Note: Descriptions are shown in the official language in which they were submitted.
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IN VITRO INDUCTION OF ANTIGEN-SPECIFIC T-CELLS
USING DENDRTfIC CELL-TUMOR CELL
OR DENDRITIC CELL-VIRAL CELL DERIVED IIVIMUNOGENS
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Serial No.
09/030,985 filed February 26, 1998, which claims priority to U.S. Serial No.
60/039,472, filed February 27, 1997.
FIELD OF THE INVENTION
The present invention generally relates to antigen-specific T-cells and
methods for making and using the same. More specifically, the present
invention
relates to antigen-specific T-cells that have been generated by co-culture
with
immunogens derived from a formulation comprising either hybridomas of
dendritic
cells and tumor cells or co-culture products of dendritic cells and tumor
cells.
Alternatively, virally infected cells can be used instead of tumor cells in
these
hybridomas and co-cultures. The use of these T-cells as prophylactic and
therapeutic agents against tumors and viral infection is also the subject of
the
present invention.
BACKGROUND OF THE INVENTION
T-cells, including cytotoxic T-lymphocytes (CTLs), are a critical
component of effective human immune responses to tumors and viral infections.
T-cell responses are sufficient to protect against tumors and viruses and can
eliminate even established cancers in murine tumor models and in humans. T-
cells
destroy neoplastic cells or virally infected cells through recognition of
antigenic
peptides presented by MHC Class I molecules on the surface of the effected
target
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cells. These antigenic peptides are degradation products of foreign proteins
present
in the cytosol of the effected cell, which are processed and presented to T-
cells
through the endogenous MHC Class I processing pathway. CTLs target tumors
through recognition of a ligand consisting of a self MHC Class I molecule and
a
peptide antigen. The development of CTL-dependent anti-tumor immunization
strategies, therefore, typically depends on both the identification of tumor
antigens
recognized by CTLs and the development of methods for effective antigen
delivery.
Although the recognition of a foreign protein in the context of the
MHC Class I molecule may be sufficient for the recognition and destruction of
effected target cells by CTLs, the induction of antigen-specific CTLs from T-
lymphocyte precursors requires additional signals. Specialized.antigen-
presenting
cells (APCs) can provide both the antigen MHC Class I ligand and the accessory
signals required in the induction phase of CTL mediated immunity. General
properties of APCs include MHC Class I and Class II expression, expression of
various adhesion molecules important for APC-lymphocyte interaction, and
expression of co-stimulatory molecules such as CD80 and CD86. APCs include,
for example, macrophages, B-cells, and dendritic cells, including cutaneous
epidermal Langerhans cells, dermal dendritic cells, and dendritic cells
resident in
lymph nodes and spleen. Dendritic cells (DCs) are believed to be the most
potent
QCs, and can induce effective CTL-dependent anti-tumor immunity. Procedures
are available to obtain significant quantities of dendritic cells from bone
marrow or
peripheral blood derived precursors.
It is likely that a tumor cell expresses a set of tumor-specific peptide
MHC complexes which can be recognized by CTLs. Progressive tumors, however,
are generally non-immunogenic, at least in part, because they are incapable of
providing co-stimulation.
Guo et al . , Science 263 : S 18-520 ( 1994), disclose tumor vaccines
generated by fusion of hepatoma cells with activated B-cells. The fusion of
the
activated B-cells and the tumor cells produce an immunogen capable of inducing
~mor-specific protective tumor immunity.
Mayordomo et al., Nature Med. 1 (12):1297-1302 (1995), disclose in
vitro culture of peptide-pulsed dendritic cells that show protection against
the
associated tumor challenge. The dendritic cells cultured in the presence of GM-
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CSF + IL-4 and transfected with chicken ovalbumin (OVA) were capable of
preventing establishment of an OVA+ tumor, but not the untransfected parental
melanoma.
Flamand et al., Eur. J. Immunol. 24:605-610 (1994), disclosed in
vitro culture of dendritic cells pulsed with a peptide antigen BCLl, and
subsequen~ t
induction of a T-cell dependent humoral response to the B-cell tumor BCLI. A
similar methodology is reported by Celluzzi et al., J. Exp. Med., 183:203-287
(1996). There, MHC I-peptide antigens were pulsed onto dendritic cells;
immunized hosts showed protective immunity to a lethal challenge by a tumor
transfected with the antigen gene.
