Note: Descriptions are shown in the official language in which they were submitted.
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AUTOLOGOUS IMMUNE CELL THERAPY: CELL COMPOSITIONS,
METHODS AND APPLICATIONS TO TREATMENT OF HUMAN DISEASE
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. application Serial
No. 08/506,668, filed July 25, 1995, by Micheal Gruenberg, entitled
PROCESS FOR PRODUCING EFFECTOR IMMUNE CELLS FOR USE IN
5 ADOPTIVE CELLULAR IMMUNOTHERAPY. Benefit of priority thereto is
herein claimed.
This application is also related to U.S. application Serial No.
08/506,173, filed July 25, 1995, by Micheal Gruenberg, entitled CELL
GROWING DEVICE FOR IN VITRO CELL POPULA TION EXPANSION.
For purposes of the U.S. national stage, the subject matter of each
of U.S. application Serial Nos. 08/506,668 and 08/506,173 is herein
incorporated by reference its entirety.
FIELD OF INVENTION
This invention is directed to methods of adoptive immunotherapy.
15 In particular, methods of autologous cell therapy are provided.
Compositions containing substantially homogeneous populations of
functionally or phenotypically defined immune cells that have been
isolated from a patient, differentiated and/or expanded ex vivo are
provided. Uses of such compositions for treating or preventing disease or
20 otherwise altering the immune status of the patient by reinfusing such
cells are also provided.
BACKGROUND OF INVENTION
~ T Iymphocytes are immune cells that are primarily responsible forprotection against intracellular pathogens and suppression or elimination
25 of certain tumors. Mature T Iymphocytes, which all express the CD3 cell
surface antigen, are subdivided into two subtypes, based on expression
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Z
of either the CD4 or CD8 surface antigen. CD4+T cells recognize
antigen presented in association with class ll major histocompatibility
complex (MHC) molecules. CD4+ cells are generally involved in
regulatory functions in immune responses by virtue of the cytokines they
5 produce. These cytokines, such as IL-2, mediate an immune cell attack
on a pathogen or an antibody attack against an invading organism.
CD8+T cells recognize antigen presented in association with
class I MHC molecules. CD8+ cells are involved in effector functions in
immune responses, such as cytotoxic destruction of cells bearing foreign
10 antigens. The cells that mediate these responses are designated
cytotoxic T Iymphocytes (CTLs). These cells, which are generally CD8+
cells (although some are CD4+) represent a mechanism for resistance to
viral infections and tumors. The effector function of CTLs is dependent
upon the cytokine production from CD4+ regulatory cells.
15 Adoptive immunotherapy
Adoptive immunotherapy is an experimental treatment method
designed to boost a patient's immune response against a virus or a
tumor. The method involves the removal of immune cells from an
individual, the forming of effector cells outside the body (ex vivo), the
20 expansion of the cells to clinically-relevant numbers and the re-infusion of
the cells into the patient. Adoptive immunotherapy protocols have not
been made commercially available and are not in widespread use because
of the extreme toxicities associated with the infusion of the interleukin-2
(IL-2) with the cells. IL-2 is used in these protocols to cause the
25 differentiation and/or expansion of effector immune cells. Immune cells
cultivated in IL-2, however, become dependent on the cytokine for con-
tinued viability and effector function, thus necessitating the infusion of IL-
2 together with the effector cells. All adoptive immunotherapy protocols
involving differentiated effector cells incorporate the use of IL-2.
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The severe toxicity associated with the use of IL-2 has limited the
application of adoptive immunotherapy to the treatment of terminally-ill
cancer patients and the treatment of viral infections in AIDS patients.
Adoptive immunotherapy and the use thereof for treating cancer
The first attempts at adoptive immunotherapy in humans employed
Iymphokine activated killer (LAK) cells, which are immune effector cells
functionally defined by their ability to Iyse fresh tumors. LAK cells are
prociuced when peripheral blood mononuclear cells are exposed to high
concentrations of IL-2 ex vivo [see, e.g., Grimm, et ak (1982) J. EXP.
Med. 155:1832]. To produce LAK cells for use in treating cancer
patients [see, U.S. Patent No. 4,690,915], leukocytes are removed from
a cancer patient and exposed to high levels of IL-2 for 3-6 days, which
causes a portion of the cells to differentiate into LAK cells. The resulting
heterogeneous population of cells is reinfused to the donor concomitant
with a high systemic dose of IL-2. As noted, the high systemic doses of
IL-2 are highly toxic and not well tolerated.
Methods in which the potency of LAK cells is increased have been
developed. It has been observed [see, e.g., U.S. Patent No. 4,849,329]
that the addition of an L-amino acid with IL-2 during the ex vivo
differentiation step increases the LAK activity of the resulting cells 4-5
fold. Administration of LAK cells with IL-2 and an ornithine
decarboxylase inhibitor enhances the effectiveness of the treatment [see,
U.S. Patent No. 5,002,879]. Exposure of Iymphocytes to an anti-CD3
monoclonal antibody (mAb) during the LAK differentiation stage of the
process produces effector cells with enhanced anti-tumor activity [U.S.
Patent No. 5,326,763], and use of IL-7, with or without IL-2, in the LAK
differentiation step can also produce more potent LAK effector cells [see,
U.S. Patent No. 5,229,115]. The administration of GM-CSF with IL-2
has also been reported to cause an increase in LAK activity [see
=
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Takahashi, et al. (1995) Jao. J. Cancer Res. 86:861]. All protocols,
however, require administration of IL-2.
Early clinical results of adoptive immunotherapy using LAK cells in
terminally-ill cancer patients, particularly those with malignant melanoma,
5 had reported response rates of 21-44% ~see, e.g., Rosenberg et aL
(1985) N. Engl. J. Med. 313:1485 and Rosenberg et aL (1987) N. Enql.
J. Med. 316:889]. Results of more recent phase ll clinical studies, while
stili showing promise, have produced a broad range of response rates
from 0-33% [see, e.a., Dillman, et ak (1991) J. Clin. Oncol. 9:1233.
10 Thompson, J.A. et ai. (1992) J. Clin. Oncol. 10:960); Foon, et aL (1992)
J. Immunother. 11:1984 and Koretz, etal. (1991) Arch. Surq. 126:898].
The differences in response rates are attributed, partly, to variations in
dosages of LAK cells and IL-2 administrated, and the differences in
tumor-killing activities of the heterogeneous populations of LAK cells
15 generated from different patients.
Methods for generating a relatively homogenous population of LAK
cells for adoptive immunotherapy have been developed [see, U.S. Patent
No. 5,057,423]. The process described in U.S. Patent No. 5,057,423
involves first purifying a population of LAK progenitor cells (LGL) from the
20 peripheral blood mononuclear cells. These LGL are then exposed to IL-2,
which causes a majority of the LGL to differentiate into LAK cells. The
resulting effector cells, known as A-LAK, have been shown to be
effective in killing human carcinoma in nude mice [see, Sacchi (1991) et
al. Int. J. Cancer 47:784; Boiardi, et al. (1994) Cancer Immunol.
25 Immunoth. 39: 193]. It is exceedingly difficult, however, to produce
sufficient numbers of A-LAK from humans. Even with the use of feeder
cells to improve ex vivo expansion, A-LAK cultures from approximately
60% of cancer patients demonstrated inadequate expansion [see,
Sedlmayr, et al. (1991) J. Immunother. 10:336].
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Another adoptive immunotherapy protocol involves the
administration of autologous tumor infiltrating Iymphocytes (TIL) to
cancer patients. TIL cells are more potent at killing tumors than LA K cells
in animal experiments, but are difficult and expensive to generate for
5 treatment of patients. TIL cells are autologous effector cells
differentiated in vivo in solid tumors [see, U.S. Patent No. 5,126,1 32,
which describes a method for generating TIL cells for adoptive
immunotherapy of cancer~. TIL cells are produced by removing a tumor
sample from a patient, isolating Iymphocytes that were infiltrating into
10 the tumor sample, growing these TIL cells ex vivo in the presence of IL-2
and reinfusing the cells to the patient along with IL-2. A 60 % response
rate in evaluable cancer patients using this protocol has been reported
[see/ Rosenberg, ~ !988! N. ~q!. J. Med. 31g:1676]. Arothcr
study reported a 23 % response rate [see, Dillman, et al. (1991) Cancer
15 68:1]. It, however, has been difficult to consistently propagate sufficient
numbers of TIL cells for use in adoptive immunotherapy protocols.
In addition, the type of immune cells derived from TIL cultures are
extremely variable. The cells recovered from tumor samples contain pure
or mixed populations of cells with differing activities and potencies.
20 Some cells are produced with MHC-restricted anti-tumor cytolytic
activity, some with non-MHC restricted anti-tumor cytolytic activity and
some without any anti-tumorcytolytic activity. Also, other than cultures
derived from melanomas, cultures of TIL cells rarely produce tumor-
specific cells from patients with solid tumors; and tumor-specific cells are
25 produced only from about 50-75 % of patients with metastatic melanoma.
Because TIL cell therapy is associated with extreme toxicity
associated with infusion of IL-2, efforts have been made to enhance the
efficacy of the treatment. For example, addition of IL-10 with IL-2 has
been shown to increase the anti-tumor function of TIL cells in mice [see,
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Yang, et al. (1 ~395) J. Immunol. 155:3897. Increasing the IL-6
concentration at the tumor site has also been shown to result in
enhanced anti-tumor activity in TIL cells from mice [see, Marcus, et al.
(1994) J. Immunoth. Emohasis Tumor Immunol. 15:105]. The anti-tumor
5 activity of TIL cells is also increased by activating tumor draining Iymph
node cells with anti-CD3 mAb in the presence of IL-1 rsee, Hammel, et aL
(1994) J. Immunoth. Emohasis Tumor Immunol. 16:1].
Because of the variability in the effector function of cells derived
from tumor infiltrates or draining Iymph nodes, effort is being invested in
10 development of methods to promote the ex vivo sensitization of tumor-
reactive immune cells for use in adoptive immunotherapy of cancer.
Tumor-antigen specific, MHC-restricted CTL from precursor cells present
in the cellular infiltrates of breast cancer patients have been produced by
incubating precursor cells with recombinant avipox MAGE-1 [a marker
15 present on a class of tumors], causing the formation of MAGE-1 specific
CTL [(MAGE-1 and other MAGE antigens are antigens expressed on
tumor cells); see Toso, et al. (1996) Cancer Research 56:16; see, also
U.S. Patent No. 5,512,444]. Another ex vivo sensitization method for
generating potent MHC-restricted CTL involves the incubation of
20 peripheral blood mononuclear cells (PBMC) from melanoma patients with
autologous, irradiated PBMC that have been pulsed with synthetic
peptides of gp100, a melanoma-associated antigen Lsee, Salgaller, et al.
(1995) Cancer Research 55:4972].
An alternative to TIL cells in adoptive immunotherapy of cancer are
25 "ALT" cells. These cells are ex vivo activated peripheral blood
Iymphocytes with CTL activity. They are activated in an IL-2-containing
supernatant derived from a previously prepared one-way mixed
Iymphocyte culture or by using cytokine-rich, autologous supernatant
harvested from a previous Iymphocyte culture stimulated with anti-CD3
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mAb. Monthly infusions of ALT cells, combined with daily oral cimetidine
(to reduce tumor-associated suppressor activity), significantly prolongs
survival and induces durable tumor responses in renal cell carcinoma and
melanoma patients [see, Graham, et ak (1993) Semin. Urol. 11 :27 and
Gold, et ak (1996) J. Surg. Res. 59:279].
Other effector immune cells have been used or proposed for
adoptive immunotherapy of cancer. For example, the PWM-AK cell has
been proposed as a possible candidate for adoptive immunotherapy of
cancer. These effector cells are pokeweed mitogen activated PBMC with
similar activity to LAK cells [see, Ohno, et ak (1994) Int. J.
Immunopharm. 16:761]. Human activated macrophages (MAK) have also
been proposed as effector cells in adoptive immunotherapy of cancer.
The MAK cells are differentiated from the peripheral blood by activation
with interferon-y (IFN-y) and have been shown to cause regression of
experimental tumors in animals, but have not shown a clear therapeutic
response in humans [see, Bartholeyns et al. (1994) Anticancer Research
14:2673]. Activated natural killer cells (ANK) have also been proposed
for use in adoptive immunotherapy of malignancies. ANK cells are
prepared by panning of peripheral blood stem cells on CD5/CD8 coated
flasks yielding a population enriched for monocytes or NK precursors and
then treating the cells with high concentrations of IL-2. A human-
derived, MHC non-restricted CTL clone (TALL-104) has also shown
promise for use in adoptive immunotherapy protocols for cancer
treatment when used in conjunction with IL-12 [see, Cesano, et al.
(1994) J. Clin. Invest. 94:1076]. Increasing interest in the use of MAK,
ANK and other mononuclear phagocytes in adoptive immunotherapy
protocois for treatment of cancer has led to the development of improved
methods to reproducibly harvest large numbers of functional human
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circulating blood monocytes by counterflow centrifugal elutriation [see,
Faradiji, et al. (1994) J. Immunol. Methods 174:297~.
An emerging adoptive immunotherapy strategy for treatment of
cancer is to isolate and/or generate antigen presenting cells such as
5 dendritic cells from a patient's blood, pulse the cells with tumor
fragments or antigenic peptides and then reintroduce the cells to the
patient tsee, Grabbe, et al. (1995) Immunol. Todav 16: 117] . Methods
for obtaining large numbers of dendritic cells from precursors in the blood
of adults have been described lsee, Romani, et al. (1994) J. EXP. Med.
180:83 and Bernhard, et al. (1995) Cancer Res. 55:1099].
Adoptive immunotherapy and the use thereof for treating viral ~lise~ses
Another application of immune cell adoptive immunotherapy is the
treatment of viral disease. Adoptive immunotherapy protocols using viral-
specific CD8 + and CD4+ effector cells have been developed for the
15 treatment of infections with CMV, EBV and HIV [see, Riddell et al. (1995)
Ann. Rev. Immunol. 13:545; van Lunzen, et al. (1995) Adv. EXP. Med.
Biol. 374:57; and Klimas, et al. (1994) AIDS 8:1073~. These protocols
involve purifying CD8 + T-cells from the peripheral blood of AIDS
patients, expanding the cells with phytohemagglutinin and IL-2 and re-
20 infusing the cells, with concomitant IL-2 infusion, to the patient [see,
Whiteside, et aL (1993) Blood 81 :2085; Klimas, et al. (1994) AIDS
8:1073; Riddell, et al. (1993) Curr. OPin. Immunol. 5:484; Torpey, et al.
(1993) Clin. Immunol. ImmunoPath. 68:263; Ho, et ai. (1993) Blood
81 :2093 and Riddell, et al. (1992) Science 257:238].
25 Methods for growing immune cells in vitro
A majority of adoptive immunotherapy protocols are hampered by
the inability to grow clinically relevant (i.e., therapeutically sufficient)
quantities of cells for infusion. An additional problem is that the
administration of high doses of IL-2 necessary to maintain LAK activity
.
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and CTL activity in vivo is associated with severe toxicity. Several
techniques have been reported for improving the growth of cells for
adoptive immunotherapy and for reducing the dosage requirement for
systemic administration of IL-2. None of these attempts to increase
activity provided a means to eliminate IL-2 from the protocol.
TIL cells activated with anti-CD3 mAb and expanded with
moderate amounts of IL-2 (100 U/ml) have been successfully used in
adoptive immunotherapy protocols using less toxic systemic doses of IL-2
[see, Goedegebuure, et al. (1995) J. Clin. Oncol. 13:1939, see, also,
Matsumura, et al. (1994) Cancer Research 54:2744]. In vivo
administration of anti-CD3 mAb with low doses of IL-2 has also been
suggested as an alternative adoptive immunotherapy strategy to lower
the requirement for systemic IL-2 [see, Nakajima, et al. (1994) Proc. Natl.
Acad. Sci. U.S.A. 91 :7889]. A method for expanding CD4+ cells with
helper and cytolytic function using immobilized anti-CD3 mAb and IL-2 in
rotary-tissue culture bags has also been described [see, Nakamura, Qt al.
(1993) Br. J. Cancer 67:865]. Co-culture of anti-tumor effector cells
activated with anti-CD3 mAb with lipopolysaccharide (LPS)-activated B-
cells has also been suggested as an alternative method for growing cells
Z0 for adoptive immunotherapy [see, Okamoto, et aL (1995) Cancer
Immunol. Immunoth. 40:173]. These cells are all subsequently expanded
with low doses of IL-2.
