus-leukemia (GVL) activity), and capable of homing to the
lymph nodes following transplantation, the method comprising: (a)
treating non-adherent peripheral blood mononuclear cells (PBMC)
with an agent capable of depleting CD4+ and/or CD56+ cells so as to
obtain CD8+ T cells; (b) contacting the CD8+ T cells with third
party dendritic cells in the presence of IL-21 for 12 hours to 5
days so as to allow enrichment of antigen reactive cells; (c)
culturing the cells resulting from step (b) with the third party
dendritic cells in the presence of IL-21, IL-15 and IL-7 for 12
hours to 3 days; and (d) culturing the cells resulting from step
(c) in the presence of IL-21, IL-15 and IL-7 in an antigen free
environment for 5-20 days so as to allow proliferation of cells
comprising the central memory T-lymphocyte (Tcm) phenotype.
[0183] The above describe protocol is typically used for
no-syngeneic transplantation and therefore the PBMC used are
typically allogeneic with respect to a subject (e.g. from an
allogeneic donor).
[0184] According to one embodiment of the invention, there is
provided a method of generating an isolated population of cells
comprising anti-third party cells having a central memory
T-lymphocyte (Tcm) phenotype, the cells being endowed with
anti-disease activity (e.g. anti-tumor cell activity), and capable
of homing to the lymph nodes following transplantation, the method
comprising: (a) treating non-adherent peripheral blood mononuclear
cells (PBMC) with an agent capable of depleting CD4+ and/or CD56+
cells so as to obtain CD8+ T cells; (b) contacting the CD8+ T cells
with non-syngeneic dendritic cells in the presence of IL-21 for 12
hours to 5 days so as to allow enrichment of antigen reactive
cells; (c) culturing the cells resulting from step (b) with the
non-syngeneic dendritic cells in the presence of IL-21, IL-15 and
IL-7 for 12 hours to 3 days; and (d) culturing the cells resulting
from step (c) in the presence of IL-21, IL-15 and IL-7 in an
antigen free environment for 5-20 days so as to allow proliferation
of cells comprising the central memory T-lymphocyte (Tcm)
phenotype.
[0185] The above describe protocol is typically used for syngeneic
transplantation and therefore the PBMC used are typically
autologous with respect to a subject (e.g. from the subject).
[0186] Thus, as mentioned, the PBMC may be syngeneic or
non-syngeneic with respect to a subject.
[0187] According to some embodiments of the invention, the
non-syngeneic PBMCs of the present invention may be allogeneic or
xenogeneic with respect to the subject (explained in further detail
hereinbelow).
[0188] The source of the PBMCs will be determined with respect to
the intended use of the cells (see further details hereinbelow) and
is well within the capability of one skilled in the art, especially
in light of the detailed disclosure provided herein.
[0189] The use of tolerance inducing cells is especially beneficial
in situations in which there is a need to eliminate graft rejection
and overcome graft versus host disease (GVHD), such as in
transplantation of allogeneic or xenogeneic cells or tissues.
[0190] Thus, according to another aspect of the present invention,
there is provided a method of treating a subject in need of a cell
or tissue transplantation, the method comprising transplanting a
cell or organ transplant into the subject and administering to the
subject a therapeutically effective amount of the isolated
population of cells.
[0191] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical
symptoms of a condition or substantially preventing the appearance
of clinical or aesthetical symptoms of a condition.
[0192] As used herein, the term "subject" or "subject in need
thereof" refers to a mammal, preferably a human being, male or
female at any age that is in need of a cell or tissue
transplantation or suffers from a disease which may be treated with
the Tcm cells. Typically the subject is in need of cell or tissue
transplantation (also referred to herein as recipient) due to a
disorder or a pathological or undesired condition, state, or
syndrome, or a physical, morphological or physiological abnormality
which is amenable to treatment via cell or tissue transplantation.
Examples of such disorders are provided further below.
[0193] As used herein, the phrase "cell or tissue transplantation"
refers to a bodily cell (e.g. a single cell or a group of cells) or
tissue (e.g. solid tissues/organs or soft tissues, which may be
transplanted in full or in part). Exemplary tissues or organs which
may be transplanted according to the present teachings include, but
are not limited to, liver, pancreas, spleen, kidney, heart, lung,
skin, intestine and lymphoid/hematopoietic tissues (e.g. lymph
node, Peyer's patches thymus or bone marrow). Exemplary cells which
may be transplanted according to the present teachings include, but
are not limited to, immature hematopoietic cells including stem
cells. Furthermore, the present invention also contemplates
transplantation of whole organs, such as for example, kidney,
heart, liver or skin.
[0194] Depending on the application, the method may be effected
using a cell or tissue which is syngeneic or non-syngeneic with the
subject.
[0195] According to an embodiment of the present invention, both
the subject and the donor are humans.
[0196] Depending on the application and available sources, the
cells or tissues of the present invention may be obtained from a
prenatal organism, postnatal organism, an adult or a cadaver donor.
Moreover, depending on the application needed the cells or tissues
may be naive or genetically modified. Such determinations are well
within the ability of one of ordinary skill in the art
[0197] Any method known in the art may be employed to obtain a cell
or tissue (e.g. for transplantation).
[0198] Transplanting the cell or tissue into the subject may be
effected in numerous ways, depending on various parameters, such
as, for example, the cell or tissue type; the type, stage or
severity of the recipient's disease (e.g. organ failure); the
physical or physiological parameters specific to the subject;
and/or the desired therapeutic outcome.
[0199] Transplanting a cell or tissue transplant of the present
invention may be effected by transplanting the cell or tissue
transplant into any one of various anatomical locations, depending
on the application. The cell or tissue transplant may be
transplanted into a homotopic anatomical location (a normal
anatomical location for the transplant), or into an ectopic
anatomical location (an abnormal anatomical location for the
transplant). Depending on the application, the cell or tissue
transplant may be advantageously implanted under the renal capsule,
or into the kidney, the testicular fat, the sub cutis, the omentum,
the portal vein, the liver, the spleen, the heart cavity, the
heart, the chest cavity, the lung, the skin, the pancreas and/or
the intra abdominal space.
[0200] For example, a liver tissue according to the present
teachings may be transplanted into the liver, the portal vein, the
renal capsule, the sub-cutis, the omentum, the spleen, and the
intra-abdominal space. Transplantation of a liver into various
anatomical locations such as these is commonly practiced in the art
to treat diseases amenable to treatment via hepatic transplantation
(e.g. hepatic failure). Similarly, transplanting a pancreatic
tissue according to the present invention may be advantageously
effected by transplanting the tissue into the portal vein, the
liver, the pancreas, the testicular fat, the sub-cutis, the
omentum, an intestinal loop (the subserosa of a U loop of the small
intestine) and/or the intra-abdominal space. Transplantation of
pancreatic tissue may be used to treat diseases amenable to
treatment via pancreatic transplantation (e.g. diabetes). Likewise,
transplantation of tissues such as a kidney, a heart, a lung or
skin tissue may be carried out into any anatomical location
described above for the purpose of treating recipients suffering
from, for example, renal failure, heart failure, lung failure or
skin damage (e.g., burns).
[0201] The method of the present invention may also be used, for
example, for treating a recipient suffering from a disease
requiring immature hematopoietic cell transplantation.
[0202] In the latter case, immature autologous, allogeneic or
xenogeneic hematopoietic cells (including stem cells) which can be
derived, for example, from bone marrow, mobilized peripheral blood
(by for example leukapheresis), fetal liver, yolk sac and/or cord
blood of the donor and which are preferably T-cell depleted CD34+
immature hematopoietic cells, can be transplanted to a recipient
suffering from a disease. Such a disease includes, but is not
limited to, leukemia such as acute lymphoblastic leukemia (ALL),
acute nonlymphoblastic leukemia (ANLL), acute myelocytic leukemia
(AML) or chronic myelocytic leukemia (CML), severe combined
immunodeficiency syndromes (SCID), including adenosine deaminase
(ADA), osteopetrosis, aplastic anemia, Gaucher's disease,
thalassemia and other congenital or genetically-determined
hematopoietic abnormalities.
[0203] It will be appreciated that the immature autologous,
allogeneic or xenogeneic hematopoietic cells of the present
invention may be transplanted into a recipient using any method
known in the art for cell transplantation, such as but not limited
to, cell infusion (e.g. I.V.) or via an intraperitoneal route.
[0204] Optionally, when transplanting a cell or tissue transplant
of the present invention into a subject having a defective organ,
it may be advantageous to first at least partially remove the
failed organ from the subject so as to enable optimal development
of the transplant, and structural/functional integration thereof
with the anatomy/physiology of the subject.
