Note: Descriptions are shown in the official language in which they were submitted.
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EPITOPE CONSTRUCTS COMPRISING ANTIGEN PRESENTING CELL
TARGETING MECHANISMS
FIELD OF THE INVENTION
The present invention relates to products and methods to elicit a targeted
immune response. In pauticular, the invention relates to constructs that carry
antigen
presenting cell - targeted epitope sequences.
BACKGROUND OF THE INVENTION
Cancer remains the second leading cause of death in the United States.
Non-surgical therapy for breast, lung, and colon, as well as many other solid
tumors, is
presently poor.
Tumor-Associated Antigens
Tumor-associated antigens (TAAs) are important biochemical marlcers of
tumor cells and include, for example, mutated cellular proteins such as
mutated tumor
suppressor gene products, oncogene products (including fusion proteins), and
foreign
proteins such as viral gene products. Non-mutated cellular proteins may also
be TAAs if
they are expressed aberrantly (e.g., in an inappropriate subcellular
compartment) or in
supernormal quantities. Given the numerous steps of cellular transformation
and
sometimes bizarre genotypes observed in cancer cells, it could be argued that
tumor cells
are likely to contain many new antigens potentially recognizable by the immune
system.
It also has been reported that different tumors of related tissue or cellular
origin may
share the same TAA (Sahasrabudhe et al., 1993, J. hlmnunol., 151:6302-6310;
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Shamamian et al., 1994, Cancer T_m_m__unol. Tm_m__unother., 39:73-83; Cox et
al., 1994,
Science 264:716; Peoples et al., 1993, J. T_m_m__unol., 151:5481-5491; Jerome
et al., 1991,
Cancer Res., 51:2908-2916; Moriol~e et al., 1994, J. Immunol., 153:5650-5658).
These
data support the possibility that specific anti-tumor immunotherapies
targeting TAAs,
such as vaccines, may be active against more than one form of cancer.
Immunotherapy of Cancer
In cancer, tumor-specific T cells that are capable of binding to and lysing
tumor cells displaying the corresponding tumor-associated epitopes or antigens
on their
cell surfaces can be derived from patients. Tumor-specific T cells are
localized at several
sites within cancer patients, including in the blood (where they can be found
in the
peripheral and mononuclear cell fractions), in primary and secondary lymphoid
tissue,
i.e., the spleen, in ascites fluid in ovarian cancer patients (tumor-
associated lymphocytes
or "TALS"), or within the tumor itself (tumor infiltrating lymphocytes or
"TILS"). Of
these T cell populations, TILs have been the most useful in the identification
of tumor
antigens and epitopes thereof.
The specificity of tumor-specific T cells is based on the ability of the T
cell receptor (TCR) to recognize and bind to a short amino acid sequence that
is
presented on the surface of the tumor cells by MHC class I and, in some cell
types, class
II molecules. In brief, these amino acid binding sequences (also termed "TAA
ligands"
or "TAA epitopes") are derived from the proteolytic degradation of
intracellular proteins
encoded by genes that are either uniquely or aberrantly expressed in tumor
cells.
TAAs presented in the context of major histocompatability antigen (MHC)
class I complexes on either the tumor cell itself or on antigen-presenting
cells (APCs) are
capable of inducing tumor-specific cytotoxic T lymphocytes (CTLs). The
presence of
co-stimulatory molecules, such as B7-1 and B7-2, on APCs and the secretion of
IL-2
promote the differentiation of recruited CD8+ lymphocytes into CTLs.
W deed, there is substantial evidence indicating that the immune system
plays a critical role in the prevention of cancer and the control of tumor
growth. This
includes the occasional observation of spontaneous tumor regression, the
correlation of
spontaneous regressions with the presence of tumor-infiltrating lymphocytes
(TILs) and
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the identification of TILs that are specific for TAAs. However, as evidenced
by the
incidence rates of cancer, the immune response is often not sufficient to
successfully
combat the tumor.
The growth and metastatic spread of tumors, to a large extent, depend on
their capacity to evade host immune surveillance and to overcome host
defenses. All
tumors express antigens that are recognized to a variable extent by the irmmme
system,
but in many cases an inadequate immune response is elicited because of partial
antigen
masking or ineffective activation of effector cells. Tumor escape from immune
effectors
is most often caused by weak irnmunogenicity of TAAs, antigen masking, or
overall
immunosuppression, a characteristic of advanced cancer. Failure of antigen
processing
or binding to MHC molecules, inadequate or low-affinity binding of MHC
complexes to
T-cell receptors, or inadequate expression of co-stimulatory adhesion
molecules in
conjunction with the antigen-presenting MHC complex may all lead to poor
immunogenicity of TAAs and impaired antitumor response.
In recent years, there has been a renewed interest in the development of
cancer vaccines. This has resulted, in part, from the identification of new
TAAs and an
increased understanding of the importance of antigen presentation and
hnnphocyte
activation. hn~nunotherapeutic strategies are now accepted as superior in
terms of the
specificity that they offer in targeting only tumor cells as opposed to the
existent
chemotherapy or radiation therapy that is more general and invasive with many
associated side effects.
Immunotherapeutic strategies have been developed that attempt to
"modulate" various aspects of the innnune response associated with cancer.
Vaccination
with immunogenic peptides, achninistration of ifa vitro expanded and activated
immune
effector cells, irc vivo effector cell expansion with cytokine therapies, or
genetic
modification of either immune effectors or tumor cells with cytolcine genes or
genes
encoding co-stimulatory molecules were shown to activate anti-tumor immune
responses.
The literature reveals that neither adoptive transfer of tumor-specific CTLs
nor specific active immunotherapy with whole tumor cells or cell-derived
preparations
leads to eradication of tumors or long-term survival in more than a minority
of patients.
In contrast, it has been demonstrated in vitro that peptides have succeeded in
priming T
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cells where cell-derived preparations have failed. It has been therefore
suggested that
epitopes recognized by multiple CTL lines would be promising candidates for
use in
peptide-based anti-tumor vaccines.
Importantly, however, while administration of synthetic peptides derived
from TAAs elicited tumor-specific immune responses iyZ vitro, attempts to
provoke anti-
tumor responses in vivo by vaccination with TAA protein or peptide fragments
have often
been unsuccessful, presumably because these protein or peptide fragments
failed to
access the cytosol of a cell and, therefore were not properly processed and
presented to
effector cells.
Taken together with the fact that TAA peptides generally bind weakly to
the MHC molecules, the data described above suggest that improving MHC binding
affinity and intracellular targeting of TAA peptide immunogens should improve
their
immunogenicity.
Several new prospects for preparation of T cell vaccines have been
suggested based on the identification and characterization of MHC-associated
peptides.
Synthetic peptides that correspond to these T cell epitopes may represent
ideal subunits
for safe vaccines. However, synthetic peptides are poor immunogens because of
their
small molecular size and very shoat sermn half life. To circumvent these
disadvantages,
a variety of recombinant proteins that carry short immunagenic epitopes have
been
described (Leclerc et al., Int. Rev. Tmmunol. 11: 123-132 (1994); Freimuth and
Steinman,
Res. Microbiol. 141: 995-1001 (1990); Evans et al., Nature 339: 385-388
(1989); Bona et
al., Chem. Tm_m__unol. 65: 179-206 (1997); Baier et al., J. Virol. 69: 2357-
2365 (1995)).
For example, genetically engineered antibody molecules can function as such
delivery
systems for T cell peptides and have the advantage of being self proteins
devoid of the
side effects sometimes associated with microbial vaccines and, moreover, have
a long
half life compared with synthetic peptides (Billetta et al., Proc. Natl. Acad.
Sci. USA 88:
4713-4717 (1991); Zaghouani et al., Science 259: 224-227 (1993); Rasmussen et
al.,
Proc. Natl. Acad. Sci. USA 2001 98:10296-301).
TAA Identification and Modification
The development of peptide-based anti-tumor immunotherapies is further
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hindered by the absence of a reliable iterative method to identify TAAs.
Current
protocols involve isolating and assaying extremely pure MHC molecules from
antigen
presenting cells (APCs) (Chicz and Urban, 1994, Immunol. Today, 15:155-160) or
the
determination of structural features of the MHC/peptide complexes using X-ray
crystallography (Meng and Butterfield, Pharm Res 19:926-32 (2002)).
Conventional
methods for culturing and subchoning of tumor-specific T cells are known in
the art.
Once a potent anti-tumor T cell population is recovered, it can be used to
identify tumor
antigens via conventional, but often tedious, expression cloning methodology
(I~awakami
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3515-3519), or assaying epitopes
produced in
phage libraries (Scott and Smith, 1990, Science 249:386-390; Cwirla et al.,
1990, Proc.
Natl. Acad. Sci., 87:6378-6382; Devlin et al., 1990, Science, 249:404-406).
In a different approach, requiring a known pathogen- or tumor-related
antigen, methods that attempt to identify the native epitope have been
developed. For
example, putative epitopes can be predicted using a computer to scan the
sequence of the
gene (antigen) for amino acid sequences that contain a "motif' or a defined
pattern of
amino acid residues associated with a particular MHC (HLA) allele. See, e.g.,
Englehard
(1994) Annu. Rev. Immunol. 12:181; Rammenesee et al. (1993) Annu. Rev.
Immunoh.
11:213. The "predicted" epitope sequences can then be synthesized and tested.
