U.S. patent application number 11/802182 was filed with the patent office on 2007-11-15 for veto cells effective in preventing graft rejection and devoid of graft versus host potential.
This patent application is currently assigned to Yeda Research And Development Co. Ltd.. Invention is credited to Massimo Martelli, Yair Reisner.
Application Number | 20070264274 11/802182 |
Document ID | / |
Family ID | 23897145 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264274 |
Kind Code |
A1 |
Reisner; Yair ; et
al. |
November 15, 2007 |
Veto cells effective in preventing graft rejection and devoid of
graft versus host potential
Abstract
A method of transplanting a transplant derived from a donor into
a recipient is disclosed. The method comprises the steps of (a)
transplanting the transplant into the recipient; and (b)
administering to the recipient a dose including non-alloreactive
anti-third party cytotoxic T-lymphocytes (CTLs), wherein the
non-alloreactive anti-third party CTLs are generated by directing
T-lymphocytes of the donor against a third party antigen or
antigens, the dose is substantially depleted of T-lymphocytes
capable of developing into alloreactive CTLs, thereby preventing or
ameliorating both graft rejection by the recipient and graft versus
host disease.
Inventors: |
Reisner; Yair; (Old Jaffa,
IL) ; Martelli; Massimo; (Perugia, IT) |
Correspondence
Address: |
Martin D. Moynihan;PRTSI, Inc.
P.O. Box 16446
Arlington
VA
22215
US
|
Assignee: |
Yeda Research And Development Co.
Ltd.
Rehovot
IL
76100
|
Family ID: |
23897145 |
Appl. No.: |
11/802182 |
Filed: |
May 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10169028 |
Jun 27, 2002 |
7270810 |
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PCT/IL00/00872 |
Dec 28, 2000 |
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11802182 |
May 21, 2007 |
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09477737 |
Jan 5, 2000 |
6544506 |
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10169028 |
Jun 27, 2002 |
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Current U.S.
Class: |
424/184.1 ;
424/201.1; 424/204.1; 424/234.1 |
Current CPC
Class: |
A61K 2035/124 20130101;
C12N 2501/23 20130101; A61K 35/28 20130101; A61K 39/001 20130101;
A61K 2035/122 20130101; A61K 2039/5158 20130101; Y02A 50/30
20180101; Y02A 50/396 20180101; C12N 5/0636 20130101; A61K 35/28
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/184.1 ;
424/201.1; 424/204.1; 424/234.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 39/02 20060101 A61K039/02; A61K 39/12 20060101
A61K039/12; A61K 39/295 20060101 A61K039/295 |
Claims
1. A method of preventing or decreasing graft rejection in a human
recipient of a transplant which is derived from a non-syngeneic
donor, the method comprising: (a) conditioning the recipient under
lethal or sublethal conditions; (b) transplanting the transplant
into the recipient; and (c) administering to the recipient a dose
of a cell preparation, wherein said cell preparation comprises
non-alloreactive anti-third party cytotoxic T-lymphocytes (CTLs),
does not comprise lymphocytes capable of developing into
anti-recipient CTLs, and does not comprise CD4+ cells and/or CD56+
cells, and wherein said cell preparation is generated by a method
which comprises: (i) directing peripheral blood lymphocytes derived
from the non-syngeneic donor against a third party antigen or
antigens, thereby generating third party antigen-treated
lymphocytes which comprise anti-third party CTLs; (ii) depleting
said peripheral blood lymphocytes derived from the non-syngenic
donor of CD4+ cells and/or CD56+ cells; and (iii) depleting, by
affinity purification, said third party antigen-treated lymphocytes
of T-lymphocytes capable of developing into anti-recipient CTLs,
thereby preventing or decreasing graft rejection in the recipient
of the transplant.
2. The method of claim 1, wherein the non-syngeneic donor is
allogeneic with respect to the recipient.
3. The method of claim 1, wherein the non-syngeneic donor is
xenogeneic with respect to the recipient.
4. The method of claim 1, wherein the non-syngeneic donor is HLA
identical with respect to the recipient.
5. The method of claim 1, wherein the non-syngeneic donor is HLA
non-identical with respect to the recipient.
6. The method of claim 1, wherein the non-syngeneic donor is
mismatched haploidentical with respect to the recipient.
7. The method of claim 1, wherein the transplant is selected from
the group consisting of cells, a tissue and an organ.
8. The method of claim 1, wherein said 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, a synthetic peptide presented
by autologous antigen presenting cells and a synthetic peptide
presented by non-autologous antigen presenting cells.
9. The method of claim 8, wherein said third party cells are
allogeneic or xenogeneic with respect to the recipient.
10. The method of claim 9, wherein said third party cells have HLA
antigens which are different from HLA antigens of the non-syngeneic
donor but which are not cross reactive with HLA antigens of the
recipient.
11. The method of claim 9, wherein said third party cells are
stimulatory cells selected from the group consisting of cells
purified from peripheral blood lymphocytes, cells purified from
spleen, cells purified from lymph nodes, cytokine-mobilized PBLs
and in vitro expanded antigen-presenting cells (APC).
12. The method of claim 1, wherein steps (a) and (b) are effected
at the same time.
13. The method of claim 1, wherein step (a) is effected prior to
step (b).
14. The method of claim 1, wherein step (a) is effected following
step (b).
15. The method of claim 1, wherein affinity purification is
effected by an antibody.
16. The method of claim 15, wherein the antibody is selected from
the group consisting of anti-CD69, anti-CD25, anti-IL-2 and
anti-INF.gamma..
17. The method of claim 1, wherein the transplant comprises
immature hematopoietic cells.
18. The method of claim 17, wherein said immature hematopoietic
cells are CD34+ cells.
19. The method of claim 17, wherein said immature hematopoietic
cells are stem cells.
20. The method of claim 1, wherein the recipient has a malignant
disease.
21. The method of claim 1, wherein the recipient has a
hematopoietic malignant disease.
22. The method of claim 1, wherein the recipient has a
lymphoma.
23. A method of treating a human recipient suffering from a disease
requiring immature hematopoietic cell transplantation, the method
comprising: (a) conditioning the recipient under lethal or
sublethal conditions; (b) administering to the recipient a dose
which comprises immature hematopoietic cells derived from a
non-syngeneic donor which is allogeneic with respect to the
recipient; and (c) administering to the recipient a dose of a cell
preparation which comprises non-alloreactive anti-third party
cytotoxic T-lymphocytes (CTLs), does not comprise T-lymphocytes
capable of developing into anti-recipient CTLs, does not comprise
CD4+ cells and/or CD56+ cells, and is generated by a method which
comprises: (i) directing peripheral blood lymphocytes derived from
the non-syngeneic donor against a third party antigen or antigens,
thereby generating third party antigen-treated lymphocytes which
comprise anti-third party CTLs. (ii) depleting said peripheral
blood lymphocytes derived from said non-syngeneic donor of CD4+
cells and/or CD56+ cells; and (iii) depleting, by affinity
purification, said third party antigen-treated lymphocytes of
T-lymphocytes capable of developing into anti-recipient CTLs,
thereby preventing or treating the recipient suffering from the
disease requiring immature hematopoietic cell transplantation.
24. The method of claim 23, wherein the non-syngeneic donor is
allogeneic with respect to the recipient.
25. The method of claim 23, wherein the non-syngeneic donor is
xenogeneic with respect to the recipient.
26. The method-of claim 23, wherein said non-syngeneic donor is HLA
identical with respect to the recipient.
27. The method-of claim 23, wherein said non-syngeneic donor is HLA
non-identical with respect to the recipient.
28. The method-of claim 23, wherein said non-syngeneic donor is
mismatched haploidentical with respect to the recipient.
29. The method of claim 23, wherein said 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, a synthetic peptide presented
by autologous antigen presenting cells and a synthetic peptide
presented by non-autologous antigen presenting cells.
30. The method of claim 29, wherein said third party cells are
allogeneic or xenogeneic with respect to the recipient.
31. The method of claim 30, wherein said third party cells have HLA
antigens which are different from HLA antigens of said
non-syngeneic donor and which are not cross reactive with HLA
antigens of the recipient.
32. The method of claim 30, wherein said third party cells are
stimulatory cells selected from the group consisting of cells
purified from peripheral blood lymphocytes, cells purified from
spleen, cells purified from lymph nodes, cytokine-mobilized PBLs
and in vitro expanded antigen-presenting cells (APC).
33. The method of claim 23, wherein said immature hematopoietic
cells are cells which are derived from a source selected from the
group consisting of bone marrow, mobilized peripheral blood, fetal
liver, yolk sac and/or cord blood.
34. The method of claim 33, wherein said cells which are derived
from mobilized peripheral blood are obtained by leukapheresis of
peripheral blood of said non-syngeneic donor after stimulation of
said non-syngeneic donor with a suitable cytokine.
35. The method of claim 23, wherein affinity purification is
effected by an antibody.
36. The method of claim 35, wherein the antibody is selected from
the group consisting of anti-CD69, anti-CD25, anti-IL-2 and
anti-INF.gamma..
37. The method of claim 23, wherein said immature hematopoietic
cells are T-cell depleted hematopoietic progenitor cells.
38. The method of claim 23, wherein said immature hematopoietic
cells are CD34+ cells.
39. The method of claim 23, wherein a cell ratio between said
cytotoxic T-lymphocytes and said immature hematopoietic cells is at
least 1 to 100.
40. The method of claim 23, wherein said immature hematopoietic
cells are stem cells.
41. The method of claim 23, wherein steps (b) and (c) are effected
at the same time.
42. The method of claim 23, wherein step (b) is effected following
step (c).
43. The method of claim 23, wherein the disease is a malignant
disease.
44. The method of claim 23, wherein the disease is a hematopoietic
malignancy.
45. The method of claim 23, wherein the disease is a lymphoma.
46. A method of producing a cell preparation for preventing or
decreasing graft rejection in a human recipient of a transplant,
wherein the recipient is conditioned under sublethal conditions,
wherein the transplant is derived from a non-syngeneic donor,
wherein the cell preparation comprises non-alloreactive anti-third
party cytotoxic T-lymphocytes (CTLs), wherein the cell preparation
does not comprise T-lymphocytes capable of developing into
anti-recipient CTLs, and wherein the cell preparation does not
comprise CD4+ cells and/or CD56+ cells, the method comprising: (i)
directing peripheral blood lymphocytes derived from the
non-syngeneic donor against a third party antigen or antigens,
thereby generating third party antigen-treated lymphocytes which
comprise anti-third party CTLs; (ii) depleting, by affinity
purification, said third party antigen-treated lymphocytes of
T-lymphocytes capable of developing into anti-recipient CTLs; and
(iii) removing CD4+ cells and/or CD56+ cells from said peripheral
blood lymphocytes derived from the non-syngeneic donor, thereby
producing the cell preparation.
