Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 022~941 1998-11-24
WO97t451~2 ~C~Sg7~9~4
PR~v~NLlON OF GRAFT-VERS~S-HOST DISEASE WIT~ T-C~LLS
INCL~DING POLYNUCLEOTIDES ENCODING NEGATIVE
SELEcTIvE M~R~ ~
This invention relates to the prevention of graft-
versus-host disease, or GVHD, in patients who have received
allogeneic bone marrow transplants. More particularly, this
invention relates to the prevention of graft-versus-host
disease in patients who are suffering ~rom relapsing or
persistent leukemia, subsequent to an allogeneic T-cell
depleted bone marrow transplant, by A~m; n~ stering to such
patients T-cells which include a polynucleotide encoding a
negative selective marker, followed by the ~m;n~ stration of
an interaction agent or prodrug to kill the T-cells, in
particular, those T-cells which are graft-versus-host
reactive, before graft-versus-host disease develops.
In addition, this invention relates to the prevention of
gra~t-versus-host disease in connection with the treatment of
diseases or disorders wherein the treatment includes bone
marrow ablation followed by the A~m; n; stration of an
allogeneic bone marrow transplant, such as a T-cell depleted
bone marrow transplant. In such a method, T-cells which
include a polynucleotide encoding a negative selective marker
also are A~m; ni stered to a patient, and an interaction agent
or prodrug is ~ml n~ stered to the patient to kill the T-
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wo97l4sl42 PCT~S97/09040
cells, in particular, those T-cells which are graft-versus-
host reactive, before graft-versus-host disease develops.
Bach~ d of the Invention
Hematological malignancies, or leukemias, such as, for
example, multiple myeloma (MM), chronic myelogenous leukemia
(CML), acute myeloid leukemia (AML), and acute lymphoblastic
leukemia (ALL), affect thousands of Americans per year. For
example, multiple myelom~ affects approximately 4 in 10~,000
Americans per year. There were 12,800 new cases in 1993.
Multiple myeloma comprises slightly more than 1% of all types
of malignancies and slightly more than 10~ of all
hematological malignancies. (Barlogie, et al., JAMA, Vol.
268, pgs. 2946-2951 (1992); Barlogie, et al., Blood, Vol. 73,
pgs. 865-879 (1989)). The median age at diagnosis is 62 and
it is more common in men than in women. (Bortin, et al.,
Transplantation, Vol. 42, pg. 29 (1986).) Patients present
with localized or disseminated disease, serum M-protein value
of usually more than 3.0 g/dl and reduced values of
uninvolved serum ;mm-lnoglobulins. There is often a
monoclonal light chain in the urine and skeletal lesions.
The plasma cell labeling index, ~2 microglobulin, and C-
reactive protein (CRP) have been shown to be independent
prognostic factors.
Allogeneic bone marrow transplantation has emerged as an
effective treatment modality for selected patients with
multiple myeloma (Bortin, et al., 1986). 268 patients have
received allotransplants in different trials. (Barlogie, et
al., Sem;n~rs in Hematology, Vol. 32, pgs. 31-44 (1995)).
Fifty percent of the patients died within one year, about 40
percent achieved a complete response, and the four year
projected event - free survival is approximately 35 percent.
Allogeneic bone marrow transplantation is thouyht to be
curative, in part, because of an anti-tumor (i.e., graft-
versus-myeloma, or GVM) effect derived from the adoptive
transfer of ~mmllnocompetent cells in the donor graft. (Gale,
--2--
CA 022~94l l998-ll-24
WO 97/4!j142 PCT~US97~090
et al., The Lancet, Vol. 2, pg. 28 (1984); Bortin, et al.,
Nature, Vol. 67, pg. 722 (1979).) The anti-~umor e~ect
which has been associated with gra~t-versus-host disease
(GVHD), appears to be mediated by both T-cell and non-T-cell
effector cell populations. GVHD, however, r~mAin~ a major
problem because o~ its morbidity and mortality.
In order to decrease the early mortality from allogeneic
bone marrow transplantation, T-cell depletion has been
employed. T-cells may be depleted from the allograft by any
of several techni~ues, including density gradient
centrifugation, soybean lectin agglutination and E-rosette
formation, centrifugal elutriation, cytotoxic drugs or
corticosteroids, anti-T-cell monoclonal antibodies, and
positive selection of CD34+ cells. (Champlin, Journal of
Hematotherapy, Vol. 2, pgs. 27-42 (1993); Reisner, et al.,
The Lancet, Vol. 2, pg8. 327-331 (1981); Waldmann, et al.,
The Lancet, Vol. 2, pgs. 483-486 (1984); Antin, et al.,
Blood, Vol. 78, pgs. 2139-2149 (1991); Soi~er, et al., J.
Clin. Oncol., Vol. lQ, pgs. 1191-1200 (1992).) Of the
techniques used currently to deplete T-cells from a marrow
graft, the most efficient is that o~ R~isnerl et al. (1981).
