U.S. patent application number 10/005747 was filed with the patent office on 2002-11-28 for immunotherapy of cancer with allogeneic lymphocytes.
Invention is credited to Slavin, Shimon.
Application Number | 20020176850 10/005747 |
Document ID | / |
Family ID | 22801017 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020176850 |
Kind Code |
A1 |
Slavin, Shimon |
November 28, 2002 |
Immunotherapy of cancer with allogeneic lymphocytes
Abstract
Methods have been discovered for treating minimal residual
disease following removal of most or a substantial fraction of
malignant cells from a cancer patient. An autologous stem cell
transplant is performed on the patient. Following partial
hematopoiesis recovery, the patient is infused with allogeneic
peripheral blood lymphocytes, either alone or in combination with
in vivo or in vitro cytokine. The infused allogeneic lymphocytes
engender an anti-malignant cell response and can be instrumental in
prevention of disease relapse.
Inventors: |
Slavin, Shimon; (Jerusalem,
IL) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
22801017 |
Appl. No.: |
10/005747 |
Filed: |
December 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10005747 |
Dec 3, 2001 |
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09310325 |
May 12, 1999 |
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10005747 |
Dec 3, 2001 |
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08735496 |
Oct 23, 1996 |
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5928639 |
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10005747 |
Dec 3, 2001 |
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08656694 |
Jun 3, 1996 |
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10005747 |
Dec 3, 2001 |
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08214944 |
Mar 17, 1994 |
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Current U.S.
Class: |
424/93.21 ;
424/93.7 |
Current CPC
Class: |
C12N 2501/23 20130101;
A61K 38/217 20130101; A61K 38/2013 20130101; A61K 38/2046 20130101;
A61K 2035/124 20130101; A61K 38/204 20130101; A61K 35/17 20130101;
A61K 38/2013 20130101; A61K 38/204 20130101; A61P 35/00 20180101;
A61K 38/217 20130101; C12N 5/0636 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 38/2046
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/93.21 ;
424/93.7 |
International
Class: |
A61K 048/00 |
Claims
What is claimed is:
1. A method of treating a human cancer patient, said patient having
undergone a malignant cell debulking procedure and being at risk
for disease relapse due to a population of residual malignant cells
that may remain viable in said patient following said debulking
procedure, comprising: a) providing a sample of stem cells from
said patient, said sample being suitable for autologous
transplantation into said patient; b) performing an autologous
transplant of said patient with said sample; c) monitoring said
patient until said patient is partially hematopoiesis recovered but
is not fully immune-reconstituted; d) administering to said patient
an HLA-compatible, allogeneic peripheral blood leukocyte
preparation having lymphocytes, in a regimen that causes a
clinically significant graft-versus-malignant cell response; and e)
monitoring said patient for levels of malignant cells deriving from
said population.
2. The method of claim 1, wherein said regimen comprises the
following steps in sequence: a) treating said patient by
administration of about 10.sup.7 cells/kg to about 10.sup.9
cells/kgof HLA-compatible, allogeneic peripheral blood lymphocytes;
b) monitoring said patient for indications of a
graft-versus-malignant cell response; and c) if no or insufficient
graft-versus-malignant cell response develops in said patient,
escalating said treatment by performing at least one procedure
selected from the group consisting of (1) administration of a
number of HLA-compatible, allogeneic peripheral blood lymphocytes
greater than the number of lymphocytes administered in step (a);
(2) administration of a number of HLA-compatible, allogeneic
peripheral blood lymphocytes at least as great as the number of
lymphocytes administered in step (a), accompanied by administration
of at least one T-cell-activating cytokine to said patient; (3)
administration of HLA-compatible, allogeneic CAL's to said patient;
and (4) administration of HLA-compatible, allogeneic CAL's,
accompanied by administration in vivo of at least one
T-cell-activating cytokine to said patient; wherein more than one
of said procedures is performed if no or insufficient
graft-versus-malignant cell response develops in said patient
following said first or subsequent procedure.
3. The method of claim 2, wherein step (a) further comprises
administration in vivo of at least one T-cell-activating cytokine
to said patient.
4. A method of treating a human cancer patient, said patient having
undergone a malignant cell debulking procedure and being at risk
for disease relapse due to a population of residual malignant cells
that may remain viable in said patient following said debulking
procedure, comprising: a) providing a sample of stem cells from
said patient, said sample being suitable for autologous
transplantation into said patient; b) performing an autologous
transplant of said patient with said sample; c) monitoring said
patient until said patient is partially hematopoiesis recovered but
is not fully immune-reconstituted; d) administering to said patient
an HLA-compatible, allogeneic peripheral blood leukocyte
preparation having lymphocytes, in a regimen that causes a mild
graft-versus-host response; and e) monitoring said patient for
levels of malignant cells deriving from said population.
5. The method of claim 4, wherein said regimen comprises the
following steps in sequence: a) treating said patient by
administration of about 10.sup.7 cells/kg to about 10.sup.9
cells/kg of HLA-compatible, allogeneic peripheral blood
lymphocytes; b) monitoring said patient for indications of a mild
graft-versus-host response; and c) if no or insufficient
graft-versus-host response develops in said patient, escalating
said treatment by performing at least one procedure selected from
the group consisting of (1) administration of a number of
HLA-compatible, allogeneic peripheral blood lymphocytes greater
than the number of lymphocytes administered in step (a); (2)
administration of a number of HLA-compatible, allogeneic peripheral
blood lymphocytes at least as great as the number of lymphocytes
administered in step (a), accompanied by administration of at least
one T-cell-activating cytokine to said patient; (3) administration
of HLA-compatible, allogeneic CAL's to said patient; and (4)
administration of HLA-compatible, allogeneic CAL's, accompanied by
administration of at least one T-cell-activating cytokine to said
patient; wherein more than one of said procedures is performed if
no or insufficient graft-versus-host response develops in said
patient following said first or subsequent procedure.
6. The method of claim 5, wherein step (a) further comprises
administration in vivo of at least one T-cell-activating cytokine
to said patient.
7. The method of claim 4, wherein said regimen comprises the
following steps in sequence: a) administering to said patient about
10.sup.7 cells/kg to about 10.sup.9 cells/kg of HLA-compatible,
allogeneic peripheral blood lymphocytes and at least one
T-cell-activating cytokine to said patient; b) monitoring said
patient for signs of a mild graft-versus-host response; c) if no or
insufficient graft-versus-host response develops in said patient,
administering about 10.sup.7 cells/kg to about 10.sup.9 cells/kg of
HLA-compatible, allogeneic CAL and at least one T-cell-activating
cytokine to said patient; and d) monitoring said patient for signs
of a mild graft-versus-host response.
8. The method of claim 4, wherein said regimen comprises the
following steps in sequence: a) administering to said patient about
10.sup.5 cells/kg to about 10.sup.9 cells/kg of HLA-compatible,
allogeneic peripheral blood lymphocytes, said HLA-compatible,
allogeneic peripheral blood lymphocytes comprising CAL, and at
least one T-cell-activating cytokine to said patient; b) monitoring
said patient for signs of a mild graft-versus-host response; c) if
no or insufficient graft-versus-host response develops in said
patient, administering about 10.sup.5 cells/kg to about 10.sup.9
cells/kg of HLA-compatible, allogeneic CAL and at least one
T-cell-activating cytokine to said patient; and d) monitoring said
patient for signs of a mild graft-versus-host response.
9. The method of claim 2, 3, 5, 6, 7 or 8 wherein said cytokine is
selected from the group consisting of IL2, IL4, IL5, IL6, IL7,
IFN.alpha., IFN.gamma. and TNF.alpha..
10. The method of claim 4, wherein said stem cells are obtained
from bone marrow.
11. The method of claim 4, wherein said stem cells are obtained
from the peripheral circulation.
12. The method of claim 4, wherein said stem cells are obtained
from fetal sources selected from the group consisting of fetal
tissue, fetal circulation and umbilical cord blood.
13. The method of claim 4, wherein said malignant cells are
leukemia cells.
14. The method of claim 4, wherein said malignant cells are
lymphoma cells.
15. The method of claim 4, wherein said malignant cells are breast
cancer cells.
16. The method of claim 1 or 4, wherein said HLA-compatible cells
are fully HLA-matched with said patient.
17. The method of claim 1 or 4, wherein said HLA-compatible cells
are at least haploidentical with said patient.
18. The method of claim 1 or 4, wherein said HLA-compatible cells
are single HLA locus-mismatched cells from a sibling of said
patient.
19. An article of manufacture comprising packaging material and a
biological cell container within said packaging material, wherein
said packaging material contains a label or package insert
indicating that said biological cell container and any contents
therein are to be used in the method of claim 1 or 4.
Description
FIELD OF THE INVENTION
[0001] This invention relates to methods of eradicating residual
tumor cells following surgical intervention, chemotherapy and/or
radiotherapy. The methods involve autologous bone marrow
transplantation followed by administration of allogeneic peripheral
blood leukocytes. More particularly, this invention relates to use
of HLA-compatible allogeneic peripheral blood lymphocytes (PBL),
following autologous bone marrow and/or peripheral blood stem cell
transplantation, to induce a graft-versus-malignant cell response.
Such a response is instrumental in prevention of disease
relapse.
BACKGROUND OF THE INVENTION
[0002] Patients with malignant hematological disorders resistant to
conventional doses of chemotherapy and/or radiation may be treated
by autologous or allogeneic bone marrow transplantation. Bone
marrow transplantation (BMT) makes it possible to administer to
patients with resistant disease high, "supra-lethal," combinations
of chemotherapy and radiation, ignoring the irreversible toxicity
of such therapeutic combinations on the normal bone marrow
compartment. Nevertheless, such "debulking" of a patient's tumor(s)
can leave a fraction of residual malignant cells that may lead to
disease relapse. Several lines of evidence suggest that a
significant proportion of the beneficial effect of allogeneic BMT
(i.e., BMT from an individual not genetically identical to the host
patient) stems from cell-mediated interactions of immune cells of
donor origin against residual tumor cells in the host that have
escaped the chemoradiotherapy debulking regimen.
[0003] Following allogeneic BMT, the incidence of relapse is
significantly lower in leukemia patients with clinical
manifestations of acute or chronic graft versus host disease
(GVHD), as compared with patients with no GVHD, indicating that
immune-mediated allogeneic interactions of immunocompetent cells of
donor origin against the host are also accompanied by graft vs.
leukemia (GVL) effects. Weiden et al., N. Engl. J. Med. 300: 1068
(1979); Weiden et al., N. Engl. J. Med. 304: 1529-33 (1981); Weiden
et al., Transplantation 13: 248-51 (1981); Barrett et al., Blood
74: 862 (1989); Sullivan et al., Blood 73: 1720 (1989); Horowitz et
al., Blood 75: 555 (1990); Slavin et al., Bone Marrow Transplant.
6: 155-61 (1990).
[0004] Higher relapse rates seem to occur in patients undergoing
allogeneic BMT with T-lymphocyte depletion for prevention of GVHD,
compared to recipients of non-T cell depleted marrow allografts,
regardless of the severity of GVHD. Horowitz et al., Blood 75: 555
(1990); Slavin et al., Bone Marrow Transplant. 6: 155-61 (1990);
Goldman et al., Ann. Inter. Med. 108: 806-14 (1988); Ringden and
Horowitz, Transplant. Proc. 21: 2989-92 (1989); Goldman et al.,
Ann. Int. Med. 108: 806 (1988). Likewise, relapse rates in patients
with acute leukemia or chronic myeloid leukemia reconstituted by
bone marrow grafts obtained from an identical twin (syngeneic
grafts) are significantly higher than in those reconstituted by
bone marrow cells obtained from an HLA-identical but non-syngeneic
sibling. Ringden and Horowitz, Transplant. Proc. 21: 2989-92
(1989). Similarly, relapse rates following transplantation of the
patient's own (autologous) marrow, even following adequate purging
in vitro for elimination of residual leukemia cells, are
significantly higher than following allogeneic BMT. Armitage, Curr.