[0205] According to one embodiment, the immature hematopoietic
cells and the isolated population of cells are derived from the
same donor.
[0206] According to one embodiment, the immature hematopoietic
cells and the isolated population of cells are derived from the
same subject.
[0207] The method of the present invention also envisions
co-transplantation of several organs (e.g. heart and lung tissues)
in case the subject may be beneficially effected by such a
procedure.
[0208] According to one embodiment, the co-transplantation
comprises transplantation of immature hematopoietic cells and a
solid tissue/organ or a number of solid organs/tissues.
[0209] According to one embodiment, the immature hematopoietic
cells and the solid organ or obtained from the same donor.
[0210] According to another embodiment, the immature hematopoietic
cells and the solid organ/tissue or organs/tissue are obtained from
different (non-syngeneic) donors.
[0211] According to one embodiment, the immature hematopoietic
cells are transplanted prior to, concomitantly with, or following
the transplantation of the solid organ
[0212] According to an embodiment, hematopoietic chimerism is first
induced in the subject by transplantation of immature hematopoietic
cells in conjunction with the Tcm cells of the present invention,
leading to tolerance of other tissues/organs transplanted from the
same donor.
[0213] According to an embodiment, the Tcm cells of the present
invention are used per se for reduction of rejection of
transplanted tissues/organs organs transplanted from the same
donor.
[0214] In a further embodiment, the cell or tissue transplant and
the isolated population of cells are derived from the same
donor.
[0215] In a further embodiment, the cell or tissue transplant is
syngeneic with the subject and the isolated population of cells are
non-syngeneic with the subject.
[0216] In a further embodiment, the cell or tissue transplant is
syngeneic with the subject and the isolated population of cells are
syngeneic with the subject.
[0217] Following transplantation of the cell or tissue transplant
into the subject according to the present teachings, it is
advisable, according to standard medical practice, to monitor the
growth functionality and immuno-compatibility of the organ
according to any one of various standard art techniques. For
example, the functionality of a pancreatic tissue transplant may be
monitored following transplantation by standard pancreas function
tests (e.g. analysis of serum levels of insulin). Likewise, a liver
tissue transplant may be monitored following transplantation by
standard liver function tests (e.g. analysis of serum levels of
albumin, total protein, ALT, AST, and bilirubin, and analysis of
blood-clotting time). Structural development of the cells or
tissues may be monitored via computerized tomography, or ultrasound
imaging.
[0218] Depending on the transplantation context, in order to
facilitate engraftment of the cell or tissue transplant, the method
may further advantageously comprise conditioning the subject under
sublethal, lethal or supralethal conditions prior to the
transplanting.
[0219] As used herein, the terms "sublethal", "lethal", and
"supralethal", when relating to conditioning of subjects of the
present invention, refer to myelotoxic and/or lymphocytotoxic
treatments which, when applied to a representative population of
the subjects, respectively, are typically: non-lethal to
essentially all members of the population; lethal to some but not
all members of the population; or lethal to essentially all members
of the population under normal conditions of sterility.
[0220] According to one embodiment, the conditioning step is
effected by conditioning the subject under supralethal conditions,
such as under myeloablative conditions.
[0221] Alternatively, the conditioning step may be effected by
conditioning the subject under lethal or sublethal conditions, such
as by conditioning the subject under myeloreductive conditions.
[0222] Examples of conditioning agents which may be used to
condition the subject include, without limitation, irradiation,
pharmacological agents, and tolerance-inducing cells (as described
herein).
[0223] Examples of pharmacological agents include myelotoxic drugs,
lymphocytotoxic drugs and immunosuppressant drugs.
[0224] Examples of myelotoxic drugs include, without limitation,
busulfan, dimethyl mileran, melphalan and thiotepa.
[0225] The method may further advantageously comprise conditioning
the subject with an immunosuppressive regimen prior to,
concomitantly with, or following transplantation of the cell or
tissue transplant.
[0226] Examples of suitable types of immunosuppressive regimens
include administration of immunosuppressive drugs, tolerance
inducing cell populations (as described in detail hereinbelow),
and/or immunosuppressive irradiation.
[0227] Ample guidance for selecting and administering suitable
immunosuppressive regimens for transplantation is provided in the
literature of the art (for example, refer to: Kirkpatrick C H. and
Rowlands D T Jr., 1992. JAMA. 268, 2952; Higgins R M. et al., 1996.
Lancet 348, 1208; Suthanthiran M. and Strom T B., 1996. New Engl.
J. Med. 331, 365; Midthun D E. et al., 1997. Mayo Clin Proc. 72,
175; Morrison V A. et al., 1994. Am J. Med. 97, 14; Hanto D W.,
1995. Annu Rev Med. 46, 381; Senderowicz A M. et al., 1997. Ann
Intern Med. 126, 882; Vincenti F. et al., 1998. New Engl. J. Med.
338, 161; Dantal J. et al. 1998. Lancet 351, 623).
[0228] Preferably, the immunosuppressive regimen consists of
administering at least one immunosuppressant agent to the
subject.
[0229] Examples of immunosuppressive agents include, but are not
limited to, methotrexate, cyclophosphamide, cyclosporine,
cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine
(sulphasalazopyrine), gold salts, D-penicillamine, leflunomide,
azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF.
alpha. blockers, a biological agent that targets an inflammatory
cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs).
Examples of NSAIDs include, but are not limited to acetyl salicylic
acid, choline magnesium salicylate, diflunisal, magnesium
salicylate, salsalate, sodium salicylate, diclofenac, etodolac,
fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac,
meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam,
sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors,
tramadol, rapamycin (sirolimus) and rapamycin analogs (such as
CCI-779, RAD001, AP23573). These agents may be administered
individually or in combination.
[0230] Regardless of the transplant type, to avoid graft rejection
and graft versus host disease, the method of the present invention
utilizes the novel anti third party Tcm cells (as described in
detail hereinabove).
[0231] According to the method of the present invention, these anti
third party Tcm cells are administered either concomitantly with,
prior to, or following the transplantation of the cell or tissue
transplant.
[0232] The anti third party Tcm cells may be administered via any
method known in the art for cell transplantation, such as but not
limited to, cell infusion (e.g. I.V.) or via an intraperitoneal
route.
[0233] Without being bound to theory, a therapeutically effective
amount is an amount of anti-third party Tcm cells efficient for
tolerization, anti-tumor effect and/or immune reconstitution
without inducing GVHD. Since the Tcm cells of the present invention
home to the lymph nodes following transplantation, lower amounts of
cells (compared to the dose of cells previously used, see for
example WO 2001/049243) may be needed to achieve the beneficial
effect/s of the cells (e.g. tolerization, anti-tumor effect and/or
immune reconstitution). It will be appreciated that lower levels of
immunosuppressive drugs may be needed in conjunction with the Tcm
cells of the present invention (such as exclusion of rapamycin from
the therapeutic protocol).
[0234] Determination of the therapeutically effective amount is
well within the capability of those skilled in the art, especially
in light of the detailed disclosure provided herein.
[0235] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
concentration or titer. Such information can be used to more
accurately determine useful doses in humans.
[0236] For example, in case of tissue transplantation the number of
anti-third party Tcm cells infused to a recipient should be more
than 1.times.10.sup.4/Kg body weight. The number of anti-third
party Tcm cells infused to a recipient should typically be in the
range of 1.times.10.sup.3/Kg body weight to 1.times.10.sup.4/Kg
body weight, range of 1.times.10.sup.4/Kg body weight to
1.times.10.sup.5/Kg body weight, range of 1.times.10.sup.4/Kg body
weight to 1.times.10.sup.6/Kg body weight, range of
1.times.10.sup.4/Kg body weight to 1.times.10.sup.7/Kg body weight,
range of 1.times.10.sup.4/Kg body weight to 1.times.10.sup.8/Kg
body weight, range of 1.times.10.sup.3/Kg body weight to
1.times.10.sup.5/Kg body weight, range of 1.times.10.sup.4/Kg body
weight to 1.times.10.sup.6/Kg body weight, range of
1.times.10.sup.6/Kg body weight to 1.times.10.sup.7/Kg body weight,
range of 1.times.10.sup.5/Kg body weight to 1.times.10.sup.7/Kg
body weight, range of 1.times.10.sup.6/Kg body weight to
1.times.10.sup.8/Kg body weight. According to a specific
embodiment, the number of anti-third party Tcm cells infused to a
recipient should be in the range of 1.times.10.sup.5/Kg body weight
to 1.times.10.sup.7/Kg body weight.
