Note: Descriptions are shown in the official language in which they were submitted.
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RAPAMYCIN RESISTANT T CELLS AND
THERAPEUTIC USES THEREOF
FIELD OF THE INVENTION
T cell-based therapies for treatment of medical conditions such as cancer,
disease due to infectious disease organisms such as viruses, autoimmune
diseases and
Graft Versus Host Disease ("GVHD") are provided. In particular, enriched
populations
selected for Thl, Th2, Tcl or Tc2 functions are selected for and controlled
when
administered to a patient in vivo.
BACKGROUND OF THE INVENTION
Ongoing advances in solid organ and hematopoietic stem cell transplantation
(HSCT), including new immunosuppressive agents and improvements in
histo compatibility matching, organ procurement, and surgical techniques, are
gradually
improving the outcome of clinical transplantation (Hariharan et al, 2000. N.
Engl. J.
Med. 342:605-12). However, chronic allograft rejection remains the prime
determinant
of long-term graft survival (Paul. L. C., 1999, Kidney International 56:783-
793).
Furthermore, stem cell graft rejection typically limits the application of
allogeneic
HSCT to those patients having an HLA-matched sibling donor, which represents a
minority of all patients that might benefit from allogeneic HSCT therapy.
Tissue transplantation between genetically non-identical individuals results
in
immunological rejection of the tissue through T cell-dependent mechanisms. To
prevent allograft rejection, immunosuppressive agents such as calcineurin
phosphatase
inhibitors and glucocorticosteroids which directly or indirectly interfere
with IL-2
signaling are administered to transplant recipients (see, e.g., Morris, P. J.,
1991, Curr.
Opin. Immunol. 3:748-751; Sigal et al., 1992, Ann. Rev. Immunol. 10:519-560;
and
L'Azou et al., 1999, Arch. Toxicol . 73:337-345). The most commonly used
immunosuppressive agents today are the calcineurin inhibitors cyclosporin A
and
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FK506, which act relatively indiscriminately by impairing T cell receptor
("TCR")
signal transduction. A third primary immune suppression drug, rapamycin, which
has
recently received FDA approval for prevention of organ transplant rejection,
acts
through a distinct mechanism of inhibition of the protein mammalian target of
rapamycin (mTOR). The biological effect of these three immunosuppressive
agents is
short-lasting, and as such, transplant recipients normally require life-long
treatment of
immunosuppressive agents to prevent transplant rejection. As a result of the
long-term
nonspecific immunosuppression, these immunosuppressive agents have many
serious
adverse effects. For example, the administration of cyclosporin A or FK506 to
a
transplant recipient results in degenerative changes in renal tubules.
Transplant
recipients receiving long-term immunosuppressive treatment have a high risk of
developing infections and tumors. For example, patients receiving
immunotherapy are
at higher risk of developing lymphomas, skin tumors and brain tumors (see,
e.g.,
Fellstrom et al., 1993, Immunol. Rev. 134:83-98).
In addition to graft rejection, immune T cells also mediate the primary cause
of
lethality after allogeneic HSCT, graft-versus-host disease (GVHD). GVHD, which
is
primarily initiated by donor CD4+ T cells expressing a Thl cytokine phenotype
characterized by IL-2 and 11FN-y secretion, manifests clinically as damage to
the skin,
intestine, liver, and immune system. To reduce the incidence and severity of
GYM),
immune suppression therapy involving either cyclosporin A or FK506 is
typically
administered, often in combination with other immune suppression agents such
as
methotrexate. This immune suppression approach to the prevention of G'VI-ID is
problematic, as significant morbidity and mortality from GVHD still occurs,
and the
immune suppression therapy reduces the potency of the allogeneic T cell-
mediated
graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) effect, and
predisposes to
multiple viral, bacterial, and fungal infections.
An alternative to immunosuppressive agents for the prevention of allograft
rejection is the blockage of specific receptors involved in T cell
costimulation. T cell
activation requires both TCR-mediated signal transduction and simultaneously
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delivered costimulatory signals. These costimulatory signals are contributed,
in part, by
the activation of the costimulatory molecule CD28, which is expressed on
resting T
cells, by CD80 (B7-1) or CD86 (B7-2) expressed on antigen presenting cells
("APCs").
The activation of the costimulatory molecule CD40, which is expressed on APCs
(i.e.,
B cells, dendritic cells, and macrophages), by CD40 ligand ("CD4OL"), which is
expressed on activated T cells, contributes to the upregulation of T cell
activation by
inducing the expression of B7-1 and B7-2 on APCs and the production of certain
chemokines and cytokines such as IL-8, MlP-1-a, TNF-a, and IL-12 (Cella et
al., 1996,
J. Exp. Med. 184:747-752: and Caux et al., 1994, J. Exp. Med. 180:1263-1272).
The
CD40/CD4OL interaction also results in the differentiation of T cells to. T
helper ("Th")
type 1 cells in part due to the expression of cytokines such as IL-12 by
dendritic cells
and macrophages.
CTLA-4 is normally expressed as a membrane-bound receptor on T cells and,
similar to CD28, binds to B7-1 and B7-2 molecules on APCs; however, signaling
of T
cells via CTLA-4 downregulates T cells. The administration of soluble CTLA-41g
is
believed to prevent allograft rejection by competing with CD28 for B7-1 and B7-
2.
Soluble CTLA-41g has been administered to transplant recipients to disrupt the
CD28/B7 interaction so that T cell costimulation is blocked and allograft
rejection does
not occur (Zheng et al., 1999,1 Immunol. 162:4983-4990; Lenschow et al., 1996,
Ann.
Rev. Immunol. 14:233-258). Unfortunately, CTLA-41g has variable efficacy, and
typically does not prevent development of chronic rejection.
Anti-CD4OL (anti-CD154) monoclonal antibodies have also been administered
to transplant recipients to prevent allogaft rejection. These antibodies
function by
blocking the interaction of CD40 on antigen presenting cells (APC) and CD4OL
on
activated T cells. It has recently been shown that graft survival achieved
through the
use of anti-CD4OL monoclonal antibodies results in a significant inhibition of
Thl type
cytokines (i.e., IL-2, IL-12, TNF-a, and IFN-a), and an increase in the levels
of the Th2
type cytokines (i.e., IL-4, and IL-10) in the graft sections (Hancock et al.,
1996, Proc.
Natl. Acad. Sci. USA 93:13967-13972). Although the administration of anti-
CD4OL
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monoclonal antibodies has been shown to result in permanent graft survival
when given
to mice in combination with donor-specific spleen cells, adverse side effects
such as
coagulation have also been shown to be associated with the administration of
anti-
CD4OL monoclonal antibodies. Initial clinical trials in adult renal transplant
recipients
receiving anti-CD4OL monoclonal antibody plus glucocorticoids were halted
because of
thromboembofic complications though the extent to which thromoboembolism was
attributable to monoclonal antibodies versus non-specific factors in the
antibody
formulation is unclear (Kawai et al., 2000, Nature Med. 6:114; and Kirk et
al., 2000,
Nature Med. 6:114). Further, in the primate renal allograft study, concomitant
use of
mainstream immunosuppressive agents such as FK-506, methylprednisolone and
mycophenolate mofetil diminished the efficacy of CD4OL (CD154) mAb, though the
exact contribution of each of the individual drugs to this reduction in
efficacy was not
determined (Kirk, A. D., 1999, Nature Medicine 5:686-693.).
Immunocompromised patients lack a fully active and effective immune system,
and are vulnerable to infection by a host of opportunistic organisms that are
effectively
controlled in a healthy individual. Cancer patients and transplant recipients
are
especially vulnerable to these infections since their therapeutic regimen
often includes
radiation and chemotherapeutic agents, which compromise the immune system.
Immunodeficient patients, such as AIDS and SCID patients, are also at high
risk from
these opportunistic pathogens. In particular, patients undergoing bone marrow
transplantation (BMT) are severely immunocompromised until their immune
systems
reconstitute. During the period prior to reconstitution, these patients are
susceptible to
serious, and sometimes fatal, virus infections caused by normally benign
viruses such as
adenovirus, cytomegaloviru.s (CMV), and Epstein-Barr virus (EBV).
In a normal individual, recognition and destruction of virally infected cells
is
performed principally by CD8+ cytotoxic T lymphocytes (CTLs). The mounting of
a
CTL immune response reguires that the viral proteins undergo intracellular
processing
to peptide fragments. Selected peptides of defined length are subsequently
presented at
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the cell surface in conjunction with MHC class I molecules. This complex
provides the
first stimulatory signal recognized by the specific cytotoxic T lymphocyte.
Processing of antigens for presentation by class I NBC involves a complex
5 cellular process (Berzofsky and Berkower, Fundamental Immunology, Third
Edition,
Paul (ed.), Raven Press, Ltd.: New York, pp. 258-259 (1993). Unlike processing
of
exogenous antigen via endosomal pathways for presentation by class II MHC,
antigen
presented by class I MHC generally must be synthesized endogenously and
processed
by a nonendosomal pathway into peptides. However, exogenous antigens can enter
the
cytoplasm for processing by the nonendosomal pathway and presentation by class
I
MHC.
No satisfactory methods presently exist for monitoring whether a transplant
graft
is being accepted or rejected by a recipient. In general, signs of cellular
damage within
the transplant tissue can be assayed. Alternatively, for tissues such as
kidney or liver,
physiological function of the transplant tissue can be assayed. Often,
however, by the
time overt signs of either cellular damage or a decrease in physiological
function are
detected, the tissue graft is already beyond rescue. This is particularly true
in the case
of such organ transplants as heart transplants, with which the first overt
signs of
rejection are often complete failure of the heart's function. Similarly, in
the setting of
allogeneic HSCT, there exist no reliable method to detect GVHD prior to the
onset of
significant end-organ impairment; oftentimes, when GVHD does develop, the
donor
immune reaction is relatively mature, and can thereby be refractory to even
the most
potent immune suppression therapies available.
Accordingly, there is a need for improved, safer immunomodulatory treatments
that have long-lasting effects for the prevention of transplant rejection or
GVHD. In
particular, there is a need for treatments that are more specific and less
toxic than the
currently available therapeutic agents.
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In addition to graft rejection and GVHD, immune T cells of autologous or
allogeneic source may play a beneficial role in mediating anti-tumor effects
and anti-
infectious disease effects, including against viral, bacterial, and fungal
processes. This
T cell biology offers the possibility that adoptive transfer of ex vivo
generated T cell
populations might be utilized in the therapy of cancer or infection. However,
full
realization of this possibility is limited by a general inability to amplify a
potent
autologous immune response against cancer or infectious disease antigens in
vivo.
= Furthermore, immune T cell therapy in the allogeneic setting is limited
by allogeneic T
cell attack against normal host tissues, which is manifested as GVHD. In the
allogeneic
anti-tumor immune therapy setting, the graft-versus-leukemia (GVL) or graft-
versus-
tumor (GVT) effect is reduced by the immune suppression drugs cyclosporine A,
FK506, corticosteroids, and methotrexate that are utilized to prevent or treat
GVHD.
Avoidance of standard GVHD prevention or treatment agents through rapamycin
administration post-transplant will predictably facilitate improved GVL and
GVT
effects, resulting in improved rates of cancer cure.
SUMMARY OF THE INVENTION
We have now found methods and systems for generating highly enriched
Thl/Tcl and Th2/Tc2 functions in a subject. These methods and systems allow
for the
preferential selection of either Thl/Tcl or Th2/Th2 functions, administration
of the
selected functions to a patient and subsequent control of these functions once
administered.
More particularly, we have shown that the generation of these functions are
attained by the addition of an immune suppression drug, rapamycin. In addition
to
enhanced purity of T cell function, the T cells generated in rapamycin also
express
molecules that improve immune T cell function such as CD28 and CD62L.
In a preferred embodiment, the invention provides a method for selecting and
expanding enriched T cell subsets, comprising co-stimulating isolated T
lymphocytes in
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vitro by adding cytokines for selecting a T cell subset followed by expansion
of the T
cell subset in the presence of rapamycin or a rapamycin derivative compound.
In another preferred embodiment, the subset of T cells is selected by
culturing T
cell subsets with cytokines. Preferably, a Thl/Tcl subset of T lymphocytes is
selected
by culturing the lymphocytes in the presence of IL-12, and a Th2/Tc2 subset of
T
lymphocytes can be preferably generated by addition of IL-4.
Preferably, the T lymphocytes are co-stimulated. The co-stimulation of T
.. lymphocytes suitably comprises initiating one or more intracellular
signaling events.
For instance, the intracellular signaling events can be initiated by culturing
the T
lymphocytes with one or more antibodies, polypeptides, polymicleotides, small
molecules, or combinations thereof. Alternatively, the intracellular signaling
events are
initiated by solid phase anti-CD3 and anti-CD28 antibodies binding to their
respective
ligands.
In a preferred embodiment, a subset of T lymphocytes is selected based on the
disease to be treated. Preferably, the T lymphocytes are cultured with
cytokines and
rapamycin to select for either a Thl/Tcl or Th2/Tc2 subset. The desired subset
is
.. expanded and re-infused into a patient suffering from or susceptible to a
disease.
Preferably the T lymphocytes are autologous lymphocytes from a patient, and/or
they
can be derived from an allogeneic donor, which may represent an HLA-matched
sibling
donor, an HLA-matched donor from a non-family member, or a partially matched
family member, such as a haplo-identical donor (parent or child). For
instance, a
patient to be treated is suffering from, or is susceptible to, cancer or
infectious disease
organisms, such as a virus. The preferred rapamycin resistant subset of
lymphocytes
that are infused into the patient are the Thl/Tcl subset.
In another preferred embodiment, the patient to be treated is suffering from,
or is
susceptible to graft-versus-host-disease (GVHD). In this instance, in which a
patient
with cancer is to receive an allogeneic HSCT, the donor T cells of preference
would be
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rapamycin resistant T cells of Th2/Tc2 phenotype, which are typically
associated with
reduced GVHD. As such, T cells from the donor would be harvested prior to
transplantation, in vitro expanded in rapamycin or a rapamycin derivative
compound to
generate a Th2/Tc2 phenotype, and subsequently administered in the setting of
the
allogeneic HSCT to allow for a beneficial allogeneic T cell effect, such as
the mediation
of GVL or GVT effects, or the prevention of stem cell graft rejection, with
reduced
GVHD.
In another preferred embodiment, the selected T cell subsets are preferably
cultured in at least about 0.01 p.M rapamycin or a rapamycin derivative
compound,
more preferably the T cell subsets are cultured in at least about 0.11.1.M
rapamycin or a
rapamycin derivative compound, most preferably the T cell subsets are cultured
in at
least about or up to 1.0 [tM, 2.0 p,M, 4.0pM, 6.0 [tM, or 10.0 p.M rapamycin
or a
rapamycin derivative compound. It is preferred that the rapamycin resistant T
cell
subset populations express surface markers such as CD28, and preferably CD62L.
= In another preferred embodiment, methods for preventing and/or treating
GVHD
in a mammal, comprise, harvesting allogeneic cells from the transplant donor;
selecting
for a subset of rapamycin resistant CD44- T cells and CD8+ T cells in vitro;
and,
administering to the mammal rapamycin resistant T cells concomitantly with
rapamycin. The subset of rapamycin resistant T cells that are administered to
a
mammal is a Th2/Tc2 subset. Preferably, the rapamycin resistant Th2 cell
subset
express CD4 and the Tc2 cell subset express CD8. Most preferably, the
rapamycin
resistant Th2/Tc2 cells express CD62L and secrete cytokines, preferably type
II
cytokines. References to rapamycin resistant T cells in inclusive of T cells
that are
resistant to rapatnycin or a rapamycin derivative compound. Typically, T cells
that are
resistant to rapamycin or a rapamycin derivative compound also will be
resistant to
rapamycin.
In another preferred embodiment, rapamycin or a rapamycin derivative
compound is co-administered with rapamycin resistant T cells to a mammal in
need of
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therapy. The dosage of rapamycin or a rapamycin derivative compound to be
administered to the mammal will be tailored to each recipient based on serum
= monitoring of rapamycin drug levels. Because of the in vitro generation,
rapamycin
exposed T cells will have a selective advantage in such an in vivo state, the
achievement
of rapamycin levels at the higher side of the therapeutic range is desirable.
Preferably,
. rapamycin and any derivative, salt, ethers and the like can be used.
In another aspect, the invention provides methods for treating a patient
suffering
from or susceptible to cancer, comprises, harvesting autologous cells from the
mammal;
selecting for a subset of rapamycin resistant CD4+ T cells and CD8+ T cells in
vitro;
and, administering to the mammal rapamycin resistant T cells concomitantly
with
rapamycin or a rapamycin derivative compound. The subset of rapamycin
resistant T
cells for treating a patient suffering from or susceptible to cancer, is
preferably a
Thl/Tcl subset and the Thl/Tcl subset expresses CD62L. Preferably, the Thl
cells
express CD4 and the Tcl cells express CD8. Preferably, the rapamycin resistant
Thl/Tcicellular subset secretes type I cytokines.
In a further aspect, the invention provides a use of T cells as disclosed
herein for
the treatment of a targeted disease or disorder, including for the treatment
of undesired
cell proliferation including cancer, infectious diseases, reduction of graft
versus host
disease (GVHD) and the like.
In a yet further aspect, the invention provides a use for the preparation of a
therapeutic composition of T cells as disclosed herein for treatment of a
targeted disease
or disorder, including for the treatment of undesired cell proliferation
including cancer,
infectious diseases, reduction of graft versus host disease (GVHD) and the
like.
Preferred methods of the invention including identifying and/or selecting a
subject (e.g. a mammal, particularly a human) that is susceptible to or
suffering from a
condition as disclosed herein such as cancer, an infectious diseases,
reduction of graft
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versus host disease (GVHD) and the like; and thereafter administering to the
identified
and selected subject a T cell composition as disclosed herein.
The invention also includes pharmaceutical compositions that comprise
5 rapamycin resistant T cells optionally admixed with a pharmaceutically
acceptable
carrier and optionally packaged together with instructions (e.g. written) for
use of the
composition for a condition as disclosed herein.
The invention also includes rapamycin resistant T cells, e.g. as may be
10 obtainable as disclosed herein such as by treating a sample of isolated
T cells
(mammalian, preferably human) with rapamycin or a rapamycin derivative
compound
and selecting a subset of rapamycin resistant T cells particularly rapamycin
resistant
CD4+ T cells and/or CD8+ T cells, typically in vitro.
Other aspects of the invention are discussed infra.
DEFINITIONS
The following definitions are provided:
As used herein, the singular forms "a", "an" and "the" include plural
referents
unless the context clearly dictates otherwise.
. As used herein, the term "infectious agent" refers to an organism
wherein
growth/multiplication leads to pathogenic events in humans or animals.
Examples of
such agents are: bacteria, fungi, protozoa and viruses.
As used herein, a "pharmaceutically acceptable" component is one that is
suitable for use with humans and/or animals without undue adverse side effects
(such as
toxicity, irritation, and allergic response) commensurate with a reasonable
benefit/risk
ratio.
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As used herein, the term "safe and effective amount" refers to the quantity of
a
component which is sufficient to yield a desired therapeutic response without
undue
adverse side effects (such as toxicity, irritation, or allergic response)
commensurate
with a reasonable benefit/risk ratio when used in the manner of this
invention. By
"therapeutically effective amount" is meant an amount of a compound of the
present
invention effective to yield the desired therapeutic response. For example, an
amount
effective to delay the growth of or to cause a cancer, either a sarcoma or
lymphoma, or
to shrink the cancer or prevent metastasis. The specific safe and effective
amount or
therapeutically effective amount will vary with such factors as the particular
condition
being treated, the physical condition of the patient, the type of mammal or
animal being
treated, the duration of the treatment, the nature of concurrent therapy (if
any), and the
specific formulations employed and the structure of the compounds or its
derivatives.
As used herein, a "pharmaceutical salt" include, but are not limited to,
mineral
or organic acid salts of basic residues such as amines; alkali or organic
salts of acidic
residues such as carboxylic acids. Preferably the salts are made using an
organic or
inorganic acid. These preferred acid salts are chlorides, bromides, sulfates,
nitrates,
phosphates, sulfonates, formates, tartrates, maleates, malates, citrates,
benzoates,
salicylates, ascorbates, and the like. The most preferred salt is the
hydrochloride salt.
As used herein, "cancer" refers to all types of cancer or neoplasm or
malignant
tumors found in mammals, including, but not limited to: leukemias, lymphomas,
melanomas, carcinomas and sarcomas. Examples of cancers are cancer of the
brain,
breast, pancreas, cervix, colon, head and neck, kidney, lung, non-small cell
lung,
melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.
The term "leukemia" refers broadly to progressive, malignant diseases of the
blood-
forming organs and is generally characterized by a distorted proliferation and
development of leukocytes and their precursors in the blood and bone marrow.
Leukemia is generally clinically classified on the basis of (1) the duration
and character
of the disease-acute or chronic; (2) the type of cell involved; myeloid
(myelogenous),
lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in
the
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number of abnormal cells in the blood-leukemic or aleukemic (subleukemic).
Accordingly, the present invention includes a method of treating leukemia,
and,
preferably, a method of treating acute nonlymphocytic leukemia, chronic
lymphocytic
leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute
promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a
leukocythemic
leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic
myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic
leukemia,
Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic
leukemia,
histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic
leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,
lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast
cell
leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic
leukemia,
myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia,
myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic
leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia,
stem cell
leukemia, subleukemic leukemia, and undifferentiated cell leukemia.
The term "sarcoma" generally refers to a tumor which is made up of a substance
like the embryonic connective tissue and is generally composed of closely
packed cells
embedded in a fibrillar or homogeneous substance. Examples of sarcomas which
can
be treated with the compositions disclosed herein, and optionally a
potentiator and/or
chemotherapeutic agent include, but not limited to a chondrosarcoma,
fibrosarcoma,
lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma,
adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic
sarcoma,
botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma,
Wilms'
tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial
sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma,
Hodgkin's
sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic
sarcoma
of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,
Kaposi's
sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant
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mesenchymoma sarcoma, parosteal sarcoma, reficulocytic sarcoma, Rous sarcoma,
serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
The term "melanoma" is taken to mean a tumor arising from the melanocytic
system of the skin and other organs. Melanomas which can be treated with the
compositions disclosed herein, and optionally a potentiator and/or another
chemotherapeutic agent include but not limited to, for example, acral-
lentiginous
melanoma, arnelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma,
S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna
melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and
superficial spreading melanoma.