Opinion in Hematol. 1993: 240-45 (1993). Thus, the less-than
optimal results with autologous BMT (ABMT) are similar to the
results seen with syngeneic marrow transplantation. All of the
above data suggests that in practical terms GVHD or GVHD-potential
correlates with a lower incidence of relapse.
[0005] Allogeneic donor cells may also play a role against
lymphoma, as shown in experimental animals, Slavin et al., Cancer
Immunol. Immunother. 11: 155-58 (1981), and humans. Phillips et
al., J. Clin. Oncol. 4: 480-88 (1986); Ernst et al., Proc. of the
4th International Conference on Lymphoma, Lugano 1990, Abstract
#P35; Chopra et al., J. Clin. Oncol. 10: 1690-95 (1992). As shown
in experimental animals, graft-versus-tumor effects (GVT), similar
to graft-versus-leukemia effects (GVL), may occur following BMT,
independently of GVHD. Moscovitch and Slavin, J. Immunol. 132:
997-1000 (1984).
[0006] Although GVHD-associated anti-leukemia effector mechanisms
may be of benefit in BMT, nevertheless GVHD represents one of the
major obstacles in allogeneic- BMT, even among perfectly
HLA-matched siblings. Acute GVHD develops in the majority of
recipients of allogeneic BMT, with clinically significant
manifestations in 26-64% of the recipients despite optimal
post-transplant immunosuppressive prophylaxis. Storb et al., Blood
73: 1729 (1989). Chronic GVHD may occur in up to 45% of long term
survivors. Storb et al., Blood 73: 1729 (1989). There is no
satisfactory therapy for patients with established GVHD and hence
patients with severe manifestations of acute or chronic forms of
the disease are prone to develop multisystem complications that may
require frequent hospitalizations, leading to poor quality of life
and occasionally serious or even fatal complications.
[0007] GVHD following allogeneic BMT can be prevented by adequate
pretransplant T-lymphocyte depletion, using no post-transplant
anti-GVHD prophylaxis. Reisner et al., In: Slavin, S (ed.),
Tolerance in Bone Marrow and Organ Transplantation. Elsevier,
Amsterdam (1984), p. .sup.293; Waldmann et al., Lancet 2: 483-85
(1984); Slavin et al., Transplant. Proc. 17: 465-67 (1985). BMT
without GVHD represents a better tolerated procedure that may
necessitate shorter hospitalization with superior subjective
immediate outcome following allogeneic BMT. In addition, the
quality of life of long-term survivors without GVHD is clearly
better than for those patients with severe acute or chronic
GVHD.
[0008] Unfortunately, the advantages of GVHD-free-allogeneic BMT
are counterbalanced by other serious complications due to untoward
effects of T-lymphocyte depletion, such as increased incidence of
graft rejection, occurring in 10-30% of recipients, as well as
increased rates of tumor relapse. Martin et al., Bone Marrow
Transplant. 3: 445 (1988); Kernan et al., Transplantation 43: 842
(1987); Waldmann et al., Lancet 2: 483-85 (1984); Slavin et al.,
Transplant. Proc. 17: 465-67 (1985). Consequently, there seems to
be no clear evidence to date for a significant overall benefit of
GVHD prevention by T-lymphocyte depletion.
[0009] Clearly, it would be a significant advance in the art to be
able to combine the benefits of minimal or controllable GVHD risk
following ABMT or ASCT with induction of graft-versus-malignant
cell response that may be associated with GVHD following allogeneic
BMT.
SUMMARY OF THE INVENTION
[0010] The present invention includes a method of treating a human
cancer patient who has undergone a malignant cell debulking
regimen. Generally the patient is considered to be at risk for
disease relapse due to a population of residual malignant cells
that may remain viable in the patient following the debulking
procedure. A sample of stem cells, taken from the patient, is
obtained and determined to be suitable for autologous
transplantation into the patient. This sample is used to perform an
autologous transplant of the patient, i.e, infusing the patient's
stem cells back into the patient. The patient is then monitored
until the patient is partially hematopoiesis recovered but is not
fully immune-reconstituted. Then, the patient is administered an
HLA-compatible, allogeneic peripheral blood leukocyte preparation
having lymphocytes, in a regimen that causes a clinically
significant graft-versus-malignant cell response. The patient is
then monitored for levels of malignant cells deriving from any
residual malignant cells that might have been present following the
original debulking procedure. This monitoring may constitute one or
more molecular or cellular assays to detect or quantify malignant
cells, may constitute a monitoring program to detect clinical signs
of relapse, or any combination of these monitoring methods.
[0011] As used herein, a clinically significant response permits,
for example, the patient to avoid relapse, substantially prolongs
the time to relapse or otherwise engenders a beneficial condition
that significantly prolongs life. A graft-versus malignant cell
response is not clinically significant unless there is some
clinical benefit to the patient. Evidence for a clinically
significant response may include, for example, absence or delay of
relapse, evidence for elimination of minimal residual disease,
i.e., elimination of disease-specific markers or, where
appropriate, elimination of markers directed to host-specific
cells.
[0012] The regimen for administration of an HLA-compatible
leukocyte preparation including lymphocytes may comprise the
following steps in sequence:
[0013] a) treating the patient by administration (e.g., infusion)
of about 10.sup.7 cells/kg to about 10.sup.9 cells/kg of
HLA-compatible, allogeneic peripheral blood lymphocytes;
[0014] b) monitoring the patient for indications of a
graft-versus-malignant cell response; and
[0015] c) if no or insufficient graft-versus-malignant cell
response develops in the patient, escalating the treatment by
performing at least one of the following procedures: (1)
administration of a number of HLA-compatible, allogeneic peripheral
blood lymphocytes greater than the number of lymphocytes
administered in step (a); (2) administration of a number of
HLA-compatible, allogeneic peripheral blood lymphocytes at least as
great as the number of lymphocytes administered in step (a),
accompanied by administration of at least one T-cell-activating
cytokine to said patient; (3) administration of HLA-compatible,
allogeneic cytokine-activated lymphocytes (CAL) to said patient;
and (4) administration of HLA-compatible, allogeneic CAL,
accompanied by administration in vivo of at least one
T-cell-activating cytokine to said patient. More than one of these
procedures can be performed if no or insufficient
graft-versus-malignant cell response develops in the patient
following the first or subsequent procedure.
[0016] In an alternative embodiment, step (a) above can be
augmented by administering, concomitant with the allogeneic
lymphocytes, at least one T-cell-activating cytokine to the
patient. Since the T-cell-activating cytokine is administered
directly to the patient, the infused allogeneic lymphocytes are
exposed to the cytokine in vivo.
[0017] The present invention also includes an alternative method of
treating a human cancer patient who has undergone a malignant cell
debulking regimen. Generally the patient is considered to be at
risk for disease relapse due to a population of residual malignant
cells that may remain viable in the patient following the debulking
procedure. A sample of stem cells, taken from the patient, is
obtained and determined to be suitable for autologous
transplantation into the patient. This sample is used to perform an
autologous transplant of the patient, i.e, infusing the patient's
stem cells back into the patient. The patient is then monitored
until the patient is partially hematopoiesis recovered but is not
fully immune-reconstituted. Then, the patient is administered an
HLA-compatible, allogeneic peripheral blood leukocyte preparation
having lymphocytes, in a regimen that causes a mild
graft-versus-host response. The patient is then monitored, as
above, for levels of malignant cells deriving from any residual
malignant cells that might have been present following the original
debulking procedure.
[0018] As used herein, the term "graft-versus-host response"
includes but is not limited to the classic clinical symptoms of
graft-versus host disease (GVHD), known to those having ordinary
skill in the art. The term "graft-versus-host response" also
includes molecular or cellular responses that correlate with the
clinical symptoms of GVHD or with the impending onset of the
clinical symptoms of GVHD.
[0019] For the above-described alternative method, the regimen for
administration of an HLA-compatible leukocyte preparation including
lymphocytes may comprise the following steps in sequence:
[0020] a) treating the patient by administration of about 10.sup.7
cells/kg to about 10.sup.9 cells/kg of HLA-compatible, allogeneic
peripheral blood lymphocytes;
[0021] b) monitoring the patient for indications of a mild
graft-versus-host response; and
[0022] c) if no or insufficient graft-versus-host response develops
in the patient, escalating the treatment by performing at least one
of the following procedures: (1) administration of a number of
HLA-compatible, allogeneic peripheral blood lymphocytes greater
than the number of lymphocytes administered in step (a); (2)
administration of a number of HLA-compatible, allogeneic peripheral
blood lymphocytes at least as great as the number of lymphocytes
administered in step (a), accompanied by administration of at least
one T-cell-activating cytokine to said patient; (3) administration
of HLA-compatible, allogeneic CAL to said patient; and (4)
administration of HLA-compatible, allogeneic CAL, accompanied by
administration in vivo of at least one T-cell-activating cytokine
to said patient. More than one of these procedures can be performed
if no or insufficient graft-versus-host response develops in the
patient following the first or subsequent procedure.
[0023] In an alternative embodiment, step (a) above can be
augmented by administering, concomitant with the allogeneic
lymphocytes, at least one T-cell-activating cytokine to the
patient. Since the T-cell-activating cytokine is administered
directly to the patient, the infused allogeneic lymphocytes are
exposed to the cytokine in vivo.
[0024] In an alternative embodiment, the regimen for administration
of an HLA-compatible leukocyte preparation including lymphocytes
may comprise the following steps in sequence:
[0025] a) administering to the patient about 10.sup.7 cells/kg to
about 10.sup.9 cells/kg of HLA-compatible, allogeneic peripheral
blood lymphocytes and at least one T-cell-activating cytokine to
the patient;
[0026] b) monitoring the patient for signs of a mild
graft-versus-host response;
[0027] c) if no or insufficient graft-versus-host response develops
in the patient, administering about 10.sup.7 cells/kg to about
10.sup.9 cells/kg of HLA-compatible, allogeneic CAL and at least
one T-cell-activating cytokine to the patient;
[0028] d) monitoring the patient for signs of a mild
graft-versus-host response;
[0029] Alternatively, CAL may be given in the initial infusion. In
this case the regimen for administration of an HLA-compatible
leukocyte preparation including lymphocytes may comprise the
following steps in sequence:
[0030] a) administering to the patient about 10.sup.5 cells/kg to
about 10.sup.9 cells/kg of HLA-compatible, allogeneic peripheral
blood lymphocytes in which at least some of the HLA-compatible,
allogeneic peripheral blood lymphocytes are CAL, and at least one
T-cell-activating cytokine to the patient;;
[0031] b) monitoring the patient for signs of a mild
graft-versus-host response;
[0032] c) if no or insufficient graft-versus-host response develops
in the patient, administering about 10.sup.5 cells/kg to about
10.sup.9 cells/kg of HLA-compatible, allogeneic CAL and at least
one T-cell-activating cytokine to the patient;
[0033] d) monitoring the patient for signs of a mild
graft-versus-host response;
[0034] In any of the above described methods, the cytokine can be,
without limitation, any one or more of the following: interleukin 2
(IL2), interleukin 4 (IL4), interleukin 5 (IL5), interleukin 6
(IL6), interleukin 7 (IL-7), interferon alpha (IFN.alpha.),
interferon gamma (IFN.gamma.) and tumor necrosis factor
(TNF.alpha.). Any of these cytokines can be a native factor
obtained from natural sources, a factor produced by recombinant DNA
methodology, a chemically synthesized polypeptide or other
molecule, or any derivative having the functional activity of the
native factor.