Although
many epitope sequences have been "predicted" from scamling full-length protein
sequences by "motif', upon testing in standard functional assays, the vast
majority of
these "predicted" epitopes failed to be imrnunogenic. Other techniques
include, for
example, peptide elution followed by database searching (Hunt et al. (1992)
Science
255:1261; Udahca et czl. (1992) Cell 69:989); isolation and identification of
the antigen
from complex antigen mixtures (Van de Wal et al. (1998) Proc. Nath. Acad. Sci.
USA
95:10050; Lamb et al. (1987) Immunology 60:1); screening expression libraries
and
subsequent database searches (Boon et al. (1994) Annu. Rev. Immunol. 12:337;
Neophytou et al. (1996) Proc. Natl. Acad. Sci. USA 93:2014; Gavin et al.
(1994) Eur. J.
Immunol. 24:2124); peptide positional scanning of combinatorial libraries
(Gundhach et
al. (1996) J. Immunol. Meth. 192:149; Bhake et al. (1996) J. Exp. Med.
184:121;
Hiemstra et al. (1997) Proc. Natl. Acad. Sci. USA 94:10313; Hemmer et al.
(1997) J.
Exp. Med. 185:165 1), and the hihce.
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More recently, certain pathogen- and tumor-related proteins have been
immunologically mimicked with synthetic peptides whose amino acid sequence
corresponds to that of an antigenic determinant domain of the pathogen- or
TAA.
Despite these advances, peptide immunogens based on native sequences generally
perform less than optimally with respect to inducing an immune response. Thus,
a need
exists for modified synthetic antigenic peptide epitopes with enhanced
immunomodulatory properties.
Combinatorial peptide and non-peptide chemistry methodologies have
provided additional tools for determining T cell epitopes. Epitopes so
determined
typically "mimic" the native epitope in that they bear a definable sequence
similarity
thereto (e.g., conservative substitutions as well as identical amino acids),
but not
necessarily absolute identity therewith. Epitope mimics can be designed by
directly
modifying the sequence of lmown epitopes or defined de novo with randomized
molecular libraries followed by database searching to identify the native
antigen. (Gavin
et al. (1994) Eur. J. hnmunol. 24:2124; Blake et al. (1996) J. Exp. Med.
184:121; Chen et
al. (1996) J. T_m_m__unol. 157:3783; Strausbauch et al. (1998) W tl. Immunol.
10:421;
Vahnori et al. (2000) J. Immunol 164(2):1125-1131; Needels et al., 1993, Proc.
Natl.
Acad. Sci. USA 90:10700-4; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA
90:10922-10926; Van der fee, 1989, Eur. J. hnmunol., 19:43-47; International
Patent
Publication No. WO 92/00252).
Thus, there is a need in the art to identify effective methods to target
tumor cells for immune clearance by generating an effective anti-tumor immune
response. In particular, there is a need in the art to develop new peptide-
based
immunogens which would induce a strong targeted anti-tumor immune response. As
outlined above, in connection with the need for new improved peptide-based
immunogens, there is also a need to develop new modified synthetic antigenic
peptides
with enhanced immunomodulatory and targeting properties over peptides derived
from
native tumor-associated antigens (TAAs).
The present invention addresses these and other needs in the art with the
discovery of an effective therapy based on the identification of non-native
epitopes
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derived from cancer antigens, and their assembly into epitope string
constructs, in
conjunction with targeted delivery of these epitope string constructs to
appropuate
antigen presenting cells (APCs), as well as the inclusion of sequences in the
constructs to
direct appropriate processing and presentation of the incorporated epitopes
within the
APCs.
SUMMARY OF THE INVENTION
The present invention provides a peptide construct comprising at least one
epitope sequence, wherein the construct also includes or is associated with an
antigen
presenting cell (APC) targeting mechanism and is capable of stimulating an
irnrnune
response such as, for example, a cytotoxic T-cell and/or helper T-cell and/or
B-cell
response.
The epitope contained in the peptide construct of the present invention can
be, for example, a CD8+ T cell epitope, a CD4+ T cell epitope, a B cell
epitope, or any
combination thereof. As disclosed herein, the epitope can be derived from any
antigen.
For example, the epitope can be derived from a cancer antigen (also termed
tumor-
associated antigen or TAA), a viral antigen, a bacterial antigen, a protozoan
antigen, or a
fungal antigen. Preferably, the epitope is derived from a TAA. In one of the
embodiments, the TAA is a carcinoembryonic antigen (CEA).
Preferably, the epitope is a non-native epitope, which binds with high
affinity to the MHC molecules and is useful for modulating innnune responses
to the
corresponding native epitope from which this non-native epitope is derived.
The non-
native epitope sequences of the present invention preferably differ from their
natural
counterparts in that they contain alterations in amino acid sequence, relative
to the native
sequence, in the MHC binding domain, thereby conferring tighter binding to the
MHC
molecule. Alternatively or in addition, they may contain mutations in the
putative T cell
receptor-binding domain, resulting in an increased affinity for the T cell
receptor (TCR).
These differences from the native sequence confer advantages in the methods of
the
present invention over the use of the native sequence, in that the non-native
epitopes of
the invention have enhanced irmnunomodulatory properties.
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Also preferably, the peptide construct of the invention is a polyepitope
construct comprising more than one epitope, wherein said epitopes are derived
from one
or more different antigens. In polyepitope constructs of the invention, at
least one
epitope is preferably a cytotoxic T cell (CTL) epitope. In a specific
embodiment, the
construct represents an epitope string with epitopes arranged consecutively.
Also preferably, each of the epitopes in the peptide construct of the
invention is flanked on at least one side, and preferably on both sides, by a
spacer
(flanking) sequence comprising an internal processing sequence. The internal
processing
sequence is designed to facilitate the endosomal and/or lysosomal processing
of the
construct once it has reached the antigen presenting cell (APC).
The APC targeting mechanism is comprised of (i) an APC targeting
sequence, which directs the construct to antigen presenting cells (APCs)
within the
subject and, optionally (ii) a vehicle, such as, e.g., a microsphere or
liposome, that also
acts as a targeting mechanism and/or preserves the viability of the construct
until it has
reached its intended APC and/or mediates the controlled release of the
construct. The
APC targeting sequence may be covalently or non-covalently attached to the
epitope.
Preferably, the APC targeting sequence is covalently attached to the N-
terminus or C-
terminus of the sequence flanking respectively the first or the last epitope
in the epitope
string. In a specific embodiment, the APC targeting sequence is derived from a
sequence
capable of mediating interaction with such cell surface proteins as, for
example, Heat
Shock Protein Receptor (HSR) such as CD91, C-type lectin receptors such as
Mannose
Receptor (MR), DEC-205 and DC-SIGN, and IgG Fc receptor (FcR) such as Fc~yRI.
In a specific embodiment, the peptide construct of the invention may be
represented by the following general formula:
N-[APC targeting sequence]-[flanking sequence with internal processing
sequence]-
[epitope 1]-[flanking sequence with internal processing sequence]-[epitope
2]...[epitope n]-C
or
N-[epitope 1]-[flanking sequence with internal processing sequence]-[epitope
2]... [epitope n]-[flanking sequence with internal processing sequence]-[APC
targeting sequence]-C
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wherein n is the number of epitopes in the construct and wherein the internal
processing
sequence may be represented by the following general formula:
[Leu and/or Asp and/or Pro]-[Xaa-Lys-Xaa-Lys-YTic]
wherein each Xaa is independently selected from any amino acid, and YTic is an
amino
acid that is susceptible to cleavage by trypsin or chymotrypsin.
The invention also provides a method for preparing the peptide construct
described above. In a specific embodiment, the construct is produced
synthetically. In
another embodiment, the construct is produced recombinantly.
In a further embodiment, the invention provides an isolated nucleic acid
molecule encoding the peptide construct described above. The invention also
provides an
expression vector comprising such nucleic acid, as well as a host cell (e.g.,
APC) and/or
recombinant non-human host comprising such nucleic acid. According to a
specific
embodiment, the expression vector comprising the nucleic acid encoding the
epitope
construct of the invention may further comprise or may be combined with aal
APC
targeting mechanism and may therefore be targeted directly to be expressed in
the APCs
of the host.
Further provided are pharmaceutical and vaccine compositions comprising
an immunogenically effective amount of the peptide construct of the invention
or a
nucleic acid encoding such peptide construct and, optionally, further
comprising a
pharmaceutically acceptable adjuvant or excipient.
Also provided herein is a method for inducing or augmenting immunity
(preferably, an antigen-specific cytotoxic T cell (CTL) irmnune response)
induced by an
antigen in a mammal comprising administering to the mammal the pharmaceutical
or
vaccine composition of the invention.
In a further embodiment, the invention provides a prophylactic and/or
therapeutic method for treating a disease in a mammal comprising administering
to the
mammal at least one dose of the pharmaceutical or vaccine composition of the
invention.
As specified herein, this method can be useful for preventing and/or treating
various
neoplastic diseases, infections, autoimmune diseases, and the life. In a
specific
embodiment, the method of the invention is employed to treat cancer.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the discovery that epitope sequences
may be incorporated into a construct that (i) delivers the epitope(s) to
antigen presenting
cells (APCs), and (ii) facilitates appropriate processing of the epitopes once
delivered to
the APCs.
Specifically, the present invention provides a peptide construct comprising
at least one epitope sequence, wherein the construct also includes or is
associated with an
antigen presenting cell (APC) targeting mechanism and is capable of
stimulating an
immune response such as, for example, a cytotoxic T-cell and/or helper T-cell
and/or B-
cell response.