The term "carcinoma" refers to a malignant new growth made up of epithelial
cells tending to infiltrate the surrounding tissues and give rise to
metastases.
Carcinomas which can be treated with the compositions disclosed herein, and
optionally
a potentiator and/or a chemotherapeutic agent include but not limited to, for
example,
acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic
carcinoma,
carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma,
alveolar
cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid
carcinoma,
basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar
carcinoma,
bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma,
chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,
cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical
carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum,
embryonal
carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale
adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,
gelatiniform
carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma
gigantocellulare,
glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid
carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline
carcinoma,
hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ,
intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma,
=
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=
Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma,
carcinoma
lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma
medullare,
medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,
carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,
carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal
carcinoma, oat cell carcinoma, carcinoma os ificans, osteoid carcinoma,
papillary
carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell
carcinoma,
pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma,
carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma
scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma,
solanoid
carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma
spongiosum,
squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma
telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,
carcinoma
tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.
Additional cancers which can be treated with the methods and compositions
according to the invention include, for example, Hodgkin's Disease, Non-
Hodgkin's
Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung
cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,
small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer,
malignant
pancreatic insulanoma, malignant carcinoid, urinary bladder cancer,
premalignant skin
lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma,
esophageal
cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer,
endometrial cancer, adrenal cortical cancer, and prostate cancer.
"Diagnostic" or "diagnosed" means identifying the presence or nature of a
pathologic condition. Diagnostic methods differ in their sensitivity and
specificity. The
"sensitivity" of a diagnostic assay is the percentage of diseased individuals
who test
positive (percent of "true positives"). Diseased individuals not detected by
the assay are
"false negatives." Subjects who are not diseased and who test negative in the
assay, are
termed "true negatives." The "specificity" of a diagnostic assay is 1 minus
the false
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positive rate, where the "false positive" rate is defined as the proportion of
those
without the disease who test positive. While a particular diagnostic method
may not
provide a definitive diagnosis of a condition, it suffices if the method
provides a
positive indication that aids in diagnosis.
5
The terms "patient" or "individual" are used interchangeably herein, and
refers
to a mammalian subject to be treated, with human patients being preferred: In
some
cases, the methods of the invention find use in experimental animals, in
veterinary
application, and in the development of animal models for disease, including,
but not
10 limited to, rodents including mice, rats, and hamsters; and primates.
"Sample" is used herein in its broadest sense. A sample comprising
polynucleotides, polypeptides, peptides, antibodies and the like may comprise
a bodily
fluid; a soluble fraction of a cell preparation, or media in which cells were
grown; a
15 chromosome, an organelle, or membrane isolated or extracted from a cell;
genomic
DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a
substrate; a
cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.
"Treatment" is an intervention performed with the intention of preventing the
development or altering the pathology or symptoms of a disorder. Accordingly,
"treatment" refers to both therapeutic treatment and prophylactic or
preventative
measures.. Those in need of treatment include those already with the disorder
as well as
those in which the disorder is to be prevented. In tumor (e.g., cancer)
treatment, a
therapeutic agent may directly decrease the pathology of tumor cells, or
render the
tumor cells more susceptible to treatment by other therapeutic agents, e.g.,
radiation
and/or chemotherapy. As used herein, "ameliorated" or "treatment" refers to a
symptom which approaches a normalized value (for example a value obtained in a
healthy patient or individual), e.g., is less than 50% different from a
normalized value,
preferably is less than about 25% different from a normalized value, more
preferably, is
less than 10% different from a normalized value, and still more preferably, is
not
significantly different from a normalized value as determined using routine
statistical
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16
tests. For example, amelioration or treatment of a patient suffering from an
infectious
disease organism, such as for example, Hepatitis B Virus, may be determined by
a
decrease of viral particles in a sample taken from a patient, as measured by,
for
example, a decrease in plaque forming units (p.f.u.).
The "treatment of neoplastic disease or neoplastic cells", refers to an amount
of
rapamycin resistant T cells, described throughout the specification and in the
Examples
which follow, capable of invoking one or more of the following effects: (1)
inhibition,
to some extent, of tumor growth, including, (i) slowing down and (ii) complete
growth
arrest; (2) reduction in the number of tumor cells; (3) maintaining tumor
size; (4)
reduction in tumor size; (5) inhibition, including (i) reduction, (ii) slowing
down or (iii)
complete prevention, of tumor cell infiltration into peripheral organs; (6)
inhibition,
including (i) reduction, (ii) slowing down or (iii) complete prevention, of
metastasis; (7)
enhancement of anti-tumor immune response, which may result in (i) maintaining
tumor
size, (ii) reducing tumor size, (iii) slowing the growth of a tumor, (iv)
reducing, slowing
or preventing invasion and/or (8) relief, to some extent, of the severity or
number of one
or more symptoms associated with the disorder.
"Treatment of an individual suffering from an infectious disease organism"
refers to a decrease or elimination of the disease organism from an
individual. For
example, a decrease of viral particles as measured by plaque forming units or
other
automated diagnostic methods such as ELISA, etc., may be used to monitor
efficacy of
treatment.
"Treatment of an individual suffering from graft-versus-host-disease or GVHD"
refers to a decrease or cessation of symptoms associated with GVITD. For
example, an
amelioration of lacy, livid maculopapular rash, jaundice, diarrhea, abdominal
pain,
hepatosplenomegaly, alopecia, bullae, desquamation of skin. Treatment or
amelioration
of GVHD results in clinical downgrading of the disease. For example, acute
GVHD,
which typically occurs in the first 100 days post-transplant, may be
classified according
to degree or "stage" of damage in the main target organs of GVHD, the skin,
intestine,
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17
and liver. For example, liver GVHD is staged from none (stage 0; bilirubin <2
mg/di)
to severe (stage 4; bilirubin > 15 mg/di) based on serum bilirubin level. Skin
GVHD is
staged based upon the percent body surface area that the rash involves, with
stage 0
having no rash and stage 4 having rash of up to 100 % body surface area with
bullae or
desquamation. Intestinal GVHD is staged based upon the volume of daily liquid
stool
output, with stage 0 being no diarrhea and stage 4 being > 1500 ml liquid
stool per day
with abdominal pain or ileus. Chronic GVHD, which typically occurs after day
100
post-transplant and can last several years post-transplant, is typically
scored based upon
number of organ sites that the chronic GVHD involves (limited chronic GVHD,
one
site; extensive chronic GVHD, two or more sites). Chronic GVHD involves the
same
organs as acute GVHD, but in addition, chronic GVHD may also affect the mucous
glands in the eyes, salivary glands in the mouth, and glands that lubricate
the stomach
lining and intestines.
As used herein, "an ameliorated symptom" or "treated symptom" refers to a
symptom which is approaches a normalized value, e.g., is less than 50%
different from
a normalized value, preferably is less than about 25% different from a
normalized
value, more preferably, is less than 10% different from a normalized value,
and still
more preferably, is not significantly different from a normalized value as
determined
using routine statistical tests.
"Cells of the immune system" or "immune cells" as used herein, is meant to
include any cells of the immune system that may be assayed, including, but not
limited
to, B lymphocytes, also called B cells, T lymphocytes, also called T cells,
natural killer
(NK) cells, natural killer T (NKT) cells, lymphokine-activated killer (LAIC)
cells,
monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets,
Langerhans
cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor-
infiltrating
(TIL) cells, gene modified immune cells including hybridomas, drug modified
immune
=
cells, and derivatives, precursors or progenitors of the above cell types.
=
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18
"Immune effector cells" refers to cells capable of binding an antigen and
which
mediate an immune response selective for the antigen. These cells include, but
are not
limited to, T cells (T lymphocytes), B cells (B lymphocytes), monocytes,
macrophages,
natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), for example CTL
lines,
CTL clones, and CTLs from tumor, inflammatory, or other infiltrates.
"Immune related molecules" refers to any molecule identified in any immune
cell, whether in a resting ("non-stimulated") or activated state, and includes
any
receptor, ligand, cell surface molecules, nucleic acid molecules,
polypeptides, variants
and fragments thereof.
"T cells" or "T lymphocytes" are a subset of lymphocytes originating in the
thymus and having heterodimeric receptors associated with proteins of the CD3
complex (e.g., a rearranged T cell receptor, the heterodimeric protein on the
T cell
surfaces responsible for antigen/AMC specificity of the cells). T cell
responses may be
detected by assays for their effects on other cells (e.g., target cell
killing, activation of
other immune cells, such as B-cells) or for the cytokines they produce_
As used herein, "allogeneic" is used to refer to immune cells derived from non-
self major histocompatibility complex donors. IILA haplotypes/allotypes vary
from
individual to individual and it is often helpful to determine the individual's
HLA type.
The HLA type may be determined via standard typing procedures.
As will be recognized by those in the art, the term "host compatible" or
"autologous" cells means cells that are of the same or similar haplotype as
that of the
subject or "host" to which the cells are administered, such that no
significant immune
response against these cells occurs when they are transplanted into a host.
As used herein, "partially-mismatched HLA", refers to HLA types that are
between about 20% to about 90% compatible to the host's HLA type.
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"CD4" is a cell surface protein important for recognition by the T cell
receptor
of antigenic peptides bound to MHC class II molecules on the surface of an
APC. Upon
activation, naive CD4 T cells differentiate into one of at least two cell
types, 'Thl cells
and 'Th2 cells, each type being characterized by the cytokines it produces.
"Thl cells"
are primarily involved in activating macrophages with respect to cellular
immunity and
the inflammatory response, whereas "T1i2 cells" or "helper T cells" are
primarily
involved in stimulating B cells to produce antibodies (humoral immunity). -CD4
is the
receptor for the human immunodeficiency virus (HIV). Effector molecules for
Thl
cells include, but are not limited to, IFN-y, GM-CSF, TNF-a, CD40 ligand, Fos
ligand,
IL-3, TNF-P, and IL-2. Effector molecules for Th2 cells include, but are not
limited to,
IL-4, IL-5, CD40 ligand, IL-3, GS-CSF, IL-10, TGF-I3, and eotaxin. Activation
of the
Thl type cytokine response can suppress the Th2 type cytokine response, and
reciprocally, activation of the Th2 type cytokine response can suppress the
Thl type
response.
A "chemokine" is a small cytokine involved in the migration and activation of
cells, including phagocytes and lymphocytes, and plays a role in inflammatory
responses.
A "cytokine" is a protein made by a cell that affect the behavior of other
cells
through a "cytokine receptor" on the surface of the cells the cytokine
effects. Cytokines
manufactured by lymphocytes are sometimes termed "lymphokines." Cytokines are
also characterized as Type I (e.g. IL-2 and LFN-y) and Type II (e.g. IL-4 and
IL-10).
By the term "modulate," it is meant that any of the mentioned activities, are,
e.g., increased, enhanced, increased, agonized (acts as an agonist), promoted,
decreased,
reduced, suppressed blocked, or antagonized (acts as an agonist). Modulation
can
increase activity more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold,
etc., over
baseline values. Modulation can also decrease its activity below baseline
values.
=
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An "epitope", as used herein, is a portion of a polypeptide that is recognized
(i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor.
Epitopes
may generally be identified using well known techniques, such as those
summarized in
Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and
references
5 cited
therein. Such techniques include screening polypeptides derived from the
native
polypeptide for the ability to react with antigen-specific antisera and/or T-
cell lines or
clones. An epitope of a polypeptide is a portion that reacts with such
antisera and/or T-
cells at a level that is similar to the reactivity of the full length
polypeptide (e.g., in an
ELISA and/or T-cell reactivity assay). Such screens may generally be performed
using
10 methods
well known to those of ordinary skill in the art, such as those described in
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory,
1988. B-cell and T-cell epitopes may also be predicted via computer analysis.
"Substrate" refers to any rigid or semi-rigid support to which nucleic acid
15
molecules or proteins are bound and includes membranes, filters, chips,
slides, wafers,
fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing,
plates,
polymers, and microparticles with a variety of surface forms including wells,
trenches,
pins, channels and pores.
20
"Immunoassay" is an assay that uses an antibody to specifically bind an
antigen
(e.g., a marker). The immunoassay is characterized by the use of specific
binding
properties of a particular antibody to isolate, target, and/or quantify the
antigen.
As used herein, the term "transplant" includes any cell, organ, organ system
or
tissue which can elicit an immune response in a recipient subject mammal. In
general,
therefore, a transplant includes an allograft or a xenograft cell, organ,
organ system or
tissue. An allograft refers to a graft (cell, organ, organ system or tissue)
obtained from
a member of the same species as the recipient. A xenograft refers to a graft
(cell, organ,
organ system or tissue) obtained from a member of a different species as the
recipient.
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The term "immune rejection," as used herein, is intended to refer to immune
responses involved in transplant rejection, as well as to the concomitant
physiological
result of such immune responses, such as for example, interstitial fibrosis,
chronic graft
artheriosclerosis, or vasculitis. The term "immune rejection," as used herein,
is also
intended to refer to immune responses involved in autoimmune disorders, and
the
concomitant physiological result of such immune responses, including T cell-
dependent
infiltration and direct tissue injury; T cell-dependent recruitment and
activation of
macrophages and other effector cells; and T cell-dependent B cell responses
leading to
autoantibody production.
The term "transplant rejection," as used herein, refers to T cell-mediated
rejection of transplant cells, organs, organ systems or tissue. In general,
such transplant
rejection generally includes accelerated, acute and chronic rejection. It is
intended that
the term, as used herein, also refer to GVHD, and the physiological results of
such a
disorder.
The term "reducing immune rejection," is meant to encompass prevention or
inhibition of immune rejection, as well as delaying the onset or the
progression of
immune rejection. The term is also meant to encompass prolonging survival of a
transplant in a subject mammal, or reversing failure of a transplant in a
subject. Further,
the term is meant to encompass ameliorating a symptom of an immune rejection,
including, for example, ameliorating an immunological complication associated
with
immune rejection, such as for example, interstitial fibrosis, chronic graft
atherosclerosis,
or vasculitis. The term is also meant to encompass induction of tolerance in a
subject
mammal that has undergone a transplant.
The term "tolerance," as used herein, refers to a state wherein the immune
system of a transplant recipient subject mammal is non-responsive to the
transplant.
This state is considered donor transplant-specific, and, as such, is
distinguished from
nonspecific immunosuppression. Operatively, the term as used herein, refers to
permanent acceptance of a graft without ongoing immunosuppression, wherein,
for
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22
example, challenge with a second graft of donor origin (especially when the
second
graft is of the same tissue as the first graft) should be accepted, and
challenge with a
third party graft should be rejected.
The term "autoimmune rejection," as used herein, refers to immune responses
involved in autoimmune disorders, and the concomitant physiological result of
such
immune responses.
The term "activated T cell," as used herein, refers to a T cell that expresses
antigens indicative of T-cell activation (that is, T cell activation markers).
Examples of
T cell activation markers include, but are not limited to, CD25, CD26, CD30,
CD38,
CD69, CD70, CD71, ICOS, OX-40 and 4-1BB. The expression of activation markers
can be measured by techniques known to those of skill in the art, including,
for
example, western blot analysis, northern blot analysis, RT-PCR,
immunofluoresceace
assays, and fluorescence activated cell sorter (FACS) analysis.
The term "resting T cell," as used herein, refers to a T cell that does not
express
T-cell activation markers. Resting T cells include, but are not limited to, T
cells which
are CD25-, CD69-, ICOS", SLAM-, and 4-1BB". The expression of these markers
can be
measured by techniques known to those of skill in the art, including, for
example,
western blot analysis, northern blot analysis, RT-PCR, immunofluorescence
assays, and
fluorescence activated cell sorter (FACS) analysis.
The term "T cell activator," as used herein, refers to any compound or factor
that
is a T cell receptor stimulatory factor, that is, induces T cell receptor
signaling.
Preferably, the compound or factor also induces co-stimulatory pathways. Non-
limiting
examples of T cell activators include, but are not limited to, anti-CD3,
antibodies
(preferably monoclonal antibodies) either alone or in conjunction with anti-
CD28
antibodies (preferably monoclonal antibodies), or mitogens such as, for
example,
phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA) or
concanavalin-A
(Con-A).
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BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graph illustrating the differential effect of CSA, FK506, and
rapamycin on the generation of murine CD4+ "Thl and Th2 cells.
Figure 2 is a graph showing supernatant ELISA results for the type II
cytokines
IL-4 and IL-10.
=
Figure 3 is a graph showing that addition of CSA, and in particular, FK506,
results in Thl cells with significantly diminisked capacity for both IL-2 and
IFN-y
secretion.
Figure 4 is a graph showing that at a high dose of rapamycin, co-stimulation
and
cytokine supplementation allowed for the expansion of either Thl or Th2
subsets
without any apparent reduction in numbers of CD4-expressing cells.
Figure 5 is a graph showing that Thl cells in each of the rapamycin
concentrations had similarly high secretion of both IL-2 and IFN-y.
Figure 6 is a graph showing that Th2 cells propagated in the 0.1 M rapamycin
concentration had preservation of capacity for secretion of the type II
cytokines
IL-5, and IL-10. .
Figure 7 is a graph showing Th2, and Tial cell expansion was greatly reduced
relative to CD3, CD28 co-stimulated control Tial/Th2 cultures.
Figure 8 is a graph showing CD3, CD2S generated Th2 cell expansion from day
0 to 6 of culture at rapamycin concentrations ramging from 0.1 M to 10.0 M.
Figure 9 is a graph showing the increas in numbers of CD4+ expressing cells in
the high dose rapamycin cultures after day 6.
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Figure 10 is a graph showing that at the highest rapamycin concentration, the
Th2 cells had an enhanced Th2 polarity on the basis of abrogation of
contaminating IL-
2 secretion and modest reduction in IFNI secretion.
Figure 11 is a graph showing Thl or Th2 cells re-stimulated in the presence of
0.111.M rapamycin, and 101.1.M rapamycin.
Figure 12 is a graph showing that CD28 was greatly increased on the CD4+ cells
that were propagated in high dose (10 ply!) rapamycin.
Figure 13 is a graph showing that Th2 cells expanded in high dose rapamycin,
had an increased expression of CD62L.
Figure 14 is a graph showing shows median cell volume changes during Thl ,
Th2, Tcl, or Tc2 expansion in the presence or absence of either 0.1 or 10.0
ft.M
rapamycin.
Figure 15 is a graph showing CD8+ Tcl/Tc2 expansion after CD3, CD28 co-
stimulation in the presence of 0.1 [tM and 10 M of rapamycin.
Figure 16 is a graph showing CTL assays using Tc2 effectors generated in the
presence or absence of rapamycin.
Figure 17 is a graph showing Tcl cells expanded in the high dose of rapamycin
lost their capacity for 1FN-7 secretion and had reduced capacity for IL-2
secretion.
Figure 18 is a graph showing that CD8+ cell expansion in high-dose rapamycin
was associated with a more naive T cell phenotype, as evidenced by increased
CD62L
expression.
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Figure 19 is a graph showing that recipients of allogeneic splenic T cell
inocula
had a significant number of alloreactive CD8+ T cells capable of IFN-y
secretion at day
7 post-BMT.
5 Figure 20 is a graph showing that recipients of splenic CD4+ and CD8+
T cells
underwent weight loss consistent with acute GVHD.
Figure 21 is a graph showing survival results in recipients of Th2 cells
generated
under either low dose or high dose rapamycin.
Figure 22 is a graph showing that administration of rapamycin-generated Th2
cells and in vivo rapamycin resulted in a greater number of Th2 cells in the
day +7
spleens than cell administration and CSA or vehicle administration.
Figure 23 shows the results of CD4+ cell expansion from n=4 normal donors
either without (left panel) or with rapamycin (1.0 i.t.M; right panel).
Figure 24 is a graph showing the growth curves of CD4+ cell in the presence of
rapamycin.
Figure 25 is a graph showing CD3, CD28 re-stimulation of rapamycin-generated
Th2 cells with or without 0.01 04 rapamycin.
Figure 26 is a graph showing that cells propagated under Th2 conditions and
rapamycin had an increased Th2 cytokine purity, as evidenced by reduction in
capacity
for 1F1\1-y secretion.
Figure 27 is a graph showing that rapamycin-generated Th2 cells had an
increased capacity for secretion of the type II cytokines 1L-4 and IL-13.
=
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26
Figure 28 is a graph showing that rapamycin-generated Th2 cells had imcreased
expression of CD62L relative to control Th2 cells; the increase in CD62L was
most
marked in Th2 cultures that were continuously exposed to rapamycin.
Figure 29 is a graph showing rapamycin-generated Th2 cells indeed hard an
increased capacity for rhodamine dye exclusion.
Figure 30 is a graph showing that rapamycin generated Th2 cells increased
MDR function.
Figure 31 is a graph showing purified naïve or memory human CD4 cells co-
stimulated either with or without rapamycin.
DETAILED DESCRIPTION OF THE INVENTION
We have now found new methods and T cell based systems in the field_ of
immune therapy against cancers, infectious diseases, reduction of graft versus
host
disease (GVHD) and the like.
As shown in the examples which follow, we have demonstrated that rap amycin
generated T cells were selectively resistant to the inhibitory effects of
rapamycin in
vivo. In this strategy, in vivo administration of rapamycin-resistant Thl,
Th2, ricl or
Tc2 cells with concomitant administration of rapamycin drug inhibits non-
cultured T
cells that may not possess the desired function and at the same time allow
preferential
expansion of the in vitro cultured T cell of optimal function. This new immune
therapy
strategy greatly amplifies the in vivo effects of immune therapeutic T cells
of a_ selected
function.