[0035] The stem cells used in the above-described methods may be
obtained from bone marrow, from the peripheral circulation, or,
where appropriate, from fetal sources such as fetal tissue, fetal
circulation and umbilical cord blood. Cancer patients treatable
with the methods of the present invention are any patients having a
pathological condition caused by malignant cells, including without
limitation leukemia, lymphoma, and breast cancer. The
HLA-compatible allogeneic cells employed in the present invention
preferably are fully HLA-matched with the patient. Alternatively
the allogeneic cells may be at least haploidentical with the
patient. If the allogeneic cells are derived from a sibling of the
patient, the cells preferably are fully HLA matched with the
patient, although some mismatch may be tolerated. For example, the
allogeneic cells from a sibling to the patient may, in some cases,
be single HLA locus-mismatched. If the allogeneic cells are derived
from an unrelated individual, preferably the cells are fully HLA
matched with the patient.
[0036] The present invention further includes an article of
manufacture comprising packaging material and a biological cell
container within the packaging material. The packaging material
contains a label or package insert indicating that the biological
cell container, and/or any contents therein, are to be used in any
of the above-described methods for treating a human cancer
patient.
BRIEF DESCRIPTION OF THE FIGURES
[0037] FIG. 1 depicts the results of adoptive transfer of spleen
cells obtained from lethally irradiated F1 mice transplanted with
10.sup.7 syngeneic bone marrow cells and 10.sup.5 BCL1 cells in
addition to 20-30.times.10.sup.6 PBL from allogeneic mice with (A,
n=40) or without (B, n=40) concomitant in vivo rhIL2 treatment
(12.times.10.sup.4 IU.times.2/day for 5 days IP). A similar group
received syngeneic (F1) PBL given with (C, n=20) or without (D,
n=20) rhIL2. A control group had adoptive transfer of spleen cells
obtained from 10 untreated F1 recipients (E, n=20).
[0038] FIG. 2 depicts the results of adoptive transfer of spleen
cells obtained from lethally irradiated F1 mice reconstituted with
10.sup.7 syngeneic bone marrow cells mixed with 10.sup.5 BCL1
cells. Cell-mediated immunotherapy consisted of intravenous
administration of increasing numbers of C57BL/6 spleen cells:
1.times.10.sup.6 (A, n=10); 3.times.10.sup.6 (B, n=10);
10.times.10.sup.6 (C, n=10) and 30.times.10.sup.6 (D, n=10). One of
three experiments is shown.
[0039] FIG. 3 depicts results of adoptive transfer of spleen cells
obtained from BALB/c mice treated with cyclophosphamide (300 mg/kg
IP), inoculated 24 hours later with 10.sup.3 BCL1 cells, and one
day later receiving 4.times.10.sup.6 syngeneic, ASTA-Z-treated bone
marrow cells. Immunotherapy consisted of a mixture of
20.times.10.sup.6 allogeneic C57BL/6 spleen and lymph node cells
either with (A, n=6) or without (B, n=6) rhIL2 treatment in vivo
(12.times.10.sup.4 IU.times.3/day for 3 days IP). Recipients of a
mixture of 20.times.10.sup.6 syngeneic BALB/c spleen and lymph node
cells treated with rhIL2 (C, n=7) and recipients of 10.sup.3 BCL1
cells only (D, n=10) served as controls.
[0040] FIG. 4 depicts the results of adoptive transfer of spleen
cells obtained from lethally irradiated F1 mice inoculated with
10.sup.5 BCL1 cells and 30.times.10.sup.6 bone marrow cells
pre-activated in vitro for 4 days with rhIL2; mice with no
additional treatment (A, n=33), mice with in vivo rhIL2 treatment
(12.times.10.sup.4 IU.times.2/day for 5 days IP) (B, n=25);
controls: recipients of 10.sup.5 spleen cells obtained from
untreated control mice inoculated with 10.sup.5 BCL1 cells (C,
n=30).
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present inventor has employed allogeneic peripheral
blood lymphocytes, alone or in combination with cytokine treatment
in vivo or in vitro, for successful elimination of minimal residual
disease following chemotherapy and/or radiotherapy. Appropriate
treatment regimens have been circumscribed by studies in laboratory
animals, and the treatment protocols have been further extended to
human patients at high risk for disease relapse.
[0042] A spontaneous, transplantable murine B-cell
leukemia/lymphoma of BALB origin (BCL1) was used to investigate
elimination of minimal residual disease (MRD) following bone marrow
transplantation. As used herein, MRD refers to a condition wherein
residual malignant cells remain viable in a patient following a
primary and/or metastatic tumor "debulking" regimen. The debulking
regimen may include surgical excision, chemotherapy or
radiotherapy, or any combination of these approaches. Such
treatment removes a significant fraction of malignant cells from
the patient, but may leave a clinically significant number of
residual malignant cells that put the patient at risk of relapse.
The term "tumor" as used herein includes all pathological
conditions involving malignant cells; this can include "solid"
tumors arising in solid tissues or organs as well as "liquid"
tumors such as leukemias and lymphomas deriving from malignant
transformation of hematopoietic cells.
[0043] In autologous bone marrow transplantation (ABMT) with human
patients, an individual receives his or her own bone marrow cells
by infusion following a tumor debulking procedure. Generally, the
bone marrow cells are taken from the patient and preserved, for
example by cryopreservation, prior to the debulking procedure. This
permits an otherwise lethal debulking regimen to be employed, e.g.,
chemotherapy or radiotherapy that severely damages or destroys the
patient's bone marrow. Following the debulking procedure, the
patient's bone marrow is reconstituted by stem cells present in the
preserved sample of bone marrow.
[0044] Stem cells capable of reconstituting a patient's immune
system can be obtained not only by direct extraction from the bone
marrow, but also from the patient's peripheral circulation
following mobilization of such cells from the bone marrow. This can
be accomplished by treatment of the patient with granulocyte colony
stimulating factor (G-CSF) or other appropriate factors that induce
movement of stem cells from the bone marrow into the peripheral
circulation. Following-mobilization, the stem cells can be
collected from peripheral blood by any appropriate cell apheresis
technique, for example through use of a commercially available
blood cell collection device as exemplified by the CS 3000.RTM.
Plus blood cell collection device marketed by the Fenwal Division
of Baxter Healthcare Corporation. Methods for performing apheresis
with the CS 3000.RTM. Plus machine are described in Willams et al.,
Bone Marrow Transplantation 5: 129-33 (1990) and Hillyer et al.,
Transfusion 33: 316-21 (1993), both publications being incorporated
herein by reference. Stem cells collected from the peripheral blood
are termed herein "peripheral blood stem cells" (PBSC). The term
"autologous stem cell transplantation" (ASCT) is used herein to
refer to any infusion into a patient of that same patient's stem
cells, derived from any appropriate source (e.g., bone marrow or
peripheral circulation). As such, ABMT, where the autologous
infused cells are extracted directly from the bone marrow of the
patient, may be considered simply one form of ASCT.
[0045] It is possible to create an experimental regime in mice that
simulates ASCT in humans. This is done through use of stem cell
donors and recipients derived from a syngeneic strain of mice. In
such strains, inbreeding has created a situation in which, for
practical purposes, mice within the strain are genetically
identical to each other. Such mice accept tissues and organs
transplanted between individuals without evidence of immune
rejection, in a manner analogous to acceptance of a patient's own
cells following ASCT. Transplantation of bone marrow-derived stem
cells between such mice is referred to herein as "syngeneic bone
marrow transplantation" (SBMT) and should be considered analogous
to ABMT and ASCT in humans.
[0046] In the present experiments, mice received a lethal dose of
total body irradiation (TBI) or, alternatively, a lethal dose of
cyclophosphamide administered intraperitoneally. Bone marrow cells
(BMC) were extracted directly from the bone marrow of syngeneic
mice. In some cases, these BMC preparations were treated with
mafosfamide (ASTA-Z) to simulate a "purging" procedure in which the
patient's stem cells, prior to ASCT, are treated to remove or
destroy at least a fraction of any contaminating malignant cells.
One day after irradiation or treatment with cyclophosphamide, the
mice received 10.sup.7 syngeneic bone marrow cells by infusion into
the lateral tail vein. To simulate MRD, 10.sup.5 BCL1 tumor cells
were added to the syngeneic BMC prior to SBMT.
[0047] Following SBMT, recipient mice received either allogeneic
cell-mediated immunotherapy (Allo-CMI) or allogeneic cell-mediated
cytokine-activated immunotherapy (Allo-CCI). Allo-CMI involved
transfer of immunocompetent allogeneic lymphocytes (i.e.,
lymphocytes from a mouse strain other than that of the recipient
mice) at various times and at various doses, administered
post-SBMT. Generally these lymphocytes represented peripheral blood
lymphocytes (PBL) or mixtures of donor spleen and lymph node cells.
Allo-CCI involved transfer of allogeneic lymphocytes pre-activated
in vitro with recombinant human interleukin 2 (rhIL2). As used
herein, the term "CAL" refers to such "cytokine-activated
lymphocytes." In some experimental protocols, the Allo-CMI or
Allo-CCI regimen was accompanied by simultaneous in vivo
administration of rhIL2 to the recipient mice, in order to
facilitate additional activation of the infused lymphocytes in
vivo.
[0048] Failure to develop leukemia in primary recipients does not
prove elimination of all BCL1 cells, since active suppression of
existing tumor cells can prevent development of overt disease in
these animals. This has been documented following allogeneic BMT
and rhIL2 therapy. Slavin et al., Cancer Immunol. Immunother. 11:
155 (1981); Slavin et al., Nat. Immun. Cell Growth Regul. 7: 180
(1988). To establish conclusive evidence for eradication of
residual malignant cells, spleen cells from the treated, or
recipient, mice were adoptively transferred to secondary syngeneic
recipients. If these secondary recipients failed to develop
leukemia, it was judged that the original Allo-CMI- or
Allo-CCI-treated mice were free of viable malignant cells, since as
few as 1-10 cells have been shown to be capable of causing disease.
Slavin et al., Cancer Res. 41: 4162 (1981); Cohen et al., J.
Immunol. 151: 4803-10 (1993).
[0049] The results of the SBMT experiments with mice suggested that
effective immunotherapy of MRD can be achieved in vivo by cell
therapy with alloreactive lymphocytes through an effect that can be
further enhanced in vivo with a short course of intermediate-dose
rhIL2. GVL-like effects were also induced by infusion of CAL
without causing any gross impairment of the hematopoietic capacity
of BMC in lethally irradiated recipients. Moreover, GVL effects
induced by allogeneic lymphocytes as well as CAL could be further
enhanced by concomitant condition of stabilized blood counts) is
attained, as indicated by acceptable levels of, for example, white
blood cells (WBC), hemoglobin (Hb) and platelets. This condition of
partial hematopoiesis recovery may be achieved in a matter of weeks
following ASCT in humans, well before full immune reconstitution
diminishes the likelihood of successful Allo-CMI and/or
Allo-CCI.
[0050] The Allo-CMI and Allo-CCI strategies developed in mice have
been adapted to human patients in a variety of protocols undertaken
by the present inventor in treating cancer patients at high risk of
disease relapse. Cancer patients having acute myelogenous leukemia,
chronic myelogenous leukemia, non-Hodgkin's lymphoma and metastatic
breast cancer have been treated with the methods of the present
invention.