A combination of mAbs against CD3 and CD28 in the presence of
lower dose IL-2 induces efficient expansion of TIL cells [see, Mulder, Q
al. (1995) Cancer Immunol Immunoth. 41:293]. Anti-tumor CTL
generated by in vitro stimulation with synthetic peptides can grow as
long as 4 months in culture with low dose IL-2 (30 u/ml) [see, Salgaller,
et al. (1995) Cancer Research 55:4972]. IL-7 has been shown to
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support the growth of CTL for prolonged periods in the absence of
repeated stimulation [see, Lynch et aL (1994) J. EXP. Med. 179:31].
Low concentrations of IL-2 have also been used to grow TIL cells in
artificial capillary culture systems [see, Freedman, et al. (1994) J.
5 Immunoth. Emphasis Tumor immunol. 16(3):198].
The need for exogenous IL-2 in expansion of immune cells has
been obviated only by genetically modifying cells [see, e.a., U.S. Patent
No. 5,470,730]. All the methods for growing genetically unmodified
cells, however, require exogenous IL-2 to promote the differentiation
and/or growth of cells for use in adoptive immunotherapy protocols. All
methods require systemic administration of IL-2 to maintain activity of
such cells.
Despite the showing of efficacy of adoptive immunotherapy in
terminally-ill patients, the severe toxicity of the systematic dosages of IL-
2 required in adoptive immunotherapy protocols, the variability in the
effector function of cell compositions derived from individual patients, as
well as the difficulties in expanding clinically-relevant numbers of effector
cells has limited the use of adoptive immunotherapy. In particular, the
need for exogenous IL-2 limits the cells used in adoptive immunotherapy
to effector cells that can perform their functions over a limited period of
time. In order to exploit the potential of this treatment method, there is a
need to overcome the need for systemic IL-2 administration, and the
difficulties in obtaining large quanti~ies of cells. Thus, there is a need for
improved adoptive immunotherapy methods.
Therefore, it is an object herein to provide such improved methods.
In particular, it is an object herein to provide methods for expanding
immune cells for use in adoptive immunotherapy protocols without the
use of exogenous IL-2. It is also an object herein to provide methods to
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generate a large array of cell compositions, including compositions
containing regulatory cells, for use in adoptive immunotherapy protocols.
It is an object herein to provide means to produce compositions
containing clinically relevant numbers of such cells. he availability of a
5 an array of cell compositions permits the design of adoptive
immunotherapy protocols for a wide variety of diseases and immune
function alterations. Therefore, it is an object herein to provide methods
for treating various disorders and altering immune function.
SUMMARY OF THE INVENTION
Compositions containing clinically relevant numbers of the immune
cells are provided. The compositions contain regulatory immune cells,
effector immune cells or combinations thereof. In particular compositions
cortaini,.g cliric311y relevart numbers of regulatory imm.u"e cells,
especially Th1 and Th2 cells, for use in adoptive immunotherapy [herein
referred to as autologous cell therapy (ACT)] are provided. Methods for
generating the compositions containing the clinically relevant numbers of
immune cells for use in adoptive immunotherapy are provided. The
methods do not require use of IL-2. As a consequence, the expanded
immune cells do not require IL-2 to retain activity or to remain viable.
Also provided are methods of treatment of disorders, including
infectious diseases and autoimmune diseases. In addition, methods of
treatment for immunosuppression permitting organ or tissue
transplantation and methods for enhancement of vaccination protocols
are provided. The treatment methods use the compositions.
The compositions of regulatory cells provide a means to alter the
immunoregulatory balance of a patient, either locally or sytemically, by
- changing the predominant regulatory cell population. Because many
disease states occur with the loss of regulated balance of the immune
system that is normally maintained by regulatory immune cells, the
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availability of clinically-relevant numbers of regulatory immune cells
provides a means to correct these imbalances. This ability offers great
potential for treating a variety of diseases.
Methods for generating clinically relevant numbers of effector
5 immune cells and of regulatory immune cells are provided. In particular,
methods for generating substantially homogeneous populations of
clinically relevant numbers of regulatory immune cells, including Th1 and
Th2 cells, as well as Th1-like and Th2-like mononuclear cell populations
are provided. Methods for generating compositions containing clinically
10 relevant numbers of effector cells, such as CTLs, LAKS and TILS, that do
not require exogenous IL-2 are provided.
Also provided are methods for producing clinically relevant
quantities (i.e., therapeutically effective numbers, typically greater than
109, preferably greater than 1 o10) of autologous specific T cell types for
15 treatment of disease states where a relative deficiency of such cells is
observed. In particular, methods for producing clinically relevant numbers
of autologous, ex vivo derived Th1 T-cells from patients with disease
states where a Th2 cytokine profile predominates such as, but not limited
to, infectious and allergic diseases; and autologous, ex vivo derived Th2
20 T-cells in Th1-dominant diseases, such as, but not limited to ,chronic
inflammation and autoimmune diseases, for use in ACT protocols. The
resulting cell compositions are provided and the use of the compositions
in ACT protocols are provided.
Also provided are clinically relevant numbers of ex vivo derived
25 antigen-specific Th2 cells sensitized to a donor organ for use in ACT
protocols designed to provide specific immunosuppression for
transplantation procedures. Clinically relevant numbers of ex vivo derived
viral-specific Th1 cells for ACT protocols designed to provide protection
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from viral infection and thus serve as a viral vaccination strategy are also
provided .
Methods of use of regulatory immune cells in autologous cell
therapy (ACT) protocols to treat and prevent human disease are provided.
5 The ACT protocols designed to alter the immunoregulatory balance of a
patient in order to treat diseases where imbalances in regulatory cells
exist. In particular, ACT protocols designed to alter the
immunoregulatory balance of a patient in order to treat diseases where
imbalances in regulatory cells exist are provided.
The methods involve collecting peripheral blood mononuclear cells
from a patient and then expanding the cells by appropriate activation and
then mitogenic stimulation with a cell surface specific proteins or proteins
under conditions whereby clinically relevant numbers of the expanded cell
type are produced [typically 109, preferably 10'~, more preferably 101'r or
15 more depending upon the cell type and ultimate application]. If the
collected cells are not differentiated in vivo or require further
differentiation, then following collection and prior to expansion, the
method includes activating and causing differentiation of the cells Q vivo
under conditions whereby at least some of the cells differentiate into
20 regulatory or effector cells or other cell types. The resulting cells are then
reinfused into the donor to effect treatment. The desired cells may be
purified prior to reinfusion to provided a more homogeneous population.
Where required, differentiation of mononuclear cells is effected by
activating the cells with a mitogen in the presence of the appropriate
25 array of cytokines. This activation can be achieved by use of agents,
such as cytokines or mitogens or other growth promoting agents under
- environmental conditions conducive to development of a particular
phenotype. For example, if the cells are activated in the presence of
IFN-y, Th1 cell differentiation will be produced. If they are activated in
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the presence of IL-4, then Th2 cell differentiation will be produced. Such
activating agents include monoclonal antibodies for polyclonal activation,
and natural or synthetic antigens for specific activation presented in the
context of MHC molecules.
Expansion is effected by growing the cells under conditions in
which high cell densities can be achieved, whereby endogenous cytokines
will be retained in the vicinity of the growing cell population, and in the
presence of one or more mitogenic monoclonal antibodies or other cell
surface specific protein, other than IL-2 or other such cytokine that will
10 require co-infusion. Such conditions are preferably achieved by growing
the cells in a hollow fiber [HF] bioreactor.
Methods for treating various disorders using the resulting cells are
also provided. In effecting these methods, cells of a type that are found
to be deficient or in low relative amounts are infused into a patient. For
1~ example, infectious diseases or tumors may be treated by collecting
peripheral blood mononuclear cells from a patient; expanding the cells
under conditions whereby a composition containing a therapeutically
effective number of cells is produced; and infusing the resulting
composition of cells into the patient. In preferred embodiments, the cells
20 are specific for unique antigens in the vicinity of the site where an effect
is desired or are specific for a pathogen or tumor being treated. In other
preferred embodiments, effector cells, such as cytotoxic CD8+
T Iymphocytes (CTLs) that are specific for the pathogen or tumor are
infused or co-infused with regulatory cells.
In addition, methods for specific immunosuppression for trans-
plantation procedures are provided. These methods involve
administration of clinically relevant numbers of ex vivo derived antigen-
specific Th2 cells sensitized to a donor organ. In preferred embodiments
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the cells are specific for alloantigens or an antigen unique to the organ or
tissue being transplanted.
Also provided are vaccination methods and compositions for use as
vaccines. In particular the vaccines are formulated from clinically relevant
5 numbers of ex vivo-derived viral-specific Thl cells or Th2 cells (or Th1-
like or Th2-like populations of cells) that upon infusion provide protection
from viral infection and thus serve as a viral vaccination strategy.
Methods of altering the immunoregulatory balance of a patient by
infusing autologous, ex vivo derived and expanded regulatory immune
10 cells are provided. This method includes the steps of collecting peripheral
blood mononuclear cells from a patient, activating the cells ex vivo under
conditions whereby at least some, even one, of the cells differentiate into
the desired regulatory cells, expanding the regulatory cells, and infusing
the expanded regulatory cells into the donor to affect the
15 immunoregulatory balance. In particular, the infusion is not accompanied
by co-infusion of a cytokine, such as IL-2.
The method above is useful for therapeutic treatment of disorders
characterized by imbalances in regulatory immune cells. Specifically, the
methods provided herein can be used to develop treatments for chronic
20 inflammation in disorders such as, but not limited to, multiple sclerosis,
rheumatoid arthritis, Crohn's Disease, autoimmune thyroid disease and
inflammatory bowel disease; chronic infectious diseases such as
infections with human immunodeficiency virus, herpes simplex virus,
cytomegalovirus and hepatovirus; allergic and other hypersensitivity
25 disorders such as asthma; and provides a method for specific
immunosuppression in organ and tissue transplant procedures and a
method to provide immunoprotection in vaccination.
In preferred embodiments, the regulatory immune cells are either
Th1, Th2 or Th3 cells with a CD4+ or CD8+ phenotype. The cells will
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preferably have a "memory" phenotype (i.e., CD45R0+, L-selectin~),
which permit the cells to traffic to sites of inflammation. These cells are
preferably made to exert their regulatory function at a localized area of
the body by selectively expanding cells specific for an unique antigen
5 present at the site the regulatory effect of the cells is desired. For
example, in the treatment of rheumatoid arthritis, regulatory cells specific
for type ll collagen, which is present only in joint tissue, are preferred. In
the treatment of diabetes for preventing rejection of transplanted islet
cells, regulatory cells specific for insulin are preferred.
In other embodiments, the cells are effector cells that have been
expanded up to clinically relevant (i.e., therapeutically effective) numbers
without the use of IL-2 to promote expansion.
Also provided is a method for expanding immune cells without the
use of exogenous IL-2. The expansion of immune cells is preferably
15 caused by the inclusion of one or more mitogenic mAb in the culture
medium. The immune cells are preferably expanded under conditions in
which they grow to high density. Such high density can be achieved by
growing the cells in hollow fiber bioreactors with the molecular weight
cut-offs of the fibers that retain endogenously produced cytokines. Such
20 molecular weigh cut-off is preferably less than 14,000 daltons, more
preferably 6000 daltons.
Also provided are methods for producing clinically relevant
populations of virally purged CD4+ cells obtained from HIV+ patients.
The resulting virally purged CD4+ cells are then reinfused into the donor
25 patient in order to effect treatment of HIV. The cells may also be co-
infused with anti-HlV effector cells.
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DETAILED DESCRIPTION OF THE I~Kt~tKRED EMBODIMENTS
A. Definitions
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as is commonly understood by one of skill
5 in the art to which this invention belongs. All patents and publications
referred to herein are, unless noted otherwise, incorporated by reference
in their entirety.
As used herein, adoptive immunotherapy or cellular adoptive
immunotherapy refers to a method of treatment involving administration
10 of immunologically active cells. The cells used in the treatment are
generally obtained by venipuncture or leukopheresis either from the
individual to be treated (autologous treatment) or from another individual
(allogeneic). For purposes herein, autologous treatment is herein referred
to as autologous cell therapy (ACT).
As used herein, autologous cell therapy [ACT] is a therapeutic
method in which cells of the immune system are removed from an
individual, cultured and/or manipulated ex vivo or in vitro, and introduced
into the same individual as part of a therapeutic treatment.
As used herein, activating proteins are molecules that when
20 contacted with a T-cell population cause the cells to proliferate. T-cells
generally require two signals to proliferate. Activating proteins thus
encompasses the combination of proteins that provide the requisite
signals, which include an initial priming signal and a second co-
stimulatory signal. The first signal requires a single agent, such as anti-
25 CD3 mAb, anti-CD2 mAb, anti-TCR mAb, PHA, PMA, and other such
signals. The second signal requires one or more agents, such as anti-
- CD28, anti-CD40L, cytokines and other such signals. Thus activating
proteins include combinations of molecules including, but are not limited
to: cell surface protein specific monoclonal antibodies, fusion proteins
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containing ligands for a cell surface protein, ligands for such cell surface
proteins, or any molecule that specifically interacts with a cell surface
receptor on a mononuclear cell and indirectly or directly causes that cell
to proliferate. For purposes herein, when expanding effector cells, the
5 activating proteins are selected from among those that are not needed to
substantially maintain cell viability and function after expansion. Thus,
for example, IL-2 iS not an activating protein for purposes herein for
effec~or celi expansion. As noted, the methods herein provide a means to
produce cells, particularly effector, that do not require IL-2, and thus, in
10 preferred embodiments, IL-2 will not be used as an activating agent.
As used herein, a mitogenic monoclonal antibody is an activating
protein that is an antibody that when contacted with a cell directly or
indirectly provides one of the two requisite signals for T-cell mitogenesis.
Generally such antibodies will specifically bind to a cell surface receptor
15 thereby inducing signal transduction that leads to cell proliferation.
Suitable mitogenic antibodies may be identified empirically by testing
selected antibodies singly or in combination for the ability to increase
numbers of a specific effector cell. Suitable mitogenic antibodies or
combinations thereof will increase the number of cells in a selected time
20 period, typically 1 to 10 days, by at least about 50%, preferably about
100% and more preferably 150-200% or more, compared to the numbers
of cells in the absence of the antibody.
As used herein, a growth promoting substance is a substance, that
may be soluble or insoluble, that in some manner participates in or
25 induces cells to differentiate, activate, grow and/or divide. Growth
promoting substances include mitogens and cytokines. Examples of
growth promoting substances include the fibroblast growth factors,
osteogenin, which has been purified from demineralized bone lsee, e.q.,
Luyten, et al. (1989~ J. Biol. Chem. 264:13377]), epidermal growth
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~ factor, the products of oncogenes, the interleukins, colony stimulating
factors, and any other of such factors that are known to those of skill in
the art. Recombinantly-produced growth promoting substances, such as
recombinantly-produced interleukins, are suitable for use in the methods
5 herein. Means to clone DNA encoding such proteins and the means to
produce biologically active proteins from such cloned DNA are within the
skill in the art. For example, interleukins 1 through 6 and others have
been cioned. Various growth promoting substances and combinatïons
thereof may be used to expand desired subpopulations of Iymphoid cells.
As used herein, a mitogen is a substance that induces cells to
divide and in particular, as used herein, are substances that stimulate a
Iymphocyte population in an antigen-independent manner to proliferate
and differentiate into effector cells or regulatory cells. Examples of such
substances include lectins and lipopolysaccharides.
As used herein, a cytokine is a factor, such as Iymphokine or
monokine, that is produced by cells that affect the same or other cells.
As used herein, a Iymphokine is a substance that is produced and
secreted by activated T Iymphocytes and that affects the same or other
cell types. Tumor necrosis factor, the interleukins and the interferons are
20 examples of Iymphokines. A monokine is a substance that is secreted by
monocytes or macrophages that affects the same or other cells.
As used herein, a regulatory immune cell is any mononuclear cell
with a defined cytokine production profile and in which such cytokine
profile does not directly mediate an effector function. A regulatory
25 immune cell is a mononuclear cell that has the ability to control or direct
an immune response, but does not act as an effector cell in the response.