[0237] Thus, the novel anti-third party Tcm cells of the present
invention may be used as adjuvant therapy for a cell or tissue
transplant (as described hereinabove). In addition the novel Tcm
cells of the present invention are also endowed with anti-disease
activity (e.g. anti-tumor cell activity, as described in further
detail hereinabove) and thus may be used per se for disease
treatment.
[0238] According to a specific embodiment, in order to obtain a
graft versus diseased cell activity (e.g. anti-tumor effect such as
anti-leukemia treatment), syngeneic cells as well as non-syngeneic
cells may be used.
[0239] Thus, the method of the present invention may be applied to
treat any disease such as, but not limited to, a malignant disease,
a disease associated with transplantation of a graft, an infectious
disease such as a viral disease or a bacterial disease, an
inflammatory disease and/or an autoimmune disease.
[0240] Diseases which may be treated using the methods of the
present invention include, but are not limited to, malignant
diseases such as leukemia [e.g., acute lymphatic, acute
lymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic
T cell leukemia, acute-megakaryoblastic, monocytic, acute
myelogenous, acute myeloid, acute myeloid with eosinophilia, B
cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic,
Friend, granulocytic or myelocytic, hairy cell, lymphocytic,
megakaryoblastic, monocytic, monocytic-macrophage, myeloblastic,
myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic,
subacute, T cell, lymphoid neoplasm, predisposition to myeloid
malignancy, acute nonlymphocytic leukemia, T-cell acute lymphocytic
leukemia (T-ALL) and B-cell chronic lymphocytic leukemia (B-CLL)],
lymphoma (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, B cell,
Burkitt, cutaneous T cell, histiocytic, lymphoblastic, T cell,
thymic), carcinoma, blastoma and sarcoma; diseases associated with
transplantation of a graft (e.g. graft rejection, chronic graft
rejection, subacute graft rejection, hyper-acute graft rejection,
acute graft rejection and graft versus host disease); infectious
diseases including, but are not limited to, chronic infectious
diseases, subacute infectious diseases, acute infectious diseases,
viral diseases (e.g. EBV, CMV, HIV), bacterial diseases, protozoan
diseases, parasitic diseases, fungal diseases, mycoplasma diseases
and prion diseases; inflammatory diseases (e.g. chronic
inflammatory diseases and acute inflammatory diseases); and
autoimmune diseases (e.g. cardiovascular diseases, rheumatoid
diseases, glandular diseases, gastrointestinal diseases, cutaneous
diseases, hepatic diseases, neurological diseases, muscular
diseases, nephric diseases, diseases related to reproduction,
connective tissue diseases and systemic diseases).
[0241] Thus, the method of the present invention can furthermore be
advantageously applied towards treating a disease in a subject
while concomitantly facilitating engraftment of a transplant of
cells or tissues syngeneic with the anti-third party Tcm cells
(e.g. in situations where the cell or tissue transplant and the
anti-third party cells are derived from the same donor).
[0242] As used herein the term "about" refers to .+-.10%.
[0243] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0244] The term "consisting of means "including and limited
to".
[0245] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0246] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0247] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0248] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0249] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0250] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0251] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0252] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0253] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan
J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn.
(1994); Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W.H. Freeman and Co., New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R.
I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
General Materials and Experimental Procedures
[0254] Peripheral Blood Mononuclear Cells (PBMC)
[0255] PBMC were isolated from whole blood of patients and from
healthy volunteers by Ficoll density gradient centrifugation. When
indicated the cells were typed for Class I HLA by serological
methods as previously described [Manual of Tissue Typing
Techniques. Washington D.C., National Institute of Allergy and
Infectious Diseases, NIH DHEW Publication 76-545, 1976, p 22].
[0256] Tumor Cell Lines
[0257] H.My2 C1R HLA A2 K66A transfectant cells and H.My2 C1R HLA
A2 w.t. transfectant B cell line were used.
[0258] C1R, a human B-cell lymphoblastoid line lacking surface HLA
A and B antigens, derived from Hmy.2 B-LCL by gamma irradiation
followed by selection for Class I monoclonal antibodies and
complement as previously described [Storkus W J, et al. Proc. Natl.
Acad. Sci. USA (1989) 86: 2361-2364] was used.
[0259] C1R-neo, a stable transfectant cell line established in 1987
by electroporation of the C1R cell line with a modified neomycin
drug-resistant eukaryotic vector, pSP65-Neo (the vector did not
carry an insert), as previously described [Grumet F C, et al. Hum.
Immunol. (1994) 40: 228-234] was used.
[0260] Dendritic Cell Generation
[0261] Monocytes were isolated by plastic adherence and cultured in
6-well plates using 3 ml of Cellgro DC medium supplemented with 1%
human serum and penicillin/streptomycin plus GM-CSF (800 IU/ml) and
IL-4 (20 ng/ml) (Peprotech, Hamburg, Germany). After 48 h of
culture, 1.5 ml of medium was added (+GM-CSF at 1600 IU/ml and IL4
at 20 ng/ml). 24 h later, non-adherent cells were harvested, and
large cells (mostly immature DC) were counted, resuspended in fresh
medium containing GM-CSF 800 IU/ml, IL-4 20 ng/ml, LPS from E. coli
055:B5 at 10 ng/ml (Sigma, Deisenhofen, Germany) and IFN.gamma.
(Peprotech, 100 IU/ml), and plated at approximately 106 DC per well
in 2 ml and incubated overnight. The next day, non-adherent cells
were discarded, and adherent DC were gently removed using cold
PBS/1% HS after incubation on ice for 20 minutes. Large cells
consisting of mature DC were counted. The cells were irradiated
with 30 Gy to avoid outgrowth of few potentially contaminating NK-
or memory T-cells and were then used for T-cell stimulation.
[0262] Isolation of Naive CD8 T-cells from PBMC
[0263] Naive CD8 T cells were isolated by initial negative
selection using a CD8 negative selection kit (Miltenyi, Bergisch
Gladbach, Germany) according to the manufacturer's instructions.
Antigen-experienced CD8+ T-cells were then depleted using CD45RO-
beads and on LD column.
[0264] Generation of Anti-3Rd Party Central Memory Human CD8
T-Cells
[0265] Naive CD8 T cells were isolated and resuspended in T-cell
medium supplemented with IL-21 (Peprotech, 30 ng/ml). Irradiated
DCs were added at a 1:4 DC:T-cell ratio with 4.times.10.sup.5
T-cells per well of a 48-well plate. Total volume of each well was
500 .mu.l.
[0266] 72 h after initiation of the culture, 500 .mu.l T-cell
medium with IL-7 and IL-15 (Peprotech, 5 ng/ml final
concentrations) were added and cells were subsequently fed every
2-3 days as outline in the results section.
[0267] GVL Assay
[0268] H.My C1R ("Neo") and H.My C1R HLA A2 K66A mutant
transfectant ("K66A") B lymphoblast line cells were obtained by
Ficoll density gradient centrifugation and were labeled with 0.15
.mu.g/ml CalceinAM (Molecular Probes, Inc, Eugene, Oreg.), a vital
dye that is released upon cell death, according to manufacturer's
instructions. Next, 2.times.10.sup.5 Calcein labeled B lymphoblast
line cells were incubated with or without anti-3.sup.rd party Tcm
for 22 hours at a 1 to 5 ratio in favor of anti-3.sup.rd party Tcms
in 24 well plates. Prior to the co culture anti-3.sup.rd party Tcm
were enriched for CD8+ T cells by a negative selection kit
(Miltenyi, Bergisch Gladbach, Germany). No exogenous cytokines were
added to the MLR. Cells were recovered and analyzed for survival by
measuring the number of surviving Calcein stained B lymphoblast
line cells by FACS. For detection of apoptosis by AnnexinV+ samples
were incubated with 5 .mu.l AnnexinV-APC (BD) for 15 minutes at
room temperature. Subsequently, unbound AnnexinV was washed out,
and samples were analyzed by FACS. To obtain absolute values of
cells, samples were suspended in constant volume and flow
cytometric counts for each sample were obtained during a constant,
predetermined period of time and were compared with flow cytometric
counts obtained with fixed volume and fixed numbers of input cells.
Survival rate are presented relative to the survival of B
lymphoblast line cells alone.
