Note: Descriptions are shown in the official language in which they were submitted.
CA 02340085 2001-02-09
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ENGINEERED ANTIGEN PRESENTING CELLS EXPRESSING, AN ARRAY OF ANTIGENS AND USES
THEREOF
Throughout this application various publications are referenced. The
disclosures of
these publications in their entireties are hereby incorporated by reference
into this
application in order to more fully describe the state o~f the art to which
this invention
pertains.
TECHNICAL FIELD OF TH)E: INVENTION
The invention relates to immunotherapy involving the activation of T
lymphocytes by
IO antigen-presenting cells that are genetically modified to express and
present, on their
_ surface an array of antigens that are differentially expressed by a target
population. The
antigen-presenting cells can be genetically modifed by transduction with
nucleic acid
sequences identified by differential screening of nucleic acid sequences
expressed in a
target population relative to a non-target population.
1 ~ BACKGROUND OF THE INVENTION
Dendritic cells, which are specialized antigen-presenting cells, can be used
to process
antigens from diseased tissue and present them to the immune system. Dendritic
cells
are leukocytes derived From bone marrow and are considerably more potent than
other
antigen-presenting cells with respect to presentation and activation of
cytotoxic T
20 lymphocytes (CTLs). Publications relevant to dendritic cells include Young
et al. 1996,
J. Exp. i~Ied. 183:7-11; Aiavaikko et ai. 1994, Am. J. Clin. Pathol. 101:761-
767;
Shunichi et aI. 1995, Cancer 75:1478-1483; Hsu et al., 1996, Nature Medicine
2:~2-~8;
Nlayordomo et al. 1995, Nature Medicine 1:1297-1302; Paglia et al. 1996, J.
Exp. Med.
183:317-322; and Boczkowski et al. 1996, J. Exp. Med. 184:46-472.
25 There is a need for a vaccine that can.boost multiple clones of CTLs to
enhance the
immune response and to prevent the escape of a tumor from immune selection.
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Accordingly, there is a need for a system for providing antigen-presenting
cells with (i)
a broader spectrum of potential anti-tumor antigens .and (ii) providing the
antigens in a
form which the antigen-presenting cells can effectively process and present.
SUMMARY OF THE iN'VENTION
The invention provides a system for priming antigen-presenting cells with a
repertoire
of antigens of a specific cell type, for example, a tumor cell or a virally-
infected cell.
Further, the approach need not be restricted to stimuuating an immune response
against
diseased tissue. It can be used to mount an immune .response against any
target tissue
and to ablate any target cell expressing a particular set of antigens.
I 0 The invention provides a method of producing at least one vector encoding
an array of
antigens for expression in an antigen-presenting cell. In one embodiment, the
method
_ comprises comparing first nucleic acid sequences expressed by a target cell
population
with second nucleic acid sequences expressed by ~z non-target cell population.
The
method further comprises selecting nucleic acid sequences preferentially
expressed by
I~ the target cell population relative to the non-target cell population, and
introducing the
selected nucleic acid sequences into at least one vector capable of directing
expression
of the selected nucleic acid sequences in an antigen-presenting cell. In one
embodiment,
the antigen-presenting cell is a dendritic cell, macrophage, B cell, monocyte
or
f bracyte. In another embodiment, the vector further comprises a dendritic
cell targeting
20 element.
In one embodiment, the first and second nucleic acid sequences are of the same
tissue of
origin. The selected nucleic acid sequences can number one or more. For
example, the
selected nucleic acid sequences can comprise at Ieast 3 different nucleic acid
sequences,
at least 5 different nucleic acid sequences, at least 7 different nucleic acid
sequences, or
25 at Ieast 9 different nucleic acid sequences. The vector can further
comprise a nucleic
acid sequence encoding an immunomodulatory cofactor. The immunomodulatorv
cofactor can be, for example, IL-2, IL-3, IL-8, OKT3, a.-interferon, y-
interferon, or
MIP-la. The vector can further encode at least one selectable marker. Examples
of
CA 02340085 2001-02-09
WO 00!09665 PCT/US99/18087
selectable markers include, but are not limited to, PLAP, GFP and neomycin
resistance.
In one embodiment, the target cell is a cancer cell. In another embodiment,
the target
cell is an infectious agent, such as a virus, a bacteritun or a parasite:
The invention additionally provides a composition comprising at least one
vector .
produced by the method described above. In one embodiment, the vector further
comprises an antigen-presenting cell targeting element. The composition can
further
comprise an antigen-presenting cell.
The invention also provides a method of producing an antigen-presenting cell
that
presents an array of antigens. The method comprises; comparing first nucleic
acid
sequences expressed by a target cell population with second nucleic acid
sequences
expressed by a non-target cell population. The method further comprises
selecting
nucleic acid sequences preferentially expressed by the target cell population
relative to
the non-target cell population, and genetically modifying an antigen-
presenting cell to
express the selected nucleic acid sequences. Also provided is an antigen-
presenting cell
1 ~ produced by the method described above or genetically modified with a
vector produced
by a method of the invention.
The invention provides a method of activating immune cells comprising
contacting an
immune cell with an antigen-presenting cell genetically modified in accordance
with the
invention. In one embodiment, the immune cell is a 'T cell. Examples of T
cells
activated by the method include, but are not limited t~o, cytotoYic T
lymphocytes (CTLs)
and helper T cells. Also provided is a method of inducing a toleragenic
response
comprising contacting an immune cell with an antigen-presenting cell
genetically
modified in accordance with the invention. In one embodiment. the immune cell
is a
helper T cell such as a Tm cell. In one embodiment o~f the method of
activating T cells
or inducing a toIeragenic response, the contacting occurs in viva.
Alternatively, the
contacting can occur ex vivv. The invention also provides immune cells, such
as CTLs
and T~ cells, activated by a method of the invention. The activated immune
cells can be
provided in the form of a composition:
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The invention provides a method of activating T cells in vivo comprising
administering
a composition comprising a vector or antigen-presenting cell of the invention
to a
subject. Also provided is a method of killing a target cell in viva comprising
administering a composition, vector or antigen-presenting cell of the
invention to a
S subject. The invention also provides methods of preventing infection,
treating cancer,
and treating a viral infection, the methods comprising administering a
composition,
vector or antigen-presenting cell of the invention to a subject.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1 depicts cell surface markers that can be used to identify dendritic
cells. A (-)
indicates a marker not present on dendritic cells that can be used in negative
selection
strategies. The (+), (-+-+) ~d (+++) respectively indicate increasingly useful
markers
present on the dendritic cell surface.
DET:~I3.ED DESCRIPTION OF T'HE I~iVE~iTIOI~I
The present invention provides a strategy for presentation of multiple
antigens in
antigen-presenting cells which can be used to generate a prophy lactic or
therapeutic
immune response against one or more target cell populations with which the
antigens
are associated. The strategy employs comparing and selecting nucleic acid
sequences
e~cpressed by target and non-target cells. By identifying nucleic acid
sequences
preferentially expressed in a target cell population anct expressing the
identified
2fl sequences in antigen-presenting cells, one can stimulate an immune
response directed at
a target cell population without being limited to previously identified
antigens. The use
of an array of antigens can elicit a more effective immune response by
directing
lymphocytes to a variety of antigen targets. This multiple antigen strategy is
advantageous both for targeting an immune response to more antigens associated
with a
2~ given disease, and for targeting an immune response to a disease for which
an effective
antigen for a particular patient or for a particular disease is not known.
4
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WO 00/09665 PCTIUS99118087
The vectors and antigen-presenting cells of the invention can be prepared
without prior
purification of individual disease-associated antigens. This is particularly
useful when a
given tissue or cell expresses multiple disease-associated antigens, or when a
target
antigen is unknown. By making use of differential screening to identify
preferentially
expressed nucleic acid sequences, one can avoid problems with lack of
specificity as
nucleic acid sequences encoding non-target antigens can be excluded.