Regulatory immune cells exert their regulatory function by virtue of the
cytokines they produce and can be classified by virtue of their cytokine
production profile. For example, regulatory immune cells that produce IL-
~ = ~
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2 and IFN-y, but do not produce IL-4 are termed "Th1" cells. Regulatory
immune cells that produce IL-4 and IL-10, but do not produce IFN-yare
termed "Th2" cells. Regulatory immune cells that produce TGF-,B, IL-10
and IFN-y, but do not produce IL-2 or IL-4 are termed "Th3" cells. Cells
5 that produce Th1, Th2 and Th3 cytokine profiles occur in CD4+ and
CD8 ~ cell populations. Cells that produce IL-2, IL-4 and IFN-y are
thought to be precursors of Th1 and Th2 cells and are designated "ThO"
cells. Popuiations of cells that produce a majority of Th1 cytokines are
designated "Th1-like"; populations producing a majority of the Th2
10 cytokines are designated Th2-like"; those producing a majority of Th3
cytokines are designated "Th3-like". Thus, each composition, although
containing a heterogeneous population of cells, will have the properties
that are substantially similar, with respect to cytokine, to the particular
Th subset.
It is understood that this list of T- cells is exemplary only, and any
other definable population, array or subtype of T cells that can be
expanded by the methods herein to clinically relevant numbers are
intended herein.
As used herein, a composition containing a clinically relevant
20 number or population of immune cells is a composition that contains at
least 109, preferably greater than 109, more preferably at least 101~ cells,
and most preferably more than 1 o10 cells, in which the majority of the
cells have a defined regulatory or effector function, such as Th1 cells or
Th2 cells or effector cells, such as LAK, TIL and CTL cells. The preferred
25 number of cells will depend upon the ultimate use for which the
composition is intended as will the type of cell. For example, if Thl cells
that are specific for a particular antigen are desired, then the population
will contain greater than 50%, preferably greater than 70%, more
preferably greater than 80%, most preferably greater than 90-95% of
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such cells. If the population results from polyclonal expansion, the
homogeneous cells will be those that are a particular type or subtype.
For uses provided herein, the cells are preferably in a volume of a liter or
less, more preferably 500 mls or less, even more preferably 250 mls or
5 less and most preferably about 100 mls or less.
As used herein, predominant means greater than about 50%.
As used herein, a combination refers to two component items,
such as compositions or mixtures, that are intended for use either
together or sequentially. The combination may be provided as a mixture
10 of the components or as separate components packaged or provided
together, such as in a kit.
As used herein, effector cells are mononuclear cells that have the
ability to directly eliminate pathogens or tumor cells. Such cells include,
but are not limited to, LAK cells, MAK cells and other mononuclear phag-
15 ocytes, TlLs, CTLs and antibody-producing B cells and other such cells.
As used herein, immune balance refers to the normal ratios, and
absolute numbers, of various immune cells that are associated with a
disease free state. Restoration of immune balance refers to restoration to
a condition in which treatment of the disease or disorder is effected
20 whereby the ratios of regulatory immune cell types and numbers thereof
are within normal range or close enough thereto so that symptoms of the
treated disease or disorder are ameliorated. The amount of cells to
administer can be determined empirically, or, preferably, by administering
aliquots of cells to a patient until the symptoms of the disease or disorder
25 are reduced or eliminated. Generally a first dosage will be at least 109-
1 o10 cells. In addition, the dosage will vary depending upon treatment
sought.
-
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As used herein, therapeutically effective refers to an amount of
cells that is sufficient to ameliorate, or in some manner reduce the
symptoms associated with a disease. When used with reference to a
method, the method is sufficiently effective to ameliorate, or in some
5 manner reduce the symptoms associated with a disease.
As used herein, mononuclear or Iymphoid cells (the terms are used
interchangeably) include Iymphocytes, macrophages, and monocytes that
are derived from any tissue in which such cells are present. In general
Iymphoid cells are removed from an individual who is to be treated. The
10 Iymphoid cells may be derived from a tumor, peripheral blood, or other
tissues, such as the Iymph nodes and spleen that contain or produce
Iymphoid cells.
As used herein, therapeutically useful subpopulations of In vitro or
ex vivo expanded mononuclear or Iymphoid cells are cells that are
15 expanded upon exposure of the cells to a growth promoting substances,
such as Iymphokines, when the Iymphoid cells are cultured ex vivo. The
therapeutically useful subpopulations are regulatory cells or effector cells
and contain clinically relevant numbers of cells, typically at least about
109 or more cells, which are preferably in a clinically useful volume (i.e.,
20 for infusion) that is one liter or less.
As used herein, a therapeutically effective number or clinically-
relevant number ex vivo expanded cells is the number of such cells that is
at least sufficient to achieve a desired therapeutic effect, when such
cells are used in a particular method of ACT. Typically such number is at
25 least 109, and more preferably 1 o10 or more. The precise number will
depend upon the cell type and also the intended target or result.
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As used herein, a hollow fiber bioreactor or hollow fiber bioreactor
cartridge contains an outer shell casing that is suitable for the growth of
mammalian cells, a plurality of semi-permeable hollow fibers encased
within the shell that are suitable for the growth of mammalian cells on or
5 near them, and the ECS, which contains the cells and the ECS cell
supernatant. The interior of the hollow fibers is called the lumen and the
area between the outside of the capillaries to the inside of the outer
housing is caiieci tne extracapiiiary space [ECS].
Tissue culture medium perfuses through the fiber lumens and is
10 also included within the shell surrounding said fibers. The tissue culture
medium, which may differ in these two compartments, contains diffusible
components that are capable of sustaining and permitting proliferation of
immune cells. The medium is provided in a reservoir from which it is
pumped through the fibers. The flow rate can be controlled varied by the
15 varying the applied pressure. The ECS or perfusing medium may
additionally contain an effective amount of at least one growth promoting
or suppressing substance that specifically promotes the expansion or
suppression of at least one subpopulation of the immune cells, such as
TIL cells or regulatory cells, in which the effective amount is an amount
20 sufficient to effect said specific expansion.
As used herein, a hollow cell fiber culture system includes of a
hollow fiber bioreactor as well as pumping means for perfusing medium
through said system, reservoir means for providing and collecting
medium, and other components, including electronic controlling, recording
25 or sensing devices. A hollow fiber bioreactor is a cartridge that contains
of a multitude of semi-permeable tube-shaped fibers encased in a hollow
shell. The terms hollow fiber reactor and hollow fiber bioreactor are
used interchangeably. A preferred device for methods is that described in
copending, allowed, U.S. application Serial No. 08/506,173.
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As used herein, ECS refers to the extra-capillary space cell
supernatant. It is the medium in which the cells in the ECS are growing.
It contains secreted cellular products, diffusible nutrients and any growth
promoting or suppressing substances, such as Iymphokines and
5 cytokines, produced by the cultured immune cells or added to the ECS or
tissue culture medium. The particular components included in the ECS is
a function not only of what is inoculated therein, but also of the
characteristics oF the selected noiiow fiber.
As used herein, tissue culture medium includes any culture medium
10 that is suitable for the growth of mammalian cells ex vivo. Examples of
such medium include, but are not limited to AlM-V, RPMI 1640, and
Iscove's medium (GIBC0, Grand Island, N.Y.). The medium may be
supplemented with additional ingredients including serum, serum proteins,
growth suppressing, and growth promoting substances, such mitogenic
15 monoclonal antibodies and selective agents for selecting genetically
engineered or modified cells.
As used herein, treatment means any manner in which the
symptoms of a condition, disorder or disease are ameliorated or otherwise
beneficially altered. Treatment also encompasses any pharmaceutical use
20 of the compositions herein.
As used herein, a vaccine is a composition that provides protection
against a viral infection, cancer or other disorder or treatment for a viral
infection, cancer or other disorder. Protection against a viral infection,
cancer or other disorder will either completely prevent infection or the
25 tumor or other disorder or will reduce the severity or duration of infection, tumor or other disorder if subsequently infected or afflicted with the
disorder. Treatment will cause an amelioration in one or more symptoms
or a decrease in severity or duration.
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As used herein, amelioration of the symptoms of a particular
disorder by administration of a particular composition refers to any
lessening, whether permanent or temporary, lasting or transient that can
be attributed to or associated with administration of the composition.
As used herein, substantially pure means sufficiently homogeneous
to appear free of readily detectable impurities as determined by standard
methods of analysis, such as flow cytometry, used by those of skill in the
art to assess such purity, or sufficiently pure such that further purification
would not detectably alter the physical and chemical properties, such as
biological activities, of the substance. Methods for purification of the
immune cells to produce substantially pure populations are known to
those of skill in the art. A substantially pure cell population, may,
however, be a mixture of subtypes; purity refers to the activity profile of
the population. In such instances, further purification might increase the
specific activity of the cell population.
As used herein, biological activity refers to the in vivo activities of
immune cells or physiological responses that result upon in vivo
administration of a cell, composition or other mixture. Biological activity,
thus, encompasses therapeutic effects and pharmaceutical activity of
ZO such cells, compositions and mixtures.
Although any similar or equivalent methods and materials can be
employed in the practice and/or tests of the methods and cells provided
herein, preferred embodiments are now described.
B. Effector and regulatory immune cells
Encounter of a host with antigen can result in either cell-mediated
or humoral classes of immune response. Regulatory immune cells control
- the nature of an immune response to pathogens [see, Mosmann, et al.
(1986) J. Immunol. 136:2348; Cherwinski, et al. (1987) J. EXP. Med.
166: 1229; and Del Prete, et al. (1991) J . Clin. Invest. 88:346] . The
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different types of responses are attributable to the heterogeneity of CD4+
T cells. CD4+ cells can be sub-divided according to their cytokine
expression profiles. These cells are derived from a common precursor,
ThO, which can produce Th1, Th2 and Th3 cytokines [see, Firestein, et
5 aL (1989) J. Immunol. 143:518]. As noted above, Thl clones produce
IL-2, INF-y, Iymphotoxin and other factors responsible for promoting
delayed-type hypersensitivity reactions characteristic of cell-mediated
Immunity. These cells do not express IL-4 or IL-5. Th1 cells promote
cell-mediated inflammatory reactions, support macrophage activation,
10 immunoglobulin (Ig) isotype switching to IgG2a and activate cytotoxic
function .
Th2 clones produce cytokines, such as IL-4,11-5, IL-6,1L-10 and IL-
13, and thus direct humoral immune responses, and also promote allergic
type responses. Th2 cells do not express IL-2 and IFN-y. Th2 cells
15 provide help for B-cell activation, for switching to the IgG1 and IgE
isotypes and for antibody production [see, e.a., Mosmann et ak (1989)
Annu. Rev. Immunol. 7:145]. Th3 cell produce IL-4, IL-10 and TGF-,~.
The cytokines produced by Thl and Th2 cells are mutually
inhibitory. Thl cytokines inhibit the proliferation of Th2 cells and Th2
20 cytokines inhibit Th1 cytokine synthesis lsee, e.a., Fiorentino, et ak
(1989) Med. 170:2081 (1989). This cross regulation results in a
polarized Thl or Th2 immune response to pathogens that can result in
host resistance or susceptibility to infection.
Development of the appropriate regulatory immune cell response
25 during infection is important because certain pathogens are most
effectively controlled by either a predominantly Th1 or Th2 type immune
response [see, e.a., Sher, Q al. (1989) Ann. Rev. Immunol. 46:111;
Scott, et aL (1991) Immunol. Todav 12:346; Sher, Q ak (1992)
Immunol. Rev. 127:183; and Urban, et ak (1992) Immunol. Rev.
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127:205]. For example, a correlation has been found between the
predominant regulatory immune response and disease susceptibility in
leprosy [see, e.a., Yamamura, et al. (1991) Science 254:277] AIDS [see,
e.q., Clerici, et al. (1993) Immunol. Today 14:107], toxoplasma [see,
5 Sher, et al. (1989) Ann. Rev. Immunol. 46:111], Hashimoto's thyroiditis
[see, e.q., Del Prete, et al. (1989) Autoimmunity 4:267], Grave's disease
[see, e.q., Turner, et al. (1987) Eur. J. Immunol. 17:1807],
transpiantation lsee, e.a., Benvenuto, et al. (1gg1) TransPlantation
51:887], type 1 diabetes [see, e.a., Foulig, et al. (1991) J. Pathol.
165:97], multiple sclerosis [see, e.q., Benvenuto, et aL (1991) Clin. EXP.
Immunol. 84:97], and rheumatoid arthritis [see, e.q., Quayle, et al.
(1993) Scand. J. Immunol 38:75].
A Th1 response in mice to protozoan, viral and fungal infection is
associated with resistance, while a Th2 response is associated with
15 disease. A Th2 response cures certain helminth infections in mice and
exacerbates viral infections. A Th2 response has been correlated with
AIDS and autoimmune disease in humans and with allergic disorders and
transplant rejection. Another regulatory cell, designated Th3, produces
high amounts of TGF-,B and can protect mice from a disease similar to
20 multiple sclerosis [see, e.q., Chen, et aL (1994) Science 265:1237].
Categorization of these responses may be empirically determined and
have been documented [for a summary see, e.a., Mosmann et aL (1996)
Immunoloqv Todav 17: 138-146].
Subsets of CD8+ T-cells also are known to secrete a Thl- or Th2-
25 cytokine pattern. Exposure of CD8+ cells to IFN-y and IL-2 direct
differentiation into Th1 cells; whereas, lL-4 induces differentiation into
Th2 cells. Th1 CD8+ cells are thought to be important effectors in the
immune response to viruses, while Th2 CD8+ cells have an immuno-
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suppressive function. Other regulatory cells can be characterized by
methods similar to those used to characterize the above-described cells.
By virtue of the cross regulation and the immune imbalances
observed in disease states, as described herein, regulatory cells should be
therapeutic for the treatment of a variety of diseases. Such use has been
demonstrated to some extent in animal models, but has not been possible
to achieve in humans. For example, administration of native T-cells and
Th2 antigen-specific clones for Actinobacillus actinomycetemcomitans, in
combination did ameliorate periodontal disease in nude rats lsee,
Eastcott, et al. (1994) Oral Microbiol. Immunol. 9:284 (1994)]. Antigen-
specific Th1 cell clones have been shown to protect against infection
with the protozoan Leishmania major, genital infection with chlamydia
trachomatis and murine candidiasis [see, Powrie, et al. (1994) J. Exp.
Med. 179:589; Igietseme, et al. (1993) et al. Regional Immunitv 5:317;
and Romani (1991) Inf. Immun. 59:4647]. In addition, Th2 cell clones
have been shown to prevent autoimmune uveoretinitis [see Saoudi, et al.
(1993) Eur. J. Immunol. 23:3096]. An antigen-specific Th2 cell clone
has been shown to suppress an animal model of multiple sclerosis [see,
Chen, et al. (1994) Science 265:1237]. Donor-specific Th2 cells can
reduce lethal graft vs. host disease in transplantation lsee, Fowler, et al.
(1994) Adv. Bone Marrow Purq. Process., Fourth Int. SvmPos., Wiley-
Liss, Inc., p. 533]. Purified T-cells with enhanced Th2 activity have also
been shown to prevent insulin-dependent diabetes-like disease in animals.
See, Fowell et aL (1993) J. Exp. Med. 177:627.
While Th2 clones have been used in adoptive transfer studies in
animals, regulatory cells, including Th1 and Th2 cells, have not been used
in ACT protocols in humans. Such protocols are limited by the inability to
differentiate and produce therapeutically effective quantities of such
regulatory cells. The methods herein, however, provide a means to
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produce such clinically relevant quantities of cells, and, thereby provide a
means to ameliorate disorders, provide vaccines, and suppress tissue or
organ rejection. The methods herein also provide a means to produce
clinically relevant quantities of relulatory and effector cells in the absence
5 of IL-2.
Also provided herein, are methods for growing cells that are
therapeutically useful for treatment of HIV infection, including treatment
of A.l.~).S. by enchancing or restoring the immune system [see, e.a.,
Examples 3 and 4].
10 C. Methods for production of regulatory cells
A method for obtaining regulatory cells for use in ACT protocols is
provided herein. A method for obtaining effector cells for use in ACT
protocols without the need for exogenous agents, such as IL-2, that
sustain the viability of such cells is also provided. The method includes
15 some or all of the following steps: (1 ) collecting mononuclear cells from
a patient; (2) treating the cells ex vivo with that agents that cause some
or all of the cells to the differentiate into desired T cell subtypes;
(3) purifying the resulting cells; and (4) expanding these cells by
contacting them with a mitogenic agent that specifically interacts with a
20 cell surface receptor. Such agents are herein preferably mitogenic
monoclonal antibodies. The expanded cells may be further purified to
select for the desired subtype.