[0051] Two of the earlier-treated patients with acute myelogenous
leukemia were given relatively low numbers (e.g., about 10.sup.4
cells/kg) of allogeneic peripheral blood lymphocytes as a first
dose, in an effort to minimize the risk of serious GVHD.
Thereafter, the patients were given escalating numbers of
allogeneic peripheral blood lymphocytes at various time intervals
to increase the chances of clinically significant graft-versus
malignant cell response. Both of these early patients (patients 1
and 2 in Example 2, set out below) relapsed and died. Following
this experience, it was apparent that administration of graded
increments of PBL, beginning with relatively low numbers, may not
be effective. It was hypothesized that the initial low dose of
allogeneic PBL produced only immunization without significant GVHD
or graft-versus-malignant cell response. Thus, later, higher doses
rhIL2 therapy in vivo, most likely due to continuous in vivo
activation of allogeneic effector cells against residual host
malignant cells.
[0052] The data further indicate that eradication of BCL1 can be
accomplished before overt clinical manifestations of GVHD in the
primary recipients would have occurred, since experiments showed
that GVL-like effects against BCL1 cells were achieved within 1-2
weeks following administration of allogeneic lymphocytes. This
implies that temporary engraftment of allogeneic effector cells may
be sufficient to induce beneficial GVL effects against MRD, without
the need for permanent residence of allogeneic effector cells,
which may put the patient at risk for severe GVHD across major
histocompatibility barriers. Moreover, as the time interval between
ASCT and the Allo-CMI/CCI treatment increases, larger numbers of
donor's PBL can be administered with less likelihood of severe
GVHD. Slavin et al., J. Exp. Med. 147: 963 (1978); Slavin et al.,
Blood 80: 535a (1992).
[0053] As such, it is useful to administer an Allo-CMI or Allo-CCI
regimen after the patient is partially hematopoiesis recovered
following the original tumor debulking procedure/ASCT. This raises
the likelihood that the allogeneic inoculum will be rejected by
reconstituting host immune cells in due time. On the other hand, it
is also desirable to undertake the Allo-CMI/Allo-CCI regimen prior
to full immune reconstitution of the patient, since the likelihood
of premature rejection of the allogeneic inoculum, prior to
beneficial GVL/GVT effects, is thereby reduced. Since full immune
reconstitution following ASCT in humans frequently requires up to
one year, the patient can be monitored until a stable clinical
condition (e.g., of allogeneic PBL, which otherwise might be
effective, are promptly rejected by the patient and rendered
ineffectual for graft-versus-malignant cell activity. Therefore, in
subsequent cases, more cells (e.g., at least about 10.sup.7
cells/kg) were administered in the initial dose. With this initial
higher dose of allogeneic cells following ASCT, satisfactory
results have been obtained in many patients within the treatment
population to date.
[0054] Allogeneic cells preferably are chosen from human leukocyte
antigen (HLA)-compatible donors. Generally, HLA-compatible
lymphocytes may be taken from a fully HLA-matched relative such as
a parent, brother or sister. However, as shown with patient No. 12
in Example 2, below, donor lymphocytes may be sufficiently
HLA-compatible with the recipient to obtain the desired result even
if a sibling donor is single-locus mismatched. If a donor is
unrelated to the recipient, preferably the donor lymphocytes are
fully HLA matched with the recipient.
[0055] In a preferred embodiment of the present invention, an
initial dose of at least about 10.sup.7 HLA-compatible allogeneic
lymphocytes/kg is given once the patient has achieved a clinically
stable condition (e.g., stabilized blood counts). Preferably, the
patient is also administered cytokine in vivo concomitant with
administration of the HLA-compatible lymphocytes,. Preferably the
cytokine is rhIL2 at a dose of about 6.times.10.sup.6 IU of
rhIL2/m.sup.2/day, by subcutaneous injection, beginning on the same
day as infusion of the allogeneic lymphocytes. Other appropriate
cytokines may be used alone or in combination with rhIL2, as long
as the desired enhancing effect is obtained. Such additional
cytokines include, without limitation, native or recombinant forms
of IL6, IL7 and alpha and gamma-interferon (INF.alpha., INF.gamma.,
respectively).
[0056] If GVHD fails to develop in a patient given these numbers of
HLA-compatible allogeneic lymphocytes with concomitant in vivo
cytokine, then the treatment regimen is escalated. Preferably this
is done by administering a second dose of allogeneic HLA-compatible
lymphocytes preactivated in vitro with cytokine. Preferably the
cytokine is rhIL2, although other appropriate cytokines may be used
alone or in combination with rhIL2, as long as the desired
activation in vitro is obtained. Such additional cytokines include,
without limitation, native or recombinant forms of IL6, IL7 and
alpha-interferon (INF.alpha.), alone or in combination with
IL2.
[0057] Prior to administration of the second dose of cells
comprising CAL, and contingent on the particular status of an
individual patient, cyclophosphamide (Cytoxan) or other appropriate
immunosuppressants may be administered to the patient to avoid
rejection of the second dose of cells. That is, the
immunosuppressant is given in a dose effective for killing host T
cells that might otherwise operate to reject the second allogeneic
inoculum; the immunosuppressant may have the added benefit of
eliminating potential host suppressor cell functions that can
interfere with the GVT effects of the infused CAL. Preferably, in
vivo cytokine is administered to the patient concomitant with the
second dose of cells (CAL).
[0058] Although the above-described preferred embodiment is
presently recommended, it is to be understood that any combination
of allogeneic peripheral blood lymphocytes, in vivo cytokine and/or
CAL is covered by the present invention, so long as the initial
dose of cells is capable of eliciting a beneficial GVT response.
This dose of cells corresponds to a number of HLA-compatible
allogeneic peripheral blood lymphocytes that elicits a host
response beyond mere immunization to a second dose of cells from
the same or similar donor. Generally, this number corresponds to at
least about 10.sup.7 allogeneic peripheral blood lymphocytes/kg.
However, it is possible that a lower dose of cells, e.g., about
10.sup.5 cells/kg, could be used if, for example, CAL were used for
the initial infusion.
[0059] Between the Allo-CMI/CCI treatments or at the conclusion of
an Allo-CMI/CCI regimen, the patient may be monitored for levels of
malignant cells, i.e., for evidence of minimal residual disease.
Such monitoring may comprise patient follow-up for clinical signs
of relapse. The monitoring may also include, where appropriate,
various molecular or cellular assays to detect or quantify any
residual malignant cells. For example, in cases of sex-mismatched
donors and recipients, residual host-derived cells may be detected
through use of appropriate sex markers such as Y
chromosome-specific nucleic acid primers or probes. In cases of
single HLA locus mismatches between donors and recipients, residual
host cells may be documented by polymerase chain reaction (PCR)
analysis of Class I or Class II loci that differ between the donor
and recipient. Alternatively, appropriate molecular markers
specific for tumor cells can be employed. For example, nucleic acid
primers and/or probes specific for the bcr/abl translocation in
chronic myelogenous leukemia, for other oncogenes active in various
tumors, for inactivated tumor suppressor genes, other
tumor-specific genes, or any other assay reagents known to be
specific for tumor cells, may be employed. Any of these or
functionally comparable procedures may be used to monitor the
patient for evidence of residual malignant cells.
[0060] Under normal circumstances, recipients of autologous or
allogeneic bone marrow grafts receive only irradiated blood
products when such products are required by the patient. These
products are irradiated in order to avoid the possibility of
engraftment of immunocompetent T-lymphocytes derived from the
donor's blood product (e.g., platelets or red blood cells). In most
institutions, irradiated blood products are also used for patients
receiving high dose conventional chemotherapy without transplant,
e.g., blood products given following induction of remission in
leukemia and lymphoma patients. Obviously, the chances for
engraftment of immunocompetent T-lymphocytes from otherwise
mismatched blood products is relatively small under normal
circumstances. However, if immunosuppression is sufficient to
permit engraftment, GVHD can be "stormy" and lethal.
[0061] In the methods of the present invention, non-irradiated
donor-type lymphocytes are used intentionally for induction of
graft-versus-malignant cell effects. The methods are structured to
produce transient engraftment, so as to induce
graft-versus-malignant cell effects that may be accompanied by mild
GVHD. Since the donor cells used in the Allo-CMI and Allo-CCI
procedures are HLA-compatible with the recipient, chances of
engraftment are better than if the donor's cells were not
functionally matched with the patient's major histocompatibility
complex. Moreover, the chance of immediate rejection on the one
hand and lethal GVHD on the other hand are relatively small because
of the HLA compatibility. As such, the Allo-CMI/CCI protocols of
the present invention provide the possibility for transient
engraftment of donor's PBL with effective GVT and with a minimal
chance for induction of severe GVHD.
[0062] The invention will be further understood with reference to
the following illustrative embodiments, which are purely exemplary,
and should not be taken as limiting the true scope of the present
invention as described in the claims.
EXAMPLE 1
Syngeneic BMT (SBMT) in Mice Followed by Allo-CMI or Allo-CCI
[0063] I. Methods
[0064] A. Mice.
[0065] BALB/c (BALB), and C57BL/6 (C57), (BALB/c x C57BL/6)F1 (F1)
mice, 2-6 months old, were purchased from the breeding colony of
the Hebrew University-Hadassah Medical School, Jerusalem. Mice were
kept under standard conditions, with acidic water (pH 2.7) and no
special protective measures. Mice were given 0.5% neomycin sulfate
in their drinking water for 2 weeks post-transplantation.
[0066] B. Murine B-Cell Leukemia (BCL1).
[0067] BCL1, a spontaneous, transplantable B-cell leukemia/lymphoma
of BALB origin is characterized by marked (up to 50 fold)
splenomegaly, accompanied by extreme peripheral blood lymphocytosis
(>200,00/mm.sup.3) and results in death of all mice inoculated
with >10-100 tumor cells. Slavin et al., Nature 272: 624 (1978);
Slavin et al., Cancer Res. 41: 4162 (1981). BCLI was maintained in
vivo in BALB mice by IV passage of 10.sup.6-10.sup.7 peripheral
blood lymphocytes (PBL) obtained from tumor bearing mice. Mice with
marked lymphocytosis in the blood were subsequently used as BCL1
cell donors for experimental mice. PBL counts for all experimental
groups were carried out weekly. Leukemia was defined as PBL counts
exceeding 20,000/mm.sup.3. At the peak of disease PBL counts
usually reached >100,000/mm.sup.3.
[0068] C. Mafosfamide (ASTA-Z).
[0069] ASTA-Z was kindly provided by Drs M. Peukert and H.
Sindermann (Astawerke, Bielefeld, Germany) as a lyophilized powder
and was freshly dissolved in saline before use. ASTA-Z has been
employed in vitro to reduce or eliminate malignant cell populations
from bone marrow preparations. Douay et al., CR Acad. Sci. Paris t.
301, Ser III, no. 6:303 (1985).
[0070] D. Conditioning with Radiation and Cyclophosphamide Prior to
BMT.
[0071] Mice were exposed to a single dose of 750 cGy total body
irradiation (TBI) from a Philips X-ray unit (250 kV 20 mA) with a
focus to skin distance of 70 cm at a dose rate of 60 cGy/min.
Alternatively, mice were conditioned with freshly dissolved
cyclophosphamide (CY) (300 mg/kg) (Taro, Israel) given
intraperitoneally (IP). Twenty-four hours later, mice received
10.sup.7 syngeneic marrow cells via the lateral tail vein.
[0072] E. Preparation of Bone Marrow Cells (BMC).
