Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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INDUCTION OF IMMUNITY
AGAINST TUMOR SELF-ANTIGENS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. ~ 119(e) to U.S.
Provisional Application No. 60/078,889, filed March 20, 1998, the contents of
which are hereby incorporated by reference.
TECHNICAL FIELD
This invention is in the field of molecular immunology and medicine. In
particular, the present invention provides compositions and methods for
inducing
an immune response to a native self antigen in a subject.
BACKGROUND
The goal of vaccination is to generate a protective immune response and
an expanded population of memory cells ready to encounter an agent identified
as
foreign, which will then elicit a potent secondary immune response. T and B
cells
are highly antigen specific and can develop into memory cells, and therefore
are
the target for a successful vaccine.
Tumor specific T cells, derived from cancer patients, will bind and lyse
... .. .. . . .. ... , , aumor cells:. This. specificity is based on their
ability to recognize short amino
acid sequences (epitopes) presented on the surface of the tumor cells by MHC
class I and class II molecules. . These epitopes are derived from the
proteolytic
degradation of intracellular proteins called tumor antigens encoded by genes
that
are either uniquely or aberrantly expressed in tumor or cancer cells.
The availability of specific anti-tumor T cells has enabled the
identification of tumor antigens and subsequently the generation of cancer
vaccines designed to provoke an anti-tumor immune response. A critical target
of
vaccines is the specialized antigen-presenting cell ("APC"), the most
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immunologically powerful of which is the bone marrow-derived dendritic cell
("DC").
DCs are potent antigen presenters that express high levels of co-
stimulatory molecules and are capable of activating both CD4+ and CD8+ naive T
lymphocytes. Results obtained in several animal models have shown that DCs
pulsed with defined tumor-associated peptides or with peptides eluted from the
surface of tumor cells are capable of inducing an antigen-specific CTL
response
resulting in protection from tumor challenge and, in some instances,
regression of
established tumors. The same type of approach has also been tested in human
clinical trials with encouraging results. For example, Hsu et al. have
reported that
four B cell lymphoma patients infused with autologous DCs pulsed with tumor-
specific idiotype protein all developed an idiotype-specific proliferative
response
accompanied by complete tumor regression in two patients and partial
regression
in a third. Hsu et al. (1996) Nature Med. 2:52. More recently, Nestle et al.
reported that melanoma patients treated with autologous DCs pulsed with tumor
lysate or a cocktail of CTL peptide epitopes, developed cell-mediated immunity
with objective clinical responses in S out of 16 patients evaluated. Nestle et
al.
( 1998) Nature Med. 4:328.
Successful cancer therapy, similar to the ones noted above, is rare. The
high incidence of failure may be due to the fact that naturally occurring
neoplasms
do not possess antigens that can serve as inducers and/or targets for a tumor
destructive immune response, although immunological reactions mediated by
either lymphocytes or antibodies to cultivated human tumors have been
reported.
Hellstrom K. and Hellstrom I. ( 1969) Adv. Cancer Res. 12:167. Indeed,
mechanisms of systemic immune tolerance to self have begun to emerge,
particularly from studies in transgenic mouse systems. Hanahan D. ( I 990)
Ann.
Rev. Cell Biol. 6:493. Mechanisms of systemic immune tolerance include
deletion of potentially autoreactive B or T cells, induction of anergy in B
and T
cells, and the poorly defined phenomenon of suppression of immune response by
suppressor cells. Houghton and Lewis, pages 37-54 in Fonni, et al., eds.
(1994)
CYTOKINE-INDUCED TUMOR IMMUNOGENICITY, Academic Press, New York.
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Thus, a need exists to overcome immune tolerance to self antigens and to
provide
an effective cancer vaccine. This invention satisfies these needs and provides
related advantages as well.
DISCLOSURE OF THE INVENTION
In the present invention, immunization is carried out with a heterologous
antigen or an altered antigen that is structurally distinct from the self
antigen or
"native" antigen yet is still capable of inducing an immune response against
the
self antigen. Such antigens are immunogenic (seen as foreign) and serve to
induce an immune response that cross-reacts with the native antigen.
In the context of cancer gene therapy, the invention comprises using
modified (altered) tumor antigens or tumor antigens derived from heterologous
species to break immunological tolerance and induce a cross-reactive immune
response against the corresponding native or self antigen. For example,
immunizing humans against the human melanoma antigen gp100 requires
breaking tolerance against a self antigen. As shown below, the use of the non
self antigen can provide protective immunity and tumor reduction in vivo.
Immunization and therapy are accomplished by any of the following methods: 1 )
administration of a vector encoding altered tumor antigen or antigen from a
heterologous species; 2) infecting dendritic cells ex vivo or in vivo with the
same
vector; or 3) use of transduced dendritic cells or APCs to stimulate
production of
an enriched population of antigen-specific immune effector cells that can be
adoptively transferred into the host.
Antigen presenting cells such as dendritic cells also are useful to expand a
population of immune effector cells that specifically recognize and lyre the
cells
presenting the heterologous antigen and its native or self counterpart. The
expanded immune effector cell populations and their use in prophylactical and
therapeutical methods also are provided herein.
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BRIEF DESCRIPTION OF THE FIGURES
Figures 1 A through 1 D show the results of immunizing mice with
syngeneic DCs. Five female C57BL/6 mice (represented by five different
symbols in the panels) were immunized with B 16 melanoma using dendritic cells
transfected with Ad vector encoding homologous mouse gp100 versus
heterologous human gp100.
Figures 2A through 2C show induction of CTL activity following
immunization with Ad2/hugp100v1 vector or Ad2/hugp100v1-transduced DCs.
Spleens from groups of 3 animals were collected 1 S days after i.v.
administration
of vehicle (Figure 2A), Ad2/hugpl00v1-transduced DCs (Figure 2B) or i.d.
delivery of Ad2/hugpl00vl vector (Figure 2C). Pooled spleen cells from each
group were re-stimulated is vitro with syngeneic SVB6KHA fibroblasts
transduced with Ad2/hugpl00v 1 and were tested for cytolytic activity after 6
days of culture. Targets consisted of B 16 cells and SVB6KHA fibroblasts
untransduced or transduced with Ad2/hugpl00v 1 or wild type Ad2 deleted for E3
(SVB6KHA-Ad202.9). Figure legend: (-1- B16; -~- SVB6KHA
Untransduced; -1- SVB6KHA-Ad2/hugp100v1; -~- SVB6HA-Ad2~2.9).
Figure 3 compares the effectiveness of immunization with DCs transduced
with Ad vector encoding various melanoma-associated antigens. The figure
shows the evaluation of the nature of the antigen. Figure legend: (-G-
Untransduced DCs; -o- Ad2/hugpl00v1 DCs; -o- Ad2mgp100 DCs; -e-
Ad2/mTRP-2 DCs). Groups of 5 C57BL/6 mice were injected i.v. with Sx105
DCs that were either untransduced or transduced with Ad2/hugp 1 OOv 1,
Ad2/mgp100 or Ad2/mTRP-2 vector. The animals were challenged 1 S days later
with a s.c, injection of 2x104 B16 melanoma cells.
Figure 4 shows the frequency of gp100-reactive splenic T lymphocytes
following immunization with Ad2/hugp 100- or Ad2/empty vector-transduced
DCs. Spleen cells from immunized mice were stimulated in vitro with a
cytotoxic
T lymphocyte peptide epitope derived from hugp 100 (open bar); or the
corresponding epitope from mgp100 (solid bar). An ovalbumin-derived epitope
4
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was used as a negative control (hatched bar). The number of T lymphocytes that
produced y-interferon upon recognition of peptide was measured by elispot.
MODES FOR CARRYING OUT THE INVENTION
Throughout this disclosure, various publications, patents and published
patent specifications are referenced by an identifying citation. The
disclosures of
these publications, patents and published patent specifications are hereby
incorporated by reference into the present disclosure to more fully describe
the
state of the art to which this invention pertains.
Definitions
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of immunology, molecular biology,
microbiology, cell biology and recombinant DNA, which are within the skill of
1 S the art. See, e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A
LABORATORY MANUAL, 2"d edition ( 1989); CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (F. M. Ausubel, et al, eds., ( 1987)): the series METHODS IN
ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J.
MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds.
(1989) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I:
Freshney, ed. ( 1987)).
As used herein, certain terms may have the following defined meanings.
As used in the specification and claims, the singular form "a", "an" and
"the" include plural references unless the context clearly dictates otherwise.
For
example, the term ''a cell" includes a plurality of cells, including mixtures
thereof.
The term "genetically modified" means containing and/or expressing a
foreign gene or nucleic acid sequence which in turn, modifies the genotype or
phenotype of the cell or its progeny. In other words, it refers to any
addition,
deletion or disruption to a cell's endogenous nucleotides.
As used herein, the term "cytokine" refers to any one of the numerous
factors that exert a variety of effects on cells, for example, inducing growth
or
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proliferation. Non-limiting examples of cytokines which may be used alone or
in
combination in the practice of the present invention include, interleukin-2
(IL-2),
stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6),
interleukin 12
(IL-12). G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF),
interleukin-1 alpha (IL-la), interleukin-11 (IL-11), MIP-la, leukemia
inhibitory
factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The present
invention also includes culture conditions in which one or more cytokine is
specifically excluded from the medium. Cytokines are commercially available
from several vendors such as, for example, Genzyme (Framingham, MA),
Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA}, R&D
Systems and Immunex (Seattle, WA). It is intended, although not always
explicitly stated, that molecules having similar biological activity as wild-
type or
purified cytokines (e.g., recombinantly produced or muteins thereof) are
intended
to be used within the spirit and scope of the invention.
1 S The term "antigen presenting cell" ("APC"), as used herein, intends any
cell which presents on its surface an antigen in association with a major
histocompatibility complex molecule, or portion thereof, or, alternatively,
one or
more non-classical MHC molecules, or a portion thereof. Examples of suitable
APCs are discussed in detail below and include, but are not limited to, whole
cells
such as macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen
presenting cells or other cell types) expressing the necessary MHC and co-
stimulatory molecules. Methods of making hybrid APCs have been described.
See, for example, International Patent Application Publication Nos. WO
98/46785
and WO 95/16775.
Dendritic cells (DCs) are potent antigen-presenting cells. It has been
shown that DCs provide all the signals required for T cell activation and
proliferation. These signals can be categorized into two types. The first
type,
which gives specificity to the immune response, is mediated through
interaction
between the T-cell receptor/CD3 ("TCR/CD3") complex and an antigenic peptide
presented by a major histocompatibility complex ("MHC") class I or II protein
on
the surface of APCs. This interaction is necessary, but not sufficient, for T
cell
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activation to occur. In fact. without the second type of signals. the first
type of
signals can result in T cell anergy. The second type of signals. called co-
stimulatory signals, is neither antigen-specific nor MHC-restricted, and can
lead
to a full proliferation response of T cells and induction of T cell effector
functions
in the presence of the first type of signals. As used herein. "dendritic cell"
is to
include, but not be limited to a pulsed dendritic cell, a foster cell . a
dendritic cell
hybrid or a genetically modified dendritic cell. Methods for generating
dendritic
cells from peripheral blood or bone marrow progenitors have been described
(Inaba et al. (1992) J. Exp. Med. 175:1157; Inaba et al. (1992) 3. Exp. Med.
176:1693-1702; Romani et al. (1994) J. Exp. Med. 180:83-93; Sallusto et al.
( 1994) J. Exp. Med. 179:1109-1118; Bender et al. ( 1996) J. Imm. Methods
196:121-13 5; and Romani et al. ( 1996) J. Imm. Methods 196:13 7-1 S 1 ).