This technique, which consistently removes 2.5-3.0 log,
clonable T-cells, first employs differential agglutination
with soybean lectin to remove mature leukocytes, including T-
cells, ~3-cells, monocytes, and granulocytes, followed by E-
rosette depletion for removal o~ residual T-cells. This
technique has been used to treat a population of over 200
HLA-matched related transplants given to lellk~m; A patients.
In this population, the incidence of Grade II acute GVHD has
been 5~, and Grades III and IV acute GVHD have not been
observed. (O'Reilly, et al., Bone Marrow TransPlantation~
Vol. 3, pgs. 3-6 (1988).)
Although T-cell depletion decreases the incidence of
n GVHD, T-cell depletion also increases the risk of early
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CA 022~94l l998-ll-24
WO97/45142 PCT~S97/09040
relapse, incomplete ;mmllnological reconstitution, gra~t
~ailure, and ~pstein-Barr Virus-related lymphoma.
The risk o~ relapse has been documented best in chronic
myelogenous leukemia. (Marmont, et al., Blood, Vol. 78, pgs.
2120-2130 (1991); Goldman, et al., Ann. Int. Med., Vol. 108,
pgs. 806-814 (1988).) Direct evidence of a graft-versus-
leukemia (GVL) effect has been ~Pmo~trated in patients with
recurrent chronic myelogenous leukemia after a T-cell
depleted bone marrow transplant by infusion of peripheral
blood mono~l~clear cells from the bone marrow donor without
any additional chemotherapy or radiotherapy, resulting in at
least a 6 Iogl0 kill of leukemic cells. (Drobyski, et al.,
Blood, Vol. 82, pgs. 2310-2318 (1993); Sullivan, et al., N.
Enql. J. Med., Vol. 320, pgs. 828-834 (1~89); Porter, et al.,
N. Engl. J. Med., Vol. 300, pgs. 100-106 (1994); Slavin, et
al., Bone Marrow Transplantation, Vol. 6, pgs. 155-161
(1990); Sosman, et al., Amer. J. Pediatr. Hem./Onc., Vol. 15,
pgs. 185-195 (1993).) Although infused peripheral blood
mononuclear cells can induce complete and long-term remission
in such patients, such therapy may be associated with GVHD.
(Bar, et al., J. Clin. Oncol., Vol. 11, pg. 513 (1993);
Drobyski, et al., 1993; Slavin, et al., 1990.)
Allogeneic ~one marrow transplantation following T-cell
depletion also has been associated with increased incidence
of bone marrow engra~tment failure. (Beatty, et al., N.
Enql. J. Med., Vol. 315, pgs. 765-771 (1985); Hale, et al.,
Txansplantation, Vol. 45, pgs. 753-759 (1988); Patterson, et
al., Br. J. Hematol., Vol. 63, pgs. 221-230 (1986).) In a
murine model for allogeneic bone marrow transplantation,
durable engraftment was observed only after addition of
potent immllnosuppressive treatment with total body
irradiation (TBI) in the conditioning regimen to deplete the
host's T-cells. (Lapidot, et al., Blood, Vol. 73, pg. 2025
(1989)). In human allotransplantation, the same problem is
CA 022~941 1998-ll-24
WO 97/45142 PCT/US97/09040
overcome by adding thiotepa and anti-thymocyte globulin to
the conditioning regimen.
The ~ml~ne de~iciency a~ter a T-cell depleted bone
marrow transplant is very pronounced as the consequence of
the intensive conditioning regimen and the absence o~ mature
donor T-cells in the transplant. It is probably prolonged by
larger differences in minor histocompatibility antigens
between donor and patient.
One o~ the consequences of the severe ;mmllne deficiency
in T-cell depleted bone marrow transplants is the increased
risk of Epstein-Barr Virus (EBV) lymphoproliferative
disorders. (Shapiro, et al., Blood, Vol. 71, pgs. 1234-1243
(1988); Zutter, et al., Blood, Vol. 72, pgs. 520-522 ~1988).)
These lymphoproli~erative disorders are o~ten unresponsive to
stAn~Ard forms of therapy. The Memorial Sloan-Kettering Bone
~arrow Transplantation Group has reported on 7 patients
developing lymphoproliferative disorder who were treated with
donor lymphocyte infusion. (Papadopoulos, et al., Blood,
Vol. 82, supp. 1:214a (~993).) All patients were recipients
o~ T-cell depleted transplants (4 related, 3 unrelated3. The
lymphoproliferative disorders developed within 6 months post
transplantation. ~iopsy spec~m~n~ ~mnn~trated a diffuse
large cell lymphoma. Four evaluable specimens were found to
be of donor cell origin. EBV DNA was detected in 5 of 5
evaluable samples by PCR. The patients received donor
leukocytes at doses providing 0.2-1.0 x 106 CD3+ cells/kg o~
patient weight. Complete pathologic and/or clinical
responses were observed in all 5 patients. These responses
were documented pathologically by 8-21 days post-infusion.
Clinical remissions were achieved within 14-30 days and have
been sustA~ without other therapy in the 3 surviving
patients. Two patients died 8 days and 16 days after
infusion from sepsis and interstitial pnell~nn~ A,
respectively. Autopsy did not reveal any evidence of
residual lymphomA. The 3 surviving patients developed GVHD.