[0269] The percentage of B lymphoblast line cells killing was
calculated by the following formula:
( 1 - The number of live B lymphoblast line cells in the assessed
well The number of live B lymphoblast line cells in the control
well ) .times. 100 ##EQU00002## The percentage of B lymphoblast
line cells undergoing specific apoptosis was calculated by the
following formula := ( % Calcein + AnnexinV + B lymphoblast line
cells in the assessed well ) - ( % Calcein + AnnexinV + B
lymphoblast line cells in the control well ) . ##EQU00002.2##
[0270] A Two Stage Magnetic Sorting Approach for Depletion of
Alloreactivity, Based on the CD137 Activation Marker
[0271] Naive CD8 T cells were stimulated with irradiated allogeneic
3rd party DC at a ratio of 6:1 in the presence of IL-21 (Peprotech,
30 ng/ml). After 14 hours of activation, CD137+ cells were
positively selected by magnetic sorting (Miltenyi, Bergisch
Gladbach, Germany). CD137+ cells were then re-stimulated with
irradiated allogeneic 3rd party DC at a ratio of 4:1 in the
presence of IL-21 (Peprotech, 30 ng/ml) until day 3. Thereafter,
the cells were expanded with 5 ng/ml IL-7 and 5 ng/ml IL-15
(Peprotech) until day 10. On day 10, cells were divided into two
test groups. In the first group cells continued to be expanded with
IL-7 and IL-15 until day 14, while cells in the second test group
were activated with irradiated host PBMC in the presence of IL-7
and IL-15 (at a ratio of 1 to 2). After 24 h, CD137+ cells were
depleted by magnetic sorting. The CD 137 depleted cells were re
plated with IL-7 and IL-15 and cultured until day 14 ("Anti
3.sup.rd CD137+ and Anti host CD137-"). On day 14, anti 3rd party
and anti-host alloreactivity was evaluated by CFSE assay against
3rd party or irradiated host PBMCs. For the CFSE assay,
1.times.10.sup.6 CFSE+ responders were incubated with or without
2.times.10.sup.6 irradiated (20 gy) PBMC stimulators for 84 h in
the presence of IL-7. After 84 h, cells were recovered and analyzed
for cell division by measuring the number of CFSE low stained CD8 T
cells (CD3+CD8+CD56-) by FACS. To obtain absolute values of cells,
samples were suspended in a constant volume and flow cytometric
counts for each sample were obtained during a constant,
predetermined period. The number of specific dividing cells=(Number
of dividing cell with APC)-(Number of dividing cell without APC).
Negative values signify that the number of dividing cells in
response to activation with host PBMC was even lower that the
number of dividing cell without any activation.
Example 1
Generation and Optimization of Human Anti-Third Party T Central
Memory (Tcm) Cells
[0272] In order to translate the mouse studies previously presented
to clinical application, the procedure was optimized for generating
human anti-3rd party cytotoxic T lymphocytes (CTLs). To that end,
different parameters were evaluated including different reagents
for the isolation of CD8 responder cells, the composition of the
stimulators and the cytokine milieu.
[0273] Potentially, as found in the previously presented mouse
model, treatment with central memory T cells (Tcm) could be
valuable either in the context of autologous [Lask A et al., Blood
(ASH Annual Meeting Abstracts), (2010) 116: 424] or in allogeneic
bone marrow transplant (BMT) [Ophir E et al., Blood. (2010)
115(10): 2095-104; Ophir E., 37th EBMT annual meeting, Apr. 3-6,
2011, Paris, France. Oral Presentation Abstract Nr: 662].
[0274] In the human autologous setting (FIG. 1A) anti-3.sup.rd
party Tcm can be administrated together with autologous BMT. The
patient's own CD8+ T cells are isolated and stimulated against
allogeneic dendritic cells from an allogeneic donor.
[0275] In the human allogeneic setting (FIG. 1B), anti-3.sup.rd
party Tcm can be transplanted together with allogeneic T depleted
BM cells. Naive CD8+ T cells originating from the allogeneic BM
donor serve as responders and 3.sup.rd party donor dendritic cells
are used as stimulators to enable the generation of host
non-reactive Tcm. In order to avoid GVHD, the 3.sup.rd party donor
is selected so as to insure that none of his HLA class I alleles
are shared with the HLA class I alleles of the host.
[0276] While in both mouse and humans, the basic envisioned
protocol similarly comprised CD8 T cell isolation followed by
stimulation against 3.sup.rd party cells (FIGS. 1A-B and 2A-B),
several other parameters had to be modified in the human protocol,
as outlined in Table 1, below.
[0277] Considering that autologous Tcm are free of GVHD risk, the
optimization of the production protocol largely concentrated on
attaining effective expansion of anti-3.sup.rd party CD8 T cells
with central memory phenotype.
[0278] As can be seen in FIG. 3A, a new protocol was developed
based on three major steps: a) Selection of CD8 T cells from PBMC;
b) Stimulation against allogeneic dendritic cells (DC) for 3 days
in the presence of IL-21; and c) Expansion in an antigen free
environment with IL-7, IL-15 and IL-21 for an additional 8
days.
[0279] Thus, in this newly developed protocol various parameters
differ from that used for the generation of mouse Tcm (Table 1 and
FIGS. 3A-B). The major differences concern the tissue of origin for
the responders and stimulators (PBMC vs. splenocytes), the
stimulators (dendritic cells vs. splenocytes), as well as the
cytokine composition.
TABLE-US-00001 TABLE 1 Comparison of autologous human protocol
versus the syngeneic mouse protocol for generation of Tcm Human
Mouse Parameters Major stages Frozen PBMC Fresh Splenocytes Tissue
of origin Responders YES NO Depletion of adhered cells (Adherence
on plastic overnight + IL-7) YES NO Depletion of CD4.sup.+,
CD56.sup.+ Cells CD8+ T cells Whole Splenocytes Final cell
composition before co-culture with stimulators Frozen PBMC Fresh
Splenocytes Tissue of origin 3.sup.rdparty YES NO Generation of
monocyte Stimulators derived mature dendritic cells Dendritic Cells
Whole Splenocytes Final cell composition before co-culture with
responders CD8+ T cells.fwdarw. Splenocytes.fwdarw. Cell
composition Co-Culture: Irradiated DCs Irradiated (Priming)
Splenocytes 3 Days 2.5 Days Length of Co culture D0: IL-21 is
added. No Cytokines! Cytokines Non adherent cells Ficoll, CD8+
positive Co culture is stopped by: are transferred to a selection
and plating new plate in a new flask. IL-7, IL-15, IL-21 IL-15
Cytokines Antigen free NO YES Positive selection of CD62L+
Expansion Cells at the end of culture
[0280] Optimization of a GMP Grade Protocol for the Generation of
Human Anti-3.sup.rd Party Tcm:
[0281] The initial attempts to develop a human protocol for the
generation of anti-3.sup.rd party Tcm was based on a recent study
by Wolff et al. [Wolff M et al., Cancer Immunol Immunother (2011)
60(2): 173-186], who described a procedure for the generation of
human antigen specific CD8 T cells with a central memory
phenotype.
[0282] The present approach was based on stimulation against
antigen pulsed DC in the presence of IL-21 for 3 days and
subsequent expansion in the presence of IL-7 and IL-15 for an
additional 8 days.
[0283] In these initial experiments which resulted in an impressive
expansion of anti-3.sup.rd party Tcm and subsequently served as a
reference for further optimization, the following steps were used:
a) CD8 T cell enrichment from PBMCs by depletion of non-CD8.sup.+
cells. (i.e., CD4.sup.+ T cells, .gamma./.delta. T cells, B cells,
NK cells, dendritic cells, monocytes, granulocytes and erythroid
cells); b) Enrichment of naive cells by depletion of activated
cells expressing CD45RO; and c) Stimulation of naive CD8 T cells
against allogeneic dendritic cells in the presence of IL-21 for 3
days followed by expansion in an antigen free environment with IL-7
and IL-15 for an additional 8 days.
[0284] The results of these initial reference experiments, shown in
FIGS. 4A-C, enabled evaluation of the role of different parameters
in the protocol, by defining the impact of each parameter on the
level of cell expansion and expression level of Tcm phenotype (as
described in detail below).
[0285] The Role of Priming with 3Rd Party DCs
[0286] Considering that autologous Tcm are free of GVHD risk by
definition, the first parameter evaluated was the role of
stimulation against a 3.sup.rd party DC. This step was originally
intended to reduce the risk for GVHD in the allogeneic setting, by
selective expansion of anti-3.sup.rd party clones in the absence of
stimulation of anti-host clones mediating GVHD.