47. The method of claim 46, wherein the non-syngeneic donor is
allogeneic with respect to the recipient.
48. The method of claim 46, wherein the non-syngeneic donor is
xenogeneic with respect to the recipient.
49. The method of claim 46, wherein step (iii) is effected by
removing both the CD56+ cells and the CD4+ cells from said
peripheral blood lymphocytes derived from the non-syngeneic
donor.
50. The method of claim 46, wherein said 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, a synthetic peptide presented
by autologous antigen presenting cells and a synthetic peptide
presented by non-autologous antigen presenting cells.
51. The method of claim 50, wherein said third party cells are
allogeneic or xenogeneic with respect to the recipient.
52. The method of claim 51, wherein said third party cells have HLA
antigens which are different from HLA antigens of the non-syngeneic
donor and which are not cross reactive with HLA antigens of the
recipient.
53. The method of claim 51, wherein said third party cells are
stimulatory cells selected from the group consisting of cells
purified from peripheral blood lymphocytes, cells purified from
spleen, cells purified from lymph nodes, cytokine-mobilized PBLs
and in vitro expanded antigen-presenting cells (APC).
54. The method of claim 46, wherein affinity purification is
effected by an antibody.
55. The method of claim 54, wherein the antibody is selected from
the group consisting of anti-CD69, anti-CD25, anti-IL-2 and
anti-INF.gamma..
56. A cell preparation for preventing or decreasing graft rejection
in a human recipient of a transplant, wherein the recipient has
been conditioned under lethal or sublethal conditions, wherein the
transplant is derived from a non-syngeneic donor wherein the cell
preparation comprises non-syngeneic donor derived non-alloreactive
anti-third party cytotoxic T-lymphocytes (CTLs), the cell
preparation comprising non-syngeneic donor derived non-alloreactive
anti-third party cytotoxic T-lymphocytes (CTLs), and wherein the
cell preparation does not comprise T-lymphocytes capable of
developing into anti-recipient CTLs and does not comprise CD4+
cells and/or CD56+ cells; the cell preparation having been
generated by a method which comprises: directing peripheral blood
lymphocytes derived from the non-syngeneic donor against a third
party antigen or antigens, thereby generating third party
antigen-treated lymphocytes which comprise anti-third party CTLs;
depleting said peripheral blood lymphocytes derived from the
non-syngeneic donor of CD4+ cells and/or CD56+ cells; and
depleting, by affinity purification, said third party
antigen-treated lymphocytes of T-lymphocytes capable of developing
into anti-recipient CTLs.
57. The method of claim 56, wherein the non-syngeneic donor is
allogeneic with respect to the recipient.
58. The method of claim 56, wherein the non-syngeneic donor is
xenogeneic with respect to the recipient.
59. The cell preparation of claim 56, wherein said 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, a synthetic peptide
presented by autologous antigen presenting cells and a synthetic
peptide presented by non-autologous antigen presenting cells.
60. The cell preparation of claim 59, wherein said third party
cells are allogeneic or xenogeneic with respect to the
recipient.
61. The cell preparation of claim 60, wherein said third party
cells have HLA antigens which are different from HLA antigens of
the non-syngeneic donor and which are not cross reactive with HLA
antigens of the recipient.
62. The cell preparation of claim 60, wherein said third party
cells are stimulatory cells selected from the group consisting of
cells purified from peripheral blood lymphocytes, cells purified
from spleen, cells purified from lymph nodes, cytokine-mobilized
PBLs and in vitro expanded antigen-presenting cells (APC).
63. The cell preparation of claim 56, wherein the cell preparation
further comprises immature hematopoietic cells derived from the
non-syngeneic donor.
64. The cell preparation of claim 63, wherein said immature
hematopoietic cells are derived from a source selected from the
group consisting of bone marrow, mobilized peripheral blood, fetal
liver, yolk sac and cord blood.
65. The cell preparation of claim 64, wherein said cells derived
from mobilized peripheral blood are obtained by leukapheresis of
peripheral blood of the non-syngeneic donor after stimulation of
the non-syngeneic donor with a suitable cytokine.
66. The cell preparation of claim 63, wherein said immature
hematopoietic cells are T-cell depleted hematopoietic progenitor
cells.
67. The cell preparation of claim 63, wherein said immature
hematopoietic cells are CD34+ cells.
68. The cell preparation of claim 63, wherein a cell ratio between
said cytotoxic T-lymphocytes and said immature hematopoietic cells
including stem cells is at least 1 to 100.
69. The cell preparation of claim 63, wherein said immature
hematopoietic cells are stem cells.
70. The method of claim 56, wherein affinity purification is
effected by an antibody.
71. The method of claim 70, wherein the antibody is selected from
the group consisting of anti-CD69, anti-CD25, anti-IL-2 and
anti-INF.gamma..
72. A method of producing a cell preparation for preventing or
decreasing graft rejection in a human recipient of a transplant,
wherein the recipient is conditioned under lethal or sublethal
conditions, wherein the transplant is derived from a non-syngeneic
donor, wherein the cell preparation comprises non-alloreactive
anti-third party cytotoxic T-lymphocytes (CTLs), wherein the cell
preparation does not comprise T-lymphocytes capable of developing
into anti-recipient CTLs, and wherein the cell preparation does not
comprise CD4+ cells and/or CD56+ cells, the method comprising: (i)
directing peripheral blood lymphocytes derived from the
non-syngeneic donor against a third party antigen or antigens,
thereby generating third party antigen-treated lymphocytes which
comprise anti-third party CTLs; (ii) depleting said peripheral
blood lymphocytes derived from the non-syngeneic donor of CD4+
cells and/or CD56+ cells; and (iii) depleting, by affinity
purification, said third party antigen-treated lymphocytes of
T-lymphocytes capable of developing into anti-recipient CTLs,
thereby producing the cell preparation.
73. The method of claim 72, wherein the non-syngeneic donor is
allogeneic with respect to the recipient.
74. The method of claim 72, wherein the non-syngeneic donor is
xenogeneic with respect to the recipient.
75. The method of claim 72, wherein step (ii) is effected by
depleting said peripheral blood lymphocytes derived from the
non-syngeneic donor of CD56+ cells or CD4+ cells.
76. The method of claim 72, wherein said 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, a synthetic peptide presented
by autologous antigen presenting cells and a synthetic peptide
presented by non-autologous antigen presenting cells.
77. The method of claim 76, wherein said third party cells are
allogeneic or xenogeneic with respect to the recipient.
78. The method of claim 77, wherein said third party cells have HLA
antigens which are different from HLA antigens of the non-syngeneic
donor and which are not cross reactive with HLA antigens of the
recipient.
79. The method of claim 77, wherein said third party cells are
stimulatory cells selected from the group consisting of cells
purified from peripheral blood lymphocytes, cells purified from
spleen, cells purified from lymph nodes, cytokine-mobilized PBLs
and in vitro expanded antigen-presenting cells (APC).
80. The method of claim 72, wherein affinity purification is
effected by an antibody.
81. The method of claim 80, wherein the antibody is selected from
the group consisting of anti-CD69, anti-CD25, anti-IL-2 and
anti-INF.gamma..
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/169,028 filed on Jun. 27, 2002, which is a
U.S. National Phase of PCT Application No. PCT/IL00/00872 filed on
Dec. 28, 2000, which is a continuation of U.S. patent application
Ser. No. 09/477,737 filed on Jan. 5, 2000, now U.S. Pat. No.
6,544,506 issued on Apr. 8, 2003. The contents of the above
Applications are incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to veto cell preparations,
methods of their manufacture and transplantation method using same
which can be used to prevent or ameliorate immune rejection of
donor organs, tissues or cells without inducing graft versus host
disease (GVHD). More particularly, the present invention relates to
a cell preparation for use in transplantation which preparation
includes cells which are functional as veto cells but which are
substantially devoid of alloreactive cells.
[0003] Transplantation of allogeneic and xenogeneic organs, tissues
and cells is commonly practiced in humans in order to alleviate
numerous disorders and diseases.
[0004] For example, bone marrow (BM) transplantation is
increasingly used to treat a series of severe diseases in humans,
such as for example, leukemia. However, bone marrow transplantation
is limited by the availability of suitable donors, since
transplanted tissues must traverse major histocompatibility
barriers which can otherwise lead to graft rejection.
[0005] In view of such limitations, several approaches for
enhancing graft acceptance have been suggested.
[0006] In one approach, cancer patients receiving autologous BM
transplantation were treated with granulocyte colony-stimulating
factor (G-CSF), resulting in mobilization of pluripotential stem
cells from the marrow to the blood thereby increasing the number of
cells which can be collected for autologous transplantation.
[0007] In another approach, major histocompatibility barriers in BM
transplantation in leukemia patients were overcome by using a very
large dose of stem cells, preferably a dose at least 3-fold greater
than conventional doses used in T-cell-depleted BM transplantation,
in particular a megadose of CD34+ hematopoietic progenitors (U.S.
Pat. No. 5,806,529 to Reisner et al.).
[0008] Although the megadose approach facilitated permanent
acceptance of allogeneic donor type skin grafts in mice.sup.1, such
an approach is not readily applicable for human transplantation
since the number of stem cells which are required to attain this
desirable goal may not be easily collected from human donors.
[0009] A difficult barrier for the engraftment of donor
hematopoietic cell transplantation arises from the marked level of
host hematopoietic and immune cells surviving mild preparatory
regimens. Several studies have shown that this challenge can be
successfully addressed in rodents by using large doses of bone
marrow cells, adequately depleted of T-cells and utilized in
conjunction with one form or another of tolerance inducing cells,
also termed as veto cells.
[0010] Veto cell activity is defined as the capacity to
specifically suppress cytotoxic T-cell precursors (CTL-p) directed
against antigens of the veto cells themselves, but not against
third party antigens.sup.2 Several veto cells or bone marrow
transplantation facilitating cells capable of suppressing cytotoxic
T-cell precursors have been described.sup.3-11
[0011] Interestingly, it has been shown that some of the most
potent veto cells are of T-cell origin, and in particular a very
strong veto activity was documented for CD8+ CTL lines or
clones.sup.12-16
[0012] The specificity of CTL veto cells was demonstrated by
several studies to be unrelated to their T-cell receptor
specificity.sup.17-19. [0013] The suppression of effector CTL-p
directed against the veto cells is both antigen-specific and
MHC-restricted. This suppression results from the unidirectional
recognition of the veto cell by the responding cytotoxic
T-lymphocytes.sup.18. Furthermore, it has been shown that this
suppression is mediated by apoptosis.sup.18, 20.