Hsu et al., Nature Medicine 2:52-58 (1996) investigated the use of
dendritic cells pulsed with tumor-specific idiotype proteins as vaccines.
Celluzzi and Falo, J. Immunol. p. 3081-3085 (1998), disclose
formulations comprising dendritic cells fused to or co-cultured with tumor
cells.
Gong et al., Nature Med. 3:558 (1997), disclose fusion of dendritic
cells with MC38 carcinoma cells.
U.S. Patent No. 5,788,963 discloses preparation of dendritic cells for
prostate cancer immunotherapy; stimulation of antigen specific T-cells by the
dendritic cells is reported. The patent appears to be Limited to exposure of
dendritic
cells to specific, defined prostate cancer antigens, or to Iysates or
fractionated
lysates as potential sources for these antigens. The patent methodologies do
not
appear to include fusion or co-culture of dendritic cells with whole tumor
cells or
with virally infected cells, or the use of undefined antigens.
U. S . Patent No. 5, 846, 827 reports methods for using peptide-loaded
antigen presenting cells for the activation of CTL. The methods appear to rely
on
the modification of antigen presenting cells in vivo by exposure to specific
identified
and isolated epitopes. There does not appear to be any teaching of the antigen
specific T-cells or methods of producing these T-cells as taught below.
None of the above articles or patents appear to teach or suggest the
generation of a unique, antigen-specific T-cell such as those disclosed
herein. In
addition, none of these offer a significant advantage of the present
invention,
namely the elimination of the need to isolate and/or identify specific
antigens.
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There is a need for cancer immunotherapy that provides protective
and therapeutic immunity to a wide variety of tumor types. A similar need
exists
for viral immunotherapy directed towards a wide variety of viral infections.
SUMMARY OF THE INVENTION
The present invention has met the above described needs by providing
antigen-specific T-cells and methods of making and using the same. Generally,
the
T-cells are prepared by co-culture of T-cells with formulations comprising
dendritic
cells and either tumor cells or virally-infected cells. One embodiment of this
invention uses a formulation comprising one or more hybridomas; each hybridoma
is further comprised of at least one dendritic cell fused to at least one of
either a
tumor cell or a virally-infected cell. Another embodiment of this invention
uses a
formulation comprising the products of co-culture of dendritic cells and
either tumor
cells or virally-infected cells. Both of these formulations produce immunogens
specific for the type of tumor or virus used in the formulation. Co-culture of
either
of these formulations with T-cells results in T-cells that are antigen-
specific; the
generated T-cells will recognize, and attack, cells expressing the particular
antigens
with which the T-cells have been co-cultured. In the case of tumor
immunotherapy,
the generated T-cells provide both protection against tumor challenge and
regression
of tumor growth. Thus, the T-cells of the present invention provide
prophylactic
resistance to tumors of the type represented by the tumor cell used in the
formulation, and also provide a therapeutic treatment for patients suffering
from
such tumors. Similarly, in the case of viral immunotherapy, the generated T-
cells
protect against the viral infection caused by the virally infected cells used
in the
formulation, and/or provide therapeutic relief for patients having such viral
infections.
Tumor cells and virally infected cells express antigens which can be
targeted by T-cells, but the tumor cells and virally infected cells themselves
do not
stimulate T-cell immunity. This is presumably because the tumor cells and
viral
cells are incapable of providing the antigen or antigens in the appropriate
context of
co-stimulation. Antigen presenting cells (APC), of which dendritic cells (DC)
are
thought to be the most potent, express a variety of co-stimulatory molecules
and
cytokines. The present invention uses formulations in which DCs are fused to
or
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are in a co-culture with either tumor cells or virally infected cells. Fusion
or co-
culture of the DCs with the tumor cells or virally infected cells causes the
antigens
to become more immunogenic by association with the DCs, which are
"professional" antigen presenting cells. The fusion products and the co-
culture
products express properties of both the DC and either the tumor or virus. The
fused cells and/or co-cultured cells are then further co-cultured with
unstimulated
T-cells. During this co-culture, the T-cell is exposed to a complete array of
antigens from either the tumor cells or virally-infected cells. The generated
T-cells
which result are then antigen-specific, and will destroy tumor cells that
express the
same or similar tumor antigens or virally-infected cells that express the same
or
similar viral antigens.