[0073] BMC were obtained from the femura, tibiae and humera of
syngeneic mice. Mononuclear cells containing 10.sup.7 BMC in 0.25
ml Hank's medium were injected into the lateral tail vein of
recipients 24 hours post-radiation.
[0074] F. Purging Procedure.
[0075] Cells were resuspended at a concentration of
20.times.10.sup.6 cells/ml in Hank's medium containing 4% human
serum albumin. ASTA-Z was then added to a final concentration of
100 .mu.g/ml. Both untreated control cells and ASTA-Z treated BMC
were incubated at 37.degree. C. for 30 minutes, washed twice in
Hank's medium and counted. Purged or unpurged BMC
(4.times.10.sup.6) were injected into BALB mice conditioned with
CY.
[0076] G. Recombinant Human Interleukin-2 (rhIL2).
[0077] rhIL2 provided as 1 mg Proleukin (3.times.10.sup.6 Cetus
Units, equivalent to 18.times.10.sup.6 International Units) was
kindly supplied by Dr. S. L. Aukerman, Cetus/Chiron, Calif., USA.
rhIL2 was initially diluted with water for injection and
subsequently rediluted with dextrose 5%. International units (IU)
are used throughout the remainder of the present application.
[0078] H. Activation of BMC by rhlL2.
[0079] BMC were cultured in 225 cm.sup.3 flasks (Corning 25160-225,
Corning Glass, Corning N.Y.) in RPMI 1640 medium (Beit Haemek,
Israel) containing L-glutamine, non-essential amino acids,
pyruvate, 10% bovine calf serum (BCS) and rhIL2 (6,000 IU/ml) for 4
days in a humidified incubator with 5% CO.sub.2 at 37.degree. C.
Following harvesting, viability was determined by the trypan blue
exclusion method.
[0080] I. Simulation of Minimal Residual Disease Following
Syngeneic Bone Marrow Transplantation.
[0081] In order to simulate minimal residual disease (MRD)
quantitatively, 10.sup.5 BCL1 cells were added to the marrow
inoculum during syngeneic bone marrow transplantation (SBMT), prior
to immunotherapy.
[0082] J. Immunotherapy by Immunocompetent Allogeneic
Lymphocytes.
[0083] Allogeneic cell-mediated immunotherapy (Allo-CMI) consisted
of adoptive transfer of immunocompetent allogeneic lymphocytes (PBL
or a mixture of donor spleen and lymph node cells) as detailed in
the results for each experiment, below. Allogeneic cell-mediated
cytokine-activated immunotherapy (Allo-CCI) consisted of adoptive
transfer of allogeneic lymphocytes pre-activated in vitro with
rhIL2 (allogeneic "CAL") In some experiments allogeneic lymphocyte
infusion was followed by subsequent in vivo activation with rhIL2,
by additional Allo-CMI with in vivo rhIL-2, or by additional
Allo-CCI with in vivo rhIL-2, respectively.
[0084] K. Detection of Residual Clonogenic BCL1 by Adoptive
Transfer Experiments.
[0085] In order to determine whether or not residual BCLI cells
were present after various treatments, 10.sup.5 spleen cells
obtained from treated mice were adoptively transferred to untreated
secondary syngeneic (BALB) recipients. Absence of leukemia (>100
days) in secondary recipients was indicative of elimination of BCL1
since as few as 1-10 cells were previously shown to cause
disease.
[0086] L. Statistical Analysis.
[0087] The significance of differences between treated and
untreated mice was calculated by the independent statistical
t-test.
[0088] II. Results
[0089] A. Induction of Allo-CMI and Allo-CCI Effects.
[0090] F1 mice were lethally irradiated (750 cGy) and transplanted
with 10.sup.7 syngeneic BMC. Following inoculation of 10.sup.5 BCL1
cells to simulate MRD, varying numbers of C57 PBL were administered
intravenously to induce GVL-like effects through allo-CMI. In order
to detect the efficacy of allo-CMI in eradicating residual BCL1
cells, aliquots of 10.sup.5 spleen cells pooled from 2-3
experimental mice were adoptively transferred to secondary normal
BALB recipients, one or two weeks post-SBMT.
[0091] FIG. 1 summarizes results obtained from three different
experiments in a total of 120 mice. Injection of
20-30.times.10.sup.6 PBL, obtained from C57 mice to induce allo-CMI
after SBMT in F1 recipients, effectively eliminated residual BCL1
cells, as none of 40 secondary adoptive BALB recipients developed
leukemia (>180 days). In contrast, leukemia developed in all 20
secondary BALB recipients inoculated with 10.sup.5 spleen cells
obtained from Fl recipients that had received 20-30.times.10.sup.6
PBL from syngeneic donors post-SBMT. Addition of rhIL2
(12.times.10.sup.4 IU.times.2/day for 5 days IP) post-transplant
did not improve the disease-free survival of secondary recipients
of 10.sup.5 spleen cells (obtained from similarly treated F1 mice)
since all 20 secondary recipient BALB mice developed leukemia (FIG.
1). Addition of rhIL2 in vivo at the same dose to recipients of
20.times.10.sup.6 allogeneic PBL for further in vivo activation of
effector cells did not induce measurable additional GVL effects
since all 40 secondary BALB recipients remained disease free
(>180 days) (FIG. 1).
[0092] B. Quantitative Effect of the Number of Effector Cells on
the Efficacy of Allo-CMI.
[0093] Anti-leukemic effects mediated by allo-CMI were cell-dose
dependent. As shown in FIG. 2, all SBMT recipients injected with
30.times.10.sup.6 C57 spleen cells completely resisted the
development of leukemia following inoculation of 10.sup.5 BCL1
cells. Injection of 10.times.10.sup.6 allogeneic spleen cells
together with 10.sup.5 BCL1 cells induced effective allo-CMI in 70%
of the secondary adoptive recipients. However, reduction of
allo-CMI inducing C57 spleen cells to 3.times.10.sup.6 or
1.times.10.sup.6 failed to eliminate residual BCL1 cells and all
secondary adoptive recipients developed leukemia (FIG. 2).
[0094] C. Induction of Allo-CMI and Allo-CMI/IL-2 Effects Following
Transplantation with ASTA-Z-Purged BMC.
[0095] The feasibility of induction of allo-CMI was investigated by
conditioning with high-dose CY followed by rescue of recipients
with ASTA-Z-purged syngencic BMC. BALB recipients received
high-dose CY (300 mg/kg IP) and were injected 24 hours later with
10.sup.3 BCL1 cells to simulate MRD. One day later, all mice
received intravenously 4.times.10.sup.6ASTA-Z treated syngeneic
BMC. Mice were divided into 3 experimental groups: the first group
(6 mice) received intravenously a mixture of allogeneic C57 spleen
and lymph node cells (20.times.10.sup.6 cells) for induction of
allo-CMI; the second group (6 mice) received identical cell therapy
with additional in vivo potentiation of GVL by rhIL2 treatment
(12.times.10.sup.4 IU.times.3/day for 3 days, IP); the third group
(7 mice) received a mixture of syngeneic spleen and lymph node
cells, with an identical in vivo rhIL2 treatment. One week later,
aliquots of 10.sup.5 cells from a pool of 2-3 spleens obtained from
each experimental group were adoptively transferred to secondary
BALB mice.
[0096] As shown in FIG. 3, all mice inoculated with spleen cells
from control mice given 10.sup.3 BCL1 cells, or mice given
syngeneic BALB lymphocytes with in vivo rhIL2, developed leukemia
and died within 40 and 60 days, respectively. Likewise, secondary
recipients of 10.sup.5 spleen cells obtained from mice that were
treated with allo-CMI, using allogeneic C57 cells alone, showed no
measurable GVL effects since all recipients developed leukemia. In
contrast, addition of rhIL2 in vivo following administration of C57
spleen and lymph node cell mixtures induced substantial
anti-leukemic effects and 50% of the secondary adoptive recipient
mice remained leukemia-free for >125 days (FIG. 3).
[0097] D. Enhancement of Immunotherapeutic Effect by in vitro
Activation of Allogeneic lymphocytes with rhIL2.
[0098] The following experiment was designed to test for potential
enhancements in efficacy of treatment by in vitro pre-activation of
allogeneic effector cells with rhIL2. Lethally irradiated (750 cGy
TBI) F1 mice were infused with 30.times.10.sup.6 C57 BMC
pre-activated in vitro for 4 days with rhIL2. BMC were mixed with
10.sup.5 BCL1 cells to simulate MRD. Results of 3 separate sets of
experiments gave similar results and therefore the data were pooled
(FIG. 4).
[0099] All F1 recipients were divided into two groups. The first
group of 33 mice received no additional treatment. Mice in the
second group (25 mice) were injected with rhIL2 (12.times.10.sup.4
IU.times.2/day for 5 days, IP) in an attempt to further increase
efficacy of cell therapy by continuous activation of
rhIL2-dependent effector cells in vivo. Aliquots of 10.sup.5 cells
obtained from a pool of spleen cells prepared from mice of either
experimental group were adoptively transferred to secondary BALB
recipients. As shown in FIG. 4, 10 of 33 secondary recipients of
spleen cells obtained from the first experimental group remained
disease-free for >150 days. Additional in vivo rhIL2 therapy in
the second experimental group further improved the Allo-CCI
effects, as 19 of 25 secondary recipients remained disease-free for
an observation period >150 days (p=0.05) (FIG. 4).
EXAMPLE 2
Autologous Stem Cell Transplantation (ASCT) in Humans Followed by
Allo-CMI and/or Allo CCI
[0100] I. Patient Treatment Protocols
[0101] Patient No. 1.
[0102] This female patient was diagnosed with acute myclogenous
leukemia (AML), French American British (FAB) classification M4,
and was in first complete remission (i.e., no evidence of disease)
at the time of autologous stem cell transplantation (ASCT). The
patient was 41 years old at the time of ASCT. Prior to ASCT, she
received a conditioning regimen of Busulfan, 4 mg/kg, days 6
through 9 pre-ASCT (days -9 to -6), as well as Cytoxan
(cyclophosphamide), 50 mg/kg, days -5 to -2, Cotrimoxazol, 10
mg/kg, days -10 to -2, Allopurinol, 300 mg/kg, days -10 to -1 and
cytosine arabinoside (Ara-C), 25 mg intrathecally.
[0103] Prior to ASCT, the autologous cells to be infused were
purged by treatment with Mafosfamide (ASTA-Z). ASTA-Z was provided
by Drs. M. Peukert and H. Sindermann (Astawerke, Bielefeld,
Germany) as a lyophilized powder and was freshly dissolved in
saline before use. Autologous cells were resuspended at a
concentration of 20.times.10.sup.6 cells/ml in Hank's medium
containing 4% human serum albumin. ASTA-Z was then added to a final
concentration of 100 ug/ml and the cells were incubated in the
ASTA-Z at 37.degree. C. for 30 min. After this, the cells were
washed twice in Hank's medium and counted. Cells were cryopreserved
and kept in liquid nitrogen until used. ASCT consisted of
2.5.times.10.sup.8 nucleated bone marrow cells/kg, infused
intravenously (IV) on day 0.
[0104] On day 1 following ASCT (day +1), the patient received
10.sup.4 T cells/kg of peripheral blood lymphocytes (PBL) from an
HLA-matched donor. On days +3, +22, +29 and +36 she received PBL
from the same donor at an equivalent dose of 10.sup.5, 10.sup.5,
10.sup.6, and 10.sup.6 T cells/kg, respectively. On day +47 she
received PBL from the same donor at an equivalent dose of 10.sup.7
T cells/kg. The patient showed no evidence of GVHD.