[0287] As illustrated in FIGS. 5A-C and 6A-B, naive CD8 T cells
grown with IL-21 in the absence of allogeneic stimulation by
dendritic cells, exhibited low proliferation level (2.7.+-.1.1% of
that exhibited by the reference control group), representing on day
7 approximately 0.4 fold expansion from day 0 (most cells died
before day 10), and maintaining their naive phenotype
(CD45RO-CD62L+, small morphology) (Tcm level was only 10% from that
of reference control group). A similar poor level of
differentiation and expansion was found when the cells were
maintained with IL-7 and IL-15 in the absence of allogeneic
stimulation by dendritic cells; under these conditions, the cells
maintained their naive phenotype (Tcm level was only 7.+-.1.6% from
that of reference control group), though some proliferation was
induced (12.+-.3.2% of the control group value, representing on day
13 approximately 6 fold expansion from day j). Thus, the role of
allogeneic third party DC was very critical for induction of the
Tcm phenotype and for robust cell expansion.
[0288] The Role of IL-21 in the Priming and Expansion Phases of
Anti-3Rd Party Tcm
[0289] Generally, both in mouse and human, conventional T cell
expansion protocols the expansion phase is performed in antigen
free environment. However, while in the mouse protocol, only IL-15
was added (FIG. 3B), in the human protocol described by Wolff et
al. (Wolff et al. 2011, supra), cell expansion was performed in the
presence of IL-7 and IL-15. Furthermore, considering that IL-21 was
shown to be beneficial if added during the initial priming phase,
the role of IL-21 was evaluated herein.
[0290] Interestingly, as shown in FIGS. 7A-C and 8A-B, while
priming of naive CD8 T cells by allogeneic DC in the presence or
absence of IL-21 had only minor effect on cell composition (data
not shown), priming in the absence of IL-21 hampered the
acquisition of Tcm phenotype (CD45RO+CD62L+) (Only 69.+-.18% of the
Tcm level in the reference control group), and also resulted in
reduced proliferation (76.+-.23% of the expansion level in the
reference control group) (FIGS. 8A-B). Even in the single
experiment out of four, in which expansion was not reduced (140% of
reference control group) the Tcm phenotype was only 35% of the Tcm
level in the reference control group, suggesting that priming in
the presence of IL-21 is important for both expansion and induction
of Tcm phenotype from the naive CD8 T cell population.
[0291] Interestingly, continuous presence of IL-21 in both the
priming phase (IL-21 alone) and in the expansion phase (together
with IL-7 and IL-15) consistently improved induction of cells of
the central memory phenotype (108.+-.1.9% of the Tcm level in the
reference control group) (FIGS. 7A-C and 8A-B). The impact of
continuous IL-21 presence on cell expansion, although clearly
leading to higher average increase (135.+-.47% of the values found
in the reference control group) was less consistent, leading in two
out of three experiments to slightly reduced expansion (95% and 81%
of reference values, respectively), while in the third experiment,
exhibiting dramatically enhanced expansion (228%), indicating that
adding IL-21 in both the priming and expansion phases might be
desirable (FIGS. 8A-B).
[0292] Composition of the 3.sup.Rd Party Stimulator Cells
[0293] The results described above show that the sequential
addition of IL-21, IL-7, and IL-15 must be accompanied by
allogeneic stimulation by monocyte derived mature DC for successful
induction of Tcm phenotype and for robust cell expansion.
[0294] To simplify the procedure, an experiment was carried out to
evaluate whether the essential allogeneic stimulation could be
delivered by irradiated PBMCs instead of monocyte derived mature DC
that requires a 4 day preparation.
[0295] As can be seen in Table 2 below, at 7 days of culture, the
efficiency of induction of Tcm phenotype by PBMC, as opposed to
monocyte derived mature DC (md-mDC), stimulators was very similar,
both when purified naive CD8 T cells (92% vs. 92%, respectively)
and when un-separated CD8 T cells served as responders (77% vs.
80%). However, the PBMC stimulators were not able to elicit the
same level of Tcm expansion compared to DCs using either naive CD8
T cells (6.75 vs. 20.5, respectively) or un-separated CD8 T cells
(1.8 vs. 16) as responders.
TABLE-US-00002 TABLE 2 The critical role of DCs as stimulators %
Tcm Fold expansion (CD3 + CD8 + from day 0 CD62L + CD45RA-) (on day
7) Group 92 20.5 Naive CD8 T cells .fwdarw.(md-mDC) 92 6.75 Naive
CD8 T cells .fwdarw. PBMC 80 16 Un-separated CD8 T cells .fwdarw.
(md-mDC) 77 1.8 Un-separated CD8 T cells .fwdarw. PBMC MLR culture
in which naive CD8 T cells or unseparated CD8 T responders were
stimulated against monocyte-derived mature DC (8:1 responder/DC
ratio) or PBMC (1:1 responder/PBMC ratio), from the same allogeneic
donor, in a medium containing IL-21 for 3 days. Thereafter, the
cells were grown with IL-7 and IL-15 until day 7 without further
activation. On day 7 of the culture, the different groups were
evaluated for percentage of Tcm using FACS analysis and for cell
numbers by trypan blue exclusion.
[0296] Thus, unlike the mouse protocol in which irradiated
splenocytes were sufficient for inducing expansion, in the human
protocol, allogeneic monocyte derived mature DC are crucial for
good expansion of Tcm cells and cannot be replaced by allogeneic
PBMC.
[0297] Defining the Optimal Responder/DC Ratio for the Induction of
Tcm Phenotype and Cell Expansion.
[0298] To define the optimal responder/DC ratio, a MLR system was
used, as described above, except that different responder/DC ratios
were tested. As can be seen in FIGS. 9A-B, when using
4.times.10.sup.5 responders, an optimal acquisition of Tcm
phenotype was attained upon addition of 50-100.times.10.sup.3 DC,
while expansion was optimal at the lowest DCs concentration.
Further experiments at lower responder/DC ratios are examined.
[0299] Defining a Final Autologous Protocol Based Exclusively on
GMP Grade Reagents.
[0300] Upon establishment of a satisfactory autologous protocol for
the generation of anti-3.sup.rd party Tcm, an experiment was
carried out to develop an equivalent procedure based solely on GMP
grade reagents currently available commercially so as enable
testing of this approach in human patients.
[0301] Depletion of Adherent Cells on Plastic Dishes.
[0302] Before optimizing the process of CD8 T cell selection, an
experiment was carried out to attain a significant initial
enrichment by removal of plastic adherent cells present in PBMC.
This process not only increases the concentration of the desired
CD8+ T cells but is also useful when processing cryopreserved human
PBMC as opposed to fresh splenocytes used in the mouse model. This
experiment revealed that overnight incubation with 10% human serum
and IL-7 allowed the thawed cells to recover before being subjected
to the magnetic enrichment process (data not shown).
[0303] Enrichment of Naive CD8 T Cells
[0304] Next, the focus was on adapting the enrichment of naive CD8
T cells to clinical grade reagents using as few antibodies as
possible
[0305] As shown in FIG. 10, representing a typical experiment, the
desired population of CD8 T cells represents 21% of the cells on
day 0 after depletion of adhered cells, while the other major
"contaminating" subpopulations include CD4 T cells (61%), B cells
(7%) and NK cells (7%). Thus, the CD4 cells represent the largest
contamination, and were previously shown to compete with CD8 T
cells; these cells therefore had to be removed. Likewise, it was
important to remove NK cells that are known to expend in IL-15
cultures. In contrast, B cells tend to die under these culture
conditions. Thus, potential depletion with anti-CD4 and anti-CD56
magnetic beads was initially evaluated with and without depletion
of CD19+ B cells.
[0306] In addition, since in PBMC of patients with B cell
malignancies, the levels of CD8 T cells are lower compared to
healthy donors, a parallel evaluation was carried out examining the
possibility of omitting the enrichment of naive CD8 T cells by
positive selection of CD45RA+ cells, as it further decreases the
number of recovered CD8 T cells.
[0307] Thus, on day -1 donor PBMC were first depleted from adherent
cells by overnight incubation in greiner-bio-one CELLSTAR tissue
culture plates (Greiner Bio-One Ltd., Stonehouse UK), specifically
designed to remove adherent myeloid cells and on day 0 non-adherent
cells were divided to four experimental groups, each subjected to a
different magnetic sorting protocol. On days 0, 7, 10 and 14 of
culture, cells were evaluated for cell composition and Tcm
phenotype by FACS analysis and for expansion by counting live cells
based on trypan blue exclusion.
[0308] As can be seen in FIG. 10, minimal magnetic cell sorting
using only anti-CD4 and anti-CD56 beads, decreased the percentage
of CD4 T and NK cells from 61% and 7% to 12% and 1%, respectively,
resulting in enrichment of CD8 T cells from 23% to 60%. However,
this procedure was associated with enhancement of B cell levels
from 7% to 24%. Adding anti-CD19 to the depletion cocktail
completely depleted the B cells, resulting in improved enrichment
of CD8 T cells (90%). Adding a second step of positive enrichment
of naive cells with anti-CD45RA increased the percent of naive
cells (CD45RO-CD45RA+, gated on CD3+CD8+) from 53% before magnetic
sorting to 91% in both groups. However, this step did not markedly
affect the final level of CD8 T cells, which increased from 60% to
66% when using anti-CD4 and anti-CD56, or from 90% to 94% when the
negative selection step also included anti-CD19.