Preferred methods of T cell subset generation are growth in in vitro T ell
culture conditions comprising the immune suppression drug rapamycin to genrate
rapamycin-resistant cells having a desiredillymphocyte function, such as for
example,
T helper cells (Thl or Th2 function) and/or cytotoxic T cells (Tcl or Tc2
function).
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Preferably, the desired T cell subset is selected for, activated, and co-
administered with
rapamycin to a patient in need of therapy. For example, patients with cancer
or
infectious disease, where a Thl or Tcl cell would provide optimal T cell
function, T
cells are harvested from the patient, activated and expanded in the identified
conditions
with rapamycin to generate rapamycin-resistant Thl or Tcl cells, then re-
infused to the
patient with simultaneous administration of rapamycin drug. This expands the
therapeutic T cell against cancer or infection, and inhibits any non-cultured
T cell in the
body that might otherwise adversely affect the therapeutic T cell response.
Alternatively, in cases of GVHD prevention or treatment, or therapy of
autoimmune disease, T cells would be harvested from the patient, expanded in
conditions containing rapamycin to generate, preferably, rapamycin resistant
Th2 or
Tc2 cells, and then re-infused to the patient with simultaneous administration
of the
rapamycin drug. Preferably, the immune therapeutic T cells are expanded to
prevent
GVHD or treat autoimmune disease, and inhibit any non-cultured T cell in the
body that
may otherwise promote GVHD or autoimmune disease.
As used herein, "therapeutic T cell" refers to the rapamycin resistant T cell
subsets, for example, Thl/Tcl and Th2/Tc2.
As used herein, the term "rapamycin" refers to rapamycin and/or structurally
modified rapamycin compounds (such structurally modified rapamycin compounds
sometimes referred to herein as rapamycin derivatives). The unmodified
compound is
the macrolide antibiotic that can be produced by Streptomyces hyhoscopius
having the
structure as disclosed e.g. in J.B. McAlpine et al. J. Antibiotics (1991)
44:688 and S.L.
Schrieber et al., J. Am. Chem. Soc., (1991) 113:7433.
That unmodified rapamycin is in general a preferred rapamycin compound and
is the compound referred to in the examples which follow. Additional suitable
and
preferred structurally modified rapamycin compounds (or rapamycin derivatives)
can be
identified through simple testing. For instance, suitable rapamycin
derivatives for
CA 02529244 2013-04-19
28
identifying resistant cells can be evaluated using in vitro assays as
described in detail in
the Examples which follow. Briefly, cells such as for example T cells are
stimulated
and cultured in the presence of cytokines till the cells reach a desired
concentration,
such as for example 2 x 106 cells. Candidate rapamycin derivative compounds
are
added to the cell culture in varying concentrations such as at least about
0.004 p.M up to
about 0.02 pM. Viable cells as determined by microscopic observations or dye
exclusion assays are counted by a Multi-Sizer Instrument (Coulter), and the
cellular
expansion, for example, CD4 expansion is plotted, as shown in Figure 1. If a
candidate
rapamycin compound results in decrease in cell populations as compared to
normal
controls and controls incubated with rapamycin, then the compound is
considered
suitable for use in the methods and compositions of the invention. Candidate
rapamycin
compounds, include, but are not limited to, tetrazole containing rapamycin
analogs
disclosed in US Patent No. 6,329,386; acyl derivatives of rapamycin disclosed
in US
Patent No. 4,316,885; mono- and di-ester derivatives of rapamycin; 27-oximes
of
rapamycin; 42-oxo analog of rapamycin; bicyclic rapamycins disclosed in US
Patent
No; 5,120,725; rapamycin dimers disclosed in US patent No. 5,120,727; silyl
ethers,
arylsulfonates and sulfamates of rapamycin disclosed in US Patent No; 5,120,
842;
sulfonates disclosed in US patent No.: 5,177,203; mono- and di-acyl
derivatives of
rapamycin; water soluble rapamycin compounds disclosed in US Patent No.:
4,650,803;
hydrogenated rapamycin derivatives such as those disclosed in US Patent No.
5,023,262.
The number of cells of desired function, administered to the patient will vary
depending on various factors such as the disease or condition to be treated,
the
condition of the patient, which should be determined via consideration of all
appropriate
factors by the practitioner. Preferably, however, about 1x106 to about 1x1012
cells of
desired function are administered to a patient, more preferably about lx108 to
about
lx10" cells of desired function are administered to a patient, and even more
preferably,
about lx109 to about lx10-1 cells of desired function are administered to an
adult
human. Most preferred, the number of cells administered are about 2.5x 109
cells.
These amounts will vary depending on the age, weight, size, condition, sex of
the
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29
patient, the type of disease to be treated, the route of administration,
whether the
treatment is regional or systemic, and other factors. Those skilled in the art
should be
readily able to derive appropriate dosages and schedules of administration to
suit the
specific circumstance and needs of the patient.
Methods of re-introducing cellular components are known in the art and include
procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et
al. and
U.S. Pat. No. 4,690,915 to Rosenberg. For example, administration of CD8+
cells of
Thl/Tcl function via intravenous infusion is appropriate.
T cells from patients are, preferably activated ex vivo, either by soluble
anti-CD3
antibody, or most preferably, are co-activated by using anti-CD3 and anti-CD28
monoclonal antibodies, either by soluble or immobilized on a solid support. A
preferred solid support are plastics, or any surface upon which antibodies can
be
immobilized, or beads, such as, for example, Dynal beads. Particularly
preferred
surface antigens for optimal co-stimulation are CD3 and/or CD28 and particular
secreted cytokines (like IL-2, IL-4, IL-10, IFN-y).
The present invention is also useful as the activation is conducted in vitro
and
the activated helper or cytotoxic T-cells are reintroduced into the patient.
Activation is
achieved by the crosslinking of the T cell receptor complex (anti-CD3 and anti-
CD28
antibodies) which increase the effectiveness of the activation. Cross linking
of the TCR
with anti-CD3 triggers a signaling cascade resulting in T cell proliferation,
cytokine
synthesis, and immune responses. However, optimal activation and proliferation
requires costimulation of CD28 receptors on T cells with anti-CD28 or B7
molecules
(CD80 and CD86). These interactions enhance proliferation and stabilization of
mRNAs for IL-2, IFN-y, TNF:a ,and granulocyte-macrophage colony stimulating
factor
(GM-CSF). Costimulation of the CD28 receptor also leads to enhanced production
of
beta chemokines RANTES, and MIP I -a. The enhanced secretion of chemokines at
the
tumor site may augment recruitment of effector cells.
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The presentation of antigen to CD8 T-cells is accomplished by major
histocompatibility complex (MEC) molecules of the Class I type. The major
histocompatibility complex (MHC) refers to a large genetic locus encoding an
extensive
family of glycoproteins which play an important role in the immune response.
The
5 MHC genes, which are also referred to as the HLA (human leukocyte
antigen) complex,
are located on chromosome 6 in humans.. The molecules encoded by MHC genes are
present on cell surfaces and are largely responsible for recognition of tissue
transplants
as "non-self'. Thus, membrane-bound MHC molecules are intimately involved in
recognition of antigens by T-cells.
MHC products are grouped into three major classes, referred to as I, II, and
IIL
T-cells that serve mainly as helper cells express CD4 and primarily interact
with Class
II molecules, whereas CD8-expressing cells, which mostly represent cytotoxic
effector
cells, interact with Class I molecules.
As used herein, the term "transplantation antigen" is used to refer to
antigenic
molecules that are expressed on the cell surface of transplanted cells, either
at the time
of transplantation, or at some point following transplantation. Generally
these antigenic
molecules are proteins and glycoproteins. The primary transplantation antigens
are
products of the major histocompatibility complex (MHC), located on chromosome
6 in
humans. The human MHC complex is also called the human leukocyte antigen (HLA)
complex. MHC antigens are divided into MHC class I antigens (in humans, this
class
includes HLA-A, -B, and -C antigens) and MHC class II antigens (in humans,
this class
includes HLA-DP, -DQ, and -DR antigens). Thus, the terms "MHC-II antigens",
"MHC class II antigens", and "MHC class II transplantation antigens" are used
interchangeably herein to refer to the class of proteins, which in humans,
includes HLA-
DP, -DQ and -DR antigens. While the terms "MHC class II genes" and "MHC-II
genes" are used interchangeably herein to refer to the genes which encode the
MHC
class II transplantation antigens. The term "MHC-II" is used herein to refer
to the gene
locus which encodes the MHC class II transplantation antigens, as well as the
group of
proteins encoded by that locus. Transplantation antigens also include cell
surface
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=
molecules other than MHC class I and II antigens. These antigens include the
following: (1) the ABO antigens involved in blood cell recognition; (2) cell
adhesion
molecules such as ICAM, which is involved in leukocyte cell-cell recognition;
and (3)
f32 -microglobulin, a polypeptide associated with the 44 kd heavy chain
polypeptide that
comprises the HLA-I antigens but is not encoded by the MHC complex. Even in
those
cases where the most complete HLA matching is correctly done, G'VHD frequently
results. It has been suggested that GVHD results, in those instance, from
alloaggression
due to minor histocompatibility antigen differences for which many authors
have
suggested the depletion of donor T cells as a means to avoid GVHD. Although
this
strategy of T cell depletion may avoid GVHD, such patients are at increased
risk for
tumor relapse, infection, and graft rejection, and as such, T cell depletion
has both
positive and negative consequences.
Class I molecules are membrane glycoproteins with the ability to bind peptides
derived primarily from intracellular degradation of endogenous proteins.
Complexes of
MHC molecules with peptides derived from viral, bacterial and other foreign
proteins
comprise the ligand that triggers the antigen responsiveness of T-cells. In
contrast,
complexes of MHC molecules with peptides derived from normal cellular products
play
a role in "teaching" the T-cells to tolerate self-peptides, in the thymus.
Class I
molecules do not present entire, intact antigens; rather, they present peptide
fragments
"loaded" onto their "peptide binding groove".
The presentation of Class I MHC molecules bound to peptide alone has
generally been ineffective in activating CD8 cells. In nature, the CD8 cells
are
activated by antigen-presenting cells, such as, for example, dendritic cells,
which
present not only a peptide-bound Class I MEC molecule, but also a
costimulatory
molecule. Such costimulatory molecules include B7 which is now recognized to
be two
subgroups designated as B7.1 and B7.2. It has also been found that cell
adhesion
molecules such as integthis assist in this process.
_ .
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32
Dendritic cells are antigen-presenting cells that are found in all tissues and
organs, including the blood. Specifically, dendritic cells present antigens
for T
lymphocytes, i.e., they process and present antigens, and stimulate responses
from naive
and memory T cells. In addition to their role in antigen presentation,
dendritic cells
directly communicate with non-lymph tissue and survey non-lymph for an injury
signal
(e.g., ischemia, infection, or inflammation) or tumor growth. Once signaled,
dendritic
cells initiate the immune response by releasing EL-1 which triggers
lymphocytes and
monocytes.
When the CD8 T-cell interacts with an antigen-presenting cell, such as a
dendritic cells, having the peptide bound by a Class I MIX and costimulatory
molecule,
the CD8 T-cell is activated to proliferate and becomes an effector T-cell.
See,
generally, Janeway and Travers, Immunobiology, published by Current Biology
Limited, London (1994).
In another preferred embodiment, rapamycin resistant T cells co-administered
with rapamycin ameliorate GVHD as determined by the change in stage of GVHD.
Preferably, graft-versus-host-disease is ameliorated by at least about 50%,
more
preferably by at least about 75%, most preferably about at least 90%, 95%,
98%, 99%,
99.9% or 100%.
In another preferred embodiment, autologous T cells from the patient are
cultured in rapamycin and/or a rapamycin derivative and under conditions to
generate a
Th2 response. Preferred conditions include the addition of cytokines such as
IL-4 and
1L-2, and rapamycin and/or a rapamycin derivative, alone. Specific conditions
are
described in the Examples which follow.
In another preferred embodiment, allogeneic donor Th2 cells are used to
supplement the allotransplant. Preferably, the allogeneic Th2 cells increase
in number
with a concomitant decrease in GVHD. .
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In another preferred embodiment, immune T cell therapy is utilized for the
treatment of a wide range of medical conditions such as cancer, disease due to
infectious disease organisms such as viruses, autoinunune diseases,
immunosuppressed
individuals, burn victims and Graft versus Host Disease.
In another preferred embodiment, the invention provides for pharmaceutical
compositions comprising rapamycin and/or a rapamycin derivative compound
and/or
rapamycin resistant T cells, rapamycin resistant stem cells and/or rapamycin
resistant
dendritic cells.
In a further aspect, the invention provides use of a rapamycin resistant T
cell; a
rapamycin resistant stem cell; a rapamycin resistant dendritic cell;
composition for the
treatment or prevention (including prophylactic treatment) of a disease or
condition as
disclosed herein, including acute GVHD, chronic GVHD, lacy, livid
maculopapular
rash, jaundice, diarrhea, abdominal pain, hepatosplenornegaly, alopecia,
bullae,
desquamation of skin; prolonging survival of a transplant in a subject mammal,
or
reversing failure of a transplant in a subject and ameliorating disorders and
symptoms
such as associated with immune rejection, including, for example, interstitial
fibrosis,
chronic graft atherosclerosis, or vasculitis; treatment of cancers such as,
leukemias,
lymphomas, melanomas, carcinomas and sarcomas; diseases caused by or otherwise
associated with a virus such as viruses of the herpes family, e.g., herpes
simplex viruses
(HSV) including herpes simplex 1 and 2 viruses (HSV 1, HSV 2), varicella
zoster virus
(VZV; shingles), human herpes virus 6, cytomegalovirus (CMV), Epstein-Barr
Virus
(EBV), and other herpes virus infections such as feline herpes virus
infections, and
diseases associated with hepatitis viruses including hepatitis B viruses (HBV)
B virus.
Examples of clinical conditions which are caused by such viruses include
herpetic
keratitis, herpetic encephalitis, cold sores and genital infections (caused by
herpes
simplex), chicken pox and shingles (caused by varicella zoster) and CMV-
pneumonia
and retinitis, particularly in immunocompromised patients including renal and
bone
marrow transplant patients and patients with Acquired Immune Deficiency
Syndrome
(ADS). Epstein-Barr virus can cause infectious mononucleosis, and is also
suggested
=
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as the causative agent of nasopharyngeal cancer, immunoblastic lymphoma and
Burldtt's lymphoma. Additional specific examples of retroviral infections
which may
be suitably treated in accordance with the invention include human retroviral
infections
such as HIV-1, HIV-2, and Human T-cell Lymphotropic Virus (HTLV) e.g. HTLV-I
or
HTLV-II infections.
In yet a further aspect, the invention provides use of a rapamycin resistant T
cell; a rapamycin resistant stem cell; a rapamycin resistant dendritic cell;
composition
for the preparation of a medicament for the treatment or prevention (including
prophylactic treatment) of a disease or condition as disclosed herein,
including acute
GVIID, chronic GVHD, lacy, livid maculopapular rash, jaundice, diarrhea,
abdominal
pain, hepatosplenomegaly, alopecia, bullae, desquamation of skin; prolonging
survival
of a transplant in a subject mammal, or reversing failure of a transplant in a
subject and
ameliorating disorders and symptoms such as associated with immune rejection,
including, for example, interstitial fibrosis, chronic graft atherosclerosis,
or vasculitis;
treatment of cancers such as, leukemias, lymphomas, melanomas, carcinomas and
=
sarcomas; diseases caused by or otherwise associated with a virus such as
viruses of the
herpes family, e.g., herpes simplex viruses (HSV) including herpes simplex 1
and 2
viruses (HSV 1, HSV 2), varicella zoster virus (VZV; shingles), human herpes
virus 6,
cytomegalovirus (CMV), Epstein-Barr virus (EBV), and other herpes virus
infections
such as feline herpes virus infections, and diseases associated with hepatitis
viruses
including hepatitis B viruses (IIBV) B virus. Examples of clinical conditions
which are
caused by such viruses include herpetic keratitis, herpetic encephalitis, cold
sores and
genital infections (caused by herpes simplex), chicken pox and shingles
(caused by
varicella zoster) and CMV-pneumonia and retinitis, particularly in
immunocompromised patients including renal and bone marrow transplant patients
and
patients with Acquired Immune Deficiency Syndrome (AIDS). Epstein-Barr virus
can
cause infectious mononucleosis, and is also suggested as the causative agent
of
nasopharyngeal cancer, inimunoblastic lymphoma and Burkitt's lymphoma.
Additional
specific examples of retroviral infections which may be suitably treated in
accordance
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with the invention include human retroviral infections such as HIV-1, HIV-2,
and
Human T-cell LymphOtropic Virus (IITLV) e.g. HTLV-I or HTLV-II infections.
Preferred methods of the invention including identifying and/or selecting a
5 subject (e.g. a mammal, particularly human) that is susceptible to or
suffering from a
condition disclosed herein, and thereafter administering to the identified and
selected
subject one or more compounds of the invention, particularly a subject that is
identified
and selected as being susceptible to or suffering from acute GVHD, chronic
GVHD,
lacy, livid maculopapular rash, jaundice, diarrhea, abdominal pain,
10 hepatosplenomegaly, alopecia, bullae, desquamation of skin; prolonging
survival of a
transplant in a subject mammal, or reversing failure of a transplant in a
subject and
ameliorating disorders and symptoms such as associated with immune rejection,
including, for example, interstitial fibrosis, chronic graft atherosclerosis,
or vasculitis;
treatment of cancers such as, leukemias, lymphomas, melanomas, carcinomas and
15 sarcomas; diseases caused by or otherwise associated with a virus such
as viruses of the
herpes family, e.g., herpes simplex viruses (HSV) including herpes simplex 1
and 2
viruses (HSV 1, HSV 2), varicella zoster virus (VZV; shingles), human herpes
virus 6,
cytomegalovirus (CMV), Epstein-Barr virus (EBV), and other herpes virus
infections
such as feline herpes virus infections, and diseases associated with hepatitis
viruses
20 including hepatitis B viruses (HBV) B virus. Examples of clinical
conditions which are
caused by such viruses include heipetic keratitis, herpetic encephalitis, cold
sores and
genital infections (caused by herpes simplex), chicken pox and shingles
(caused by
varicella zoster) and CMV-pneumonia and retinitis, particularly in
irnrnunocompromised patients including renal and bone marrow transplant
patients and
25 patients with Acquired Immune Deficiency Syndrome (AIDS). Epstein-Barr
virus can
cause infectious mononucleosis, and is also suggested as the causative agent
of
nasopharyngeal cancer, immunoblastic lymphoma and Burkitt's lymphoma.
Additional
specific examples of retroviral infections which may be suitably treated in
accordance
with the invention include human retroviral infections such as HIV-1, HIV-2,
and
30 Human T-cell Lymphotropic Virus (HTLV) e.g. HTLV-I or HTLV-II
infections:
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In a preferred embodiment, T cell function is selected based on the cell type
that
is generated by the immune system in response to that disease. For example,
the
immune system effectively responds to a viral, bacterial, and fungal infection
by
generating a Thl/Tcl cell subset; an effective immune response to other
infections may
require the generation of a Th2/Tc2 response. It is also possible that both a
Thl/Tcl
and Th2/Tc2 immune response may be optimal in some treatment settings, so as
to
invoke both cellular and antibody arms of the immune response. From work by
Mossrnan and Coffman (Mossmann T. R., Coffmann R. L.: Thl and Th2 cells:
Different patterns of lympholdne secretion lead to different functional
properties. Ann.
Rev. Immunol. 1989, 7: 145-173), growth factors known as cytokines produced by
T
helper or CD4F T cells in both human and murine systems were classified into
two
subsets, Thl and Th2. These were characterized by their functions in
regulating various
types of immune responses. Cytokines produced by Thl cells [interleukin (IL)-
2,
interferon-alpha, interferon-gamma, tumor necrosis factor-alpha (TNF-a), IL-
12]
stimulated strong cellular immunity whereas Th2 cytokines [IL-4, IL-5, IL-6,
IL-10, IL-
13] were important for eliciting humoral (antibody) responses in vivo.
Cytokines
produced by non-CD4+ T cells have been shown to be important in in vivo
responses.
In particular, the cytotoxic or CD8+ T cells can also be subdivided into two
subgroups,
Tcl and Tc2, which correspond to the same subsets in T helper cells (Carter L.
L.,
Dutton R. W.: Type 1 and Type 2: a functional dichotomy for all T cell
subsets. Curr.
Opin. Immunol. 1996, 8: 336-342). This has led to the current nomenclature
being
generalized from Thl/Th2 to Type 1/Type 2 to reflect more closely the response
generated by particular cytokines, rather than the cell types that produces
them.
In vitro T cell cytotoxic assays are well known to those skilled in the art.
In
general, cytotoxicity is measured in a 5 hr 51Sodium chromate (51Cr ) release
assay.
Target cells, that is cells that are recognized by the T cells are plated in
flat-bottomed
microtiter plates and incubated at 37 C overnight. The targets are washed and
labeled
the next day with 5ICr at 3-7 C. 51Cr is taken up by the target cells, either
by endocytosis
or pinocytosis, and is retained in the cytoplasm. The wells containing target
cells are
washed, and then T cells, referred to as "effector cells" are plated at
different E:T ratios
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and incubated overnight at 37 C. Cytolysis is a measure of the 51Cr released
from the
target cells into the supernatant due to destruction of the target cells by
the effector
cells. The microtiter plates are centrifuged at 1000 rpm for 10 minutes and an
aliquot of
about 50 fd to about 100 ftl is removed and the level of radioactivity is
measured the
next day by a gamma counter and the percent specific lysis calculated.
Percent specific lysis is measured by using the formula:
(51Cr released from the target cells) - (spontaneous 51Cr released from the
target cells)/
(maximum 51Cr released from the tnrget cells) - (spontaneous 51Cr released
from the target cells)
x100
The spontaneous 51Cr released from the target cells is measured with tumor
cells =
to which no effector cells have been added. Maximum 51Cr released from the
target
cells is obtained by adding, for example, 1M HC1 and represents the total
amount of
51Cr present in the cytoplasm of the target cell.