The epitope contained in the peptide construct of the present invention can
be, for example, a CD8+ T cell epitope, a CD4+ T cell epitope, a B cell
epitope, or a
combination thereof. As disclosed herein, the epitope can be derived from any
antigen.
For example, the epitope can be derived from a cancer antigen (also termed
tumor-
associated antigen or TAA), a viral antigen, a bacterial a~itigen, a protozoan
antigen, or a
fungal antigen. Preferably, the epitope is derived from a TAA. In one of the
embodiments, the TAA is carcinoembryonic antigen (CEA).
The present invention also provides novel non-native epitopes designed
for enhanced binding to MHC molecules and useful for modulating immune
responses to
the corresponding native peptides from which they are derived. Preferably, the
synthetic
antigenic peptide epitope sequences of the present invention differ from their
native
counterparts in that they contain alterations in amino acid sequence, relative
to the native
sequence, in the MHC class I binding domain, thereby conferring tighter
binding to the
MHC. Alternatively or in addition, they contain mutations in the putative T
cell receptor
(TCR)-binding domain, resulting in an increased affinity for the TCR. These
differences
from the native sequence confer advantages in the methods of the present
invention over
the native sequence, in that the synthetic antigenic peptide epitopes of the
invention have
enhanced irnmunomodulatory properties.
The constructs of the invention comprise at least one epitope sequence, or
a combination of epitope sequences, which may be the same or different.
Preferably, the
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peptide construct of the invention is a polyepitope construct comprising more
than one
epitope, wherein said epitopes are derived from one or more different
antigens. In
polyepitope constructs of the invention, at least one epitope is preferably a
cytotoxic T
cell (CTL) epitope. In a specific embodiment, the construct represents an
epitope string
with epitopes arranged consecutively.
Also preferably, each of the epitopes in the peptide constructs of the
invention is flanked on at least one side, and preferably on both sides, by a
spacer
(flanking) sequence comprising an internal processing sequence. The internal
processing
sequence is designed to facilitate the endosomal and/or lysosomal processing
of the
construct once it has reached the antigen presenting cell (APC).
Specific residues that may be included in the internal processing sequence
include, but are not limited to, arginine (Arg, R), leucine (Leu, L),
phenylalanine (Phe,
F), aspartic acid (Asp, D), lysine (Lys, K), or proline (Pro, P). In a
preferred
embodiment, the internal processing sequence includes one or more Arg, Asp,
and Pro
residues. As endosomes and lysosomes possess proteolytic activity with
substrate
specificity similar to trypsin and chymotrypsin (see Hershko et al., Ann. Rev.
Biochem.
67: 425-79 (1998)), the epitope sequences included in the constructs of the
present
invention are preferably bounded by amino acid residues that act as substrates
for those
enzymes, such as lysine (Lys, K) or arginine (Arg, R) for trypsin, and
phenylalanine
(Phe, F), tryptophan (Trp, W), or tyrosine (Tyr, Y~ for chymotrypsin.
In a specific embodiment, the internal processing sequence may further
include a sequence signaling ubiquitination and/or an additional targeting
signals) for
transport to ER, endosomes or lysosomes. According to a specific embodiment,
such
additional targeting signal may contain one or more of the residues identified
above for
the internal processing sequence and is followed by an "internal sequence"
which
contains at least one lysine residue, and preferably two lysine residues at
positions 3 and
17 of the internal sequence. Immediately following or adjacent to the internal
sequence
is the sequence of the epitope. One or more such additional targeting and
ubiquitination
sequences may be incorporated into the construct of the invention, the one or
more
targeting sequences being positioned in between each epitope sequence. With
regard to
the additional targeting sequences described above, reference is made to
Bachmair et al.,
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Science, 1986, 234: 179-186; Dantuma et al., Nature Biotech., 2000, 18: 538-
43; Velders
et al., J. Irnmunol., 2001, 166: 5366-5373; Toes et al., Proc. Natl. Acad.
Sci. USA, 1997,
94: 14660-14665; Thomson et al., J. Virol., 1998, 72: 2246-2252; Ishioka et
al., J.
T_m_m__unol., 1999, 162: 3915-3925; Thomson et al., J. Immunol., 1998, 160:
1717-1723;
and International Patent Publication Nos. WO 01/19408, WO 01/47541 amd WO
97/35021.
According to the present invention, a "targeting mechanism" is a system
that is capable of directing the constructs of the invention to APCs and
facilitating proper
internal processing of the constructs once inside the APCs. Specifically, the
APC
targeting mechanism is comprised of (i) an APC targeting sequence, which
directs the
construct to the APCs within the subject and, optionally (ii) a vehicle, such
as, e.g., a
microsphere or liposome, that also acts as a targeting mechanism andlor
preserves the
viability of the construct until it has reached its intended APC and/or
mediates the
controlled release of the construct.
The APC targeting sequence may be covalently or non-covalently attached
to the epitope. Preferably, the APC targeting sequence is covalently attached
to the N-
terminus or C-terminus of the sequence flanking respectively the first or the
last epitope
in the epitope string. In a specific embodiment, the APC targeting sequence is
derived
from a sequence capable of mediating interaction with 511Ch cell surface
proteins as, for
example, Heat Shoclc Protein Receptor (HSR) such as CD91, C-type lectin
receptors such
as Mannose Receptor (MR), DEC-205 and DC-SIGN, and IgG Fc receptor (FcR) such
as
Fc~yRI. In the Example 1 provided below, the APC targeting sequence is
incorporated
into the constructs at the C-terminus and comprises the gp96 sequence which
interacts
with the N-terminal p80 fragment of the alpha subunit of CD91, which is a
receptor for
heat shock proteins (see Binder et al., Nature Immunol. 1: 151-55 (2000)).
Accordingly, as used herein, the teen "construct" refers to a molecule
consisting of at least one epitope sequence and at least one APC targeting
sequence,
which may be associated with the epitope sequence either covalently or non-
covalently.
In a specific embodiment, the peptide construct of the invention may be
represented by the following general formula:
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N-[APC targeting sequence]-[flanking sequence with internal processing
sequence]-
[epitope 1]-[flanking sequence with internal processing sequence]-[epitope
2] ... [epitope n]-C
or
N-[epitope 1]-[flanking sequence with internal processing sequence]-[epitope
2]...[epitope n]-[flanking sequence with internal processing sequence]-[APC
targeting sequence]-C
wherein n is the number of epitopes in the construct and wherein the internal
processing
sequence may be represented by the following general formula:
[Leu and/or Asp and/or Pro]-[Xaa-Lys-Xaa-Lys-YTic]
wherein each Xaa is independently selected from any amino acid, and YTic is an
amino
acid that is susceptible to cleavage by trypsin or chymotrypsin.
As described above, in addition to the APC targeting sequence, the
targeting mechanism of the invention may include incorporating the constructs
of the
present invention into vehicles such as microspheres or liposomes that are
internalized,
e.g., endocytosed, phagocytosed or pinocytosed, by the APCs, and then further
processed
by endosomes and/or lysosomes within the cells. In this regard, reference is
made to U.S.
Patent No. 6,312,731, which relates to a composition for inducing or
potentiating an
immune response, comprising an antigen and/or a bioactive agent encapsulated
in a
polymeric composition consisting of a polymer present in an amount sufficient
to provide
structural integrity to the composition, and a rapidly biodegradable
component, a rapidly
dissolving component, a rapidly swelling component, or a component that causes
osmotic
rupture of the encapsulated polymeric composition.
Methods of Making Pepetide Constructs of the Invention
The invention also provides methods for preparing the peptide constructs
described above. Such methods employ conventional techniques. Because the
epitope-
containing peptide constructs of the present invention will generally be short
sequences,
they can be routinely prepared by chemical synthesis using standard
techniques.
Particularly convenient are solid phase peptide synthesis techniques (See,
e.g., Steward
and Young, eds. (1968) "Solid Phase Peptide Synthesis" Freemantle, San
Francisco,
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Calif.). Automated peptide synthesizers are commercially available (such as
those
manufactured by Perkin Elmer/Applied Biosystems, Inc., Model 430A or 431A,
Foster
City, Calif., USA.), as are the reagents required for their use. The peptides
can also be
prepared using recombinant techniques known to those of skill in the ai-t
using the host
cells and nucleic acid vector systems described below.
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
other standard
teclmuque 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. Alternatively, affinity tags such as hexa-His (Invitrogen), Maltose
binding
domain (New England Biolabs), influenza coat sequence (I~olodziej et al.
(1991)
Methods Enzymol. 194:508-509), and glutathione-S-transferase can be attached
to the
peptides of the invention to facilitate purification by passage over an
appropriate affinity
column. Isolated peptides can be physically characterized using such
techniques as
proteolysis, nuclear magnetic resonance, and x-ray crystallography.
Further provided are pharmaceutical and vaccine compositions comprising
an immunogenically effective amount of the peptide construct of the invention
or the
nucleic acid encoding such polypeptide construct and, optionally, further
comprising a
pharmaceutically acceptable adjuvant or excipient.
Also provided herein is a method for inducing or augmenting immunity
(preferably, an antigen-specific cytotoxic T cell (CTL) immune response)
induced by an
antigen in a mammal comprising administering to said mammal the pharmaceutical
or
vaccine composition of the invention.
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In a further embodiment, the invention provides a prophylactic and/or
therapeutic method for treating a disease in a mammal comprising administering
to said
mammal at least one dose of the pharmaceutical or vaccine composition of the
invention.