[0309] After the magnetic cell sorting, all four groups were primed
with allogeneic dendritic cells in the presence of IL-21 for 3
days, and thereafter, the cells were expanded in an antigen free
environment until day 14, in the presence of IL-21, IL-7, and
IL-15. As a control group for non-expanded cells, naive CD8 T cells
enriched by a depletion step using anti-CD4, anti-CD56, and
anti-CD19, and a positive enrichment step using anti-CD45RA, were
maintained in an antigen free environment in the presence of only
IL-7 until day 14.
[0310] Interestingly, while on day 0, the groups treated or
untreated with anti-CD19 exhibited markedly different levels of CD8
T cells, this difference was abolished as early as day 7 (FIG. 11),
and also when tested on day 14 (FIG. 12), likely due to selective
death of B cells in the culture. Similarly, the positive selection
of CD45RA+ cells after the initial CD8 T cell purification only
marginally contributed to the final enrichment of CD8+ T cells with
a Tcm phenotype. Thus, all four groups showed similar levels of the
desired cells with a minor advantage for the two groups also
enriched for naive cells by anti CD45RA.
[0311] This initial result shown above in a typical experiment was
further analyzed by comparing average results attained in the
control group exposed to optimal cell isolation reagents
("CD4-CD56-CD19-, CD45RA+") to the other groups in which an attempt
to eliminate the use of anti-CD19 or anti CD45RA was done.
[0312] Thus, the average percent of CD8 T cells (FIG. 13A), and
more importantly, the percent Tcm (FIG. 13B) in all the
experimental groups when calculated as a percent of the level
attained in the optimal control group were very similar.
[0313] In contrast, marked differences were found in the average
expansion of each cell preparation when tested on day 10 of
culture, ranging from 65.6.+-.0.5% to 105.2.+-.6.8% (FIG. 14A).
However, the differences in expansion capacity were counteracted by
the reduced yield associated with the second purification step
(FIG. 14B).
[0314] As can be seen in FIG. 14C, showing the final calculated
yield of Tcm at day 10, the addition of a second step of positive
enrichment of naive cells with anti-CD45RA only decreased the final
yield of Tcm cells, from 141% to 123%, when using initial depletion
with anti-CD4 and anti-CD56, or from 157% to 100%, when the
negative selection step also included anti-CD19.
[0315] Collectively these results suggest that the protocol based
on minimal use of reagents for the isolation of CD8 T cells, namely
negative selection with anti-CD4 and anti-CD56, is satisfactory for
clinical application in the autologous setting.
Example 2
Large Scale Preparation of Human Anti-3.sup.Rd Party Tcm in Plastic
Bags Using GMP Grade Reagents
[0316] To simulate the conditions anticipated when using patients
own PBMC for the generation of autologous Tcm, initially two large
scale leukaphersis procedures were performed, from two normal
donors, and a large number of mononuclear cells were cryopreserved
(divided into several batches). Each batch was used for one large
scale experiment. In the first experiment, several technical
problems were encountered including difficulty in the generation of
DCs from the frozen bags according to the Wurzburg protocol and
sourcing of a new GMP grade IL-15 for which the biological activity
was unclear. These problems, which resulted in a very poor CD8 T
cell expansion (around 3 folds), were corrected in the subsequent
experiments by using the presently described protocol for DCs (as
described above) and by using the appropriate concentration of
IL-15 (i.e. 300 U/ml).
[0317] As can be seen in FIG. 15, when PBMC of the same donor were
generated in two large scale experiments against two different
3.sup.rd party DCs, similar CD8 T cell expansion was attained
ranging from 26.8 to 31.0 folds at day 11. Considering that at this
day the cells exhibited a linear growth it is likely that further
expansion could be attained at later time points. However, this
level of expansion is satisfactory as it allows potential
administration of up to 3.times.10.sup.7 cells per Kg body weight.
Interestingly, while at day 0 it was found that the leukaphersis
preparation was largely contaminated with CD14+ monocytes and CD20
B cells, these cells disappear upon cell culture and the final cell
composition at day 12 comprised 94% and 98% CD8+CD3+ T cells,
respectively (FIGS. 16A-B)
[0318] Importantly, Tcm phenotype attained in the two cultures
against different DCs were within the range of the small scale
experiments although some variability occurred (FIGS. 17A-B). Thus,
while at day 5 in both experiments high level of Tcm phenotype (77%
and 71%, respectively) was found, this level declined more
significantly in the culture against the 2.sup.nd DC donor upon the
9th day (65% vs. 46%) and day 12 (62% vs. 35%). This variability
which was not observed significantly in the small scale experiments
could be explained in part by a relative difficulty to remove the
DCs on day 5, as they are less likely to adhere to the plastic bag
compared to their adhesion to the plates used in the small scale
experiments. Thus, the longer presence and stimulation with the DCs
could lead to a more pronounced transition from a Tcm to a Teff
phenotype.
Example 3
GVL Potential of the Human Anti-3.sup.rd Party Tcm Against an
Established Cell Line
[0319] Considering that in the autologous setting the potential use
of anti-3.sup.rd party Tcm is solely for eradicating residual tumor
cells (in the allogeneic setting it also serves to enhance
engraftment of the BM cells) it is important to develop a
straightforward assay for cytotoxic capacity ex-vivo, which could
be used for quality control prior to infusion of the Tcm to the
patient. To that end, a TCR independent assay was used based on the
demonstration by Lask et al. [Lask A et al., J. Immunol. (2011)
187(4):2006-14] that a MHC mutated line not recognizable by TCR due
to this mutation, can still be killed by anti-3.sup.rd party CTLs
through their TCR independent killing mechanism. Clearly, if
applicable also for human Tcm, such a killing modality could serve
to distinguish the Tcm killing from that exhibited by NK cells.
[0320] To address this question, a mixed lymphocyte reaction (MLR)
was carried out with anti-3.sup.rd party Tcm targeting B-cell
lymphoma and plasma cell leukemia cell lines and the percent
apoptotic cells was measured after 22 hours. As can be seen in FIG.
17C, representing a typical experiment, marked GVL reactivity was
exhibited by the Tcm.
[0321] In a different experiment, CD8 T cells were first enriched
by extensively depleting non CD8 T cells (i.e., CD4+ T cells,
.gamma./.delta. T cells, B cells, NK cells, dendritic cells,
monocytes, granulocytes, and erythroid cells) using magnetic bead
sorting. As can be seen in FIG. 18, representing a typical
experiment, the percent of contaminating NK and NKT cells was very
low (below 0.1% for NK cells, and below 1.9% for NKT cells) for all
four groups tested.
[0322] The highly purified CD8 T cells were then incubated with two
types of cells: a) H.My C1R HLA-A2 K66A mutant cell line (K66A) to
demonstrate TCR independent killing, and b) H.My C1R (Neo), a
B-cell lymphoblastoid line lacking surface HLA A and B antigens and
therefore insensitive to killing by Tcm via a mechanism which
requires interaction between the CD8 molecule on the Tcm and the
.alpha.3 domain on MHC of the target leukemia cells.
[0323] As shown in FIGS. 19A-D, marked killing of the K66A mutated
target cells compared to the MHC-1-deficient H.My C1R (Neo) cells
was exhibited by anti-3.sup.rd party Tcm. Thus, human Tcm similarly
to human anti-3.sup.rd party CTLs can kill B cell tumor cells
through a TCR independent killing mechanism, which, in contrast to
NK mediated killing that requires MHC expression on the target
cells.
[0324] Most importantly, when further analyzed by comparing average
results attained in the control group exposed to optimal cell
isolation reagents ("CD4- CD56- CD19-, CD45RA+") to the other
groups in which the use of anti-CD19 or anti CD45RA (FIGS. 19A-D)
was reduced, showed that the percent TCR independent killing of the
H.My C1R HLA-A2 K66A mutant cell line in all the experimental
groups (calculated as a percent of the level attained in the
optimal control group) was very similar (FIG. 20) (P>0.05 when
comparing all three test groups to the reference control
group).
[0325] Collectively, these results suggest that the GVL reactivity
exhibited by cells isolated with a minimal use of reagents for the
isolation of CD8 T cells, is not inferior to that associated with
more extensive isolation protocols.