The invention provides a composite preparation of vectors encoding antigens
for
expression in antigen-presenting cells, which cells can then be used to
activate T cells of
a subject in vivo, ex vivo, ar in vitro, without specifi~~ prior knowledge of
the relevant
disease-associated antigens iri that subject. A composite preparation can be
used to
activate a variety of T cells, directed against more than one antigen and
against more
than one disease or target cell population.
Definitions
All scientific and technical terms used in this application have meanings
commonly
used in the art unless otherwise specified. As used in this application, the
following
words ar phrases have the meanings specified.
As used herein, "target cell population" means one or more cells sharing at
least one
common feature, the elimination or protection of which is desired. Examples of
a target
cell include, but are not limited to, an infectious agent such as a virus,
bacterium or
parasite, a cell which is susceptible to autoimmune al:tack, and a disease
cell such as a
cancer cell, including highly metastatie cancer cells and low metastatic
cancer cells. As
used in the context of a target cell, "infectious agent'' includes both the
infectious agent
itself and cells infected by the agent. For example, when the target cell is a
virus, the
target cell may be the virus alone, a virally infected cell or both.
As used herein, "preferentially expressed" refers to nucleic acid sequences
that are
present in substantially greater amounts in a target sample as compared to a
non-target
sample. A substantially greater amount can.be at least about 20% more. In one
CA 02340085 2001-02-09
WO 00/09665 PCT/US99/18087
embodiment, substantially greater is about 50% more. In another embodiment,
substantially greater is about twice as much. In a preferred embodiment,
substantially
greater is at least about I O times as much. In more preferred embodiments,
substantially
greater is at least about 20, 50 or 100 times as much. The relationship
between the
target and non-target samples is selected to optimize the identif cation of
sequences
encoding antigens relatively specific to the target cells. For example, a
target cell can
be a colon carcinoma cell and a corresponding non-target cell would be a non-
cancerous
colon cell. In another example, the target cell can be a highly pathogenic
virus and the
non-target cell a less pathogenic virus. Differential screening between such
target and
f0 , non-target populations can yield nucleic acid sequences encoding antigens
associated
with a particular disease or pathogen, without requiring knowledge of the
relevant
antigens.
As used herein, "vector" means a construct which is capable of delivering, and
preferably expressing, one or more genes) or sequence{s) of interest in a host
cell.
I~ Examples of vectors include, but are not limited to, viral vectors, naked
DNA or RNA
expression vectors, DIVA or RNA expression vectors associated with cationic
condensing agents, DNA or RNA expression vectors encapsulated in liposomes,
and
certain eukaryotic cells, such as producer cells.
As used herein, "expression control sequence" means a nucleic acid sequence
which
20 directs transcription of a nucleic acid. An expression control sequence can
be a
promoter, such as a constitutive or an inducibIe promoter, or. an enhaneer.
The
expression control sequence is operably linked to the; nucleic acid sequence
to be
transcribed.
The term "nucleic acid" refers to a deoxyribonucIeotide or ribonucIeotide
polymer in
25 either single- or double-stranded form, and unless otherwise limited,
encompasses
known analogs of natural nucleotides that hybridize to nucleic acids in a
manner similar
to naturally-occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid
sequence optionally includes the complementary sequence.
CA 02340085 2001-02-09
WO 00/09665 PCT/US99/18087
As used herein, "antigen-presenting cell" or "APC;" means a cell capable of
handling
and presenting antigen to a lymphocyte. Examples of APCs include, but are not
limited
to, macrophages, Langerhans-dendritic cells, follicular dendritic cells, B
cells,
monocytes, fibroblasts and fibrocytes. Dendritic cells are a preferred type of
antigen
presenting cell. Dendritic cells are found in many non-lymphoid tissues but
can migrate
via the afferent lymph or the blood stream to the T'-dependent areas of
lymphoid organs.
In non-lymphoid organs, dendritic cells include Langerhans cells and
interstitial
dendritic cells. In the lymph and blood, they include afferent lymph veiled
cells and
blood dendritic cells, respectively. In lymphoid organs, they include lymphoid
dendritic
ID cells and interdigitating cells. As used herein, each ofthese cell types
and each of their
progenitors is referred to as a "dendritic cell," unless otherwise specified.
As used herein, "antigen-presenting cell targeting element" means a molecule
which is
capable of specifically binding an antigen-presenting cell, such as a
dendritic cell. A
targeting element specifically binds a dendritic cell when a biological effect
is seen in
1 ~ that cell type after binding of the targeting element and ics ccmpiement.
or, when there
is greater than a IO fold difference, and preferably I=reater than a 2~, SO or
100 fold
difference between the binding of the coupled targeting element to dendritic
cells and
non-target cells. Generally, it is preferable that the targeting element bind
to antigen-
presenting cells with a KD of less than 10'511~I, preferably less than 10'6 M,
more
20 preferably less than I0'' M, and most preferably less than 10'8 M (as
determined by
Scatchaxd analysis (Scatchard, 1949, Ann. ~1.Y. Acad. Sci. ~I:660-672)).
Suitable
targeting elements are preferably non-immunogenic, not degraded by
proteolysis, and
not scavenged by the immune system. Particularly preferred targeting elements
have a
half life (in the absence of a clearing agent) in an animal of between 10
minutes and 1
25 week. Examples of dendritic cell surface markers, against which antibodies
(or antigen
binding domains derived therefrom) can be generated to produce dendritic cell
targeting
elements, include, but are not limited to, those depicted in Figure 1 {e.g.,
CDl, CDi la,
CDl lc, CD23, CD%S, CD32, CD40, CD4~, CD~4, t~D58, NiHC Class I, MHC Class II,
Niac-l, Mac-2, Mac-3).
7
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As used herein, "immunomodulatory cofactor" includes a factor which, when
expressed
in APCs, causes the immune response to an antigen presented by the APC to be
enhanced in quality or potency from that which would have occurred in the
absence of
the cofactor. The quality or potency of a response may be measured by a
variety of
assays known in the art including, for example, in virno assays which measure
cellular
proliferation (e.g.,'H-thymidine uptake), and in vitro cytotoxicity assays
(e.g., which
measure s'Cr release; Warner et aI. 1991, AIDS Res. and Human Retroviruses
4:645-
655). In alternative embodiments, an. immunomodulatory cofactor is, rather
than being
encoded by the expression vector, added exogenousIy before, concurrently with,
or after
I0~ administration of the vector. Examples of immunomodulatory cofactors
include, but are
not limited to cytokines and chemokines, such as IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-
7, IL-8, iL-9, IL-10, IL-11, IL-12, IL-I3, IL-14, IL-1~~,~OKT3, a-interferon,
~i-
interferon, y-interferon, MIP-la (LD-78), granulocyte~-macrophage colony-
stimulating
factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), tumor necrosis
factors (TNFs), CD3, CD8, ICAl4i-1, ICAVI-2, LFA-l, LFA-3, and other proteins
such
as HLA Class I molecules, HLA Class II molecules, E.7, B7-?, /3~-
microglobulin,
chaperones, and MHC linked transporter proteins or a~zaIogs thereof. The
choice of
irnmunomodulatory cofactor is based on the therapeutic effects of the factor.
Preferred
immunomodulatory cofactors include a.-interferon, y-interferon and IL-2.
l~Fucleic Acid Sequences
The invention provides nucleic acid sequences encoding an array of antigens
that are
preferentially expressed in a target cell population and which can be used to
genetically
modify antigen-presenting cells (APCs). The invention further provides a
method for
preparing these nucleic acid sequences. The nucleic acid sequences can be
prepared by
comparing first nucleic acid sequences expressed by a target cell population
with second
nucleic acid sequences expressed by a non-target cell population. Nucleic acid
sequences preferentially expressed by the target cell population relative to
the non-target
cell population are then selected. This method provides nucleic acid sequences
that can
be used to express an array of antigens in APCs without prior knowledge of the
antigens
CA 02340085 2001-02-09
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and without compromising specificity for the target cell population. Nucleic
acid
sequences include DNA, RNA, and synthetic derivatives thereof.