[0014] Blocking experiments conducted with anti-CD8 or anti class I
antibodies indicated that the elimination of host anti-donor CTL-p
is induced via an interaction of CD8 molecules on the CTL veto
cells with the a3 domain of class I molecules on the host
CTL-p's.sup.18. Support to this observation was provided by studies
in which CD8 cDNA was introduced into clones lacking CD8.sup.18.
Further support was provided by experiments which demonstrated that
CD8 molecules on the veto cells can directly induce apoptosis in
effector cells.sup.21. More recently, Asiedu et al. demonstrated
that antibody mediated cross linking of CD8 on primate bone marrow
veto cells, leads to an increased TGF.beta. production which
induces apoptosis in the effector cells.sup.22. Alternatively, it
has been suggested that mouse bone marrow veto cells can induce
apoptosis via Fas-Fas-L interaction.sup.23.
[0015] The studies described hereinabove demonstrated that veto
T-cell preparations can greatly facilitate graft tolerance in bone
marrow transplantation. However, the veto T-cell preparations are
inadequate for generating graft tolerance since such preparations
still include a substantial amount of alloreacting donor T-cells
which can lead to GVHD, thus limiting the successful implementation
of this approach.
[0016] Reisner et al., .sup.24, 28 describe the preparation of
non-alloreactive anti-third party CTLs which can be used to enhance
graft acceptance in mice. However, following the publication of
this abstract and a more careful study, it was realized that the
CTL preparation described therein is not depleted of T-cells
capable of developing post transplantation into anti-host CTLs
inflicting GVHD. Thus, this approach per se is not applicable for
application in humans.
[0017] There is thus a widely recognized need for, and it would be
highly advantageous to have, a novel veto cell preparation devoid
of alloreactivity which can be used for substantially reducing
rejection of transplanted organs, tissues or cells without
generating GVHD, thereby leading to durable tolerance towards the
transplanted, organs tissues or cells.
SUMMARY OF THE INVENTION
[0018] According to one aspect of the present invention there is
provided a method of transplanting a transplant derived from a
donor into a recipient, the method comprising the steps of (a)
transplanting the transplant into the recipient; and (b)
administering to the recipient a dose including non-alloreactive
anti-third party cytotoxic T-lymphocytes (CTLs), wherein the
non-alloreactive anti-third party CTLs are generated by directing
T-lymphocytes of the donor against a third party antigen or
antigens, the dose is substantially depleted of T-lymphocytes
capable of developing into alloreactive CTLs, thereby preventing or
ameliorating both graft rejection by the recipient and graft versus
host disease.
[0019] According to another aspect of the present invention there
is provided a method of treating a recipient suffering from a
disease requiring immature hematopoietic cell transplantation, the
method comprising the steps of (a)
[0020] conditioning the recipient under sublethal, lethal or
supralethal conditions; (b) administering to the recipient a first
dose including immature hematopoietic cells including stem cells
from an allogeneic or xenogeneic donor; and (c) administering to
the recipient a second dose including non-alloreactive anti-third
party cytotoxic T-lymphocytes (CTLs), wherein the CTLs are
generated by directing T-lymphocytes derived from the donor against
a third party antigen or antigens, the second dose is substantially
depleted of T-lymphocytes capable of developing into alloreactive
CTLs, thereby preventing or ameliorating both graft rejection and
graft versus host disease.
[0021] According to yet another aspect of the present invention
there is provided a method of producing non-alloreactive anti-third
party cytotoxic T-lymphocytes (CTLs), the method comprising the
step of directing T-lymphocytes against a third party antigen or
antigens, and substantially depleting T-lymphocytes capable of
developing into alloreactive CTLs.
[0022] According to still another aspect of the present invention
there is provided a cell preparation for transplantation to a
recipient, the cell preparation comprising donor derived
non-alloreactive anti-third party cytotoxic T-lymphocytes (CTLs)
directed against a third party antigen or antigens, the cell
preparation being substantially depleted of T-lymphocytes capable
of developing into alloreactive CTLs.
[0023] According to an additional aspect of the present invention
there is provided a cell preparation for transplantation to a
recipient, the cell preparation comprising (a) donor derived
immature hematopoietic cells including stem cells; and (b) donor
derived, non-alloreactive anti-third party cytotoxic T-lymphocytes
(CTLs) directed against a third party antigen or antigens, the cell
preparation being substantially depleted of T-lymphocytes capable
of developing into alloreactive CTLs.
[0024] According to further features in preferred embodiments of
the invention described below, the dose, T-lymphocytes or
preparation is substantially depleted of CD4 T cells and/or CD56
natural killer cells.
[0025] According to still further features in the described
preferred embodiments depletion of T-lymphocytes capable of
developing into alloreactive CTLs is effected by deprivation of a
factor which is (i) required for CTLs maturation; and (ii) secreted
by maturing CTLs.
[0026] According to still further features in the described
preferred embodiments the factor is a cytokine.
[0027] According to still further features in the described
preferred embodiments the cytokine is IL2.
[0028] According to still further features in the described
preferred embodiments depletion of T-lymphocytes capable of
developing into alloreactive CTLs is effected by affinity labeling
followed by label based separation.
[0029] According to still further features in the described
preferred embodiments depletion of T-lymphocytes capable of
developing into alloreactive CTLs is effected by affinity
purification.
[0030] According to still further features in the described
preferred embodiments the donor is selected from the group
consisting of an allogeneic donor either HLA identical or HLA
non-identical and a xenogeneic donor.
[0031] According to still further features in the described
preferred embodiments the recipient is a human.
[0032] According to still further features in the described
preferred embodiments the recipient and the donor are both
humans.
[0033] According to still further features in the described
preferred embodiments 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
and a purified protein. In a presently preferred embodiment the
third party antigen are synthetic peptides which constitute viral
antigens and which are presented by autologous antigen presenting
cells pulsed therewith.
[0034] According to still further features in the described
preferred embodiments the viral antigen is an EBV or a CMV
antigen.
[0035] According to still further features in the described
preferred embodiments the purified protein is ovalbumin.
[0036] According to still further features in the described
preferred embodiments the third party cells are allogeneic or
xenogeneic cells with respect to the recipient.
[0037] According to still further features in the described
preferred embodiments the allogeneic cells have HLA antigens
different from that of the donor but which are not cross reactive
with the recipient HLA antigens.
[0038] According to still further features in the described
preferred embodiments the allogeneic cells are stimulatory cells
selected from the group consisting of cells purified from
peripheral blood lymphocytes (PBLs), spleen or lymph nodes,
cytokine-mobilized PBLs and in vitro expanded antigen-presenting
dendritic cells (APC).
[0039] According to still further features in the described
preferred embodiments the immature hematopoietic cells including
stem cells are derived from the bone marrow, mobilized peripheral
blood, fetal liver, yolk sac and/or cord blood of the donor.
[0040] According to still further features in the described
preferred embodiments the mobilized peripheral blood cells are
obtained by leukapheresis of peripheral blood of the donor after
stimulation with a suitable cytokine.
[0041] According to still further features in the described
preferred embodiments the immature hematopoietic cells are T-cell
depleted hematopoietic progenitor cells.
[0042] According to still further features in the described
preferred embodiments the T-cell depleted hematopoietic progenitor
cells are CD34+ progenitor hematopoietic cells.
[0043] According to still further features in the described
preferred embodiments a cell ratio between the cytotoxic
T-lymphocytes and the immature hematopoietic cells including stem
cells is at least 1 to 100, preferably 1.5 to 100.
[0044] According to still further features in the described
preferred embodiments the cell number of the cytotoxic
T-lymphocytes infused to a recipient is more than
5.times.10.sup.6/Kg body weight and the immature hematopoietic
cells including stem cells (CD34 cells) is more than
1.0.times.10.sup.6/Kg body weight.
[0045] According to still further features in the described
preferred embodiments steps of transplanting and administering are
effected at the same time, or alternatively, the step of
transplanting is performed either prior to, or after the step of
administering.
[0046] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
veto cells which are highly effective in preventing graft
rejection, yet are devoid of graft versus host disease
potential.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The invention is 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 the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
re, no attempt is made to show details of the invention in more
detail than is necessary for a fundamental understanding of the
invention, the description taken with the drawings making apparent
to those skilled in the art how the several forms of the invention
may be embodied in practice.
[0048] In the drawings:
[0049] FIG. 1 is a graph depicting the level of anti-host CTLp in
donor anti-third party CTL lines prepared with and without IL2
starvation.
[0050] FIG. 2a is a histogram depicting the veto activity of
non-alloreactive anti third-party CTLs. The Figure shows activity
of anti-third party CTLs as a function of veto cell concentration
as compiled from 17 different experiments. Specific lysis exhibited
by responder cells (C3H/Hej) was documented upon stimulation
against Balb/c (solid squares) or SJL (.mu.open squares)
splenocytes, in the presence of different concentrations of cells
from a Balb/c anti third-party (C57BL/6) CTL line.
[0051] FIG. 2b is a graph depicting the veto effect of
non-alloreactive anti third party CTLs as a function of effective
veto/responder cell ratios. The killing of responder cells (CTL
progenitors) (C3H/Hej) stimulated against Balb/c (solid circle) or
SJL (open circle) splenocytes, was tested in the presence of
different concentrations of cells from a Balb/c anti third party
(C57BL/6) CTL line.
[0052] FIG. 3 is a graph depicting the veto activity of anti third
party CTLs as a function of the time of veto cell addition. The
percent killing of responder cells (C3H/Hej) stimulated against
Balb/c splenocytes by a Balb/c anti third party (C57BL/6) CTL line
(At a 1:50 veto/responder cell ratio), was tested as a function of
the time of CTL addition.
[0053] FIG. 4 is a histogram depicting the inhibition of the veto
effect by a monoclonal anti-CD8 antibody. Anti-CD8 mAb (anti
Ly-2.2) was added at different concentrations to mixed lymphocyte
reaction (MLR) which consisted of responder cells (C3H/Hej)
stimulated against Balb/c splenocytes, and cells from a Balb/c
anti-third party (C57BL/6) CTL line (At a 1:50 veto/responder cell
ratio).
[0054] FIGS. 5a-c depict the specific deletion of responder CTL-p
directed against the H-2 antigen of the veto cells. Splenocytes
from 2c transgenic mice (H-2.sup.b) bearing transgene TCR specific
against H-2.sup.d (1B2.sup.+) were stimulated in MLR against Balb/c
splenocytes (H-2.sup.d). The frequency of 1B2.sup.+ CD8 T cells
before (5a) and after addition to the MLR culture of Balb anti-C3H
(5b) or SJL anti-C3H (5c) CTLs, was determined by FACS 5 days after
initiation of the MLR.
[0055] FIG. 6a is a histogram depicting the role of Fas ligand
(Fas-L) and perforin in the veto activity of anti-third party CTLs.