[0326] The killing of autologous B-CLL tumor cells by Tcm in-vivo
are done, using a Hu/SCID model previously employed for the
demonstration of such B-CLL killing by anti-3.sup.rd party
CTLs.
Example 4
Generation of Allogeneic Human Anti-3.sup.rd Party Tcm Cells
[0327] Initiation of a New GMP Grade Approach to Minimize Risk of
GVHD when Using Allogeneic Human Anti-3.sup.rd Party Tcm
[0328] As previously demonstrated in a mouse model, anti-3.sup.rd
party Tcm could be very useful for tolerance induction in
allogeneic BMT [Ophir E. et al. Blood. (2010) 115(10):2095-104]. In
this case, naive CD8+ T cells originating from the allogeneic BM
donor serve as responders, and 3.sup.rd party donor dendritic cells
(DC) are used as stimulators to enable the generation of host
non-reactive Tcm cells. In order to avoid GVHD, the 3.sup.rd party
donor is selected so as to ensure that none of his HLA class I
alleles are shared with the HLA class I alleles of the host.
[0329] Nonetheless, considering that human patients may be more
prone to GVHD than inbred mice, clinical translation of this
approach must be pursued with caution. Additional allo-depleting
steps, such as photo-depletion or selection of activated cells at
the end of the anti-third party allo-stimulation period, might be
required in order to further reduce the risk of GVHD.
[0330] Modifications of the Autologous Human Protocol (for
Allogeneic Protocol)
[0331] As shown in FIGS. 21-22, the protocol for generating Tcm for
the allogeneic setting differs from the protocol for the autologous
setting in two major steps: [0332] a) Selection of CD45RA+ cells
following the isolation CD8 T cells. Memory T-cells have lower
activation threshold than naive T cells that can cause non-specific
cytokine-driven expansion of the memory T-cell fraction. These
cells may include clones that cross-react with host antigens, thus
increasing the risk for GVHD induction. In order to minimize the
effect caused by the difference in percentage of naive T cells
between different human donors, and to reduce the risk for GVHD,
naive CD8 T (CD45RA+CD8+) cells were used as the source for the
generation of Tcm cells. [0333] b) Removal of potentially host
reactive T cells at the end of the culture, by depletion of CD137+
activated CD8 T cells.
[0334] Extension of the IL-7 and IL-15 Deprivation Period
[0335] As described for the autologous cultures (hereinabove), the
present inventors have observed that naive CD8 T cells exposed to
IL-7 and IL-15 proliferate in an antigen independent manner. On the
other hand, naive CD8 T cells exposed to IL-21 in the absence of
allogeneic stimulation did not proliferate, and did not even
survive beyond day 7 of culture. Therefore, delaying the addition
of IL-7 and IL-15 from day 3 to day 7 could potentially lead to a
selective depletion of anti-host clones not responsive to the
3.sup.rd party stimulators.
[0336] In order to define the optimal timing for the addition of
cytokine vis-a-vis alloreactivity depletion, naive CD8 T cells were
stimulated with irradiated allogeneic 3rd party DC at a ratio of
4:1 in the presence or absence of IL-21 for 7 days. Thereafter, the
cells received no further activation and were expanded with IL-7
and IL-15 (FIG. 23B), IL-15 and IL-21 (FIG. 23C), or IL-15 alone
(FIG. 23D) until day 13; the resulting cell populations were
compared to the naive CD8 T cells cultured according to the
reference control group, expanded as described for the autologous
setting (incubation on d (0-3) with IL21 and DC; on d(3-13),
addition of IL7+IL15 (FIG. 23A).
[0337] Using the same sequence of cytokine addition as the
reference control group but with different timing, namely, IL-21
addition was extended from 3 days to 7 days, and IL-7 and IL-15
were added on day 7 and not on day 3, hindered the expansion of the
cells (FIG. 24A) (proliferation only to 54.+-.7% of that exhibited
by the reference control group). However, induction of central
memory phenotype was similar (FIG. 24B) (99.+-.14.8% of that
exhibited by the reference control group).
[0338] As shown, removing IL-7 and extending the addition of IL-21
to the end of the culture, reduced expansion of the cells (FIG.
24A) (60.+-.13% of the proliferation exhibited by the reference
control group), as well as decreased central memory phenotype
acquisition (82.+-.6.8% of the Tcm level exhibited by the reference
control group, FIG. 24B).
[0339] Priming of naive CD8 T cells using 7 days cytokine
deprivation, followed by addition of only IL-15 from day 7
drastically reduced the expansion potential of the cells (only
5.+-.1.3% proliferation of that exhibited by the reference control
group), and also decreased central memory phenotype acquisition
(FIG. 24B) (68.+-.26% of the Tcm level exhibited by the reference
control group).
[0340] The most critical parameter, namely depletion of host
reactive clones (tested with appropriate donors who are completely
distinct in HLA Class I from the 3.sup.rd party cells used for
stimulation) are examined. Further experiments to optimize the
cytokine deprivation period are also carried out.
[0341] A Two Stage Magnetic Sorting Approach for Depletion of
Alloreactivity, Based on THE CD137 Activation Marker
[0342] An elegant way to deplete anti-host clones based on the
3.sup.rd party concept may be achieved by a two stage magnetic
sorting technique, comprising the following CD137 selection
steps:
[0343] a. Positive selection of anti-3.sup.rd party specific clones
at the beginning of the culture.
[0344] b. Depletion of anti-host specific clones near the end of
the culture.
[0345] Recently, CD137 has been described to be a suitable marker
for antigen-specific activation of human CD8.sup.+ T cells, as
CD137 is not expressed on resting CD8.sup.+ T cells and its
expression is reliably induced after 24 hours of stimulation.
[0346] In order to evaluate this approach, naive CD8 T cells were
stimulated with irradiated allogeneic 3rd party DC in the presence
of IL-21. After 14 hours of activation, CD137+ cells were
positively selected by magnetic sorting. CD137+ cells were then
re-stimulated with irradiated allogeneic 3rd party DC in the
presence of IL-21 until day 3. Thereafter, the cells were expanded
with IL-7 and IL-15 until day 10 and, and were then activated with
irradiated host PBMC in the presence of IL-7 and IL-15. After 24
hours of activation, CD137+ cells were depleted by magnetic
sorting. The CD 137 depleted cells were re-plated with IL-7 and
IL-15 and cultured until day 14. On selected days, cells were
evaluated for cell numbers by trypan blue exclusion and percentage
of Tcm (CD62L+CD45RO+) within the CD8 T cell population using FACS
analysis. Frequency of anti-3.sup.rd party and anti-host
alloreactive cells was evaluated by CFSE assay against 3.sup.rd
party or host irradiated PBMCs. These results were compared to
those attained in the control group stimulated in the presence of
IL-21 for 3 days and thereafter expanded with IL-7 and IL-15
("reference control group").
[0347] Thus, as can be seen in FIG. 25, while immediately after
enrichment for naive CD8 T cells (day 0), only 0.7% of the total
CD8 T cells expressed CD137+, upon activation against 3.sup.rd
party DC in the presence of IL-21 for 14 h, the percentage of CD8 T
cells expressing CD137+ from the total CD8 T cell compartment
increased to 8.3% as opposed to 2.5% in the absence of DC
stimulation. Magnetic sorting of this subpopulation of activated
cells led to marked enrichment of CD137+ cells (85%, respectively)
and the level of CD62L+CD8 T cells in the total CD8 T cell
compartment drastically decreased from 84% to 14% (data not
shown).
[0348] As shown in Table 5, below, this positive selection was
associated with reduced cell recovery. Thus, on day 0, the yield
from PBMC depleted of adherent cells after enrichment for naive CD8
T cells was 7.6% and on day 1, after the positive selection for
CD137+, the yield decreased to 0.25% (3.3% of 7.6%). When evaluated
on day 7 of culture, the test group of CD8 T cells subjected to
positive selection of CD137+ cells highly resembled the reference
control group in the percent of Tcm cells (67% vs. 70%,
respectively), and this similarity between the groups in percent
Tcm was also maintained on day 10 of culture (54% vs. 52%,
respectively) (FIG. 26).