As will be appreciated by one skilled in the art, therefore, the present
invention obviates the need to identify specific antigens that elicit a T-cell
response
by providing T-cells that are generated or engineered to recognize the
antigens with
which they are co-cultured. By delivering and co-culturing the entire array of
antigens produced by a tumor cell or a virally infected cell with the T-cells,
a
mechanism is provided for broad, polyvalent immunotherapy.
It is therefore an object of the present invention to provide antigen-
specific T-cells.
It is a further object of the present invention to provide a
pharmaceutical composition comprised of antigen-specific T-cells.
It is a further object of the present invention to provide methods for
treatment of a patient with antigen-specific T-cells.
Yet another object of the invention is to provide methods for the
prophylactic and/or therapeutic treatment of cancer and viral infections.
Another object of the present invention is to provide antigen-specific
T-cells which can be used to identify tumor or viral antigens.
These and other objects of the invention will be more fully
understood from the following description of the invention.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates flow labelling patterns showing the efficiency of
association between tumor cell components and dendritic cells, as described in
Example 3.
Figure 2 shows the % specific lysis of peptide loaded cells, as
described in Example 4.
Figure 3 shows the % lysis of tumor cells, as described in Example
4.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to methods for. generating tumor
antigen-specific T-cells by combining dendritic cells from a patient with
tumor cells,
preferably from the same patient, in vitro; adding T-cells from the patient to
the
combination of DCs and tumor cells; culturing the T-cell/DC/tumor cell
mixture;
and harvesting the T-cells from the co-culture. The present invention is
further
directed to similar methods using virally-infected cells rather than tumor
cells,
which would result in viral antigen-specific T-cells. T-cells prepared
according to
either of these methods are also within the scope of the present invention.
The T-
cells prepared according to the present invention have demonstrated efficacy
in
providing both prophylactic and therapeutic relief to patients at risk for, or
suffering
from, tumors or viral infections.
The first step of the present methods involves combining dendritic
cells from a patient with either tumor cells or virally-infected cells.
Preferably, the
tumor cells or virally infected cells are from the same patient. The cells can
be
combined in any manner known in the art. Preferred for use in the present
methods
are fusions of the DC with the afflicted cell and co-culture of the DC with
the
afflicted cell. As used herein, the terms "afflicted cell" or "afflicted
cells" refer
generally to either tumor cells or virally infected cells; the afflicted cells
will vary
from application to application depending on the particular type of T-cell
that is to
be generated and the type of immunotherapy desired.
In one embodiment, therefore, the DC and afflicted cell are fused to
form a hybridoma. As will be appreciated by those skilled in the art, a
hybridoma
is a physical combination of at least two different kinds of cells. At least
two
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different hybridomas fall within the scope of the present invention--namely a
hybridoma between at least one DC and one tumor cell, and a hybridoma between
at least one DC and at least one virally infected cell.
According to one embodiment of the present invention, one or more
S dendritic cells are fused to one or more tumor cells to form hybridomas or
fusion
products in the first step of the present methods. Any starting ratio of
dendritic
cells to tumor cells can be used. For example, the starting ratio can be
anywhere
from 1:10 to 10:1, 1:100 to 100:1, or even higher. In a preferred embodiment,
this
fusion product is made with a starting ratio of dendritic cells to tumor cells
of about
6:1; this starting ratio was found to yield a sufficient number of DC/tumor
cell
hybridomas. Preferably, the DCaumor cell ratio includes a higher number of
DCs,
as a higher number of DCs increases the probability that the tumor cells will
become fused to at least one DC. As will be appreciated by one skilled in the
art,
one or more DCs can become fused to one or more tumor cells. Thus, the
hybridomas prepared in the first step of the methods can have a range of
DC:tumor
cell ratios. For example, when starting with an DCaumor cell ratio of 6:1, the
resulting hybridomas could have an DC:tumor cell ratio of anywhere from about
1:1 to 10:1 or more.