As specified herein, this method can be useful for preventing and/or treating
various
neoplastic diseases, infections, autoimmune diseases, and the like. In a
specific
embodiment, the method of the invention is employed to treat a cancer. The
constructs of
the present invention are preferably designed to elicit an anti-tumor immune
response in
an effort to decrease the rate of tumor growth, cause tumor regression,
increase the time
to relapse, and decrease mortality.
General Definitions
The terms used in this specification generally have their ordinary
meanings in the art within the context of this invention and in the specific
context where
each term is used. Certain terms are discussed below, or elsewhere in the
specification,
to provide additional guidance to the practitioner in describing the
compositions and
methods of the invention and how to male and use them.
The terms "cancer," "neoplasm," and "tumor" are used interchangeably
and refer to cells that have undergone a malignant tra~zsformation that makes
them
pathological to the host organism. Primary cancer cells (that is, cells
obtained from near
the site of malignant transformation) can be readily distinguished from non-
cancerous
cells by well-established techniques, particularly histological examination.
The
definition of a "cancer cell", as used herein, includes not only a primary
cancer cell, but
any cell derived from a cancer cell ancestor. This includes metastasized
cancer cells, and
in vitro cultures and cell lines derived from cancer cells. When referring to
a type of
cancer that normally manifests as a solid tumor, a "clinically detectable"
tumor is one that
is detectable on the basis of tumor mass; e.g., by such procedures as CAT
scan, magnetic
resonance imaging (MRI), X-ray, ultrasound, or palpation. Biochemical or
immunologic
findings alone rnay or may not be insufficient to meet this definition.
The term "vaccine" refers to a composition that can be used to elicit
protective immunity in a recipient (e.g., a composition comprising the peptide
construct
of the invention or the nucleic acid encoding such construct). It should be
noted that to
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16
be effective, a vaccine of the invention can elicit immunity in a portion of
the population,
as some individuals may fail to mount a robust or protective immune response,
or, in
some cases, any immune response. This inability may stem from the individual's
genetic
background or because of an immunodeficiency condition (either acquired or
congenital)
or immunosuppression (e.g., treatment with immunosuppressive drugs to prevent
organ
rejection or suppress an autoimmune condition). Efficacy can be established in
animal
models.
The term "DNA vaccine" is an informal term of art, and is used herein to
refer to a vaccine delivered by means of a recombinant vector. An alternative,
and more
descriptive term used herein is "vector vaccine" (since some potential
vectors, such as
retroviruses and lentiviruses are RNA viruses, and since in some instances non-
viral
RNA instead of DNA is delivered to cells through the vector). Generally, the
vector is
administered ifa vivo, but e.~ vivo transduction of appropriate antigen
presenting cells,
such as dendritic cells (DC), with administration of the transduced cells i~
vivo, is also
contemplated.
The term "immunotherapy" refers to a treatment regimen based on
activation of an antigen-specific immune response. A vaccine administration
can be one
form of immunotherapy.
''hnmune response" broadly refers to the antigen-specific responses of
lymphocytes. Any substance that can elicit an immune response is said to be
"immunogenic" and is referred to as an "irrnnunogen". An immune response of
this
invention can be humoral (via antibody activity) or cell-mediated (via T cell
activation)
or both.
As used herein, the term "immunogenic" means that the agent (e.g.,
protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, or
combination
thereof) is capable of eliciting a humoral or cellular immune response, and
preferably
both, when administered to an animal having an immune system. An immunogenic
peptide is also antigenic. A molecule is "antigenic" when it is capable of
specifically
interacting with an antigen recognition molecule of the irmnune system, such
as an
immunoglobulin (antibody) or T cell receptor (TCR). Antigenic ligand sequences
that
specifically interact with antibodies, MHC class I, and MHC class II molecules
are
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17
described herein. An antigenic portion of a peptide, also called herein an
epitope, can be
that portion that is immunodominant for antibody or T cell receptor
recognition, or it can
be a portion used to generate an antibody to the molecule by conjugating the
antigenic
portion to a carrier polypeptide for irnrnunization. A molecule that is
antigenic need not
itself be immunogenic, z.e., capable of eliciting an immune response, without
a carrier,
adjuvant or excipient.
Antigens and Epitopes of the Invention
The term "antigen" refers to any agent (e.g., protein, peptide,
polysaccharide, glycoprotein, glycolipid, nucleic acid, or combination
thereof) that, when
introduced into a host having an immune system (directly or upon expression as
in, e.g.,
DNA vaccines), is recognized by the immune system of the host and is capable
of
eliciting a specific immune reaction.
The terms "tumor-associated antigen (TAA)" and "tumor-specific antigen
(TSA)" are used interchangeably and refer to an antigenic peptide that is
associated with
a tumor. As described in the Background Section, TAAs include, for example,
mutated
cellular proteins such as mutated tumor suppressor gene products, oncogene
products
(including fusion proteins), and foreign proteins such as viral gene products.
Non-
mutated cellular proteins may also be TAAs if they are expressed aberrantly
(e.g., in an
inappropriate subcellular compartment) or in supernormal quantities.
The term "epitope" or "antigenic determinant" refers to any portion of an
antigen recognized either by B cells, or T cells, or both. Preferably,
interaction of an
epitope with an antigen recognition site of an immunoglobulin or TCR involves
antigen-
specific immune recognition.
CTL epitope sequences, T helper cell sequences, and B cell epitope
sequences may be included in the constructs of the invention. The epitopes
used in
immunogenic (e.g., vaccine) compositions of the instant invention can be
derived from
any antigen present in a eukaryotic cell (e.g., tumor, parasite, fungus),
bacterial cell, viral
particle, or any portion thereof.
Examples of preferred antigens of the present invention include tumor-
associated antigens (TAAs) such as ErbB receptors, Melan A [MART1], gp100,
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18
tyrosinase, TRP-1/gp 75, and TRP-2 (in melanoma; for additional examples, see
also a
list of antigens provided in Storkus and Zarour, Forum (Genova), 2000 Jul-Sep,
10(3):256-270); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small
cell
carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin [MUC-1] (in
breast,
pancreas, colon, and prostate cancers); prostate-specific antigen [PSA] (in
prostate
cancer); carcinoembryonic antigen [CEA] (in colon, breast, lung, thyroid, and
gastrointestinal cancers), PlA tumor antigen (e.g., as disclosed in
International Patent
Publication No. WO 98/56919), and such shared tumor-specific aaltigens as MADE-
2,
MAGE-4, MADE-6, MAGE-10, MAGE-12, BALE-1, CAGE-1,2.,8, CAGE-3 to 7,
LAGS-1, NY-ESO-1/LAGE-2, NA-88, and GnTV (see, e.g., International Patent
Publication No. WO 98/56919).
In a specific embodiment, the constructs of the present invention include
at least one epitope derived from carcinoembryonic antigen (CEA). CEA is
associated
with neoplasms of epithelial origin, including carcinomas of the
gastrointestinal tract,
breast, lung, and thyroid, and therefore, constructs of the invention that
include CEA
epitopes may be used to threat neoplasms of epithelial origin.
Other antigens of the invention include but are not limited to (i) protozoan
antigens such as those derived from Plasf~aoa'iuyn sp., T~xoplasfna sp.,
Pneumoeystis
caf°inii, Leislamania sp., and Tyypa~iosoma sp.; (ii) viral protein or
peptide antigens such
as those derived fiom influenza virus (e.g., surface glycoproteins
hemagluttinin (HA) and
neuraminidase (NA) or the nucleoprotein (NP) as described in Bodmer et al.,
Cell,
52:253, 1988 and Tsuji et al., J. Virol. 72: 6907-6910, 1998 or NP CTL
epitopes as
described in Gould et al., J. Virol., 65:5401, 1991; Mw-ata et al., Cell
Immunol., 173:96-
107, 1996, and PCT Application No. WO 98/56919); irmnunodeficiency virus
(e.g., a
simian immunodeficiency virus (SIV) antigen [e.g., SIV-env CTL epitope as
disclosed in
PCT Application No. WO 98/56919], or a human immunodeficiency virus antigen
(HIV-
1) such as gp120 CTL epitopes as disclosed, e.g., in PCT Application No. WO
98/56919], gp160, p18 antigen [e.g., CD8+ T cell epitopes and gp41 CTL
epitopes as
disclosed, e.g., in PCT Application No. WO 98/56919], Gag p24 CD8+ T cell
epitopes,
Gag p17 CD8+ T cell epitopes, Tat, Pol, Nef CTL epitopes as disclosed, e.g.,
in PCT
Application No. WO 98/56919], and Env CTL epitopes as disclosed, e.g., in PCT
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19
Application No. WO 98/56919]; herpesvirus (e.g., a glycoprotein, for instance,
from
feline herpesvirus, equine herpesvirus, bovine herpesvirus, pseudorabies
virus, canine
herpesvirus, herpes simplex virus (HSV, e.g., HSV tk, gB, gD), herpes zoster
virus,
Marek's Disease Vinus, herpesvirus of turkeys (HVT), cytomegalovirus (CMV), or
Epstein-Barr virus); hepatitis C virus; human papilloma virus (HPV); human T
cell
leukemia virus (HTLV-1); bovine leukemia virus (e.g., gp51,30 envelope
antigen); feline
leukemia virus (FeLV) (e.g., FeLV envelope protein, a Newcastle Disease Virus
(NDV)
antigen, e.g., HN or F); rous associated virus (such as RAV-1 envy; infectious
bronchitis
virus (e.g., matrix and/or preplomer); flavivirus (e.g., a Japanese
encephalitis virus (JEV)
antigen, a Yellow Fever antigen, or a Dengue virus antigen); Morbillivirus
(e.g., a canine
distemper virus antigen, a measles antigen, or rinderpest antigen such as HA
or F); rabies
(e.g., rabies glycoprotein G); parvovirus (e.g., a canine parvovirus antigen);
hepatitis C
virus (HCV); poxvirus (e.g., an ectromelia antigen, a canary poxvirus antigen,
or a fowl
poxvirus antigen such as chicken pox virus varicella zoster antigen);
infectious bursal
disease virus (e.g., VP2, VP3, or VP4); Hantaan virus; mumps virus, and
measles virus;
(iii) bacterial antigens such as Mycobacte>~iutyt tubey~culosis-specific
(e.g., Bacillus
Calinette-Guerin [BCG] - 381~D protein; antigen 85 complex [as described in
I~lein et al.,
J. Infect. Dis., 183:928-34, 2001], see also a list of antigens in I~lein and
McAdam, Arch.