"Co-stimulatory molecules" are molecules involved in the interaction
between receptor-ligand pairs expressed on the surface of antigen presenting
cells
1 S and T cells. "Co-stimulatory activity" was originally defined as an
activity
provided by bone-marrow-derived accessory cells such as macrophages and
dendritic cells, the so called "professional" APCs. Several molecules have
been
shown to enhance co-stimulatory activity. These are heat stable antigen (HSA)
(Liu Y. et al. (1992) J. Exp. Med. 175:437), chondroitin sulfate-modified MHC
invariant chain (Ii-CS) (Naujokas M.F. et a1. (1993) Cell 74:257).
intracellular
adhesion molecule 1 (ICAM-1) (Van Seventer G.A. (1990} J. Immunol.
144:4579), B7-1 and B7-2B70 (Schwartz R.H. (1992) Cell 71:1065) and B7's
counter-receptor CD28 or CTLA-4 on T cells (Freeman et al. (1993) Science
262:909; Young et al. ( 1992) J. Clin. Invest 90: 229; and Nabavi et al. (
1992)
Nature 360:266). Other important co-stimulatory molecules are CD40, CD54,
CD80, CD86. As used herein, the term "co-stimulatory molecule'' encompasses
any single molecule or combination of molecules which, when acting together
with a peptide/MHC complex bound by a TCR on the surface of a T cell, provides
a co-stimulatory effect which achieves activation of the T cell that binds the
peptide. The term thus encompasses B7, or other co-stimulatory molecules) on
an antigen-presenting matrix such as an APC, fragments thereof (alone,
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complexed with another molecule(s), or as part of a fusion protein) which,
together with peptide/MHC complex, binds to a cognate ligand and results in
activation of the T cell when the TCR on the surface of the T cell
specifically
binds the peptide. Co-stimulatory molecules are commercially available from a
variety of sources, including, for example, Beckman Coulter. It is intended.
although not always explicitly stated, that molecules having similar
biological
activity as wild-type or purified co-stimulatory molecules (e.g.,
recombinantly
produced or muteins thereof) are intended to be used within the spirit and
scope of
the invention.
The term "antigen" is used in its broadest sense and includes minimal
epitopes and chimeric molecules in addition to isolated full length proteins.
A
"self antigen", also referred to herein as a "native antigen", is an antigenic
peptide
that induces little or no immune response in the subject due to self tolerance
to the
antigen. An example of a self antigen is the melanoma antigen gp100. The
antigen of this vaccine is "heterologous" (i.e., allogeneic or a homologe from
an
isolated species, e.g., a murine antigen administered to a human patient) or
an
"altered antigen" as compared to the corresponding native self antigen. The
heterologous or altered antigen also can be made by chemical synthesis.
The term "immune effector cells" refers to cells capable of binding an
antigen or which mediate an immune response. These cells include, but are not
limited to, T cells, B cells, monocytes, macrophages, NK cells and cytotoxic T
lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor,
inflammatory, or other infiltrates. Certain diseased tissues express specific
antigens and CTLs specific for these antigens have been identified. For
example,
approximately 80% of melanomas express the antigen known as gp100.
The term "immune effector molecule" as used herein, refers to molecules
capable of antigen-specific binding, and includes antibodies, T cell antigen
receptors, and MHC Class I and Class II molecules.
A "naive" immune effector cell is an immune effector cell that has never
been exposed to an antigen.
s
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As used herein, the term "educated. antigen-specific immune effector cell"
is an immune effector cell as defined above, which has encountered antigen and
which is specific for that antigen. An educated, antigen-specific immune
effector
cell may be activated upon binding antigen. "Activated" implies that the cell
is no
longer in Go phase, and begins to produce cytokines characteristic of the cell
type.
For example, activated CD4+ T cells secrete IL-2 and have a higher number of
high affinity IL-2 receptors on their cell surfaces relative to resting CD4+ T
cells.
A peptide or polypeptide of the invention may be preferentially recognized
by antigen-specific immune effector cells, such as B cells and T cells. In the
context of T cells, the term "recognized" intends that a peptide or
polypeptide of
the invention, comprising one or more synthetic antigenic epitopes, is
recognized,
i.e., is presented on the surface of an APC together with (i.e., bound to) an
MHC
molecule in such a way that a T cell antigen receptor (TCR) on the surface of
an
antigen-specific T cell binds to the epitope wherein such binding results in
activation of the T cell. The team "preferentially recognized" intends that a
polypeptide of the invention is substantially recognized, as defined above, by
a T
cell specific for an antigen. Assays for determining whether an epitope is
recognized by an antigen-specific T cell are known in the art and are
described
herein.
The term "syngeneic" or "autologous" as used herein, indicates the origin
of a cell. Thus, a cell being administered to an individual (the "recipient")
is
autogeneic if the cell was derived from that individual (the "donor") or a
genetically identical individual. An syngeneic cell can also be a progeny of
an
syngeneic cell. The term also indicates that cells of different cell types are
derived from the same donor or genetically identical donors. Thus, an effector
cell and an antigen presenting cell are said to be syngeneic if they were
derived
from the same donor or from an individual genetically identical to the donor,
or if
they are progeny of cells derived from the same donor or from an individual
genetically identical to the donor.
Similarly, the term "allogeneic" as used herein, indicates the origin of a
cell. Thus, a cell being administered to individual (the "recipient') is
allogeneic if
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the cell was derived from an individual not genetically identical to the
recipient;
in particular, the term relates to non-identity in expressed MHC molecules. An
allogeneic cell can also be a progeny of an allogeneic cell. The team also
indicates that cells of different cell types are derived from genetically non-
identical donors, or if they are progeny of cells derived from genetically non-
identical donors. For example, an APC is said to be allogeneic to an effector
cell
if they are derived from genetically non-identical donors.
A "hybrid" cell refers to a cell having both antigen presenting capability
and also expresses one or more specific antigens. In one embodiment, these
hybrid cells are formed by fusing, in vitro, APCs with cells that are known to
express the one or more antigens of interest.
The term "culturing" refers to the in vitro propagation of cells or
organisms on or in media of various kinds. It is understood that the
descendants
of a cell grown in culture may not be completely identical (either
morphologically, genetically, or phenotypically) to the parent cell. By
"expanded" is meant any proliferation or division of cells.
A "subject" is a vertebrate, preferably a mammal, more preferably a
human. Mammals include, but are not limited to, murines, simians, humans, farm
animals, sport animals, and pets.
As used herein, "expression" refers to the process by which
polynucleotides are transcribed into mRNA and translated into peptides,
polypeptides, or proteins. If the polynucleotide is derived from genomic DNA,
expression may include splicing of the mRNA, if an appropriate eukaryotic host
is
selected. Regulatory elements required for expression include promoter
sequences to bind RNA polymerase and transcription initiation sequences for
ribosome binding. For example, a bacterial expression vector includes a
promoter
such as the lac promoter and for transcription initiation the Shine-Dalgarno
sequence and the start codon AUG (Sambrook et al. (1989) Supra ). Similarly,
an
eukaryotic expression vector includes a heterologous or homologous promoter
for
RNA polymerase II, a downstream polyadenylation signal, the start codon AUG,
and a termination codon for detachment of the ribosome. Such vectors can be
io
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obtained commercially or assembled by the sequences described in methods well
known in the art, for example, the methods described below for constructing
vectors in general.
The terms "major histocompatibility complex" or "MHC" refers to a
complex of genes encoding cell-surface molecules that are required for antigen
presentation to T cells and for rapid graft rejection. In humans, the MHC
complex
is also known as the HLA complex. The proteins encoded by the MHC complex
are known as "MHC molecules" and are classified into class I and class II MHC
molecules. Class I MHC molecules~include membrane heterodimeric proteins
made up of an a chain encoded in the MHC associated noncovalently with (32-
microglobulin. Class I MHC molecules are expressed by nearly all nucleated
cells and have been shown to function in antigen presentation to CD8+ T cells.
Class I molecules include HLA-A, -B. and -C in humans. Class II MHC
molecules also include membrane heterodimeric proteins consisting of
noncovalently associated a and ~i chains. Class II MHC are known to
participate
in antigen presentation to CD4'' T cells and, in humans, include HLA-DP, -DQ,
and DR. The term "MHC restriction" refers to a characteristic of T cells that
permits them to recognize antigen only after it is processed and the resulting
antigenic peptides are displayed in association with either a self class I or
class II
MHC molecule. Methods of identifying and comparing MHC are well known in
the art and are described in Allen M. et al. (1994) Human Imm. 40:25;
Santamaria
P. et al. (1993) Human Imm. 37:39 and Hurley C.K. et a1. (1997) Tissue
Antigens
50:401.
The term "peptide" is used in its broadest sense to refer to a compound of
two or more subunit amino acids, amino acid analogs, or peptidomimetics. The
subunits may be linked by peptide bonds. In another embodiment, the subunit
may be linked by other bonds, e.g. ester, ether, etc. As used herein the term
"amino acid" refers to either natural and/or unnatural or synthetic amino
acids,
including glycine and both the D or L optical isomers, and amino acid analogs
and
peptidomimetics. A peptide of three or more amino acids is commonly called an
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oligopeptide if the peptide chain is short. If the peptide chain is long, the
peptide
is commonly called a polypeptide or a protein.
A "composition" is intended to mean a combination of active agent and
another compound or composition, inert (for example, a solid support, a
detectable agent or label) or active, such as an adjuvant.
As used herein, ''solid phase support" or "solid support" used
interchangeably, is not limited to a specific type of support. Rather a large
number of supports are available and are known to one of ordinary skill in the
art.
Solid phase supports include silica gels, resins, derivatized plastic films,
glass
beads, cotton, plastic beads, and alumina gels. As used herein, "solid
support"
also includes synthetic antigen-presenting matrices, cells, and liposomes. A
suitable solid phase support may be selected on the basis of desired end use
and
suitability for various protocols. For example, for peptide synthesis. solid
phase
support may refer to resins such as polystyrene (e.g., PAM-resin obtained from
Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE~ resin (obtained from
Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories),
polystyrene resin grafted with polyethylene glycol (TentaGel~, Rapp Polymere,
Tubingen, Germany) or polydimethylacrylamide resin (obtained from
MilligenBiosearch, California).
A "pharmaceutical composition" is intended to include the combination of
an active agent with a Garner, inert or active, making the composition
suitable for
diagnostic or therapeutic use in vitro, in vivo or ex vivo.
As used herein, the term "pharmaceutically acceptable Garner"
encompasses any of the standard pharmaceutical carriers, such as a phosphate
buffered saline solution, water, and emulsions, such as an oil/water or
water/oil
emulsion, and various types of wetting agents. The compositions also can
include
stabilizers and preservatives. For examples of carriers, stabilizers and
adjuvants,
see Martin, REMINGTON'S PHARM. scl., 15th Ed. (Mack Publ. Co.. Easton (
1975)).
An "effective amount" is an amount Buff cient to effect beneficial or
desired results. An effective amount can be administered in one or more
administrations, applications or dosages.
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The term "immunomodulatory agent" as used herein, is a molecule, a
macromolecular complex, or a cell that modulates an immune response and
encompasses a synthetic antigenic peptide of the invention alone or in any of
a
variety of formulations described herein; a polypeptide comprising a synthetic
antigenic peptide of the invention; a polynucleotide encoding a peptide or
polypeptide of the invention; a synthetic antigenic peptide of the invention
bound
to a Class I or a Class II MHC molecule on an antigen-presenting matrix,
including an APC and a synthetic antigen-presenting matrix (in the presence or
absence of co-stimulatory molecule(s)); a synthetic antigenic peptide of the
invention covalently or non-covalently complexed to another molecules) or
macromolecular structure; and an educated, antigen-specific immune effector
cell
which is specific for a peptide of the invention.