Any type of DCs can be used. DCs, it will be appreciated, are one
type of antigen presenting cell ("APC"), which is a cell capable of presenting
antigens. DCs are found throughout the body, and include cutaneous epidermal
Langerhans cells, dermal dendritic cells, dendritic cells located in the lymph
nodes
and spleen, and dendritic cells derived through in vitro culture of
precursors.
Dendritic cells can be obtained from a host by any means known in the art. For
example, dendritic cells can be obtained from bone marrow according to the
methods of Celluzzi, et al., J. Fxp. Med. 183:283-287 (1996). DCs can also be
obtained from peripheral blood and skin. Blood-derived DCs are the preferred
DCs
for use in the present invention.
Any type of tumor cells can be used, including but not limited to,
tumor cells obtained from patients including melanomas, lung cancers, prostate
cancers, breast cancers, colon cancers and cervical cancers. Tumor cells for
which
single-cell suspensions of auto-tumor may be obtained are preferred. In
addition,
while autologous tumor cells are preferred, allogenic tumor cell lines can
also be
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used as the source of tumor antigen. It is well known in the art that certain
tumor
antigens can be found to be present in tumor cells obtained from more than one
patient. These are often referred to as "shared" tumor antigens. In the
present
invention, allogenic tumor cells can be used as a source of antigen as these
cells can
contain "shared" tumor antigens also present in the patient's tumor. For use
in the
present invention, the tumor cells can be treated before or after use in the
present
formulations by, for example, radiation or similar treatment.
According to another embodiment of the present invention, one or
more DCs are fused to one or more virally infected cells. As with the DC/tumor
cell fusion, any starting ratio of DCs to virally infected cells can be used.
Preferably, this ratio is sufficient to allow for the fusion of all of the
virally infected
cells to one or more DC. Any virally infected cells can be used, including but
not
limited to, cells infected with influenza virus, human immunodeficiency virus
(HIV), cytomegalovirus (CMV), human papilloma virus (HPV) and herpes simplex
virus (HSV).
The hybridomas or fusion products of the present invention can be
formed by any method known in the art. In a preferred embodiment, the DC-tumor
cell or DC-virally infected cell hybridoma is formed by fusing the two types
of cells
together with polyethylene glycol (PEG). Generally, this method involves
adding
DCs and afflicted cells to the same container and centrifuging the cell
suspensions
to form a pellet. Approximately 1 ml of a 50 % PEG solution heated to about 37
°C
should be gradually added to the pellet. The pellet/PEG is gradually diluted
with
PBS while gentle stirring is applied. The fused cells are then washed by
centrifugation and the supernatant decanted, to form the fusion product.
The first step in the methods of the present invention can also be
achieved by combining DCs and afflicted cells in vitro, and co-culturing the
mixture, rather than fusing the two cell types together. As used herein, "co-
culture" refers to the culturing together of at least two different types of
cells, here
DCs and afflicted cells alone or in further combination with unstimulated T-
cells.
A co-culture of DCs and either tumor cells or virally infected cells can be
prepared
by simply culturing the DCs with either the tumor or virally infected cells;
that is,
the two cell types can simply be mixed and co-incubated together. Although any
method of co-culturing cells known in the art can be used, in a preferred
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methodology the two cell types are combined and centrifuged to form a pellet.
The
pellet is then diluted with a culture medium, preferably RPMI or AIM S, and
incubated overnight at about 37 °C in a 5 % COZ incubator. As with the
hybridoma
formulation, any ratio of DC to afflicted cell can be used; a ratio of between
about
1:100-100:1 is preferred, a ratio of between about 1:10-10:1 is more preferred
and
the ratio of about 6:1 is most preferred. It will be appreciated by one
skilled in the
art that the co-culture product can be used in the methods of the present
invention
without a selection step.
The terms "products of co-culture" or "co-culture products)", as
used herein, refer to matter resulting from co-culture of afflicted cells and
DCs and
can include, for example, tumor cells and dendritic cells which have become
fused
together, dendritic cells which have internalized or become associated with
antigenic
tumor components or other components of tumor cells, tumor cells which have
internalized or become associated with dendritic cell components relevant to
antigen-presentation function, or subcellular components derived from any of
the
cells described above. It will be understood, therefore, that these terms
reflect that
during co-culture a stimulator cell complex is produced which contains tumor
antigen and molecules necessary for antigen presentation to T-cells. The same
applies to use of virally infected cells.