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WO97/45142 PCT~S97/09040
Tiberghien, et al., in a series of abstracts and in a
journal article, (Tiberghien, et al., Proceedinqs of the
American Association ~or Cancer Research, Vol. 34, pg. 338,
abstract 2011 (March 1993); Tiberghien, et al., J. Cell,
Biochem., Supp. 17E, pg. 234, abstract SZ223 ~1993);
Tiberghien, et al., Nouv. Rev. Fr. d~Hematol, Vol. 35, pg.
329 (1993); Tiberghien, et al., First Meetinq of the
European Workinq GrouP on Human Gene Transfer and Therapy,
Abstract Book, pg. 44 (November 19, 1993); Tiberghien, et
al., Blood, Vol 84, No. 4, pgs. 1333-1341 (August 15, 1994))
disclose the transduction o~ T-cells with a retroviral vector
including the Herpes Simplex Virus thymidine kinase gene.
Ganciclovir treatment of the transduced T-cells resulted in
a growth inhibition of these cells which was greater than
80~. The ganciclovir had no e~ect on control (non-
transduced) T-cells. Tiberghien, et al., then state that T-
cells may be transduced with the Herpes Simplex Virus
thymidine kinase (TK) gene ex vivo, and then be administered
to a patient with hematopoietic stem cells. If the patient
develops GVHD, ganciclovir may be ~m;n;stered to the patient
in order to deplete the transduced T-cells.
SummarY of the Invention
It is an o~ect o~ the present invention to treat
patients with relapsed or persistent leukemia after a T-cell
depleted allogeneic bone marrow transplant. Such patients
are given T-cells which have been genetically engineered to
include a polynucleotide encoding a negative selective
marker. After the cells have r~m~i n~ in the patient ~or an
amount of time sufficient to provide a therapeutic effect, an
interaction agent or prodrug is ~mi n; stered to the patient,
whereby the genetically engineered T-cells are killed, and
the development o~ GVHD is prevented. Alternatively, if GVHD
develops be~ore the predetermined time o~ ~m; n~ stration o~
the interaction agent, the interaction agent may be
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~m; n; stered to the patient at the time of development of
GVHD, whereby the GVHD is treated through the killing of the
transduced T-cells.
It is another object of the present invention to prevent
graft-versus-host disease in patients being treated for
diseases or disorders wherein the treatment includes bone
marrow ablation followed by the A~mi ni stration of a T-cell
depleted bone marrow transplant. Such diseases or disorders
include, but are not limited to, solid tumor malignancies,
and acquired or genetic immllnologic or hematopoietic
diseases. During the treatment, T-cells genetically
engineered with a polynucleotide encoding a negative
selective marker are administered to the patient. After the
cells have remained in the patient ~or an amount o~ time
sufficient to provide a therapeutic effect, an interaction
agent or prodrug is administered to the patient, whereby the
genetically engineered T-cells are killed, there~y preventing
graft-versus-host disease.
Detailed Descri~tion of the Invention
In accordance with an aspect of the present invention,
there is provided a method of preventing graft-versus-host
disease in a host that is being treated for a disease or
disorder which is treatable by ~mi ni stering T-cells to a
host, such as, for example, a relapsed or persistent
leukemia. The method comprises ~mi n; stering to a host T-
cells genetically engineered to include a polynucleotide
encoding a negative selective marker or "suicide" gene. The
cells are ~m~n; ~tered in an amount ef~ective and remain in
the host for a period of time effective to provide a
therapeutic ef~ect in the host. After the T-cells have
r~m~;nPd in the host for a period of time sufficient to
provide a therapeutic effect in the host, and prior to the
occurrence of graft-versus-host disease, an interaction agent
~ or prodrug is ~m~ ni stered to the host. The interaction
agent is ~m; n; stered to the host in an amount effective to
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kill the genetically engineered T-cells, in particular, those
T-cells which are graft-versus-host reactive, i.e., capable
of providing a graft-versus-host effect, thereby preventing
the occurrence of graft-versus-host disease in the host.
The term '1polynucleotide" as used herein means a
polymeric form of nucleotide of any length, and includes
ribonucleotides and deoxyribonucleotides. Such term also
includes single- and double-stranded DNA, as well as single-
and double-stranded RNA. The term also includes modified
polynucleotides such as methylated or capped polynucleotides.
The polynucleotide encoding the negative selective
marker may be contained within an appropriate expression
vehicle which is transduced into the T-cells. Such
expression vehicles include, but are not limited to,
eukaryotic vectors, prokaryotic vectors (~uch as, for
example, bacterial vectors), and viral vectors.
In one alternative em~odiment, the polynucleotide
encoding the agent, or an expression vehicle cont~; ni ng the
polynucleotide encoding the agent, is contained within a
liposome.