The term "modulate an immune response" includes inducing (increasing,
eliciting) an immune response; and reducing (suppressing) an immune response.
I S An immunomodulatory method (or protocol) is one that modulates an immune
response in a subject.
As used herein, the term "inducing an immune response in a subject" is a
term well understood in the art and intends that an increase of at least about
2-
fold, more preferably at least about 5-fold, more preferably at least about 10-
fold,
more preferably at least about 100-fold, even more preferably at least about
500-
fold, even more preferably at least about 1000-fold or more in an immune
response to an antigen (or epitope) can be detected (measured), after
introducing
the antigen (or epitope) into the subject, relative to the immune response (if
any)
before introduction of the antigen (or epitope) into the subject. An immune
response to an antigen (or epitope), includes, but is not limited to,
production of
an antigen-specific (or epitope-specific) antibody, and production of an
immune
cell expressing on its surface a molecule which specifically binds to an
antigen (or
epitope). Methods of determining whether an immune response to a given antigen
(or epitope) has been induced are well known in the art. For example, antigen-
specific antibody can be detected using any of a variety of immunoassays known
in the art, including, but not limited to, ELISA, wherein, for example,
binding of
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an antibody in a sample to an immobilized antigen (or epitope) is detected
with a
detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig
antibody). Immune effector cells specific for the antigen can be detected any
of a
variety of assays known to those skilled in the art, including, but not
limited to,
S ; ~ Cr-release assays, 3N-thymidine uptake assays or induction of cytokine
release.
As used herein, the term "a disease or condition related to a population of
CD4+ or CD8+ T cells" is one which can be related to a population of CD4+ or
CD8+ T cells, such that these cells are primarily responsible for the
pathogenesis
of the disease; it is also one in which the presence of CD4+ or CD8+ T cells
is an
indicia of a disease state; it is also one in which the presence of a
population CD4+
or CD8+ T cells is not the primary cause of the disease, but which plays a key
role
in the pathogenesis of the disease; it is also one in which a population of
CD4+ or
CD8+ T cells mediates an undesired rejection of a foreign antigen. Examples of
a
condition related to a population of CD4+ or CD8+ T cells include, but are not
limited to, autoimmune disorders, graft rejection, immunoregulatory disorders,
and anaphylactic disorders.
As used herein, the terms "neoplastic cells", "neoplasia", "tumor", "tumor
cells", "cancer" and "cancer cells", (used interchangeably) refer to cells
which
exhibit relatively autonomous growth, so that they exhibit an aberrant growth
phenotype characterized by a significant loss of control of cell proliferation
(i.e.,
de-regulated cell division). Neoplastic cells can be malignant or benign.
"Suppressing" tumor growth indicates a growth state that is curtailed when
compared to growth without treatment or prevention as described herein. Tumor
cell growth can be assessed by any means known in the art, including, but not
limited to, measuring tumor size, determining whether tumor cells are
proliferating using a 3H-thymidine incorporation assay, or counting tumor
cells.
"Suppressing" tumor cell growth means any or all of the following states:
slowing, delaying, and stopping tumor growth, as well as tumor shrinkage.
"Host cell" or "recipient cell" is intended to include any individual cell or
cell culture which can be or have been recipients for vectors or the
incorporation
of exogenous nucleic acid molecules, polynucleotides and/or peptides (or
14
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polypeptides). It also is intended to include progeny of a single cell, and
the
progeny may not necessarily be completely identical (in morphology or in
genomic or total DNA complement) to the original parent cell due to natural,
accidental, or deliberate mutation. The cells may be procaryotic or
eucaryotic,
S and include but are not limited to bacterial cells, yeast cells, animal
cells, and
mammalian cells, e.g., marine, rat, simian or human.
The term "isolated" means separated from constituents, cellular and
otherwise, in which the polynucleotide, peptide, polypeptide, protein,
antibody, or
fragments thereof, are normally associated with in nature. For example, with
respect to a polynucleotide, an isolated polynucleotide is one that is
separated
from the 5' and 3' sequences with which it is normally associated in the
chromosome. As is apparent to those of skill in the art. a non-naturally
occurring
polynucleotide, peptide, polypeptide, protein. antibody, or fragments thereof,
does
not require "isolation" to distinguish it from its naturally occurring
counterpart.
In addition, a "concentrated" "separated" or "diluted" polynucleotide,
peptide,
polypeptide, protein, antibody, or fragments thereof, is distinguishable from
its
naturally occurring counterpart in that the concentration or number of
molecules
per volume is greater than "concentrated" or less than "separated" than that
of its
naturally occurring counterpart. A polynucleotide, peptide, polypeptide,
protein,
antibody, or fragments thereof, which differs from the naturally occurring
counterpart in its primary sequence or for example, by its glycosylation
pattern,
need not be present in its isolated form since it is distinguishable from its
naturally
occurring counterpart by its primary sequence, or alternatively, by another
characteristic such as glycosylation pattern. Although not explicitly stated
for
each of the inventions disclosed herein, it is to be understood that all of
the above
embodiments for each of the compositions disclosed below and under the
appropriate conditions, are provided by this invention. Thus, a non-naturally
occurring polynucleotide is provided as a separate embodiment from the
isolated
naturally occurring polynucleotide. A protein produced in a bacterial cell is
provided as a separate embodiment from the naturally occurnng protein isolated
from a eukaryotic cell in which it is produced in nature.
IS
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As used herein, the term ''comprising" is intended to mean that the
compositions and methods include the recited elements, but not excluding
others.
"Consisting essentially of" when used to define compositions and methods,
shall
mean excluding other elements of any essential significance to the
combination.
Thus, a composition consisting essentially of the elements as defined herein
would not exclude trace contaminants from the isolation and purification
method
and pharmaceutically acceptable carriers, such as phosphate buffered saline,
preservatives, and the like. "Consisting of shall mean excluding more than
trace
elements of other ingredients and substantial method steps for administering
the
compositions of this invention. Embodiments defined by each of these
transition
terms are within the scope of this invention.
This invention provides improved cancer vaccines and methods of using
the vaccines to induce an immune response to a native self antigen in a
subject.
As shown in the experimental examples below, the compositions and methods of
this invention provide protective immunity against growth of tumor cells in
vivo
and a means to inhibit the growth of tumors in vivo. The methods also induce
tumor reduction of established tumors in vivo. For purposes of immunization,
heterologous/altered antigens can be delivered to antigen-presenting cells as
protein/peptide or in the form of polynucleotide encoding the protein/peptide.
Antigen-presenting cells (APCs), as defined above, include but are not limited
to
dendritic cells (DCS), monocytes/macrophages, B-lymphocytes or other cell
types) expressing the necessary MHC/co-stimulatory molecules. The methods
described below focus primarily on DCS which are the most potent, preferred
APCs.
This invention also provides an isolated novel heterologous or altered
antigen that is capable of inducing an immune response against a self antigen
in a
subject, an isolated nucleic acid encoding the antigen, as well as vectors and
host
cells containing the nucleic acids. Methods of replicating and expressing the
isolated nucleic acids also are within the scope of this invention. Vectors
and
methods for in vitro and in vivo transduction are briefly described below and
are
well known in the art. The incorporation and expression of the exogenous
nucleic
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acid can be confirmed using RT-PCR, Northern and Southern blotting analysis.
Sambrook et al. ( 1989) Supra.
The methods of the invention are exemplified below. Melanoma-
associated antigens (MAAs) were used to transduce marine DCs which were then
tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce
protective immunity against B16 melanoma tumor cells. Dendritic cells derived
from bone marrow displayed surface markers characteristic of DCs and were
functionally active in vitro as determined in a mixed lymphocyte reaction and
as
indicated by their ability to induce primary antigen-specific proliferation of
syngeneic T lymphocytes. The DCs were efficiently transduced with adenovirus
type 2 (Ad2) based vectors while remaining phenotypically and functionally
intact. Immunization of C57BL/6 mice with DCs transduced with Ad vector
encoding the non-self human gpI00 melanoma antigen (Ad2/hugp100) elicited
the development of gp100-specific CTLs capable of lysing syngeneic fibroblasts
transduced with Ad2/hugp 100 as well as B 16 cells expressing endogenous
marine
gp100. The induction of gp100-specific CTLs was associated with long-term
protection against lethal subcutaneous challenge with B 16 cells.
Although this invention is exemplified using heterologous gp100
melanoma tumor antigen, any heterologous or altered antigen is useful in the
methods described herein.
For example, polypeptides and the polynucleotides encoding antigens of
this invention can be, in one embodiment, the heterologous counterpart or an
altered antigen of previously characterized tumor-associated antigens such as
MUC-1 (Henderson et al. (1996) Cancer Res. 56:3763); MART-1 (Kawakami et
al. (1994) Proc. Natl. Acad. Sci. 91:3515; Kawakami et al. (1997) Intern. Rev.
Immunol. 14:173; Ribas et al. (1997) Cancer Res. 57:2865); HER-2/neu (U.S.
Patent No. 5,550,214); MAGE (PCT/LTS92/04354); HPV 16, 18E6 and E7
(Ressing et al. (1996) Cancer Res. 56(1):582; Restifo (1996) Current Opinion
in
Immunol. 8:658; Stern (1996) Adv. Cancer Res. 69:175; Tindle et al. (1995)
Clin.
Exp. lmmunol. 101:265; van Driel et al. (1996) Annals of Medicine 28:471); CEA
(U.S. Patent No. 5,274,087); PSA (Lundwall, A. (1989) Biochem. Biophys.
CA 02322624 2000-09-06
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Research Communications 161: I I 51 ); prostate membrane specific antigen
(PSMA) (lsraeli et al. (1993) Cancer Research 53:227}; tyrosinase (U.S. Patent
Nos. x.530,096 and 4,898,814; Brichard et al. (1993} J. Exp. Med. 178:489);
tyrosinase related proteins 1 or 2 (TRP-1 and TRP-2); NY-ESO-1 (Chen et al.
(1997) Proc. Natl. Acad. Sci. U.S.A. 94:1914), or the GA733 antigen (U.S.
Patent
No. 5,185,254).
Aiso within the scope of this invention is an heterologous or altered
antigen corresponding to an epitope or wild-type antigenic peptide
corresponding
to a yet unidentified protein. A common strategy in the search for tumor
antigens
is to isolate tumor-specific T-cells and attempt to identify the antigens
recognized
by these cells. In patients with cancer, specific CTLs have been derived from
lymphocytic infiltrates present at the tumor site. Weidmann et al., supra.
These
TILs are unique cell population that can be traced back to sites of disease
when
they are labeled with indium and adoptively transferred. Alternatively, large
libraries of putative antigens can be produced and tested. Using the "phage
method" (Scott and Smith (1990) Science 249:386; Cwirla et al. (1990) Proc.
Natl. Acad. Sci. 87:6387; and Devlin et al. (1990) Science 249:404), very
large
libraries can be constructed. Another approach uses primarily chemical
methods.
of which the Geysen method (Geysen et al. ( 1986) Mol. Immunol. 23:709; and
Geysen et al. (1987) J. immunol. Method 102:259) and the method of Fodor et
al.
( I 991 ) Science 251:767, are examples. Furka et al. ( 1988) 14th Inter.
Cong. Bio.
Vol. 5, Abst. FR:OI 3; Furka ( 1991 ) Inter. J. Peptide Protein Res. 37:487),
Houghton (U.S. Patent No. 4,683,211, issued December 1986) and Ratter, et al.
(U. S. Patent No. 5,010,175, issued April 23, 1991 ) describe methods to
produce a
mixture of peptides.
In a further aspect of this invention, Solid-PHase Epitope REcovery
("SPHERE", described in PCT WO 97/35035) described below, can be used to
identify tumor antigens and altered antigens corresponding to self antigens.