Following preparation of either the fusion product or the co-culture of
DCs and afflicted cells, T-cells from the patient should then be added to the
dendritic cell/afflicted cell combination. The T-cells, like the DCs and, in a
preferred embodiment the afflicted cells, should all derive from the same
patient.
Thus, the DCs, afflicted cells, and T-cells are preferably all autologous, and
should
be obtained from the patient who will ultimately be treated with the T-cells
generated according to the present methods. The use of autologous cells
eliminates
the need to identify and characterize the specific antigens unique to various
tumor
cells and virally infected cells, which can vary from patient to patient and
from
illness to illness. The T-cells prepared according to the present methods have
the
capability of targeting the particular antigens generated by each host, since
they
themselves have been co-cultured with those antigens. In addition, if using
allogenic tumor cells, "shared" antigens will be present in the co-culture.
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The term "T-cell" as used herein will be understood by those skilled
in the art. The term includes, but is not limited to CD8+ or CD4+ T-cells
capable
of lysing target cells or providing effector or helper functions, such as
cytokine
secretion, which can result in the death of target cells or the generation or
enhancement of anti-target effector activity. The invention is not intended to
be
limited to these examples, however, and any T-cells known in the art can be
used
according to the present invention. Preferably, the T-cells are unstimulated T-
cell
precursors.
T-cells can be obtained from any suitable source, including, but not
limited to spleen tissue, lymph nodes, peripheral blood, tumors, ascitic
fluid,
dermal biopsies, and CNS fluids. Any method for harvesting T-cells from the
host
can be used. For example, Ficoll-Paque (commercially available from Pharmacia)
centrifuged peripheral blood mononuclear cells (PBMC) can be used.
Alternatively,
purified CD4+ or CD8+ T-cells isolated by immunoaffinity procedures, such as
through the use of MACs or dyna-beads, can be used. Appropriate methods for
obtaining T-cells are taught, for example, in Tilting, et al., J. Immunol.
160:1139-
1147 ( 1998) .
The harvested T-cells should then be added to the media containing
the fusion product or co-culture product of the dendritic cells and afflicted
cells. A
ratio of 10:1 to 100:1 T-cells:dendritic cells is preferred, although any
other
suitable ratio is also within the scope of the invention.
The mixture of the T-cells, dendritic cells, and either tumor cells or
virally infected cells should then be cultured. The cells can be cultured for
seven to
ten days in culture medium with restimulation at seven to ten-day intervals
with
identically prepared DC/afflicted cell stimulators. Cultures may be maintained
indefinitely using this protocol. Culture medium is preferably RPMI or AIM-V
medium supplemented with FCS or HABS (0-10%) and interleukin-2 (5-100 IU/ml).
Approximately five to ten days after the last stimulation, T-cells are
analyzed for
cytotoxicity and cytokine-release assays, or after three to five days from the
last
stimulation for proliferation-based assays.
Harvesting the T-cells from the co-culture can be accomplished by
any means known in the art. For example, harvesting can be simply accomplished
by pipet transfer of cultured cells from the flask or plate into a centrifuge
tube.
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After centrifugation, cells are recovered from the pellet and used in the
readout
assays (i. e. , cytotoxicity, cytokine release and proliferation assays). More
specifically, CD4+ and CD8+ T-cells can be selectively recovered using the
immunoaffinity procedures discussed above. Typically, after 2-3 rounds of in
vitro
stimulation only T-cells are in the cultures. The T-cells can be used in any
variety
of manners, including in methods for treating patients, as probes for specific
antigens, and in methods of establishing animal models useful in the study of
the
immunological arts.
The present invention is therefore also directed to a method of
effecting immunotherapy in a host comprising combining dendritic cells from a
patient with afflicted cells in vitro; adding unstimulated T-cell precursors
from the
patient to the dendritic cell/afflicted cell combination; co-culturing the
mixture;
harvesting the T-cells from the mixture; and administering to a mammalian host
an
effective amount of harvested T-cells. As used herein, the terms "host",
"mammalian host" and "patient" refer to the organism from which the relevant
cells
are being extracted and/or who is being treated by the present methods,
including
but not limited to humans. It will be understood that "immunotherapy" includes
the
prophylactic treatment of a patient susceptible to tumors, such as a patient
in a high
risk group for certain types of cancer; "immunotherapy" also includes the
therapeutic treatment of a tumor-bearing patient. DCs and tumor cells as
described
above can be used. It will be further appreciated that the type of tumor cell
used
will depend on the type of cancer for which the treatment is being
administered.