Methods of comparing sets of nucleic acid sequences and selecting
differentially
expressed sequences ase known in the art. The comparing can be performed using
nucleic acid sequences isolated from samples, such as from one or more cell
types, or it
can be performed by comparing sequence data obtained, for example, from a
public
database such as GenBank, EMBL, or DNA Database of Japan (DDBJ). For example,
when two nucleic acid samples are to be compared, such as sequences from a
target cell
. population and sequences from a non-target cell population, each sample can
be derived
from either an isolated pool of nucleic acid sequences, database sequence
information,
any other source of nucleic acid sequence information, or a combination of two
or more
sources. Conventional techniques for differential display of mRNA and
differential
screening of a cDNA library are described in F. 11~f. ,Ausubel et al., eds.,
1996, Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., unit 5.8 and sections
5.8.6,
5.8.14.
One example of detecting differential expression of genes is described in U.S.
Patent
No. 5,677,125, which relates to a method of detecting and diagnosing pre-
invasive
breast cancer. In this example, epithelial cells are isolated from a sample of
abnormal
breast tissue which exhibits histological or cytological characteristics of
pre-invasive
breast cancer, and mRNA is isolated from the sample. One or more abnormal
breast
tissue cDNA libraries is prepared from the isolated tnRNA. One or more cDNA
libraries is then prepared by similar means from a sample of normal breast
tissue. The
cDNA of the abnormal and normal libraries is compared to detect the expression
of at
least one gene in the abnormal sample which is different from that expressed
in the
normal sample. A similar approach can be applied to obtain an array of nucleic
acid
sequences preferentially expressed by a target cell population relative to a
non-target
cell population for introduction into APCs.
9
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The non-target cell can be a normal cell or it can be a diseased cell, but
having a
different spectrum of antigens from the target cell. For example, it may be
desirable to
obtain antigens specif c for a particular stage in disease. Thus, the non-
target cell could
be a cell in a different stage of the target disease. ArA example of a non-
target cell
population is one or more selected from stage l and :>tage 2 cells in cervical
carcinoma.
Antigen-presenting cells can be provided that present antigens specific to a
desired
stage, such as stage 3 cervical carcinoma. In another example, the target cell
population
is a highly metastatic colon cancer cell line and the non-target cell
population is a low
metastatic colon cancer cell line. Differential screening of nucleic acid
sequences
expressed by the two cell lines can be used to select sequences encoding
antigens
specific to highly metastatic colon cancer cells. When the non-target cell is
a normal
cell, differential screening eliminates or reduces the nucleic acid sequences
common to
normal cells, thereby avoiding an immune response directed at antigens present
on
normal cells. When the non-target cell is a normal cell, differential
screening eliminates
i~ or reduces Nucleic acid sequences common to normal cells, thereby avoiding
an immune
response directed at antigens present on normal cells. Examples of target!non-
target cell
combinations suitable for differential screening include, but are not Limited
to,
K.~II2L4A cells (high metastatic colon cancer line; iVlorikawa et al., 1988,
Cancer Res.
48:1943)/KM12C (low metastatic colon cancer line; Trforikawa et al., 198$,
Cancer Res.
48:6863); MDA-MB-231 cells (high metastatic breast cancer line; Brinkley et
al., 1980,
Cancer Res. 40:3118)II~ICF-7 cells (ron-metastatic breast cancer line: Yang et
al., 199$,
Anticancer Res. I8{lA):53-59); and MV-522 cells (high metastatic lung cancer
line;
Varki et al., 1987, Int. J. Cancer 40:46)/UCP-3 cells (low metastatic lung
cancer cell
line; Varki et aL, 1987, supra).
Differential screening can also provide various tailored combinations of
antigen-
presenting cells. One combination of APCs of the present invention encompasses
antigens relating to associated pathologies. Far examl>le, HIV-infected
patients are
often infected with CMV. Accordingly, antigen-presenting cells can be prepared
expressing genes preferentially associated with HIV-infected cells, and a
second
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preparation can be prepared in which the antigen-presenting cells express
antigen
preferentially expressed by C1~IV-infected cells. These two preparations can
be
combined and administered to a subject providing simultaneous treatment for
both
pathologies. Examples of other associated pathologies wherein preparations of
antigen-
presenting cells can be combined include HIV and Epstein-Barr virus (EBV)
positive
lymphoma; and HIV and hepatitis C virus (HCV).
Another example in which separate preparations can be combined provides APCs
for
targeting a tumor or tumors of unclear origin. In thus case, antigen-
presenting cells
genetically modified to express nucleic acid sequ.erxces from a spectrum of
tumor types
can be used to elicit a broad anti-tumor immune re:;ponse.
A third instance in which separate preparations can be combined provides for
targeting a
disease associated with a different spectrum of anti;;ens in different
patients.
Preparations from various patients that encompass most or all of the
repertoire of
antigens associated with the disease in most or all patients can be combined
and
1S administered to a subject without having to first determine what antigens
are expressed
in that individual subject's disease.
The combinations can be prepared at the nucleic acid level. In this case,
preferentially
expressed nucleic acids are combined and then introduced into antigen-
presenting cells
in vitro. Alternatively. preferentially expressed nucleic acids can be used to
prepare
separate antigen-presenting cells which then can be combined to provide a
composite
antigen preparation.
In one embodiment of the invention, mRNA preferentially expressed by the
target cell
population is used to obtain nucleic acid such as cDNA encoding full-length
sequences.
Nucleic acid sequences so obtained can be used to identify differentially
expressed
2S antigens associated with the target cell population. ~~ne can then test
antigens so
obtained to determine which are most effective as immunogens.
CA 02340085 2001-02-09
WO OOJ09665 PCT/US99J18087
In one example of testing an antigen's immunogen:icity, periplierai blood
cells are
removed from a subject. Dendritic cells are then isolated and either pulsed
with the
antigen to be tested or genetically modified to express the test antigen. The
pulsed or
modified dendritic cells can then be used to stimulate the subject's T cells
in vitro. The
ability of the modif ed dendritic cells to stimulate T cells is indicative of
immunogenicity of the test antigen.
Vectors
The invention additionally provides vectors containing nucleic acid sequences
selected
by differential screening as described above. The sf;lected sequences can be
introduced
IO into at least one vector. Preferably the vector is capable of directing
expression of the
selected nucleic acid sequences in an antigen-presenting cell, such as a
dendritic cell. A
vector can encode a single antigen or multiple antigens. When multiple
antigens are
encoded by a single expression vector, the antigens can be derived from the
same or
different cell types. Alternatively, a composition comprising more than one
vector,
I S each encoding one or more antigens associated with a cell type the same as
or different
from those encoded by other vectors, can also be prepared.
In one embodiment, the invention provides a methoc! of producing at least one
vector
encoding an array of antigens for expression in an antigen-presenting cell. In
one
embodiment, the method comprises comparing first nucleic acid sequences
expressed
20 by a target cell population with second nucleic acid sequences expressed by
a non-target
cell population. The method further comprises selecting nucleic acid sequences
preferentially expressed by the target cell population relative to the non-
target cell
population, and introducing the selected nucleic acid sequences into at least
one vector
capable of directing expression of the selected nucleic acid sequences in an
antigen-
25 presenting cell. In one embodiment, the antigen-presE:nting cell is a
dendritic cell,
macrophage, B cell, monocyte or fibrocyte. In another embodiment, the vector
further
' comprises a dendritic cell targeting element.
12
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WO 00/09665 PCTIUS99/18087
In one embodiment of the method, the first and second nucleic acid sequences
are of the
same tissue of origin. The selected nucleic acid sequences can number one or
more.
For example, the selected nucleic acid sequences can comprise at least 3
different
nucleic acid sequences, at least 5 different nucleic acid sequences, at least
7 different
nucleic acid sequences, or at least 9 different nucleic acid sequences. The
vector can
further comprise a nucleic acid sequence encoding an immunomodulatory
cofactor. The
immunomodulatory cofactor can be, for example, IL-2, IL-3, IL-8, OKT3, a-
interferon,
y-interferon, or MIP-la. The vector can further encode at least one selectable
marker.
Examples of selectable markers include, but are not limited to, PLAP (U.S.