Anti-third party CTLs generated from wild type C3H splenocytes were
compared to CTLs generated from Fas-L deficient gld/C3H or from
perforin deficient PO splenocytes. Cultures for veto determination
consisted of Balb anti-C3H MLR (solid column) while Balb anti-SJL
served as a control for background inhibition (open column). In the
case of PO CTLs, cultures for veto determination consisted of C3H
anti-PO MLR (solid column) while C3H anti-SJL served as a control
(open column).
[0056] FIG. 6b is a histogram depicting the reversal of veto
activity in Fas-L deficient CTLs by gene transfer of Fas-L. Anti
third-party CTLs generated from wild type C3H splenocytes, were
compared to CTLs generated from Fas-L deficient gld/C3H, before, a,
and after transfection with Fas-L containing retroviral vector, b,
or with a mock vector, c.
[0057] FIG. 7a is a histogram depicting the role of Fas in the veto
activity of anti-third party CTLs. Responder CTL progenitors of
wild type C3H or of Fas deficient lpr/C3H background, were compared
for their sensitivity to the veto effect of Balb anti-C57BL/6 CTLs.
Cultures for veto determination consisted of C3H anti-Balb MLR
(solid columns) or lpr/C3H anti-SJL (open columns) which served as
a control for determining background inhibition.
[0058] FIG. 7b is a histogram depicting the effect of a Fas-L
antagonist on the veto effect of CTLs. Cultures for veto
determination consisted of Balb anti-C3H MLR (solid columns) or
Balb anti-SJL (open column) which served as a control for
determining background inhibition.
[0059] FIGS. 8a-d depict the deletion of anti-H2.sup.d T-cells by
non-alloreactive anti-third party CTLs. Splenocytes from 2c
transgenic mice (H-2.sup.b) bearing transgene TCR specific against
H-2.sup.d were stimulated in MLR against Balb/c splenocytes
(H-2.sup.d). Expression of Fas on CD8 transgenic (1B2.sup.+) T
cells was analyzed by FACS before MLR (FIG. 8a) and at 48 hr (FIG.
8b). Deletion of Fas.sup.+1B2.sup.+ CD8 T-cells following addition
to the MLR culture of Balb anti-C3H (FIG. 8c) or SJL anti-C3H (FIG.
8d) CTLs, was determined at 48 hrs after initiation of the MLR.
[0060] FIG. 9 is a graph depicting the relationship between cells
per culture and the % of non-responding cells of unmanipulated
peripheral blood lymphocytes (PBLs) taken from a donor (R) and
stimulated against potential host type PBL (patient No. 38). The
specific killing of HLA-unrelated cell types (patient No. 40) was
demonstrated to be negligible (square), while killing was
maintained at high potency against the original stimulators
(diamond and circle).
[0061] FIG. 10 is a graph depicting the relationship between cells
per culture and the percent of non-responding cells of a CTL line
established from PBL of donor R (described in FIG. 1) by
stimulation against third party EBV transformed stimulators (named
S). When stimulating the cells of this CTL line against the host
type PBL (patient No. 38), the specific killing was demonstrated to
be negligible (circle) as is compared to that found against control
cells (patient No. 40, non-specific background) (square).
[0062] FIGS. 11a-b are bar graphs demonstrating the veto activity
of human anti-third party CTLs. FIG. 11a shows the inhibition of
specific lysis at 1:25 and 1:50 veto to effector cell ratio,
compared to a control experiment (FIG. 11b) using the same veto
cells in a non-relevant MLR, directed against an irrelevant
stimulator bearings different HLA antigens.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0063] The present invention is of veto cell preparations, methods
for their production and their use in transplantations, which veto
cells can be used to induce durable tolerance of donor organs,
tissues or cells without inducing GVHD. Specifically, the present
invention relates to veto cell preparations for use in
transplantation, which veto cell preparations include cells which
are functional as veto cells but which are substantially devoid of
alloreactive cells and as such, when introduced into a recipient,
these veto cells prevent graft rejection without inducing GVHD.
[0064] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0065] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description. 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.
[0066] Early studies in murine models and more recent clinical data
in heavily pre-treated Leukemia patients have shown that escalation
of hematopoietic progenitor cells can overcome major genetic
barriers and enable rapid and durable engraftment of haploidentical
3-loci mismatched transplants without inducing GVHD. In vitro
studies suggest that veto cells within the CD34 progenitors
population likely mediate this facilitating effect.
[0067] The "megadose" concept was also shown recently to be useful
for tolerance induction in sublethally irradiated mice by
overcoming the marked resistance presented by the large number of
lymphocytes surviving the sublethal conditioning. Allogeneic
chimera generated by transplantation of a megadose of Sca1.sup.+
Lin.sup.- cells, permanently accept allogeneic donor type skin
grafts.
[0068] However, the numbers required to attain this desirable goal
may not be easily collected from human donors.
[0069] Non-alloreactive, Peanut Agglutinin negative thymocytes from
(host.times.donor) F1 which are non-alloreactive by virtue of their
genetic composition, were also shown to enhance engraftment of
T-cell depleted BM.
[0070] More recently these cells were shown to synergise with
murine Sca1.sup.+ Lin.sup.- cells so as to reduce the minimal
number of cells required to achieve induction of substantial mixed
chimerism in the sub-lethal mouse model.
[0071] In humans, non-alloreactive T-cells can be generated by
purging interleukin-2 receptor (CD25) MLR reactive T-cells or by
anergy induction upon incubation with CTLA-4. However, these
approaches, which make use of T-cell stimulation with the very
antigens against which tolerance is desired, might involve some
risk for generating committed CTLs, if any of the CTL progenitors
escape deletion or anergy induction. Once such anti-host CTLs are
generated, it is very difficult to suppress their alloreactivity in
vivo.
[0072] An alternative approach is based on earlier studies which
showed that CD8.sup.+ CTL clones possess extremely high veto
activity.
[0073] Donor derived T-lymphocytes exposed to a third party antigen
proved useful in generating CTLs effective in preventing or
ameliorating graft rejection, however, such CTL preparations
included a substantial fraction of donor derived T-lymphocytes
which could develop into alloreactive donor CTLs, inducing
GVHD.
[0074] Thus, all prior art approaches which utilize veto cells of
T-cell origin fail to provide adequate solution for inducing graft
acceptance since such veto cell preparations include a substantial
amount of alloreactive T-cells which induce GVHD in the host
following administration.
[0075] While reducing the present invention to practice, donor
anti-third party CTLs which are depleted of anti-host
alloreactivity were generated. In the preferred embodiments of the
invention cell preparations are also substantially depleted of CD4
T cells and/or CD56 natural killer cells, preferably both.
[0076] As is further described in Examples 1 and 2 of the Examples
section below, depletion of cells having a potential of becoming
alloreactive (anti-host) CTLs from a donor anti-third party CTLs
culture involved, in the specific example provided, initial IL-2
starvation which resulted in apoptosis of non-induced T-cells
present in the culture. Studies utilizing such mouse and human CTL
preparations which were performed in-vitro, demonstrated that such
non-alloreactive CTL preparations are depleted of cells having the
potential of maturing into anti-host CTLs.
[0077] Thus, according to one aspect of the present invention there
is provided a method of transplanting a transplant derived from a
donor into a recipient.
[0078] As used herein the terms "transplant" or "graft" refers to
either allogeneic or xenogeneic transplants or grafts including,
but not limited to whole organs, such as for example, kidney,
heart, liver or skin; tissues, such as, for example, tissues
derived from an organ such as a liver; or cells, such as, for
example, immature hematopoietic cells.
[0079] As used herein the term "allogeneic" refers to as being from
the same species. As such an "allograft" is a transplant between
two individuals of the same species which individuals display
strong (unrelated individuals) or weak (haploidentical siblings)
histocompatibility differences.
[0080] As used herein the terms "xenogeneic" or "heterogeneic"
refer to as being from two different species. As such a "xenograft"
or a "heterograft" is a transplant between two individuals of a
different species.
[0081] Thus, the method according to the present invention, which
is further described and exemplified hereinbelow, can be utilized
for transplantation of an organ such as a kidney or a heart to a
recipient suffering from, for example, renal or heart failure, or
for the transplantation of liver, lung or skin tissue to a
recipient suffering from hepatic or lung failure or skin damage
(e.g., burns).
[0082] The method described below can also be used, for example,
for treating a recipient suffering from a disease requiring
immature hematopoietic cell transplantation.
[0083] In the latter case, immature 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 malignant disease. Such a disease can be, 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), and severe combined
immunodeficiency syndromes (SCID), including adenosine deaminase
(ADA), osteopetrosis, aplastic anemia, Gaucher's disease,
thalassemia and other congenital or genetically-determined
hematopoietic abnormalities.
[0084] Regardless of the transplant type, to avoid graft rejection,
the method according to the present invention also utilizes a novel
veto cell preparation which includes non-alloreactive anti-third
party cytotoxic T-lymphocytes (CTLs). Such CTLs are generated by
conditioning donor derived T-cells against third party antigens.
This CTL cell preparation functions in reducing graft rejection by
the recipient. Further detail of such a CTL preparation is given in
Examples 1-3 of the Examples section. Such preparations are
depleted of T-lymphocytes capable of developing into alloreactive
CTLs, thereby preventing or ameliorating both graft rejection by
the recipient and graft versus host disease.
[0085] As used herein the phrase "non-alloreactive" refers to
having substantially no reactivity against the donor or
recipient.
[0086] According to the method of the present invention, these CTLs
are administered either concomitantly, prior to, or following the
transplantation of the donor transplant.
[0087] In the case of hematopoietic cell transplantation, the cell
ratio between the cytotoxic T-lymphocytes and the immature
hematopoietic cells including stem cells is at least 1 to 100,
preferably, in the range of 1 to 1-5 to 1, more preferably about.
Preferably, the cell number of the cytotoxic T-lymphocytes infused
to a recipient is more than 5.times.10.sup.6/Kg body weight and the
immature hematopoietic cells including stem cells (CD34 cells) is
more than 1.0.times.10.sup.6/Kg body weight.
[0088] As used herein the phrase "third party antigen or antigens"
refers to antigens which are not present in either the donor or
recipient.
[0089] For example, third party antigens can be antigens of
viruses, such as for example, Epstein-Barr virus (EBV) or
cyto-megalo virus (CMV) or of third party cells (cells not from the
donor or recipient). Viral antigens can be presented by cells
(e.g., cell line) infected therewith or otherwise made to express
viral proteins. 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. 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.
[0090] Third party cells can be either allogeneic or xenogeneic
with respects to the recipient. Preferably, 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 CTLs generated against
such cells are not reactive against transplant or recipient
antigens.