For example, if the patient is being treated to provide prophylactic
resistance to
melanoma, melanoma cells should be used. The patient's own melanoma cells, or
allogenic melanoma cells containing shared antigens, should be used.
"Immunotherapy" also includes both the prophylactic treatment of a patient
prior to
a viral infection and the therapeutic treatment of patients having a viral
infection.
Any DCs and virally infected cells as described above can be used. Again, the
type
of virally infected cell used will vary depending on the viral infection for
which
treatment is being provided.
An effective amount of the T-cells generated according to the present
methods should be administered to the host being treated. As used herein, the
term
"effective amount" refers to that amount of T-cells to achieve the desired
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unotherapy, such as the amount of T-cells which will bring about the desired
level of prophylactic resistance or therapeutic relief to a patient.
As will be appreciated by those skilled in the art, the effective
amount will differ from patient to patient depending on such variables as
whether
the use is prophylactic or therapeutic, the size and/or severity of the tumor
or
tumors, the type and/or severity of the viral infection, the size and weight
of the
patient, and the like. Even a minimal dosage of antigen-specific T-cells would
provide a benefit to a patient. It is within the skill of one practicing in
the art to
determine the effective amount for each patient; a typical maximum dosage per
treatment would be about 10" T-cells. Higher or lower doses can be used
depending on the patient being treated. The number of treatments will also
depend
on the patient being treated, the illness being treated, the patient's
response to
treatments, and the like. Again, it is within the skill of the practitioner to
determine
the appropriate number of treatments.
The T-cells can be introduced to the host by any means known in the
art including but not limited to the use of a pharmaceutical composition
prepared
with the T-cells. For example, the T-cells can be combined with a suitable
pharmaceutical carrier. Any suitable pharmaceutical carrier can be used, as
long as
compatibility problems do not arise. The preferred carriers are saline,
phosphate
buffered saline (PBS) and media which contains T-cell growth factors. The T-
cell
compositions can be administered, for example, intravenously, intramuscularly,
intralymphatically, intradermally, subcutaneously and intratumorally.
The T-cells of the present invention can also be utilized to identify
tumor or viral antigens. It will be appreciated that when tumor cells are used
to
stimulate T-cells, the T-cells will be useful in the identification of tumor
antigens,
and when virally infected cells are used to stimulate T-cells the T-cells will
be
useful in identifying viral antigens. For example, T-cells may be used as
indicator
reagents to identify specific tumor peptides or tumor gene products, as taught
by
Storkus et al. , J. Immunol. 151: 3719-3727 ( 1993) and van der Bruggen et al.
,
Science 254:1643-1647 (1991). Antigen presenting cells (such as dendritic
cells or
transformed cell lines) may be loaded with fractionated peptides extracted
from
tumor cells (Storkus et al., 1993) and then analyzed for reactivity
(cytotoxicity,
proliferation, cytokine release, etc.) with T-cells prepared according to the
current
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invention. Those peptide pools recognized by T-cells may then be analyzed
using
mass spectroscopy or Edman degradation sequencing methods to identify the
sequence of individual peptides within the pool. Synthetic peptides conforming
to
these sequences may then be generated and tested for T-cell reactivity, with
positively recognized peptides constituting potential vaccine components.
Alternatively, tumor-derived DNA or cDNA may be transfected into DC or
transformed cell lines (van der Bruggen, 1991) and the resulting transfectants
screened for their ability to be recognized by T-cells obtained by the methods
of the
current invention. Transfected DNA extracted from targets recognized by T-
cells
may then be sequenced, with the resulting tumor-associated gene/gene product
constituting a potential gene vaccine component. In addition, peptides derived
from
these gene products may also serve as components of peptide-based vaccines and
therapies .