Patent
Application Serial No. 09/006,298, filed January l 3, 1998); GFP and neomycin
resistance. In one embodiment, the target cell is a cancer cell. In another
embodiment,
the target cell is an infectious agent, such as a virus, a bacterium or a
parasite.
Vectors of the invention can be used to genetically modify APCs such as
dendritic cells
either in vivo or in vitro. Several ways of geneticaIfty modifying APCs are
known,
including transduction with a viral vector either directly ur via a retrovi:al
producer cell,
calcium phosphate precipitation, fusion of the recipient cells with bacterial
protoplasts
containing the DNA, treatment of the recipient cells. with liposomes
containing the
DNA, DEAF dextran, receptor-mediated endocytosis, electroporation, micro-
injection,
and many other techniques known to those of skill. See, e.g.. Methods in
Enzymology,
185, Academic Press, Inc., San Diego, CA (D.V. Gc~eddel, ed.) 1990, or M.
Krieger,
Gene Transfer and Expression -- A Laboratory :Manual, Stockton Press, New
York, NY,
1990, and the references cited therein, as well as Berger and KimmeI, Guide to
Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc.,
San
Diego, CA (Berger); Sambrook et al. Molecular Cloning - A Laboratory Manual
(2nd
ed.) 1-3 1989; and Current Protocols in Molecular P.~ioiogy, F.;~i. Ausubel et
al., eds.,
Greene Publishing Associates, Inc. and John WiIey & Sons, Inc., (1994
Supplement),
WO 93124640; Mannino and Gouid-Fogerite 1988, Biotechniques 6(7):682-691;
IJ.S.
' Patent No. 5,279,833; WO 91/06309; and Felgner et al. Proc. Natl. Acad. Sci.
USA
84:7413-7414 I 987.
13
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a - WO 00/09665 PCT/US99118087
Examples of viral vectors include, but are not limited to retrovii~al vectors
based on, e.g.,
HSV, HIV, marine retroviruses, gibbon ape Leukemia virus and other viruses
such as
adeno-associated viruses (AAVs) and adenoviruses. (Miller et ai. 1990, Mol
Cell Biol.
10:4239; J. Kolberg 1992, NIH Res. 4:43, and Corneas et al. 1991, Hum. Gene
Ther.
2:215). Widely used retroviral vectors include those based upon marine
leukemia virus
(MuLV), gibbon ape Leukemia virus (GaLV), ecotropic retroviruses, human
immunodeficiency virus (HIV), and combinations. :>ee, e.g. Buchscher et aI.
1992, J.
Virol. 66(5}:2731-2739; 3ohann et aI~. 1992, J. Virol. 66(5):1635-1640;
Sommerfelt et aI.
1990, Virol. 176:58-59; Wilson et aI. I989, J. Virol. 63:2374-2378; Miller et
al. 1991, J.
I O Virol. 65:2220-2224, and Rosenberg and Fauci 1993 in Fundamental
Immunology,
Third Edition, W.E. Paul (ed) Raven Press, Ltd., New York and the references
therein;
Miller et al. 1990, Mol. Cell. BioI. 10:4239; R. Kolbe;rg 1992, J. NIH Res.
4:43; and
Cornetta et ai. I991, Hum. Gene Ther. 2:215.
Marine vectors comprising Gibbon Ape Leukemia Virus (GaLV) envelopes can be
used
to transduce many mammalian cells. See, Joha,nn et al. i 992, supra. The same
receptor
is used by sinuan sarcoma associated virus (SSAV), a strain ofGaLV (Sommerfelt
et al.
1990, supra). The construction of hybrid virions having GaL V envelope
proteins has
been demonstrated (Wilson et al. 1989, supra; Miller et a1. 1991, supra). Any
of these
vectors and methods of making retroviral clones can be applied to the present
invention.
GaLV retroviraI packaging cell Lines can be used to provide infectious
replication-
defective hybrid virions for use in gene transfer in humans, hamsters, cows,
cats, dogs,
monkeys, chimpanzees, macaques, primates, and other species whose cells have
host
cell receptors for GaLV envelope proteins.
Additional examples of vectors include, but are not limited to adenoviral
vectors, AAV
vectors, pox viral vectors (B. Moss, 1992, Current Topics in Microbiology and
Immunology 158:25-38) including vaccinia, fowl pox, and canary pox,
recombinant
influenza viral vectors {A. Garcia-Sastre and P. Palese 1995, Biologicals 23:1
X-I78) or
non-viral gene delivery techniques (F. Leedley 1994, Biotechnology 5:626-636).
AAV-
based vectors can be used to transduce cells with seIecied nucleic acids,
e.g., in the in
l4
CA 02340085 2001-02-09
' WO 00/09665 PCT/US99118087
vitro production of nucleic acids and peptides, and in in vivo and ex vivo
gene therapy
procedures. See, West et aI. 1987, Virology 160:38-47; U.S. Patent No.
4,797,368; WO
93/24641; Kotin 1994, Human Gene Therapy 5:793-801; and Muzyczka 1994, J.
Clin.
Invest. 94:1351.
In vitro amplification techniques suitable for amplifying sequences to be
subcioned into
an expression vector are known. Examples of such in vitro amplification
methods,
including the polymerase chain reaction (PCR), Iigase chain reaction (LCR),
Q~i-
replicase amplification and other RNA polymerase mediated techniques (e.g.,
NASBA),
are found in Berger, Sambrook et al. 1989, Molecular Cloning - A Laboratory
Manual
I O (2nd Ed) I-3; and U.S. Patent No. 4,683,202; PCR Protocols A Guide to
Methods and
Applications (Innis et al. gds) Academic Press Inc. ~>an Diego, CA 1990;
Arnheim &
Levinson (October l, 1990) C&EN 36-47; J. NIH Res. 1991, 3:81-94; Kwoh et al.
1989, Proc. Natl. Acad. Sci. USA 86:1173; Guatelli et al. 1990. Proc. Natl.
Acad. Sci.
USA 8 7: I 874; Lomeli et al. 1989, 3. Clin. Chem. ~~: i 826; handegren et al.
1988,
IS Science 241:1077-1080; Van Brunt 1990, Biotechnology 8:291-294; Wu and
Wallace
1989, Gene 4:560; Barriner et al. 1990, Gene 89:11 T; and Sooknanan and
lrialek 1995,
Biotechnology 13:563-564. Improved methods of cloning in vitro amplifed
nucleic
acids are described in U.S. Patent No. 5,426,039.
Antigen-Presenting Cells
20 Antigen-presenting cells (APCs) process antigen and present it to a
lymphocyte. The
invention provides APCs that present an array of antigens. The APCs are
genetically
modif ed to express nucleic acid sequences preferentially expressed by a
target cell
population relative to a non-target cell population. Examples of APCs include,
but are
not limited to, macrophages, Langerhans dendritic cells, follicular dendritic
cells, B
25 cells, monocytes, fibroblasts and fibrocytes. in a preferred embodiment,
the APC is a
dendritic cell. Selection of the optimal APC can vary, however, with the
antigen to be
presented (Butt and Bevan 1998, J. Immunol. 160:21.30-2144).
CA 02340085 2001-02-09
WO 00/09665 PCT/US99718087
Dendritic cells have an unusual dendritic shape, are motile, and efficiently
cluster and
activate T cells that are specific for cell surface stimuli. Typically,
dendritic cells in
non-lymphoid organs, such as Langerhans cells and interstitial cells, become
veiled
cells (cells which continually extend and retract large lamellipodia) in the
afferent
Iymph and blood which migrate to lymphoid tissues, where they can be isolated
as
dendritic or interdigitating cells.
Dendritic cells initiate T-dependent responses from quiescent lymphocytes.
Once
sensitized, T cells interact with other antigen presenting cells. Dendritic
cell antigen
processing activity is regulated. Fresh cells, i.e., cells cultured for less
than a day,
isolated from skin or lymphoid organs present native; proteins. After that
time, they do
not process antigens. in addition, dendritic cells are not actively
phagocytic.