Other cytotoxicity assays such as the labeling of target cells with tritiated
thymidine (3H-TdR) may also be used. 3H-TdR is taken up by target cells into
the
nucleus of the cell. Release of 3H-TdR is a measure of cell death by DNA
fragmentation. The assay is conducted as above except the incubation period is
at least
about 48 hours and 5011.1 to about 100 1.t1 of the supernatant is measured by
a beta-
counter in the presence of at least about 1 ml of scintillation fluid.
Calculation of
percent specific lysis is performed using the above formula.
T cell proliferation assays are used to determine class II MHC antigen
recognition. Briefly, target cells are irradiated so that they do not
proliferate. The
source of the target cells can be allogeneic or autologous cells. CD4+ T cells
are
incubated with the irradiated target cells in the presence of3H-TdR. The CD4+
Tcells
react against the Class II MHC by proliferating. Proliferation is measured by
the
amount of3H-TdR that is taken up by the proliferating T cells as compared to
normal
control cells.
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The search for additional immunosuppressive agents for preventing transplant
rejection and for the treatment of autoimmune and inflammatory disorders
occupies
considerable attention in the pharmaceutical industry. Since cytokines such as
interferon-gamma and tumor necrosis factor-alpha play a critical role in
transplant
rejection and in the pathophysiology of autoimmune disorders, much effort has
been
invested in the development of agents that suppress their production,
secretion and/or
end-organ effect.
While not being bound by any theory, the methods described herein, which
ameliorate organ transplant rejection and/or GVBD, are believed due to an
increase in
Th2/Tc2 function. This is surprising and contrary to the teachings in the
prior art
whereby, immunosuppressants are used to suppress immune responses, thereby,
preventing prevent organ rejection or GVHD.
Without being bound by any theory, one potential mechanism that may
contribute to the observed rapamycin-associated changes in human Th2 cell
generation
is preferential utilization of the multi-drag resistance (MDR) pump in cells
of more
naive phenotype. That is, previous data indicates that human naive CD45RA
cells
express increased MDR, and to this extent, such cells may be intrinsically
more
resistant to rapamycin effects. To initiate investigation into this
possibility, Th2 cells
expanded with or without rapamycin were evaluated for their ability to exclude
an
MDR substrate, rhodamine. This evaluation was performed by flow cytometry in
the
presence or absence of an MDR pump inhibitor (results in Figure 29). As this
figure
shows, rapamycin-generated Th2 cells indeed had an increased capacity for
rhodamine
dye exclusion. This enhanced MDR function in the rapamycin-generated Th2 cells
was
significantly abrogated by the MDR blocking agent.
Graft-versus host disease, the reaction of the donor immune system in
allogeneic
transplantation against the-tissue of the recipient, is initiated by a T-cell
reaction. Such
T cells, in addition to causing GVHD, can also mediate a beneficial graft-
versus-
leukemia/lymphoma (GVL) effect or graft-versus-tumor (GVT) effect to eradicate
the
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malignant clone. GVHD occurs at 3 different time points after transplantation,
involving different organs and with different clinical and histopathological
pictures.
Hyper-acute and acute GVHD develop during and after engraftment till day + 100
post-
transplant; an acute inflammation of the recipient's tissue especially
involving skin, soft
tissue of the whole gastro-intestinal-tract, liver and biliary tract system.
In accordance
with the degree of skin involvement, amount of diarrhea and the value of
ALT/AST and
bilirubin four different grades are defined as 0-W. Acute GVHD 11 typically
needs an
intensification of the immunosuppressive therapy and grade III/IV are often
refractory
to high dose immunosuppression. Chronic GVHD typically develops after day
+100,
and usually ensues directly from acute GVHD or during the reduction of the
immunosuppression. Histologically, the tissue of chronic GVIED shows no
inflammation but does show a fibrotic or sclerotic appearance. Skin, liver and
the GI-
tract tissue are involved and additionally: eyes, sino bronchial-system, lung,
pancreas or
vagina. A reduced quality of life is the result of decreased organ functions.
Therefore,
avoiding refractory acute and chronic GVHD is the main goal of the rapamycin
resistant
T cell based therapy before and after transplantation. The added advantage is
that the
associated increase of risk of infection is not observed as is the case with
treatment with
immunosuppressive agents, as the rapamycin resistant T cells are fully
functional. (See
the examples which follow).
In another preferred embodiment, the rapamycin resistant T cells express
CD62L. CD62L mediates lymphocyte homing to high endothelial venules of
peripheral
lymphoid tissue and leukocyte rolling on activated endothelium at inflammatory
sites.
CD62L is expressed on the surfaces of most peripheral blood B cells, T cells,
monocytes and granulocytes express CD62L. However, some NK cells express
CD62L; some spleen lymphocytes, bone marrow lymphocytes, bone marrow myeloid
cells and thymocytes express CD62L; and, certain hematopoietic malignant cells
express CD62L.
T cells at different stages of maturation or differentiation express surface
molecules indicative of that stage or differentiation. For example, memory T
cells
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express CD45R0+. Memory T cells can be expanded (proliferated) without the
need of
specific antigenic stimulation to maintain the clonal size. Naïve T cell
repertoires
express CD45RA+. For example, to evaluate the frequency of resting T cells
with
memory phenotype that could be stimulated by cytokines to grow, limiting
dilution
5 experiments can be performed. CD45R0 CD4+ resting T cells can be
cultured with IL-
2 alone or in combination with TNF-a and IL-6, in the presence of autologous
irradiated macrophages and anti-DR antibodies to prevent autoreactive
responses.
Systemic memory T cells are characterized according to the cell surface
10 expression of certain known antigens. Typically, these cells are
positive for CD4, and
lack expression of CD45RA, and integrin a4137. They are further characterized
by
expression of CCR4. A subset of cells of interest are common leukocyte antigen
positive (CLA+). Verification of the identity of the cells of interest may be
performed
by any convenient method, including antibody staining and analysis by
fluorescence
15 detection, ELISA, etc., reverse transcriptase PCR, transcriptional
amplification and
hybridization to nucleic acid microarrays, etc. Some memory T cells associated
with
the skin are known to express CLA. Thus, any type of cell can be identified
when
necessary.
20 Other systemic memory cells are triggered to adhere to endothelial ICAM-
1, by
LFA-1 binding. These adhesion molecules are implicated in graft rejection,
psoriasis,
and arthritis. In a preferred embodiment, systemic memory T cells are killed
by the co-
administration of rapamycin to a patient that has received an organ, tissue or
cell
transplant. Without, wishing to be bound by theory, removal of memory T cells
25 decreases a cell mediated immune rejection of an allograft. However,
rapamycin or a
rapamycin derivative compound can be administered together with other agents
such as
for example, CCR4 blocking agents that prevents triggering of LFA-1 mediated
adhesion is useful in the inhibition of graft rejection by preventing the
accumulation of
memory T cells at the site Of graft implantation; preventing intra-islet
infiltration by T
30 cells to inhibit development of insulin-dependent diabetes mellitus;
blocking infiltration
of T cells into the central nervous system to treat multiple sclerosis and
other
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demyelinating diseases; blocking the accumulation of T cells in the synovial
joints of
patients suffering from rheumatoid arthritis; accumulation of memory T cells
to
influence immune responsiveness, and the like.
=
Immune cells express a variety of cell surface molecules which can be detected
with either monoclonal antibodies or polyclonal antisera. Immune cells that
have
undergone differentiation or activation can also be enumerated by staining for
the
presence of characteristic cell surface proteins by direct immunofluorescence
in fixed
smears of cultured cells.
T lymphocytes, at whichever stage of maturity and cell differentiation
expressing CD62L can be identified. For example, one such method is by
measuring
cell phenotypes. The phenotypes of immune cells and any phenotypic changes can
be
evaluated by flow cytometry after immunofluorescent staining using monoclonal
antibodies that will bind membrane proteins characteristic of various immune
cell types.
A second means of assessing cell differentiation is by measuring cell
function.
This may be done biochemically, by measuring the expression of enzymes,
mRNA's,
genes, proteins, or other metabolites within the cell, or secreted from the
cell.
Bioassays may also be used to measure functional cell differentiation or
measure
specific antibody production directed at a patient's tumor, tumor cell lines
or cells from
fresh tumors.
Preferably, rapamycin or a rapamycin derivative enhances the generation of
other therapeutic cells such as, for example, dendritic cells, pluripotent
stem cells, or
hematopoietic stem cells. Rapamycin-generated dendritic cells would, for
example,
improve cellular immune therapy strategies, as the dendritic cells can be
pulsed with
tumor or infectious disease antigens to more optimally generate an effective T
cell
immune response. Purified dendritic cells can be pulsed with (exposed to)
antigen, to
allow them to take up the antigen in a manner suitable for presentation to
other cells of
the immune systems. Antigens are classically processed and presented through
two
-
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42
pathways. Peptides derived from proteins in the cytosolic compartment are
presented in
the context of Class I MHC molecules, whereas peptides derived from proteins
that are
found in the endocytic pathway are presented in the context of Class II MHC.
However, those of skill in the art recognize that there are exceptions; for
example, the
response of CD8+ tumor specific T cells, which recognize exogenous tumor
antigens
expressed on MHC Class I. A review of MHC-dependent antigen processing and
peptide presentation is found in Germain, R. N., Cell 76:287 (1994).
Numerous methods of pulsing dendritic cells with antigen are known; those of
skill in the art regard development of suitable methods for a selected antigen
as routine
. experimentation. In general, the antigen is added to cultured dendritic
cells under
conditions promoting viability of the cells, and the cells are then allowed
sufficient time
to take up and process the antigen, and express antigen peptides on the cell
surface in
association with either Class I or Class II MHC, a period of about 24 hours
(from about
18 to about 30 hours, preferably 24 hours). Dendritic cells may also be
exposed to
antigen by transfecting them with DNA encoding the antigen. The DNA is
expressed,
and the antigen is presumably processed via the cytosolic/Class I pathway.
The present invention provides methods of using therapeutic compositions
comprising activated, antigen-pulsed dendritic cells. The use of such cells in
conjunction with soluble cytokine receptors or cytokines, or other
immunoregulatory
molecules is also contemplated. The inventive compositions are administered to
stimulate an allogeneic immune response, and can be given by bolus injection,
continuous infusion, sustained release from implants, or other suitable
technique.
Typically, the cells will be administered in the form of a composition
comprising the
antigen-pulsed, activated dendritic cells in conjunction with physiologically
acceptable
carriers, excipients or diluents. Such carriers will be nontoxic to recipients
at the
dosages and concentrations employed. Neutral buffered saline or saline mixed
with
serum albumin are exemplary appropriate diluents.
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=
43
Ex Vivo Culture ofDendritic Cells
A procedure for ex vivo expansion of hematopoietic stem and progenitor cells
is
described in U.S. Pat. No. 5,199,942. Other suitable methods are known in the
art. Briefly,
ex vivo culture and expansion comprises: (1) collecting CD34+ hematopoietic
stern and
progenitor cells from a patient from peripheral blood harvest or bone marrow
explants; and
(2) expanding such cells ex vivo. In addition to the cellular growth factors
described in U.S.
Pat. No. 5,199,942, other factors such as flt3-L, IL-1. IL-3 and c-kit
ligancl, can be used.
Stem or progenitor cells having the CD34 marker constitute only about 1% to
= 3% of the mononuclear cells in the bone marrow. The amount of CD34+ stem
or
progenitor cells in the peripheral blood is approximately 10- to 100-fold less
than in
bone marrow. Cytokines such as flt3-L may be used to increase or mobilize the
numbers of dendritic cells in vivo. Increasing the quantity of an individual's
dendritic
Peripheral blood cells are collected as described in the Examples which follow
or, alternatively, can be using procedures known in the art such as, for
example,
apheresis procedures. See, for example, Bishop et al., Blood, vol. 83, No. 2,
pp. 610-
616 (1994). Briefly, peripheral blood progenitor cells (PBPC) and peripheral
blood
stem cells (PBSC) are collected using conventional devices, for example, a
Haemonetics Model V50 apheresis device (Haemonetics, Braintree, Mass.). Four-
hour
collections are performed typically no more than five times weekly until
approximately
6.5x108 mononuclear cells (MNC)/kg are collected. The cells are suspended in
standard
media and then centrifuged to remove red blood cells and neutrophils. Cells
located at
the interface between the two phases (the buffy coat) are withdrawn and
resuspended in
HBSS. The suspended cells are predominantly mononuclear and a substantial
portion
of the cell mixture are early stem cells.
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44
A variety of cell selection techniques are known for identifying and
separating
CD34+ hematopoietic stem or progenitor cells from a population of cells. For
example,
monoclonal antibodies (or other specific cell binding proteins) can be used to
bind to a
marker protein or surface antigen protein found on stem or progenitor cells.
Several
such markers or cell surface antigens for hematopoietic stem cells (i.e., flt-
3, CD34,
My-10, and Thy-1) are known in the art, as are specific binding proteins. -
In one method, antibodies or binding proteins are fixed to a surface, for
example, glass beads or flask, magnetic beads, or a suitable chromatography
resin, and
contacted with the population of cells. The stem cells are then bound to the
bead
matrix. Alternatively, the binding proteins can be incubated with the cell
mixture and
the resulting combination contacted with a surface having an affinity for the
antibody-
cell complex. Undesired cells and cell matter are removed providing a
relatively pure
population of stem cells. The specific cell binding proteins can also be
labeled with a
fluorescent label, e.g., chromophore or fluorophore, and the labeled cells
separated by
sorting. Preferably, isolation is accomplished by an immunoaffinity column.
Immunoaffinity columns can take any form, but usually comprise a packed bed
reactor. The packed bed in these bioreactors is preferably made of a porous
material
having a substantially uniform coating of a substrate. The porous material,
which
provides a high surface area-to-volume ratio, allows for the cell mixture to
flow over a
large contact area while not impeding the flow of cells out of the bed. The
substrate
should, either by its own properties, or by the addition of a chemical moiety,
display
high-affinity for a moiety found on the cell-binding protein. Typical
substrates inclu_de
avidin and streptavidin, while other conventional substrates can be used.
In one useful method, monoclonal antibodies that recognize a cell surface
antigen on the cells to be separated are typically further modified to present
a biotin
moiety. The affinity of biotin for avidin thereby removably secures the
monoclonal
antibody to the surface of a packed bed (see Berenson, et al., J. Immunol.
Meth., 91:11,
CA 02529244 2013-04-19
1986). The packed bed is washed to remove unbound material, and target cells
are
released using conventional methods. Immunoaffinity columns of the type
described
above that utilize biotinylated anti-CD34 monoclonal antibodies secured to an
avidin-
coated packed bed are described for example, in WO 93/08268.
5
An alternative means of selecting the quiescent stem cells is to induce cell
death
in the dividing, more lineage-committed, cell types using an antimetabolite
such as 5-
fluorouracil (5-FU) or an alkylating, agent such as 4-hydroxycyclophosphamide
(4-
HC). The non-quiescent cells are stimulated to proliferate and differentiate
by the
10 addition of growth factors that have little or no effect on the stem
cells, causing the non-
stem cells to proliferate and differentiate and making them more vulnerable to
the
cytotoxic effects of 5-FU or 4-HC. See Berardi et al., Science, 267:104
(1995).
15 Isolated stem cells can be frozen in a controlled rate freezer (e.g.,
Cryo-Med,
Mt. Clemens, Mich.), then stored in the vapor phase of liquid nitrogen using
dimethylsulfoxide as a cryoprotectant. A variety of growth and culture media
can be
used for the growth and culture of dendritic cells (fresh or frozen),
including serum-
depleted or serum-based media. Useful growth media include RPM!, TC 199,
Iscoves
20 modified Dulbecco's medium (Iscove, et al., F. I Exp. Med., 147:923
(1978)), DMEM,
Fischer's, alpha medium, NCTC, F-10, Leibovitz's L-15, MEM and McCoy's.
Particular
nutrients present in the media include serum albumin, transferrin, lipids,
cholesterol, a
reducing agent such as 2-mercaptoethanol or monothioglycerol, pyruvate,
butyrate, and
a glucocorticoid such as hydrocortisone 2-hemisuccinate. More particularly,
the
25 standard media includes an energy source, vitamins or other cell-
supporting organic
compounds, a buffer such as HEPES, or Tris, that acts to stabilize the pH of
the media,
and various inorganic salts. A variety of serum-free cellular growth media is
described
in WO 95/00632. The collected CD34r cells are cultured with suitable
cytokincs. for example,
as described herein. CD34' cells then are allowed to differentiate and commit
to cells of the
30 dendritic lineage. These cells are then further purified by flow
cytometrv or similar means,
using markers characteristic
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46
_ .
of dendritic cells, such as CD1a, HLA DR, CD80 and/or CD86. The cultured
dendritic
cells are exposed to an antigen, for example, an allogeneic class I HLA
molecule,
allowed to process the antigen, and then cultured with an amount of a CD40
binding
protein to activate the dendritic cell. Alternatively, the dendritic cells are
transfected
with a gene encoding an allogeneic HLA class I molecule or immune related
receptors,
and then cultured with an amount of a CD40 binding protein to activate the
antigen-,
presenting dendritic cells.
The activated, antigen-carrying dendritic cells are them administered to an
individual in order to stimulate an antigen-specific immune response. The
dendritic
cells can be administered prior to, concurrently with, or subsequent to,
antigen
administration. Alternatively, T cells may be collected from the individual
and exposed
to the activated, antigen-carrying dendritic cells in vitro to stimulate
antigen-specific T
cells, which are administered to the individual.
Rapamycin-generated pluripotent stem cells would have particular application
for stem cell therapy, which includes for example, the treatment of a wide
variety of
diseases such as Parkinson's Disease, post cerebral vascular accident
neurological
deficiency, type I diabetes mellitus, and post myocardial infarction
deficiency.
Rapamycin-generated hematopoietic stem cells would have particular application
to the
use of hematopoietic stem cell transplantation, which includes therapeutic
application
for the treatment of immune deficiency syndromes, auto-immune disease,
hematologic
malignancy, and solid tumors. In each of these embodiments detailed in this
invention,
the relevant starting cell population is, for example, precursor monocytes or
hematopoietic stem cells in the case of dendritic cell therapy. Preferably,
highly
purified pluripotent stem cells if the desired cell is for use in stem cell
therapy.
Preferably, CD34+ hematopoietic stem cells are used in the case of
hematopoietic stem
cell therapy.
The cells are placed into in vitro culture conditions, described herein, in
the
presence of rapamycin. In each case the cell culture in the presence of
rapamycin is
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47
performed in the presence of suitable cytokines. For example, fc>r dendritic
cell
expansion in the presence of rapamycin, the dendritic cells are preferably
propagated in
cytokines such as IL-4 and GM-CSF. In the case of pluripotent or hematopoeitic
stem
cell culture in the presence of rapamycin, cytokine additives to tl-ke culture
comprise, for
example, recombinant stem cell factor, IL-3, IL-6, GM-CSF, G-CSF, IL-7, or
other
recombinant cytokines.
Any cell can be used in the methods of the invention, including but not
limited
to, stem cells, thymocytes, precursor cells and the like. A precursor cell
population
includes cells of a mesodermal derived cellular lineage, more particularly of
hematopoietic lineage, endothelial lineage, muscle cell lineage, epithelial
cell lineage
and neural cell lineage.
A "precursor cell" can be any cell in a cell differentiation pathway that is
capable of differentiating into a more mature cell. As such, the term
"precursor cell
population" refers to a group of cells capable of developing into a_ more
mature cell. A
precursor cell population can comprise cells that are totipotent, cells that
are pluripotent
and cells that are stem cell lineage restricted (i.e. cells capable of
developing into less
than all hematopoietic lineages, or into, for example, only cells or erythroid
lineage).
As used herein, the term "totipotent cell" refers to a cell capable c=f
developing into all
lineages of cells. Similarly, the term "totipotent population of cells" refers
to a
composition of cells capable of developing into all lineages of cells. Also as
used
herein, the term "pluripotent cell" refers to a cell capable of developing
into a variety
(albeit not all) lineages and are at least able to develop into all
hematopoietic lineages
(e.g., lymphoid, erythroid, and thrombocytic lineages). For example, a
pluripotent cell
can differ from a totipotent cell by having the ability to develop into all
cell lineages
except endothelial cells. A "pluripotent population of cells" refers to a
composition of
cells capable of developing into less than all lineages of cells but a_t least
into all
hematopoietic lineages. ,As such, a totipotent cell or composition of cells is
less
developed than a pluripotent cell or compositions of cells. As used herein,
the terms
"develop", "differentiate" and "mature" all refer to the progression of a cell
from the
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48
stage of having the potential to differentiate into at least two different
cellular lineages
to becoming a specialized cell. Such terms can be used interchangeably for the
purposes of the present application.
As used herein, the term "population" refers to cells having the same or
different
identifying characteristics. The term "lineage" refers to all of the stages of
the
development of a cell type, from the earliest precursor cell to a completely -
mature cell
(i.e. a specialized cell).
Preferred cells within a stem cell population of the present invention include
cells of at least one of the following cellular lineages: hematopoietic cell
lineage,
erythroid lineage, endothelial lineage, leukocyte lineage, thrombocyte
lineage, erythroid
lineage (including primitive and definitive erythroid lineages), macrophage
lineage,
neutrophil lineage, mast cell lineage, megakaryocyte lineage, natural killer
cell lineage,
eosinophil lineage, T cell lineage, endothelial cell lineage and B cell
lineage.
Various techniques may be employed to separate the cells by initially removing
cells of dedicated lineage. Monoclonal antibodies are particularly useful for
identifying
markers associated with particular cell lineages and/or stages of
differentiation.
If desired, a large proportion of terminally differentiated cells may be
removed
by initially using a "relatively crude" separation. For example, magnetic bead
separations may be used initially to remove large numbers of lineage committed
cells.
Desirably, at least about 80%, usually at least 70% of the total hematopoietic
cells will
be removed.
Procedures for separation may include but are not limited to, magnetic
separation, using antibody-coated magnetic beads, affinity chromatography,
cytotoxic
agents joined to a monockinal antibody or used in conjunction with a
monoclonal
antibody, including but not limited to, complement and cytotoxins, and
"panning" with
_ .
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49
antibody attached to a solid matrix, e.g., plate, elutriation or any other
convenient
technique.