[0091] According to another preferred embodiment of the present
invention the allogeneic third party cells are stimulatory cells
selected from the group consisting of cells purified from PBL,
spleen or lymph nodes, cytokine-mobilized PBLs and in vitro
expanded antigen-presenting dendritic cells (APC).
[0092] Third party antigens can be presented on the cellular, viral
or bacterial surfaces or derived and/or purified therefrom.
Additionally, a viral antigen can be displayed on an infected cell
and a cellular antigen can be displayed on an artificial vehicles
such as a liposome.
[0093] 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.
[0094] According to the present invention, the CTLs are preferably
generated against third party cells or viruses or virally infected
or viral peptides presenting cells and as such, the third party
antigens utilized by the present invention are various native
cellular or viral antigens or a combination of both which are
displayed on the surface of the cell or virus.
[0095] Utilizing cells, viruses, virally infected or viral 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
CTLs 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
procedure. Furthermore, when CTLs are directed against viral
infected cells, it is plausible to obtain at least some graft
versus cancer cells activity due to cross reactivity between viral
antigens and cancer cell associated or specific antigens.
[0096] Thus, as already mentioned above, the method according to
this aspect of the present invention is effected by transplanting
the transplant into the recipient along with a non-alloreactive
anti-third party cytotoxic T-lymphocytes (CTLs) depleted of
T-lymphocytes capable of developing into alloreactive CTLs, thereby
preventing or ameliorating both graft rejection by the recipient
and graft versus host disease.
[0097] Since the non-alloreactive anti-third party CTLs are
generated by directing T-lymphocytes derived from the donor against
third party antigen or antigens, the resultant CTLs prevent or
ameliorate graft rejection by the recipient by trapping and killing
recipient CTL progenitors (responders) which are formed against the
transplant. Furthermore, since the CTLs are generated against third
party antigens, such CTLs are not active against recipient tissues
or against the donor transplant. Since the anti-third party CTLs
are depleted of T-lymphocytes capable of developing into
alloreactive CTLs, they prevent or ameliorate graft versus host
disease.
[0098] According to a preferred embodiment of the present invention
the method of transplanting a donor transplant further includes an
additional step in which the recipient is conditioned under
sublethal, lethal or supralethal conditions prior to
transplantation.
[0099] Such conditioning is dependent on the nature of the
transplant and the condition of the recipient. The recipient may be
conditioned under sublethal, lethal or supralethal conditions, for
example, by total body irradiation (TBI) and/or by treatment with
myeloablative and immunosuppressive agents according to standard
protocols. For example, a sublethal dose of irradiation is within
the range of 1-7.5 Gy TBI, a lethal dose is within the range of
7.5-9.5 Gy TBI and a supralethal dose is within the range of
9.5-16.5 Gy TBI. Examples of myeloablative agents are busulphan,
dimethyl mileran and thiotepa, and of immunosuppressive agents are
prednisone, methyl prednisolone, azathioprine, cyclosporine,
cyclophosphamide, fludarabin, etc.
[0100] According to the method of the present invention, the
recipient is preferably conditioned under sublethal conditions.
[0101] As already stated numerous times, the non-alloreactive
anti-third party CTLs according to the present invention are
substantially depleted of T-lymphocytes capable of developing into
alloreactive CTLs, and are therefore said to be "non-alloreactive".
As such they differ and are advantageous over prior art veto cells.
Depletion of T-lymphocytes capable of developing into alloreactive
CTLs is of particular advantage since the presence of alloreactive
cells in the recipient can lead to the development of GVHD.
[0102] A non-alloreactive anti-third party CTLs cell preparation is
generated as described above by directing donor derived
T-lymphocytes against third party antigens and the preparation is
depleted of T-lymphocytes capable of developing into alloreactive
CTLs.
[0103] According to one preferred embodiment of the present
invention the depletion of T-lymphocytes capable of developing into
alloreactive CTLs is effected by depriving T-lymphocytes cultured
in the presence of third party antigens of a factor which is (i)
required for CTLs maturation or protection from apoptosis; and (ii)
secreted by maturing CTLs. Under such culturing conditions
T-lymphocytes capable of developing into alloreactive CTLs undergo
apoptosis, wherein maturing CTLs present in the culture survive
factor deprivation since such cells self secrete (autocrine) this
factor.
[0104] The factor according to the teachings of the present
invention can be for example, a cytokine, such as, but not limited
to, IL2. IL2 depravation of CTL preparations is described in detail
in Examples 1-3 of the Examples section below.
[0105] According to an embodiment of the present invention,
depletion of T-lymphocytes capable of developing into alloreactive
CTLs is effected by affinity labeling followed by label based
separation. Thus, when stimulated against host antigens, a
fluorescently labeled anti-CD69 or anti-CD25 antibody which
specifically binds the unique activation antigen of T-lymphocytes
and/or a fluorescently labeled anti-IL2 or anti-.gamma.INF which
specifically binds cells secreting these cytokines, can be used to
separate anti-host T-lymphocytes from anti-third party CTLs,
thereby deplete T-lymphocytes capable of developing into
alloreactive CTLs. Such specific labeling can be used to select
anti-third party CTL precursors prior to IL-2 starvation or as a
substitute for IL-2 starvation.
[0106] Such specific labeling can be used to select anti-third
party CTL precursors prior to IL-2 starvation or as a substitute
for IL-2 starvation.
[0107] According to still further features in the described
preferred embodiments depletion of T-lymphocytes capable of
developing into alloreactive CTLs is effected by affinity
purification.
[0108] For example, a substrate including an antibody or a ligand
capable of specifically binding a cell surface molecule displayed
only by T-lymphocytes but not by CTLs or vice versa, can be used to
effectively deplete T-lymphocytes capable of developing into
alloreactive CTLs from the CTL preparation.
[0109] The affinity substrate according to the present invention
can be a column matrix such as, for example agarose, cellulose and
the like, or beads such as, for example, magnetic beads onto which
an anti-T-lymphocyte or an anti-CTLs antibody, as is described
above, is immobilized.
[0110] Thus, according to this aspect of the present invention,
depletion of T-lymphocytes capable of developing into alloreactive
CTLs, can be effected via column chromatography or magnetic bead
separation.
[0111] The purpose in growing donor T cells sensitized against
third party antigen or antigens is to achieve a T cell preparation
depleted of alloreactive clones directed against host antigens. As
is described herein, the procedure includes two major steps. In the
first, IL-2 starvation is used to effect partial depletion of cells
which cannot produce their own IL-2, in the second major step
anti-third party clones are expanded vigorously with optimal doses
of IL-2 so as to further deplete remaining anti host clones. In
general, CD8 CTLs are directed against antigens presented in the
context of HLA class 1, while CD4 helper T cells recognize antigens
in the context of HLA Class II. The latter cells lack veto activity
and therefore the procedure described herein leads to minimal
generation of such cells. However, it was experimentally found that
even when using stimulation protocols favoring generation of CTLs
against third party antigens, it is difficult to achieve high
levels of CD8 CTLs at the end of the culture, unless CD4 T cells
and CD56 natural killer (NK) cells are removed 10-16 days after the
IL2 starvation period (e.g., prior to stimulation with high levels
of IL-2). Thus in a preferred embodiment of the present invention
CD8 cells purification is effected by either negative selection for
removing CD4 and CD56 cells or by positive selection with for
retaining CD8 cells. Such selection can be effected, for example,
using affinity solid supports including anti-CD8 antibodies (for
positive selection) or anti-CD4 antibodies and anti CD56 antibodies
(for positive selection). The solid support can be a column, beads
or magnetic beads. Alternatively, such selection can be effected,
for example, using affinity labeling followed by FACS separation
employing fluorescently labeled anti-CD8 antibodies, anti-CD4
antibodies and/or anti CD56 antibodies.
[0112] According to yet an additional aspect of the present
invention there is provided a method of transplanting a transplant
derived from a donor into a recipient. The method according to this
aspect of the present invention is identical to the method
described hereinabove with the exception that the veto cells
utilized are of non T-lymphocyte origin.
[0113] A veto cell preparation according to this aspect of the
present invention, includes donor derived, genetically modified
non-T-cells expressing recombinant Fas ligand and recombinant CD8
antigen.
[0114] As is described in Example 2 of the Examples section, while
reducing the present invention to practice it was uncovered that
CTLs can veto effectively CTL progenitors only if both Fas-L and
CD8 are co-expressed on the former CTLs.
[0115] As such, donor derived non-T-lymphocyte circulatory cells,
such as, for example, monocytes, neutrophilles or basophilles, can
also serve as effective veto cells provided these cells express and
display CD8 and Fas-L.
[0116] To express these proteins in such cells, the coding
sequences of Fas-L and CD8 subunit .alpha. (Gene Bank accession
Nos.: U11821 and M12825, respectively, both are incorporated herein
by reference) including appropriate leader sequences are isolated
and each is ligated into a suitable expression cassette of an
expression vector construct.
[0117] The vector constructs can then be co-introduced into the
cell by any one of a variety of methods known in the art. Such
methods can be found generally described in Sambrook et al.,
"Molecular Cloning: A Laboratory Manual", Cold Springs Harbor
Laboratory, New York, 1989, Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md., 1989,
Chang et al., "Somatic Gene Therapy", CRC Press, Ann Arbor, Mich.,
1995, Vega et al., "Gene Targeting", CRC Press, Ann Arbor Mich.
(995), "Vectors: A Survey of Molecular Cloning Vectors and Their
Uses", Butterworths, Boston Mass., 1988, and Gilboa et al.,
Biotechniques 4 (6): 504-512, 1986, and include, for example,
stable or transient transfection, lipofection, electroporation,
biolistic bombardment and infection with recombinant viral vectors.
[0118] Each nucleic acid construct according to this aspect of the
present invention includes a promoter for regulate the expression
of Fas-L or CD8. Such promoters are known to be cis-sequence
elements required for transcription as they serve to bind DNA
dependent RNA polymerase which transcribes sequences present
downstream thereof. [0119] The promoter of choice that is used in
conjunction with this aspect of the invention can be any promoter
suitable for expression in mammalian cells. Preferably the promoter
utilized is a strong, constitutive promoter capable of expressing
high levels of the transcripts. In addition, to further increase
transcriptional activity the vector construct of the present
invention may also include a transcriptional enhancer element.
[0120] The vector construct according to this aspect of the present
invention preferably further includes an appropriate selectable
marker. It will be appreciated that since two independent
constructs are utilized herein, each construct includes a unique
selectable marker, such that cell transformed with both constructs
can be selected for. Alternatively, a single vector harboring both
genes, each with its own expression regulatory sequences, is
employed.