In one preferred embodiment, the expression vehicle is
a viral vector. Viral vectors which may be employed include
DNA virus vectors (such as adenoviral vectors, adeno-
associated virus vectors, Herpes Virus vectors, and vaccinia
virus vectors), and RNA virus vectors (such as retroviral
vectors). When an RNA virus vector is employed, in
constructing the vector, the polynucleotide encoding the
negative selective marker is in the form of RNA. When a DNA
virus vector is employed, in constructing the vector, the
polynucleotide encoding the negative selective marker is in
the form of DNA .
In one embodiment, the viral vector is a retroviral
vector. Examples of retroviral vectors which may be employed
include, ~ut are not limited to, Moloney Murine Leukemia
Virus, spleen necrosis virus, and vectors derived from
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retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma
Virus, avian leukosis virus, human ~mmllnode~iciency virus,
myeloproliferative sarcoma virus, and m~mm~ry tumor virus.
The vector is preferably an infectious, replication
inco~petent retrovirus particle.
Retroviral vectors are useful as agents to mediate
retroviral-mediated gene transfer into eukaryotic cells.
Retroviral vectors are generally constructed such that the
majority of sequences coding for the structural genes of the
virus are deleted and replaced by the gene(s) of interest.
Most often, the structural genes (i.e., gag, pol, and env),
are removed from the retroviral backbone using genetic
engineering techniques known in the art.
The removal o~ the gag, pol and env genes results in a
vector backbone, comprised of a 5' LT~, a packaging signal,
one or more cloning sites, into which the heterologous gene
or genes of interest can be introduced, and a 3' LTR. The
preferred vector backbone is the Gl vector backbone, which is
disclosed in McLachlin, et al., Viroloqy, 195:1-5 (1993) and
in PCT Patent Application No. WO 91/10728 for "Novel
Retroviral Vectors," published on July 25, 1991.
The heterologous gene or genes are incorporated into the
proviral backbone by st~n~rd techniques to form the
retroviral vector. Techniques for the preparation of
retroviral vectors are disclosed in PCT application WO
91/10728 as well as the following articles: Arm~nt~no, et
al., ~. Virol., 61:1647-1650 (1987), Bender, et al., J.
Virol., 61:1639-1646 (1987), and Miller, et al.,
Biotechniques, 7:980-990 (1989). The most straightforward
constructions are ones in which the structural genes of the
retrovirus are replaced by a single gene which then is
transcribed under the control o~ the viral regulatory
sequences within the long terminal repeat (LTR). Retroviral
vectors have also been constructed which can introduce more
than one gene into target cells. Usually, in such vectors
_g_
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WO97/45142 PCT~S97/09040
one gene is under the regulatory control of the viral LTR,
while the second gene is expressed either o~f a spliced
message or is under the regulation of its own, internal
promoter. Suitable promoters include the SV40 promoter, the
hllm~n cytomegalovirus (CMV) promoter, the beta-actin
promoter, the alpha fetoprotein promoter, and any promoter
naturally associated with any heterologous gene of interest.
Additionally, a polycistronic vector can be created by using
an internal ribosome entry site.
The retroviral vectors may be in the ~orm of a plasmid,
a segment of viral RNA, or a segment of proviral DNA. For
the present invention, the preferred retroviral vector is
GlTKlSvNa, which is disclosed in PCT Patent Application No.
WO 95/06486, published on March 9, 1995, entitled Treatment
of ~llm~n Tumors by Genetic Transformation of Human Tumor
Cells.
The retroviral vector is introduced into a packaging
cell to form a producer cell. Packaging cells provide the
gag, pol, and env genes in trans, which permits the packagin~
of the retroviral vector into a recombinant retrovirus that
is infectious but replication defective. The vectors are
transferred into the packaging cells ~y st~n~rd gene
transfer techniques, which include transfection,
transduction, calcium phosphate precipitation,
electroporation, and liposome-mediated DNA transfer.
Examples of packaging cells that may be used include, but are
not limited to, the PE501, PA317, Psi-2, Psi-AM, PA12, T19-
14X, VT-19-17-H2, Psi-CRE, Psi-CRIP, GP+E-86, GP+envAM12,
PG13, and DAN cell lines. A preferred producer cell line for
the present invention for the production of recombinant
retrovirus is the producer cell line designated
PA317/GlTKlSvNa, which is disclosed in PCT application WO
95/06486.
Ne~ative selective markers include, but are not limited
to, viral thymidine kinases such as Herpes Simplex Virus
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thymidine kinase, cytomegalovirus thymidine kinase, and
varicella-zoster virus thymidine kinase; xanthine-guanine
phosphoribos~l transferase; and cytosine ~e~m~n~se.
The retroviral vectors cont~; n; n~ the polynucleotide
encoding the negative selective marker are transduced into T-
cells. In general, from about 106 to about 109, preferably
from about 107 to about lo8 T-cells are transduced with the
retroviral vectors, which may be contained in from about 2 ml
to about 500 ml of retro~iral supernatant having a titer of
from about 105 cfu/ml to about 109 cfu/ml, pre~erably fro~
about 2X106 cfu/ml to about 1x108 cfu/ml.