After identification and cloning of an altered antigen, the antigen or
epitope can be expressed and purified for presentation to APC using the
methods
disclosed herein. In a further embodiment. the full-length native antigen can
be
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selectively modified to encode or present the altered epitope using methods
known in the art, e.g. PCR directed mutagenesis. Sambrook et al., supra.
This invention further provides methods to elicit CD4+ and CD8+ T cells
responses in a subject. The induction of this immune response also is a means
to
assay a positive response to the therapy. The presence of a large number of T-
cells in tumor has been correlated with a prognostically favorable outcome in
some cases (Whiteside and Parmiani (1994) Cancer Immunol. Immunother.
39:15). Woolley et al. (1995} Immmunology 84:55, has shown that implantation
of polyurethane sponges containing irradiated tumor cells can efficiently trap
anti-
tumor CTLs (4-times greater than lymph fluid, 50-times greater than spleen or
peripheral blood). Following activation with T-cell cytokines in the presence
of
their appropriately presented recognition antigen, TILs proliferate in culture
and
acquire potent anti-tumor cytolytic properties. Weidmann et al. ( 1994) Cancer
Immunol. Immunother. 39:1. Assays to detenmine T cell response are well known
in the art and any method that will compare T cell number and activity prior
to
and subsequent to therapy can be utilized. In addition, the induction of co-
stimulatory cytokines by the heterologous/altered antigen could also stimulate
pre-existing anergic or low affinity self reactive CTL clones.
When the method is practiced in vitro as a screen to identify antigenic
peptides and nucleic acids of the invention, induction of cytotoxic T
lymphocytes
capable of lysing host tumor cells indicates that the antigenic peptide and/or
nucleic acid of the screen is a potential therapeutic agent.
The methods of this invention can be further modified by co-administering
more than one heterologous/altered antigen and/or an effective amount of a
cytokine or co-stimulatory molecule or other transgene to the subject.
The antigen is administered to the subject either as a nucleic acid coding
for the peptide/protein or by administering APC presenting the antigen. In one
embodiment, the APC is a dendritic cell which includes, but is not limited to
a
pulsed or genetically modified dendritic cell. When the method is practiced in
vitro, the APC may be a foster antigen presenting cell. Methods of presenting
the
antigen to the APC are described herein.
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The APC can be further genetically modified to co-express a cytokine
alone, or in combination with a co-stimulatory molecule or other transgene.
The APC expressing a heterologous and/or altered antigen also can be
used to expand and isolate a population of immune effectors which. in turn,
are
useful for adoptive immunotherapy alone or as an adjuvant to the methods
described above. As above, cytokines and/or co-stimulatory molecules or
nucleic
acids encoding them, can be co-administered with the immune effector cells.
Alternatively, the immune effector cells can be genetically modified to
express a
foreign nucleic acid encoding a cytokine or co-stimulatory molecule. Prior to
administration in vivo, the immune effector cells are screened in vitro for
their
ability to lyse tumor cells.
Furthermore. the invention provides a method for cloning the cDNA and
genomic DNA encoding such protein by generating degenerate oligonucleotides
probes or primers based on the sequence of the epitope. Compositions
comprising
the nucleic acid and a carrier, such as a pharmaceutically acceptable carrier,
a
solid support or a detectable label, are further provided by this method as
well as
methods for detecting the sequences in a sample using methods such as Northern
analysis, Southern analysis and PCR.
Further provided by this invention are therapeutic and diagnostic
comprising oligopeptide sequences determined according to the foregoing
methods. Compositions comprising the oligopeptide sequence and a carrier, such
as a pharmaceutically acceptable carrier, a solid support or a detectable
label, are
further provided by this method as well as methods for detecting the
oligopeptide
sequence in a sample using methods such as Western analysis and ELISA.
Harlow and Lane ( I 989) supra.
Materials and Methods
Identification of Tumor Associated Antigens
Any conventional method, e.g., subtractive library, comparative Northern
and/or Western blot analysis of normal and tumor cells, Expression Cloning,
Serial Analysis Gene Expression "SAGE" (U.S. Patent No. 5,695,937) and Solid
CA 02322624 2000-09-06
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PHase Epitope REcovery "SPHERE" (described in PCT WO 97/35030, can be
used to identify putative antigens for use in the subject invention.
SAGE analysis can be employed to identify the antigens recognized by
expanded immune effector cells such as CTLs. SAGE analysis involves
identifying nucleotide sequences expressed in the antigen-expressing cells.
Briefly, SAGE analysis begins with providing complementary deoxyribonucleic
acid (cDNA) from ( 1 ) the antigen-expressing population and (2) cells not
expressing that antigen. Both cDNAs can be linked to primer sites. Sequence
tags are then created, for example, using the appropriate primers to amplify
the
DNA. By measuring the differences in these tags between the two cell types,
sequences which are over expressed in the antigen-expressing cell population
can
be identified.
Expression cloning methodology as described in Kawakami et al. ( 1994)
PNAS 91:3515, also can be used to identify a novel tumor-associated antigen.
Briefly, in this method, a library of cDNAs corresponding to mRNAs derived
from tumor cells is cloned into an expression vector and introduced into
target
cells which are subsequently incubated with cytotoxic T cells. One identifies
pools of cDNAs that are able to stimulate the CTL and through a process of
sequential dilution and re-testing of less complex pools of cDNAs one is able
to
derive unique cDNA sequences that are able to stimulate the CTL and thus
encode
the cognate tumor antigen.
An antigen identification method, SPHERE, is described in PCT WO
97/35035. Briefly, an empirical screening method for the identification of MHC
Class I-restricted CTL epitopes is described that utilizes peptide libraries
synthesized on a solid support (e.g., plastic beads) where each bead contains
approximately 200 picomoles of a unique peptide that can be released in a
controlled manner. The synthetic peptide library is tailored to a particular
HLA
restriction by fixing anchor residues that confer high-affinity binding to a
particular HLA allele (e.g., HLA-A2) but contain a variable TCR epitope
repertoire by randomizing the remaining positions. Roughly speaking, 50 96-
well
plates with 10,000 beads per well will accommodate a library with a complexity
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of approximately 5 X 10'. In order to minimize both the number of CTL cells
required per screen and the amount of manual manipulations, the eluted
peptides
can be further pooled to yield wells with any desired complexity. Based on
experiments with soluble libraries, it should be possible to screen 10'
peptides in
96-well plates (10,000 peptides per well) with as few as 2 X 106 CTL cells.
After
cleaving a percentage of the peptides from the beads and incubating them with
S~Cr-labeled APCs (e.g., T2 cells) and the CTL line(s), peptide pools
containing
reactive species can be determined by measuring S~Cr-release according to
standard methods known in the art. Alternatively, cytokine production (e.g.,
interferon-y) or proliferation (e.g., incorporation of 3H-thymidine) assays
may be
used. After identifying reactive 10,000-peptide mixtures, the beads
corresponding
to those mixtures are separated into smaller pools and distributed to new 96-
well
plates (e.g., 100 beads per well). An additional percentage of peptide is
released
from each pool and re-assayed for activity by one of the methods listed above.
Upon identification of reactive 100-peptide pools, the beads corresponding to
those peptide mixtures are redistributed at 1 bead per well of a new 96-well
plate.
Once again, an additional percentage of peptide is released and assayed for
reactivity in order to isolate the single beads containing the reactive
library
peptides. The sequence of the peptides on individual beads can be determined
by
sequencing residual peptide bound to the beads by, for example, N-terminal
Edman degradation or other analytical techniques known to those of skill in
the
art.
In vitro confirmation of the immunogenicity of a putative antigen of this
invention can be confirmed using the method described below which assays for
the induction of CTLs.
After isolation of the epitope or antigen, it can be expressed and purified
using methods known in the art.
Alternatively, muteins of the antigen as well as allogeneic and antigens
from a different species, of previously characterized antigens are useful in
the
subject invention. For example, MART1 and gp100 are melanocyte
differentiation antigens specifically recognized by HLA-A2 restricted tumor-
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WO 99/46992 PCTNS99/06039
infiltrating lymphocytes (TILs) derived from patients with melanoma. and
appear
to be involved in tumor regression (Kawakami Y. et al. (1994) PNAS USA
91:6458 and Kawakami Y. et al. ( 1994) PNAS USA 91:91:3515). Recently, the
mouse homologue of human MART-1 has been isolated. The full-length open
S reading frame of the mouse MART1 consists of 342 bp, encoding a protein of I
13
amino acid residues with a predicted molecular weight of ~13 kDa. Alignment of
human and murine MART1 amino acid sequences showed 68.6% identity.
The murine homologe of gp100 has also been identified. The open
reading frame consists of 1,878 bp, predicting a protein of 626 amino acid
IO residues which exhibits 75.5% identity to human gp100.
Additional antigens include, but are not limited to HER-2/neu (U.S.
Patent No. 5,550,214); MAGE (PCT/LJS92/04354); HPV 16, 18E6 and E7
(Ressing et al. (1996) Cancer Res. 56(1):582; Restifo (1996) Current Opinion
in
Immunol. 8:658; Stern (1996) Adv. Cancer Res. 69:175; Tindle et al. (1995)
Clin.
15 Exp. Immunol. 101:265; and van Driel et al. ( I 996) Annals of Medicine
28:471 );
CEA (U.S. Patent No. 5,274,087); PSA (Lundwall A. (1989) Biochem. Biophys.
Research Communications 161:1151 ); prostate membrane specific antigen
(PSMA) (Israeli et al. (1993) Cancer Research 53:227); tyrosinase (U.S. Patent
Nos. 5,530,096 and 4,898,814, and Brichard et al. (1993) J. Exp. Med.
178:489);
20 tyrosinase related proteins 1 or 2 (TRP-1 and TRP-2); NY-ESO-1 (Chen et al.
(1997) PNAS 94:1914); or the GA733 antigen (U.S. Patent No. 5,185,254).
In vitro confirmation of the immunogenicity of a putative antigen of this
invention can be confirmed using the method described below which assays for
the generation of CTLs.
Isolation, Culturing and Expansion of APCs, Including Dendritic Cells
Various methods to isolate and characterize APCs including DCs have
been known in the art. At least two methods have been used for the generation
of
human dendritic cells from hematopoietic precusor cells in peripheral blood or
bone marrow. One approach utilizes the rare CD34+ precursor cells and
stimulate
them with GM-CSF plus TNF-a. The other method makes use of the more
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abundant CD34- precursor population, such as adherent peripheral blood
monocytes, and stimulate them with GM-CSF plus IL-4 (see. for example,
Sallusto et al. ( 1994). supra).
In one aspect of the invention, the method described in Romani et al
S ( 1996), supra; and Bender et al ( 1996), supra is used to generate both
immature
and mature dendritic cells from the peripheral blood mononuclear cells (PBMC)
of a mammal, such as a murine, simian or human. Briefly, isolated PBMC are
pre-treated to deplete T- and B-cells by means of an immunomagnetic technique.
Lymphocyte-depleted PBMC are then cultured for 7 days in RPMI medium,
supplemented with 1% autologous human plasma and GM-CSF/IL-4, to generate
dendritic cells. Dendritic cells are nonadherence as opposed to their monocyte
progenitor. Thus, on day 7, non-adherent cells are harvested for further
processing.
The dendritic cells derived from PBMC in the presence of GM-CSF and
IL-4 are immature, in that they can lost the nonadherence property and revert
back
to macrophage cell fate if the cytokine stimuli are removed from the culture.
The
dendritic cells in an immature state are very effective in processing native
protein
antigens for the MHC class II restricted pathway (Romani et al. ( 1989) J.
Exp.