1. Collecting mononuclear cells
Mononuclear cells (i.e., Iymphocytes and monocytes) can be
25 obtained from a variety of sources, including, but not limited to,
peripheral blood, Iymphoid tissue, biopsy tissue or from body cavity
Iavage procedures. Preferably, the cells are obtained by simple
venipuncture (50-500 ml). When larger numbers of cells are required,
they may be obtained by a Iymphapheresis procedure. The mononuclear
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cells can be purified from the blood using Ficoll-Hypaque density gradient
centrifugation or any other suitable method.
a . Ex Vivo Differel ~ Iidt.~.,
Many studies have indicated that different antigens can cause a
5 selective induction of distinct immunoregulatory cell subsets, causing the
development of either a humoral or cell-mediated immune response.
Furthermore, many disease states are the result of the predominance of
the certain cell types. Recent advances in the understanding of the
mechanisms regulating the differentiation of T-cell subsets allows the
10 generation of selected subsets ex vivo.
Several factors, including the dose of antigen, the type of antigen
presenting cell and the MHC haplotype of an individual can affect the
differentiation of specific types of regulatory immune cells. Various
cytokines are also able to affect the type of regulatory response that
15 develops in a person. For example, it is known that the presence of IL-4
during initial T-cell activation gives rise to Th2-like cells [see, Hsieh, et ak(1992) Proc. Natl. Acad. Sci. U.S.A. 89:6065 and Paliard, et al. (1988)
et ak J. Immunol. 141:849]. Conversely, activation of cells in the
presence of IL-12 or interferon-gamma leads to the formation of Th1-like
20 cells [see, Sedar, et aL (1993) Proc. Natl. Acad. Sci. U.S.A. 90:10188].
Accordingly, in a preferred embodiment, the mononuclear cells
collected in the first step of the present process are next activated in the
presence of IL-12, interferon-gamma or IL-4 to cause the development of
Th1 or Th2 cells, respectively. To enhance the differentiation of
25 regulatory cells, antibodies to IL-12 and/or interferon-gamma can be used
to promote Th2 responses, while antibodies to IL-4 can be used to
promote the differentiation of Th1 cells. Antibodies or other proteins
specific for the IL-12, interferon-gamma or IL-4 receptor on T-cells could
also be used to provide a signal in place of the Iymphokines. The cells
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can be activated either non-specifically with chemical agents such as PHA
and PMA or with monoclonal antibodies such as anti-CD3 or anti-CD2.
Preferably, they are activated specifically with natural or man-made
protein antigens added to the medium, processed and presented by APC
to T-cells. It may be necessary in some cases to vaccinate the patient
prior to blood collection in order to increase the starting number of
antigen-specific cells. Another strategy is to oral tolerize patients prior to
blood collection. In cases where the cells generated are specific for a
known antigen, the antigen may also be used after the cell reinfusion as a
booster to increase the desired regulatory cells in vivo. Additional
strategies for effecting Th1 cell differentiation is to activate cells in the
presence of aB7.2 mAb or TGF-,B. Th2 differentiation also can be
promoted by activating cells in the presence of one or more of agents,
such as, one or more of the following: aB7.1 mAb, low antigen doses
and CTLA4/lg fusion protein (CTLA4 is a ligand for CD28). CD28 is
expressed on T-cells and antigen presenting cells.
The type of regulatory cells generated should be determined from
animal models of the disease. It is known that not all regulatory cells
within a classification are alike. For example, some Th2 cells secrete high
levels of IL-4 and low levels of IL-10, while others have increased levels
of IL-5. Other regulatory cells produce IL-10 and interferon-gamma.
Regulatory cells termed "Th3" cells secrete TGF-,~ and are deemed
preferential for treatment of multiple sclerosis.
b. Regulatory Cell Isolation
Most techniques for isolation of immune cell subsets are based on
the reactivity of mAb against T-cell surface antigens. Positive selection
- can be achieved by fluorescent-activated cell sorting [see, Reinherz, et al.
(1979) Proc. Natl. Acad. Sci. U.S.A. 76:4061]. Various panning
techniques where specific mAb are bound to plastic plates to capture the
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desired T-cell subsets can also be used. See, Lum, et aL t1982) Cell
Immunol. 72: 122.
Panning techniques can be used for negative selection as well,
depleting unwanted subsets with specific mAb [see, e.g., Engleman, et
al. (1981) J. Immunol. 127:2124]. The use of magnetic polymer beads
coated with mAb is a preferred method to isolate highly purified,
functionally intact Iymphoid cell populations by positive and negative
selection [see, e.s:l., Lea, et ak (1985) Scand. J. Immunol. 22:207; Lea,
et al. (1986) Scand. J. Immunol. 23:509) and Gaudernack, et al. (1986)
J. Immunol. Methods 90:179].
Since an antibody has not yet been described that can distinguish
regulatory immune cell subsets, efforts must be made to enhance the
desired population by purifying on the basis of certain cell surface
proteins. For example, CD30 positive [see, Manetti, et al. (1994) J. Exp.
Med. 180:2407], CD27 negative [see, Elson, et al. (1994) Int. Immunol.
6:1003] and CD7 negative [see, Autran, et al. (1995) J. Immunol.
154: 1408] cell populations have been shown to have the majority of Th2
cells. Also, repeatedly contacting the cells with anti-CD28 mAb is
another method for enhancing Th2 cells.
Another strategy for purification of regulatory cells is to expand the
cells in the presence of agents known to inhibit the growth of the
unwanted subset(s) of cell. Such agents include dexamethasone,
colchicine, CTLA4/lg fusion protein and progesterone, which inhibit Th2
cell growth. TGF-,B inhibits Th1 cell growth.
c. Regulatory Cell Expansion
Methods for expanding purified T-cells to clinically relevant
numbers ex vivo without the use of exogenous IL-2 are provided herein.
Although IL-2 could be used in the present methods, it is preferably to
grow cells without the addition of this cytokine. Cells exposed to IL-2 ex
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vivo may become dependent on the presence of IL-2 to maintain their
viability and function, requiring the systemic infusion of IL-2 with the
cells to the patient. Because the systemic infusion of IL-2 is known to be
extremely toxic to patients, it is best to avoid the necessity for this
cytokine.
In order for T-cells to proliferate, they require two separate signals.
The first signal is generally delivered through the CD3/TCR antigen
complex on the surface of the cells. The second is generally provided
through the IL-2 receptor. In order to bypass the IL-2 signal,
combinations of mAb are used. Preferably, the mAb are in the soluble
phase or immobilized on plastic or magnetic beads, in order to simplify
the cell harvesting procedure.
(i) First signal
To provide the first signal, it is preferable to activate cells with
mAb to the CD3/TCR complex, but other suitable signals, such as, but
not limited to, antigens, super antigens, polyclonal activators, anti-CD2
and anti-TCR antibodies, may be used. Other suitable agents can be
empirically identified. Immobilized or cross-linked anti-CD3 mAb, such as
OKT3 or 64.1, can activate T-cells in a polyclonal manner [see, Tax, et al.
(1983) Nature 304:445]. Other polyclonal activators, however, such as
phorbol myristate acetate can also be used [see, e.g., Hansen, et al.
(1980) Immunogenetics 10:247].
Monovalent anti-CD3 mAb in the soluble phase can also be used to
activate T-cells [see, Tamura, et al. (1992) J. Immunol. 148:2370].
Stimulation of CD4+ cells with monovalent anti-CD3 mAb in the soluble
form is preferable for expansion of Th2 cells, but not Th1 cells [see,
deJong, et al. (1992) J. Immunol. 149:2795]. Soluble heteroconjugates
of anti-CD3 and anti-T-cell surface antigen mAb can preferentially
activate a particular T-cell subset [see, Ledbetter, et al. (1988) Eur. S.
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lmmunol. 18:525]. Anti-CD2 mAb can also activate T-cells [see, Huet, et
al. (1986) J. Immunol. 137:1420]. Anti-MHC class ll mAb can have a
synergistic effect with anti-CD3 in inducing T-cell proliferation [see,
Spertini, et al. (1992) J. Immunol. 149:65]. Anti-CD44 mAb can activate
5 T-cells in a fashion similar to anti-CD3 mAb. See, Galandrini, et al.
(1993) J. Immunol. 150:4225].
For purposes herein, monoclonal antibodies to anti-CD3 are
preferred. Anti-CD3 is used because Cl:)3 is adjacent to the T-cell
receptor. Triggering of CD3, such as by monoclonal antibody interaction,
10 causes concomitant T cell activation.
(ii) Second signal
To then cause proliferation of such activated T cells, a second
signal is required. A variety of mAb singly or in combination can provide
the second signal for T-cell proliferation. Anti-lL-4R mAb (specific for the
15 interleukin-4 receptor molecule) can enhance the proliferation of the Th2
cells [see, Lindquist, et al. (1993) J. Immunol. 150:394]. Immobilized
ligands or mAb against CD4, CD8, CD11a (LFA-1), CD49 (VLA),
CD45R0, CD44 and CD28 can also be used to enhance T-cell
proliferation [see, Manger, et al. (1985) J. Immunol. 135:3669; Hara, et
20 al. (1985) J. Exro. Med. 161:1513; Shimizu, etal. (1990) J. Immunol.
145:59; and Springer, (1990) Nature 346:425]. Cell surface proteins
that are ligans to B-cells are preferred targets for Th2 cell proliferation,
while macrophage ligands are preferred for Th1 cell proliferation.
Anti-CD28 mAb in combination with anti-CD3 or anti-CD2 induces
25 a long lasting T-cell proliferative response [see, Pierres, et al. (1988) Eur.
J. Immunol. 18:685]. Anti-CD28 mAb in combination with anti-CD5
mAb results in an enhanced proliferative response ~hat can be sustained
for weeks lsee, Ledbetter, et al. (1985) J. Immunol. 135:2331]. Anti-
CD5 mAb alone can also provide a second signal for T-cell proliferation
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[see, Vandenberghe et aL (1991) Eur. J. Immunol. 21:251]. Other mAb
known to support T-cell proliferation include anti-CD45 and CD27 [see,
Ledbetter, et ak (1985) J. Immunol. 135:1819 and Van Lier, et aL (1987)
J. Immunol. 139: 1589].
To determine the combination of mAbs or proteins that optimally
induce sustained regulatory cell proliferation, a screening procedure using
combinations of these mAbs or proteins is used. The cells are incubated
with various combinations of these substances and screened for growth
by analysis of 3H-thymidine incorporation or equivalent methods. The
10 group demonstrating the best growth characteristics is selected for use in
the medium.
(iii) Ex~ io~ -
!n order to expand purified T-ce!!s to c!inica!!y re!evant numbers of
up to 100 billion (10l1), the cells should be grown to high density. This
15 can be achieved using any suitable means, including, but not limited to:
stirred tank fermentors, airlift fermentors, roller bottles, culture bags, and
other bioreactor devices. Hollow fiber bioreactors are presently preferred.
Hollow fiber bioreactors permit cells to be cultured to the required high
densities in a minimal volume. This reduces the amount of monoclonal
20 antibodies, serum and medium required in the production process. In
addition, selection of fibers with molecular weight cut-offs of 6000
daltons will allow continuous feeding and waste product removal while
retaining cell derived cytokines in the culture space. These cytokines,
such as IL-2 and IL-4, promote and sustain cell viability and proliferation.
T-cells, like most mammalian cells, will grow to a maximum density
of 1 x 106 cells/ml in tissue culture. Thus, a total of 100 liters of culture
- medium would be required to support 100 billion cells. In addition, the
100 liters of medium would have to be replenished regularly to maintain a
proper nutrient/waste product balance necessary to keep the cells viable.
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A method would aiso be required to keep the 100 liters of medium
saturated with oxygen.
Hollow fiber technology for cell culture is well known [see, e.a.,
U.S. Patent Nos. 4,220,725, 4,206,015, 4,200,689, 3,883,393, and
5 3,821,087; see, also, U.S. Patent No. 4,391,912; U.S. Patent No.
4,546,083; U.S. Patent No. 4,301,249; U.S. Patent No. 4,973,558, U.S.
Patent No. 4,999,298; and U.S. Patent No. 4,629,686] and is used to
achieve issue-like cell densities in culture li.e.. densities of greater than
about 108 cells/mll. The original hollow fiber bioreactor contains a
housing with a plurality of artificial capillary hollow fiber membranes. The
capillaries extend between an inflow opening at one end of the device
and an outflow opening at the other. The capillaries have selectively
permeable walls though which dissolved medium components can
diffuse. The lumen and ECS are separated by potting material at the
inflow and outflow openings. The housing also contains ports for access
to the ECS enabling cells to be inoculated into the ECS lsee, e.a., U.S.
Patent Nos. 3,821,087; 3,883,393 and 4,220,725, 4,206,015,
4,200,689, 3,883,393, and 3,821,087; see, also Knazek, et al. (1972)
Science 178:65].
Hollow fiber technology permits cells to grow to densities 100-fold
greater than cell densities [1 x 108 cells/ml or greater~ observed in
conventional cell culture. Thus, only one liter of culture volume is
required to generate 100 billion cells. The reduced cell volume would
also decrease the amount of human serum and soluble mAb required in
the expansion process. In addition, high cell densities provide
environments that are a closer approximation to in vivo condition.
The hollow fiber bioreactor is a component of a hollow fiber cell culture
system. A typical hollow fiber cell culture system, such as the
CELLMAXTM 100 hollow fiber cell culture system (Cellco Advanced
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Bioreactors, Inc., MD) contains a standard glass medium bottle, which
serves as the reservoir, stainless steel/Ryton gear pump, an autoclavable
hollow fiber bioreactor, which contains the fibers and shell casing in
which cells are cultured, and medical grade silicone rubber tubing, or
5 other connecting means, which serves as a gas exchanger to maintain
the appropriate pH and P02 of the culture medium. All components are
secured to a stainless steel tray of sufficiently small dimensions to enable
tour such systems to fit within a standard tissue culture incubator
chamber. The pump speed and automatic reversal of flow direction are
10 determined by an electronic control unit which is placed outside of the
incubator and is connected to the pump motor via a flat ribbon cable
which passes through the gasket of the incubator door. The pump motor
is magnetically coupled to the pump and is lifted from the system prior to
steam autoclaving.
The preferred HF bioreactor system for use herein is described in
copending, allowed, U.S. application Serial No. 08/506,173.
2. Preferred Hollow Fiber System for Large Scale T-Cell
Cultures
A HF system that closely emulates in vivo conditions thereby
20 permitting T-cells to grow to densities of over 1 x 1 O7 cells/mls,
preferably 1 x 108 cells/ml, that uses fibers with a low molecular weight
cutoff to retain mitogenic mAbs and serum components, and that does
not have gradient formation problems, is described in copending, allowed,
U.S. application Serial No. 08/506,173. This HF device allows outflow
25 of the lumenal flow to be completely blocked. This leads to equal
perfusion of nutrients along the entire length of the hollow fiber
capillaries. It also includes an oxygen feed on the ECS of the bioreactor to
provide desired oxygen delivery characteristics.
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Artificial kidney cartridges [CD Medical of Hialeah, FLl having a
length of 14 inches, an ECS volume of volume of 120 ml, and a
molecular weight cutoff (MWC) of 6,000 daltons were selected as the
hollow fiber bioreactors for use in the hollow fiber processing apparatus.
To ensure equai distribution of nutrients across the entire length of these
low MWC cartridges, an automatic on/off solenoid valve was placed on
the outflow opening of the bioreactor. When the solenoid is in the "off'
position, medium is prevented from exiting the bioreactor. Instead, the
medium ultrafiltrates to the cells in the ECS equally to all points of the
10 bioreactor. The medium then passes out of the bioreactor through the
ports. Ultrafiltration of nutrients is more physiological and therefore more
desirable for maintenance of dense cultures of cells [see, e.q., Swaab et
al. (1974) Cancer Res. 34:2814; and Davis et ab (1974) Chem. Enq.
J. 7:213].
To remove the metabolic waste from the cells in the ECS, the
solenoid valve is switched to the "on" position and the medium is
returned at a controlled pressure to the ECS through the eist ports. The
medium then moves radially into the lumen. Finally, the medium is
carried out the outflow opening.
The hollow fiber system permits the medium that ultrafiltrates from
the lumen to the ECS (Cycle 1) to be automatically replenished with
oxygen and for the levels of glucose, lactate and carbon dioxide to be
adjusted. This reconditioned medium is then returned to the ECS when
the solenoid valve is opened in Cycle 2. The same adjustments are
25 conducted for medium on the lumenal side of the bioreactor. In this
manner, oxygen diffusion limitations can be overcome as oxygen is
supplied to the lumen and the ECS of the bioreactor, eliminating diffusion
across the hollow fiber capillaries as the sole means of oxygen transfer.