Dendritic cells can be isolated and prepared using conventional techniques
such as those
described in Tedder and Jansen, i 99 i, Current Protocols in Im~-nunology,
John Vv'iley &
Sons, unit 7.32. Other methods for obtaining and identifying dendritic cells
are
described in WO 98/1 »?9; WO 98/01 X38; WO 9811 ~6I ~; and W O 98/14561. For
example; human dendritic cells can be isolated from blood mononuclear cells by
first
enriching a peripheral cell population for dendritic cells by depletion of T
cells and
adherent cells. Density gradient centrifugation of the preparation over
metrizamide is
used to isolate low buoyant density cells. This population has virtually no
lymphocytes
and contains 20-80% dendritic cells. The puray of de:ndritic cells can be
determined by
a variety of techniques, including hemacytometer counting, immunostaining
after
cytocentrifugation onto glass slides and immunofluo>:escence staining with
flow
cytometric analysis. Flow cytometry is preferred, however, for optimal
quantitation and
consistency.
Young et aL nave identified dendritic cell colony-forming units among normal
human
CD34~- (positive for this hematopoietic stem cell marker) bone marrow
progenitors that
give rise to pure dendritic cell colonies {Young et al. 199, J. Exp. Med.
182:1111-
1120). Addition of c-kit-ligand to GM-CSF- and TNF-a.-supplemented suspension
of
16
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CD34+ bone marrow cells expands dendritic cell colony-forming units almost 100-
fold
by 14 days. These colony-derived dendritic cells ane potent stimulators of T
cells.
Partially enriched populations of epidermal Langerhans cells, wherein
Langerhans cells
may comprise up to about 60% of the total cell population, may be readily
prepared.
Keratinocytes can be depleted from marine tissue using a-thy-1 (a monoclonal
antibody) and complement plus adherence. Enriched preparations of human
Langerhans
cells can be prepared by substituting an anti-CD 1 antibody for a-thy-1. In
culture,
neither mouse nor human Langerhans cells are active antigen-presenting cells
until after
1-3 days in culture, after which time they enlarge, express more MHC Class II
and cell
adhesion molecules, and lose Fc receptors, fully resembling blood and lymphoid
dendritic cells. Cell populations containing more than 90% dendritic cells
have been
obtained from human blood, where, without enrichment, fewer than 0. I % of the
white
cells are dendritic cells. Such enrichment can be achieved by successive
depletion of T
cells, monocytes, and B plus NK cells to yield an initial population ranging
from 30-
1 ~ 60°,% dendritic cells. Greater purity is then obtained by panning
or fluorescence
activated cell sorting (FACS) using a monoclonal antibody, especially to
CD4~RA, that
selectively reacts to contaminants (P. Freudenthal, et al. 1990, Proc. Natl.
Acad. Sci.
(USA) 87:7698-7702). To enrich for dendritic cells generally, selection for
low
buoyant density, non-adherence to plastic in culture (especially after one or
more days),
and absence of markers found on other cells is performed. Such methods deplete
other
cell types, but do not positively select dendritic cells.
Dendritic cells express a distinct pattern of markers on their cell membranes.
Figure 1
illustrates this pattern by indicating the presence or absence of several
distinct cell
surface markers. Other markers which can be used u~ positively or negatively
select for
dendritic cells include ICAM-I (CD34}, LFA-3 (CDSB), and CD l lb. Dendritic
cells
isolated from human or mouse blood, but not skin, express CD l la or LFA-1. In
skin,
the immunostimulatory effect of dendritic cells may be enhanced by cyrtokines,
particularly by GM-CSF.
~r
CA 02340085 2001-02-09
WO 00/49665 PCT/US99/18087
Dendritic cells, or other APCs, can be selected to obtain a population
comprised
substantially of dendritic cells, i.e., greater than about 50% dendritic
cells, more
preferably greater than about 75% dendritic cells, more preferably still
greater than
about 90% dendritic cells, with greater than about 9~5% dendritic cells being
particularly
preferred. The antigen-presenting cells are preferably isolated from the
subject into
which the activated T cells are to be active ("autologous" therapy).
Alternatively, the
cells can be obtained from a donor or a cell bank (e.g., a blood bank).
The invention provides a method for preparing antigen-presenting cells that
present an
array of antigens. APCs can be genetically modified! using one or more-vectors
of the
invention. Antigen-presenting cells are transduced with vectors encoding an
array of
antigens. These antigens are then expressed in the cells, processed by the
cells, and the
relevant processed antigen fragment is routed to the cell surface where it can
be
presented.
The culture of cells used in conjunction with the present invention, including
cell lines
and cultured cells from tissue or blood samples, including dendritic cells is
well known
in the art. Freshney (Culture of Animal Cells, a Il~ianual of.Basic Technique,
third
edition WIley-Liss, New York (1994)) and the references cited therein provides
a
general guide to the culture of cells.
T Cells
T cells can be isolated and activated in vitro by contact with an antigen-
presenting cell.
Several such techniques are well-known. The expression of surface markers
facilitates
identification and purification of T cells. Methods oiE identification and
isolation of T
cells include FACS, incubation in flasks with fixed antibodies which bind the
particular
cell type and panning with magnetic beads.
T cells and dendritic cells are characterized by expression of particular
markers an the
surface of the cell, and lack of expression of other markers. For instance,
dendritic cells
express MHC molecules and costimulatory molecules (e.g., B7-l and B7-2), a
lack of
18
CA 02340085 2001-02-09
WO OOI09665 PCT/US99/t808'7
markers specific for granulocytes, NK cells, B cells, and T cells. In the
mouse, some,
but not all, dendritic cells express 33D1 (dendritic cells from spleen and
Peyer's patch,
but not skin or thymic medulla), NLDC 145 (denalritic cells in skin and T-
dependent
regions of several lymphoid organs and CD 11 c) ((~D I I c also reacts with
macrophage).
T cells are positive for various markers depending on the particular subtype,
most
notably CD4 and CDB.
CeII isolation or immunoassays for detection of cells during cell purifcation
can be
performed in any of several configurations, e.g., reviewed in Ivlaggio (ed.)
1980,
Enzyme Immunoassay CRC Press, Boca Raton, Florida; Tijan 198, "Practice and
IO Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and
Molecular Biology, Elsevier Science Publishers B.V., Amsterdam; Harlow and
Lane,
supra; Chan (ed.} 1987, Immunoassay: A Practical Guide Academic Press.
Orlando, FL;
Price and Newman (eds.) I991, Principles and Pracaice of Immunoassays Stockton
Press, NY; and Ngo (ed.) I988, Non-isotopic Immunoassays Plenum Press, NY. For
a
1 ~ review of immunoiogical and immunoassay procedures in general, see Stizes
and Te:r
(eds.) 1991. Basic and Clinical Immunology (7th ed.). For a discussion of how
to make
antibodies to selected antigens, see, e.g., Coligan I991, Current Protocols in
Immunology WileylGreene, N.Y.; and Harlow and Lane 1989, Antibodies: A
Laboratory Manual Cold Spring Harbor Press, N.Y"; Stites et al. (eds.) Basic
and
20 Clinical Immunology (~.th ed.).
Mast preferably, cells are isolated and characterized by flow cytometry
methods using
fluorescence activated flow cytometry (FACS). A «ride variety of flow-
cytometry
methods are known. For a general overview of FAGS see, for example, Abbas et
al.
1991, Cellular and Molecular Immunology; W.B. Sounders Company, particularly
2~ chapter 3, and Kuby 1992, Immunology, W.H. Freeman and Company,
particularly
chapter 6.
!9
CA 02340085 2001-02-09
WO 00/09655 PCTIUS99118087
Methods
The invention provides methods for using antigen-presenting cells that present
an array
of antigens. In one embodiment, the invention provides a method of activating
immune
cells in vivo. Examples of immune cells include, but are not limited to, T
cells,
including cytotoxic T lymphocytes and helper T cells. In one embodiment, the
method
comprises administering to a subject a vector encoding an array of antigens
preferentially expressed by a target cell population as compared with a non-
target
population. The vector is preferably constructed so as to be capable of
directing
. _ expression of the preferentially expressed antigens in an antigen-
presenting cell.