[0105] Patient No. 2.
[0106] This female patient was diagnosed with AML, FAB M5, and was
in first complete remission at the time of ASCT. The patient was 42
years old at the time of ASCT. Prior to ASCT, she received a
conditioning regimen of Busulfan, 4 mg/kg, (days -9 to -6), as well
as Cytoxan, 50 mg/kg, days -5 to -2, Cotrimoxazol, 10 mg/kg, days
-10 to -2, Allopurinol, 300 mg/kg, days -10 to -1 and Ara-C, 25 mg
intrathecally.
[0107] Prior to ASCT, the autologous bone marrow cells to be
infused were purged by treatment with ASTA-Z. Autologous cells were
resuspended at a concentration of 20.times.10.sup.6 cells/ml in
Hank's medium containing 4% human serum albumin. ASTA-Z was then
added to a final concentration of 100 ug/ml and the cells were
incubated in the ASTA-Z at 37.degree. C. for 30 min. After this,
the cells were washed twice in Hank's medium, counted and
cryopreserved. ASCT consisted of 1.times.10.sup.8 nucleated bone
marrow cells/kg, infused IV on day 0.
[0108] On day +1, the patient received 10.sup.4 T cells/kg of PBL
from an HLA-matched donor. On days +8, +18 and +26 she received PBL
from the same donor at an equivalent dose of 10.sup.5, 10.sup.6,
and 10.sup.7 T cells/kg, respectively. On day +80 she received PBL
from the same donor at an equivalent dose of 10.sup.7 T cells/kg,
with 3.times.10.sup.6 IU of rhIL-2/m.sup.2/day for 3 days, by
subcutaneous injection beginning on day +80. The patient showed no
evidence of GVHD.
[0109] Patient No. 3.
[0110] This female patient was diagnosed with AML, FAB M3, and was
in first complete remission at the time of ASCT. The patient was 32
years old at the time of ASCT. Prior to ASCT, she received a
conditioning regimen of Busulfan, 4 mg/kg, (days -9 to -6), as well
as Cytoxan, 50 mg/kg, days -5 to -2, Cotrimoxazol, 10 mg/kg, days
-10 to -2, Allopurinol, 300 mg/kg, days -10 to -1 and Ara-C, 25 mg
intrathecally. ASCT consisted of non-purged bone marrow cells, and
0.79.times.10.sup.8 nucleated cells/kg were infused IV on day 0. On
day +1, the patient received PBL from an HLA-matched donor at an
equivalent dose of 10.sup.7 T cells/kg. On day +1, she also
received 6.times.10.sup.6 IU of rhIL-2/m.sup.2 by subcutaneous
injection. No GVHD was observed.
[0111] Patient No. 4.
[0112] This male patient was diagnosed with AML, FAB M2, and was in
first complete remission at the time of ASCT. The patient was 23
years old at the time of ASCT. Prior to ASCT, he received a
conditioning regimen of Busulfan 4 mg/kg, days -9 to -6, Cytoxan 60
mg/kg, days -3 to -2, Thiotepa 5 mg/kg/day, days -5 to -4,
Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg,
days -10 to -1 and Ara-C, 25 mg intrathecally. ASCT consisted of
non-purged 1.37.times.10.sup.8 nucleated bone marrow cells/kg,
infused IV on day 0. On day +1, the patient received PBL from an
HLA-matched donor at an equivalent dose of 10.sup.7 T cells/kg. On
day +1, he also received 6.times.10.sup.6 IU of rhIL-2/m.sup.2 by
subcutaneous injection. GVHD was suspected (Grade I) in the
skin.
[0113] Patient No. 5.
[0114] This male patient was diagnosed with chronic myelogenous
leukemia (CML) in chronic phase. The original CML karyotype was
positive for the Philadelphia chromosome (Ph+). The patient was in
chronic phase (CP) and was Ph- at the time of ASCT. The patient was
51 years old at the time of ASCT. Prior to ASCT, he received a
conditioning regimen of total body irradiation (TBI) 200 cGY/day,
days -5 to -3, and Cytoxan 60mg/kg, days -2 to -1. ASCT consisted
of non-purged 0.5.times.10.sup.8 nucleated bone marrow cells/kg,
infused IV on day 0.
[0115] On day +71, as soon as blood counts had stabilized (WBC:
4,200/mm.sup.3; Hb: 11.5 g %; platelets: 133,000/mm.sup.3), the
patient received 3.times.10.sup.7 T cells/kg of PBL from an
HLA-matched brother. No GVHD was observed; hence, the allo-CMI
regimen was escalated. On day +10.sup.7, the patient received
4.6.times.10.sup.7 T cells/kg of PBL from the same donor. Starting
on day +10.sup.7, he also received 6.times.10.sup.6 IU of
rhIL-2/m.sup.2/day for 3 days, by subcutaneous injection. No GVHD
was observed. On day +240, after the Ph+ karyotype had reappeared,
the patient received 4.95.times.10.sup.7 cells/kg of
cytokine-activated lymphocytes ("CAL") from the same donor.
Starting on day +240, he also received 6.times.10.sup.6 IU of
rhIL-2/m.sup.2/day for 3 days, by subcutaneous injection. The CAL
were produced by exposing the donor's PBL to 6,000 IU/ml rhIL-2 for
four days in culture.
[0116] Patient No. 6.
[0117] This female patient was diagnosed with AML, FAB M2, and was
in first complete remission at the time of ASCT. The patient was 50
years old at the time of ASCT. Prior to ASCT, she received a
conditioning regimen of Busulfan 4 mg/kg, days -9 to -6, Cytoxan 60
mg/kg, days -3 to -2, Thiotepa 5 mg/kg/day, days -5 to -4,
Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg,
days -10 to -1 and Ara-C, 25 mg intrathecally. ASCT consisted of
non-purged 0.64.times.10.sup.8 nucleated bone marrow cells/kg
infused IV on day 0. On day +58, as soon as blood counts had
stabilized (WBC: 4,400/mm.sup.3; Hb: 9.5 g %; platelets:
66,000/mm.sup.3), the patient received 5.times.10.sup.7 T cells/kg
of PBL from an HLA-matched sister. On day +86 the patient received
a second dose of 6.1.times.10.sup.7 T cells/kg of PBL from the
HLA-matched sister. Starting on day +86, she also received
6.times.10.sup.6 IU of rhIL-2/m.sup.2/day for 3 days, by
subcutaneous injection. No GVHD was observed.
[0118] Patient No. 7.
[0119] This male patient was diagnosed with CML. The original CML
karyotype was Ph+. The patient was in chronic phase (CP) and 50% of
his marrow cells were Ph+ (i.e., the patient was Ph+) at the time
of ASCT. The patient was 47 years old at the time of ASCT. Prior to
ASCT, he received a conditioning regimen of TBI 200 cGY/day, days
-5 to -3, and Cytoxan 60mg/kg, days -2 to -1. ASCT consisted of
non-purged 0.98.times.10.sup.8 nucleated bone marrow cells/kg,
infused IV on day 0. On day +55, as soon as blood counts had
stabilized (WBC: 6,900/mm.sup.3; Hb: 12.0 g %; platelets:
248,000/mm.sup.3), the patient received 4.times.10.sup.7 T cells/kg
of PBL from an HLA-matched sister. No GVHD developed. On day +77,
the patient received 2.8.times.10.sup.7 cells/kg of PBL from the
HLA-matched sister. Starting on day +77, he also received
6.times.10.sup.6 IU of rhIL-2/m.sup.2/day for 3 days, by
subcutaneous injection. No GVHD was observed.
[0120] Patient No. 8.
[0121] This male patient was diagnosed with non-Hodgkin's lymphoma
(NHL), Burkitt-like, and was in a second partial remission at the
time of ASCT. As used herein, the term "partial remission"
indicates at least a 50% response (i.e., at least a 50% reduction
of lymphoma cell mass) but with continued evidence of disease. The
patient was 36 years old at the time of ASCT. Prior to ASCT, he
received a conditioning regimen of etoposide, 200 mg/M.sup.2/day,
days -6 to -3, thiotepa, 40 mg/m.sup.2/day, days -5 to -2, Ara-C,
200 mg/m.sup.2/day, days -4 to -1, Cytoxan, 60 mg/kg/day, day -3,
and melphalan, 60 mg/m.sup.2/day, days -2 to -1.
[0122] ASCT consisted of 0.74.times.10.sup.8/kg viable bone marrow
nucleated cells plus 2.36.times.10.sup.8/kg viable peripheral blood
stem cells. Subcutaneous GM-CSF, 5 ug/kg/day, was administered from
day +1 to day +18. Prior to ASCT, the autologous cells were purged
with Dynal magnetic beads coated with anti-CD19 for elimination of
residual lymphoma cells.
[0123] On day +90, the patient received 5.times.10.sup.7 cells/kg
of PBL from an HLA-matched brother. The patient showed no signs of
GVHD following this first cell infusion. Polymerase chain reaction
(PCR) analysis using two VNTR loci (VNTR Variable Number of Tandem
Repeats) revealed no evidence of circulating donor-specific cells.
On day +124, the patient received 5.times.10.sup.7 cells/kg of PBL
from the same donor. This was followed by three days outpatient
treatment with 6.times.10.sup.6 IU of rhIL-2/m.sup.2/day, by
subcutaneous injection, beginning on day +124.
[0124] Fifty days later (day +174) the patient developed
pancytopenia, and bone marrow biopsy revealed severe hypocellular
marrow with increased numbers of large granular lymphocytes and
plasma cells. Lymphocytes with a similar morphology were found in
the blood. Repeated PCR using two different VNTR loci showed
partial engraftment of donor cells on day +191 and 100% engraftment
of donor cells on day +211. An allogeneic BMT was then performed by
infusing the patient with 3.4.times.10.sup.8/kg of the donor's
marrow cells; no post-transplant anti-GVHD prophylaxis was
administered. The patient had rapid three-lineage engraftment with
normal platelet counts after 14 days and normal hemoglobin after 26
days. WBC normalized after 10 days with 70% neutrophils, 5%
monocytes and 25% lymphocytes. Large granular lymphocytes
disappeared from the blood. On day 14 following allogeneic BMT, the
patient showed minimal signs of acute GVHD with involvement of skin
and oral cavity. There was no intestinal or liver involvement.
Since then the patient has continued to experience grade I/II
mucocutaneous GVHD, partially controlled with steroids and
cyclosporin A.
[0125] Patient No. 9.
[0126] This male patient was diagnosed with NHL, follicular mixed
IV A, and was in a second partial remission at the time of ASCT.
The patient was 39 years old at the time of ASCT. Prior to ASCT, he
received a conditioning regimen of etoposide, 200 mg/M.sup.2/day,
days -6 to -3; thiotepa, 40 mg/m.sup.2/day, days -5 to -2; Ara-C,
200 mg/m.sup.2/day, days -4 to -1; Cytoxan, 60 mg/kg/day, day -3;
and melphalan, 60 mg/m.sup.2/day, days -2 to -1.
[0127] ASCT consisted of 0.5.times.10.sup.8 nucleated bone marrow
cells/kg, infused IV on day 0. Prior to ASCT, the autologous cells
were purged with Dynal magnetic beads coated with anti-CD19 for
elimination of contaminating tumor cells.