Med. 169:1169.
Further maturation of cultured dendritic cells is accomplished by culturing
for 3 days in a macrophage-conditioned medium (CM), which contains the
necessary maturation factors. Mature dendritic cells are less able to capture
new
proteins for presentation but are much better at stimulating resting T cells
(both
CD4+ and CD8+) to grow and differentiate.
Mature dendritic cells can be identified by their change in morphology,
such as the formation of more motile cytoplasmic processes; by their
nonadherence; by the presence of at least one of the following markers: CD83,
CD68, HLA-DR or CD86; or by the loss of Fc receptors such as CD 115
(reviewed in Steinman ( 1991 ) Annu. Rev. Immunol. 9:271.)
More specifically, the method requires collecting an enriched collection of
white cells and platelets from leukapheresis that is then further fractionated
by
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countercurrent centrifugal elutriation (CCE) (Abrahamsen, T.G. et al. ( 1991 )
J.
Clin. Apheresis. 6:48-53). Cell samples are placed in a special elutriation
rotor.
The rotor is then spun at a constant speed of, for example, 3000 rpm. Once the
rotor has reached the desired speed, pressurized air is used to control the
flow rate
of cells. Cells in the elutriator are subjected to simultaneous centrifugation
and a
washout stream of buffer that is constantly increasing in flow rate. This
results in
fractional cell separations based largely but not exclusively on differences
in cell
size.
Quality control of APC and more specifically DC collection and
confirmation of their successful activation in culture is dependent upon a
simultaneous mufti-color FACS analysis technique which monitors both
monocytes and the dendritic cell subpopulation as well as possible contaminant
T
lymphocytes. It is based upon the fact that DCs do not express the following
markers: CD3 (T cell); CD14 (monocyte); CD16, 56, 57 (NK/LAK cells); CD19,
20 (B cells). At the same time, DCs do express large quantities of HLA-DR,
significant HLA-DQ and B7.2 (but little or no B7.1 ) at the time they are
circulating in the blood (in addition they express Leu M7 and M9, myeloid
markers which are also expressed by monocytes and neutraphils).
When combined with a third color reagent for analysis of dead cells,
propridium iodide (PI), it is possible to make positive identification of all
cell
subpopulations (see Table 1 ):
TABLE 1
. " . .....,,_. FACS analysis of fresh peripheral cell subpopulations
Color # 1 Color #2 Color #3
Cocktail HLA-DR _PI
3/14/16/19/20/56/57
Live Dendritic Negative Positive Negative
cells
Live Monocytes Positive Positive Negative
Live NeutrophilsNegative Negative Negative
Dead Cells Variable Variable Positive
Additional markers can be substituted for additional analysis:
Color #1: CD3 alone, CD14 alone, etc.; Leu M7 or Leu M9; anti-Class I,
etc.
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WO 99/46992 PCTNS99/06039
Color #2: HLA-Dq, B7.1, B7.2, CD25 (IL2r), ICAM, LFA-3, etc.
The goal of FACS analysis at the time of collection is to confirm that the
DCs are enriched in the expected fractions, to monitor neutrophil
contamination,
and to make sure that appropriate markers are expressed. This rapid bulk
collection of enriched DCs from human peripheral blood, suitable for clinical
applications, is absolutely dependent on the analytic FRCS technique described
above for quality control. If need be, mature DCs can be immediately separated
from monocytes at this point by fluorescent sorting for "cocktail negative"
cells.
It may not be necessary to routinely separate DCs from monocytes because, as
will be detailed below, the monocytes themselves are still capable of
differentiating into DCs or functional DC-like cells in culture.
Once collected, the DC rich/monocyte APC fractions (usually I 50 through
190) can be pooled and cryopreserved for future use, or immediately placed in
1 S short term culture.
Alternatively, others have reported that a method for upregulating
(activating) dendritic cells and converting monocytes to an activated
dendritic cell
phenotype. This method involves the addition of calcium ionophore to the
culture
media convert monocytes into activated dendritic cells. Adding the calcium
ionophore A23187, for example, at the beginning of a 24-48 hour culture period
resulted in uniform activation and dendritic cell phenotypic conversion of the
pooled "monocyte plus DC" fractions: characteristically, the activated
population
becomes uniformly CD14 (Leu M3) negative, and upregulates HI,A-DR, HLA- ~ ~ ~~
DQ, ICAM-1, B7.1, and B7.2. Furthermore this activated bulk population
functions as well on a small numbers basis as a further purified.
Specific combinations) of cytokines have been used successfully to
amplify (or partially substitute) for the activation/conversion achieved with
calcium ionophore: these cytokines include but are not limited to G-CSF, GM-
CSF, IL-2, and IL-4. Each cytokine when given alone is inadequate for optimal
upregulation.
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In one embodiment. the APCs and cells expressing one or more antigens
are autologous. In another embodiment, the APCs and cells expressing the
antigen are allogeneic, i.e., derived from a different subject.
Presentation of Antigen by the APC
Peptide fragments from antigens must first be bound to peptide binding
receptors (major histocompatibility complex class I and class II molecules)
that
display the antigenic peptides on the surface of the APCs. Palmer E. and
Cresswell (1998) Annu. Rev. Immunol. 16:323 and Germain R.N. (1996)
Immunol. Rev. 151:5. T lymphocytes produce an antigen receptor that they use
to
monitor the surface of APCs for the presence of foreign peptides. The antigen
receptors on CD4+ T cells recognize antigenic peptides bound to MHC class II
molecules whereas the receptors on CD8+ T cells react with antigens displayed
on
class I molecules. For a general review of the methods for presentation of
exogeneous antigen by APC, see Raychaudhuri and Rock {1998) Nature
Biotechnology 16:1025.
For purposes of immunization, antigens can be delivered to antigen-
presenting cells as protein/peptide or in the form of polynucleotides encoding
the
protein/peptide ex vivo or in vivo. The methods described below focus
primarily
on DCs which are the most potent, preferred APCs.
Several different techniques have been described to produce genetically
modified APCs. These include: ( 1 ) the introduction into the APCs of
palynucleotides that express antigen or fragments thereof; (2) infection of
APCs
with recombinant vectors to induce endogenous expression of antigen; and (3)
introduction of tumor antigen into the DC cytosol using liposomes. (See,
Boczkowski D. et al. ( 1996) J. Exp. Med. 184:465; Rouse et al. ( 1994) J.
Virol.
68:5685; and Nair et al. ( 1992) J. Exp. Med. 175:609). For the purpose of
this
invention, any method which allows for the introduction and expression of the
heterologous or non-self antigen and presentation by the MHC on the surface of
the APC is within the scope of this invention.
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WO 99/46992 PCT/US99/06039
Several techniques have been described for the presentation of exogenous
protein and/or peptide by the APC. These techniques are briefly described
below.
Antigen Pulsing
Pulsing is accomplished in vitrvlex vivo by exposing APCs to antigenic
protein or peptide(s). The protein or peptides) are added to APCs at a
concentration of I-10 g,m for approximately 3 hours. Paglia et al. (1996) J.
Exp.
Med. 183:317, has shown that APC incubated with whole protein in vilrv were
recognized by MHC class I-restricted CTLs, and that immunization of animals
with these APCs led to the development of antigen-specific CTLs in vivo.
Protein/peptide antigen can also be delivered to APC in vivo and presented
by the APC. Antigen is preferably delivered with adjuvant via the intravenous,
subcutaneous. intranasal, intramuscular or intraperitoneal route of delivery.
Grant
E.P. and Rock K.L. (1992) J. Immunol. 148:13; Norbury, C. C. et al. (1995)
Immunity 3:783; and Reise-Sousa C. and Germain R.N. (1995) J. Exp. Med.
182:841.
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Antigen Painting
Another method which can be used is termed ''painting''. It has been
demonstrated that glycosyl-phosphotidylinositol (GPI)-modified proteins
possess
the ability to reincorporate themselves back into cell membranes after
purification. Hirose et al. (1995) Methods Enzymol. 250:582; Medof et al.
(1984)
J. Exp. Med. 160:1558; Medof (1996) FASEB J. 10:574; and Huang et al. (1994)
Immunity 1:607, have exploited this property in order to create APCs of
specific
composition for the presentation of antigen to CTLs. Expression vectors for
~i2-
microglobulin and the HLA-A2.1 allele were first devised. The proteins were
expressed in Schneider S2 Drosophila melanogaster cells, known to support GPI-
modification. After~purification, the proteins could be incubated together
with a
purified antigenic peptide which resulted in a trimolecular complex capable of
efficiently inserting itself into the membranes of autologous cells. In
essence,
these protein mixtures were used to "paint" the APC surface, conferring the
ability to stimulate a CTL clone that was specific for the antigenic peptide.
Cell
coating was shown to occur rapidly and to be protein concentration dependent.
This method of generating APCs bypasses the need for gene transfer into the
APC
and permits control of antigenic peptide densities at the cell surfaces.
Foster Antigen Presenting Cells
Foster APCs are derived from the human cell line 174xCEM.T2, referred
to as T2, which contains a mutation in its antigen processing pathway that
restricts
the association of endogenous peptides with cell surface MHC class I molecules
(Zweerink et al. (1993) J. Immunol. 150:1763). This is due to a large
homozygous deletion in the MHC class II region encompassing the genes TAP 1,
TAP2, LMPI, and LMP2, which are required for antigen presentation to MHC
class 1-restricted CD8+ CTLs. In effect, only "empty" MHC class I molecules
are
presented on the surface of these cells. Exogenous peptide added to the
culture
medium binds to these MHC molecules provided that the peptide contains the
allele-specific binding motif. These T2 cells are referred to herein as
"foster"
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APCs. They can be used in conjunction with this invention to present the
heterologous. altered or control antigen.
Transduction of T2 cells with specific recombinant MHC alleles allows for
redirection of the MHC restriction profile. Libraries tailored to the
recombinant
S allele will be preferentially presented by them because the anchor residues
will
prevent efficient binding to the endogenous allele.
High level expression of MHC molecules makes the APC more visible to
the CTLs. Expressing the MHC allele of interest in T2 cells using a powerful
transcriptional promoter (e.g., the CMV promoter) results in a more reactive
APC
(most likely due to a higher concentration of reactive MHC-peptide complexes
on
the cell surface).
Expansion of Immune Effector Cells
In one embodiment, the present invention makes use of these APCs to
1 S stimulate production of an enriched population of antigen-specific immune
effector cells. The antigen-specific immune effector cells are expanded at the
expense of the APCs, which die in the culture. The process by which naive
immune effector cells become educated by other cells is described essentially
in
Coulie ( 1997) Molec. Med. Today 3: 261. The substantially pure populationof
educated, antigen-specific immune effector cells produced by this method are
useful to cause tumor regression.
The APCs (e.g., DCs) presenting the heterologous/altered antigen are
mixed with naYve immune effector cells. Preferably, the cells may be cultured
in
the presence of a cytokine, for example IL2. Because dendritic cells secrete
potent immunostimulatory cytokines, such as IL-12, it may not be necessary to
add supplemental cytokines during the first and successive rounds of
expansion.
In any event, the culture conditions are such that the antigen-specific immune
effector cells expand (i. e., proliferate) at a much higher rate than the
APCs.
Multiple infusions of APCs and optional cytokines can be performed to further
expand the population of antigen-specific cells.
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In one embodiment, the immune effector cells are T cells. In a separate
embodiment, the immune effector cells can be genetically modified by
transduction with a transgene coding for example, IL-2, IL-11 or IL-13.
Methods
for introducing transgenes in vitro, ex vivo and in vivo are well known in the
art.
See Sambrook et al. ( 1989) supra.