[0121] The vector construct according to the present invention
preferably further includes an origin of replication in mammalian
cells and an appropriate selectable marker and origin of
replication for propagation in E. coli (i.e., shuttle vector). The
vector construct of the present invention can be utilized for
transient expression (i.e., no genomic integration), or for
integration into the genome of transformed cells and expression
therefrom. The construct according to this aspect of the present
invention can be, for example, a plasmid, a bacmid, a phagemid, a
cosmid, a phage, a virus or an artificial chromosome.
[0122] It will be appreciated that CD8 and Fas-L can also be
expressed from a single vector construct as a single chimeric
transcript, provided this transcript includes an IRES sequence for
directing the translation of a second polypeptide encoded by the
transcript.
[0123] Thus according to this aspect of the present invention the
method of transplanting a donor transplant utilizes a veto cell
preparation of genetically modified non-T-cells expressing
recombinant Fas ligand and recombinant CD8 antigen.
[0124] It will be appreciated that such a veto cell preparation is
inherently advantageous for inducing tolerance of transplants since
such a preparation does not include cells of T-lymphatic origin and
therefore, such veto cells inherently cannot induce GVHD.
[0125] In addition, since such cells are derived from the donor
they serve as excellent traps for CTL progenitors generated by the
recipient as a response to the transplant and as such prevent or
ameliorate transplant/graft rejection.
[0126] Thus, this aspect of the present invention describes an
alternative approaches to generating non-alloreactive anti-third
party veto cells. By expressing Fas-L and CD8, in a variety of
cells, these cells can serve as veto cells for antigens against
which tolerance induction is desired.
[0127] Similarly to the anti-third party CTLs described by other
aspects of the present invention, these `artificial` veto cells
could help reduce the effective CD34 megadose requirements in
mismatched leukemia patients and may lead to safe mismatched
hematopoietic transplants in patients for whom the risk of
supralethal radio-chemotherapy is not justified, such as patients
with thalassemia, sickle cell anemia and several enzyme
deficiencies.
[0128] Furthermore, induction of substantial durable chimerism can
be used, according to the present invention, to induce tolerance
towards organ or tissue transplants, or as a prelude for adaptive
cell therapy in cancer patients.
[0129] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0130] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0131] 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 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 1-111 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.
Example 1
Generation of Donor Anti-Third Party Mouse CTLs
IL-2 Starvation Improves Deletion of Anti-Host CTL Precursors
[0132] Non-myeloablative conditioning protocols are currently
explored in elderly leukemia patients who have HLA identical
donors. Engraftment is mediated in these patients by the large
number of T-cells present in the unseparated peripheral blood
progenitor preparations used for transplantation. Thus, these
transplants are associated with limited GVHD and with anti-host
reactivity leading to myeloablation of host hematopoiesis. However,
for patients who do not have a fully matched donor, T-cell toxicity
is more pronounced and rather unacceptable. Removal of alloreactive
T-cells from the transplant inoculum is likely to weaken the
engraftment potency and to result in a high rate of graft
rejection. As such, and as described in this Example, anti-third
party CTL preparations devoid of cells capable of maturing into
anti-host CTLs were established in order to reduce the rate of
rejection and to prevent induction of GVHD.
[0133] While reducing the present invention to practice and as is
specifically shown herein, the inventors have uncovered that CTL
veto cell preparations of a donor type, can be effectively depleted
of alloreactivity against host cells by stimulation against third
party stimulators by initial exogenous IL-2 starvation. This
depletion is due to the fact that only activated CTL precursors
(CT's) are capable of surviving the IL-2 starvation in the primary
culture. Evidently, this CTL preparation depleted of alloreactivity
against the host could potentially facilitate the induction of
donor type chimerism without GVHD in sub-lethally irradiated
recipients.
[0134] In addition, this study, examines the mechanisms responsible
for the veto activity mediated by non-alloreactive anti-third party
CTLs.
[0135] Materials and Methods:
[0136] Animals: 6-12 week old female mice were used. C3H/HeJ,
BALB/c, C57BL/6, gld/C3H/HeJ, lpr/C3H/HeJ were obtained from the
Roscoe B. Jackson Memorial Laboratory (Bar Harbor, Me.). A breeding
pair of Transgenic H-2.sup.b mice expressing the TCR-ab from the
CTL clone 2C with specificity for L.sup.d, was kindly provided by
Janko Nikolic-Zugic, (Sloan-Kettering, NY). Progeny from these Tg
mice and PO mice were bred at the Weizmann Institute Animal
Breeding Center (Rehovot, Israel). All mice were kept in small
cages (five animals in each cage) and fed sterile food and acid
water containing cyprofloxacin (20 .mu.g/ml).
[0137] Preparation of non-alloreactive donor anti-third party CTLs:
Splenocytes of Balb/c mice (donor responders) were harvested and
single cell suspensions were prepared. The cell suspensions were
treated with Tris-buffered ammonium chloride to remove red cells
and the isolated mononuclear cells (2.times.10.sup.6/ml) were
stimulated with irradiated (20 Gy) C57BL/6 or C3H/HeJ (third party
stimulators) splenocytes (2.times.10.sup.6/ml), and treated with
Tris-buffered ammonium chloride. Responders and stimulators were
incubated in a complete tissue culture medium (CTCM) at 37.degree.
C. in a 5% CO.sub.2/air Heraeus incubator. 5 days following
seeding, 20 U/ml of human rhIL-2 (Eurocetus, Milan, Italy) were
added to the mixed lymphocyte reaction culture every 24 hours, 2
days later the medium was replaced by a fresh culture medium. 10
days following seeding, the MLR cultures were harvested,
fractionated on Ficol-Paque plus (Amersham Pharmacia Biotech AB,
Sweden), analyzed by FACS for their CD8 level and added to the MLR
cultures at different cell ratios, as described hereinbelow in the
results section.
[0138] MLR cultures and cytotoxicity assay: Spleen cells of C3H/Hej
mice (responders) were harvested and single cell suspensions were
prepared as described above. The cells (2.times.10.sup.6/ml) were
then stimulated with irradiated (20 Gy) Balb/c splenocytes
(2.times.10.sup.6/ml), or with 2.times.10.sup.6/ml irradiated (20
Gy) SJL splenocytes. Four-six replicates per group were cultured in
96-well U bottomed plates in 0.2 ml CTCM, for 6 days at 37.degree.
C. (5% CO.sub.2 atmosphere). ConA blasts generated from SJL or
Balb/c spleen cells (2 days in the presence of 2 mg
ConA/2.times.10.sup.6 cells/ml) were labeled with .sup.51Cr (NEN,
Boston Mass.). Cell-mediated lysis assay was performed using
variable numbers of MLR effector cells and 5000 target cells in
96-well V-bottom plates. .sup.51Cr release was measured after a 4-h
incubation at 37.degree. C. Results are expressed as a specific
lysis calculated as follows: % specific
lysis=100.times.((experimental release-spontaneous
release)/(maximum release-spontaneous release)). The standard
deviation (SD) of replicate values were consistently less than 10%
of the mean.
[0139] Veto activity of non-alloreactive donor anti-third party
CTLs: To determine whether mouse non-alloreactive donor anti-third
party CTLs possess veto activity, spleen cells from C3H/HeJ mice
(2.times.10.sup.6/ml) were incubated for 6 days with irradiated (20
Gy) allogeneic spleen cells (2.times.10.sup.6/ml) from Balb/c (H-2
matched) or SJL (H-2 mismatch) mice. Non-alloreactive donor
anti-third party Balb/c CTLs were added to the MLC at a 1:100, 1:50
and 1:10 veto:responder cell ratio. The killing activity of the
responder CTL-p was determined by .sup.51Cr-release assay.
[0140] Inhibition of the veto effect of CTLs by anti-CD8 mAb: Veto
anti-third party CTLs from Balb/c origin were added to the MLR
described above, at a final concentration of 2%. Anti-CD8
monoclonal antibody (kindly provided by Uli Hamerling,
Sloan-Kettering, NY) directed against Ly-2.2 antigen expressed
selectively on the veto cells and not on the effector cells, was
added to the MLR at different concentrations as described in
results, and the inhibition of the veto effect was monitored.
[0141] Deletion of anti-H2.sup.d T-cells by non-alloreactive
anti-third party CTLs: Spleen cells of 2C Transgenic H-2.sup.b mice
expressing the TCR-ab with specificity for L.sup.d mice (kindly
provided by Janko Nikolic-Zugic, Sloan-Kettering, NY), were treated
as described above. The cells (2.times.10.sup.6/ml) were then
stimulated with irradiated (20 Gy) Balb/c splenocytes
(2.times.10.sup.6/ml) in the presence of 2% or 20% veto anti-third
party CTLs originated from Balb/c or from SJL (background control)
splenocytes. Cultures containing 20% or 2% veto CTLs were continued
for 48 hours or 5 days, respectively, in 6-well plates. The
deletion of transgenic T-cells was monitored by cytofluorometry,
measuring the level of 2C transgenic cells specifically stained by
the 1B2 antibody directed against the clonotypic anti-H-2L.sup.d
TCR.
[0142] Cytoflowmetry: FACS analysis was performed using a modified
Becton Dickinson FACScan. Fluorescence data were collected using
3-decade logarithmic amplification on 25-50.times.10.sup.3 viable
cells as determined by forward-light-scatter intensity. Cells were
stained with a CD8a (Ly-2)-FITC, CD3e-PE, CD95 (Fas)-FITC
(Pharmingen) CD4-Qantum Red (sigma), 1B2 biotinated (kindly
provided by Janko Nikolic-Zugic, Sloan-Kettering, NY), R-PE
streptavidin (Jackson Immuno. Research Lab. Inc.)
[0143] Vectors for gene transfer: PLNGFP-mock vector was
constructed by subcloning gene encoding enhanced green fluorescence
protein (EGFP, Clontech) from pEGFP-N1 (Clontech) into EcoRI and
HpaI sites of pLXSN provided by AD Miller, St. Jude Children's
Research Hospital, Memphis, Tenn.) PLNGFPFas-L was also a
derivative of pLXSN and was constructed by subcloning of a fragment
containing EGFP gene, internal ribosome entry site (IRES) and
murine Fas-L cDNA into BamHI and XhoI sites of pLXSN
(LTR-EGFP-IRS-mFas-L-SV40p-NeoR).
[0144] Packaging of retroviral vectors and virus supernatant
production: Plasmid DNA was transfected into ecotropic murine
retroviral vector packaging cell line GP-E86 and stably transfected
cells were selected with G418. High titter packaging cell lines
were isolated by screening individual colonies. Retrovirus
supernatant was prepared according to standard procedure.