Once the T-cells are transduced with the retroviral
vectors including the polynucleotide encoding the negative
selective marker, the T-cells are administered to a host
suffering from a relapsed or chronic leukemia. The host is
an ~n;m~l host, and in particular is a m~mm~lian host,
including human and non-human primate hosts. The transduced
T-cells are administered by means known to those skilled in
the art, includin~ intravascular administration, such as
intra~enous or intraarterial ,~mi n~ strationi or by
intraperitoneal ~m~ni stration. In one embodiment, the
transduced T-cells are ~ministered as a bolus in~usion. The
transduced T-cells are ~m~n~stered in an amount effective to
provide a therapeutic ef~ect, i.e., in an amount èf~ective to
treat the relapsed or persistent leukemia in the host. In
general, the transduced T-cells are A~ministered in an amount
of from about 105 cells/kg to about 109 cells/kg, preferably
~rom about 2x105 cells/kg to about lX107 cells/kg.
The transduced T-cells are administered in conjunction
with an acceptable pharmaceutical carrier, such as, for
example, saline solution, or agueous buffers, such as
phosphate buffers, Tris buffers, Plasmalyte A (Baxter), or
lactated Ringer's solution. The selection of a suitable
pharmaceutical carrier is deemed to be apparent to those
skilled in the art from the teachings cont~n~ herein.
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Alternatively, the transduced T-cells may be frozen in an
acceptable cryopreservation medium (such as, for example, a
medium including phosphate buffered saline, 5~ DMSO, and
human albumin) until the cells are administered to the host.
Prior to administration, the cells and medium are thawed, and
the cells and medium are ~m~ ni stered to the host upon
thawing.
After a period of time sufficient to allow the
transduced T-cells to provide a therapeutic effect in the
host, but prior to the development of graft-versus-host
disease, an interaction or chemotherapeutic agent is
~m~n; stered to the host in an amount effective to kill the
transduced T-cells, and in particular, to kill transduced
proliferating graft-versus-host-reactive T-cells, i . e., T-
cells which are capable of providing a gra~t-versus-host
effect, thereby preventing the development of graft-versus-
host disease.
Preferably, the interaction agent is A~mi n; stered when
the T-cells which are graft-versus-host reactive ~whereby
such T-cells which are capable of providing a graft-versus-
host effect through the recognition of MHC Class I antigens
of the host cells) are in a proliferative phase, and a
portion of T-cells such as, for example, some of those T-
cells which provide a graft-versus-leukemia effect or which
provide an anti-viral effect, are in a ~uiescent or non-
proliferative phase. The interaction agent, or prodrug, when
~m; ni stered at such a time, will provide for the killing of
the proliferating T-cells which are c~pAhle of providing a
graft-versus-host effect, whereas the non-proliferating T-
cells, which include a portion of T-cells which provide a
graft-versus-leukemia effect or an anti-viral effect, will
survive the ~m; n~ stration of the interaction agent or
prodrug. Thus, the ma~ority of the T-cells responsible for
causing GVHD are ablated preferentially, while other T-cells
capable of providing a graft-versus-leukemia effect or an
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anti-viral effect remain. In general, the interaction agent
is administered at a period of time of from about 10 days to
about 50 days, preferably from about 14 days to about 28
days, more pre~erably at 21 days, after the administration of
the transduced T-cells.
When the negative selective marker is a viral thymidine
kinase, such as those hereinabove described, the interaction
or chemotherapeutic agent or prodrug preferably is a
nucleoside analogue, for example, one selected from the group
consisting of ganciclovir, acyclovir, and 1-2-deoxy-2-~luoro-
~-D-arabinofuranosil-5-iodouracil (FIAU). Such interaction
agents are utilized efficiently by the viral thymidine
kinases as substrates, and such interaction agents thus are
incorporated lethally into the DNA of the transduced T-cells
expressing the viral thymidine kinases, and in particular,
proliferating T-cells which are gra~t-versus-host reactive,
thereby resulting in the death of the transduced T-cells.
When the negative selective marker is cytosine
~m~n~e~ a preferred interaction agent or prodrug is 5-
fluorocytosine. Cytosine ~P~mln~e converts 5-fluorocytosine
to 5-fluorouracil, which is highly cytotoxic. Thus, the
transduced T-cells which express the cytosine ~eAm~n~e gene
convert the 5-fluorocytosine to 5-fluorouracil and are
killed.
The interaction agent or prodrug is ~m; n; stered in an
amount effective to provide for the death of the transduced
T-cells. The interaction agent is ~m;n;stered preferably by
systemic ~mi n~ stration, such as by intravenous
~m;n~ stration. In general, the interaction agent is
~; n~ stered in an amount of from about 2 mg/kg/day to about
10 mg/kg/day, preferably about 10 mg/kg/day, for a period of
from about 3 to 18 days, preferably for about 5 days. In a
preferred embodiment, the interaction agent or prodrug is
~mln~ stered in an amount of 5 mg/kg every 12 hours for a
period of 5 days.
A
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In the event the host develops gra~t-versus-ho~t disease
prior to the desired time that one wishes to kill the
transduced T-cells, the interaction agent is ~mi ni stered
upon the development of the gra~t-versus-host disease,
thereby treating the graft-versus-host disease in the host.