TABLE-US-00003 TABLE 5 Comparison of proliferation and final cell
number e d c b a Group (7.6) .times. (61) = 463% 61 7.6% Reference
control (0.25) .times. (72) = 18% 72 (3.3% from Anti-3rd CD137+
7.6%) = 0.25% and Anti host CD137- Naive CD8 T cells were
stimulated with irradiated allogeneic 3rd party DC at a ratio of
4:1 in the presence of IL-21 for 3 days. The cells received no
further activation thereafter and were expanded with IL-7 and IL-15
until day 14 ("Reference control group"). Alternatively, naive CD8
T cells were stimulated with irradiated allogeneic 3rd party DC at
a ratio of 5.7:1 in the presence of IL-21. After 14 hours of
activation, CD137+ cells were positively selected by magnetic
sorting. CD137+ cells were then re-stimulated with irradiated
allogeneic 3rd party DC in at a ratio of 4:1 in the presence of
IL-21 until day 3. Thereafter, the cells were expanded with IL-7
and IL-15 until day 10. On day 10, were activated with irradiated
host PBMC in the presence of IL-7 and IL-15 (at a ratio of 1 to 2).
After 24 h, CD137+ cells were depleted by magnetic sorting. The
CD137 depleted cells were re plated with IL-7 and IL-15 and
cultured until day 14 ("Anti 3.sup.rd CD137+ and Anti host
CD137-"). On the indicated days, cells were counted by trypan blue
exclusion a = Yield after enrichment of na+e+,uml ive CD8 T cells
(Represented as percent of starting number of PBMC-adhered cells).
b = Yield after activation with 3rd party DCs and enrichment of
CD137+ CD8 T cells (Represented as percent of starting number of
PBMC-adhered cells). c = Fold expansion from day 0 at day 13. d =
Fold expansion from day 0 at day 14. e = Final cell number =
(Yield) .times. (Fold Expansion from day 0) (Represented as percent
of starting number of PBMC-adhered cells).
[0349] Moreover, when cell composition (% CD8 T cells, % NK cells
and % NKT cells) was evaluated on days 7 and 10 of culture, the
test group of CD8 T cells subjected to positive selection of CD137+
cells, highly resembled the reference control group in its cell
composition (Table 6, below).
TABLE-US-00004 TABLE 6 Enrichment of anti-3.sup.rd party specific
CD8 T cells by positive selection of CD137+ cells does not
drastically change cell composition (NKT 16+) (NKT 56+) (NK16+)
(NK56+) CD3+ CD3+ CD3- CD3- (CD8 T cells) CD16+ CD56+ CD16+ CD56+
CD3 + CD8+ Day 7 2.6 5.6 1.3 2 92.5 Reference control group 5.2 7.8
3.8 3.2 88 anti-3rd party CD137+ Day 10 2.8 8.3 3.2 2.1 91.8
Reference control group 3.6 6.3 4 4.1 87 anti-3rd party CD137+
Naive CD8 T cells were stimulated with irradiated allogeneic 3rd
party DC at a ratio of 4:1 in the presence of IL-21 for 3 days.
Thereafter, the cells received no further activation and were
expanded with IL-7 and IL-15 until day 10 ("Reference control
group"). Alternatively, naive CD8 T cells were stimulated with
irradiated allogeneic 3rd party DC at a ratio of 5.7:1 in the
presence of IL-21. After 14 hours of activation, CD137+ cells were
positively selected by magnetic sorting. CD137+ cells were then
re-stimulated with irradiated allogeneic 3rd party DC at a ratio of
4:1 in the presence of IL-21 until day 3. Thereafter the cells were
expanded with IL-7 and IL-15 until day 10. Cells were evaluated for
cell composition by FACS analysis.
[0350] On the other hand, as shown in FIG. 27, the test group of
CD8 T cells subjected to positive selection of CD137+ cells,
exhibited superior expansion potential in comparison to the
reference control group at both time points (35 vs. 7 fold
expansion from day 0 on day 7, respectively, and 119 vs. 34 fold
expansion from day 0 on day 10, respectively). On day 10, the group
of CD8 T cells subjected to positive selection of CD137+ cells was
divided into two test groups. In the first group, cells continued
to be expanded with IL-7 and IL-15 until day 14 ("Anti 3.sup.rd
CD137+"), while cells in the second test group were activated with
irradiated host PBMC in the presence of IL-7 and IL-15. After 24 h
of activation CD 137+ cells were depleted by magnetic sorting. The
CD 137 depleted cells were then re plated with IL-7 and IL-15 and
cultured until day 14 ("Anti 3.sup.rd CD137+ and Anti-host
CD137-").
[0351] When evaluated on day 13, CD8 T cells from the test group
subjected to positive selection of CD137+ continued to exhibited
superior expansion potential in comparison to the reference control
group at both time points (134 vs. 61 fold expansion,
respectively). In contrast, CD8 T cells from the test group
subjected to both anti-3rd party positive selection of CD137+ and
depletion of anti-host CD137+ cells, exhibited lower expansion
potential when evaluated on day 14 (72 fold expansion) (FIG. 27)
indicating that cell expansion between days 11 to 14 could not
compensate for the loss of cells caused by the depletion of CD137+
anti-host specific alloreactive T cells.
[0352] As shown in FIG. 28, when CD137 expression was evaluated on
day 10, only 0.5% of the total CD8 T cells compartment in the CD8 T
cells subjected to positive selection of CD137+ cells expressed
CD137+. Thus, the CD8 T cells in this group down-regulated
considerably the expression of CD137 (from 85% on day 1 to only 0.5
on day 10).
[0353] However, after 24 h of activation with irradiated host PBMC
(at a 1 to 2 ratio, in favor of the host PBMC), the percent of CD8
T cells expressing CD137+ from the total CD8 T cell compartment
increased to 16%. Depletion of these CD137+ cells by magnetic
sorting decreased the percent of CD8 T cells expressing CD137 of
the total CD8 T cell compartment, from 16% to 3%.
[0354] Final analysis of residual anti-host alloreactivity was
performed on day 14, by comparing the level of CFSE retaining cells
upon stimulation against host PBMC as opposed to 3.sup.rd party
PBMC in the presence of IL-7.
[0355] As shown in FIG. 29, the number of cells specifically
dividing after stimulation with 3.sup.rd party PBMC was
approximately 3 times higher in the group subjected to the CD137
based positive and negative selection compared to the reference
control group (2259 vs.741 dividing cells, respectively). Most
importantly, the removal of CD 137+ cells towards the end of the
culture, completely prevented proliferation in response to host
PBMC, in contrast to the reference control group which exhibited
detectable proliferation (134 dividing cells). Interestingly, the
group undergoing positive selection of cells activated against
3.sup.rd party without removal of anti-host clones at the end of
the culture, exhibited higher level of host reactive cells compared
to the control group, indicating potential cross reactivity between
the MHC allotypes of host and 3.sup.rd party stimulators (although
deliberately mis-matched by HLA typing.) Thus, while the importance
of anti-host depletion step at the end of the culture is clearly
indicated, further studies are required to evaluate the potential
role of the first positive selection of anti-3.sup.rd party
activated cells.
[0356] However, as shown in Table 5, above, the successful
depletion of anti-host specific clones by the two stage CD137 based
magnetic sorting, affords on the whole lower cell recovery at the
end of the culture (18% vs. 463%, represented as percent from input
number of PBMC-adherent cells, respectively).
[0357] Collectively, this preliminary experiment indicates that
depletion of alloreactivity by two-stage magnetic sorting, based on
the CD137 activation marker, is feasible and might be incorporated
into the present protocol for generating host non-reactive
allogeneic Tcm cells. Encouraging attributes indicated are: 1) the
high expression levels induced by the allogeneic activation upon
positive selection were completely down-regulated on day 10,
allowing for another allo-activation against host antigens. 2) Cell
composition and percent of Tcm cells were not drastically affected
by the magnetic sorting, based on the CD137 activation marker. 3)
The removal of CD137+ cells towards the end of the culture,
completely prevented proliferation in response to host PBMC, in
contrast to the reference control group, which exhibited detectable
proliferation (134 dividing cells). Current studies include: 1) the
use of FcR blocking before the positive selection step. 2) Using
Host DC instead of PBMC for more effective detection of host
reactive cells at the end of the culture. 3) Adding more clinically
available activation markers like CD25 or IFN gamma capture to the
depletion step.
CONCLUSIONS
[0358] The use of CD137 depletion at the end of Tcm generation
might afford a feasible approach to further deplete these cells of
alloreactivity.
[0359] Attempts to continue refining the use of CD137 depletion in
conjunction with CD25 depletion, both of which are available as GMP
reagents, are explored.
[0360] In addition, experiments are carried out to minimize
potential cross reactivity by using artificial APC bearing only one
HLA-I allele.
[0361] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0362] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
into the specification, to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated herein by reference. In
addition, citation or identification of any reference in this
application shall not be construed as an admission that such
reference is available as prior art to the present invention. To
the extent that section headings are used, they should not be
construed as necessarily limiting.
* * * * *