Techniques providing accurate separation include but are not limited to, flow
cytometry, which can have varying degrees of sophistication, e.g., a plurality
of color
channels, low angle and obtuse light scattering detecting channels, impedance
channels,
etc.
In another preferred embodiment, cells are isolated and purified cell from a
sample, patient or donor individual and are used in functional assays to
determine any
properties of the cells. Depending on the isolated and purified cellular
population,
appropriate functional assays known in the art can be conducted. For example,
if the
population of cells are T cells specific for a desired antigen such as a tumor
antigen,
cytotoxic T cell assays, T cell proliferation assays, cytokine profiles,
determination of
surface antigens for T cell maturity or memory T cells, etc., can be carried
out.
Isolation of cells useful in the present invention are well known in the art.
For
example, peripheral blood mononuclear cells (PBMCs) can be obtained from a
subject
and isolated by density gradient centrifugation, e.g., with Ficoll/Hypaque.
Specific cell
populations can be depleted or enriched using standard methods. For example,
monocytes/macrophages can be isolated by adherence on plastic. T cells or B
cells can
be enriched or depleted, for example, by positive and/or negative selection
using
antibodies to T cell or B cell surface markers, for example by incubating
cells with a
specific primary monoclonal antibody (mAb), followed by isolation of cells
that bind
the rnAb using magnetic beads coated with a secondary antibody that binds the
primary
mAb. Peripheral blood or bone marrow derived hematopoietic stem cells can be
isolated by similar techniques using stem cell-specific mAbs (e.g., anti-CD34
mAbs).
Specific cell populations can also be isolated by fluorescence activated cell
sorting
according to standard methods. Monoclonal antibodies to cell-specific surface
markers
known in the art and many are commercially available.
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If desired, a large proportion of terminally differentiated cells may be
removed
by initially using a "relatively crude" separation. For example, magnetic
bead.
separations may be used initially to remove large numbers of lineage committd
cells.
Desirably, at least about 80%, usually at least 70% of the total hematopoietic
cells can
5 be removed.
Procedures for separation may include but are not limited to, magnetic
separation, using antibody-coated magnetic beads, affinity chromatography,
cytotoxic
agents joined to a monoclonal antibody or used in conjunction with a
monoclamal
10 antibody, including but not limited to, complement and cytotoxins, and
"pannimg" with
antibody attached to a solid matrix, e.g., plate, elutriation or any other
conveniient
technique.
Techniques providing accurate separation include but are not limited tr>, flow
15 cytometry, which can have varying degrees of sophistication, e.g., a
plurality c> f color
channels, low angle and obtuse light scattering detecting channels, impedance
channeIs,
etc.
In one preferred embodiment, rapamycin resistant allogeneic cells are
20 administered to a patient. Allogeneic cells may be derived from any
person and
comprise both CD4+ and CD8+ T cells. Cells are treated with the desired
cytolcines and
rapamycin prior to administering to a patient.
An advantage of the present invention is that the peripheral pool of memory T
25 cells (CD45R0+) are susceptible to rapamycin or a rapamycin derivative
compound and
are inhibited, thereby decreasing the risk of GVHD. Conversely, the naive T
repertoire (CD45RA) is maintained. For example, to evaluate the frequency of
resting
T cells with memory phenotype that could be stimulated by cytokines to grow,
limiting
dilution experiments can be performed. CD45R0+ CD4+ resting T cells can be
cultured
30 with IL-2 alone or in combination with TNF-a and IL-6, in the presence
of autologous
irradiated macrophages and anti-DR antibodies to prevent autoreactive
responss. The
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51
in vitro expansion of immune T cells with a more naive phenotype may be
particularly
applicable to the therapy of autoimmune disease. In a therapeutic plan,
patients with
autoimmune disease may undergo apheresis to isolate T cells, have T cells
expanded in
rapamycin to enrich for a naive T cell phenotype, receive immune depleting
chemotherapy to eliminate autoreactive T cell clones in vivo, and then receive
infusion
of in vitro generated T cells from immune reconstitution with a T cell source
less likely
to reconstitute autoirnmunity (T cells with characteristics more typical of
naïve T cells;
i.e., CD28+, CD621).
The allogeneic cells contained in the medicament of the invention may assume
any formation. For example, the allogeneic cells suspended in an adequate
solution
may be used. The solution containing the allogeneic cells can desirably be
used as an
injection or drip-feed solution. Especially, an injection or drip-feed
solution, which is
prepared by suspending the allogeneic cells in physiological saline and so on
containing
about 0.01% to 5% of human serum albumin. The allogeneic cells or the
preparations
containing them may be frozen and kept in their frozen state so as to be used
for
remedying or preventing various disease. Cryopreservation should be performed
under
liquid nitrogen conditions, preferably in solutions that preserve immune T
cell function,
such as reduced DMSO concentrations of 5% and addition of cryopreservant
molecules
such as pentastarch.
When the medicaments according to the invention can desirably be administered
to a patient by an intravenous drip, arterial injection, local injection and
the like. The
desirable dosage of the medical solution varies in accordance with the way or
place of
the administration thereof. However, it is commonly desirable to administer at
least
about 50 to about 500 ml of the medical solution containing the allogeneic
cells in the
aforesaid ratio to the patient. It is preferable that the medical solution is
administered
one time a day to one time a month. In any event, at least one administration
of the
medicament comprising the allogeneic cells should be made. In the allogeneic
setting,
the T cells are administered at the time of the HSCT (within 24 hours of stem
cell
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52
infusion), and can be administered at the time of any other donor T cell
infusion, for
example, at the time of a donor lymphocyte infusion (DLI).
The dosage of the allogeneic cells contained, as the main ingredient, in the
medicament of the invention, may be arbitrarily decided in accordance with the
condition of the patient and/or the clinical procedure. In general, about
1x102 to about
1x109 allogeneic cells per kilogram of patient's weight may be used.
The extraction of cells from the donor may be performed any way, for example,
by blood collection, pheresis, or other possible operations. It is desirable
to draw blood
from the vein of the donor, and add heparin or citric acid to the blood thus
drawn to
prevent blood coagulation. The blood of the order of 0.01 ml to 100 ml is
generally
drawn in one blood extraction operation, but the amount of the blood to be
drawn is not
limited in the invention. Taking into consideration the physical burden of the
donor,
labors involved in collecting the blood, and troublesome operations for
separating the
lymphocyte cells, it is desirable to draw the blood by 5 ml to 10 ml,
preferably 10 ml to
ml in one blood extraction operation. For most clinical applications, harvest
of
sufficient numbers of autologous or allogeneic T cells will require an
outpatient
apheresis procedure.
The operation for separating the lymphocyte cells from the blood drawn in the
aforementioned manner may be accomplished by a known method for separating
lymphocyte cells such as a discontinuous density gradient centrifugation
method which
is performed by using sucrose or lymphocyte separating agents on the market.
Alternatively, the apheresis product can be subjected to counterflow
centrifugal
elutriation as a mechanism to enrich for lymphocyte populations. Furthermore,
such
lymphocytes can be enriched for the desired T cell subset by negative or
positive
selection using antibodies and selection beads or selection columns.
The type of the anti-CD3 antibodies.used in the invention is not limited to a
specific antibody, as far as the antibody makes for proliferation and
activation of the
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53
desired lymphocyte cells. The anti-CD3 antibodies used for stimulating the
lymphocyte
cells are possibly yielded in organisms or organic cells by use of refined CD3
molecules.
As the culture medium solution for cultivating the desired cells, there may be
used a culture medium derived from a living organism or a culture medium
composed
by mixing amino acid, vitamins, nucleic acid base and the like with
equilibrium salt
solution. For example, as the culture medium, RPMI-1640, DMEM, IMDM,
X-Vivo 15, or X-Vivo 20 or the like are preferable. In particular, the culture
medium of
X-Vivo 20 is particularly recommended for expansion of human T cells under the
conditions identified here. Such media is further supplemented by the addition
of 5%
autologous plasma, or 5% human A/B serum. These culture medium components
applicable to the invention are commercially available.
The cultivation of the desired cells may be fulfilled by common cell-
cultivating
methods. For example, it can be carried out in a CO2 -incubator at a CO2
concentration
of about 1% to about 10%, preferably about 5%, at a temperature of 30 C. to
40 C.,
most preferred at about 37 C.
The number of days which the cultivation takes place is not specifically
restricted, but it is desirable to allow about 2 to about 20 days. For the
human
condition, a period of about 20 days appears sufficient to achieve the desired
T cell
cytoldne phenotype and to achieve clinically relevant T cell numbers. Such
cells appear
to be stable, with appropriate re-stimulation with anti-CD3 and anti-CD28
molecules,
for several weeks after day 20, and such an expansion may prove valuable in
some
circumstances that require increased cell number or further in vitro
modifications. For
example, it may be desirable to first initiate a polyclonal expansion in
rapamycin to
alter the T cell phenotype towards a nave T cell character, and then to
perform further
stimulations in an antigen:specific manner in an attempt to enhance reactivity
to cancer
or infectious disease antigens. Within the period for the cultivation, it is
best to observe
the conditions of the cells under a microscope and take count of the number of
cells so
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54
as to suitably adjust the amount of the culture medium solution by adding the
solution.
The proliferation of the cells does not appreciably take place within about 1
to about 2
days after commencement of the cultivation, but is generally observed about 3
to six
days after the commencement. When the cells are satisfactorily proliferated,
the color of
the culture medium solution will be changed from orange to yellow. The culture
medium is supplemented at about 0.1 to about 5 times the culture solution
initially
given. It is preferred to monitor the cell number and median cell volume by
Coulter
Multisizer evaluation daily, as this approach allows accurate determination of
T cell
expansion and T cell activation.
The invention has been described in detail with reference to preferred
embodiments thereof. However, it will be appreciated that those skilled in the
art, upon
consideration of this disclosure, may make modifications and improvements
within the
spirit and scope of the invention. The following non-limiting examples are
illustrative
of the invention.
EXAMPLES
Materials and Methods
Purification of T cells
Murine CD4+ splenic T cells from C57B1/6 mice were purified to > 98% purity
by negative selection using anti-macrophage, anti-B cell, anti-CD8 cell, and
anti-
granulocyte antibodies (StemCell Technologies; murine CD4+ T cell enrichment
procedure). Murine CD4 cells were plated at a concentration of 0.2 x 106
cells/ml in
RPMI-1640 media supplemented with 10% fetal calf serum (Gemini Bioproducts).
Activation of T cells and Culture Conditions
CD4+ cells were stimulated with magnetic beads (tosylated beads; Dynal) that
were coated with anti-murine CD3 (PharMingen) and anti-murine CD28
(PharMingen)
at a T cell to bead ratio of 1:3. Media in the Thl condition consisted of
recombinant
murine IL-12 (2.5 ng/ml; R and D Systems), anti-murine IL-4 neutralizing
antibody
(clone 111; 10 micrograms/n-4 recombinant human IL-2 (20 I.U./m1; Chiron),
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recombinant human IL-7 (20 ng/ml; Peprotech), and the anti-oxidant N-acetyl
cysteine
(NAC; 3.3 M). Media in the Th2 condition consisted of recombinant murine IL-4
(1000 I.U./m1; Peprotech), recombinant human IL-2 (20 I.U./m1), recombinant
human
IL-7 (20 nem% and 3.3 M NAC.
5
Immune suppression molecules cyclosporine A (CSA), FK506, and rapamycin
were purchased from Sigma and reconstituted according to the manufacturers
instructions, with rapamycin and FK506 being tested at 0.004 NI and 0.02 M
concentrations and CSA being tested at 0.04p.M and 0.2 NI concentrations.
Media
10 containing IL-2, IL-7, NAC, and the particular immune suppression agent
was added to .
maintain cell concentration at between 0.2 and 1.0 x 106 cells/ml throughout
the culture
interval. Cells were counted by a Multi-Sizer Instrument (Coulter), and CD4
expansion
is plotted, as shown in Figure 1.
15 Lymphocyte Harvest and T Cell Isolation from Human Donors
After determination that the donor is HLA-matched with recipient, the donor
undergoes a 2 to 5 liter apheresis procedure using a CS-3000 or an equivalent
machine.
The apheresis product is subjected to counterflow centrifugal elutriation by
standard
operating procedures of the N1H Department of Transfusion Medicine, Cell
Processing
20 Section. The lymphocyte fraction of the elutriation product (120 to 140
fraction) is
depleted of B cells by incubation with an anti-B cell antibody (anti-CD20;
Nexell) and
an anti-CD8 antibody (Nexell) and sheep anti-mouse magnetic beads (Dynal;
obtained
through Nexell) by standard operating procedures using the MaxCep Device
(Nexell).
Flow cytometry will be performed to document that CD8+ T cell contamination is
< 1%.
25 The resultant CD4-enriched donor lymphocyte product can be cryopreserved
in aliquots
of 50 to 200 x 106 cells/vial. Sterility of the population is not tested at
this early stage
of the Th2 cell generation procedure; such testing occurs after final co-
culture of donor
CD4 cells.
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56
Peripheral Blood Stem Cell Harvest from Donor
Immediately following lymphocyte harvest, the donor will receive filgrastim as
an outpatient (10 pg/kg/day each morning; subcutaneously) for 5, 6, or 7 days.
The
donor should take the filgrastim as early as possible upon awakening in the
morning.
This is especially important on days 5, 6, and 7 of the injections.
Apheresis is typically performed on days 5 and 6 of this regimen. On some
occasions, sufficient numbers of CD34+ 'cells might be obtained with a single
apheresis
on day 5; on other occasions, it may be necessary to perform apheresis on days
5,6, and
7 to reach the target CD34+ cell number (?_ 4 x 106 per kg). The donor is
instructed to
take filgrastim for the complete 7 day period, unless notified by the
transplant team that
adequate CD34+ cells were harvested before day 7. If? 3 x 106 CD34+ cells per
kg are
harvested after apheresis on days 5, 6, and 7, no further mobilization or
apheresis is
performed, and the patient is eligible to receive the stem cell transplant
with that dose of
CD34+ cells. In the event that less than 3 x 106 CD34+ cells per kg are
harvested after
apheresis on days 5, 6, and 7, the donor will be given two weeks of rest, and
then will
be re-treated with filgrastim followed by repeat peripheral blood stem cell
harvesting.
A 15 to 25 liter large volume whole blood pheresis is performed via a 2-armed
approach
or via a temporary central venous catheter in the femoral position using the
Baxter
CS3000P1us, Cobe Spectra, or an equivalent instrument. This procedure
typically takes
4 to 6 hours.
The apheresis procedure typically uses ACD-A anti-coagulant; alternatively,
partial anti-coagulation with heparin may be utilized. The apheresis product
can be
cryopreserved and stored at ¨180 degrees Celsius in a solution containing
Plasmalyte A,
Pentastarch, human serum albumin, DMSO, and preservative free heparin (10
U/m1).
The concentration of CD34+ cells in the apheresis product is determined by
flow
cytometry, and the number of CD34+ cells in each cryopreserved bag calculated.
If the
donor and host are ABO in- compatible, red blood cells will be depleted from
the stem
cell product by standard protocols.
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In Vitro Generation of Donor CD4+ Th2 Cells
Cryopreserved donor CD4 T cells are resuspended to a concentration of 0.3 x
106 cells per ml. Media consist of X-Vivo 20 supplemented with 5% heat-
inactivated
autologous plasma. The donor CD4+ T cells are cultured in filtered flasks at
37 C in
5% CO2 humidified incubators. At the time of culture initiation, T cells are
stimulated
with anti-CD3/anti-CD28 coated magnetic beads (3 to 1 ratio of beads to T
cells). At
the time of co-culture initiation and on day 2 of culture, the following
reagents are
added: recombinant human IL-4 (Shering IL-4; 1000 I.U. per ml), and
recombinant
human IL-2 (purchased from Chiron Therapeutics; 20 I.U. per m1). After day 2,
cells
are maintained at a concentration of 0.25 to 1.0 x 106 cells per ml by the
addition of
fresh X-Vivo 20 media supplemented with autologous plasma (5%), 1L-2 (20
I.U./ml),
and IL-4 (1000 I.U./m1). The median cell volume is determined using a
Multisizer II
instrument (Coulter). When the T cell volume approaches 500 fl (acceptable
range of
650 to 350), the T cells are restimulated with anti-CD3/anti-CD28 beads;
typically, this
time of restimulation will be after 8 to 12 days of culture. Bead
restimulation is at a
bead to T cell ratio of 3:1. T cell concentration is 0.2 x 106 cells/ml. Media
consists of
X-Vivo 20 supplemented with autologous plasma (5%), IL-2 (20 I.U./m1), and IL-
4
(1000 I.U./m1). After bead restimulation, CD4 cells are maintained at a
concentration
of 0.25 to 1.0 x 106 cells per ml by the addition of fresh X-Vivo 20 media
supplemented
with autologous plasma (5%), IL-2 (20 I.U./m1), and IL-4 (1000 I.U./m1).
Rapamycin
(commercially available oral solution; Sirolimus, Wyeth-Ayerst) is added to
the Th2
culture condition at day 0 at a concentration of 1 micromolar. For some donors
who are
particularly sensitive to the effects of rapamycin, it may be necessary to
initiate culture
in lower doses of rapamycin, such as 0.01 to 0.1 micromolar. When the Th2
culture
media is expanded for the purposes of cytokine addition or maintenance of cell
concentration at 0.2 to 1.0 x 106 cells/ml, the media added to culture should
be replete
with rapamycin, and contain a concentration of rapamycin between 0.01 and 1.0
micromolar. The highest concentration of rapamycin that allows CD4 Th2 cell
expansion should be utiliied. In the case of Th2 generation in rapamycin, it
is typically
not necessary to restimulate the CD4 cells with anti-CD3 and anti-CD28, as the
cells
have attained a purified Th2 phenotype after only one round of CD3, CD28 co-
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stimulation. This methodology therefore allows rapid and uncomplicated
generation of
Th2 cells and represents a technical advance that allows Th2 generation with
reduced
reagent utilization and reduced labor.
When the CD4 cell mean cell volume approaches 500 fl (acceptable range of
650 to 350), the cells are harvested and cryopreserved.
The following is the minimal phenotypic requirements of any particular Th2
cell
culture to qualify for cryopreservation with subsequent administration:
1. Presence of predominately CD4+ T cells by flow cytometry (greater than
70% CD4+ T cells, and less than 5% contaminating CD8+ T cells).
2. In addition, the cryopreserved product is tested for
sterility with both
fungal and bacterial cultures, through the ongoing testing done on cell
products
processed in the NIH Department of Transfusion Medicine. In addition, the CD4
Th2
cell product is tested for endotoxin content by the limulus assay. Cell
products positive
for fungal, bacterial, or endotoxin content are discarded.
Transplant Procedure: Allogeneic Peripheral Blood Stem Cell Transplantation
a) On day 0, the patient receives the cryopeserved PBSC.
b) The cryopreserved PBSC product is thawed and administered intravenously
immediately. The target dose of the PBSC is > 4 x 106 CD34+ cells per kg.
However, if donor apheresis on days 5, 6, and 7 yields a total of > 3 x 106
CD34+ cells
per kg, this level of CD34+ cell dose is also allowed.
(c) No steroids are allowed in the management of DMSO-related toxicities
(chills, muscle aches) that may occur immediately after cellular infusion
(diphenhydramine and meperidine are allowed).
(d) In the case of rapamycin generated Th2 cells, standard GVHD
prevention strategy may involve either the standard calcineurin inhibitor
drugs cycliisporine A or FK506, or most preferably, in vivo rapamycin.
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Transplant Procedure: Donor Th2 Cell Administration
a) On day 1 of the transplant procedure, the cryopreserved donor Th2 cells
are thawed and immediately administered intravenously.
b) No steroids are allowed in the management of DMSO-related toxicities
(chills, muscle aches) that may occur immediately after cellular infusion
(diphenhydramine and meperidine are allowed).
c) The determination of whether a Th2 cell infusion is safe will be based
on
the presence or absence of hyperacute GVHD and of any grade 4 or 5 toxicity
attributable to the Th2 cells that occurs in the first 14 days post-
transplant.
d) Hyperacute GVHD is defmed as a severe level of acute GVHD (grade HI
or IV) that occurs within the first 14 days post-transplant.
e) The initial three patients to be enrolled to Th2 cell dose
level #1 (5 x 106
Th2 cells/kg). If no hyperacute GVHD or grade 4 or 5 toxicity attributable to
the Th2
cells is observed in these initial three patients, then it will be determined
that this dose
level is safe, and accrual to dose level #2 will commence. If hyperacute GVHD
or
grade 4 or 5 toxicity attributable to the Th2 cells is observed in any of the
initial three
patients, then accrual to dose level #1 will be expanded to include a total of
six patients.
If two patients in six develop hyperacute GVHD or a grade IV toxicity related
to the
Th2 cells, then it will be determined that dose level #1 is not safe, and
further accrual to
the study will stop at that point. If only one of the six patients experiences
such an
adverse effect, then it will be determined that dose level #1 is safe, and
accrual will
proceed to dose level #2.
Three patients may then be enrolled to Th2 cell dose level #2(2.5 x 107
Th2 cells/kg). The same accrual and stopping rules will apply to this dose
level as those
used for dose level #1. As such, either three or six patients will be accrued
to dose level
#2.
If it is determined that Th2 cell dose level #2 is safe, accrual to the final
dose level #3 will start (Th2 cell dose of 1.25 x 108 cells/kg). Six patients
in total will
be evaluated on dose level #3. If more than one patient on dose level #3
develops
hyperacute GVHD or grade 4 or 5 toxicity attributable to the Th2 cells, then
accrual to
dose level #3 will stop.
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h) In the phase II component of this study, eighteen (18)
additional patients
will receive Th2 cells at either dose level #2 or level #3. To help ensure
that the Th2
cells continue to be safely administered in this expanded cohort, the same
accrual and
stopping rules pertaining to severe toxicity attributed to the 'Th2 cells will
be continued.
5 Specifically, 24 total patients (6 in the Phase I cohort, 18 in the
expanded Phase II
cohort) will be evaluated at either Th2 cell dose level #2 or level #3.