An effector cell population suitable for use in the methods of the present
invention can be autologous or allogeneic, preferably autologous. When
effector
cells are allogeneic, preferably the cells are depleted of alloreactive cells
before
use. This can be accomplished by any known means, including. for example, by
mixing the allogeneic effector cells and a recipient cell population and
incubating
them for a suitable time, then depleting CD69+ cells, or inactivating
alloreactive
cells, or inducing anergy in the alloreactive cell population.
Hybrid immune effector cells can also be used. Immune effector cell
hybrids are known in the art and have been described in various publications.
See, for example, International Patent Application Nos. WO 9$!46785 and WO
95/16775.
The effector cell population can comprise unseparated cells, i.e., a mixed
population, for example, a PBMC population, whole blood, and the like. The
effector cell population can be manipulated by positive selection based on
expression of cell surface markers, negative selection based on expression of
cell
surface markers, stimulation with one or more antigens in vitro or in vivo,
treatment with one or more biological modifiers in vitro or in vivo,
subtractive
stimulation with one or more antigens or biological modifiers, or a
combination of
any or all of these.
Effector cells can obtained from a variety of sources, including but not
limited to, PBMC, whole blood or fractions thereof containing mixed
populations,
spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells
obtained by
leukopharesis, biopsy tissue, lymph nodes, e.g., lymph nodes draining from a
tumor. Suitable donors include an immunized donor, a non-immunized (naive)
donor, treated or untreated donors. A "treated" donor is one that has been
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exposed to one or more biological modifiers. An "untreated" donor has not been
exposed to one or more biological modifiers.
Methods of extracting and culturing effector cells are well known. For
example, effector cells can be obtained by leukopharesis, mechanical apheresis
using a continuous flow cell separator. For example, lymphocytes and monocytes
can be isolated from the buffy coat by any known method, including, but not
limited to, separation over Ficoll-HypaqueTM gradient, separation over a
Percoll
gradient, or elutriation. The concentration of Ficoll-HypaqueTM can be
adjusted to
obtain the desired population, for example, a population enriched in T cells.
Other methods based on affinity are known and can be used. These include, for
example, fluorescence-activated cell sorting (FACS), cell adhesion, magnetic
bead separation. and the like. Affinity-based methods may utilize antibodies,
or
portions thereof, which are specific for cell-surface markers and which are
available from a variety of commercial sources, including, the American Type
Culture Collection (Manassas, VA). Affinity-based methods can alternatively
utilize ligands or ligand analogs, of cell surface receptors.
The effector cell population can be subjected to one or more separation
protocols based on the expression of cell surface markers. For example, the
cells
can be subjected to positive selection on the basis of expression of one or
more
cell surface polypeptides, including, but not limited to, "cluster of
differentiation"
cell surface markers such as CD2, CD3, CD4, CDB, TCR, CD45, CD45R0,
CD45RA, CD1 lb, CD26, CD27, CD28, CD29, CD30, CD31, CD40L; other
markers associated with lymphocyte activation, such as the lymphocyte
activation
gene 3 product (LAG3), signaling lymphocyte activation molecule (SLAM),
TI/ST2; chemokine receptors such as CCR3, CCR4, CXCR3, CCRS; homing
receptors such as CD62L, CD44, CLA, CD146. a4~37, aE(37; activation markers
such as CD25, CD69 and OX40; and lipoglycans presented by CD1. The effector
cell population can be subjected to negative selection for depletion of non-T
cells
andlor particular T cell subsets. Negative selection can be performed on the
basis
of cell surface expression of a variety of molecules, including, but not
limited to,
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B cell markers such as CD19, and CD20; monocyte marker CD14; the NK cell
marker CD56.
An effector cell population can be manipulated by exposure. in vivo or in
vitro. to one or more biological modifiers. Suitable biological modifiers
include,
but are not limited to, cytokines such as IL-2, IL-4, IL-10, TNF-a. IL-12, IFN-
y;
non-specific modifiers such as phytohemagglutinin (PHA), phorbol esters such
as
phorbol myristate acetate (PMA), concanavalin-A, and ionomycin; antibodies
specific for cell surface markers, such as anti-CD2, anti-CD3, anti-IL2
receptor,
anti-CD28; chemokines, including, for example, lymphotactin. The biological
modifiers can be native factors obtained from natural sources, factors
produced by
recombinant DNA technology, chemically synthesized polypeptides or other
molecules, or any derivative having the functional activity of the native
factor. If
more than one biological modifier is used, the exposure can be simultaneous or
sequential.
The present invention provides compositions comprising immune effector
cells, which may be T cells, enriched in antigen-specific cells, specific for
a
peptide of the invention. By "enriched" is meant that a cell population is at
least
about 50-fold, more preferably at least about 500-fold, and even more
preferably
at least about 5000-fold or more, enriched from an original naive cell
population.
The proportion of the enriched cell population which comprises antigen-
specific
cells can vary substantially, from less than 10% up to 100% antigen-specific
cells.
If the cell population comprises at least 50%, preferably at least 70%, more
preferably at least 80%, and even more preferably at least 90%, antigen-
specific
immune effector cells, specific for a peptide of the invention, then the
population
is said to be "substantially pure". The percentage which are antigen-specific
can
readily be determined, for example, by a 3H-thymidine uptake assay or cytokine
release assay in which the effector cell population (for example, a T-cell
population) is challenged by an antigen-presenting matrix presenting an
antigenic
peptide of the invention.
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Assaying Antigen-Specificity
An in vitro system will be needed to test or confirm which version of the
modified or heterologous tumor antigen is most likely to be immunogenic in
humans or the test subject. In this system, DCs will be used to present
antigen to
autologous peripheral blood lymphocytes. The DCs can be pulsed or transduced.
Various culture conditions have been described that will support the
generation of
effector cells in cultures of DCs and lymphocytes. After several rounds of
stimulation, the effector cells generated are tested for their ability to
recognize
native tumor antigen. Fewer rounds of stimulation may be required for antigens
with high immunogenic potential. Both T helper (CD4+) and cytolytic effector
cells (CD8+) can be elicited. The development of TAA-specific cells can be
measured by several methods including proliferation or cytokine production
(e.g.
TNF-a, interferon-y) upon exposure to TAA or lysis of TAA-expressing target
cells as assessed by release of various intracellular labels/markers such as
5 ~ Chromium or lactose dehydrogenase (LDH). The antigen that induces the
strongest response (in particular cytolytic activity) against the native human
antigen would then be selected for immunization purposes.
In addition to previously identified and characterized antigens and
epitopes, the methods of this invention also can use newly identified antigens
which can be identified as exemplified below.
Production of Epitope or Antigen
Most preferably, heterologous/altered antigens and peptides of the present
invention can be synthesized using an appropriate solid state synthetic
procedure.
Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco,
Calif. (1968). A preferred method is the Merrifield process. Merrifield,
Recent
Progress in Hormone Res. 23:451 (1967). The antigenic activity of these
peptides
may conveniently be tested using, for example, the assays as described herein.
Once an isolated peptide of the invention is obtained, it may be purified by
standard methods including chromatography (e.g.. ion exchange, affinity, and
sizing column chromatography), centrifugation, differential solubility, or by
any
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WO 99/46992 PCTNS99/06039
other standard technique for protein purification. For immunoaffinity
chromatography. an epitope may be isolated by binding it to an affinity column
comprising antibodies that were raised against that peptide, or a related
peptide of
the invention, and were affixed to a stationary support.
S Alternatively, affinity tags such as hexa-His (Invitrogen), Maltose binding
domain (New England Biolabs), influenza coat sequence (Kolodziej et al. ( 1991
)
Methods Enzymol. 194:508), and glutathione-S-transferase can be attached to
the
peptides of the invention to allow easy purification by passage over an
appropriate
affinity column. Isolated peptides can also be physically characterized using
such
techniques as proteolysis, nuclear magnetic resonance, and x-ray
crystallography.
Also included within the scope of the invention are antigenic peptides that
are differentially modified during or after translation, e.g., by
phosphorylation,
glycosylation, crosslinking, acylation, proteoIytic cleavage, linkage to an
antibody
molecule. membrane molecule or other ligand, (Ferguson et al. ( I 988) Ann.
Rev.
Biochem.57:285).
Another aspect of the invention encompasses isolated nucleic acid
sequences that encode the novel antigenic peptides described herein. With
regard
to nucleic acid sequences of the present invention, "isolated" means: an RNA
or
DNA polymer, portion of genomic nucleic acid, cDNA, or synthetic nucleic acid
which, by virtue of its origin or manipulation: (i) is not associated with all
of a
nucleic acid with which it is associated in nature (e.g. is present in a host
cell as a
portion of an expression vector); or (ii) is linked to a nucleic acid or other
chemical moiety other than that to which it is linked in nature; or (iii) does
not
occur in nature. By "isolated" it is further meant a nucleic acid sequence:
(i)
amplified in vitro by, for example, polymerase chain reaction (PCR); (ii}
synthesized by, for example, chemical synthesis; (iii) recombinantly produced
by
cloning; or (iv) purified, as by cleavage and gel separation.
The nucleic acid sequences of the present invention may be characterized,
isolated, synthesized and purified using no more than ordinary skill. See
Sambrook et al. ( 1989) supra.
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Compositions
This invention also provides compositions containing any of the above-
mentioned proteins, muteins, polypeptides, nucleic acid molecules, vectors,
cells
antibodies and fragments thereof, and an acceptable solid or liquid carrier.
When
the compositions are used pharmaceutically, they are combined with a
"pharmaceutically acceptable carrier" for diagnostic and therapeutic use.
These
compositions also can be used for the preparation of medicaments for the
diagnosis and treatment of diseases such as cancer.
Tumor Protection in Animal Models
The murine B 16 melanoma model was used. In this model, CS'7BL/6 mice
were immunized with bone marrow-derived DCs transduced with an Ad vector
encoding either human gp 100 (Ad/hugpl 00) or mouse gp 100 (Ad/mgp100). Mice
immunized against heterologous human gp100 developed a protective immune
response and were resistant to a lethal subcutaneous challenge of B 16
melanoma
cells (syngeneic tumor cell line that expresses gp100). In contrast, mice
immunized with homologous mouse gp100 failed to mount a protective immune
response against B 16 melanoma cells and developed tumors at the site of B 16
cell
injection. This finding illustrates the difficulty in breaking tolerance
against a self
antigen (mouse gp100). The corresponding heterologous antigen from a different
species (human gp100), however, is likely to contain several Class I and Class
II-
associated epitopes that will be recognized as foreign and elicit CD8+ and
CD4+ T
cell responses, respectively. The induction of cross-reactive CTLs that
recognize
both the heterologous and homologous self antigen can then lead to lysis of
host
tumor cells.
Unfortunately, this type of animal model cannot be used to test the
efficacy of modified or heterologous tumor antigens being considered for use
in
humans since mice and humans recognize different epitopes, primarily as a
result
of differences in their MHC molecules. It may be possible, however, to use the
allogeneic human peripheral blood lymphocyte - severe combined
immunodeficiency mouse (Hu-PBL-SCID) model. SCID mice lack mature B and
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T lymphocytes and can be reconstituted with human PBLs. It may be possible to
immunize such mice with test antigen to induce a response in adoptively
transferred human PBLs and evaluate protection against challenge with a human
tumor cell line (Mosier et al. (1988) Nature 335:256; Parney et al. (1997)
Human
Gene Therapy 8:1073; and Albert et al. (1997) J. Immunol. 159:1393).
Another possibility is immunization of HLA-A2.1 transgenic mice to
reproduce the immune reactivity of HLA-A2 individuals (Wentworth et al. (1996)
Eur. J. Immunol. 26:97).