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For large-scale growth of regulatory immune cells hollow fiber
bioreactors that have improved fluid dynamics to reduce gradient
formation are preferable [see, e.a., U.S. Patent No. 4,804,628, see,
especially, allowed copending U.S. application Serial No. 08/506,173] are
5 presently preferred. The hollow fiber bioreactors that have such
improved fluid dynamics are best suited for the large-scale growth of
regulatory immune cells.
In preferred embodiments, mitogenic monoclonal antibodies are
coated onto the hollow fiber surafce in order to deliver the proper signals
10 necessary to cause the immune cells to divide.
D. Effector cell expansion
Effector cells are mononuclear cells that have the ability to directly
eliminate pathogens or tumor cells. Such cells include, LAK cells, TlLs,
CTLs and antibody-producing B cells and other such cells. These cells
15 are produced by first treating cells collected from a patient in manner
known to lead to differentiation of such cells. For example, TIL cells are
produced by culturing solid tumor tissue obtained by biopsy in IL-2 and/or
other agents that lead to TIL production. The cells are then activated and
expanded in the presence of mitogenic agents, such as monoclonal
20 antibodies specific for cell surface receptors or other agents, as described
above for the regulatory cells.
In accord with the methods provided herein, the cells are not
exposed to exogenous IL-2 (or any other agent upon which the cells will
become dependent for in vivo activity or survival) and reinfusion is not
25 accompanied by co-infusion of IL-2.
E. Selection of Immune Cell Phenotype
Depending on the site of action at which a regulatory effect of
infused cells is required (or at which effector cells are required), different
cell phenotypes may be required. Lymphocytes recirculate extensively
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throughout the body and then localize in tissues and Iymphoid organs.
This is accomplished by an array of adhesion molecules on Iymphocytes
and counter-receptors on the vascular endothelium, extracellular matrix
and epithelium. Recent studies have identified several of the specific
5 receptor/ligand interactions that mediate Iymphocyte trafficking.
Infused cells that need to migrate out of circulation (e.q., to sites
of inflammation) must have the capacity to move through extracellular
matrix (ECM) of various compositions. For example, subendothelial
basement membrane presents a barrier rich in type IV collagen, laminin
10 and heparan sulfate proteoglycans. The ECM of the interstitium contains
collagens I and lll, as well as various glycosaminoglycans such as
hyaluronic acid. Fibronectin and vitronectin are also encountered in
basement membrane and interstitium. Immune cells can be loaded into
columns containing these materials in order to screen for cells capable of
15 migration through the interstitium.
It is also know that cells with a "memory" phenotype (i.e.,
CD45RA-, CD45R0+, CD29+, CD11a+, CD44+, CD54+, CD58+, L-
selectin-) will accumulate non-specifically at sites of chronic
inflammation. Cells that express L-selectin are least likely to migrate and
20 should be used when the desired regulatory effect is required in the
Iymphatic organs.
Growing out cells with a defined antigen specificity may also be
desired in order to prevent non-specific immunoregulation. Antigens
should be selected that are unique to the site a regulatory effect is
Z5 desired or to the disease-causing antigen(s).
F. Practice of the therapeutic methods
The therapeutic methods herein are designed to produce
compositions containing clinically relevant ~at least 109, preferably 101~,
cells or more] populations of regulatory immune cells and/or effector
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immune cells for autologous infusion for treatment. The methods herein
do not rely or use any agents for expansion that must be present after
expansion to maintain cell viability or activity. In particular, expansion
does not require or use IL-2. As a result, re-infusion of the cells does not
require or use IL-2, thereby obviating toxicity and other problems
associated with IL-2 infusion.
The compositions preferably contain substantially homogeneous
populations of cells, such as Thl cells or Th1-like cells, in which the
cytokine profile is predominantly one type of cell (i.e., greater than about
50%). The compositions can contain regulatory immune cells, effector
cells or both. In all instances the compositions contain clinically relevant,
i.e., a therapeutically effective, numbers of cells.
Such compositions can be used therapeutically to restore an
immune cell imbalance. Immune cell imbalances are common in many
disease states. For example, a predominance of Th1 regulatory immune
cells has been reported in autoimmune diseases such as rheumatoid
arthritis [see, Simon, et al. (1994) Proc. Natl. Acad. Sci. U.S.A.
91:8562]; type I diabetes [see, Foulis, et ak (1991) J. Pathol. 165:97];
systemic inflammation [see, Brod, et aE (1991) J. Immunol. 147:810];
inflammatory bowel syndrome tNiessner et ak (1995) Clin. EXP. Immunol.
101 :428]; Grave's disease [see, de Carli, et al. (1993) J. Clin. Endocr.
Metab. 77:1120]; Sjogren's syndrome [see, Oxholm, et ak (1992)
Autoimmunitv 12: 185]; primary systemic vasculitis [Grau (1990) Eur.
Cvtokine Netw. 1 :203]; and rejected autografts [see, Benvenuto, et al.
(1991) Transplantation 51:887]. A predominance of Th2 regulatory
immune cells has been reported in AIDS [see, Romagnani,et al. (1994)
Res. Immunol. 145:611]; candidiasis [see, Puccetti, et aE (1995) Trends
in Microbioloqy 3:237]; tuberculosis [Zhang, et al. (1995) Infect. Immun.
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63:3231]; and allergy [see, Romagnani, et al. (1994~ Curr. OPin.
Immunol. 6:838].
Also, the polarized Th1 and Th2 responses in humans to different
antigens are known to play a role in protection, but also result in
5 immunopathology. The methods provided herein can be used to correct
pathologic Th1 and Th2 responses by infusing autologous regulatory cells
of the subset in short supply, thereby adjusting the ratios and absolute
numbers. Since Thl and Th2 cells have cross-regulatory properties, large
infusions of the subset in short supply can counter-act the pathologic
10 effects of an imbalanced response. Some examples of the use of these
methods and cells for treating several disease are provided. It is
understood that the following are exemplary uses; any condition in which
a pathologic T cell response is observed in which the ratios or amounts of
particular subsets of T cells are outside the normal range can be treated
15 by infusion of the T cell subset(s) that is in relatively short supply.
1. ~d",;.~ dliOI,
The compositions of cell can be administered by any suitable
means, including, but not limited to, intravenously, parenterally, or
locally. The particular mode selected will depend upon the particular
Z0 treatment and trafficking of the cells. Intravenous administration is
presently preferred. Typically, about 101~-10'1 cells can be administered
in a volume of a 50 ml to 1 liter, preferably about 50 ml to 250 ml., more
preferably about 50 ml to 150 ml, and most preferably about 100 ml.
The volume will depend upon the disorder treated and the route of
25 adminstration. The cells may be administered in a single dose or in
several doses over selected time intervals in order to titrate the dose,
particularly when restoration of immune system balance is the goal.
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2. Tre~l~,.el,l of autoimmune disorders
The methods and composition of regulatory cell provided herein
may be used to treat disorders that have an underlying autoimmune basis
or component.
a. Treallne.. l of Rheumatoid Arthritis (RA)
RA is an immunologically mediated, chronic inflammatory disease
characterized by synovial inflammation and autoantibodies. While the
underlying cause of RA is unknown, it is well agreed upon that a fault in
immune regulation is a principal factor contributing to the disease
10 pathogenesis. Regulated control of normal immune responses are largely
the result of interactions between, and the cytokine production of,
macrophages, T-cells and B-cells.
Disease activity in RA patients has been positively correlated with
the cytokine production of activated macrophages. In an inflamed joint,
15 macrophages produce large amounts of pro-inflammatory cytokines
which include IL-1, IL-6, IL-8, TNF-a and GM-CSF. These cytokines act
to recruit Thl memory cells to the joint and stimulate rheumatoid factor
(RF) production leading to pannus formation and joint destruction.
Treatment protocols which decrease the levels of proinflammatory Th1
20 cytokines in RA have been shown to result in clinical improvement.
The cytokines IL-4 and IL-10 are known to down-regulate macro-
phage activation and inhibit their production of IL-1, IL-6, IL-8 and TNF-a.
IL-4 is also capable of suppressing the uncontrolled proliferation of
synoviocytes, which is a major pathological feature of RA. IL-4 and IL-10
25 are produced by Th2 cells, which are virtually absent from the RA joint.
Rather, RA joints have an abundance of Th1 cells.
Accordingly, RA can be treated by generating large numbers of
autologous, ex vivo derived Th2 cells from RA patients by the methods
provided herein. The resulting cells, preferably in amounts greater than
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109, more preferably 1 olo, are re-infused into the patient to thereby
suppress the chronic inflammatory lesions. Th2 cells of memory
phenotype are preferred, since memory cells are most likely to migrate to
the site of inflammation. In addition, the cells can be infused in an
activated state; infiltrating T-cells in RA have been shown to have 5-6
fold increases in HLA-DR expression and 2-5 fold increases in VLA-1
expression, both of which are activation markers.
It is also preterred that the infused Th~ cells only exert their
regulatory action in the joints, so as to prevent a systemic
immunosuppressive effect. Since the eliciting antigen is unknown in RA,
the Th2 cells used should be specific for unique joint antigens ~e.q., Type
Il collagen or proteoglycan].
b. Tredl".e.,l of Multiple Sclerosis (MS)
MS is an autoimmune disease characterized by central nervous
system inflammation and demyelination. The regulation of cytokine
spectrum and production in MS is thought to have a decisive influence on
disease outcome. Collective data has shown that Thl-associated
cytokines, such as TNF-a, Iymphotoxin, interleukin-12 and interferon-y
promote disease, while cytokines from Th2 cells, such as IL-10, limit
disease. In addition, TGF-,l3 has been shown to be a disease
downregulator. Studies in animal models of MS [experimental
autoimmune encephalomyelitis (EAE)] have determined that a regulatory
cell producing IL-10 and TGF-,I~, termed "Th3n, has the greatest effect
suppressing the development and inducing recovery from disease.
Accordingly, the methods herein can be used to generate
therapeutic quantities of Th3 cells from MS patients for use in autologous
cell therapy. Since recovery from disease is associated with inrilLI~Ling
cells which produce IL-10 and TGF-,~, the ex vivo derived Th3 cells should
preferably have a memory phenotype in order to enhance migration to the
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inflammatory lesions. In addition, in order to make the
immunosuppressive effect of the cells specific for the inflammatory
lesions, cells specific for myelin or encephalitogenic epitopes of myelin
antigens (e.q., myelin basic protein or proteolipid protein) should be used.
c. I ,rlal"."atc,ry Bowel Disease (IBD)
IBD is a chronic inflammatory condition of the gastrointestinal
tract. The etiology and pathogenesis of IBD is not known. Crohn's
disease (CD) and ulcerative colitis (UC) are thought to be mediated by an
abnormal or uncontrolled T-cell reaction to one or more common gut
constituents. Active CD and UC are characterized by increases in Th1-
like cytokines, with little to no detectable Th2-like cytokines.
Accordingly, the methods provided herein can be used to generate
autologous Th2 cells for infusion in IDB patients. Preferably, the infused
cells will express the integrin, a4"~7. This integrin has been shown to be
the ligand for mucosal addressin cell adhesion molecule-1 found on
Peyer's patch high endothelial venules, which occur in the gastrointestinal
tract. Lymphocytes which express a4"B7 will traffic to and are retained
in mucosal organs. The gut mucosa is the site of chronic inflammation in
IBD .
d. Tredl",e-~L of Insulin-Dependent Diabetes Mellitus
(IDDM)
IDDM results from the autoimmune destruction of pancreatic islet
,~ cells by the host immune system. The destruction of islet cells is
known to be mediated by T-cells. The NOD mouse is a spontaneous
model of human IDDM. Islet transplantation as an isograft in these mice
can produce normoglycemia and prevent and reverse early complications
of diabetes. Host inflammatory responses, however, eventually lead to
destruction of the islet transplants and disease recurrence. Analysis of
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these inflammatory responses has shown that graft specific Th1 cells
mediate rejection, while Th2 cells are protective.
There is evidence that isograft and allograft rejection is mediated
by Th1 cells and can be suppressed by Th2 cells. Th1 cells have been
5 shown to actively promote diabetes in NOD mice. Inhibition of Th1
cytokines leads to protection of islet isografts in NOD mice, Recently, it
has been shown that the systemic administration of Th2 cytokines (IL-4
and IL-10) and adoptive transfer of an islet-specific Th3 clone can inhibit
syngeneic islet graft rejection in these animals. Furthermore, Th2-like
10 responses have been shown to be protective in models of allogeneic
organ and tissue transplantation.
Accordingly, the methods herein can be used to generate clinically
relevant numbers of Th2 ceils for infusion in IDDM patients that will
protect against rejection of transplanted allogeneic islet cells. Preferably,
15 the Th2 cells will be specific for the allogeneic antigens on the
transplanted islets. Alternatively, Th2 cells specific for insulin can be
used. Insulin-specific Th2 cells could also be used to treat early diagnosed
IDDM patients to prevent islet destruction, as well as used in high risk
patients as a vaccine to prevent or at least retard development of the
20 diabetes.
e. Treatment of other auloi~,.."une ~ise~ses
Th1-mediated autoimmune diseases, such as, but not limited to,
autoimmune thyroid diseases, anti-tubular basement membrane disease
(kidney) Sjogren's syndrome, ankylosing spohdylitis, ureoretinitis and
25 others, can be treated by administration of compositions containing a
clinically relevant, typically 109-10'1, Th2 cells or a Th2-like composition.
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3. Transplantation
Th2 cell ACT can be used as an immunosuppressive strategy
permitting organ and tissue transplantation. For example, Th2 cytokines
have been correlated with non-rejecting heart allografts, while Thl
5 cytokines correlate with rejection. The same is has been observed for
renal allografts and mouse orthotopic liver allografts and skin allografts.
Adoptively transferred Th2 cells suppress skin allograft rejection and also
allow allogeneic engraftment of spleen cells in sublethally irradiated mice
as well as suppress lethal GVHD (graft vs. host disease). T-cell mediated
10 alloreactivity has been shown to be central in the pathogenesis of GVHD
and graft rejection.
Accordingly, the methods provided herein can be used to generate
autologous Th2 cells for infusion in patients scheduled for organ or tissue
transplant. Preferably, the Th2 cells will be specific for the alloantigens
15 or an antigen unique to the organ or tissue being transplanted.
4. A - ~, a Disorders
Th2 cells appear to have a crucial role in initiating eosinophil
infiltration which causes eczematous reactions in patients with atopic
dermatitis, and airway hyper-responsiveness and pulmonary eosinophilia
20 in allergic asthma. Furthermore, atopic patients (patients with hayfever,
dust and food allergies) have a preferential activation of Th2 cells.
Recent evidence has shown that treatments that suppress Th2
development in vivo have profound inhibitory effects on allergen-induced
airway changes and other atopic responses. Accordingly, since Th1
25 cytokines are known to inhibit Th2 responses, the methods herein can be
used to generate large numbers of autologous Th1 cells for infusion into
~ atopic patients. Preferably, these cells will be specific for the allergen.
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5. Infectious D;.,eases and Cancer
An excess of Th2 cells is correlated with most infectious diseases,
including viral, fungal, yeast, parasitic and mycobacterial infection. In
order to change the regulatory balance in favor of cell-mediated immunity,
5 Th1 cells could be infused into these patients. Prior art ACT protocols
have used TIL and LAK effector cells and methods that use pathogen- or
tumor cell-specific CTLs. These effector cells would not be expected to
work properly in an immunocompromised host.
The co-infusion of Th1 regulatory cells should provide the "help"
10 necessary for the effector cells to perform their function and thus
improve these therapies. Infusion of Th1 cells alone could provide
sufficient help in vivo to drive endogenous CD8 + effector cells.
Accordingly, the methods herein could be used to generate large
numbers of autologous Th1 cells for infusion into patients with infectious
15 diseases or cancers. Preferably, the cells will be specific for antigens
unique to the pathogen or tumor. The Th1 cells can also be infused with
pathogen or tumor-specific cytolytic cells.
Of particular interest herein, are methods for treatment of HIV
infection. Methods for producing virally purged CD4+ cells are provided.
20 In preferred embodiments, the cells are expanded under conditions in
which Th1 cell differentiation is promoted. The resulting cells are
reinfused into the donor HIV patient, whereby immunity will be restored.
In other embodiments, these cells are reinfused with expanded effector
cells, particularly effector cells that are specifically targeted against HIV
25 infected cells.