Preferably, the antigen-presenting cell is a dendritic cell. The vector can be
further
modified, as described above, to encode an immunonaodulatory cofactor. The
target cell
can be, for example, a cancer cell, virus, bacterium, parasite, or other
disease-associated
cell. Upon administration, the vector transduces an APC in the subject,
thereby
genetically modifying an APC in vivo. The genetically modified APC is then
available
I S to contact and activate an immune cell.
In another embodiment, the method of activating immune cells comprises
contacting an
immune cell with an antigen-presenting cell genetically modified to express an
array of
antigens preferentially expressed by a target cell population as compared with
a non-
target population. Preferably, the antigen-presenting cell is a dendritic
cell. The
contacting can occur in vivo or ex vivo. Examples of eliciting an in vitro CTL
response
are provided by S. Nair et aL, 1993, 3. Virol. 68:685 and F.J. Rouse et al.,
199.4, J.
Virol. 68:6685. Example of stimulating an in vivo T c~elI response are
provided by A.
Porgador et aL, 1996, J. lmmunol. 156(8):2918-2926; E.C. McICinney and J.W.
Streilein, 1989, J. Immunol. 143:1560; and H. Takahashi et al., 1993, Int.
Immunol.
5:849. Far in vivo contact, the APC is administered to a subject.
Alternatively; immune
cells are isolated from a subject and brought into contact with the APC in
vitro or ex
vivo. The immune cells, such as T cells, are activated c.~x vivo and then can
be
reintroduced into the subject or provided to a different subject where they
can then
come in contact with target cells in that subject. Techniques for adoptive
CA 02340085 2001-02-09
WO 00/09665 PCTIUS99/18087
immunotherapy of cancer are described and reviewed in Chang, A.E. and S. Shu,
1996,
Crit. Rev. Oncol. Hematol. 22(3j:213-228; and Kradin, R.K., 1993, in
Therapeutic
Applications of Interleukin-2, Atkins, M.B. and J.V~. Mier, eds., Marcel
Dekker, Inc.
NY, pp. 217-232.
The activation of immune cells by the above methods can, in some embodiments,
be
used to kill target cells. Thus, the invention provides a method for killing a
target cell
comprising contacting an immune cell with an APC'. genetically modified in
accordance
with the invention. The contacting can be effected inn vivo by administering a
genetically
modif ed APC or by administering a vector of the invention which transducer an
APC
within the subject, which then contacts an immune cell. Alternatively, the
contacting
can occur in vitro. Immune cells activated in vitro b;y contact with
genetically modified
APCs can be administered to a subject.
Alternatively, the selection of vectors or APCs can be designed to obtain a
toleragenic
response, for example, by contacting the APC with a T~ cell. Thus, the
invention
additionally provides a method of inducing a toleragenic response comprising
contacting an immune cell with an APC genetically modified in accordance with
the
invention. Inducing a toleragenic response can be useful for such applications
as
treatment of autoimmune disorders and inhibiting rejection of foreign tissue,
such as
transplant tissue or autologous cells which have been genetically modif ed
with foreign
material. As with the methods for killing a target cell, the methods for
inducing a
toleragenic response can be effected by APCs genetically modified in vitro or
in vivo.
The invention additionally provides a method of preventing disease such as
infection or
cancer comprising administering to a subject a composition comprising a vector
or
APC of the invention. Also provided is a method of treating disease such as
cancer or
infection comprising administering to a subject a composition comprising a
vector or
APC of the invention. Examples of infections include, but are not limited to,
viral,
bacterial and parasitic infections. Examples of cancers include, but are not
limited to,
melanoma, glioma, and cancers of the colon, breast, prostate, lung and Liver.
21
CA 02340085 2001-02-09
WO 00/09665 PCT/US99118087
Compositions
The invention provides compositions which are useful for treating and
preventing
disease, such as cancer or infection. In one emboaliment, the composition is a
pharmaceutical composition. The composition can comprise a therapeutically or
prophyiactically effective amount of a vector and/or antigen-presenting cell
of the
invention, as described above. The composition can optionally include a
carrier, such as
a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers
are
determined in part by the particular composition being administered, as well
as by the
particular method used to administer the composition. Accordingly, there is a
wide
variety of suitable formulations of pharmaceutical compositions of the present
invention. Most typically, quality controls (microbiology, clonogenic assays,
viability
tests), are performed and the cells are reinfused back to the patient.
preceded by the
administration of diphenhydramine and hydracorti;>one. See, for example :Vi.
Korbling,
et aI. 1986, Blood 6 i:~29-X32 and Haas et al. 1990, EYp. Hematol. 1 Q:94-98.
1 ~ Formulations suitable far parenteral administration, such as, for example,
by
intraarticular (in the joints), intravenous, intramuscular, intradermal,
intraperitoneal, and
subcutaneous routes, and carriers include aqueous isotonic sterile injection
solutions,
which can contain antioxidants, buffers, bacteriostats, and solutes that
render the
formulation isotonic with the blood of the intended recipient. and aqueous and
non-
aqueous sterile suspensions that can include suspending agents, solubilizers,
thickening
agents, stabilizers, and preservatives. Intravenous or intraperitoneal
administration is a
preferred method of administration.
Methods Of Administration
In one embodiment of the invention, a patient infected with a virus such as
HIV-1 or
suffering from a cancer such as a melanoma can be treated by administering
genetically
modified antigen-presenting cells, or by using genetically modified antigen-
presenting
cells to activate a population of T cells against the infection or cancer, and
introducing
the T cells back into the patient. Thus, the present invention provides a
method of
22
CA 02340085 2001-02-09
WO 00/09665 PCT/US99118087
producing cytotoxic T cells in vitro, ex vivo or in vavo. In another
embodiment, the
patient is treated by administering at least one vector encoding an array of
antigens,
wherein the vector includes a dendritic cell target element. The patient's own
dendritic
cells can then be genetically modified in vivo.
S T cells such as CD8+ CTLs activated in vitro can be introduced into a
subject where
they are cytotoxic against target cells bearing antigenic peptides
corresponding to those
the T cells are activated to recognize on class I MHC molecules. These target
cells are
typically cancer cells, or infected cells which express unique antigenic
peptides on their
MHC class I surfaces.
Similarly, helper T cells (e.g., CD4+ T cells), which recognize antigenic
peptides in the
context of MHC class II, are also stimulated by genetically modified antigen-
presenting
cells (e.g., dendritic cells), which can comprise antigenic peptides both in
the context.of
class I and class li MHC. These helper T cells also stimulate an immune
response
against a target cell. As with cytotoxic T cells, helper T cells are
stimulated with the
I5 genetically modified antigen-presenting cells in vitro ar in vivo. In one
embodiment, a
toieragenic response is generated by administration of genetically modified
APCs via
the stimulation of Ta,, cells.
Arrays of antigens are preferably associated with diseases selected from the
group
consisting of cancer, a hyperproliferative disease, a bacterial infection, a
parasitic
infection, and a viral infection. Diseases suitable for treatment using an
immunostimulation strategy include: viral infections, such as those caused by
HBV (see
WO 93lIS207), HCV (see WO 93/15207), HPV (se:e WO 9210524$, WO 90/10459,
EF'O 133,123), Epstein-Barr Virus (see EPO 173,254; JP 1,128,788; and U.S.
Patent
Nos. 4,939,088 and S,I72,414), Feline Leukemia Virus (see W0 93/09070, EPO
2S 377,842, WO 90108832, and WO 93109238), Feline Immunodeficiency Virus (U.S.
Patent No. 5,037,753, WO 92/15684, WO 90/13573, and JP 4,126,085), HTLV I and
II,
' and HIV (see WO 91/02805); cancers, such as melanoma, cervical carcinoma,
colon
23
CA 02340085 2001-02-09
WO 00/09bb5 PCT/US99/I8087
carcinoma, renal carcinoma, breast cancer, ovarian cancer, prostate cancer,
leukemias;
and heart disease.
Bacterial infections that may be treated include, bui; are not limited to,
pneumonia,
sepsis, tuberculosis, and Staphylococcus infections, among others.