Vectors Useful in Genetic Modifications
In general, genetic modifications of cells employed in the present
invention are accomplished by introducing a vector containing a polypeptide or
transgene encoding a heterologous or an altered antigen. A variety of
different
gene transfer vectors, including viral as well as non-viral systems can be
used.
Viral vectors useful in the genetic modifications of this invention include,
but are
not limited to adenovirus, adeno-associated virus vectors, retroviral vectors
and
adeno-retroviral chimeric vectors. APC and immune effector cells can be
modified using the methods described below or by any other appropriate method
known in the art.
Construction of Recombinant Adenoviral Vectors or Adeno-Associated Virus
Vectors
Adenovirus and adeno-associated virus vectors useful in the genetic
modifications of this invention may be produced according to methods already
taught in the art. (see, e.g., Karlsson et al. (1986) EMBO 5:2377; Carter
(1992)
Current Opinion in Biotechnology 3:533; Muzcyzka (1992) Current Top.
Microbiol. lmmunol. 158:97; and GENE TARGETING: A PRACTICAL APPROACH
( 1992) ed. A. L. Joyner, Oxford University Press, NY). Several different
approaches are feasible. Preferred is the helper-independent replication
deficient
human adenovirus system.
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The recombinant adenoviral vectors based on the human adenovirus 5
(Virology 163:614, 1988) are missing essential early genes from the adenoviral
genome (usually E 1 A/E 1 B). and are therefore unable to replicate unless
grown in
permissive cell lines that provide the missing gene products in traps. In
place of
the missing adenoviral genomic sequences, a transgene of interest can be
cloned
and expressed in cells infected with the replication deficient adenovirus.
Although adenovirus-based gene transfer does not result in integration of the
transgene into the host genome (less than 0.1 % adenovirus-mediated
transfections
result in transgene incorporation into host DNA), and therefore is not stable,
adenoviral vectors can be propagated in high titer and transfect non-
replicating
cells. Human 293 cells, which are human embryonic kidney cells transformed
with adenovirus E 1 A/E 1 B genes, typify useful permissive cell lines.
However,
other cell lines which allow replication-deficient adenoviral vectors to
propagate
therein can be used, including HeLa cells.
Additional references describing adenovirus vectors and other viral vectors
which could be used in the methods of the present invention include the
following: Horwitz, M.S., Adenoviridae and Their Replication, in Fields, B.,
et al.
(eds.) vIROLOGY, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham F.
et al., pp. 109-128 in METHODS IN MOLECULAR BIOLOGY, Vol. 7: GENE TRANSFER
AND EXPRESSION PROTOCOLS, Murray E. (ed.) Humana Press, Clifton, N.J. (1991 );
Miller N. et al. (1995) FASEB Journal 9:190; Schreier H (1994) Pharmaceutica
Acta
Helvetiae 68:145; Schneider and French (1993) Circulation 88:1937; Curiel D.T.
et al. (1992) Human Gene Therapy 3:147; Graham F.L. et al., WO 95/00655; Falck-
Pedersen WO 95/16772; Denefle P. et al. WO 95/23867; Haddada H. et al. WO
94/26914; Perncaudet M. et al. WO 95/02697; and Zhang W. et al. WO 95/25071.
A variety of adenovirus plasmids are also available from commercial sources,
including, e.g., Microbix Biosystems of Toronto, Ontario (see, e.g., Microbix
Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996).
See also, the papers by Vile et al. ( I 997) Nature Biotechnology 15:840; and
Feng
et al. (1997) Nature Biotechnology, 15:866, describing the construction and
use of
adeno-retroviral chimeric vectors that can be employed for genetic
modifications.
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Additional references describing AAV vectors which could be used in the
methods of the present invention include the following: Kotin R. ( 1994) Human
Gene Therapy 5:793; Flotte T.R. et al. (1995) Gene Therapy 2:357; Allen J.M.
WO 96/17947; and Du et al. (1996) Gene Therapy 3:254.
APCs can be transduced with viral vectors encoding a relevant antigen.
The most common viral vectors include recombinant poxviruses such as vaccinia
and fowlpox virus (Bronte et al. (1997) PNAS 94:3183; and Kim et al. (1997) J.
Immunother. 20:276} and, preferentially, adenovirus (Arthur et al. ( 1997) J.
Immunol. 159:1393; Wan et al. (1997) Human Gene Therapy 5:1355; and Huang
et al. (1995) J. Virol. 69:2257). Retrovirus also may be used for transduction
of
human APCs (Mann et ai. (1996) J. Virol. 70:2957}.
In vitro%x vivo, exposure of human DCs to adenovirus (Ad) vector at a
multiplicity of infection (MOI) of 500 for 16-24 hours in a minimal volume of
serum-free medium reliably gives rise to transgene expression in 90-100% of
DCs. The efficiency of transduction of DCs or other APCs can be assessed by
immunofluorescence using fluorescent antibodies specific for the.tumor antigen
being expressed (Kim et al. (1997) J. Immunother. 20:276). Alternatively, the
antibodies can be conjugated to an enzyme (e.g. HRP) giving rise to a colored
product upon reaction with the substrate. The actual amount of antigen being
expressed by the APCs can be evaluated by ELISA.
Transduced APCs can subsequently be administered to the host via an
intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route
of
delivery.
In vivo transduction of DCs, or other APCs, can be accomplished by
administration of Ad (or other viral vectors) via different routes including
intravenous, intramuscular, intranasal, intraperitoneal or cutaneous delivery.
The
preferred method is cutaneous delivery of Ad vector at multiple sites using a
total
dose of approximately 1x10'°-lx 10'2 i.u. Levels of in vivo
transduction can be
roughly assessed by co-staining with antibodies directed against APC markers)
and the antigen being expressed. The staining procedure can be carried out on
biopsy samples from the site of administration or on cells from draining lymph
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nodes or other organs where APCs (in particular DCs) may have migrated
(Condon et al. ( 1996) Nature Med. 2:1122; Wan et al. ( 1997) Human Gene
Therapy 8:1355). The amount of antigen being expressed at the site of
injection
or in other organs where transduced APCs may have migrated can be evaluated by
S ELISA on tissue homogenates.
Although viral gene delivery is more efficient, DCs can also be transduced
in vitro%x vivo by non-viral gene delivery methods such as electroporation,
calcium phosphate precipitation or cationic lipid/plasmid DNA complexes
(Arthur
et al. ( 1997) Cancer Gene Therapy 4:17). Transduced APCs can subsequently be
administered to the host via an intravenous, subcutaneous, intranasal,
intramuscular or intraperitoneal route of delivery.
In vivo transduction of DCs, or other APCs, can potentially be
accomplished by administration of cationic lipid/plasmid DNA complexes
delivered via the intravenous, intramuscular, intranasal, intraperitoneal or
cutaneous route of administration. Gene gun delivery or injection of naked
plasmid DNA into the skin also leads to transduction of DCs (Condon et al.
( 1996) Nature Med. 2:1122; and Raz et al. ( 1994) PNAS 91:9519).
Intramuscular
delivery of plasmid DNA may also be used for immunization (Rosato et al.
(1997)
Human Gene Therapy 8:1451.
The transduction efficiency and levels of transgene expression can be
assessed as described herein.
Administration Methods
Dendritic cells derived from peripheral blood of a subject such as a human
patient are transduced with adenovirus vector encoding the tumor antigen using
a
multiplicity of infection of 200-500. Approximately 24 hours after infection,
the
transfected dendritic cells ( 10 x 10' cells) are administered to the patient
iv or
subcutaneously. The process is repeated 3-4 weeks later with up to 6
administrations of dendritic cells. Since it is possible to freeze dendritic
cells and
administer thawed cells, the subject does not have to be leukopharesed each
time.
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The agents identified herein as effective for their intended purpose can be
administered to subjects having tumors or to individuals susceptible to or at
risk
of developing a tumor by inducing an immune response against the tumor. When
the agent is administered to a subject such as a mouse, a rat or a human
patient,
the agent can be added to a pharmaceutically acceptable carrier and
systemically
or topically administered to the subject. To determine patients that can be
beneficially treated, a tumor regression can be assayed. Therapeutic amounts
can
be empirically determined and will vary with the pathology being treated, the
subject being treated and the efficacy and toxicity of the therapy.
Administration i» vivo can be effected in one dose, continuously or
intermittently throughout the course of treatment. Methods of determining the
most effective means and dosage of administration are well known to those of
skill in the art and will vary with the composition used for therapy, the
purpose of
the therapy, the target cell being treated, and the subject being treated.
Single or
multiple administrations can be carried out with the dose level and pattern
being
selected by the treating physician. Suitable dosage formulations and methods
of
administering the agents can be found below.
The agents and compositions of the present invention can be used in the
manufacture of medicaments and for the treatment of humans and other animals
by administration in accordance with conventional procedures, such as an
active
ingredient in pharmaceutical compositions.
More particularly, an agent of the present invention also referred to herein
as the active ingredient, may be administered for therapy by any suitable
route
including nasal, topical (including transdermal, aerosol, buccal and
sublingual),
parental (including subcutaneous, intramuscular, intravenous and intradermal)
and
pulmonary. It will also be appreciated that the preferred route will vary with
the
condition and age of the recipient, and the disease being treated.
Adoptive Immunotherapy and Vaccines
The expanded populations of antigen-specific immune effector cells of the
present invention also find use in adoptive immunotherapy regimes and as
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vaccines. Thus, tumors expressing the antigen can be eradicated using the
methods and compositions described herein.
Adoptive immunotherapy methods involve, in one aspect, administering to
a subject a substantially pure population of educated, antigen-specific immune
effector cells made by culturing naive immune effector cells with APCs as
described above. Preferably. the APCs are dendritic cells. In one embodiment,
the adoptive immunotherapy methods described herein are autologous. In this
case, the APCs are made using cells isolated from a single subject. The
expanded
population also employs T cells isolated from that subject. Finally, the
expanded
population of antigen-specific cells is administered to the same patient.
In a further embodiment, APCs or immune effector cells are administered
with an effective amount of a stimulatory cytokine, such as IL-2 or a co-
stimulatory molecule.
The following examples are intended to illustrate, but not limit the
invention.
Experimental Examples
Animals and cell lines
Female C57BL/6 mice were purchased from Taconic (Germantown, NY)
and were used at 8-12 weeks of age. Syngeneic SV40-transformed fibroblasts
(SVB6KHA) have been described elsewhere (Gooding L.R. (1979) J. Immunol.
122:1002) and were a gift from Dr. Linda Gooding (Emory University, Atlanta,
GA). The B16.F10 melanoma cell line syngeneic to C57BL/6 mice was obtained
from the National Cancer Institute (Bethesda, MD). For injection, B 16.F 10
cells
(1.5-2x104 cells) were resuspended in phosphate-buffered saline (PBS) and
delivered to the abdomen subcutaneously (s.c.) in a 100 pl volume. Tumor size
was measured with electronic digital calipers 3 times per week starting around
day
10. Tumors >3 mm2 in size were scored as positive.
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Adenoviral vectors
All recombinant adenovirus (Ad) vectors used were derived from Ad
serotype 2 from which the EI region was deleted and replaced with an
expression
cassette containing a cytomegalovirus (CMV) promoter driving expression of the
transgene. The vector encoding ~i-Galactosidase (Ad2/~iGal-4) and human gp100
(Ad2/hugp100v1) contained intact E3 and E4 regions (Armentano D. (1997) J.
Virol. 71:2408 and Zhai Y. (1996) J. Immunol. 156:7001. The vector encoding
marine gp 100 (Ad2/mgp 100) or vector lacking a transgene (Ad2/empty vector),
possessed an intact E3 region with an E4 region modified by removal of all
open
reading frames and replacement with the E4 open reading frame 6 and protein IX
moved from its original location (Armentano D. (1985) Human Gene Therapy
6:1343). Finally, the Ad vector encoding marine tyrosinase-related protein 2
(Ad2/mTRP-2) contained an intact E4 region but was deleted for E3. The E2
region was left intact in all vectors.