Other infectious diseases that can be treated with Th1 cell
compositions include, but are not limited to: influenza viruses, polio
virus, leukemia viruses, hepatitis viruses, respiratory synctial virus, herpes
viruses, retroviruses Epstein-Barr virus, syphillis (Treponema pallidum),
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cutaneous T-cell Iymphoma (mycosis fungoides), Rhodococcus equi
(intracellular respiratory pathogen), hypersensitivity pneumonitis,
onchocercal keratitis (river blindness), burn victims, chlamydia
trachomatis, mycobacterium avium, candida albicans, coxackievirus,
5 Leishmania major infection, cryptococcal infection and Bordetella
pertussis respiratory infection.
Infectious diseases that can be treated with Th2 cell compositions
include, but are not limited to: filarial nematode (parasite), Plasmodium
chaboudi chaboudi (malaria), and Borrelia burgdofi (spriochete) infections.
Also of interest herein, are methods of treatment of cancer. In
preferred embodiments, methods for treatment of renal cell carcinoma are
provided. Transformed renal cells express heat shock protein hsp70.
Consequently, hsp70-specific Th1 cells could serve as a cytokine delivery
vehicle to increase local concentrations of IL-2 and IFNy in the tumor,
15 thereby promoting anti-tumor effector cell function, activity and/or
proliferation .
Th1 cells can also be used to mediate tumor regression in cancers
including melanoma, breast cancer, head and neck cancer, prostate
cancer and lung cancer. These is evidence that for certain tumors, a
20 Th2 rsponse may mediate regression.
6. Vaccination
The development of effective vaccine strategies for intracellular
pathogens, including, but not limited to, bacteria, viruses and parasites, is
one of the major frontiers of medical research. Research centers on
25 antigens from pathogenic organisms and adjuvants that can elicit a Th1-
like response in patients. It is known that a Th1 response is protective
- for infectious pathogens. Th1 responses are weak or non-existent in
some patients with most vaccine protocols. Other research focuses on
eliciting an IgA antibody response, which is thought to be protective
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against organisms that enter the body through muscous membranes. An
IgA response is mediated by Th2 cells. To better control the type of
immune response a patient will elicit to a vaccine, the methods herein
provide a means for ex vivo vaccination (i.e., the addition of the vaccine
5 antigen(s) to patient mononuclear cells ex vivo, whereby thecells are
activated under conditions that promote the desired regulatory cell
differentiation .
The methods provided herein can be used to withdraw blood from
a patient, expose the isolated mononuclear cells to the vaccine antigen in
10 the presence of IL-12 and/or IFN-yand/or IL-4, and expand the Th1 or
Th2 cells for reinfusion. Preferably, the cells used will have a memory
phenotype so they will provide long-term protection. CD4+ and CD8 +
Th1 or Th2 cells could be generated alone or in combination.
The following examples are included for illustrative purposes only
15 and are not intended to limit the scope of the invention.
EXAMPLE 1
Screening mitogenic monoclonal antibodies
This example demonstrates a method for identifying antibodies that
are suitable for expanding T-cell subsets, either singly or in combinations
20 thereof.
In order to determine co-stimulatory signals required for T-cell
subset proliferation, cells are incubated with various monoclonal
antibodies (mAb) and their proliferation determined in 3H-thymidine
incorporation assays. To exemplify this procedure, the following
25 experiments were conducted.
Monoclonal Ab to CD3 (64.1, IgG2a) and anti-CD5 (10.2, IgG2a)
were gifts from J. Ledbetter (Bristol Meyers, Seattle) and the mAb to
CD28 (Kolt-2, IgG1) was a gift from K. Sagawa (Kurume University,
Kyushu, Japan). These mAb were purified from ascites fluids on protein
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A sepharose columns. All other mAbs were purchased from PharMingen
~San Diego, CA). All mAbs were dialyzed against phosphate buffered
saline and filtered through sterile 0.45,um filters.
Goat anti-mouse affinity purified antibody (Tago, Burlingame, CA)
5 was immobilized on plastic 96 well tissue culture plates. The antibody
was dissolved in sodium borate buffer ~pH 8.6) at a concentration of
10,ug/ml and 100,ul was placed in each weli. Plates were washed three
times with RPMI-1640 with 10% normal human serum. Celis were
labelled with anti-CD3 mAb (1,ug/ml) on ice for 15 minutes prior to
10 plating. 50,000 cells were plated in each well. Co-stimulatory mAbs
were added in the soluble phase at 1 ,~rg/ml. The cells were cultured at
37~ C in an atmosphere of 5% C02. After 88 hours of culture, cells were
pulsed with 1 ,uCi of 13H]- thymidine (specific activity of 2 Ci/mole, New
England Nuclear). Eight hours later, cells were harvested with a PHD cell
15 harvester (Cambridge Technology, Cambridge, MA) and the radioactivity
on the filter papers counted on a liquid scintillation counter (LS1701,
Beckman) .
The results of mAb addition to purified CD4+ and CD8+ cells
from a normal individual are shown below. Results are shown as mean
Z0 counts per minute (cpm) of four replicates. Standard errors were always
less than 10% .
Stimulation CD4 + CD8 +
medium alone 320 484
anti-CD3 582 541
anti-CD3 + anti-18,450 17,222
CD5
anti-CD3 + anti-20,400 18,641
- CD28
anti-CD5 450 246
anti-CD28 826 821
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These data demonstrate that anti-CD5 and CD28 are capable of
providing a co-stimulatory signal for T-cell proliferation in CD4+ and
CD8 + subsets when the cells are activated with anti-CD3. The results
of combining anti-CD5 and CD28 are shown below:
Stimulation CD4 + CD8 +
medium 428 ~24
anti-CD3 585 508
anti-CD3 + anti-CD5 13,4~10,0~0
anti-CD3 + anti-CD28 14,62812,821
anti-CD3 + anti-CD5 + anti-CD28 25,248 29,804
anti-CD3 + IL-2 (10 U/ml) 11,42812,401
These results show that the combination of anti-CD5 and anti-
CD28 as co-stimulatory signals in CD3 activated, purified T-cells induces
15 a greater proliferative response than either mAb alone. In addition, the
combined mAbs generated a proliferative response without addition of
IL-2.
The effect of various mAbs (second signal) on purified CD8 + cells
from a normal donor used in conjunction with anti-CD3 or anti-CD2 (first
20 signal) was also tested. These results are shown below:
Stimulation aCD3 aCD2 Medium
aCD5 206 193 155
aCD8 787 578 640
aCD1 la 949 830 840
aCD27 844 2 788
aCD28 1928 529 640
aCD44 779 477 498
aCD45RO 3199 1878 1978
IL-2 4347 1834 nd
Medium 289 217 212
.
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These results demonstrate that anti-CD3 as the first signal delivers
a more powerful proliferative stimulus than anti-CD2. Anti-CD45R0 and
anti-CD28 mAbs appear to deliver the strongest second or co-stimulatory
signals when used with anti-CD3.
Combinations of these antibodies were tested on anti-CD3
activated, ex vivo generated CD8 + cytolytic cells specific for the
MAGE-3 antigen on melanoma cells. These results are shown below:
an~i- anti-CD27 anti-CD28 anti-
CD 11 a CD45R0
anti-CD1 la ----- 1365 1116 1208
anti-CD27 1365 ----- 374 973
anti-CD28 1116 374 ----- 948
anti-CD45R0 665 973 948 ----
Combinations including anti-CD11a provided the strongest prolifer-
15 ative signals for these cells. None of these combinations provided veryexceptional growth. This sometimes occurs in CD8 + CTL, which are
unable to produce sufficient endogenous cytokines. Co-culturing of these
cells with autologous CD4+, however, enhanced the proliferation of
these cells with mAb stimulation. This probably resulted from the~0 increased endogenous production of IL-2, as well as IFN-y and IL-7.
EXAMPLE 2
CD4+and CD8+ T-cells from Normal Donor
This example demonstrates that polyclonally activated CD4+ and
CD8+ regulatory T-cell subsets can be expanded without IL-2 to clinically
25 relevant numbers from a starting number of about 1 x 106 cells using the
disclosed methods.
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A. Collecting mononuclear cells
Mononuclear cells from normal donors were obtained from source
leukocyte packs (Interstate Blood Bank, Inc.). The leukopack cells were
diluted 1:1 with Hank's Buffered Salt Solution (HBSS) without calcium
5 (Ca2+) or magnesium (Mg2+) and 30 to 35 ml of the diluted cells were
placed over 12 ml of Ficoll-Hypaque and the tube centrifuged at 1500
RPM at room temperature. The buffy coat layer containing Iymphocytes
and monocytes was transterred by Pasteur pipette to a clean 50 ml
centrifuge tube and washed three times with HBSS. The cells were then
10 resuspended in RPMI-1640 medium supplemented with 10% human
serum, 25 mM HEPES buffer, 2.0 mM glutamine, 1.0 mM sodium
pyruvate, 0.1 mM non-essential amino Acids, 2 x 10-~ M 2-
mercaptoethanol, 10 IU of penicillin G and 100 mg/ml streptomycin
sulfate (cRPMI). The monocytes were depleted by adherence to plastic
15 T-cell flasks incubated overnight at 37~C in an atmosphere of 5% C02
and 100% humidity.
B. Precursor cell purification
T-cell subsets were purified with immunomagnetic bead
technology. GAM-coated beads (Dynal, Inc.) were washed twice with
20 HBSS and incubated overnight on a rotating wheel at 4~C in HBSS with
1% normal human serum in order to block nonspecific binding. The non-
adherent cells were incubated with either anti-CD4 or anti-CD8 mAb at
pre-titered concentrations on ice for 30 minutes. Labelled cells were
washed twice and resuspended in cRPMI at 10 cells/ml. The beads were
2~; added to the cells at a bead/cell ratio of 2:1 and mixed well. This mixture
was gently centrifuged at 500 RPM for 1 minute at 4~ C. The bead/cell
mixture was then resuspended by gently inverting the centrifuge tube.
The tube was then placed on a rotating wheel for 30 minutes at 4~ C
The bead/cell mixture was then diluted 5 fold with cRPMI and placed on a
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cobalt salarium magnet. The supernatant was aspirated and rosetted and
the procedure repeated. The rosettes were incubated for 24 hours in
cRPMI at 37~C in an atmosphere of 5% CO2. After 24 hours, the
majority of cells detached from the beads and the beads were removed
5 by placing the solution back on the magnet. The resulting cells were
greater that 98% pure CD4+ or CD8+ T-cells as assessed by flow
cytometry.
(1. Ex ~ivo ~Iffere~lidlio~
The purified CD4+ cells were divided into twoeparate groups of 1
10 million cells each. The first group was activated with immobilized
anti-CD3 mAb in the presence of 400 U/ml of IL-4 and 10,Llg/ml of anti-
lFN-y mAb and anti-CD28 mAb. This first group (ThZ) was expanded
under these Gonditions .f~r another 10 days. The second group wa~
activated with immobilized anti-CD3 in the presence of 25 U/ml of IL-12
15 and 150 U/ml of IFN-y, and anti-CD28 mAb. These cells were harvested
and washed after 6 days of culture.
D. Regulatory cell expal-;,;o..
One million of each of the purified T-cell subsets were labelled for
30 minutes on ice with anti-CD3 mAb (64.1, IgG2a). 2.5 X 105 cells of
20 the purified CD4+ and CD8+ cells were suspended in 1 ml of cRPMI and
plated into 4 separate wells of a 24-well plate coated with goat anti-
mouse (GAM) polyclonal antibody. Purified anti-CD5 (10.2, IgG2a) and
anti-CD28 (KOLT-2, IgG1) mAb were added to the wells at a final
concentrations of 200 ng/ml. The cells were then incubated at 37~C in
25 an atmosphere of 5% CO2.
After 3 days, 1 ml of cRPMI with 200 ng/ml of anti-CD5 and anti-
CD28 was added to the wells. After 6 days, the wells were harvested,
pooled and washed twice in cRPMI. The viable cells were counted and
resuspended in cRPMI at 1 x 106 cells/ml and incubated in T-flasks for 48
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hours at 37~C. The cells were then harvested, washed twice, labelled
with anti-CD3 mAb on ice for 30 minutes and inoculated into the extra
capillary space of a GAM-coated mini-hollow fiber bioreactor with 200
ng/ml of anti-CD28 an danti-CD5 mAb. The cells were harvested,
5 washed and counted after 14 days.
1. Mini-Hollow Fiber Bioreactor
A mini-hollow fiber device was constructed to expand immune
effector cells. The device had four mini-hollow fiber units in parallel. The
hollow fibers (CD Medical, Hialeah, FL) had a 9 ml extracapillary volume
10 and the fibers had molecular weight cut offs of 10,000 daltons. The
hollow fibers were coated with GAM polyclonal antibody. Coating was
accomplished by dissolving GAM polyclonal antibody, at a concentration
of 10 mg/ml, in sodium borate buffer (pH 8.6) and inoculating the sterile
solution into the extracapillary space (ECS) of the hollow fiber
15 bioreactors. The lumenal and ECS ports were then sealed and the
bioreactors placed on a rotating plate and incubated at 4~C for 24 hours.
Prior to use, the bioreactors were washed with phosphate buffered saline
with 1% normal human serum.
The flow path included an integration vessel, pump and
20 oxygenation cartridge. Luminal flow rates ranged between 100 and 400
ml/minute and were increased manually proportionate with the cell
growth in the bioreactors. The pH and temperature were continually
monitored and controlled by microprocessor. The pH was adjusted and
maintained at 7.2 by altering the speed of fresh medium fed into the
25 integration vessel and the percent C02 in the oxygenation cartridge. The
temperature was controlled to 37~C by adjusting the wattage to a
heating coil wrapped around the integration vessel.
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2. Single Large Hollow Fiber Bioreactor
The cells recovered from the mini hollow fiber device were
incubated in T-flasks at 1 x 107 cells/ml in cRPMI without mAb
stimulation for 48 hours. The cells were then labelled with anti-CD3 mAb
5 and inoculated into a GAM-coated large hollow fiber bioreactor [see,
copending allowed U.S. application Serial No. 08/506,173, discussed
abovel with 200 ng/ml of anti-CD5 and anti-CD28 mAb. The cells were
harvested, washed and counted after 14 days.
3. 8-Cartridge Hollow Fiber Bioreactor
The cells recovered from the single large hollow fiber bioreactor
[see, copending allowed U.S. application Serial No. 08/506,173,
discussed above] were incubated for 48 hours in a 10 liter spinner flask
at 107 cells/ml in cRPMI without mAb stimulation. The cells were then
labelled with anti-CD3 mAb and inoculated into each of the 8 GAM-
15 coated hollow fiber bioreactors with 200 ng/ml of anti-CD5 and anti-
CD28 mAb. After 14 days, the cells were harvested, washed and
counted .
E. Results
Clinically relevant numbers of cells were produced as follows:
DayCD4+ (Thl)CD4+ ITh2) CD8+ Culture Vessel
01 x1 o6 celis1 x1 o6 cells1 x1 o6 cells24-well plate
61.3x107 cells7.2x106 cells9.8x106 cells24-well plate
81.0x107 cells6.5x106 cells6x 106 cellsMini-HF
221.3x109 cells1.0xlO9cells1.2x109 cellsMini-HF
241.1x109 cells1.0x109cells1.1x109 cells1-large HF
381.4x10t~ cells1.0x10l~cells1.2x101~ cells1-large HF
401.3x10l~ cells1.0xlO~~cells1.0x10'~ cells8-Large HF
541.1x101l cells 1.0x1011 cells 9.9x101~ cells 8-Large HF
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Therefore, compositions containing clinically relevant numbers of T-
cell subsets can be produced.
EXAMPLE 3
Virus-purged CD4+ Thl-cells from HIV+ ~d~ t
This example demonstrates that clinically-relevant numbers of
virus-purged CD4+ Th1-cells can be generated by the methods herein for
use as an ACT for A.l.D.S. The cells were purged of active virus by
selection of CD4 antigen and were polyclonally activated and again
selected for CD4 antigen to purge of latent virus.
A. Obtaining Mononuclear Cells
An HIV+ patient, identified by a routine blood screening procedure
confirmed by Western Blot analysis, in WHO stage IV was the donor for
this study. The patient underwent a leukopheresis procedure for
collection of peripheral blood mononuclear cells.
B. Regulatory cell purification
CD4+ cells were isolated by positive selection on immunomagnetic
beads as described above. The CD4f cells were then activated in
24-well plates with immobilized anti-CD3 mAb and in the presence of
40 U/ml of interferon-y (IFN-y). After 24 hours in culture, the cells were
harvested, washed and re-selected for CD4 on immunomagnetic beads.