Immunol. Ther. Exp. (Warsz.), 47:313-320, 1999), Liste~ia t~tayaocytogea2es-
specific (e.g.,
as disclosed in Finelli et al., T_m_m__unol. Res., 19:211-223, 1999),
Salntottella typhii-
specific, Shigella flexinet°i-specific, staphylococcus-specific,
streptococcus-specific,
pneumococcus-specific (e.g., PspA [see PCT Publication No. WO 92/14488]),
Neisse~ia
gofzof°~laea-specific, Bot~t~elia-specific (e.g., OspA, OspB, OspC
antigens of Bof°Yelia
associated with Lyme disease such as Bot°relia bmgdorfet-i, Bot~Yelia
af~elli, and Boj°>~elia
ga>~i3tii [see, e.g., U.S. Patent No. 5,523,089; PCT Application Nos. WO
90/04411, WO
91/09870, WO 93/04175, WO 96/06165, W093/08306; PCT/IJS92/08697; Bergstrom et
al., Mol. Microbiol., 3: 479-486, 1989; Johnson et al., Infect. and Immun. 60:
1845-1853,
1992; Johnson et al., Vaccine 13: 1086-1094, 1995; Tlae Sixth Ifttef~national
Confe~en.ee
oft Lyme Bof~j~eliosis: Pt~ogt°ess o>2 tlae Developtne>zt of Lyt~ae
Disease Tlaccitte, Vaccine
13: 133-135, 1995]), A. pef°tussis-specific, S. par°athyphoid A
and B-specific, C.
diphtlzeriae-specific, C. tetatzus-specific, C. botulittunz-specific, C.
pef°if °ifagens-specific,
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A. anthracis-specific, A. pestis-specific, Y choler°a-specific, H.
influerzzae-specific, T.
palladium-specific, Chlamydia trachornatis-specific (e.g., as disclosed in
I~im et al., J.
T_mmunol., 162:6855-6866, 1999), and pseudomonas-specific proteins or
peptides; and
(iv) fungal antigens such as those isolated from candida (e.g., 65kDa
mamloprotein
[MP65] from Caradida albicans), trichophyton, or ptyrosporum.
The foregoing list of antigens is intended as exemplary, as the antigen of
interest can be derived from any animal or human pathogen or tumor. With
respect to
DNA encoding pathogen-derived antigens of interest, attention is directed to,
e.g., U.S.
Patent Nos. 4,722,848; 5,174,993; 5,338,683; 5,494,807; 5,503,834; 5,505,941;
5,514,375; 5,529,780; U.K. Patent No. GB 2 269 820 B; and PCT Publication Nos.
WO
92/22641; WO 93/03145; WO 94/16716; WO 96/3941; PCT/US94/06652. With respect
to antigens derived from tumor viruses, reference is also made to Molecular-
Biology of
Tumor Viruses, RNA Turnor Tlir°uses, Second Edition, Edited by Weiss et
al., Cold Spring
Harbor Laboratory Press, 1982. For a list of additional antigens useful in the
compositions of the invention see also Stedman's Medical Dictionary (24th
edition,
1982).
To provide additional antigen-derived B and T cell epitopes for use in the
constructs of the present invention, these epitopes can be identified by one
or a
combination of several methods well known in the art, such as, for example, by
(i)
fragmenting the antigen of interest into overlapping peptides using
proteolytic enzymes,
followed by testing the ability of individual peptides to bind to an antibody
elicited by the
full-length antigen or to induce T cell or B cell activation (see, e.g., Janis
Kuby,
Immunology, pp. 79-80, W. H. Freeman, 1992); (ii) preparing s5mthetic
peptides, the
sequences of which are segments or analogs of a given antigen (see, e.g.,
Alexander et
al., Immunity, 1: 751-61, 1994; Hammer et al., J. Exp. Med., 180: 2353-8,
1994), or
constructs based on such segments, or analogs linked or fused to a carrier or
a
heterologous antigen and testing the ability of such synthetic peptides to
elicit antigen-
specific antibodies or T cell activation (e.g., testing their ability to
interact with MHC
class II molecules both in vitro and in vivo [see, e.g., O'Sullivan et al., J.
Immunol., 147:
2663-9, 1991; Hill et al., J. hnmunol., 147: 189-197, 1991]); for
determination of T cell
epitopes, peptides should be at least 8 to 10 amino acids long to occupy the
groove of the
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21
MHC class I molecule and at least 13 to 25 amino acids long to occupy the
groove of
MHC class II molecule, preferably, the peptides should be longer; these
peptides should
also contain an appropriate anchor motif which will enable them to bind to
various class I
or class II MHC molecules with high enough affinity and specificity to
generate an
immune response (see Bocchia et al., Blood, 85: 2680-2684, 1995; Englehard,
Ann. Rev.
T_m_m__unol., 12: 181, 1994); (iii) sequencing peptides associated with
purified MHC
molecules (see, e.g., Nelson et al., Proc. Natl. Acad. Sci. USA, 94:628-33,
1997); (iv)
screening a peptide display library for high-affinity binding to MHC class II
molecules,
TCR, antibodies raised against a full-length antigen, etc. (see, e.g., Hammer
et al., J. Exp.
Med., 176:1007-13, 1992); (v) computationally analyzing different protein
sequences to
identify, e.g., hydrophilic stretches (hydrophilic amino acid residues are
often located on
the surface of the protein and are therefore accessible to the antibodies)
and/or high-
affinity TCR or MHC class II allele-specific motifs, e.g., by comparing the
sequence of
the protein of interest with published structures of peptides associated with
the MHC
molecules (Mallios, Bioinformatics, 15:432-439, 1999; Mililc et al., Nat.
Biotechnol., 16:
753-756, 1998; Brusic et al., Nuc. Acids Res, 26: 368-371, 1998; Feller and de
la Cruz,
Nature, 349: 720-721, 1991); (vi) performing an x-ray crystallographic
analysis of the
native antigen-antibody complex (Jams Kuby, Immunology, p. 80, W. H. Freeman,
1992), and (vii) generating monoclonal antibodies to various portions of the
antigen of
interest, and then ascertaining whether those antibodies attenuate if2
vits°o or in vivo
growth of the pathogen or tumor from which the antigen was derived (see U.S.
Patent No.
5,019,384 and references cited therein).
Non-Native Epitopes and Methods for Their Generation
A "native" or "wild-type" or "natural" or "self' epitope is an epitope,
which has been isolated from a natural biological source, and which can be
recognized by
the immune system.
An "altered" or "non-natural" or "non-native" or "modified" or
"synthetic" epitope is one having a primary sequence that is different from
that of the
corresponding native epitope. Although using epitopes with non-native peptide
sequences may induce immune responses that are not specific for the intended
target,
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22
according to the present invention, the use of non-native epitopes is
preferred for the
following reasons. First, it is likely that native peptide sequences derived
from antigens
(e.g., tumor-associated antigens (TAAs) or self antigens in autoimmune
disorders) may
be recoguzed by the immune system as self and patients may be tolerized to
these
peptide sequences. Second, many native peptide sequences do not bind with high
affnuty
to the MHC molecules. As it has been shown that the strength of the immune
response to
a particular epitope is related to the binding affinity of the epitope to the
MHC class I
complex (i.e., stronger immune responses can be induced by peptides that bind
with
higher affinity to the MHC molecules), it is likely that the immune response
to native
epitopes will be lower than the immune responses to non-native epitopes that
bind with
higher affinity to the MHC molecules.
Non-native peptide epitopes can be produced using any method known in
the art. The following provides non-limiting examples of such methods. In
addition,
modifications or combinations of any of the following methods can be used.
The non-native epitopes may be derived from native epitopes (e.g., using
site-directed mutagenesis) by modifying them in any way known in the art, as
long as the
modification does not completely prevent their ability to generate an immune
response.
In particular, the constructs of the invention may have one or more amino acid
substitutions, deletions, or inseutions. For example, such amino acid
substitutions may
include substitutions of functionally equivalent amino acid residues. One or
more amino
acid residues can be substituted by another amino acid of a similar polarity
that acts as a
functional equivalent resulting in a silent alteration. Substitutes for an
amino acid may be
selected from other members of the class to which the amino acid belongs. For
example,
the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,
valine,
proline, phenylalanine, tryptophan and methionine. The polar neutral amino
acids
include glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine. The
positively charged (basic) amino acids include arginine, lysine and histidine.