Accrual and
stopping rules will be applied after each cohort of six patients. Therefore,
if at any
point, the frequency of severe toxicity attributable to the Th2 cells exceeds
1/6, 2/12,
3/18, or 4/24, then accrual to that treatment arm will be stopped.
Example I: CD4+ TI21/Th2 Modulation.
Use of the calcineurin inhibitors cyclosporine A or FK506 is a standard
component of immune suppression after allogeneic PBSCT. Given the known role
of
Thl/Th2 biology in the modulation of immunity post-SCT, it is an important
goal to
identify any differential influence of these two agents on the Thl/Th2
balance. In
addition to CSA and FK506, rapamycin is an immune suppression agent that has
been
studied in murine models, and more recently, in clinical trials of allogeneic
PBSCT.
Rapamycin, by binding to the mammalian target of rapamycin, controls multiple
aspects
of T cell metabolism, including phosphorylation of Rb protein with subsequent
regulation of cyclin dependent kinases and control of protein translation via
the 14-3-3
pathway. As such, the mechanism of action of rapamycin stands in stark
contrast to that
of CSA and FK506, which work primarily through inhibition of cytokine and
other
molecule mRNA transcription. To this extent, a thorough evaluation and
comparison of
these three molecules as they relate to the modulation of Thl/Th2 biology is
warranted,
particularly as it relates to immunity in the allogeneic PBSCT context.
In Figure 1, results are shown that illustrate the differential effect of CSA,
FK506, and rapamycin on the generation of murine CD4+ Thl and Th2 cells.
Murine
CD4 cells were purified, timulated in a polyclonal manner with anti-CD3 and
anti-
CD28 antibodies, and propagated in culture conditions that promote either Thl
or Th2
differentiation. For Thl cultures, media was supplemented with IL-12, antibody
to IL-
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4, IL-2, IL-7, and the cell death inhibitor N-acetyl cysteine; for Th2
cultures, media was
supplemented with IL-4, IL-2, IL-7, and NAC. As figure 1 shows, control ml and
Th2
cell cultures expanded approximately 2 to 3 logs in six days. In contrast, Thl
or Th2
expansion in the presence of either 0.004 M or 0.02 M of FK506 was
associated with
a dramatic reduction in CD4 cell expansion under these optimized conditions;
in both
Thl or 'Th2 conditions, there was only a two to three fold increase in CD4
cell number
in the presence of FK506. Presence of CSA at either 0.04 KM or 0.2 ItM
concentrations
yielded a similar inhibition of Thl and Th2 expansion. As such, there did not
appear to
be any preferential Th2 or Thl generation with the calcineurin inhibitors.
Remarkably,
at concentrations of rapamycin that in the literature have previously been
associated
with T cell inhibition (0.004 NI and 0.02 ELM), no significant inhibition in
CD4+ T cell
expansion under either Thl or Th2 conditions, was observed. In fact, Thl and
Th2
expansion under the 0.02 INA concentration of rapamycin actually resulted in
an
increased CD4 cell number relative to the Thl and Th2 control cultures.
Example 2: Evaluation of Immunosuppressive agents on Th2 Responses.
To evaluate for Th2-bias relative to the three immune suppression agents, CSA,
FK506, and rapamycin, on day 6 after Th2 expansion, the CD4 cells were
harvested
from culture, washed, normalized to a concentration of 0.5 x 106 cells/ml, and
re-
stimulated with anti-CD3 and anti-CD28 for supernatant generation. On day 6 of
culture, T cells were harvested, washed, normalized to a concentration of 0.5
x 106
cells/ml, and re-stimulated with anti-CD3 and anti-CD28 (3:1 bead to T cell
ratio) for
24 hours to generate a supernatant. Culture supernatants were tested for IL-4
and IL-10
content by a two site ELISA (BioSource), with experimental samples scored
relative to
a standard curve generated from evaluation of recombinant murine IL-4 and IL-
10. Cell
culture labels along the x-axis represent cytokine and immune suppression
agent
conditions during the initial six days of T cell generation; there were no
cytokines or
immune suppression agents added during the time of 24 hour supernatant
generation.
Figure 2 shows supernatant ELISA results for the type II cytolcines IL-4 and
IL-
10. Th2 expansion in the presence of CSA, and in particular, in the presence
of FK506,
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reduced the capacity of the CD4 cells to produce IL-4 and IL-10 relative to
the control
Th2 culture. In contrast, Th2 expansion in the presence of rapamycin resulted
in Th2
cells with similar IL-4 and M-10 secretion relative to the control. As such,
rapamycin,
but not CSA or FK506, facilitated or maintained Th2 cell generation, both with
regards
to CD4+ cell expansion and effector Th2 cytokine production.
Example 3: Evaluation of Immunosuppressive agents on Thl Responses. -
To evaluate the effect of these three agents, CSA, FK506, and rapamycin, on
potential Thl-bias, the 'Thl cultured cells were also re-stimulated with anti-
CD3, anti-
CD28, and the supernatant was tested for the type I cytokines IL-2 and IFN-y.
Murine
CD4+ cells were expanded under the Thl culture condition using anti-CD3, anti-
CD28
co-stimulation. On day 6 of culture, T cells were harvested, washed,
normalized to a
concentration of 0.5 x 106 cells/ml, and re-stimulated with anti-CD3 and anti-
CD28 (3:1
bead to T cell ratio) for 24 hours to generate a supernatant. Culture
supernatants were
tested for IL-2 and TIN-y content by alwo site ELISA (BioSource). Experimental
samples scored relative to a standard curve generated from evaluation of
recombinant
murine IL-2 and IFNI. Cell culture labels along the x-axis represent cytokine
and
immune suppression agent conditions during the initial six days of T cell
generation;
there were no cytokines or immune suppression agents added during the time of
24 hour
supernatant generation.
As Figure 3 shows, CSA, and in particular, FK506, resulted in Thl cells with
significantly diminished capacity for both IL-2 and IFN-y secretion. In marked
contrast, ml expansion in the presence of rapamycin resulted in a dramatic
increase in
Thl cell capacity for IFN-y secretion, and a nominal increase in IL-2
secretion capacity.
As such, rapamycin, but not CSA or FK506, facilitated or maintained Thl cell
generation, both with regards to CD4+ expansion and effector Thl cytokine
production.
In sum, these results also indicate that rapamycin, although it has been
associated with a
type II cytokine immune shift upon in vivo administration, does not appear to
induce a
Thl to Th2 shift directly upon CD4+ cells. This observation implies that
rapamycin
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induced type 11 promotion may operate indirectly, for example, through its
actions on
APC modulation.
Given these results that rapamycin unexpectedly preserved or enriched for
Thl/Th2 polarity, a five-fold higher concentration of rapamycin, 0.1 ItM, was
evaluated. Murine CD4+ cells were co-stimulated_ with anti-CD3 and anti-CD28
coated
magnetic beads under Thl or Th2 conditions at previously tested rapamycin
concentrations (0.008 pM to 0.02 M), and a relatively high concentration (0.1
p.M).
Cell expansion was monitored over the six day culture by Multi-Sizer
evaluation, and
plotted on a log scale. Figure 4 shows that even at this higher dose of
rapamycin, the
method of optimized co-stimulation and cytokine supplementation disclosed
herein,
allowed for the expansion of either Thl or Th2 subsets without any apparent
reduction
in CD4 cell yield.
Example 4: Evaluation of Thl and Th2 Responses Generated in Rapamycin.
The Thl or Th2 populations generated in the 0.1 pM rapamycin concentration
were also evaluated. Murine CD4+ cells were expanded with anti-CD3, anti-CD28
coated magnetic beads under the Thl or the Th2 culture conditions in the
absence or
presence of rapamycin (0.008 pM to 0.1 pM), as denoted on the x-axis of this
figure.
On day 6 of culture, the T cells were harvested, washed, and restimulated with
fresh
CD3, CD28 coated beads (3:1 bead to T cell ratio) in media not containing
cytokines or
immune suppression agent. A 24 hour culture supernatant was generated and
tested for
IL-2 and LFN-7 cytokine content by two site ELISA (BioSource) in reference to
a
standard curve.
As Figure 5 shows, 'Thl cells in each of the rapamycin concentrations had
similarly high secretion of both IL-2 and IFNI. With respect to Th2 cell
expansion in
rapamycin, it was observed that expansion in the 0.1 pM rapamycin
concentration was
associated with elimination of the "contaminating" quantities of IL-2
secretion that
were present in the lower dose rapamycin cultures and the control Th2
cultures. As
such, the higher dose of rapamycin was associated with an improved Th2
phenotype, as
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defined by reduced IL-2 secretion. In contrast, Figure 6 shows that Th2 cells
= propagated in the 0.1 it.M rapamycin concentration had preservation of
capacity for
secretion of the type II cytoldnes IL-4, IL-5, and IL-10. Thl cells propagated
in 0.1 ..M
rapamycin did not have an increased capacity for type II cytokine secretion.
These
results thus further confirm that, in our system, Thl or Th2 polarization can
be
maintained or even enhanced in the presence of relatively high rapamycin
concentrations.
=
In Figure 6, murine CD4+ cells were expanded with anti-CD3, anti-CD28 coated
magnetic beads under the Thl or the Th2 culture conditions in the absence or
presence
of rapamycin (0.008 iuM to 0.1 itM), as denoted on the x-axis of this figure.
On day 6
of culture, the T cells were harvested, washed, and restimulated with fresh
CD3, CD28
coated beads (3:1 bead to T cell ratio) in media not containing cytokines or
immune
suppression agent. A 24 hour culture supernatant was generated and tested for
type II
cytokine content (IL-4, IL-5, and IL-10) by two site ELISA (BioSource) in
reference to
a standard curve.
Example 5: Cytokine Production after Rapamycin Exposure without Co-stimulation
To determine whether CD28 signaling, perhaps through up-regulation of
survival molecules such as bc1-2 family members or activation of the AKT
pathway,
might account for the observed capacity to overcome the expected rapamycin
immune T
cell suppression effect, the following experiments were conducted. The
experiments
were performed evaluating the polarizing cytokine conditions and rapamycin
exposure
after activation without co-stimulation through use of beads conjugated with
only anti-
CD3 antibodies. Murine CD4 T cells were expanded with magnetic beads
conjugated
with only the T cell receptor activating antibody anti-CD3 or with beads
conjugated
with both anti-CD3 and anti-CD28 (denoted in figure by Thl or Th2 condition):
The
condition receiving only anti-CD3 stimulation was performed either with or
without the
addition of rapamycin (0.02 p.M concentration). Cell expansion was monitored
over the
= 30 six day culture by Multi-Sizer evaluation, and plotted on a log scale.
- -
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=
As Figure 7 shows, Th2, and in particular, Thl cell expansion was greatly
reduced relative to CD3, CD28 co-stimulated control Th1/Th2 cultures. However,
addition of rapamycin to "signal 1 only" generated Thl or Th2 cultures did not
result in
a significant decrease in CD4+ cell yield. This result suggests, at least at
the 0.02 p.M
5 rapamycin concentration tested, that CD28 in this system may not provide
a specific T
cell activation or survival signal for the abrogation of the expected
rapamycin T cell
inhibition effects.
Example 6: Evaluation of Thu/ Th2 Differentiation Generated in High Doses of
10 Rapamycin.
Higher dose levels of rapamycin during Thl/Th2 differentiation were evaluated.
In the first panel of Figure 8, murine CD4+ cells were expanded in the Th2
culture
condition using anti-CD3 and anti-CD28 coated magnetic beads, with culture
performed
either in the absence or presence of rapamycin (0.1 FM to 101.11\4). Cell
expansion was
15 monitored over the six day culture by Multi-Sizer evaluation, and
plotted on a log scale.
In figure 8 (second panel), CD4+ cells in each of the Th2 culture conditions
were
replated with normalization of T cell concentrations, and further expanded in
media
containing both the Th2 culture condition additives and rapamycin at the same
20 concentrations as during culture initiation. Cell expansion was
monitored from day 6 to
day 9 of culture by Multi-Sizer evaluation, and plotted on a log scale.
Figure 8 shows CD3, CD28 co-stimulation, generated Th2 cell expansion from
day 0 to 6 of culture at rapamycin concentrations ranging from 0.1 p.M to 10.0
pt..M (left
25 panel). As this figure shows, at both the 2.5 p.M and 10 ftM
concentration, Th2 cell
expansion was reduced approximately one log relative to the control culture.
To
evaluate whether this rapamycin-associated reduction in Th2 cell expansion was
a
progressive or transient process, cultures were each normalized for cell
number on day
6 of culture, and propagated an additional three days in cytokine replete
media (no
30 further CD3, CD28 re-stimulation; Figure 8, right panel). As this panel
shows, the Th2
cultures initiated and continued in the higher concentration of rapamycin, had
an
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increase in CD4+ cell empansion during day 6 to day 9 culture interval
relative to the
control Th2 culture. The increased CD4+ expansion in the high dose rapainycin
cultures
was also observed after day 6 anti-CD3 and anti-CD28 re-stimulation of
cultures (with
ongoing cytoldne and rapamycin addition; result in Figure 9).
These co-stimulated Th2 cultures generated in concentrations of rapamycin
ranging from 0.1 to 10.0 p..M were also evaluated for their Thl/Th2 cytokine
secretion
capacity (Figure 10). As this figure shows, at the highest rapamycin
concentration, the
Th2 cells had an enhanced Th2 polarity on the basis of abrogation of
contaminating IL-
2 secretion and modest reduction in IFN-y secretion. Such high-dose rapamycin
generated Th2 cells had full preservation of the Th2-type cytoldnes associated
with
more proximal Th2 effector function, namely IL-4 and IL-5. In contrast, such
cells had
a significant reduction in the Th2-type cytoldnes associated with more distal
Th2
effector function, IL-10 and IL-13. In sum, these results indicate that the
high-dose
rapamycin facilitated generation of a 'Th2 cell of enhanced purity (less Th1
contaminating elements) that was more proximal in its state of Th2
differentiation.
To further evaluate the issue of rapamycin resistance in the murine Thl or Th2
cultures, control effector Thl or Th2 cells (day 6 of culture) or rapamycin-
generated
'Thl or Th2 effectors were re-stimulated with anti-CD3, anti-CD28 in the
presence or
absence of rapamycin (Figure 11). As this figure shows, control Thl or Th2
effectors
re-stimulated in the presence of 0.1 p.M rapamycin, and in particular, 101.1.M
rapamycin,
had significantly reduced secondary expansion capacity. In contrast, Th2 cells
generated in either 0.1 ji.M or 10 RM rapamycin had similar secondary
expansion in
either unsupplemented media or media supplemented with 0.1 p.M rapamycin. This
result indicates that, at the 0.1 ItivI rapamycin concentration, the rapamycin-
generated
Th2 cells are relatively resistant to the T cell inhibition compared to
control Th2
effectors. However, secondary anti-CD3, anti-CD28 re-stimulation in the
presence of
10 p.M rapamycin resulted in significant inhibition of Th2 expansion even in
the 'Th2
culture initially propagated in the 10 p.M rapamycin concentration.
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In the high dose rapamycin generated Th2 cultures, the purity of cytokine
polarity and the pattern of individual Th2 cytokine member secretion suggested
that the
CD4 cells emanating from such cultures possessed a more naïve phenotype. To
address
the possibility that high dose rapamycin might be selecting for a more naive
CD4 cell in
culture, an evaluation of surface markers characteristic of naive vs. memory
function
was conducted. One such functional marker is CD28 itself, which is present on
nearly
all naive CD4+ cells, only to be reduced during the end stages of memory CD4-1-
effector
differentiation. Figure 12 demonstrates that CD28 indeed was greatly increased
on the
CD4+ cells propagated in high dose rapamycin. This result demonstrates that
rapamycin, and in particular, the high dose rapamycin conditions, select for a
more
naive CD4 + cell phenotype that expands during CD3, CD28 co-stimulation and
thereby
attains an increased purity of Th2 polarity.
To further evaluate this, another cell surface molecule was measured. CD62L
that functionally helps determine naive vs. memory CD4 cell function. CD62,
which is
primarily expressed by more naive CD4 cells, dictates T cell lymph node homing
capacity rather than tissue-based effector function. As figure 13 shows, Th2
cells
expanded in rapamycin, in particular, high dose rapamycin, had an increased
expression
of CD62L. Another cell surface molecule evaluated in these cultures was CD4OL.
CD4OL is not so much a characteristic of naive vs. memory status, but rather
is a marker
for Thl/Th2 polarity. That is, in our prior results, Thl-type cells have
significantly
increased CD4OL relative to Th2-type cells. This association is of importance
in light
of the role of CD4 cell CD4OL expression in up-regulation of IL-12 production
in
dendritic cell populations. A significant reduction in Th2 cell CD4OL
expression in the
= Th2 cultures propagated in rapamycin, in particular, high dose rapamycin was
observed.
In sum, Th2 generation in high-dose rapamycin provided a more pure Th2
profile, both
on the basis of reduced contamination with IL-2 and LEN-7 secretion and
reduced
CD4OL expression.
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Example 7: CD8+ Tcl/Tc2 Modulation
Both low- and high-dose rapamycin was evaluated on the generation of murine
Tcl and Tc2 populations after CD3, CD28 co-stimulatory conditions. As a first
step in
this direction, T cell activation patterns of nnurine CD4+ and CD8+ T cells
under
Thl/Th2 or Tcl/Tc2 differentiation conditions were evaluated. In prior
studies, we
have demonstrated that median cell volume, as measured by Coulter counting, is
a
surrogate marker for T cell activation as it orrelates with other events such
CD25
and CD69 up-regulation. Figure 14 shows rnedian cell volume changes during
Thl,
Th2, Tcl, or Tc2 expansion in the presence or absence of either 0.1 or 10.0 pM
rapamycin. Without rapamycin, each T cell_ subset has a dramatic increase in
median
cell volume after CD3, CD28 co-stimulatiora. With rapamycin addition, even at
the 10
ItM condition, maximal median cell volum in each T cell subset was not reduced
relative to the control T cells. However, in the CD4 Thl and Th2 cultures,
there was a
more rapid return of median cell volumes towards the basal levels in high dose
rapamycin. In contrast, there was not such a dramatic rapamycin-associated
reduction
in median cell volume in the CD8+ Tel or T'c2 conditions. This result suggests
that
CD4+ T cells may be more amenable to modulation by high-dose rapamycin than
CD8+
T cells.
Similar to results with CD4 Thl/Tlx_2 generation, CD8+ Tel/Tc2 expansion
after
CD3, CD28 co-stimulation was nominally rduced at the 0.1 ILM rapamycin
concentration, with more significant reductions occurring at 10 KM of
rapamycin
(Figure 15).
Example 8: Effects of Rapamycin on Cytota7cic T cells
To evaluate whether rapamycin exposure influenced CD8+ cytotoxic effector
function, chromium release assays were performed (Figure 16 shows CTL assays
using
Tc2 effectors generated in the presence or absence of rapamycin). As this
figure shows,
the Tc2 cells propagated in high dose rapam_ycin had reduced lytic capacity
through the
fas pathway, as evidenced by their reduced capacity to lyse L1210-fas
transfected tumor
targets under conditions of calcium neutraliation (left panel). Similarly, Tc2
cells
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propagated in high dose rapamycin had a reduced capacity to lyse the P815
tumor target
in a heteroconjugate assay in calcium-replete conditions, an assay that
reflects granule-
mediated killing function. In sum, Tc2 cells generated in high dose rapamycin
had
reduced granule and fasL killing function relative to Tc2 cells propagated in
low-dose
or no rapamycin.
In contrast to Th1/Th2 differentiation in high dose rapamycin, where either
phenotype could be attained without evidence for Th1/Th2 bias, the results
indicate that
CD8 expansion in rapamycin may favor a type II cytokine bias. That is, as
Figure 17
shows, Tel cells expanded in the high dose of rapamycin lost their capacity
for IFN-y
secretion and had reduced capacity for IL-2 secretion. Similarly, Tc2 cells
expanded in
high dose rapamycin lost their capacity for IFN-y secretion. In marked
contrast, Tc2
cell secretion of the type II cytokines IL-4, IL-5, and IL-10 was not reduced
by the high
dose rapamycin condition. As such, similar to the Th2 cell culture in high
dose
rapamycin, the purity of Tc2 cells can be increased (on the basis of reduction
in
contaminating type I cytokine secretion) by high dose rapamycin exposure. It
is
interesting to note that loss of IFN-y secretion in the rapamycin-generated
Tcl culture
was not associated with induction of Tcl cell type II cytokine secretion, and
therefore
does not indicate a simple rapamycin-associated Ti to T2 shift in polarity.
Similar to
the case with CD4+ Th2 cells, CD8 expansion in high-dose rapamycin was
associated
with a more naïve T cell phenotype, as evidenced by increased CD62L expression
(Figure 18).
Example 9: Evaluation of Rapatnycin In Vitro and In Vivo: GVHD and GVT
In prior studies, allogeneic donor Th2 cells were associated with reduced
GVHD, and could modulate GVHD induced by urnnanipulated donor CD4+ and CD8+
T cells. In light of the results shown herein, that rapamycin enhanced Th2
purity of co-
stimulated donor Th2 cells, it is likely that rapamycin-generated Th2 cells
may have
enhanced in vivo capacity to modulate GVHD. Furthermore, since the rapamycin-
generated cells maintained resistance to rapamycin inhibition relative to
unmanipulated
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T cells it was hypothesized that in vivo rapamycin may allow selective
expansion of the
Th2 cells relative to other, unmanipulated donor T cells.
In a murine model of fully MHC-mismatched transplantation, the in vivo effect
5 of co-stimulated control 'Th2 cells relative to rapamycin-generated Th2
cells was
evaluated. This model involves transfer of C57B1/6 (B6) bone marrow and
splenic
CD4+ and CD8+ T cells into lethally irradiated Fl hosts (C57BI/6 x Balb/c).