T-cells can also be generated according to the present methods for
use in preparing animal models. Such models would be useful, for example, in
studying the various effects of various types of immunotherapy on the host
being
treated. For example, T-cells may be induced in vivo in mice as outlined by
Celluzzi and Falo, J. Immunol. 3081-3085 (1998), with tumor vaccination and
therapy being the outcome. Alternatively, using the current invention, antigen-
specific T-cells may be generated ex vivo, with the expanded cells adoptively
transferred into tumor bearing mice or patients, or virally-infected patients.
Since
murine models allow for both protective and therapeutic systems to be
evaluated,
this approach serves to define surrogate systems in which to validate the
potential
clinical efficacy of various treatment methodologies.
When using the present T-cells, the immunotherapy of the present
invention results in the induction of tumor specific lytic activity in an
immunized
mammalian host. That is, prophylactic or therapeutic treatment will be
specific for
the type of tumor used in the DC-tumor cell hybridoma or co-culture. Such
immunization protects patients from tumor challenge and/or results in
regression of
established tumors. Similarly, prophylactic or therapeutic treatment will be
specific
for the type of virus used, and will protect patients from viral challenge
and/or will
result in reduction of viral infection. Thus, the present methods of
immunotherapy
eliminate the need to isolate and characterize individual antigens. The
present
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invention therefore provides a rapid, inexpensive and efficient technology for
generating antigen-specific T-cells in vitro. These T-cells can be used as
immunotherapy through the adoptive transfer of autologous antigen-specific T-
cells
in patients suffering from tumors or viral infections.
EXAMPLES
The following examples are intended to illustrate the invention, and
should not be construed as limiting the invention in any way. The mice used in
the
examples were female C57BL/6 mice, 5-8 weeks old and were obtained from the
Jackson Laboratory in Bar Harbor, Maine. B16 is a C57BL/6 melanoma (H-2b)
obtained from ATCC, Rockville, Maryland, and 3LL is a lung carcinoma also
available from ATCC. Cell lines were maintained in DME containing 10% FCS
and antibiotics. Monoclonal antibodies used to deplete cell subsets were
prepared
from the hybridomas GK 1.5 (anti-CD4, ATCC T1B 207), 2.43 (anti-CDB, ATCC
T1B 210), 30-H12 (anti-Thy 1.2, ATCC T1B107), B220 (anti-B cell surface
glycoprotein, ATCC T1B 146), and NK1.1, obtained from W. Chambers,
University of Pittsburgh School of Medicine.
Example 1 - Fusion of DCs and Tumor Cells
Dendritic cells were prepared from bone marrow as generally
described in Celluzzi et al., J. Exp. Med. 183:283-287 (1996) using GM-CSF as
described in the reference. Briefly, bone marrow cells were depleted of
lymphocytes and cultured at 5 x 105 cells/ml in 10 % FCS-containing RPMI 1640,
obtained from Irvine Scientific, Santa Ana, California, with granulocyte
macrophage-colony stimulating factor (GM-CSF), in a concentration of 103 U/ml,
obtained from Sigma Chemical Company, St. Louis, Missouri. Loosely adherent
cells were collected on day 6 for fusion. Between about 50 and 75 % of the DCs
expressed CD86 (B7.2) and Class II MHC (I-A+) antigens, as determined by flow
cytometry .
Day 6 DCs were fused with either B16 or 3LL cells at a ratio of 6:1,
DC to tumor cells, using polyethylene glycol at 37°C. After
washing by
centrifugation, fused cells were cultured overnight at 37°C in RPMI
1640 (10%
FCS).
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Example 2 - Preparation of Dendritic Cell and Tumor Cell Co-cultures
Dendritic cells were prepared according to the methods of
Example 1. Day 6 dendritic cells were then used to form a co-culture with
either
B16 cells or 3LL cells. Each DC/tumor cell co-culture was prepared by placing
DCs into test tubes with the respective tumor cells. A pellet of cells was
formed by
centrifugation. The pellet was then diluted with RPMI (10% FCS) and incubated
overnight at about 37°C in a 5% COZ incubator. The ratio of DCsaumor
cells was
about 6:1. The products of the co-culture were prepared to further evaluate
whether tumor antigens were present in close association with DCs and to
evaluate
if soluble factors released from the tumors were present on the DCs.