The
negatively charged (acidic) amino acids include aspartic acid and glutamic
acid.
Additionally, one or more amino acid residues can be substituted by a
nonclassical amino acid or chemical amino acid analogs. Non-classical amino
acids
include but are not limited to the D-isomers of the common amino acids, alpha-
amino
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23
isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic
acid, 2-
amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline,
hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-
butylalanine,
phenylglycine, cyclohexylalanine, beta-alanine, fluoro amino acids, designer
amino acids
such as beta methyl amino acids, C-a-methyl amino acids, N-cx methyl amino
acids, and
amino acid analogs in general. Peptides of the invention may also comprise
various
"designer" amino acids (e.g., (3-methyl amino acids, C-a methyl amino acids,
and N-a,-
methyl amino acids, etc.) to convey special properties to peptides.
Additionally, by
assigning specific amino acids at specific coupling steps, peptides with a-
helices, [3-
turns, (3-sheets, y-turns, and cyclic peptides can be generated. Generally, it
is believed
that a-helical secondary structure or random secondary structure is preferred.
The following non-classical amino acids may be incorporated in the
peptides of the invention in order to introduce particular conformational
motifs: 1,2,3,4-
tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991) J. Am. Chem.
Soc.
113:2275-2283); (2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,
(2R,3S)-
methyl-phenylalanine and (2R,3R)-methyl-phenylalanine; 2-aminotetrahydro-
naphthalene-2-carboxylic acid (Landis (1989) Ph.D. Thesis, University of
Arizona);
hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et czl. (1989) J.
Takeda
Res. Labs 43:53-76), histidine isoquinoline carboxylic acid (Zechel et al.
(1991) Int. J.
Pep. Protein Res. 43), and HIC (histidine cyclic urea) (Dharanipragada).
The following amino acid analogs and peptidomimetics may be
incorporated into a peptide to induce or favor specific secondary structures:
LL-Acp (LL-
3-amino-2-propenidone-6-carboxylic acid), a (3-turn inducing dipeptide analog
(Kemp et
al. (1985) J. Org. Chem. 50:5834-5838); (3-sheet inducing analogs (Kemp et al.
(1988)
Tetrahedron Lett. 29:5081-5082); (3-tum inducing analogs (Kemp et al. (1988)
Tetrahedron Lett. 29:5057-5060); a-helix inducing analogs (Kemp et al. (1988)
Tetrahedron Lett. 29:4935-4938); y-turn inducing analogs (Kemp et al. (1989)
J. Org.
Chem. 54:109:115); and analogs provided by the following references: Nagai and
Sato
(1985) Tetrahedron Lett. 26:647-650; DiMaio et al. (1989) J. Chem. Soc. Perkin
Trans. p.
1687; also a Gly-Ala turn analog (Kahn et al. (1989) Tetrahedron Lett.
30:2317); amide
bond isostere (clones et al. (1988) Tetrahedron Lett. 29:3853-3856); tretrazol
(Zabrocki
CA 02478930 2004-09-10
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24
et al. (1988) J. Am. Chem. Soc. 110:5875-5880); DTC (Samanen et al. (1990)
Int. J.
Protein Pep. Res. 35:501:509); and analogs taught in Olson et al. (1990) J.
Am. Chem.
Sci. 112:323-333 and Garvey et al. (1990) J. Org. Chem. 56:436.
Conformationally
restricted mimetics of beta turns and beta bulges, and peptides containing
them, are
described in U.S. Patent No. 5,440,013.
In addition, the present invention envisions preparing peptides that have
more well-defined structural properties, and the use of peptidomimetics, and
peptidomimetic bonds, such as ester bonds, to prepare peptides with novel
properties. In
another embodiment, a peptide may be generated that incorporates a reduced
peptide
bond, i.e., Rl--CH2 NH--R2, where Rl, and R2 are amino acid residues or
sequences. A
reduced peptide bond may be introduced as a dipeptide subunit. Such a molecule
would
be resistant to peptide bond hydrolysis, e.g., protease activity. Such
molecules would
provide ligands with unique function and activity, such as extended half lives
ifz vivo due
to resistance to metabolic breakdown, or protease activity.
In a specific embodiment, non-native epitopes are derived from native
epitopes recombinantly by introducing post-translational modifications e.g.,
by
phosphorylation, glycosylation, cross-linking, acylation, proteolytic
cleavage, linkage to
an antibody molecule, membrane molecule or other epitope (Ferguson et al. Aml.
Rev.
Biochem. 1988, 57:285-320).
For generation of large numbers of new non-native epitope sequences,
bacteriophage "phage display" libraries (10~-10~ chemical entities) can be
constructed
(Scott and Smith (1990) Science 249:386-390; Cwirla et al. (1990) Proc. Natl.
Acad. Sci.
USA 87:6378-6382; Devlin et al. (1990) Science 249:404-406). Other methods
involve
primarily chemical synthesis methods, of which the Geysen method (Geysen et
al. (1986)
Molecular Immunology 23:709-715; Geysen et al. (1987) J. Imtnunologic Method
102:259-274) and the method of Fodor et al. ((1991) Science 251:767-773) are
examples.
See also Furka et al. (1988) 14th International Congress of Biochemistry,
Volume 5.
Abstract FR:013; Furka, (1991) Int. J. Peptide Protein Res. 37:487-493). See
also U.S.
Pat. Nos. 4,631,211 and 5,101,175, which describe methods to produce a mixture
of
peptides. Other methods which can be employed involve use of synthetic
libraries
(Needels et al. (1993) Proc. Natl. Acad. Sci. USA 90:10700-4; Ohlmeyer et al.
(1993)
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Proc. Natl. Acad. Sci. USA 90:10922-10926; International Patent Publication
No. WO
92/00252) and tecluuques based on cDNA subtraction or differential display
(Hedricl~ et
al. (1984) Nature 308:149; Lian and Pardee (1992) Science 257:967). Another
technique
which can be used is the "pepscan" technique (Van der Zee (1989) Eur. J.
Lm_m__unol.
19:43-47), in which several dozens of peptides are simultaneously synthesized
on
polyethylene rods arrayed in a 96-well microtiter plate pattern, similar to an
indexed
library in that the position of each pin defines the synthesis history on it.
Peptides are
then chemically cleaved from the solid support and supplied to irradiated
syngeneic
thymocytes for antigen presentation. A cloned CTL line can then be tested for
reactivity
in a proliferation assay monitored by 3H-thymidine incorporation.
In a specific embodiment non-natural epitopes of the present invention are
generated from a combinatorial library of oligopeptides attached to solid
phase supports
using SPHERETM technology, which is described in the International Patent
Publication
No. WO 97/35035, U.S. Patent Nos. 6,528,060 and 6,338,945, and Published U.S.
Application No. 2002/0164346. This approach utilizes combinatorial peptide
libraries
synthesized on polystyrene beads wherein each bead contains a pure population
of a
unique peptide that can be chemically released from the beads in discrete
aliquots. The
peptides attached to a single bead have essentially the same amino acid
sequence. The
synthesis history of each peptide bead may be recorded on each solid support
in a code of
inert molecular tags, such that beads of interest can be rapidly and
efficiently decoded. A
photocleavable crosslinker allows release of some of the oligopeptide by
exposure to UV
light. Molecular tags, if present, remain covalently bound to the beads for
post-assay
analysis. Released peptide from pooled bead arrays are screened using methods
to detect
T cell activation, including, for example, 3H-thymidine incorporation (for
CD4+ or CD8+
T cells), SICr-release assay (for CTLs), or IL-2 production (for CD4+ T cells)
to identify
peptide pools capable of activating a T cell of interest. By utilizing an
iterative peptide
pool/releasing strategy, it is possible to screen more than 107 peptides in
just a few days.
Analysis of residual peptide on the corresponding positive beads (>100 pmoles)
allows
rapid and unambiguous identification of the epitope sequence.
As specified above, non-native epitopes of the invention may bind to
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26
MHC molecules with higher affinity than the corresponding native epitopes.
Binding of
the epitopes of the invention to MHC molecules can be measured by methods that
are
known in the art and include, but are not limited to, calculating the affinity
based on an
algorithm (see, for example, Parker et al. (1992) J. hrmmnol. 149:3580-3587);
and
experimentally determining binding affinity (see, e.g., Tan et al. (1997) J.
T_m_m__unol.
Meth. 209:25-36). For example, the relative binding of a peptide to an MHC
molecule
can be measured on the basis of binding of a radiolabeled standard peptide to
detergent-
solubilized MHC molecules, using various concentrations of test peptides
(e.g., ranging
from 100 rnM to 1 nVI). For example, MHC class I heavy chain and (32-
microglobulin
may be co-incubated with a fixed concentration (e.g., 5 nM) of radiolabeled
native
(control) peptide and various concentrations of a corresponding non-native
peptide for a
suitable period of time (e.g., 2 hours to 72 hours) at room temperature in the
presence of a
mixture of protease inhibitors. The percent MHC-bound radioactivity is
determined by
gel filtration. The ICSO (concentration of test peptide which results in 50%
inhibition of
binding of control peptide) is calculated for each peptide.
As specified above, non-native epitopes of the invention may also bind to
TCRs with higher affinity than the corresponding native epitopes. Methods for
determining binding affinity to TCRs are k~zown in the art and include, but
are not limited
to, those described in al-Ramadi et al. (1992) J. Imunol. 155(2):662-673; and
Zuegel et
al. (1998) J. Immunol. 161(4):1705-1709.