[0128] At week 8 post-ASCT, the patient received 5.times.10.sup.7 T
cells/kg of PBL from an HLA-matched sister. At week 12 post-ASCT,
the patient received 5.times.10.sup.7 T cells/kg of PBL from the
same donor. Starting at week 12, on the same day as the
administration of the second Allo-CMI treatment, the patient also
received 6.times.10.sup.6 IU of rhIL-2/m.sup.2/day for 3 days, by
subcutaneous injection. No GVHD was observed. At week 16 post-ASCT,
the patient received 0.5.times.10.sup.7 CAL/kg from the HLA-matched
sister. The CAL were produced by exposing the donor's PBL to 6,000
IU/ml rhIL-2 for four days in culture. No GVHD was observed.
[0129] Patient No. 10.
[0130] This female patient was diagnosed with NHL, mixed
cellularity II A, and was in a second complete remission at the
time of ASCT. The patient was 36 years old at the time of ASCT.
Prior to ASCT, she received a conditioning regimen of etoposide,
200 mg/m.sup.2/day, days -6 to -3, thiotepa, 40 mg/m.sup.2/day,
days -5 to -2, Ara-C, 200 mg/m.sup.2/day, days -4 to -1, Cytoxan,
60 mg/kg/day, day -3, and melphalan, 60 mg/m.sup.2/day, days -2 to
-1.
[0131] ASCT consisted of 0.94.times.10.sup.8 non-purged nucleated
bone marrow cells/kg plus 3.9.times.10.sup.7 peripheral blood stem
cells mobilized by G-CSF prior to collection with the CS 3000.RTM.
Plus blood cell separator (Baxter Healthcare Corporation, Fenwal
System Catalogue No. 4R4538). Cells were infused IV on day 0.
[0132] At week 10 post-ASCT, as soon as blood counts had stabilized
(WBC: 4,300/mm.sup.3; Hb: 11.2 g %; platelets: 116,000/mm.sup.3),
the patient received 3.times.10.sup.7 T cells/kg of PBL from an
HLA-matched brother. At week 15 post-ASCT, the patient received
4.1.times.10.sup.7 T cells/kg of PBL from the same donor. Starting
at week 15, on the same day as the administration of the second
Allo-CMI treatment, the patient also received 6.times.10.sup.6 IU
of rhIL-2/m.sup.2/day for 3 days, by subcutaneous injection. At
week 23 post-ASCT, the patient received 5.times.10.sup.7 CAL/kg
from the HLA-matched brother. The CAL cells were produced by
exposing the donor's PBL to 6,000 IU/ml rhIL-2 for four days in
culture. Starting at week 23, on the same day as the administration
of the Allo-CCI treatment, the patient also received
6.times.10.sup.6 IU of rhIL-2/m.sup.2/day for 3 days, by
subcutaneous injection. No GVHD was observed.
[0133] Patient No. 11.
[0134] This female patient was diagnosed with NHL, immunoblastic,
and was in a second complete remission at the time of ASCT. The
patient was 21 years old at the time of ASCT. Prior to ASCT, she
received a conditioning regimen of etoposide, 200 mg/m.sup.2/day,
days -6 to -3; thiotepa, 40 mg/m.sup.2/day, days -5 to -2; Ara-C,
200 Mg/M2/day, days -4 to -1; Cytoxan, 60 mg/kg/day, day -3; and
melphalan, 60 mg/M.sup.2/day, days -2 to -1.
[0135] ASCT consisted of 3.82.times.10.sup.8 non-purged bone marrow
cells/kg plus 1.29.times.10.sup.8 peripheral blood stem cells
mobilized with G-CSF prior to collection with the CS 3000.RTM. Plus
blood cell separator. Cells were infused IV on day 0.
[0136] At week 10 post-ASCT, the patient received 5.times.10.sup.7
T cells/kg of PBL from an HLA-matched sister. At week 15 post-ASCT,
the patient received a second infusion of 5.times.10.sup.7 T
cells/kg of PBL from the HLA-matched sister. At week 19 post-ASCT,
the patient received a third infusion of 5.times.10.sup.7 T
cells/kg of PBL from the same donor. No GVHD was observed, but the
patient did not accept the suggestion that she receive in vivo
rhIL2. At week 30 post-ASCT, the patient received a fourth infusion
of 5.times.10.sup.7 T cells/kg of PBL from the HLA-matched sister.
Starting at week 23, on the same day as the administration of the
fourth Allo-CMI treatment, the patient also received
6.times.10.sup.6 IU of rhIL-2/m.sup.2/day for 3 days, by
subcutaneous injection. No GVHD has been observed to date.
[0137] Patient No. 12.
[0138] This female patient was diagnosed with NHL, diffuse large
cell, and was in a condition of relapse at the time of ASCT. The
patient was 21 years old at the time of ASCT. Prior to ASCT, she
received a conditioning regimen of etoposide, 200 mg/m.sup.2/day,
days -6 to -3; thiotepa, 40 mg/m.sup.2/day, days -5 to -2; Ara-C,
200 mg/m.sup.2/day, days -4 to -1; Cytoxan, 60 mg/kg/day, day -3;
and melphalan, 60 mg/m.sup.2/day, days -2 to -1.
[0139] ASCT consisted of 1.8.times.10.sup.8 non-purged mononuclear
bone marrow cells/kg, infused IV on day 0.
[0140] At week 6 post-ASCT, as soon as blood counts had stabilized
(WBC: 4,400/mm.sup.3; Hb: 11.7 g %; platelets: 150,000/mm.sup.3),
the patient received 5.times.10.sup.7 T cells/kg of PBL from a
single locus-mismatched sister. At week 10 post-ASCT, the patient
received a second infusion of 5.times.10.sup.7 T cells/kg of PBL
from the same donor. Starting at week 10, on the same day as the
administration of the second Allo-CMI treatment, the patient also
received 6.times.10.sup.6 IU of rhIL-2/m.sup.2/day for 3 days, by
subcutaneous injection. No GVHD was observed.
[0141] Patient No. 13.
[0142] This male patient was diagnosed with AML, FAB M5, and was in
first complete remission at the time of ASCT. The patient was 46
years old at the time of ASCT. Prior to ASCT, he received a
conditioning regimen of Busulfan 4 mg/kg, days -9 to -6; Cytoxan 60
mg/kg, days -3 to -2; Thiotepa 5 mg/kg/day, days -5 to -4;
Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg,
days -10 to -1 and Ara-C, 25 mg intrathecally. ASCT consisted of
1.39.times.10.sup.8 non-purged bone marrow cells infused IV on day
0. At week 11, the patient received 3.86.times.10.sup.7 T cells/kg
of PBL from an HLA-matched brother. No GVHD developed.
[0143] Patient No. 14.
[0144] This female patient was diagnosed with AML, FAB M3, and was
in second partial remission at the time of ASCT. The patient was 12
years old at the time of ASCT. Prior to ASCT, she received a
conditioning regimen of Busulfan 4 mg/kg, days -9 to -6; Cytoxan 60
mg/kg, days -3 to -2; Mitoxantrone 6 mg/m.sup.2/day, days -5 to -4;
Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg,
days -10 to -1 and Ara-C, 25 mg intrathecally. ASCT consisted of
1.14.times.10.sup.8 non-purged marrow cells infused IV on day 0. At
week 16, the patient received 2.times.10.sup.7 T cells/kg of single
locus-mismatched PBL from her mother. At week 20, the patient
received 1000 mg/m.sup.2 Cytoxan in order to improve the efficacy
and temporal engraftment of a second infusion of the donor's PBL.
Twenty four hours later, the patient received 5.times.10.sup.7 T
cells/kg from the same donor. Starting on the same day as the
administration of the second Allo-CMI treatment, the patient also
received 6.times.10.sup.6 IU of rhIL-2/m.sup.2/day for 3 days, by
subcutaneous injection. No GVHD has been noted thus far.
[0145] Patient No. 15.
[0146] This male patient was diagnosed with AML, FAB MS, and was in
first complete remission at the time of ASCT. The patient was 61/2
years old at the time of ASCT. Prior to ASCT, he received a
conditioning regimen of Busulfan 4 mg/kg, days -9 to -6; Cytoxan 60
mg/kg, days -3 to -2; Thiotepa 5 mg/kg/day, days -5 to -4;
Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg,
days -10 to -1 and Ara-C, 25 mg intrathecally. ASCT consisted of
2.7.times.10.sup.8 non-purged marrow cells infused IV on day 0. At
week 14, the patient received 5.times.10.sup.7 T cells/kg of single
locus-mismatched PBL from his father. No GVHD has developed, and
the patient is scheduled to receive a second dose of allo-CMI plus
in vivo rhIL2.
[0147] Patient No.16.
[0148] This male patient was diagnosed with NHL, mixed large and
small cell, in the lymph nodes, in February of 1993. There was also
heavy bone marrow involvement. He received chemotherapy (12 courses
of MACOP-B), but relapsed in the lymph nodes and bone marrow in
October of 1993. He underwent additional chemotherapy and was in a
second partial remission at the time of ASCT, with recurrence in
the marrow. The patient was 45 years old at the time of ASCT. Prior
to ASCT, he received a conditioning regimen consisting of
etoposide, 200 mg/m.sup.2/day, days -6 to -3; thiotepa, 40
mg/m.sup.2/day, days -5 to -2; Ara-C, 200 mg/m.sup.2/day, days -4
to -1; Cytoxan, 60 mg/kg/day, day -3; and melphalan, 60
mg/m.sup.2/day, days -2 to -1.
[0149] ASCT consisted of 2.15.times.10.sup.8 non-purged
G-CSF-mobilized peripheral blood stem cells, infused IV on day
0.
[0150] At week 7 post-ASCT, the patient received 3.times.10.sup.7 T
cells/kg of PBL from an HLA-matched sister. At week 11 post-ASCT,
the patient received a second infusion of 3.times.10.sup.7 T
cells/kg of PBL from the same donor. Starting at week 11, on the
same day as the administration of the second Allo-CMI treatment,
the patient also received 6.times.10.sup.6 IU of rhIL-2/m2/day for
3 days, by subcutaneous injection. No GVHD has been observed to
date.
[0151] Patient No. 17.
[0152] This female patient was diagnosed with metastatic breast
cancer, with metastases to the liver and spine. She underwent a
right partial mastectomy with axillary lymph node dissection
showing involvement of 18/35 lymph nodes. The patient was 43 years
old at the time of ASCT. Prior to ASCT, she received a conditioning
regimen of carboplatin, 200 mg/m.sup.2/day, days -7. to -4;
thiotepa, 60 mg/m.sup.2/day, days -6 to -4; etoposide, 200
Mg/m.sup.2/day, days -5 to -3; and melphalan, 60 mg/m.sup.2/day,
days -4 to -3.
[0153] ASCT consisted of 3.7.times.10.sup.8 peripheral blood stem
cells cells/kg, infused IV on day 0. The stem cells were collected
after mobilization with G-CSF (7.5 mg/kg/day for 5 days) using
three collections with the CS 3000.RTM. Plus blood cell separator.
Following ASCT, the patient recovered with no complications and was
discharged on day 20 post-ASCT.
[0154] A fully HLA-matched (A, B, DR and mixed lymphocyte reaction
(MLR)-negative) sibling was chosen as donor of PBL for Allo-CMI. At
week 8 post-ASCT, as soon as the patient achieved a stable clinical
condition (WBC: 2,700/mm.sup.3; Hb: 9.1 g %; platelets:
56,000/mm.sup.3), the patient received 5.times.10.sup.7 T cells/kg
of PBL from the HLA-matched sibling. Starting at week 8, on the
same day as the administration of the first Allo-CMI treatment, the
patient also received 6.times.10.sup.6 IU of rhIL-2/m2/day for 3
days, by subcutaneous injection. No GVHD has been observed to
date.
[0155] Patient No. 18.