[0145] Gene transfer into splenocytes of Fas-L mutated C3H-gld
mice: 50 u/ml of human rhIL-2 were added to a CTL culture generated
from "gld" spleen cells. Two days following addition, the
stimulated CTLs were resuspended in RPMI-1640 containing 5 mg/ml
protamine sulfate and 50 u/ml rhIL-2 and were incubated with viral
supernatant which was added daily for 3 days. Forty eight hours
following the last addition of the viral supernatant, the infected
cells were selected in G418 (450 mg/ml) in the presence of rhIL-2
(50 u/ml) for one week and then tested for their veto activity in
MLR as described above.
Experimental Results:
[0146] Veto activity of anti-third party CTLs: To evaluate the
effect of IL2 starvation on anti-host reactivity of
non-alloreactive anti-third party CTLs, Balb/c (H-2.sup.d)
splenocytes were stimulated, in the presence or absence of
recombinant human IL-2 (rhIL-2), against irradiated (20 Gy) C57BL/6
(H-2.sup.b, third party) splenocytes in MLR culture for 4 days. On
the second day or fifth day of the cell culture rhIL-2 was added
and the cells were kept for additional six days. After culture,
these cells were harvested and tested for their CTL-p frequency
against the intended host (C3H) or the third party used for
stimulation (C57BL/6). As can be seen in FIG. 1, following IL2
starvation for 4 days no anti-host CTLp could be detected while a
significant frequency of anti-host CTLp was documented when IL2 was
added to the MLR culture on the second day.
[0147] To evaluate the veto activity of such non-alloreactive C,
Balb/c H-2.sup.d splenocytes were stimulated against irradiated (20
Gy) C57BL/6H-2.sup.b (third party) splenocytes in MLR culture for
10 days, adding rhIL-2 only on the fifth day of the cell culture.
Following culturing, these cells were harvested and tested for
their veto activity in MLR cultures of C3H/Hej H-2.sup.k responder
cells (host type) stimulated against irradiated splenocytes from
Balb/c (donor type), or in an SJL culture (non-relevant control,
H-2.sup.S type). The inhibition of killing activity exerted by the
veto cells on the responder cells was determined 6 days post
culturing by the .sup.51Cr release assay.
[0148] As can be seen in FIG. 2a (17 different experiments) and in
FIG. 3 (one representative experiment), an addition of cells
generated in a Balb/c CTL line directed against third party cells
and cultured in the absence of IL-2, inhibits the killing activity
of the responder cells (C3H/Hej) which were stimulated against
Balb/c splenocytes. In contrast, the addition of cells from the
same CTL line to responder cells (C3H/Hej) that were stimulated
against SJL splenocytes, did not affect the responder's killing
activity. The specificity revealed by these results suggests that
the non-alloreactive CTLs directed against third party, indeed
possess veto activity.
[0149] The dose response curve shown in FIG. 2b suggests that the
anti-third party CTLs possess a very marked veto activity,
attaining complete inhibition at a 1:50 veto/responder cell ratio.
Addition of veto cells at 5 fold higher concentrations leads to
non-specific elimination of CTL-p's directed at targets not sharing
the donor type H-2 determinants (data not shown).
[0150] Optimal specific inhibition is achieved upon addition of
anti-third party CTLs, (at a 1:50 veto/responder cell ratio),
between day 0 and day 2. Thereafter, the veto effect is markedly
reduced (FIG. 3). Thus, as previously suggested, veto cells
probably inhibit or delete the responder cells at the precursor
level.sup.2. Once these CTL precursors develop into differentiated
CTLs the veto cells can no longer exert their effect.
[0151] In addition, by using monoclonal antibodies directed against
the CD8a Ly-2.2 allele expressed on the anti-third party CTLs but
not on the responder cells, confirmed previous observations 18 that
the CD8 molecules of the veto cells participate in the deletion of
the responder cells (FIG. 4).
[0152] The role of Fas-Fas-L apoptosis: The role of apoptosis in
responder cell deletion was confirmed by two approaches: (i) the
addition of the apoptosis inhibitor (BD-FmK) to the MLR culture led
to a complete inhibition of the veto effect (data not shown); and
(ii) the addition of anti-third party CTLs of Balb/c (H-2.sup.d)
origin, to an MLR culture of TCR transgenic C57B1/6 responders
bearing the 2C TCR directed against H-2.sup.d, led to a marked and
specific deletion of the transgenic responders upon stimulation
against Balb/c stimulators (FIGS. 5a-c).
[0153] Considering that two distinct major apoptosis mechanisms,
namely perforin mediated or Fas-Fas-L mediated apoptosis have been
previously described, two different non-alloreactive anti-third
party CTL lines from splenocytes of Fas-L deficient strain
(C3H-gld, H-2.sup.k) or from splenocytes of perforin deficient mice
(PO, H-2.sup.bd) were prepared. C3H-gld or PO-splenocytes were
stimulated against irradiated C57BL/6H-2.sup.b or DBA/1H-2.sup.q
splenocytes, respectively, as described for wild type splenocytes
(culturing for 10 days, without the addition of exogenous rhIL-2
during the first 4 days). The use of veto cells of gld-C3H/Hej
(H-2.sup.k origin) and of PO(H-2.sup.bd origin) necessitated
changes in the design of the MLR responders and stimulators
utilized. Thus, the gld mutant anti-third party CTLs were added to
bulk MLR cultures in which Balb/c responders were stimulated
against C3H/Hej splenocytes. The PO knockout anti-third party CTLs
were added to bulk MLR cultures in which C3H/Hej responders were
stimulated against PO or SJL splenocytes.
[0154] As can be seen in FIG. 6a, the anti-third party CTLs
originating from C3H-gld lacked veto activity, whereas the CTLs
originating from perforin deficient mice did exhibit significant
veto activity.
[0155] Additional evidence to the role of Fas-L in the veto effect
was afforded by gene transfer experiments in which gld-anti-third
party CTLs were transfected with Fas-L, using a retroviral vector.
As can be seen in FIG. 6b such transfected CTLs exhibited marked
veto activity compared to mock-infected anti-third party CTLs.
[0156] Using a similar approach, the role of Fas in the veto
activity induced by anti-third party CTLs was investigated. As can
be seen in FIG. 7a, when Fas deficient C3H-lpr splenocytes were
stimulated against Balb/c in the primary MLR culture, the addition
of Balb/c anti-third party CTLs failed to inhibit the primary CTLs.
In contrast, these cells exhibited veto activity when added to
primary MLR cultures of wild type C3H responders. Similarly, a Fas
antagonist (Fas fusion protein) also inhibited the veto effect of
anti-third party CTLs (FIG. 7b).
[0157] FACS analysis of Fas during a typical MLR of 2C splenocytes
against Balb/c stimulators (FIGS. 8a-d), revealed that Fas
expression on CD8.sup.+ 1B2.sup.+ T-cells is markedly enhanced at
48 hr (FIG. 8b). Addition of Balb anti-C3H (FIG. 8c), but not SJL
anti-C3H CTLs (FIG. 8d), led to a marked deletion of the Fas.sup.+
activated CD8 T cells, illustrating the potent veto activity and
the remarkable specificity by which the anti-third party veto CTLs
expressing Fas-L .sup.25, 29 are capable of selectively recognizing
and killing Fas.sup.+ T-cells aimed against their H-2 antigens.
[0158] Altogether these results suggest that the veto effect of
non-alloreactive anti-third party CTLs is mediated by the
simultaneous expression of CD8 and Fas-L on the veto CTLs and the
expression of Fas on the effector CTL-p's directed against the
antigens presented by the veto cells.
Example 2
Generation of Donor Anti-Third Party Human CTLs Depleted of
Anti-Host CTL Precursors
[0159] Following the demonstration described above that anti-third
party CTLs can be depleted of anti-host CTLp an attempt to apply
this approach to human settings has been undertaken. It will be
appreciated in this respect, and it is well accepted among art
scholars, that it is not at all predictable that an immunogenic
approach effective in mice would have similar effectiveness in
human beings.
[0160] Materials and Methods:
[0161] Establishment of anti-third party CTLs: Anti-third party
CTLs were prepared by stimulating normal peripheral blood
lymphocytes (PBL) derived from a human donor against an
Epstein-Barr virus (EBV) transformed cell line of known HLA type
(hereinafter stimulator A). PBLs were cultured in growth complete
medium+10% FCS (CM-RPMI 1640+2 mM L-glutamine+100 U/ml
penicillin+0.1 mg/ml streptomycin+2 mM HEPES+1 mM sodium
pyruvate+0.1 mM non-essential amino acids+5.times.10.sup.-5 M
.beta.-mercaptoethanol) at 2.times.10.sup.6 cells/ml and were
stimulated with 5.times.10.sup.4 cells/ml irradiated (10,000 rad)
stimulators.
[0162] Following 10 days of co-culture, the cells were harvested
and live cells were isolated on Ficoll-Paque gradients and
re-stimulated, 5.times.10.sup.6 cells/ml with 1.5.times.10.sup.5
cells/ml of stimulators. Four days later the cultures were treated
with hrIL-2 at 20 U/ml, for the first time.
[0163] Thereafter, the cultures were treated three times per week,
each time with 20 U/ml rhIL-2. The third treatment also included
the addition of irradiated stimulators at a ratio of 4:1.
[0164] The CTL line, originally stimulated against stimulator S was
tested for is CTL-p responders against the original stimulator S or
against stimulator No. 38 which represents the intended host.
[0165] The cells from this anti-third party CTL line were cultured
either with stimulator S or with stimulator 38, at 1.times.10.sup.6
cell/ml 1:1 for 5 days. The cells were then harvested from the bulk
culture and serial dilutions of CTL responder cells
(4.times.10.sup.4 to 0.2.times.10.sup.3) per well, were prepared
with the original irradiated stimulators. Each well contains
10.sup.5 of irradiated (30 Gy) stimulator 38 or 3.times.10.sup.4
(100 Gy) irradiated stimulator S. The cultures were incubated for 7
days in complete medium+1%+20 U/ml rhIL-2.
[0166] Cytotoxicity was assayed with 5.times.10.sup.3 51Cr labeled
blasts (target cells) from S, No. 38 and No. 40 (control for
non-specific killing) donors.
[0167] Normal unseparated PBLs from the original donor of the cell
line were tested in parallel.
[0168] Experimental Results:
[0169] Non-alloreactive human CTL lines were established in-vitro
by stimulating PBLs with EBV transformed cell line of known HLA
type. While a significant level of anti-host CTLp was demonstrated
in untreated PBL of donor A (FIG. 9), it was found that the
anti-third party CTL line that was generated from PBL of the same
donor A was markedly depleted of such cells (FIG. 10). These
results indicate that the CTL preparation of this Example is
depleted of T-cells having the potential of maturing into anti-host
CTLs (alloreactive CTLs) and is therefore advantageous as an agents
for enhancing graft acceptance in humans.