Immune Effector Cells
The term "irn~nune effector cells" refers to cells capable of binding an
a~ltigen and which thereby 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). The activation of T cells by professional antigen
presenting cells
(APCs) leads to their proliferation and the differentiation of their progeny
into armed
effector T cells which can act on any target cell that displays antigen on its
surface.
Effector T cells can mediate a variety of functions. One set of important
functions is the
killing of cells, e.g., cancer cells or cells infected by viruses or bacteria,
by CD8+ CTLs,
and the activation of macrophages by TH1 cells, which together make up cell-
mediated
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27
immunity. In addition, B cells are activated by TH2 cells to produce different
types of
antibody, thus driving the humoral immune response.
The term "antigen presenting cells" or "APCs" refers to a class of cells
capable of presenting one or more antigens in the form of antigen-MHC
complexes
recognizable by specific effector cells of the immune system, and thereby
inducing an
effective cellular immune response against the antigen or antigens being
presented. While
many types of cells may be capable of presenting antigens on their cell
surface for T-cell
recognization, only professional APCs have the capacity to present antigens in
an
efficient amount and further to activate T-cells for CTL responses. APCs
include, for
example, macrophages, B-cells and dendritic cells (DCs).
The term "dendritic cells" or "DCs" refers to a diverse population of
morphologically similar cell types found in a variety of lymphoid and non-
lymphoid
tissues (Steinman (1991) Ann. Rev. Immunol. 9:271-296). DCs constitute the
most
potent and preferred APCs in the organism. A subset, if not all, of DCs are
derived from
bone marrow progenitor cells, circulate in small numbers in the peripheral
blood and
appear either as immature Langerhans' cells or terminally differentiated
mature cells.
DCs are professional APCs that efficiently capture antigens in the peripheral
tissues and
process these antigens to form MHC-peptide complexes. After antigen uptake,
these
immature DCs acquire the unique capacity to migrate from the periphery to the
T cell
areas of secondary lymphoid organs. As the cells travel, they mature and alter
their
profile of cell surface molecules, to attract resting T cells and present
their antigenic load
(Shaw et al., (1986) Nature 323, 262-264; Adema et al., (1997) Nature 387, 713-
717;
Banchereau and Steinman, (1998) Nature 392, 245-252). While the DCs can be
differentiated from monocytes, they possess distinct phenotypes. For example,
a
particular differentiating marker, CD14 antigen, is not found in DCs but is
possessed by
monocytes. Also, mature DCs are not phagocytic, whereas the monocytes are
strongly
phagocytosing cells. It has been shown that DCs provide all the signals
necessary for T
cell activation and proliferation.
T cells recognize proteins only when they have been cleaved into smaller
peptides and are presented in a complex called the "major histocompatability
complex
(MHC)" located on the another cell's surface. The terms "major
histocompatability
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28
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 is also known as the "human leukocyte antigen" or "HLA" complex. The
proteins encoded by the MHC are known as "MHC molecules" and are classified
into
class I and class II MHC molecules. Class I MHC includes membrane
heterodimeric
proteins made up of an a chain encoded in the MHC noncovalently linked with
the ~32-
microglobulin. Class I MHC molecules are expressed by nearly all nucleated
cells axed
have been shown to function in antigen presentation to CD8+ T cells, i.e., T
cells which
have the CD8 protein on their surface. CD8+ T cells, bind specifically to the
MHC class
Ilpeptide complexes via the T cell receptor (TCR). This leads to cytolytic
effector
activities. Class I molecules include HLA-A, B and C in humans. Class II MHC
molecules also include membrane heterodimeric proteins consisting of non-
covalently
associated a and ~i chains. Class II MHC complexes are found only on APCs and
are
used to present peptides from antigens which have been endocytosed by APCs. T
cells
which have the CD4 protein on their surface, i. e., CD4+ T cells, bind to the
MHC class
II/peptide complexes via TCR. This leads to the synthesis of specific
cytokines which
stimulate an immune response. Class II MHCs in humans include HLA-DP, DQ, and
DR. Those of skill in the art are familiar with the serotypes and genotypes of
the HLA
(see Rammensee, H.Ci., Bachmann, J., and Stevanovic, S. MHC Ligands and Peptid
Motifs (1977) Chapman & Hall Publishers; Schreuder et al., The HLA dictionary,
Tissue
Antigens 1999, 54:409-437). To be effectively recognized by the immune system
via
MHC class I presentation, an antigenic polypeptide has to contain an epitope
of at least
about 8 to 10 amino acids, while to be effectively recognized by the irmnune
system via
MHC class II presentation, ajz antigenic polypeptide has to contain an epitope
of at least
about 13 to 25 amino acids. See, e.g., Fundamental Imn2uraology, 3ra Edition,
W.E. Paul
ed., 1999, Lippincott-Raven Publ.
The term "native antibodies" or . "immunoglabulins" refers to usually
heterotetrameric glycoproteins of about 150,000 daltons, composed of two
identical light
(L) chains and two identical heavy (H) chains. In most classes of
immunoglobulin
molecules, each light chain is linked to a heavy chain by one covalent
disulfide bond,
while the number of disulfide linkages varies between the heavy chains of
different
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29
immunoglobulin isotypes. Each heavy and light chain also has regularly spaced
intrachain disulfide bridges. Each heavy chain has at one end a variable
domain (VH)
followed by a number of constant domains. Each light chain has a variable
domain (VL)
at one end and a constant domain at its other end; the constant domain of the
light chain
is aligned with the first constant domain of the heavy chain, and the light
chain variable
domain is alig~led with the variable domain of the heavy chain. Particular
amino acid
residues are believed to form an interface between the light and heavy chain
variable
domains (Clothia et al., J Mol. Biol., 186: 651-663, 1985; Novotny and Haber,
Proc.
Natl. Acad. Sci. USA, 82: 4592-4596, 1985).
The term "antibody" or "Ab" is used in the broadest sense and specifically
covers not only native antibodies but also single monoclonal antibodies
(including
agonist and antagonist antibodies), antibody compositions with polyepitopic
specificity,
as well as antibody fragments (e.g., Fab, F(ab')2, scFv and Fv), so long as
they exhibit the
desired biological activity.
The term "cross-reactive" is used to describe compounds of the invention
which are functionally overlapping. More particularly, the immunogenic
properties of a
native epitope and/or immune effector cells activated thereby are shared to a
certain
extent by the non-native epitope derived therefrom such that the non-native
epitope is
"cross-reactive" with the native epitope and/or the immune effector cells
activated
thereby. For purposes of this invention, cross-reactivity may be manifested at
multiple
levels: (i) at the epitope level, e.g., the non-native epitopes can bind the
TCR of and
activate native epitope-specific CTLs; (ii) at the T cell level, i.e., non-
native epitopes of
the invention bind the TCR and activate a population of T cells (distinct from
the
population of native epitope-specific CTLs) which can effectively target and
lyse cells
displaying the native epitope; and (iii) at the antibody level, e.g., "anti"-
non-native
epitope antibodies can detect, recognize and bind the native epi~pe and
initiate effector
rliechanisms in an immune response.
Therapeutic Definitions
As used herein, the term "inducing an immune response in a subj ect" is a
term well understood in the art and means that an increase in an immune
response to an
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antigen (or epitope) can be detected or measured, after introducing the
construct into the
subject, relative to the ixmnune response (if any) before introduction of the
construct into
the subject. 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 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).
The term "treat" is used herein to mean to relieve or alleviate at least one
symptom of a disease or condition in a subject. Within the meaning of the
present
invention, the term "treat" may mean to prolong the prepatency, i.e., the
period between
infection or neoplastic transformation and clinical manifestation of a
disease. The term
"protect" is used herein to mean prevent or treat, or both, as appropriate,
the development
or continuance of a disease in a subject. Within the meaning of the present
invention, the
disease is selected from the group consisting of malignancy (e.g., solid or
blood tumors
such as sarcomas, carcinomas, gliomas, blastomas, pancreatic cancer, breast
cancer,
ovarian cancer, prostate cancer, lymphoma, leukemia, melanoma, etc.),
infection (e.g.,
viral, bacterial, parasitic, or fungal) and an autoimmune disease (e.g., most
forms of
arthritis, ulcerative colitis, asthma, or multiple sclerosis). For example, if
the epitope(s)
incorporated into the construct of the invention is(are) derived from CEA
(carcinoembryonic antigen), which is associated with ~eoplasms of epithelial
origin,
including carcinomas of the gastrointestinal tract, breast, lung, and thyroid,
the construct
of the invention may be administered to prevent or treat neoplasms of
epithelial origin in
a subject in need of such treatment. Therefore, the intended use of the
constructs of the
invention will largely depend on the origin of the epitope(s). Prophylactic
administration
of the vaccine can protect the recipient subject at risk of developing such a
cancer, e.g., as
determined from family history.
The phrase "pharmaceutically acceptable", as used in connection with
compositions of the invention, refers to molecular entities and other
ingredients of such
compositions that are physiologically tolerable and do not typically produce
untoward
reactions when administered to a human. Preferably, as used herein, the term
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31
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal or a
state government or listed in the U.S. Pharmacopeia or other generally
recognized
pharmacopeia for use in mammals, and more particularly in humans.
The terms "adjuvant" and "immunoadjuvant" are used interchangeably in
the present invention and refer to a compound or mixture that may be non-
immunogenic
when administered to a host alone, but that augments the host's immune
response to
another antigen when administered conjointly with that antigen.