[0156] This male patient was diagnosed with metastatic breast
cancer, with metastases to the bone marrow, sternum and vertebrae
T.sub.4 to T.sub.7. He underwent lumpectomy with axillary lymph
node dissection showing involvement of 16/18 lymph nodes. The
patient was 36 years old at the time of ASCT. Prior to ASCT, he
received a conditioning regimen of carboplatin, 200 mg/m/dlay, days
-7 to -4; thiotepa, 60 mg/m.sup.2/day, days -6 to -4; etoposide,
200 mg/m.sup.2/day, days -5 to -3; and melphalan, 60
mg/M.sup.2/day, days -4 to -3.
[0157] ASCT consisted of 1.81.times.10.sup.8 peripheral blood stem
cells cells/kg, infused IV on day 0. The stem cells were collected
after mobilization with G-CSF (7.5 mg/kg/day for 5 days) using
three collections with the CS 3000.RTM. Plus blood cell separator.
Following ASCT, the patient recovered with no complications and was
discharged.
[0158] A fully HLA-matched (A, B, DR and mixed lymphocyte reaction
(MLR)-negative) brother was chosen as donor of PBL for Allo-CMI. At
week 5 post-ASCT, as soon as the patient achieved a stable clinical
condition (WBC: 11,000/mm.sup.3; Hb: 11.5 g %; platelets:
201,000/mm.sup.3), the patient received 2.3.times.10.sup.7 T
cells/kg of PBL from the HLA-matched brother. Starting at week 5,
on the same day as the administration of the first Allo-CMI
treatment, the patient aIlso received 6.times.10.sup.6 IU of
rhIL-2/m.sup.2/day for 3 days, by subcutaneous injection. No GVHD
has been observed to date.
[0159] Portions of the above-described patient treatment protocols
are summarized in Table 1, below. Patients are grouped according to
disease (AML, CML, NHL and breast cancer).
1TABLE 1.sup.a Patient Status Allo Cells No. Pre-ASCT Cond. Regimen
Type ASCT (T Cells/Kg) AML: 1 1CR BU/CY ABMT: Purged PBL 10.sup.4 -
10.sup.7 (day +1 to +47) 2 1CR 13U/CY ABMT: Purged PBL 10.sup.4 -
10.sup.7 (day +1 to +80) 3 1CR BU/CY ABMT: Non-Purged PBL 10.sup.7
+ IL2 (day +1) 4 1CR BU/CY + TT ABMT: Non-Purged PBL 10.sup.7 + IL2
(day +1) 6 1CR BU/CY + TT ABMT: Non-Purged 1. PBL 2. PBL + IL2 13
1CR BU/CY + TT ABMT: Non-Purged 1. PBL 14 2PR BU/CY + MX ABMT:
Non-Purged 1. PBL 2. Cytoxan/PBL + IL2 15 1CR BU/CY + TT ABMT:
Non-Purged 1. PBL CML: 5 CP(Ph.sup.-) CY/TBI ABMT: Non-Purged 1.
PBL 2. PBL + IL2 3. CAL + IL2 7 CP(Ph.sup.+) CY/TBI ABMT:
Non-Purged 1. PBL 2. PBL + IL2 NHL: 8 2PR ETACM ABMT + PBSC: Purged
1. PBL 2. PBL + IL2 3. Allogeneic BMT (due to marrow aplasia) 9 2PR
ETACM ABMT: Purged 1. PBL 2. PBL + IL2 3. CAL + IL2 10 2CR ETACM
ABMT + PBSC: Non-Purged 1. PBL. 2. PBL + IL2 3. CAL + IL2 11 2CR
ETACM ABMT + PBSC: Non-Purged 1. PBL 2. PBL 3. PBL 4. PBL + IL2 12
Relapse ETACM ABMT: Non-Purged 1. PBL 2. PBL + IL2 16 2PR ETACM
ABMT + PBSC: Non-Purged 1. PBL BREAST CANCER: 17 Metastatic CTEM
PBSC: Non-Purged 1. PBL + IL2 18 Metastatic CTEM PBSC: Non-Purged
1. PBL + IL2 .sup.aTerms used in the Table are defined as follows:
1CR, 2CR: First or second complete remission, respectively. 2PR:
Second partial remission CP(Ph.sup.-): Chronic phase, no
cytogenetic evidence of Philadelphia chromosome. CP(Ph.sup.+):
Chronic phase, cytogenetic evidence for presence of Philadelphia
chromosome. BU/CY: Conditioning regimen of Busulfan, 4 mg/kg, days
6 through 9 pre-ASCT (days -9 to -6), as well as Cytoxan
(cyclophosphamide), 50 mg/kg, days -5 to -2, Cotrimoxazol, 10
mg/kg, days -10 to -2, Allopurinol, 300 mg/kg, days -10 to -1 and
cytosine arabinoside (Ara-C), 25 mg intrathecally. BU/CY + TT:
Conditioning regimen of Busulfan 4 mg/kg, days -9 to -6, Cytoxan 60
mg/kg, days -3 to -2, Thiotepa 5 mg/kg/day, days -5 to -4,
Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg,
days -10 to -1 and Ara-C, 25 mg intrathecally. BU/CY + MX:
Conditioning regimen of Busulfan 4 mg/kg, days -9 to -6; Cytoxan 60
mg/kg, days -3 to -2; Mitoxantrone 6 mg/m.sup.2/day, days -5 to -4;
Cotrimoxazol, 10 mg/kg, days -10 to -2, Allopurinol, 300 mg/kg,
days -10 to -1 and Ara-C, 25 mg intrathecally. CY/TBI: Conditioning
regimen of total body irradiation (TBI) 200 cGY/day, days -5 to -3,
and Cytoxan 60 mg/kg, days -2 to -1. ETACM: Conditioning regimen of
etoposide, 200 mg/m.sup.2/day, days -6 to -3; thiotepa, 40
mg/m.sup.2/day, days -5 to -2; Ara-C, 200 mg/m.sup.2/day, days -4
to -1; Cytoxan, 60 mg/kg/day, day -3; and melphalan, 60
mg/m.sup.2/day, days -2 to -1. CTEM: Conditioning regimen of
carboplatin, 200 mg/m.sup.2/day, days -7 to -4; thiotepa, 60
mg/m.sup.2/day, days -6 to -4; etoposide, 200 mg/m.sup.2/day, days
-5 to -3; and melphalan, 60 mg/m.sup.2/day, days -4 to -3. ABMT:
Infusion of stem cells extracted directly from the bone marrow.
PBSC: Enfusion of stem cells taken from peripheral blood following
mobilization from the bone marrow. PBL, CAL: Without further
numerical designation, PBL (peripheral blood lymphocytes) and CAL
(cytokine-activated lymphocytes) refer to one infusion of
.gtoreq.10.sup.7 cells/kg of patient body weight. For patient No.'s
5-18, the first infusion of allogeneic cells was done only after
attainment of partial hematopoiesis reconstitution. Cytoxan/PBL:
Cyclophosphamide (Cytoxan) administered prior to infusion of
.gtoreq.10.sup.7 cells/kg of PBL.
[0160] II. Results
[0161] The above-described patients have been followed for various
lengths of time. The disease status of the patients is summarized
below:
[0162] Patient No. 1 relapsed after 6 months and died at 9 months
after ASCT.
[0163] Patient No. 2 relapsed after 12 months and died at 19 months
after ASCT.
[0164] Patient No. 3 is alive and well over 34 months after
ASCT.
[0165] Patient No. 4 is alive and well over 24 months after
ASCT.
[0166] Patient No. 5 is alive and well over 23 months after ASCT,
showing no signs of the Philadelphia chromosome (Ph-) by
cytogenetic analysis and being negative by PCR for the bcr/abl
translocation characteristic of the Philadelphia chromosome. The
patient is receiving 9.times.10.sup.6 units of IFN.alpha. (Roferon
A, S.C.) every day.
[0167] Patient No. 6 is alive and well over 23 months after ASCT
with no evidence of disease.
[0168] Patient No. 7 is alive and well over 21 months post-ASCT.
Currently, the patient is on IFIN.alpha. treatment, and is
hematologically normal with no clinical sign of disease.
[0169] Patient No. 8, currently at nearly 2 years after initial
ASCT and over one year following allogeneic BMT, is alive and well
with no evidence of lymphoma. The patient has mild cutaneous
chronic GVHD with Karnofsky score of 100%
[0170] Patient No.9 was alive and well until 18 months after ASCT.
At 19 months he has lymphadenopathy and is being evaluated for
relapse.
[0171] Patient No. 10 is alive and well over 15 months after ASCT
with no evidence of disease.
[0172] Patient No. 11 is alive and well over 13 months after ASCT
with no evidence of disease.
[0173] Patient No. 12 underwent relapse at 3 months post-ASCT and
died at 5 months post-ASCT.
[0174] Patient No. 13 underwent relapse at 4 months post-ASCT,
before receiving allo-CMI with rhIL2. He is alive and further
immunotherapy is planned.
[0175] Patient No. 14 is alive and well with no evidence of disease
over 5 months post-ASCT.
[0176] Patient No. 15 is alive and well with no evidence of disease
over 4 months post-ASCT.
[0177] Patient No. 16 is alive and well with no evidence of disease
3 months post-ASCT.
[0178] Patient No. 17 is alive and well with no evidence of disease
2 months post-ASCT.
[0179] Patient No. 18 is alive and well with no evidence of disease
1.5 months post-ASCT.
[0180] The results reported above indicate that Allo-CMI and
Allo-CCI may be the most rational and practical approaches for
eradication of residual malignant cells. GVT effects induced by
administration of allogeneic lymphocytes may be further enhanced by
administration in vivo of rhIL2.
EXAMPLE 3
Utility of Cytokines Other than IL2
[0181] Cytokines used in the following experiments were obtained as
follows: (1) rhIL2 was provided by Dr. C. R. Franks (EuroCetus BY,
Amsterdam, The Netherlands) as a lyophilized powder in 1 mg vials
contianing 18.times.10.sup.6 international units (IU). (2)
Recombinant interferon-gamma (rIFN.gamma.) was provided by Roussel
Uclaf (Romainville, France) as a lyophilized powder containing
2.times.10.sup.7 U/mg. (3) Recombinant human IL-6 (rIL-6) was
kindly provided by Dr. M. Revel and Dr. O. Laub (InterPharm
Laboratories, Rehovot, Israel) in a concentration of 1.13 mg/ml
protein containing 43.times.10.sup.6 IU/ml. (4) Recombinant human
IL-7 (rhIL-7) was provided by Pepro Tech (New Jersey, USA) as
lyophilized powder and was reconstituted to 100 mg/ml.
[0182] Lymphocytes were preactivated in vitro with rhIL2 or with
combinations of rhIL2 and other cytokines for four days at
37.degree. C. Concentrations of cytokines used for in vitro
incubation of lymphocytes were as follows: (a) rhIL2: 6000 IU/ml;
(b) rIL6: 100-1000 U/ml; (c) rIL7: long/ml; and (d) IFNy: 1000
U/ml. Anti-tumor effects of CAL collected post-culturing were
assayed against natural killer (NK)-sensitive K562 tumor cells and
NK-resistant Daudi tumor cells. The in vitro toxicity was evaluated
by specific chromium release following incubation of effector cells
with chromium-labeled tumor cells. Results are presented below as
lytic units per 10.sup.6 effector cells, determined for 30% lysis
of 5.times.10.sup.3 target cells:
2 Cytotoxicity (Lytic Units/10.sup.6 Cells) Anti-Daudi Anti-K562
IL2 alone 48 53 IL2 + IL6 + IL7 + IFN.gamma. 210 129
* * * * *