Thus, the method o~ the present invention enables one to
treat a patient suffering from a relapsing or chronic
hematological malignancy or leukemia by ~m;nl~tering T-cells
to the patient, and to prevent the occurrence of graft-
versus-host disease by killing the T-cells after the T-cells
have provided a desired therapeutic e~fect. heukemias which
may be treated with the transduc~d T-cells include, but are
not limited to, multiple myeloma (MM), myelodysplastic
syndrome, chronic myelogenous leukemia (CML), chronic
lymphocytic leukemia (CLL), acute myeloid leukemia (AML),
acute lymphoblastic leukemia ~ALL), non-Hodgkin~s lymphoma,
Hodg~in~s disease, and myelofibrosis.
An advantage of the present invention is that the T-
cells which are genetically engineered to include a
polynucleotide encoding a negative selective marker are safer
than T-cells which are not genetically engineered to be
employed in the treatment of relapsed or chronic leukemia
because the major cause of mortality and morbidity, graft-
versus-host disease, may be prevented by the ~m;n~stration
of a prodrug or interaction agent. Becau~e graft-versus-host
disease is a consequence of the degree of mismatch between
the donor and patient, the method of the present invention
expands the number o~ patients that are eligible for an
allogeneic bone marrow transplant to include older patients
and more genetically disparate donors, including HhA non-
identical ~iblings and matched, unrelated donors.
In addition, the timing of the administration of the
interaction agent or prodrug is such that the majority of the
GVHD reactive cells (i.e., the T-cells that are responsible
for causing GVHD) are ablated preferentially, thus leaving
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WO 97/45142 PCTflJS97~09(140
the patient with the greater part of the ;mmllnologic T-cell
repertoire. It is likely that at least a part of the graft-
versus-leukemia reactive cells and T-cells which are reactive
with antigens other than those associated with GVHD, will
survive the ~m; n~ stration of the interaction agent or
prodrug, thus helping to decrease the ~requency of EBV
lymphom~ and infections.
In addition to the treatment of leukemia, the method
hereinabove described may be applied to the treatment of
other diseases and disorders. For example, the method may be
applied to the treatment of solid tumor malignancies,
especially in cases where the tumor has metastasized and
tumor cells are ~ound in the bone marrow. During such
treatment, patients would undergo a high dose chemotherapy
treatment with or without total body irradiation followed by
rescue with an allogeneic T-cell depleted bone marrow
transplant. T-cells genetically engineered with a
polynucleotide encoding a negative selective marker or
suicide gene would be ~m; ni fitered to the patient as
hereinabove described, and GVHD would be prevented by the
A~m; n; stration of the interaction agent or prodrug. In this
embodiment, the T-cells provide a graft-versus-tumor (GVT)
effect. Solid tumor malignancies which may be treated in
accordance with this method include, but are not limited to,
breast cancer, neuroblastoma, testicular carcinoma, ovarian
and uterine car~;nom~, and soft tissue sarcomas.
In addition, the hereinabove described method of the
present invention may be employed in the treatment of any
acquired or genetic ;mmllnologic or hematopoietic disease in
which treatment thereof includes bone marrow ablation
followed by the A~m; n; stration of a T-cell depleted bone
marrow transplant. Such diseases include, but are not
limited to, AIDS, severe combined ;m~lln~ deficiency (S~ID),
Wiskott-Aldrich syndrome, disorders of lymphocyte function,
disorders of myeloid function, aplastic ~n~m; ~, Fanconi's
CA 022~941 1998-11-24
wo97l4sl42 PCT~S97/09040
anemia, thalassemia, sickle cell anemia, enzyme de~iciencies
(including Gaucher's disease, the mucopolysaccharidoses, and
the leukodystrophies), and osteopetrosis, a disease which is
characterized by a deficiency in bone marrow cells which
break down older bone. In these diseases, there is no gra~t-
versus-leukemia or graft-versus-tumor effect. The
genetically engineered T-cells, in this em~odiment, would be
responsible for improved bone marrow engra~tment, and ~or
decreasing the incidence of EBV lymphoma. GVHD is prevented
by administration of the prodrug or interaction agent.
Thus, in accordance with another aspect of the present
invention, there is provided a method of treating a disease
or disorder in a host (which may be a m~mm~lian host,
including human and non-human prim--ate hosts) wherein
treatment o~ the disease or disorder in the host includes
ablating the bone marrow of the host, ~ollowed by the
administration o~ a T-cell depleted ~one marrow transplant to
the host. The method comprises ablating the bone marrow o~
the host. A T-cell depleted bone marrow transplant then is
~mi ni stered to the host. The host then is ~mi n~ stered T-
cells genetically engineered to include a polynucleotide
encoding a negative selective marker, which may be selected
from those hereinabove described. Prior to the occurrence of
graft-versus-host disease, an interaction agent or prodrug
(such as those hereinabove described) is ~m~n; stered to the
host in an amount effective to kill the genetically
engineered T-cells in the host.
Diseases or disorders which may be treated in accordance
with this method include, but are not limited to, those
hereinabove described. The polynucleotide encoding the
negative selective marker may be contained in an appropriate
expression vehicle such as those hereinabove described,
including viral vectors such as retroviral vectors.