The positively-selected cells were labelled with anti-CD3 mAb and plated
at 25,000 cells/well in a GAM-coated 96-well plate in cRPMI. Anti-
CD28 mAb and IFN-y was added to the wells at a concentration of
1 ,ug/ml and 40 U/ml, respectively. After 7 days, supernatant from each
well was tested for p24 antigen with a commercial ELISA assay (Dupont).
All negative wells were pooled, relabelled with anti-CD3 mAb and re-
plated at 25,000 cells/well in a GAM-coated 96-well plate in cRPMI with
anti-CD28 mAb.
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C. Regulatory cell expansion
The cells were expanded as described in Example 2 above, except
that only anti-CD28 mAb was used as a co-stimulatory agent.
D. Results
6.3 x 1 o10 cells were grown over a 72 day period. The cells were
negative for p24 antigen and were capable of producing IL-2 and IFN-y,
but little or no IL-4. The cells were also shown to be capable of providing
help for t\ll<:-function in a dose-dependent manner. The cells were
reinfused into the patient. Reinfusion of these cells into the HIV+ patient
should be a treatment for A.l.D.S.
EXAMPLE 4
HlV-specific CD8+ cells from a HIV+ donor
This example demonstrates that antigen-specific CTL can be
purified and expanded from an individual with a viral infection.
A. Ol,ldi.,;.. g Effector Cells
3 x 1 o8 mononuclear cells were obtained by leukaphoresis from a
stage IV A.l.D.S. patient. CD8+, CD25+ cells were purified by two
rounds of selection on immunomagnetic beads.
B. Expansion of Effector Cells
Approximately 2 x 1 o6 cells were recovered and expanded in a 24-
well plate coated with anti-CD3 mAb and with soluble anti-CD28 mAb.
After 6 days, the cells were washed (x 2) and inoculated into mini-
hollow fiber bioreactors. After 18 days in the mini-hollow fiber units, the
cells were washed, counted and allowed to rest 2 days before inoculation
into a cartridge of the large hollow fiber bioreactor under the same
conditions as described in Example 2 above.
~ After 16 days, the cells were harvested, washed and allowed to
rest for 2 days. The viable cells were then inoculated into the 8-cartridge
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hollow fiber bioreactor system and cultured under the same conditions as
described in example 2 above.
C. Results
6 x 101~ viable cells were harvested after 20 days. The cells
5 showed significant Ag-specific CTL activity against infected autologous
cells.
These cells can be reinfused into the patient as a treatment for
A.l.D.S. In addition, these can be co-infused with virally-purged CD4+,
produced as described in EXAMPLE 3.
EXAMPLE 5
Antigen-specific Th2-like cells from a normal donor
This example demonstrates that antigen-specific Th2-like CD4+
cells can be derived from a normal individual and expanded to clinically
relevant numbers.
A. Ol~l ' .i.,g regulatory Cells
50 ml of blood was collected into a heparinized syringe, using
sterile technique, from an HIV- volunteer. Peripheral blood mononuclear
cells (PBMC) were separated by Ficoll-Hypaque density gradient
centrifugation. The PBMC were cultured in 10 ml T-flasks at 2 x 106
20 cells/ml and pulsed with gpl20 antigen in cRPMI that contained
1.0 ,ug/ml of anti-lFN-y mAb and 20 U/ml of IL-4. After 2 days, the
blasts were collected by selection of CD25 on immunomagnetic beads.
The blasts were allowed to rest for 72 hours and were than re-stimulated
with gp-120 pulsed, autologous monocytes and immediately cloned in
25 soft agar. The small number of cells that survived and grew out as
colonies (1/150,000) were enriched in Ag-specific cells that produced IL-
4 and IL-10 and little IFN-y upon stimulation, and, thus, were Th2-like in
cytokine profile.
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B. Expansion of Effector Cells
The cells were expanded as described in Example 2 and grew to
9 x 1 ol~ cells in 62 days.
EXAMPLE 6
5 Differentiation of Th2 cells from Precursors in RheL..,dt~i~ Arthritis
Peripheral Blood
While T cell cytokine expression is very low in rheumatoid arthritis
[RA), the absenGe of Th2 facto!s (e.g., !L-a and !1 13~ is especia!!y
striking. Since Th2 cytokines suppress production of pro-inflammatory
10 cytokines, metalloproteinases and rheumatoid factor, their relative
absence in RA could contribute to disease perpetuation. The lack of Th2
cells in synovium suggests that this differentiation pathway might be
defective in RA. To determine if Th2 precursors are present in RA, the
ability of peripheral blood RA CD4+ T cells to differentiate into ThO (IL-4
15 + IFN-A), Th1 (IFN-A, no IL-4) and Th2 cells (IL-4, no IFN-A) in vitro was
studied .
Purified CD4+ T cells were cultured in the presence of immobilized
aCD3 antibody, alL-12 and IL-4 for 3 d. Cells were then washed and
stimulated with PMA and ionomycin in the presence of monensin for 6 hr.
20 The cytokine phenotype was determined using 2-color flow cytometry on
permeabilized cells with alL-4 and,l3IFN-A monoclonal antibodies. The
results are shown as percent cells + standard error (se); "n" values are in
parentheses .
T~al~ l Th2(%) ThO(%) Thl(%)
- 25 RA (9) aCD3 0.68iO.19 0.44iO.11 10.38i2.61
Normal (6) 0.56iO.08 0.55iO.17 11.07i2.89
RA (4) aCD2+1L-4 1.43iO.32 0.29iO.09 4.68iO.9l
Normal (5) 1.50iO.26 1.69iO.56 13.27i2.46
RA (6) aCD3+alL-12+1L-4 3.03+0.92 1.68+0.44 12.51 +3.15
30 Normal (3) 1.45+0.35- 0.72+0.36 7.30+0.84
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These data indicate that similar numbers of Th2 cell precursors are
present in the peripheral blood of normals and patients with RA.
Furthermore, the mature Th2 cell population can be significantly increased
5 (p<0.05) with IL-4 and a-lL-12 antibody. Hence, a specific Th2
precursor defect does not account for the cytokine profile in the joint.
This raises the possibility that novel therapeutics could be developed
invoiving the administration of ex vivo differentiated and expanded Th2
cells.
EXAMPLE 7
HIV+ Lymphocyte Proliferation
The ability of PBL from HIV+ donors to proliferate in response to
the polyclonal activator PHA-P and immobilized anti-CD3 mAb was
compared with PBL from a normal donor (Table 1). PBL from HIV +
15 donors exhibited a marked suppression in the ability to respond to either
mitogenic signals when compared to PBL from normal donors.
Table 1. Comparison of Proliferative Response of Normal
and HIV + PBL to Mitogenic Factors
PBL Source Medium Alone PHA-P Immobilized
(1 ng/ml) anti-CD3 mAb
normal donors 1,446 i 241 25,813 + 1200 27,206 i 1891
HIV+ donors 2,041 i421 5,680i460 4,204i56Z
Peripheral blood iymphocytes (PBL) isolated over Ficoll-Hypaque
25 were plated at 50,000 cells/well in 96-well flat bottom culture plates.
Cells were pulsed after 88 hours of stimulation with medium alone, PHA-
P or immobilized anti-CD3 mAb with t3H]-thYmidine for eight hours and
the average mean and standard error of quadruplicate samples for six
normal and six HIV+ individuals is shown in cpm.
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To determine if purified T-cell subsets from HIV + donors were
capable of responding to mitogenic stimuli in the absence of activator,
the following study was conducted. PBL from six normal and six HIV+
individuals (same individuals as used in the experiments shown in
5 Table 1 ) were incubated in plastic tissue culture dishes for 24 hours at
37~ C in an atmosphere of five percent C02 in air. The CD4+ and CD8 +
T-cell subsets were purified using positive selection on immunomagnetic
beads as described previously. The results are shown in Table 2.
Table Z. Prolirerdli~re Response of Normal and HIV+ T-Cell Subsets to
1 0 Mitogens
(purity %)CD4+ Medium l.. o' ' ~~ anti- PMA
CD3 + IL-2
(99.5) Normal donors 1,841 i320 42,186i3444 35,920i3420
(98.8) HIV + donors 1,346 i 230 29,212 i 1841 31,440 i 6210
15 (purity %) CD8+
(98.8) Normal donors 1,925i421 12,420i821 10,920+1104
(98.4) HIV+ donors 1,212il68 10,861 i948 6,155i718
T-cell subsets isolated by positive selection on immunomagnetic beads from six
20 normal and six HIV+ donors. Average purities are shown in parenthesis. The cells were
plated at 50,000 cells per well in 96 well flat bottom tissue culture plates in CRPMI and
10 percent NHS pulsed for eight hours with 1 ~Ci [3H] - thymidine after 88 hours of
stimulation with either medium alone, immobilized anti-CD3+ IL-2 (10 u/ml) or PMA
(0.5 ng/ml). Results are shown as the average cpm and standard errors, Each group was
25 performed in triplicate.
The results indicate that a significant T-cell proliferative response is
possible from HIV+ donors. The CD4+ cell response to anti-CD3+ IL-2
of HIV+ donor cells was approximately 30 percent less than for the
30 normal donors, but still significantly higher than the medium alone
control. The CD8 + cells of HIV + donors responded nearly the same to
anti-CD3 + IL-2 as did normal cells. The CD8 + response of normal and
HIV+ donor cells was significantly less than that observed in CD4+
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cells. These results indicate that purified T-cell subsets from HIV+
donors are capable of responding to mitogenic signals.
To demonstrate that mitogenic mAbs could provide the second
signal for T-cell proliferation in anti-CD3 activated T-cells from HIV+
5 donors the following experiments were performed. T-cells purified from
PBL of HIV+ donors were isolated using AET-treated SRBC. The anti-
CD3 activated T-cells were exposed to soluble anti-CD8 alone, anti-CD5
alone and a combination of anti-CD28 and anti-CD5. The results are
shown in Table 3.
Table 3 Prcl~r~ralion Response of T-Cells from HIV+ Donors
to Mitogenic mAbs
Stimulation cpm + SEM
medium 1,810 + 130
anti-CD3 2,338 + 144
anti-CD3 + IL-2 11,882 + 35
anti-CD3 + anti-CD28 13,334 + 300
anti-CD3 + anti-CD5 3,629 + 102
anti-CD3 + anti-CD5 + anti-CD28 12,882 + 69
T-cells purified by the AET-treated SRBC E-rosetting procedure
(99.6 percent CD3+) were isolated from PBL of an HIV+ donor. The
cells were plated at 50,000 per well in a 96 well flat bottom tissue
culture plate in cRPMI and 10 percent NHS. The cells were activated
25 with immobilized anti-CD3 mAb and stimulated with either IL-2 (10 u/ml),
soluble anti-CD28 mAb (200 ng/ml) soluble anti-CD5 (200 ng/ml) or a
combination of soluble anti-CD8 and anti-CD5. Cells were pulsed for eight
hours with 1 u Ci [3H]- thymidine after 88 hours of stimulation. Results
are shown as cpm and standard error from a single donor. Each
30 treatment group was run in guadruplicate.
Anti-CD28 was as effective as IL-2 in providing the second signal
to purified T-cells from an HIV+ donor. Anti-CD5 had no effect alone or
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in combination with anti-CD28 while augmenting the proliferative
response in T-cells from normal donors.
Minimum Cell Density Required for Flol rerali~e Response.
In order to determine the minimum cell density required for the
5 immobilized anti-CD3/soluble anti-CD28 system to cause 7-cells from
HIV~ donors to proliferate, the following study was conducted.
T-cells from an HIV + donor and a normal donor were purified using
~he AET-treated SRBC E-rosette procedure described earlier. Purities ot 1-
cells were 99.4 percent for the HIV+ donor and 99.2 percent for the
10 normal donor. The T-cells were serially diluted from a starting
concentration of I x 106 cells/ml and plated onto 96 well plates. Final cell
count/well ranged from 100,000 to 1,000. All experimental groups were
studied in quadruplicate. The results are shown in Table 4.
Table 4. Minimum Cell Density Required for
15 T-Cell Proliferative Response in the
Anti-CD3/Anti-CD28 System
HlV+Donor Normal Donor
# Cells/Well Medium Anti-CD3 Medium Anti-CD3
Anti-CD28 Anti-CD28
100,000 1,628i42 22,842+462 1,042i214 52,820i428
50,000 1,822 i 120 14,920 i 108 1,944 i 108 29,642 i 262
25,000 1,206 i 24 8,444 i 48 1,496 i 51 14,322 i 125
10,000 1,828 + 18 2,420 i 186 1,684 i 49 6,246 i 68
5,000 1,484 i 56 1,848 i 342 1,544 i 32 4,820 i 320
1,000 1,741 i85 1,296+260 1,821 i74 1,948i146
T-cells purified by an E-rosetting procedure using AET-treated SRBC from a
normal and an HIV+ donor were tested for their ability to respond to immobilized anti-
CD3 mAb and 200 ng/ml of soluble anti-CD28 mAb. T-cells were cultured for 88 hours
30 with anti-CD3/anti-CD28 or medium alone and then pulsed with [3H]- thymidine for an
additional eight hours. Results are shown as cpm i standard error. All treatment groups
were run in duplicate. A single donor was used in each treatment group.
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T-cells from the HIV+ donor exhibited significant proliferative
response in the anti-CD3/anti-CD28 system at cell densities above 2.5 x
105 cells/ml (25,000 cells per well). T-cells from the normal donor were
capable of responding down to a density of 5 x 104 cells/ml (5,000
cells/well). The proliferative response of T-cells from the HIV+ donor
was approximately 50 percent less than the T-cells from the normal
donor.
Hl~ Purge Method
H9 Continuous Cell Line. In order to reconstitute the Immune
system of an AIDS patient, large numbers of CD4+ cells are required.
Since these cells harbor latent and active HIV-1, a method is required that
will isolate a viral-free starting population of CD4+ cells. If the purging
method is not 100 percent effective, the virus will quickly take over the
culture as it is stimulated to replicate by activation of the host cell.
To demonstrate the feasibility of purging CD4+ cells from AIDS
patients of HIV-1, an HlV-infected continuous cell line was used. The cell
line, H9 (gift from Dr. Gallo, NIH, deposited under ATCC No. CRL 8543),
is a cloned CD4+ human Iymphocyte line. It grows continuously in
culture and can also continuously propagate HIV-1.
Z0 p24 ELISA. A commercial kit (Dupont) was used to assay the
amount of virus in the cell cultures and monitor the efficiency of the
purging experiments. The kit can detect one viral particle in 5,000 cells.
The test uses highly specific rabbit polyclonal antibodies to HIV p24 core
antigen. These antibodies are immobilized on a 96-well plate. The
antibodies capture p24 antigen that is released into the supernatant of a
cell culture after treatment with five percent triton-X to Iyse the cells.
The captured p24 core antigen is then complexed with anti-p24
biotinylated polyclonal antibodies. The complexes are probed with a
streptavidin-HRP (horseradish peroxidase) conjugate. The complexes are
-
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detected by incubation with orthophenyldiamine-HCI (ORD) which
produces a yellowish color proportional to the amount of HIV p24 antigen
captured. The absorbance of each well was determined on a microplate
reader (Dynatech, Minireader ll) and calibrated against the absorbance of
5 known values of p24 antigen. To increase the sensitivity of the test, test
cells were co-cultured with PHA-activated, normal Iymphocytes.
Results
The theory used for the purging protocol is based on known
phenotypic behavior of infected cells. HIV + cells with active virus will
10 express the env gene products gpl20 and gp41 on their cell surfaces.
Since it was reported that HIV+ cells with active virus internalize their
CD4 receptors, positive selection of CD4 was tested.
H9 cells not infected with HIV-1 are 85 percent CD4+ (H9-)
whereas infected H9 cells (H9+) are four percent CD4+ as determined
15 by flow cytometry. An experiment was designed where 10 million H9
cells were mixed in the following ratios:
(1) 10 percent H9+ and 90 percent H9-;
(2) 30 percent H9+ and 70 percent H9-:
(3) 60 percent H9+ and 30 percent H9-; and
20 (4) 80 percent H9+ and 20 percent H9-
Cells from each group were positively selected for CD4 with
immunomagnetic beads. A sample of the positively selected cells were
tested for p24 with the commercial ELISA test ~no co-cultivation).
Results are shown in Table 5.