After
parental transplantation with this GVHD-inducing inocula, recipient mice were
further
injected i.v. with host-type breast cancer cells, the TS/A cell line
(spontaneously arising
10 tumor of balb/c origin). On day 7 after BMT, n=5 mice were killed per
treatment
group, splenic T cells were isolated, re-stimulated with either syngeneic B6
or
allogeneic Fl dendritic cells in vitro, and cytokine secretion (IFN-y) from
splenic CD8+
T cells was evaluated by Miltenyi cytokine capture assay. The absolute number
of
splenic CD8+,1FN-y+ donor T cells was then calculated per spleen, with this
result
15 being a biologic endpoint for acute GVED biology. As Figure 19 shows
(left panel),
recipients of the allogeneic splenic T cell inocula had a significant number
of
alloreactive CD8+ T cells capable of IFN-y secretion at day 7 post-BMT. Other
treatment groups received the same splenic T cell inoculate and additional
donor Th2
cells that were either co-stimulated in the presence or absence of rapamycin
(in this
20 experiment, 0.1 JIM rapamycin). As Figure 19 shows, recipients of
additional donor
Th2 cells had reduced in vivo generation of allospecific CD8+IFN-y cells,
indicating
Th2 down-modulation of GVHD. The level of reduction in CD8+IFN-y secretion was
comparable in recipients of Th2 or rapamycin-generated Th2 populations. In
addition,
Th2 recipients were also evaluated for IFN-y secretion from the expanded Th2
cells 7
25 days after in vivo transfer in the GVHD/GVT model (Th2 cells were
identified by flow
cytometry on the basis of their expression of the congenic marker, Ly5.1). As
figure 19
shows (right panel), Th2 cells propagated in rapamycin had a reduced capacity
for IFN-
y secretion after in vivo transfer relative to conventional co-stimulated Th2
cells. This
reduced Th2 cell IFN-y secretion in rapamycin-generated Th2 recipients was
observed
30 with syngeneic DC re-stimulation, which likely reflects true in vivo
activation in the
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GVHD model, and with potential for WN-y secretion upon allogeneic DC
stimulation.
These data thus indicate that rapamycin-generated Th2 cells maintained a anore
pure
Th2 function in vivo in the GVHD model as determined by reduced IFN-y
secretion.
In a separate experiment, conventional Th2 cells or Th2 cells expaxided in low
dose (0.1 M) or high dose (10 M) rapamycin were evaluated in the same
GVHD/GVT model, with weight loss, histology, and survival as experimal
endpoints. As Figure 20 shows, recipients of splenic CD4+ and CD8+T cells
underwent
weight loss consistent with acute GVHD. Also shown in this figure is the TS/A
control
group that .received tumor and no donor splenic T cells; weight loss in this
group is thus
attributed to tumor (pulmonary metastasis): Supplementation of splenic T cell
inoculate
with conventional co-stimulated Th2 cells resulted in a modest amelioration of
acute
GVHD-related weight loss. Importantly, recipients of Th2 cells generated under
either
low dose or high dose rapamypin had a more dramatic reduction in GVHD-related
weight loss. This result indicates that rap amycin-generated Th2 cells were
more
effective at GVHD modulation.
Figure 21 shows survival results from this experiment, with n=10 rinice in
each
group evaluated for survival. As this figure shows, each recipient of
carcitioma cells
and no donor T cells (TS/A control) died of tumor within one month post-EMT.
In this
experiment, which was carried out at a modest irradiation dose of 1050 cG-y,
the GVHD
control group receiving splenic T cells did not undergo lethality in spite of-
the dramatic
pattern of progressive GVHD-induced weight loss. In this treatment cohort,
there was a
significant GVT effect based on increased survival, with deaths in this group
attributed
to GVHD. Recipients of additional Th2 cells, and in particular, Th2 cells
expanded in
rapamycin, had preservation of a component of the GVT effect. The potency of
this
GVT effect, however, was reduced relative to the GVHD control, as deaths
occurring in
these treatment cohorts were attributable to tumor relapse. As such, these
data indicate
that co-stimulated Th2 cells reduce GVHD (rapamycin-generated Th2 >
conventional
'Th2) and that Th2 modulation of GVHD reduced but did not abrogate the potency
of
the GVT effect. Further experiments evaluating these treatment cohorts are
being
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conducted at higher radiation doses. GVHD-related lethality is generated in
the control
groups and a survival advantage in recipients of the rapamycin-generated Th2
cells is
shown.
Example 10: Preferential Expansion of Rapamycin Resistant T cells in vivo..
As a further strategy for Th2 modulation of acute GVHD, it was evaluated
whether rapamycin-generated Th2 cells might be preferentially expanded by in
vivo
rapamycin administration. To this extent, the GVHD/GVT model was utilized,
with
splenic T cell inoculate supplemented with rapamycin-generated donor Th2 cells
(0.1
p,M concentration). In addition, recipient mice were injected daily with
either
rapamycin, cyclosporin A, or CMC vehicle from day 0 to day +7 post-BMT. As
Figure
22 shows (left panel), administration of rapamycin-generated Th2 cells and in
vivo
rapamycin resulted in a greater number of Th2 cells in the day +7 spleens than
cell
administration and CSA or vehicle administration. Importantly, as Figure 22
(tight
panel) indicates, this enhanced Th2 cell expansion in vivo was associated with
a net
reduction in the capacity of post-BMT splenic T cells to secrete IFN-y. In
sum, these
results indicate that rapamycin generated Th2 cells, which have a more pure
Th2
phenotype and a more naïve phenotype, have a greater capacity for GVHD
modulation;
this Th2 modulation can be further optimized by in vivo rapamycin
administration.
Example I I : Evaluation of Rapamycin in Human CD4+ Cells
To evaluate whether a similar rapamycin biology exists in human CD4 cells, and
to initiate a translation of rapamycin-generated Th2 cells into clinical
trials, experiments
of human CD4 cell co-stimulation in the presence or absence of rapamycin were
performed. Figure 23 shows the results of CD4 expansion from n-,4 normal
donors
either without (left panel) or with rapamycin (1.0 p.M; right panel). In vitro
conditions
consisted of anti-CD3, anti-CD28 co-stimulation with IL-4 and IL-2. As this
figure
shows, without rapamycin, a three to four log CD4 Th2 cell expansion occurred
over 20
days in culture. In contraa, addition of rapamycin was associated with an
initial
significant reduction in CD4 cell numbers in the first six days of culture,
followed by a
period of CD4 expansion. This pattern of CD4 cell contraction/expansion
appeared
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consistent with an initial CD4 cell selection process, followed by a period of
rapamycin-
resistant expansion. To begin evaluating this possibility, we expanded n=4
donor
cultures for six days in the presence of rapamycin, and then either continued
to
propagate the cultures in rapamycin (Figure 24, left panel) or in the absence
of
rapamycin (Figure 24, right panel). As this figure shows, the post day 6 CD4
cells
expanded significantly whether maintained with or without rapamycin (without
rapamycin > with rapamycin). Experiments were then performed to evaluate
whether
rapamycin-generated Th2 cells were indeed relatively resistant to rapamycin,
and to
evaluate whether cross-resistance to. CSA existed (Figure 25). As this figure
shows,
CD3, CD28 re-stimulation of rapamycin-generated Th2 cells with or without 0.01
ft.M
rapamycin yielded a similar degree of CD4 cell expansion. This concentration
of
rapamycin was shown in the same experiment to significantly reduce expansion
of
conventionally propagated Th2 cells. In contrast, rapamycin-generated Th2
cells were
sensitive to inhibition by CSA at concentrations of either 0.2 or 0.04 p.M. As
such, the
rapamycin-generated Th2 cells appear to have resistance to further rapamycin
challenge, without cross-resistance to CSA.
Example 12: Purity of Th1/Th2 cells
The human Th2 cultures were additionally evaluated for issues of Thl/Th2
purity. As Figure 26 shows, cells propagated under Th2 conditions and
rapamycin had
an increased Th2 cytokine purity, as evidenced by reduction in capacity for
1FN-y
secretion. This increased Th2 purity (reduced IFN- y) was observed simply by
an initial
day 0 to day 6 rapamycin exposure, and was more fully realized by continued
presence
of rapamycin in the Th2 culture. This result indicated that rapamycin may
operate
initially by some CD4 cell subset selection mechanism (acute process), and
additionally
by a more chronic mechanism that maintains Th2 purity. Surprisingly, rapamycin
generated Th2 cells had a dramatic reduction in IL-2 secretion, with this
effect
occurring during the initial six days of rapamycin exposure. Concomitant with
these
reductions in type I cytokine contaminations, Figure 27 demonstrates that
rapamycin-
generated 'Th2 cells had an increased capacity for secretion of the type II
cytokines IL-4
and IL-13. In sum, rapamycin enhanced the ability of CD28 co-stimulation and
_
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cytokines to generate human Th2 cells, both on the basis of reducing Th1
cytokines and
increasing Th2 cytokines.
Similar to results in the murine system, rapamycin-generated Th2 cells in the
human system displayed a more naive CD4 cell phenotype relative to
conventionally
co-stimulated Th2 cells. As Figure 28 shows, rapamycin-generated Th2 cells had
increased expression of CD62L relative to control Th2 cells; the increase in
CD62L was
most marked in Th2 cultures that were continuously exposed to rapamycin. Also
similar to the murine studies, human rapamycin-generated Th2 cells also
expressed
significantly reduced CD4OL relative to control Th2 cells; because CD4OL is a
molecule preferentially expressed on ml cells, this observation further
supports the
conclusion that rapamycin facilitates generation of a human Th2 cell with
enhanced
purity.
Example 13: Rapamycin Treated Naïve CD4+ T cells
In an attempt to tie these results together, we predicted that naive CD4 cells
would be more resistant to rapamycin, and would therefore exhibit a higher
cloning
efficiency after co-stimulation during rapamycin exposure. To this extent,
naive or
memory human CD4 cells were purified by flow sorting, and co-stimulated either
with
or without rapamycin (results in Figure 31). As this figure shows, naive
sorted CD4
cells had only a nominal reduction in CD4 expansion in rapamycin relative to
the
= control culture (-25% reduction in CD4 yield). In contrast, memory
CD45R0+ sorted
cells not only had reduced expansion to CD28 co-stimulation, but also had a
more
.significant degree of rapamycin-associated reduction in CD4+ T cell expansion
(¨ 50%
reduction). Together, these results show that naive CD4 + T cells are more
resistant to
rapamycin inhibition, perhaps in part through their increased expression of
MDR
molecules, which results in co-stimulation of CD4+ T cells that have a more
naive
effector phenotype and a greater capacity for Th2 polarization.
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Example 14: Reduction of GVHD by Th2 cells.
=
Materials and Methods
Lymphocyte Harvest and T Cell Isolation from Donor
(a) After determination that donor is HLA-matched with recipient, donor
will
5 undergo a 2 to 5 liter apheresis procedure using a CS-3000 or an
equivalent machine.
(b) Apheresis product will be subjected to counterflow centrifugal
elutriation by the
standard operating procedure of the NIEI DTM.
(c) The lymphocyte fraction of the elutriation product (120 to 140
fraction) will be
depleted of B cells by incubation with an anti-B cell antibody (anti-CD20;
Nexell) and
10 an anti-CD8 antibody (Nexell) and sheep anti-mouse magnetic beads
(Dynal; obtained
through Nexell) by a standard operating procedure of the NIEI DTM using the
MaxCep
Device (Nexell). Flow cytometry will be performed to document that CD8+ T cell
contamination is < 1%.
(d) The resultant CD4-enriched donor lymphocyte product will be
cryopreserved
15 using an NIH DTM protocol in aliquots of 50 to 200 x 106 cells/vial.
Sterility of the
population will not be tested at this early stage of the Th2 cell generation
procedure;
such testing will occur after final co-culture of donor CD4 cells.
Peripheral Blood Stem Cell Harvest from Donor
20 a) Immediately following lymphocyte harvest, the donor will receive
filgrastim as
an outpatient (10 tg/kg/day each morning; subcutaneously) for 5,6, or 7 days.
The
donor should take the filgrastim as early as possible upon awakening in the
morning.
This is especially important on days 5, 6, and 7 of the injections.
b) Apheresis will typically be performed on days 5 and 6 of this
regimen. On some
25 occasions, sufficient numbers of CD34+ cells might be obtained with a
single apheresis
on day 5; on other occasions, it may be necessary to perform apheresis on days
5, 6, and
7 to reach the target CD34+ cell number (_?_ 4 x 106 per kg). The donor will
be
instructed to take filgrastim for the complete 7 day period, unless notified
by the
transplant team that adequate CD34+ cells were harvested before day 7.
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c) If? 3 x 106 CD34+ cells per kg are harvested after apheresis on days 5,
6, and 7,
no further mobilization or apheresis will be performed, and the patient will
be eligible
to receive the stem cell transplant with that dose of CD34+ cells.
d) In the event that less than 3 x 106 CD34+ cells per kg are harvested
after
apheresis on days 5,6, and 7, the donor will be given two weeks of rest, and
then will
be re-treated with filgrastim followed by repeat peripheral blood stem cell
harvesting.
e) A 15 to 25 liter large volume whole blood pheresis will be performed in
the NII-I
DTM via a 2-armed approach or via a temporary central venous catheter in the
femoral
position using the Baxter CS3000Plus, Cobe Spectra, or an equivalent
instrument. This
procedure typically takes 4 to 6 hours.
Apheresis procedure will typically use ACD-A anti-coagulant; alternatively,
partial anti-coagulation with heparin may be utilized.
The apheresis product will be cryopreserved and stored at ¨180 degrees Celsius
in a solution containing Plasmalyte A, Pentastarch, human serum albumin, DMSO,
and
preservative free heparin (10 U/ml).
h) The concentration of CD34+ cells in the apheresis product will be
determined by
flow cytometry, and the number of CD34+ cells in each cryopreserved bag
calculated.
i) If the donor and host are ABO incompatible, red blood cells will be
depleted
from the stem cell product by standard DTM protocols.
In Vitro Generation of Donor CD4+ Th2 Cells
a) Cryopreserved donor CD4+ T cells will be resuspended to a
concentration of 0.3
x 106 cells per ml. Media will consist of X-Vivo 20 supplemented with 5% heat-
inactivated autologous plasma.
b) The donor CD4+ T cells will be cultured in filtered flasks at 37 C in
5% CO2
humidified incubators. At the time of culture initiation, T cells will be
stimulated with
anti-CD3/anti-CD28 coated magnetic beads (3 to 1 ratio of beads to T cells).
c) At the time of co-culture initiation and on day 2 of culture, the
following
reagents will be added: recombinant human IL-4 (obtained through cross-filing
on
CTEP IND of Shering IL-4; 1000 I.U. per nil), and recombinant human IL-2
(purchased
from Chiron Therapeutics; 20 I.U. per m1).
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d) After day 2, cells will be maintained at a concentration of 0.25 to 1.0
x 106 cells
per ml by the addition of fresh X-Vivo 20 media supplemented with autologous
plasma
(5%), IL-2 (20 I.U./m1), and IL-4 (1000 I.U./m1).
e) The median cell volume will be determined using a Multisizer II
instrument
5. (Coulter). When the T cell volume approaches 500 fl (acceptable range of
650 to 350),
the T cells will be restimulated with anti-CD3/anti-CD28 beads; typically,
this time of
restimulation will be after 8 to 12 days of culture.
0 Bead restimulation will be at a bead to T cell ratio of 3:1. T
cell concentration
will be 0.2 x 106 cells/ml. Media will again consist of X-Vivo 20 supplemented
with
autologous plasma (5%), IL-2 (20 I.U./m1), and IL-4 (1000 I.U./m1).
g) After bead restimulation, CD4 cells will be maintained at a
concentration of 0.25
to 1.0 x 106 cells per ml by the addition of fresh X-Vivo 20 media
supplemented with
autologous plasma (5%), IL-2 (20 I.U./m1), and IL-4 (1000 I.U./m1).
h) When the CD4 cell mean cell volume approaches 500 fl (acceptable range
of
650 to 350), the cells will be harvested and cryopreserved by the NIH DTM
method in
protocol-relevant quantities for administration on study. It is anticipated
that the total
time of CD4 cell culture will be 15 to 20 days.
i) If an adequate numbers of CD4 cells is obtained, then such cells may be
available for administration on this protocol as a Th2 infusion.
j) The following will be the minimal phenotypic requirements of any
particular
Th2 cell culture to qualify for cryopreservation with subsequent
administration:
1. Presence of predominately CD4+ T cells by flow cytometry (greater than
70%
CD4+ T cells, and less than 5% contaminating CD8+ T cells).
2. In addition, the cryopreserved product will be tested for sterility with
both
fungal and bacterial cultures, through the ongoing testing done on cell
products
processed in the NIH Department of Transfusion Medicine. In addition, the CD4
Th2
cell product will be tested for endotoxin content by the limulus assay. Cell
products
positive for fungal, bacterial, or endotoxin content will be discarded.
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* Pre-transplant Induction Chemotherapy
a) After cell products have been harvested from the patient, chemotherapy
will be administered as an outpatient. All patients will receive at least one
cycle of
induction chemotherapy, even if their CD4 count is less than 50 cells per Id
at the time
of study entry. At this point in the protocol or earlier at the time of cell
harvesting,
placement of permanent central venous access may be requested.
Cycle 1 of Induction Chemotherapy
Drug Dose Days
Fludarabine 25 mg/m2 per day IV Days
1,2,3
Infusion over 30 minutes,
Daily for 3 days
Etoposide 50 mg/m2 per day continuous IV Days
1,2,3
Infusion over 24 hours, =
Daily for 3 days
Doxorubicin 10 mg/m2 per day continuous IV Days
1,2,3
Infusion over 24 hours,
Daily for 3 days
Vincristine 0.5 mg/m2 per day continuous IV Days
1,2,3
Infusion over 24 hours,
Daily for 3 days
Cyclophosphamide 600 mg/m2 IV Infusion over 2 hr. Day 4
Prednisone 60 mg/m2 per day orally,
Days 1,2,3,4
daily for 4 days
Filgrastim 10 ug/kg per day subcutaneously Daily
from day 5
Until ANC > 1000/u1
for two days
b) Fludarabine will be administered i.v. at a dose of 25 mg/m2 per day for
three days
(days 1, 2, and 3). Fludarabine will administered over a 30 minute interval.
Steroids
should not be used as an anti-emetic during this chemotherapy regimen.
d) Cyclophosphamide will be administered i.v. at a dose of 600 mg/m2 over
30
minutes on day 4.
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d) Etoposide will be administered at a dose of 50 mg/m2 per day by continuous
intravenous infusion for three days (days 1,2, and 3).
e) Doxorubicin will be administered at a dose of 10 mg/m2 per day by
continuous
intravenous infusion for three days (days 1,2, and 3).
0 Vincristine will be administered at a dose of 0.5 mg/m2 per day by
continuous
intravenous infusion for three days (days 1,2, and 3).
g) Prednisone will be administered at a dose of 60 mg/m2 per day orally for
four days
(days 1, 2, 3, and 4).
h) Filgrastrim will be initiated on day 5 at a dose of 10 pg/kg/day; G-CSF
will be =
continued until the ANC is greater than 1000 cells per ul on two consecutive
days.
Determination ofNumber of Cycles of Induction Chemotherapy
(a) Because the primary purpose of the induction chemotherapy is to
establish
severe host immune T cell depletion prior to the allotransplant, the number of
induction
chemotherapy cycles administered will be determined by the severity of immune
T cell
depletion observed.
(b) The CD4 count will be measured by flow cytometry in the interval of day
15 to
day 21 of the fludarabine/EPOCH chemotherapy. If there are 50 or more CD4
cells per
pi of blood during this interval, further cycles of induction chemotherapy
will be
administered (in an attempt to achieve greater immunosuppression prior to
transplantation). However, a maximum of three cycles of induction chemotherapy
will
be administered.
(c) Patients will receive the second cycle of chemotherapy on day 22 after
the first
cycle was initiated. However, an additional two weeks of recovery time before
administration of the second cycle may be provided if medically indicated (for
example,
for delay in neutrophil recovery, documented infection, or other complication
resulting
from the induction chemotherapy regimen).
(d) If there are less than 50 CD4 cells per ul of blood when measured
within days
15 to 21 after fludarabine/EPOCH administration, then that patient will
receive the
transplant preparative regimen.
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(e) If a patient develops neutropenia of less than 500 PMN's per I
for more than
seven days during any cycle of induction chemotherapy, the patient will
receive no
further induction chemotherapy. At that point, they will receive the
transplant
preparative regimen (even if the CD4 count is not less than 50 cells per I).
5 (f) A maximum of three cycles of induction chemotherapy can be
administered.
Patients will then proceed to the preparative regimen chemotherapy (even if
the CD4
count is still greater than 50 cells per 1).
(g) If a patient develops progressive disease at any point during
induction
chemotherapy cycles, such a patient will proceed to the transplant preparative
regimen
=
10 (independent of the CD4 count).
Determination of Cycle 2 and Cycle 3 Dose Escalation
a) If the first cycle of induction chemotherapy does not reduce the CD4
count to a
value below 50 cells per I and does- not result in febrile neutropenia or
prolonged
15 neutropenia as evidenced by two consecutive bi-weekly ANC values less
than 500 cells
per I, then the next cycle of induction chemotherapy may be dose escalated.
b) Dose escalation will consist of a 20% escalation in the daily dose of
fludarabine,
etoposide, adriamycin, and cyclophosphamide.
c) If a third cycle of chemotherapy is required (CD4 count still greater
than 50) and
20 febrile neutropenia or two timepoints of ANC less than 500 did not occur
after cycle 2,
then the third cycle of induction chemotherapy may be administered at a
further 20%
escalation of doses administered for cycle 2.
Dose Reduction of Pre-transplant Induction Chemotherapy
25 (a) In the event that more than one patient experiences a period of
neutropenia
(ANC less than 500 per 1) for more than 10 days, the etoposide, doxorubicin,
vincristine, and prednisone will be reduced from three days to two days of
administration. The doses of these medications will remain unchanged. In the
event of
this change, the cyclophos-phamide and filgrastim will be given on day 3.
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(b) The
same schedule modification described in subsection a) (above) will be
performed if any grade IV toxicity by the NCI Common Toxicity Criteria is
observed in
more than one patient.