Adenoviral particles were gradient-purified as previously described
(Armentano D. (1985) Human Gene Therapy 6:1343) and titers were determined
by end-point dilution on 293 cells using fluorescent isothiocyanate (FITC)-
conjugated anti-hexon antibody (Rich D.P. (1993) Human Gene Therapy 4:461).
Preparation o bone marrow-derived dendritic cells
Dendritic cells (DCs) were prepared from bone marrow essentially as
' described by Inaba et al. (Inaba K. (1992) J. Exp. Med. 176:1693). Briefly,
bone
marrow was flushed from the tibias and femurs of C57BL/6 mice and depleted of
erythrocyte with commercial lysis buffer (Sigma, St. Louis, MO). Bone marrow
cells were then treated with a cocktail of antibodies (Pharmingen, San Diego,
CA)
directed against CD8 (clone 53-6.7), CD4 (clone GK1.5), CD45RJB220 (clone
RA3-6B2), Ly-6G/Gr-1 (clone RB6-8C5) and Ia (clone KH74) followed by rabbit
complement (Accurate Chemical and Scientific Corporation, Westbury, N.Y.) to
deplete lymphocytes, granulocytes and Ia+ cells. The remaining cells were
cultured for 6 days in 6-well plates in RPMI-1640 medium (Gibco, Grand Island,
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NY) supplemented with 100 Ulml penicillin. 100 pg/ml streptomycin, 2 mM
glutamine, 10% fetal calf serum (FCS) and 100 ng/ml recombinant mouse GM-
CSF (Genzyme, Cambridge, MA). Loosely adherent DCs were then collected,
replated in 100 mm dishes and cultwed in the same medium for another 24 hours
S after removal of contaminating non-adherent cells. This final DC population
was
then collected for FACS analysis and transduction with Ad vector.
For analysis of surface markers, DCs were first incubated with unlabeled
antibodies (Pharmingen) specific for the major histocompatibility complex
(MHC) Class I (clone AF6-88.5) and Class II (clone AF6-120.1 ) molecules, the
co-stimulatory molecules B7.1 (CD80; clone IG 10) and B7.2 (CD86; clone GL-
1), the adhesion molecule ICAM-1 (CD54; clone 3E2}, the integrin CD 11 c
(clone 3E2) and the myeloid swface marker CD13 (clone R3-242). The cells
were then counterstained with FITC-conjugated antibodies specific for the
primary antibody. FACS analysis of the stained cells was performed on an EPICS
Profile Analyzer from Coulter.
Transduction of DCs with Ad vector was conducted in 6-well plates with
4x106 DCs/well in a 3 ml volume of RPMI-1640 medium containing 10% FCS
and 100 ng/ml GM-CSF. Virus was added to the wells at a multiplicity of
infection (MOI) of 500 and the DCs were collected after 18-24 hows of
incubation. For injection, transduced DCs were washed and resuspended in a 100
~tl volume of PBS and delivered either s.c. to the abdomen or intravenously
{i.v.)
into the tail vein as specified in the text.
Cytotoxic T cell assay
To evaluate levels of cytotoxic T lymphocyte (CTL) activity, spleen cells
from mice in the same treatment group (3 mice/group) were pooled and
stimulated
in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2 vector at an
MOI of 100 for 24 hours. Cells were cultwed in 24-well plates containing 5x106
spleen cells and 0.8-1.5x105 stimulator fibroblasts per well in a 2 ml volume.
Cytolytic activity was assayed after 6 days of incubation. Target cells
consisted
of B 16 melanoma cells and fibroblasts untransduced or transduced with virus
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an MOI of 100 for 48 hours. Targets were treated with 100 U/ml recombinant
mouse y-interferon (Genzyme) for 24 hours labeled with S~Chromium (S1-Cr;
New England Nuclear) overnight (30 p.Ci/105 cells) and plated in round bottom
96
well plates at 5x103 cells/well. Effector cells were added at various
effectoraarget
(E:T) cell ratios in triplicate. The total reaction volume was kept constant
at 200
pl/well. After 5 hours of incubation of effector and target cells at
37°C/S% C02,
26 pl of cell-free supernatant was collected from each well and counted in a
MicroBeta Trilux Liquid Scintillation Counter (Wallac Inc., Gaithersburg, MD).
The amount of S~Cr spontaneously released was obtained by incubating target
cells in medium alone and the total amount of S~Cr incorporated was determined
by adding 1 % Triton X-100 in distilled water. The percentage lysis was
calculated as follows:
Lysis = (Sample cpm) - (Spontaneous cpm) x 100
(Total cpm) - (Spontaneous cpm)
ELISPOT assay
The frequency of splenic T lymphocytes reactive with gp 100 was
evaluated in an ELISPOT assay. Spleen cells from mice immunized with
Ad2/hugp100- or Ad2/empty vector-transduced DCs (4 mice/group) were pool
and stimulated with H-26-restricted CTL epitopes derived from human gp100
(KVPRNQDWL), marine gp100 (EGSRNQDWL) or ovalbumin as a negative
control (SIINFEKL). (For human and marine gp100 peptides, see Overwijk et al.
1998} 3: Exp: :Med: :188: 277-286; for ovalbumin peptide, see Brossart et al.
(1997) J. Immunol. 158: 3270-3276.)
After 4 hours of stimulation with peptide, the spleen cells were transferred
to 96-well nitrocellulose filter plates coated with y-interferon-specific
antibodies.
After 40 hours of incubation, the cells were removed by washing and
biotinylated
antibodies against y-interferon were added to the wells. The subsequent
addition
of streptavidin-alkaline phosphatase gave rise to dark spots corresponding to
y-
interferon-producing cells.
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Experimental Results
Characterization of bone-n:arrow derived dendritic cells and transduction by
adenovirus vectors
Dendritic cells (DCs) derived from mouse bone marrow exhibited the
veiled dendrite morphology typical of DCs and displayed a characteristic set
of
DC surface markers (Crowley M. (1989) Cell. Immunol. 118:108) as determined
by FACS analysis (Table 2). The cells expressed high levels of the major
histocompatibility (MHC) Class I and Class II molecules, the co-stimulatory
molecules B7.1 and B7.2. the ICAM-I adhesion molecule, the integrin CD1 Ic and
the CD13 myeloid surface marker. Transduction of DCs with recombinant Ad2-
based vectors was achieved reproducibly with an efficiency of 90% or greater.
Transduction did not affect the distribution of DC surface markers
significantly
except for a reproducible increase in levels of MHC Class I molecules (Table
2).
Table 2. FACS analysis of dendritic cell surface markers
DC sample B7.1 B7.2 MHC MHC ICA CD CD
1 13
I II MI Ic
Untransduced80 77 41 70 96 81 80
Transduced 84 83 85 79 94 71 74
Results shown are the percentage of bone marrow-derived DCs ,staining positive
, . , . ,
20- for each marker..
DCs were untransduced or transduced with Ad2/~iGal-4.
Induction of tumor-specirc cytotoxic T lymphocyte response by transduced
dendritic cells
The ability of DCs to induce a cytotoxic T lymphocyte (CTL) response
against a melanoma-associated antigen (MAA) was evaluated in vivo. DCs were
transduced with an Ad vector encoding human gp 100 (Ad2/hugp 1 OOvI), a
differentiation antigen that is expressed by most melanomas but is also
present in
normal melanocytes and pigmented cells of the retina. Ad2/hugp100v1-
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transduced DCs (5x105) were administered intravenously (i.v.) to C57BL/6 mice
and, 1 ~ days later, spleens were collected for assessment of CTL activity.
Separate groups of mice were also treated with vehicle as a negative control
or
with the Ad2/hugp100v1 vector itself for comparison. The vector was delivered
under conditions previously determined to be optimal for immunization (3 x 109
i.u, intradermally).
After in vitro re-stimulation with syngeneic fibroblasts transduced with
Ad2/hugp 1 OOvI, effector splenocytes were tested for cytolytic activity
against
'~Cr-labeled target fibroblasts that were either untransduced or transduced
with
Ad2/hugpl00v1 or wild-type (WT) E3-deleted Ad (Ad2~2.9). The CTLs were
also tested against B16 tumor cells, a cell line originally derived from a
spontaneously arising melanoma in C57BL/6 mice which expresses the murine
equivalent of human gp100.
As expected, mice treated with vehicle failed to develop any significant
1 S CTL activity against any of the targets (Figure 2A). Mice immunized with
transduced DCs developed high levels of CTL activity against target
fibroblasts
infected with the Ad2/hugp100v1 vector. Interestingly, the bulk of the CTL
response appeared to be directed against the hugp100 transgene product rather
than adenoviral proteins) since there was very little lysis of fibroblasts
infected
with WT Ad (Figure 2B).
Mice immunized i.d. with the Ad2/hugp100v1 vector itself, developed
robust but comparatively lower levels of CTL activity against Ad2/hugp 1 OOv 1-
transduced fibroblasts. Furthermore, in contrast to the response obtained with
transduced DCs, a significant proportion of the CTL response appeared to be
specific for Ad antigen as indicated by the greater level of lysis of
fibroblasts
infected with WT Ad (Figure 2C). Importantly, CTLs from mice immunized with
transduced DCs and, to a lesser extent, with Ad vector, were both able to lyse
B 16
tumor cells indicating that the CTLs raised against human gp100 also
recognized
the endogenous mouse gp100 expressed by the tumor cells (Figures 2B and 2C).
a~
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Immunization with Ad Lector-Transduced DCs Induces Anti-Tumor Protection
Groups of 5 C57BL/6 mice were immunized against the gp100 melanoma
antigen with an intravenous injection of 5 x 10i bone marrow-derived dendritic
cells (DCs) transfected with adenovirus vector encoding mouse gp100 (Ad2/mgp
100 DCs) or human gp100 (Ad2/hugp100 DCs). Uninfected DCs served as a
negative control. Two weeks after immunization, the mice were challenged with
a subcutaneous injection of 2 x 104 B 16 melanoma cells and tumor growth was
monitored over time. The results which are shown in Figures I A through 1 D
and
3 indicate that immunization of the mice with the heterologous human gp100
antigen was more effective than immunization with the homologous mouse gp100
antigen in inducing protective immunity against B16 melanoma cells.
Cross-reactivity between human and marine gp100 CTL epitopes
ELISPOT analysis on spleen cells from mice immunized with
Ad2/hugp100-transduced DCs confirmed the presence of splenic T lymphocytes
specific for a dominant CTL epitope from human gp100 (Figure 4). Importantly,
the CTLs, which were raised against hugp100, showed cross-reactivity against
the
corresponding marine gp100 epitope. This finding is in agreement with the
observation of cross-reactivity at the CTL level (Figure 2) and the resistance
of
human gp100-immunized mice to challenge with B16 melanoma cells positive for
marine gp 100 (Figures 1 A through 1 D and 3).
As expected, spleen cells from mice immunized with Ad2/hugp100-
transduced DCs did not display any significant reactivity against a known CTL
epitope from ovalbumin and spleen cells from mice that received DCs transduced
with Ad2/empty vector did not show any significant reactivity against any of
the
peptides (Figure 4).
It is to be understood that while the invention has been described in
conjunction with the above embodiments, that the foregoing description and the
following examples are intended to illustrate and not limit the scope of the
invention. For example, any of the above-noted compositions andlor methods can
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be combined with known therapies or compositions. Other aspects, advantages
and modifications within the scope of the invention will be apparent to those
skilled in the art to which the invention pertains.
49