Parasitic infections that can be treated include, but are not limited to,
malaria (caused by
protozoa of the genus Plasmodium, and include P. ~alciparum, P. malariae, P.
ovate,
and P. vivax), sleeping sickness (caused by trypanosomes), and river
blindness.
Viral infections that can be treated include, but are not limited to, those
caused by
hepatitis A, hepatitis B, hepatitis C, non-A, non-B hepatitis, hepatitis delta
agent, CMV,
I O Epstein-Barr virus, HTLV I, HTLV II, FeLV, FIV, and HIV I.
Treatment includes prophylaxis and therapy. Prophylaxis or treatment can be
accomplished by a single direct injection at a single time point or multiple
time paints.
Administration can also be nearly simultaneous to multiple sites.
Patients or subjects include mammals, such as human, bovine, equine, canine,
feline,
1 ~ porcine, and ovine animals.
In one embodiment, T cells or antigen-presenting cells are administered
directly to the
subject to produce T cells active against a target cancerous, infected, or
other cell type.
Administration of these is by any of the routes normally used For introducing
a cell into
ultimate contact with a mammal's blood or tissue cells. In another embodiment,
at least
20 one vector encoding an array of antigens is administered.
Compositions are typically administered in vivo via ;parenteral (e.g,
intravenous,
subcutaneous, and intramuscular} or other traditional direct routes, such as
buccallsublingual, rectal, oral, nasal, topical, (such as transdermal and
ophthalmic),
vaginal, pulmonary, intraarteriai, intraperitoneal, int~raocular, or
intranasal routes or
25 directly into a specific tissue, such as the liver, bone marrow, or into
the tumor in the
case of cancer therapy. Non-parenteral routes are discussed further in WO
96120732.
24
CA 02340085 2001-02-09
WO 00/09665 PCT/US99/t808~
The cells or vectors are administered in any suitable manner, often with
pharmaceutically acceptable carriers: Suitable methods of administering cells
in the
context of the present invention to a patient are available, and, although
more than one
route can be used to administer a particular cell composition, a particular
route can often
provide a more immediate and more effective reaction than another route.
The dose of cells (e.g., activated T cells, or dendritic cells) administrated
to a patient, in
the context of the present invention should be sufficient to effect a benef
cial therapeutic
response in the patient over time, or to inhibit growth of cancer cells, or to
inhibit
infection. Thus, cells are administered to a patient: in an amount sufficient
to elicit an
effective immune response to the specific antigens andlor to alleviate;
reduce, cure or at
least partially arrest symptoms and/or complications from the disease or
infection. An
amount adequate to accomplish this is defined as a. "therapeutically effective
dose."
The dose will be determined by the activity of the 'T cell or antigen-
presenting cell
produced and the condition of the patient, as well ~~s the body weighs or
surface areas of
1 ~ the patient to be treated. The size of the dose also will be determined by
the existence.
nature, and extent of any adverse side effects that accompany the
administration of a
particular cell in a particular patient. In determining the effective amount
of the cell to
be administered in the treatment or prophylaxis of diseases such as AIDS or
cancer
(e.g., metastatic melanoma, prostate cancer, etc.), the physician needs to
evaluate
circulating plasma levels, CTL toxicity, progression of the disease, and the
production
of immune response against any introduced cell type.
Generally at least about I O'~ to 106 and typically, between I x 108 and 1 x
I0'° cells are
infused intravenously or intraperitoneally into a 70 kg patient over roughly
60-I20
minutes. Intravenous infusion is preferred. Vital signs and oxygen saturation
by pulse
oximetry are closely monitored. Blood samples are obtained 5 minutes and I
hour
following infusion arid saved for analysis. Ceil reim~usions are repeated
roughly every
month for a total of IO-12 treatments in a one year,period. After the first
treatment,
infusions can be performed on an outpatient basis at the discretion of the
clinician. If
CA 02340085 2001-02-09
WO 00!09665 PCTNS99/38087
the reinfusion is given as an outpatient, the participant is monitored for at
least 4 hours
following the therapy.
For administration, cells of the present invention can be administered at a
rate
determined by the LD-50 (or other measure of toxicity) of the cell type, and
the side-
s effects of the cell type at various concentrations, as applied to the mass
and overall
health of the patient. Administration can be accom~Iished via single or
divided doses.
The cells of this invention can supplement other treatments for a condition by
known
conventional therapy, including cytotoxic agents, nucleotide analogues and
biologic
_ response modif ers. Similarly, biological response nnodifiers are optionally
added for
treatment. For example, the cells axe optionally administered with an
adjuvant, or
cytokine such as GM-CSF, IL-12 or IL-2.
Administration by many of the routes of administration described herein or
otherwise
known in the art may be accomplished simply by direct administration using a
needle,
catheter or related device, at a single time point or at multiple time points.
In addition,
I5 an "administration" of a gene delivery vehicle (or ex vivo transduced
cells, for that
matter) at a given time point includes administration to one or more areas, or
by one or
more routes. In certain embodiments of the invention, one or more dosages is
administered directly in the indicated manner: intravenously at dosage greater
than or
equal to 103, 1 O5, 10', 109, 10'° or I0" cfu; intraarterially at
dosages greater than or
equal to 10', 10',10', 109, I0'° or 10" cfu; intrasmuss:ularly at
dosages greater than or
equal to 10'; I05, I0', I09, 10'° or 10" cfu, with dosages of
10'° or 10" cfu being
preferred; intradermally at dosages greater than or equal to 103, I05, 10',
109, 10'° or 10"
cfu; pulmonarily at dosages greater than or equal to 10', 105, 10', 109,
10'° or 10" cfu;
subcutaneously at dosages greater than or equal to 103, 105, 10', 109,
10'° or 10" cfu,
with dosages of 109, 10'° or 10" cfu being preferred; .interstitially
at dosages greater
than er equal to 103, I05, IO', 108, 10g, 10'° or I O" cfw; with
dosages of 108, 109, 10'° or
i 0" cfu being preferred; into a lymphoid organ such ~~.s the spleen, a
tonsil, or a lymph
node at dosages greater than or equal to 10', I05, I0', 108, 109, 10'°
or I O" cfu; into a
tumor at dosages greater than or equal to 103, 10a, 105, I05, 10', 108, I09,
10'° or 10" cfu,
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WO 00/09b65 PCT/US99/18087
with dosages of i O8, 109, 10'° or 10" cfu being preferred; and into
the afferent Iymph at
dosages greater than or equal to 10', 105, 10', 10g, 1.09, l0t° or IO"
cfu. Far purposes of
the convenience, "cfu" shall also refer to non-viral particles, such that one
efu is
equivalent to one-non-viral particle.
EXAMPLES
The following examples are presented to illustrate the present invention and
to assist
one of ordinary skill in making and' using the same. The examples are not
intended in
any way to otherwise limit the scope of the inventic>n.
Example 1: Preparation of dendritic cells genericaliv modified to present an
array
of antigens
Dendxitic cells are isolated according to Basic Protocol I described in Tedder
and
Jansen, 1997, Current Protocols in Immunology, John Wiley and Sons, 7.32. I.
Cell
lines from a matched pair of target and non-target cell types are obtained. A
matched
pair of cell types includes, for example, cell lines derived from the same
tissue of origin,
I S such as prostate, and differing in a targeted feature. An example of a
target cell is a
prostate cancer cell. An example of a matched pair of cell types is prostate
cells
differing in their metastatic potential. Nucleic acid sequences preferentially
expressed
in prostate cancer tissue are described in United States patent application
serial number
60/088,877, filed June I i, 1998, the entire contents of which are
incorporated herein by
reference. The dendritic cells are then transduced with the preferentially
expressed
nucleic acid sequences using conventional techniques, such as those described
in WO
97124447, the entire contents of which are incorporated herein by refexence.
Successful
txansduction of the human dendritic cells can be confirmed by in vitro T cell
priming
(Albert et al., 1998, Nature 392:86-89; Nair et al., 1998, Nature
Biotechnology
16(4):364-369; Antigen Processing and Presentation, in Current Protocols in
Immunology; Wiley, New York, 1998). Candidate immunogenic tumor-associated
antigen sequence arrays can be determined by screening tmansduced dendritie
cells with
responding T cells obtained from peripheral blood err dLN of tumor-beaming
patients.