[0170] Conclusions:
[0171] Prior art approaches, which make use of T-cell stimulation
with the very antigens against which tolerance is desired, might
involve some risk for generating committed CTLs, if any of the
CTL-p escape deletion or anergy induction. Once such anti-host CTLs
are generated, it is very difficult to suppress their
alloreactivity in vivo.
[0172] A more recent approach, demonstrated that a "megadose" of
cells can be applied to induce tolerance in sublethally irradiated
mice, and as such effectively overcome the marked resistance
presented by the large number of lymphocytes surviving the
sublethal conditioning. However, the number of cells required to
attain this desirable goal may not be easily collected from human
donors.
[0173] Non-alloreactive anti-third party CTLs which can be used to
enhance graft acceptance in mice were also shown, however, it was
soon realized that such CTL preparations are not depleted of
T-cells capable of developing post transplantation into anti-host
CTLs inflicting GVHD. The present research demonstrates that donor
anti-third party CTLs can be depleted of anti-host alloreactivity
if exogenous IL-2 is not provided during the initial days of the
bulk culture with the stimulator cells. Such non-alloreactive CTLs
can enhance engraftment, without GVHD, of, for example, T-cell
depleted BM originating from the same donor.
[0174] The present study further uncovers the mechanism responsible
for the marked potent veto activity of such anti-third party
CTLs.
[0175] By utilizing anti-CD8 antibodies, the present study
demonstrates that the expression of CD8 on the CTLs is crucial for
CTL-p recognition.
[0176] By using anti-third party non-alloreactive CTLs generated
from spleen cells of different mutant mice the present study
demonstrates that the veto activity of these cells is mediated by
apoptosis via the Fas-Fas-L system and not by the perforin
mechanism.
[0177] Thus, the results of the present study suggest that
simultaneous expression of both CD8 and Fas-L on the CTL surface
and the expression of Fas on the effector CTL-p is a prerequisite
for the veto activity.
Example 3
The Primate Model
[0178] Materials and methods:
[0179] Monkeys: Female cynomolgus monkeys (aged 1.5-2 years)
weighing 1.5-2.5 kg, are used as bone marrow recipients and MLC
mismatched male cynomolgus monkeys weighing 6-9 kg are used as
donors.
[0180] Conditioning regimen: A sublethal irradiation-based protocol
utilized is as follows: Fludarabine (FLU) 40 mg/m.sup.2 from day-9
(prior to transplantation) to day-5 (total amount 200 mg/m.sup.2);
Rabbit anti-thymocyte globulin (ATG) 5 mg/kg from day-7 to -3 and
6.5 or 7 Gy TBI (dose rate 0.15 Gy/min) delivered on day-1.
[0181] Peripheral blood mononuclear cells collection: RhG-CSF (20
mg/kg/day) is administered to donor monkeys over a period of five
days in two daily subcutaneous injections. Two leukapheresis
procedures are performed between days 4 and 5.
[0182] Donors and leukapheresis: Healthy male cynomologus donors
are subjected to a double or triple set of 100-minute leukapheresis
on a Cobe Spectra cell separator, as described by Hillyer et al.
[reference No. 27] with the following minor modifications: acid,
citrate, dextrose (ACD)-to-blood ratio is 12:14 and centrifuge
speed is 1,600 rpm.
[0183] Peripheral blood mononuclear cell processing: Peripheral
blood progenitor cells (PBPC) preparations are depleted of
T-lymphocytes using the E-rosetting technique, as previously
described 26. Stem cells are then purified by positive selection by
using an avidin-biotin immunoadsorbtion column (CEPRATE SC System,
CellPro, Bothell, Wash.), according to the manufacturer's
instructions. The number of CD34.sup.+ cells is measured both in
whole blood and in the leukapheresis product by flow cytometry. The
T-lymphocytes before and after T-cell-depletion are also measured,
by using a phycoerythrin-coupled anti-CD2 monoclonal antibody.
Aliquots are taken for differential cell counts, mAb staining and
GFU-GM assay at each stage of processing.
[0184] Supportive care: Monkeys are cared for in laminar air-flow
cages until the neutrophils count recovered to 1.times.10.sup.9/L.
All monkeys receive cyproxin and amphotericin B as gut preparation,
combined antibiotics as prophylaxis against bacterial infection
since the day of transplant, and fluconazole as antifungal
prophylaxis. Acyclovir is administered for preventing viral
infection. Whole blood or component blood products are transfused
according to monkey's hematocrit and platelet count. All blood
products are irradiated with 25-30 Gy and filtered before
transfusion.
[0185] Engraftment and chimerism determination: Time to engraftment
is assessed by determining the day after transplant on which
monkeys displayed a level of 0.5 neutrophils.times.10.sup.9/L and
25.times.10.sup.9 platelets/L independent of transfusion support.
Chimerism is assessed by quantitative polymerase chain reaction
(PCR). Briefly, the PCR products are subjected to 3% MetaPhor
agarose (FMC) gel. The density of each band for Y specific DNA and
competitor DNA is measured by using computerized densitometer and
the target (Y specific)/competitor DNA (T/C) ratio for each sample
is calculated. At the point where Y specific and competitor DNA are
in equivalence (i.e., ratio=1.0), the starting amount of Y specific
DNA prior to PCR is equal to the known starting amount of
competitor DNA.
[0186] Generation of donor-anti-third party CTLs: A slightly
modified procedure is used for preparation of monkey donor
anti-third party CTLs. Peripheral blood cells of donor and third
party monkeys are fractionated on Ficoll-Paque plus and the
isolated mononuclear cells, 1.times.10.sup.6/ml of responder cells
and 2.times.10.sup.6/ml irradiated (20 Gy) third party stimulators,
are cultured for 10 days in CTCM medium. Four days hours after
seeding, human rhIL-2 (20 U/ml, Eurocetus, Milan, Italy), is added
every 24 hours.
Example 4
Preparation of Human CD8+ CTLs is Enhanced by Removal of CD4+ and
CD56+ Cells
[0187] It was observed that the level of CD8+ cells in the CTL
cultures prepared essentially as described above is extremely
variable. Therefore, selection of CD8+ cells by removal (negative
selection) of CD4+ and CD56+ cells, using antibodies linked to
magnetic beads. As can be seen in FIGS. 11a-b the cells obtained by
this procedure exhibited very potent veto activity at a ratio of
1:25 and 1:50 veto to effector cell ratio.
[0188] Treatment of donor cells: Anti third party CTLs were
prepared by stimulating donor peripheral blood lymphocytes against
EBV transformed human derived cell line of a known HLA type
(stimulators) in 24-well plates. The peripheral blood lymphocytes
were cultured in growth complete medium supplemented with 10% fetal
calf serum (CM-RPMI 1640, 2 mM L-glutamine, 100 Units/ml
penicillin, 0.1 mg/ml streptomycin, 2 mM Hepes, 1 mM sodium
pyruvate, 0.1 mM non essential amino acids, 5.times.10.sup.-5 M
.beta.2-mercaptoethanol) at 1.times.10.sup.6 cells/ml and
stimulated with 2.5.times.10.sup.4 cells/ml irradiated (10,000 Rad)
stimulators, in 250 ml flasks at a volume of 50 ml.
[0189] After 10 days of co-culture, the cells were harvested and
live cells were isolated on ficoll gradient and re-stimulated, the
cells were resuspended to a concentration of 5.times.10.sup.5
cells/ml and were re-stimulated with 1.5.times.10.sup.5 stimulator
cells/ml.
[0190] Four days later, the cells were negatively selected for CD4+
and CD56+ cells with MACS beads (Miltenyi Biotec). The unbound
cells were resuspended at 1.times.10.sup.6 cells/ml and
re-stimulated with 0.33.times.10.sup.6 stimulator cells/ml. At this
stage (following two weeks in culture) the cells were treated with
interleukin-2 (IL-2, Proleukin) at 600 Units/ml.
[0191] Thereafter, the cultures were treated three times per week,
each time with 600 U/ml IL-2. The third time also included the
addition of irradiated stimulators at a ratio of 4:1. Please note
that during the first two weeks of culture, no IL-2 was added to
the cells.
Example 5
Experiments in Human
Case Report
[0192] Treatment of donor cells: Anti third party CTLs were
prepared by stimulating donor peripheral blood lymphocytes against
EBV transformed human derived cell line of a known HLA type
(stimulators), as described in Example 4 above. The CTLs obtained
were infused to a patient as described bellow in order to tolerize
the immune system of the patient, so as to facilitate engraftment
of stem cells thereto.
[0193] The patient was diagnosed on May 1998 with mantle cell
lymphoma. The patient was treated with 4 courses of PROMACE
resulting in partial remission. During December 1998 the patient
was treated with a high dose of Cytoxan (CTX) and autologous
peripheral blood stem cells were harvested and cryopreserved.
During March 1999 the patient received an autologous transplant
after conditioning with TBI- and thiotepa. Thereafter, complete
remission has been achieved.
[0194] During October 1999 the patient relapsed with
lympho-plasmacytoid infiltration of the bone marrow. IgG/k serum
levels were treated by plasmapheresis and dexametasone until July
2000.
[0195] During October 2000 the patient received an allogeneic mega
dose stem cell transplant from a mis-matched haploidentical child.
The patient was conditioned with melphalan 70 mg/sqm on day -12,
thiotepa 7 mg/kg body weight on day -11, fludarabin 40 mg/sqm from
day -9 to day -6, Fresenius ATG 2.5 mg/kg body weight from day -9
to day -6. On day -1 the anti-third party CTLs were infused at a
cell dose of 6.1.times.10.sup.6 CTL/Kg recipient body weight. On
day 0 the donor stem cells were infused at a dose of
9.7.times.10.sup.6 CD34+ cells per Kg body weight (processed by
CliniMACS and including 40,000 CD3T cells/Kg body weight).
Conditioning as well as CTL infusion was well tolerated despite the
pre-transplant cardio-pulmonary compromission of this old
patient.
[0196] Neutrophil engraftment (500/mm.sup.3) was first found on day
+8, 980/mm.sup.3 on day +11, 2,760/mm.sup.3 on day +14, and
5,370/mm.sup.3 on day +21.
[0197] Platelet reconstitution (35,000/mm.sup.3) was first achieved
on day +18.
[0198] CMV antigenemia became positive (only 4 cells) on day +40
(Patient was not under prophylactic treatment).
[0199] Gammapathy on electrophoresis, typical of Lymphoma patients,
was reduced to normal levels (before transplant--33.8%, 21% on day
+20 post transplant).
[0200] IgG level was reduced to normal levels (before
transplant--2330 mg/dl, 1550 mg/dl post transplant).
[0201] FISH analysis on day +20 post transplant revealed that all
the cells in the peripheral blood and in the bone marrow were of
donor origin.
[0202] 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. All
publications cited herein are incorporated by reference in their
entirety.
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