Transplant Procedure: Preparative Regimen
a) On day 22 after the final cycle of induction chemotherapy, patients will
be
eligible to receive the following transplant preparative regimen. Therefore;
day 22 of
the final induction chemotherapy cycle will be transplant day ¨6. However, in
cases
where additional recovery time is required (for example, due to prolonged
neutropenia,
documented infection, or other medical complications of the induction
regimen), an
additional two weeks of recovery time may be utilized prior to initiation of
the
transplant preparative regimen.
Transplant Preparative Regimen
Drug Dose Days
Fludarabine 30 mg/m2 per day IV Infusion
Transplant Days ¨6,-5,-4,-3
over 30 minutes, daily for 4 days
Cyclophosphamide 1200 mg/m2 per day IV Infusion
Transplant Days ¨6,-5,-4,-3
over 2 hours, daily for 4 days
Mesna 1200 mg/m2 per
day by continuous Transplant Days ¨6,-5,-4,-3
IV Infusion, daily for 4 days
(start 1 hr before cyclophosphamide)
b) Fludarabine will be administered i.v. over 15 to 30 minutes at a dose of
30
mg/m2/day on days ¨6, -5, -4, and ¨3.
c) Cyclophosphamide will be administered at a dose of 1200 mg/m2/day over a
two
hour infusion on days ¨6, -5, -4, and ¨3.
d) Mesna will be administered at a dose of 1200 mg/m2 per day by continuous
i.v.
infusion on days ¨6, -5, -4, and ¨3. The mesna should be started one hr prior
to the
cyclophosphamide. Bag #1 of the mesna will be 150 mg/m2 in 250 ml over a 3 hr
infusion (thus stopping when cyclophosphamide ends). Then, mesna will be given
at
1200 mg/m2 in 500 ml over 24 hour infusion, for four days (days ¨6, -5, -4,
and ¨3).
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Transplant Procedure: GYHD Chemoprophylaxis With Cyclosporine (CSA)
a) Cyclosporine will be be initiated on the day ¨1 before the
transplant.
CSA will be administered by iv. infusion at a dose of 2 mg/kg. CSA will
administered
12 hours, with each infusion administered over a 2 hour period.
b) In the first two weeks post-transplant, CSA dose may be modified to
achieve
adequate steady-state CSA levels. Once this intravenous dose is established
and the
patient is able to tolerate oral feedings (typically by day 14 post-
transplant), then CSA
will be switched to the oral formulation. Conversion of CSA to the oral
formulation is
typically performed by multiplying the adequate iv. dose by a factor of 1.5 to
2Ø
Patients will then be maintained on oral CSA on a 12 hour schedule, with a
goal to
achieve steady state trough CSA levels of 200 ng/ml CSA (acceptable range: 150
to 250
ng/ml). =
c) This dose of CSA will continue until day 100 post-transplant, at
which point it
will be gradually tapered as long as the level of GVBD is less than grade 2.
Taper will
consist of a 5 to 10% dose reduction each week (patient will then be taken off
of CSA
by day 180 post-transplant).
Taper Step Days post-BMT CSA Dosage (mg/kg/dose)
Taper Step 1 101-107 95% of Maintenance Dose (M.D.)
Taper Step 2 108-114 90% of M.D.
Taper Step 3 115-121 85% of M.D.
Taper Step 4 122-128 80% of M.D.
Taper Step 5 129-135 70% of M.D.
Taper Step 6 136-142 60% of M.D.
Taper Step 7 143-149 50% of M.D.
Taper Step 8 150-156 40% of M.D.
Taper Step 8 157-163 30% of M.D.
Taper Step 10 164-170 20% of M.D.
Taper Step 1 1 171-180 10% of M.D.
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=
Transplant Procedure: Allogeneic Peripheral Blood Stem Cell Transplantation
a) On day 0, the patient will receive the cryopreserved PBSC.
b) The cryopreserved PBSC product will be thawed and administered
intravenously
immediately. The target dose of the PBSC is? 4 x 106 CD34+ cells per kg.
However,
if donor apheresis on days 5, 6, and 7 yields a total of? 3 x 106 CD34+ cells
per kg, this
level of CD34+ cell dose will also be allowed.
(a) No steroids will be allowed in the management of DMSO-related
tokicities
(chills, muscle aches) that may occur immediately after cellular infusion
(diphenhydramine and meperidine are allowed).
Transplant Procedure: Donor Th2 Cell Administration
a) On day 1 of the transplant procedure, the cryopreserved donor Th2 cells
will be
thawed and immediately administered intravenously.
b) No steroids will be allowed in the management of DMSO-related toxicities
(chills, muscle aches) that may occur immediately after cellular infusion
(diphenhydramine and meperidine are allowed).
c) The determination of whether a Th2 cell infusion is safe will be based
on the
presence or absence of hyperacute GVHD and of any grade 4 or 5 toxicity
attributable
to the Th2 cells that occurs in the first 14 days post-transplant.
d) For this study, hyperacute GVHD will be defined as a severe level of
acute
GVHD (grade 111 or IV) that occurs within the first 14 days post-transplant.
e) The initial three patients will be enrolled to 'Th2 cell dose level
#1 (5 x 106 Th2
cells/kg). If no hyperacute GVHD or grade 4 or 5 toxicity attributable to the
Th2 cells
is observed in these initial three patients, then it will be determined that
this dose level
is safe, and accrual to dose level #2 will commence. If hyperacute GVHD or
grade 4 or
5 toxicity attributable to the Th2 cells is observed in any of the initial
three patients,
then accrual to dose level #1 will be expanded to include a total of six
patients. If two
patients in six develop hyperacute GVHD or a grade IV toxicity related to the
Th2 cells,
then it will be determined that dose level #1 is not safe, and further accrual
to the study
will stop at that point. If only one of the six.patients experiences such an
adverse effect,
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84
then it will be determined that dose level #1 is safe, and accrual will
proceed to dose
level #2.
Three patients may then be enrolled to Th2 cell dose level #2(2.5 x 107 Th2
cells/kg). The same accrual and stopping rules will apply to this dose level
as those
used for dose level #1. As such, either three or six patients will be accrued
to dose level
#2.
If it is determined that Th2 cell dose level #2 is safe, accrual to the final
dose
level #3 will start (Th2 cell dose of 1.25 x 1.08 cells/kg). Six patients in
total will be
evaluated on dose level #3. If more than one patient on dose level #3 develops
hyperacute GVHD or grade 4 or 5 toxicity attributable to the 'Th2 cells, then
accrual to
dose level #3 will stop.
h) In the phase II component of this study, eighteen (18) additional
patients will
receive 'Th2 cells at either dose level #2 or level #3. To help ensure that
the Th2 cells
continue to be safely administered in this expanded cohort, the same accrual
and
stopping rules pertaining to severe toxicity attributed to the Th2 cells will
be continued.
Specifically, 24 total patients (6 in the Phase I cohort, 18 in the expanded
Phase II
cohort) will be evaluated at either Th2 cell dose level #2 or level #3.
Accrual and
stopping rules will be applied after each cohort of six patients. Therefore,
if at any
point, the frequency of severe toxicity attributable to the Th2 cells exceeds
1/6, 2/12,
3/18, or 4/24, then accrual to that treatment arm will be stopped.
Treatment of Persistent Disease Post-transplant: DLI and other therapy
(a) Patients with persistent or progressive malignant disease post-SCT will
be
eligible to receive donor lymphocytes ("delayed lymphocyte infusion" or DLI).
DLI
may be administered alone or after chemotherapy administration.
(b) Donor lymphocytes will be collected by apheresis, either in steady
state (no
donor therapy) or after G-CSF mobilization. The donor product may be enriched
for
lymphocytes by Ficoll-Hypaque procedure as per NTH DTM protocol.
Alternatively, in
cases where additional donor stem cells are desired, the donor product may be
administered without lymphocyte purification. DLI may be sequentially
administered,
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with initial dosing at 1 x 106 CD3+ T cells per kg, and with subsequent dose
increases
to 1 x 107 and 1 x 108 per kg.
(c) Alternatively, persistent or progressive disease may be treated
with any
approved therapy thought to be in the best standard care of the patient, such
as
5 chemotherapy, cytokine therapy, or monoclonal antibody therapy.
Alternatively,
patients with relapse may receive therapy on other NCI protocols.
Evaluation of Pre-transplant Induction Chemotherapy Cycles
a) Blood samples (10 cc in green-top heparinized tube) will be drawn to
evaluate
10 the effects of the combination fludarabine/EPOCH regimen on host immune
depletion.
b) This sample should be drawn just prior to each cycle of induction
chemotherapy (within six days of the next cycle).
c) Experiments will consist of flow cytometry to detect depletion of
lymphoid
versus myeloid subpopulations during induction chemotherapy.
Determination of Donor/Host Chimerism Post-Transplant
a) Blood samples (10 cc in green-top heparinized tube) will be drawn to
evaluate
the extent of donor versus host chimerism post-transplant. Samples will be
sent to the
Milwaukee Blood Banking Center for VNTR-PCR analysis of chimerism. If a result
of
mixed chimerism is obtained at day 15 post-transplant, subsequent draws may be
increased to 60 ml of blood so that cell sorting experiments can be performed
to
evaluate chimerism in cell subsets.
b) Timepoints for chimerism analysis will be at day 15, day 30, and day 100
post-
transplant. After day 100, chimerism may be determined if clinically indicated
(in the
setting of disease relapse).
c) Chimerism will be evaluated by a PCR-based assay, performed by the
Milwaukee Blood Banking Center.
. 30
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86
Statistical Section
a) One objective of this study is to establish a safe and feasible dose of
donor Th2
cells to administer after allogeneic PBSCT. Once such a determination is made,
eighteen (18) additional patients will be treated at that Th2 cell dose level
in order to
gain more safety information relating to Th2 cells, and to determine the
incidence and
severity of acute GNU) associated with allogeneic SCT containing Th2 cells.
b) The 18 additional Th2 recipients will either be treated at Th2 dose
level #2 (25 x
106/kg) or at Th2 dose level #3 (125 x 106/kg). The incidence of grade H to IV
acute
GVHD at Th2 dose level #2 is 2/6; results from Th2 dose level #3 are not
known, as
accrual to this cohort has just now been initiated. If Th2 dose level #3 is
associated
with unacceptable toxicity (more than 1/6 incidence of severe toxicity) or
significant
GVHD (more than 2/6 cases of grade II to IV acute GVHD), the additional 18
patients
will be treated on 'Th2 dose level #2. If recipients of Th2 dose level #3 have
0/6 or 1/6
cases of severe toxicity and 0/6, 1/6, or 2/6 cases of grade II to IV acute
GVHD, the
additional 18 patients will be treated at dose level #3. In the event that the
high dose of
Th2 cells can not be consistently generated, then the phase II component of
accrual may
be initiated at dose level #2.
c) The incidence and severity of acute GVHD in the cohort of 24 patients
receiving
Th2 cells at dose level #2 or #3 will be determined, and compared to the
initial protocol
cohort of 19 patients receiving transplantation without Th2 cells. In this
protocol, we
hypothesize that recipients of the Th2 cells will have reduced GVHD relative
to non-
Th2 recipients. In the cohort of non-Th2 recipients, the incidence of grade II
to grade
IV acute GVHD was 12/19. Based on this experience, one can conclude that the
true
rate of grade II to IV GVHD without Th2 cells is approximately 60%. In this
protocol,
we hypothesize that the expanded cohort of n=24 Th2 recipients will have a
significantly reduced incidence of grade II to IV acute GVHD. Based on our
current
results, we predict that the incidence of grade II to IV acute GVHD will be
reduced
from 60% without Th2 cells to 20% with Th2 cells. The predicted power to
detect a
Th2-mediated reduction in gradeII to IV acute GVHD from 60% to 20% in the
expanded Th2 cohort will depend on the incidence of grade II to IV GVHD
observed on
that arm during the phase I aspect of patient accrual. Using a two-tailed
conditional
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87
power statistical analysis at the p = 0.05 level, accrual of 18 additional
subjects to a Th2
cell treatment arm will provide either 72%, 87%, or 95% power to detect a Th2-
mediated reduction in the incidence of grade II to IV GVIM from 60% to 20%.
Specifically, the initial incidence, from the phase I accrual, of grade II to
IV acute
GVHD for the Th2 cell dose selected for the phase II component will be either
2/6, 1/6,
or 0/6. For these conditions, the statistical power for detecting a reduction
in grade II to
IV GVHD from 60% to 20% would be 72%, 87%, or 95%, respectively.
d) To help ensure that the Th2 cells continue to be safely administered in
the
expanded cohort, the same accrual and stopping rules pertaining to severe
toxicity
attributed to the Th2 cells will be continued. Specifically, 24 total patients
(6 in the
Phase I cohort, 18 in the expanded Phase II cohort) will be evaluated at
either Th2 cell
dose level #2 or level #3. Accrual and stopping rules pertaining to severe
toxicity
attributable to Th2 cells will be applied after each cohort of six patients.
Therefore, if at
any point, the frequency of severe toxicity attributable to the Th2 cells
exceeds 1/6,
2/12, 3/18, or 4/24, then accrual to that treatment arm will be stopped.
e) An additional accrual and stopping rule pertaining to acute GVHD will be
utilized in the expanded Phase II cohort. The incidence of grade II to IV
acute GVHD
in non-Th2 recipients was 12/19, or 63%. In the expanded cohort of Th2
recipients, the
incidence of grade ll to IV acute G'VHD will be calculated on an ongoing basis
and
reviewed at the weekly protocol meeting. If at any point in protocol
implementation the
incidence of grade II to IV acute GVIID in Th2 recipients is 60% or greater,
then
further accrual to the protocol will be stopped. Up to 2/6 cases of grade II
to IV acute
GVHD will be allowed for expansion of Th2 accrual to the phase II component.
Therefore, it is possible that the phase II component of the Th2 accrual may
be stopped
after 4 patients (in the event that each develops grade II to IV acute GVHD).
Results
We demonstrated in murine models that Th2-mediated regulation of GVHD is
not associated with an increased rate of graft rejection- In these studies,
supplementation of marrow allografts with Th2 cells represents a strategy for
reducing
the detrimental aspect of allogeneic T cell administration (GVHD) while
preserving the
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beneficial ability of donor T cells to prevent allograft rejection. In this
clinical protocol,
the G-CSF mobilized allograft contains approximately 1 x 108 CD4+ T cells per
kg of
=
recipient weight. In the phase I aspect of this protocol, patients will
receive additional
donor Th2 cells, with the final Th2 cell dose being 1.25 x 108 Th2 cells per
kg of
recipient weight. As such, this design allows for a safety evaluation for
administering
donor Th2 cells in a dose range that we hypothesize would be associated with a
reduction in GVHD (a 1:1 ratio of unmodified donor CD4+ T cells to donor' Th2
cells).
Through administration of Th2 cells on the day following peripheral blood stem
cell
transplantation, we hypothesize that the unmanipulated T cells contained in
the
mobilized stem cell product will maintain their ability to prevent graft
rejection but will
have a reduced capacity to induce severe acute GVHD.
Complete Donor Engrafiment and Development of GVHD
In this pilot study, we will utilize an induction chemotherapy regimen
consisting
of fludarabine in combination with the agents contained in the EPOCH regimen.
The
primary purpose of administering this chemotherapy cycle is to achieve a high
level of
host immunosuppression prior to allotransplantation. Our murine data indicate
that very
severe levels of host T cell depletion are required for the engraftnient of
fully-MHC
mismatched allografts after fludarabine-based chemotherapy. As such, the
development
of induction chemotherapy regimens which induce severe host T cell depletion
without
myeloablation is a highly desirable goal. To develop such therapies, we have
attempted
to reduce the CD4 count to less than 50 cells per ftlprior to administration
of the
transplant preparative regimen. This level of host CD4+ T cell depletion is
associated
with significant immunosuppression and a reduced ability to reject allogeneic
cells in
patients with B cell malignancy.
Seven patients were treated with this fludarabine and EPOCH induction
chemotherapy regimen prior to allogeneic PBSCT. In each case, we have noted a
marked reduction in patient T cells, and have also observed either stable
disease or
partial disease responses to the chemotherapy. As such, we have observed that
the
induction chemotherapy regimen to be utilized on this pilot study achieves two
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important conditions prior to allogeneic PBS CT: immune depletion for the
purpose of
preventing graft rejection, and reduction or stabilization in malignant
disease.
After such induction chemotherapy, patients receive preparative regimen
chemotherapy consisting of fludarabine and cyclophosphamide. In the initial
six
patients treated with this regimen, rapid and complete donor engraftment has
been
observed in all recipients (98 to 100% donor elements by day 14 post-
transplant). As
=
such, this immunoablative induction and preparative regimen chemotherapy is
very
effective for the prevention of allogeneic stem cell graft rejection in the
non-
myeloablative transplant setting.
Allogeneic PBSCT in the Treatment of Leukemia and Lymphoid Neoplasia
Allogeneic bone marrow transplantation represents a potentially curative
treatment for patients with multiple hematologic and lymphoid malignancies.
The
allogeneic graft-versus-leukemia effect contributes to disease remission in
acute
lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic
leukemia,
chronic myelogenous leukemia, indolent and high-grade non-Hodgkin's lymphoma,
Hodgkin's lymphoma, multiple myeloma, and myelodysplastic syndrome. Because
the
EPOCH regimen has an established response rate in patients with chemotherapy-
refractory lymphoid malignancy, such patients will be eligible for this
protocol. The
addition of fludarabine to EPOCH may further improve the anti-tumor effects of
this
regimen. However, the activity of fludarabine and EPOCH chemotherapy in
patients
with leukemia is not known. As such, patients with leukemia (AML,
myelodysplasia,
ALL, and CML) will be candidates for this protocol.
Allogeneic SCT With Th2 Cells: Initial Phase I Results
In this protocol, donor CD4 cells are cultured in vitro to enhance Th2
differentiation and are administered on day 1 post-SCT. In the initial Th2
cohort (5 x
106 cells/kg; n=3), no serious adverse events attributable to the Th2 cells
were
identified. Acute GVIID grade II (n=2) and grade III (n=1) were observed_ As
such,
there was no apparent decrease in acute GVHD in this first Th2 dose cohort. In
the
_ .
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second Th2 dose cohort (25 x 106 cells/kg; n=6), the initial patient entered a
pathologic
complete remission from refractory bulky lymphoma, but died of DIC and shock
at day
22 post-SCT (had grade II clinical GVHD). Subsequent patients at Th2 level #2
engrafted with full donor chimerism without significant toxicity, and appear
to have
5 reduced acute GVHD [grade 0 acute GVHD (n=4); liver only acute GVHD grade
III
(n=1)]. Th2 recipients have had rapid recovery of hematopoiesis, with full
donor
chimerism; the Th2 cells thus do not appear to impair engraftment. Anti-tumor
responses have been observed in refractory malignancy patients, including a
molecular
CR in a patient with accelerated phase CML. Because this Th2 dose level #2
cohort has
10 achieved alloengraftment with documented anti-tumor responses and
limited GVHD
(2/6 grade II-W acute GVHD), this cohort is a candidate for evaluation in the
phase II
aspect of the protocol.
The Th2 level #3 is about 125 x 106 cells/kg; n=6. If the safety and
feasibility of
15 dose level #3 is demonstrated in the initial six subjects, 18 additional
subjects will be
treated with Th2 cells at dose level #3 (125 x 106 cells/kg). In the event
that dose level
#3 results in more than 1/6 Th2-related adverse events or more than 2/6 cases
of grade
II to IV acute GVHD, the additional 18 subjects will be treated at the already
established Th2 cell dose level #2 (25 x 106 cells/kg). As such, 24 total
patients will be
20 treated with a defined dose of Th2 cells, either 25 or 125 x 106/kg. The
rate and
severity of acute GVHD in these 'Th2 recipients will be compared to the
initial protocol
cohort that did not receive Th2 cells (12/19 with grade II to III acute GVHD).
- 30
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=
Table 2: Overall Study Design:
6/6 HLA-matched (a) Apheresis with Elutriation
(lymphocyte
Sibling Donor harvest)
(b) In Vitro Th2 Generation, cryopreservation
("C) G-CSF treatment, stem cell harvest.
Transplant Recipient
Induction Chemotherapy (1 to 3 cycles; 21 d cycles; cycles 2 and 3 may be
administered
in a dose-escalated fashion, as detailed on page 16-18 of protocol)
(a) Fludarabine, 25 mg/Itn2 per day (30 nun. i.v.infusion); days 1-3
(b) Etoposide, 50 mg/e per day (continuous infusion); days 1-3
(c) Doxorubicin, 10 mg/m2 per day (continuous infusion); days 1-3
(d) Vincristine, 0.5 mg/m2 per day (continuous infusion); days 1-3
(e) Cyclophosphamide, 600 mg/m2 (30 min. i.v.infusion); day 4
(f) Prednisone, 60 mg/m2per day orally; days 1-4
(g) Filgrastirn, 10 ug/kg per day, subcutaneously; days 5 onward until ANC >
5000/u1
Transplant Preparative Regimen Chemotherapy
a) Fludarabine, 30 mg/m2 per day (30 min. i.v.infusion), daily for 4 days;
days ¨6, -5, -4, -3 of SCT
b) Cyclophosphamide, 1200 mg/m2 per day IV over 2 his, daily for 4 days;
days ¨6, -5, -4, -3 of SCT
c) Mesna, 1200 mg/m2 per day by continuous IV infusion, daily for 4 days;
days¨ti, -5, -4, -3 of SCT
Allogeneic SCT: Phase I/II Evaluation of Th2 Cells (Sequential Enrollment)
: Treatment Allocation
Day 0: Mobilized PBSCT (>4 x 106 CD34/kg)
=
Cyclosporine A GVHD Prophylaxis
`11r
Th2 Cell Administration (Day 1 of PBSCT)
Phase I Th2 Phase I Th2 Phase I Th2
Dose Level 1 Dose Level 2 Dose Level 3 Phase II Th2 Arm
(5 x 106/kg) (25 x 106/kg) (125x 106/kg) (25 or 125 x 106/kg) ,
3 patients 6 patients 6 patients 18 patients
=
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92
The scope of the claims should not be limited by the preferred embodiments
and examples, but should be given the broadest interpretation consistent with
the
description as a whole.