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WO 00/09665 PCT/US99/18087
Once a pattern of reactivity is found in.a particular tumor type, the relative
efficacy of
these antigens can be evaluated using marine homoiogs and syngeneic marine
tumor
models that express one or more of these antigens. Example 2 describes one
method for
evaluating e~cacy.
Example 2: Dendritic cell-based immunother~y for the treatment of metastatic
tumors in combination with systemic ProIeukin~ IL-2
Genetically modified dendridc cells of the invention can be used to treat
tumors, alone
or in combination with other therapies. This example shows how to evaluate the
contribution of dendritic cells genetically modified using marine homologs of
nucleic
I O acid sequences identified by the methods of Example I to systemic
Proleukin IL-2
immunotherapy on marine syngeneic tumors. The C57Bl6 derived B i 6-1 10 lung
metastasis model, CT-26 marine colon model or the marine 3LL lung model can be
.
used to generate the most effective treatment regimen. These tumor models are
poorly
immunogenic and are differentially responsive to single agent Proleuitin
therapy, as
1.~ measured by both survival and tumor load.
The research strategy can be used to define the optimal mixture of dendritic
cells and
systemic Proleukin iL-2, e.g. a lower amount of IL-2 to maintain a high
objective
response rate while lowering toxicities, and to develop a dendritic cell plus
systemic IL-
2 regimen for application to a lung and/or colon marine syngeneic tumor model
that is
20 resistant to single agent IL-2 therapy.
Experimental Design:
Marine splenic dendritic cells are isolated and characterized as described by
Girolomoni
et al. i 990, J. Immunol. 145(9):2820-26. First, spleens from either naive or
day 7 tumor
bearing mice are removed, minced, and digested in Hank's Balanced Salt
Solution
25 (HBSS) with 40 mg coilagenase (Sigma, St. Louis, MO) for 1 hour. Cells are
then
filtered over I00 Lun nylon mesh; and washed. Red blood cells are lysed with
0.8~%
Ammonium chloride, 0.1 % KHCOZ, 0.004% EDTA (ACK Lysis buffer); pellet
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w _ ~ WQ 00/09665 PCT/US99/18087
resuspended to 3 x 10' cellslml in 1.035 Percoll (lPharmacia biotech) and
underlay with
an equal volume of I .075 gJml Percoll. The suspension is centrifuged at 2200
rpm for
20 minutes at 4°C. The band is harvested at interface, washed twice
with HBSS, and
resuspended to S x 106 cells/ml in complete culture medium and incubated at
37°C for
S 90 min. Nonadherent cells are removed and discarded. Fresh complete culture
medium
is added, and culture continued for 18-24 hr at 37°C. Gentle pipetting
dislodges splenic
dendritic cells.
CeIIs harvested from the spleen cell cultures are further enriched by
overlaying 2 mls of
S x 1061m1 cells onto a 3 ml layer of 14.5% Metrizamide-CM solution in a 1 S
ml
I O centrifuge tube. The gradient is centrifuged at 2000 rpm for I S min at
4°C, the band
harvested, and washed, counted, and resuspended to S x I06 ceils/mI in
complete
medium.
An aliquot can be removed for phenotyping using the following 3-color staining
protocol ("DC" refers to dendritic cells):
I S Tube 1 CD3/Isotype Isotype control
Tube 2 CD31CD41CD8 CD4/CD8 T cells
Tube 3 CD4S/I-AbICD86 CD vs monocytes
Tube 4 CD4SICD801CD40 DC vs monocytes
Tube S CD4S/CD 1 I b (Mac- I )ICD DC vs monocytes
11 c
20 Tube 6 CD4SICD4SR(B22))/CD44 DC vs B cells
Bone marrow derived dendritic cells (BM-DC) are isolated from erythrocyte
depleted
mouse bone marrow cells cultured for seven days in complete medium
supplemented
with 10 ng/m1 GM-CSF and 10 ng/ml IL-4. On day seven, BM-DC are harvested by
2S gentle pipetting and further enriched by 14.5% (by weight) metrizamide
(Sigma, St.
Louis, MO) complete medium gradients. The Iow density interface containing the
BM-
DC are collected by gentle pipette aspiration. The BM-DC are washed twice with
complete medium, enumerated (purity >90%, positive co-expression of MHC Class
II,
CD40, CD80, CD86 and CD 1 I c).
29
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CA 02340085 2001-02-09
WO 00109665 PCT/LJS99/18087
Genetically modified dendritic cells prepared as described in Example 1 and/or
2 are
then injected either before (protective) or after (therapeutic) tumor
challenge. For
protection, an intravenous {iv) tumor challenge is introduced 1 week following
the last
immunization (days -15, -8, 0). For therapy, immunizations are given at days
4, 8, and
12 post-iv tumor challenge; with or without Proleukin.
Proleukin therapy is administered as follows:
For protection: 10 mg/kg iv for 5 days starting with second immunization.
For therapy: 10 mg/kg/day iv for 5 or 10 days starti~.lg on day 2 post-iv
tumor challenge.
Experiment #I : Single agent Proleukin therapy (10 mice/group):
B I 6-F 10: 50-85,000 cells iv DO
Proleukin: iv twice per day for seven days, beginning at Day 3
Sacrifice and count lung metastases
Group 1 B 16 PBS control
2 B I 6 2.5 mg/kg Proleukin
3 B i 6 3.5 mg/kg Proleukin
4 B 16 5 mg/kg Proleukin
Experiment #2: Take best Proleukin regimen {~2/I0 dead mice with >50%
reduction in lung metastases).
Group 1 B 16 PBS
2 B 16 Proleukin
3 B 16 modified DC alone
4 B 16 modif ed DC + Proieukin
5 B 16 DC- alone
6 B 16 DC- + proleukin
7 B 16 irradiated B 16 alone
(50,000/iv)
CA 02340085 2001-02-09
WO 00/09665 PCTIUS99l18087
8 B I 6 irradiated B I 6 + Proleukin
Experiment #3: Take most active DC regimen {double single agent Proleukin lung
metastases reduction)
Group 1 B 16 PBS
2 B 16 Proleukin
3 B I6 modified DC alone
4 B I6 modif ed DC + Proleu~kin
5 B 16 modified DC + 75% Proleukan
6 B 16 modified DC + 50% Proleukin
I O 7 B 16 modified DC + 25% Proleukin
The potency of the genetically modified dendritic cells (DC) can be evaluated
with a
short term culture immune function assay, based on the antigen-induced
expression of
CD69. Briefly, whole blood andler spleen cells from naive, tumor-bearing, and
DC-
immunized mice are prepared as single cell preparations, depleted of
macrophages by
I ~ adherence, and resuspended to 5 x I 0 6 cells/ml. Five hundred microliters
of various
DC sources {x-irradiated) are titrated into 12 x 75 mm polypropylene tubes
containing
0.5 ml of the monocyte-depleted responding cells. T'he mixed cells are
cultured for 24
hours at 37°C, with a 6 hr timepoint taken for analysis. The cultures
contain Brefeldin
for the last 4 hours of culture. The cultures are then surfaced-stained with
20 CD3/CD4/CD69 or CD3/CD8/CD69. Duplicate cultures are permeabilized, and
stained
with CD3/CD4/IFNy, CD3/CD4/IL-4, CD3/CD8lIFNy, and CD3/CD8/IL-4. Reagents
for TNFa and GM-CSF are used to further defne the CD8 reactivity to the
genetically
modified DC.
DC loaded with baculovirus-produced mouse gp 100 (cloned out from B 16F I O)
are used
25 as a positive control,
Serum from the immunized mice can be obtained, pelleted, heat-inactivated, and
stared
at -70°C for future analysis of anti-tumor binding activity,
cytokinelchemokine content,
sIL-2R, and other inflammatory molecules.
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WO 00/09665 PCTIUS99/18087
The foregoing detailed description provides exemplary information about the
invention.
Those skilled in the art will appreciate that modifications can be made
without
diverging from the spirit and purpose of the invention,
32