The genetically engineered T-cells may be ~mi n~ stered
to the host in an amount effective to provide a therapeutic
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WO 97145142 PCT/US97/091}40
effect. Such amount may be as hereinabove described. The
exact amount of genetically engineered T-cells to be
administered is dependent upon a variety of factors,
including the age, weight, and sex of the patient, the
disease or disorder being treated, and the extent and
8 everity thereof.
A~ter a period of time sufficient to allow the
genetically engineered T-cells to provide a therapeutic
effect in the host, but prior to the development of gra~t-
versus-host disease, the prodrug or interaction agent i5
~m; n i stered in an amount, which may be as hereinabove
described, which is effective in killing the genetically
engineered T-cells, and in particular, those genetically
engineered T-cells capable of providing a graft-versus-host
effect, thereby preventing the development of graft-versus-
host disease. The period of time after the ~mi ni stration of
the genetically engineered T-cells at which the interaction
agent is ~m~ n~ stered may be as hereinabove described.
EXAMPLES
The invention now will be described with respect to the
following examples; however, the scope of the present
invention is not intended to be limited thereby.
ExamDle 1
~-~ in~ stration of T-cells trAn~llce~ with a
retroviral vector includinq a HerPes SimPlex Virus
th~ i~i~e ki~ase ~ene to ~atients sufferinq
from Per istent or relaPsinq multi~le
mYeloma, iollowed bY ~m;n~ Qtration 0~ ~anciclo~ir
Collection of donor lymphocytes
50 to 100 ml of donor blood are collected, yielding at
least 2.5-5x107 cells. The blood is mixed with an equal
volume of sterile phosphate buffered saline (PBS) and
subjected to gradient separation using Ficoll (Lymphoprep,
Nycomed), and the mononllclear cells are separated. The cells
then are washed in sterile PBS three times. The cells are
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WO97/45142 PCT~S97/09040
re~u~pended in serum-free AIM-V medium containing 100
units/ml penicillin and 100 ~g/ml streptomycin tPen-strep~
Gibco-BRL) at a density of lx106 cells/ml with l~gtml anti-CD3
antibody (Orthoclone OKT3, Ortho-Biotech), and incubated at
37~C in 5% CO2 for 24 hours. Recombinant human Interleukin-2
(Chiron Corporation) is ~ at 1,500 units/ml and cells are
cultured at 37~C in 5% CO2 for a period of up to 7 days. When
the lymphocytes are in the exponential proliferation phase,
they are transduced with the retroviral vector.
Transduction of lymPhocytes with GlTKlSvNa
Approximately 1 X 108 lymphocytes are transduced with the
retroviral vector GlTKlSvNa, which includes the Herpes
Simplex Virus thymidine kinase (TK) gene and a neomycin
resistance gene. Such transduction is accomplished by adding
to the lymphocytes 100 ml of viral supernatant cont~i ni ng
from 2x108 cfu to lX109 cfu of the retroviral vector. The
retroviral vector GlTKlSvNa is described further in PCT
Application No. W095/06486, published March 9, 1995. Fresh
viral supernatant is added daily for a total of three days,
along with additional amounts of Interleukin - 2 and
prot~m;ne sulfate to achieve final Interleukin - 2
concentrations of 1,500 units/ml and protamine sulfate at
5~g/ml. The cells are incubated at 37~C, 5% CO2 for 24 hours
after each addition of supernatant and protaminè sulfate.
Twenty-four hours after the third transduction, the
cells are resuspended at a density of 1 X 106 cells/ml in
fresh medium contAining Pen-Strep and 1,500 units/ml of
Interleukin-2 and incubated for 3 to 5 days. Geneticin then
is added at an active concentration of 300~g/ml, and cells
are selected for three days, are centrifuged and then are
re~uspended in AIM-V medium contA;ning Interleukin-2 and
cultured until an adequate number of cells are obt~inP~. In
general, the cells are cultured for at least an additional
four days.
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WO 97145142 PCT/US97fag~4
The efficiency of transduction and selection is measured
by a ganciclovir killing assay. One to two percent of the
transduced ce~ls are sampled for the ganciclovir killing
assay. (A m~n;mllm of 2 X 106 cells are re~uired to perform
the assay.) The assay is per~ormed by suspending transduced
lymphocytes in 5.0 ml of AIM-V (Gibco-~RL) medium cont~i nl ng
1,50~ units/ml recombinant Interleukin-2 in tissue culture
flasks. A total of three flasks are prepared. Ganciclovir
is added to two of the flasks, one at a concentration of 20~M
and the second at 50 ~M . The third flask is used as a normal
control. The cells are incubated at 37~C, in 5~ CO2 for five
days. The percentage of live cells (trypan blue negative)
are counted after 3 and 5 days of culture in ganciclovir.