Table 5 Purge of H9 Cells Infected with HIV-1.
p24 before CD4 removal p24 after CD4 removal
- 0%H9+0.03 ng 0.01 ng
10%H9+0.25 ng 0.00 ng
30%H9+ 0.58 ng 0.00 ng
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p24 before CD4 removalp24 after CD4 removal
60%H9+ 0.94 ng 0.03 ng*
80%H9+ 1.36 ng 0.03 ng*
100%H9+ 2.14 ng 0.09 ng
*same as negative control
The continuous cell line H9 infected HIV-1 (H9+) and non-infected
H9 ~H9-) were mix~d at various ratios. Cells ~xpressing tl~e C;)4 sur~ace
antigen were purged from the mixture using specific mAbs and
10 immunomagnetic beads. The amount of p24 antigen in the cultures was
determined before and after the purge process.
All groups with the exception of the 100 percent H9 + group were
successfully purged of virus below the detectable limits of this assay. To
determine if the negative fractions would continue to be viral-free the
15 cells were incubated for 20 days in 24-well plates with 3 x 106 indicator
cells (normal Iymphocytes activated with PHA for 72 hours) In cRPMI and
109 NHS. Fresh indicator cell were added again on day seven. On days
seven, 14 and 20, 1 x 108 cells from each group were Iysed with triton-
X and assayed for p24. The results are shown in Table 6.
Table 6. Co-Cultivation of Viral Purged H9 Cells with Indicator Cells
Day 10% H9+ 30% H9+ 60% H9+ 80% H9+
0 0.00 ng 0.00 ng 0.03 ng 0.03 ng
7 0.04 ng 0.14 ng 0.20 ng O.Z9 ng
14 0.09 ng 0.23 ng 0.38 ng 0.32 ng
25 20 0.25 ng 0.53 ng 0.59 ng 0.38 ng
H9+ cells mixed with H9- cells at various ratios were purged of CD4+ cells
using immunomagnetic beads. The H9- fractions were co-cultured with PHA-stimulated
Iymphocytes. The fractions were tested for presence of p24 viral antigen at days zero,
30 seven, 14 and 20.
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These results indicate that the original viral purge was not 100
percent effective and virus can still exist below the level of sensitivity of
the assay. In a further attempt to develop a viral-free culture, 1 x 1 o6
cells from each group were serially diluted and plated at 500 cells per
5 well in 2,000 wells of 24-well plates. The cells were allowed to expand
for 14 days and then were co-cultured with indicator cells for 20 days as
before. Cell samples were analyzed for p24 antigen after 20 days as
described earlier; The results are shown in Table 7.
Table 7. Co-Culture of Viral-Purged H9 Cells with indicator Cells
After Plating at 500 Cells/Well
Group% of Positive Wells*
1 0%H9 + 16%
30%H9 + 32%
1560%H9 + 26%
80%H9 + 32.5%
*any value over the ne~li.,e control
H9+ cells mixed with H9- cells at various ratios, purged of CD4+ cells and
cultured for 20 days with PHA-stimulated indicator Iymphocytes were serially diluted to
500 cells per well of a 24-well plate. The cells were allowed to expand for 14 days and
assayed for p24 viral antigen. The percent of wells from each ratio of H9 + to H9- cells
that were positive for p24 is shown.
Those results showed that virally-infected cells could be eliminated
after positive selection by serial dilution. To further validate this
procedure, the negative wells were pooled and cultured with indicator
cells for another 20 days. All groups remained negative for p24 antigen
(data not shown). Thus, the combination of positively selecting CD4+
30 cells followed by serial dilution, should be useful as a viral purge method.
- To further test the sensitivity of the assay system, two-fold serial
dilutions were made from H9+ cells from 500 cells/well to less than one
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cell/well (defined as a two-fold dilution beyond one cell/well). The results
are shown in Table 8.
Table 8. Serial Dilution of H9 + Cells to Test Sensitivity of p24 Antigen
Assay.
Positive Control H9+ Cells
CO~C~.. I.al;~l~ ng/ml Absorbance Concentration Absorbance
0.25 1.03 > 8 cells/well over
0.125 Q.5~ 8 ce!ls/we!! 1.53
0.0625 0.30 4 cells/well 0.89
0.0313 0.15 2 cellslwell 0.53
0.0157 0.04 1 celllwell 0.24
0.0 ng/ml 0.03 < 1 cell/well 0.10
Absorbance of known concentrations of p24 antigen in a commercial ELISA
15 (Dupont) were compared with absorbance of cell Iysates from an HIV-1 infected continuous cell line - H9.
These results indicate that the assay is extremely sensitive; it is
able to detect p24 in c one cell/well down to 0.0157 ng/ml
concentration.
20 Viral Purge from HIV+ Donor
The H9 studies indicated that positive selection of CD4+ cells
combined with serial diiution could isolate a viral-free subpopulation of
cells. The process can be monitored with great sensitivity by a
commercial p24 assay. This process, however, does not address the
25 purging of latent virus from the cells. In order for latent virus to
proliferate, the host cell must be activated. The immobilized anti-CD3
system has proven to be an effective activator of these cells. After
activation, the viral-free cells must be protected or they will soon become
infected just as the indicator cells do in the p24 assay. Anti-CD4 mAb
30 was used to protect uninfected CD4+ cells.
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Materials and Methods
Lymphocytes were Isolated from the AIDS patient following
leukaphoresis as described above. A sample of unfractionated cells were
tested for p24 in a co-cultivation test for 20 days. Similar samples were
5 tested after macrophage adherence, CD4 positive selection and CDB
positive selection. CD4+ cells were activated in 24-well plates on
immobilized CD3 mAb. Soluble anti-CD28 was added to the medium and
the cells were harvested after seven days. The CD4+ cells were then
again labelled with anti-CD4 and positively selected for with GAM-coated
10 immunomagnetic beads. The positively selected cells were relabelled with
anti-CD3 and placed on GAM-coated 96-well plates at 25,000 cells/well.
Anti-CD28 was added to the growth medium.
After seven days, supernatant from each well was tested for p24
antigen. All the negative wells were pooled and again subjected to CD4
15 positive selection with immunomagnetic beads. The positively selected
cells were relabelled with anti-CD3 mAb and plated again at 25,000 cells
per well. Anti-CD28 was added to the medium and the wells were tested
for p24 again after seven days. Negative wells were again pooled and
expanded as described previously for normal Iymphocytes with the
20 exception of only anti-CD28 and the addition of anti-CD4 (leu 3a, Becton
Dickinson) to protect the cells from any residual virus. The cells were
expanded to over ten million and a one-million cell aliquot was harvested
for co-cultivation with indicator cells, p24 readlngs of cell Iysate was
taken after 20 days.
25 Results
Results are shown in Table 9.
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Table 9 Viral-Purge of Lymphocytes from HIV+ Donor.
p24 Levels
PBL (before adherence) 0.32 ng
PBL (after adherence) 0.28 ng
CD4+ 0.24 ng
CD8+ 0.00 ng
Amount of p24 antigen recovered from a one million cell Iysate of HIV+ cells
before removai of macrophages by adherence to plastic T-flasks, after the removal ot
10 macrophages, after positive selection of CD4+ cells and CD8+ cells.
The CD4+ cells were plated at 25,000 cells per well of a 96-well
plate and expanded for seven days on immobilized anti-CD3 mAb and
soluble anti-CD28 mAb. Each well was then assayed for p24 antigen.
Results are shown in Table 10.
15 Table 10 Detection of HIV-1 In Wells of Expanded
CD4+ Cells Purified from HIV+ Donor.
# of Wells# Greater than% Negative
Background
Group 1133 24 82%
Group 2108 18 83%
20 Group 3141 29 79%
Amount of p24 antigen recovered from wells of 96-well plates with 25,000
CD4+ cells purified from the peripheral blood of an AIDS patient and expanded for
seven days on immobilized anti-CD3 mAb and soluble anti-CD28 mAb. Each group
25 represents the results of a separate purification from the same patient.
The percent negative wells was very consistent. The cells from the
negative wells were pooled and propagated with immobilized anti-CD3
and anti-CD28, anti-CD4 was added to protect uninfected cells. All cells
were plated at 2.5 x 105 cells/well in 24-well plates. The number of
30 CD4+ cells recovered after six days in culture is shown in Table 1 1.
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Table 11 Pooled CD4+ Cells Purged of
Active and Latent Virus Expanded 6 Days.
Day Group 1 Group 2 Group 3
o 3.3 x 106 2.1 x 106 3.6 x 106
6 12.4 x 106 11.8 x 106 11.4 x 106
CD4+ cells purged of active and latent virus were expanded in 24-well plat~3s.
Cells were harvested and counted after six days in culture with immobilized anti-CD3
mAb and anti-CD28 mAb.
The cells from the 24-well plates were pooled and incubated in
spinner flasks for three days. They were then relabelled with anti-CD4
and rosetted with GAM-coated immunomagnetic beads. 1 x 106
positively selected cells were co-cultured with indicator cells for 20 days.
The cell Iysates for all three groups were negative for p24 (data not
15 shown). These results demonstrate that this method is capable of
producing a viral-free fraction of CD4+ cells from the peripheral blood of
AIDS patients.
The cells from the three groups were pooled and relabelled with
anti-CD3 mAb and inoculated into 2 GAM-coated cartridges of a min-
20 hollow fiber device with 200 ng/ml of anti-CD28 mAb. After Z1 days of
culture, 1.7 x 108 cells were harvested. Three days after harvest, the
cells were relabelled with anti-CD3 mAb and inoculated into a single
GAM-coated cartridge on the large scale device with 200 ng/ml of anti-
CD28 mAb. After 21 days of culture, 1.1 x 10'~ cells were harvested.
25 Three days after harvest, these cells were relabelled with anti-CD3 mAb
and inoculated into 8 GAM-coated cartridges on the large-scale device
with 200 ng/ml of anti-CD28 mAb. After 18 days of culture, 6.4 x 1C)~~
CD4+ cells were recovered. The cells were negative for p24.
- CD4+ Functional Studies
To demonstrate that CD4+ cells isolated and propagated by this
process were still capable of normal function, their ability to enhance NK
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activity was assessed. Patients with AIDS are known to have reduced NK
function. Some reports have shown that exogenous IL-2 can significantly
enhance NK-function of AIDS patients in-vitro. This study demonstrated
that adding the expanded viral-purged CD4+ cells was effective.
5 Materials and methods
The NK-sensitive cell line K562 was used as the target cell. The
cells were chromium labelled by suspension at a concentration of 1 x 107
cells/ml in cRPMI containing 100 ,uCi/mi of [~lCr] sodium chromate (New
England Nuclear, Boston, MA) for 60 minutes at 37~C. The cells were
10 then washed twice, resuspended at 5 x 104 cells/ml in 100 ,ul aliquots
into wells of round-bottomed 96-well plates.
Monocyte depleted Iymphocytes from AIDS patients suspended at
5 x 106 cells/ml were added to wells containing the target cells in 50 ,ul
aliquots. An additional 50 ~l of medium or CD4+ cells was added to each
15 well such that the effector:target ratio without CD4+ cells was 50:1.
After a one hour incubation at 37~C In five percent C~2 at 100
percent humidity, the plates were centrifuged at 800 x g for 12 minutes
and 100 ,ul aliquots of each well were harvested and counted on a liquid
scintillation counter. Percent Iysis of each target cell was determined by
20 the equation:
% Iysis = CPmtest - CPmcontrol/CPmmax - CPmcontrol x 100, where
cpmteSt indicates chromium counts per minute released in the presence of
Iymphocytes, cpmcOntrol indicates release of the presence of medium alone,
and cpmmaX indicates release in the presence of BRIS-35 detergent
25 (Sigma, St. Louis, M0).
Each test was performed in quadruplicate. Significance of percent
Iysis was determined by comparing mean cpmteSt with mean cPmcontrol by
student's t-test. Results are shown in Table 12.
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Table 12. NK-Activity of Lymphocytes from AIDS Pdlie~ll S~rple ~~ented
with Autologous, Viral-Purged CD4+ Cells.
Results % Lysis
AIDS Iymphocytes alone 26.2 + 6.5%
AIDS Iymphocytes +1 IL-2 (10 U/ml) 54.5 + 6.8%
AIDS Iymphocytes + CD4+ (1000) 33.4 i 7.0%
AIDS Iymphocytes + CD4+ (5000) 48.8 i 3.5%
AIDS Iymphoeytes + CD4+ (10,0Q01 ~i~.5 + 5%
AIDS Iymphocytes + CD4+ (50,000) 64.2 + 9.5%
Normal Iymphocytes alone 60.2 i 6.4%
Normal Iymphocytes + IL-2 (10 U/ml) 73.5 + 6.5%
NK-activity of a single AIDS patient after reconstruction with autologous, viral-
purged CD4+ cells. The number of added cells is noted in parentheses. Results are
5 eXp!eSSed 2S the me2n *SE of quadrup!icate ~amp!e~.
The NK-activity of AIDS patients of 26.2 + 6.5% was significantly
lower than the 60.2 + 6.4% for normal controls. The addition of IL-2
significantly increased NK-activity in normal and AIDS patients, but had a
much greater effect in AIDS. The addition of 1,000 autologous CD4+
20 cells did not significantly increase NK-activity. Addition of 5,000 and
10,000 CD4+ cells significantly increased activity to normal levels.
Addition of 50,000 CD4+ had the same effect as 10,000 cells.
These results evidence that the CD4+ cells isolated and expanded
by this protocol are able to produce IL-2. These results also support the
25 evidence that large numbers of these CD4+ cells infused back to the
patient should restore immunological function.
Purification of HlV-Specific T-cells
HlV-specific class l-restricted T-cells are known to be present in the
blood of AIDS patients; they are presumed to be a subset of CD8 +,
30 CD28 +, CD 1 1-, CD25 + Iymphocytes. These are in vivo activated
(CD25 + same as IL2R+) Tc (CD28+ same as 9.3). To isolate these
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cells, a series of positive selection steps were conducted using CD8
~leu 2a, Becton Dickinson), CD28 (KOLT-2 gift from K. Sagawa), and
CD25 (IL-2R, Coulter) mAbs and GAM-coated immunomagnetic beads.
Positive selection occurred in the following order: CD8, CD28, and
5 finally, CD25. A subset of the isolated cells should be HlV-specific. The
other in vivo T-cells in this group may also be of therapeutic importance;
they may be specific for other adventitious agents afflicting the patient.
Al~)S patients usually had a high percentage of CD25 + cells. In six
patients tested, the mean CD25+ cells were 14 + 8% compared to six
10 normal controls at 3 + 2.5%.
CD8 + Functional Studies
The CD8+ CD28 + CD25+ T-cells isolated from an AIDS patient
and expanded to 5.3 x 101~ cells were tested for their ability to Iyse HIV-
infected autologous CD4 + Iymphocytes. The target Iymphocytes were
15 expanded viral-free CD4+ cells from the same patient from whom the
effector cells were isolated. The CD4+ cells were activated on
immobilized anti-CD3 at 5 x 105 cells/ml in one ml cRPMI on a 24-well
plate. One ml of H9+ supernatant containing 109 U/ml IL-2 was added
to each well. The CD4+ cells were harvested from the wells after
ZO incubation at 37~C in five percent CO2 at 100 percent humidity for four
days.
The cells were labelled with 5lCr using the same procedure as
described for K562 target cells. All cells were plated in round-bottomed
96-well plates at effector:target ratios of 100:1, 50:1, and 25:1. Percent
25 Iysis was determined as described earlier. Each test was performed In
triplicate. Results are shown in Table 13.
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Table 13 CD8 +, CDZ8 +, CD25 + Killer T-Cells Isolated from HIV +
Patient, Ability to Lyse Autologous HIV Infected Cells
Cell: Target Ratio % Lysis
100:1 21.0 i 8.0%
50:1 9.O i 3.5%
25:1 3.5 i 2.0%
CD8 +, CD28 +, CD25 + Tc isolated from an AIOS patient were tested for their
10 ability to Iyse autologous CD4+ cells infected with Hl-1. Percent Iysis was calculated
from a 51Cr-release assay.
These results indicate significant effector function. The low
percentage Iysis was probably due to a combination of a low percentage
of targets infected with HIV (74 percent remained CD4+) and a high
1 5 background.
Although the present invention has been described with reference
to preferred embodiments, workers skilled in the art will recognize that
changes may be made in form and detail without departing from the spirit
20 and scope of the invention. Since modifications will be apparent to those
of skill in this art, it is intended that this invention be limited only by the
scope of the appended claims.