Example 3 - Efficient of Fusion and Co-culture
To determine the efficiency of fusion and co-culture, each of the cell
types -- DCs, B16 and 3LL -- was stained with a different lipophilic
fluorochrome
before fusion and analyzed using flow cytometry. The tumor cells were stained
with Di0 while the DCs were stained with DiI, both of which were obtained from
Molecular Probes, Inc., Eugene, Oregon. After extensive washing, cells were
fused or co-cultured and allowed to incubate overnight at 37°C.
Harvested cells
were then fixed in 2 % paraformaldehyde and the forward and side scatter
patterns
measured on a Becton Dickinson Facstar Plus with Argon/HeNe duel laser,
available from Becton Dickinson Immunocytometry Systems, San Jose, California.
The scatter pattern of each cell type is depicted in Figure 1.
Individual cell staining shows two distinct patterns of DCs (DiI) which shift
up
(upper left quadrant of Figure lA) or B16 tumor cells (Di0) which shift right
(lower right quadrant of Figure 1B) as compared to unstained controls (not
shown).
When cells were co-cultured (Figure 1C) or fused (Figure 1D), the patterns
shifted
both to the right and up, indicating that those cells were doubly stained.
This upper
right quadrant was used as the indicator of the "efficiency" of the
association of
tumor antigens with dendritic cells. Cells found in the upper left or lower
right
quadrants were regarded as singly stained cells and were not included in the
measure. By these standards, the association efficiency was quite high,
ranging
from about 70 % in the co-cultured group to about 53 % in the fusion group of
the
total gated cells.
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Example 4 - Pr~aration of Antiee~pecific T-Cells
Melanoma was resected from a human HLA-A2+ patient, digested to
produce a single cell suspension, and cryopreserved. Dendritic cells were
derived
from peripheral blood by culture in AIM V medium containing rhIL-4 and
S rhGmCSF, following the procedure generally set forth by Tiiting, et al. , J.
Immunol., 160:1139-1147 (1998). At day 7, tumor cells were thawed, irradiated
(15,000 tad), and added to the seven day DC culture. The tumor cells and DCs
were allowed to "co-incubate" for 24 hrs at 37°C. The DCaumor cell
ratio was
approximately 10:1. Co-cultured cells were harvested by pipette and irradiated
at
3,000 tads. Harvested cells were added to T-cell cultures as stimulators on a
weekly basis at a ratio between about 10:1 and 100:1 (T-cells:DCs). After 45
days
T-cells were assessed for their ability to lyse tumor peptide pulsed T2 cells
(Fig. 2)
or tumor cell line targets (Fig. 3). Lysis in the cytotoxic assay was
performed
using standard procedures. Targets were HLA-A2 matched allogeneic melanoma
targets (i.e., Me1526). Figure 2 demonstrates that T-cell lines not normally
recognized can become recognized when loaded with peptides that derive from
proteins expressed by melanoma cells (such as MART-1, gp100, tyrosinase,
etc.).
The results illustrated in Figure 2 demonstrate that the DC-tumor stimulators
of the
present invention can be used to drive the expansion of T-cells that recognize
a wide
array of tumor-associated antigens. The same concept would apply with DC-
virally
infected cell stimulators against viral associated antigens. Thus, the T-cells
of the
present invention have a broad range of reactivity; that is particularly
applicable in a
clinical setting, since tumor cells or virally infected cells may attempt to
modulate
their expression of individual antigens based on immune selective pressure in
vivo.
Figure 3 demonstrates the results obtained when the T-cells of the present
invention
were evaluated for their ability to lyse an HLA-A2 matched melanoma target
(Me1526). The T-cells killed the target effectively; this killing could be
blocked by
antibodies that bind to the HLA-2 molecule (BB7.2 and W6/32), demonstrating
the
HLA-A2 class 1 restriction of T-cells in these expanded T-cell cultures. The T-
2
cell line is an HLA-A2 matched target. The results demonstrated in the figures
therefore support HLA-A2 restricted CTL activity directed against both intact
melanoma and defined peptide epitopes presented by HLA-A2.
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Whereas particular embodiments of this invention have been
described above for purposes of illustration, it will be evident to those
skilled in the
art that numerous variations of the details of the present invention may be
made
without departing from the invention as defined in the appended claims.