If the ganciclovir kill at a concentration of 50~M is
less than 85~, the cells are reselected in Geneticin as
described above. Cells are cryopreserved and infused only if
the ganciclovir kill is greater than or equal to 85~. In
order to provide enough cells for two infusions (if needed),
an ade~uate number of cells initially is collected, expanded,
and cryopreserved for each patient. The transduced
lymphocytes also are studied for subset analysis (CD3, CD4,
CD8, CD19, and CD56) by FACS prior to in~usion.
In~usion of transduced lYm~hocytes and ~mi n~ stration of
qanciclovir.
Infusion of the transduced lymphocytes is given to
patients who have undergone an allogeneic bone marrow
transplant with T-cell depletion if they show evidence of
persistent disease 90 days after transplantation, or
measurable relapse at anytime. Patients with persistent
disea~e have greater than 20~ plasma cells in a bone marrow
aspirate or biopsy, and/or presence of serum M-component, and
no reduction in the M-component in the last 6 weeks, and/or
Bence Jones Proteinuria with no reduction in the last 6
weeks.
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WO 97/45142 PCT/US97/09040
Each of three patients receives the transduced
lymphocytes in an amount of 1 ~ 106 lymphocytes/kg. One to
50 ml of lymphocytes is ~m~n; stered as a bolus infusion, and
each patient is observed for 4 hours thereafter.
Twenty-one days after the patients are given the
transduced lymphocytes, or if graft-versus-host disease
(GVHD) develops earlier, the patients are given ganciclovir
~Cytovene, Syntex Corporation, Palo Alto, California) by
intravenous infusion in an amount of 5 mg/kg every 12 hours
daily for 5 days. Patients with a complete response or a
partial response with continued reduction in measurable
disease at forty-two days after the injection of the
transduced lymphocytes will not receive a second lymphocyte
in~usion until disease plateau or progression is observed.
Complete response should include, for a m~n~mllm of two
weeks, the following: (i) absence in urine and serum of M-
components by ~mmllnofixationi (ii) bone marrow which is
adequately cellular (i.e. ~20%) with less than 3~ plasma
cells by ;mml-nost~n~ng; (iii) no elevation in serum calcium
level; and (iv) no new bone lesions nor enlargement of
existing lesions. Partial response requires, for at least 4
weeks, the following: (i) reduction of serum M-component by
at least 5%; (ii) reduction of urinary M-protein to less than
200 mg/24 hrs. and to less than 10~ of pretreatment values;
and (iii) no new lytic bone lesions or soft tissue
plasmacytoma. Implov~ lt also should include a reduction of
serum paraprotein level and urinary light chain excretion by
25-50% compared with baseline values, and a decrease of bone
marrow infiltration by plasma cells by 25-50% compared with
baseline values.
Patients with a partial response without a continued
decline in M-protein will receive, forty-two days after the
first infusion of transduced lymphocytes, the same number of
transduced lymphocytes as during the first infusion.
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WO 97/45142 PCT/U~97/09040
Ganciclovir is not administered unless the patient develops
GVHD.
For this example, dose levels are as follows:
DOSE hEVEL LYMPHOCYTE INFUSION DOSE
r (LYMPHOCYTES/KG)
1 X 106
II 5 X 106
III 1 X 107
IV 2 X 107
V 5 X 10'
Also, for purposes of this example, GVHD is graded as
follows:
CLINICAL GRADING OF ACUTE GV~D
~n~ - 5KIN ~VER :.G~T
. : }MP-AIRMENT
O .0 0 0 0
I + to ++ 0 0 ~
II + to +++ + or + +
III ++to+++ ++to+++ ++to+++ ++
IV ++to++++ ++to++++ ++to++++ +++
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WO 97/45142 PCT/US97/09040
STAGING BY ORGAN SYSTEM
Sl~ 6 Ol~ BOD5F Sl~RFACE Ar3~Ci~
<25
++ 25-50 ~'
+++ ~5 0
+ ~ + + BI~LLAE, DES QUAMATI ON
IiI~ ~ ~RTr,JR~INt~%~
+ 2 - 3
3.1 - 6
+++ 6 . 1 - 15
++++ ~15
:GT}T . :: nT~ DA~
+ ~500
++ ~1, 000
~++ ~1, 500
++ ++ PAIN/ ILEUS
If the first three patients do not achieve a complete
response or partial response, and do not develop Grade III or
Grade IV GVHD, the dose of ly~phocytes is escalated from 1 X
o6 lymphocytes/kg to 5 X lo6 lymphocytes/kg (i.e., dose level
II).
The treatment plan for each dose level is identical to
the one described above for dose level I (i.e., 1 X lo6
lymphocytes/kg). Six additional patients are entered at the
dose level at which one of three patients responds, or at one
dose below the level at which toxicity is observed.
The disclosure of all patents, publications (including
published patent applications), and database entries
referenced in thi~ application are specifically incorporated
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WO 97145142 PCT/~JS97/(J9040
herein by re~erence in their entirety to the same extent as
if each such individual patent, publication, and database
entry were specifically and individually indicated to be
incorporated by reference.
It is to be understood, however, that the scope of the
present invention is not to be limited to the speci~ic
embodiments described above. The invention may be practiced
other than as particularly described and still be within the
scope of the accompanying claims.