The adjuvant of the invention can be administered as part of a
pharmaceutical or vaccine composition comprising an antigen or as a separate
formulation. The adjuvants of the invention include, but are not limited to,
oil-emulsion
and emulsifier-based adjuvants such as complete Freund's adjuvant, incomplete
Freund's
adjuvant, MF59, or SAF; mineral gels such as aluminum hydroxide (alum),
aluminum
phosphate or calcium phosphate; microbially-derived adjuvants such as cholera
toxin
(CT), pertussis toxin, Escherichia coli heat-labile toxin (LT), mutant toxins
(e.g., LTK63
or LTR72), Bacille Calnaette-Crzcer~ifi (BCG), Conyraebacte~°iuna
parvufya, DNA CpG
motifs, muramyl dipeptide, or monophosphoryl lipid A; particulate adjuvants
such as
immunostimulatory complexes (ISCOMs), liposomes, biodegradable microspheres,
or
saponins (e.g., QS-21); cytokines such as IFN-°y, IL-2, IL-12 or GM-
CSF; synthetic
adjuvants such as nonionic block copolymers, muramyl peptide analogues (e.g.,
N-acetyl-
muramyl-L-threonyl-D-isoglutamine [thr-MDP], N-acetyl-nor-muramyl-L-alanyl-D-
isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1'-2'-
dipalmitoyl-
sn-glycero-3-hydroxyphosphoryloxy]-ethylamine), polyphosphazenes, or synthetic
pol3mucleotides, and surface active substances such as lysolecithin, pluronic
polyols,
polyanions, peptides, hydrocarbon emulsions, or keyhole limpet hemocyanins
(KLH).
Preferably, these adjuvants are pharmaceutically acceptable for use in humans.
Within the meaning of the present invention, the term "conjoint
administration" is used to refer to administration of an immune adjuvant and
an antigen
simultaneously in one composition, or simultaneously in different
compositions, or
sequentially.
The term "excipient" applied to pharmaceutical or vaccine compositions of
the invention refers to a diluent or vehicle with which an antigen-containing
compound
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32
and/or an adjuvant is administered. Such pharmaceutical excipients can be
sterile liquids,
such as water and oils, including those of petroleum, animal, vegetable or
synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water or
aqueous solution, saline solutions, and aqueous dextrose and glycerol
solutions are
preferably employed as excipients, particularly for injectable solutions.
Suitable
pharmaceutical excipients are described in "Remington's Pharmaceutical
Sciences" by
E.W. Martin, lSth Edition.
The term "protective immunity" refers to an immune response in a host
animal (either active/acquired or passive/innate, or both) which leads to
inactivation
andlor reduction in the load of an antigen and to generation of immunity (that
is acquired,
e.g., through production of antibodies), which prevents or delays the
development of a
disease upon repeated exposure to the same or a related antigen. A "protective
immune
response" involves hwnoral (antibody) immunity or cellular irmnunity, or both,
effective,
e.g., to reduce a tumor burden in an immunized (vaccinated) subject or to
eliminate or
reduce, or slow the increase in, the load of a pathogen or infected cell (or
produce any
other measurable alleviation of the infection). Within the meaning of the
present
invention, protective immunity may be partial. W specific embodiments of the
invention,
protective immunity is reflected by any improvement in any condition or
symptom being
treated, including any one of the followings a slowing of disease progression,
increasing
length to relapse, decreased rate of tumor growth, tumor regression, decreased
mortality,
etc.
As used herein, the term "augment the immune response" means
enhancing or extending the duration of the irnlnune response, or both.
The phrase "enhance immune response" within the meaning of the present
invention refers to the property or process of increasing the scale and/or
efficiency of
irmnunoreactivity to a given antigen, said immunoreactivity being either
humoral or
cellular immunity, or both. An immune response is believed to be enhanced, if
any
measurable parameter of antigen-specific immunoreactivity (e.g., antibody
titer, T cell
production) is increased at least two-fold, preferably ten-fold, most
preferably thirty-fold.
The term "modulate an immune response" includes inducing (increasing
or eliciting) an immune response; and reducing (suppressing) an immune
response. An
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33
immunomodulatory method (or protocol) is one that modulates an immune response
in a
subj ect.
The term "therapeutically effective" applied to dose or amount refers to
that quantity of a compound or pharmaceutical composition or vaccine that is
sufficient
to result in a desired activity upon administration to a mammal in need
thereof. As used
herein with respect to pharmaceutical compositions or vaccines, the term
"therapeutically
effective amount/dose" is used interchangeably with the term "ixnmunogenically
effective
amount/dose" and refers to the a~nount/dose of a compound (e.g., an epitope
presented as
part of the construct of the invention) or pharmaceutical composition or
vaccine that is
sufficient to produce an effective immune response upon administration to a
mammal.
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
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), interleulcin 3, colony stimulating factor (GM-CSF),
interleukin-1 alpha
(IL-lI), interleulcin-11 (IL-11), MIP-11, leukemia inhibitory factor (LIF), c-
lcit ligand,
thrombopoietin (TFO) and flt3 ligand. The present invention also includes
culture
conditions in which one or more cytolcine is specifically excluded from the
medium.
Cytolcines are commercially available from several vendors such as, for
example,
Genzyme (Framingham, MA), Genetech (South San Francisco, CA), Amgen (Thousand
Oaks, CA), R&D Systems (Mimleapolis, MN) and Ixnrnunex (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.
The term "subject" as used herein refers to an animal having an immune
system, preferably a mammal (e.g., rodent such as mouse). In particular, the
term refers
to humans.
The term "about" or "approximately" usually means within 20%, more
preferably within 10%, and most preferably still within 5% of a given value or
range.
Alternatively, especially in biological systems (e.g., when measuring an
immune
response), the term "about" means within about a log (i.e., an order of
magnitude)
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34
preferably within a factor of two of a given value.
Molecular Biology Related Definitions
The teen "isolated" means separated from constituents, cellular and
otherwise, in which the nucleic acid molecule, peptide, polypeptide, protein,
or fragments
thereof, are normally associated with in nature. For example, with respect to
a nucleic
acid molecule, an isolated nucleic acid 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 slcill in the art, a non-naturally occurring polynucleotide, peptide,
polypeptide,
protein, or fragments thereof, does not require "isolation" to distinguish it
from its
naturally occurring counterpart. In addition, a "concentrated", "separated" or
"diluted"
nucleic acid molecule, peptide, polypeptide, protein, or fragments thereof, is
distinguishable from its naturally occurring counterpa~.-t 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 nucleic acid molecule,
peptide,
polypeptide, protein, 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 occurnng nucleic acid is provided as a
separate
embodiment from the isolated naturally occurring nucleic acid. A protein
produced in a
bacterial cell is provided as a separate embodiment from the naturally
occurring protein
isolated from a eukaryotic ceii.W which it is produced in nature.
''coding sequence'' of aw sequence "e~nco~ci~ng" ati expression product,
such~"as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence
that, when
expressed, results in the production of that RNA, polypeptide, protein, or
enzyme, i.e.,
the nucleotide sequence encodes an amino acid sequence for that polypeptide,
protein or
enzyme. A coding sequence for a protein may include a start codon (usually
ATG) and a
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stop codon.
The term "gene", also called a "structural gene" means a DNA sequence
that codes for or corresponds to a particular sequence of amino acids which
comprise all
or part of one or more proteins, and may or may not include regulatory DNA
sequences,
such as promoter sequences, that determine for example the conditions under
which the
gene is expressed. The transcribed region of a gene can include 5'- and 3'-
untranslated
regions (UTRs) and introns in addition to the translated (coding) region.
A "promoter sequence" is a DNA regulatory region capable of binding
RNA polynerase in a cell and initiating transcription of a downstream (3'
direction)
coding sequence. For purposes of defining the present invention, the promoter
sequence
is bounded at its 3' terminus by the transcription initiation site and extends
upstream (5'
direction) to include the minimum number of bases or elements necessary to
initiate
transcription at levels detectable above background. Within the promoter
sequence will
be found a transcription initiation site (conveniently defined for example, by
mapping
with nuclease S1), as well as protein binding domains (consensus sequences)
responsible
for the binding of RNA polymerase.
A coding sequence is "under the control" of or "operably (or operatively)
associated with" transcriptional and translational control sequences in a
cell. RNA
polymerase transcribes the coding sequence into mRNA, which is then trans-RNA
spliced (if it contains introns) and translated into the protein encoded by
the coding
sequence. "Operatively associated" refers to a juxtaposition wherein the
elements are in
an arrangement allowing them to function in concert.
The terms "express" and "expression" mean allowing or causing the
information in a gene or DNA sequence to become manifest, for example
producing a
protein by activating the cellular functions involved in transcription and
translation of a
corresponding gene or DNA sequence. A DNA sequence is expressed in or by a
cell to
form an "expression product" such as a mRNA or a protein. The expression
product
itself, e.g. the resulting mRNA or protein, may also be said to be "expressed"
by the cell.
"'An expression product can be chai~~ter~zed as intracellular, extracellular
or secreted. The
term "intracellular" means something that is inside a cell. The term
"extracellular"
means something that is outside a cell. A substance is "secreted" by a cell if
it appears in
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36
significant measure outside the cell, from somewhere on or inside the cell.
"Conditions
that permit expression" irz vitro are culture conditions of temperature
(generally about
37°C), humidity (humid atmosphere), carbon dioxide concentration to
maintain pH
(generally about 5% COZ to about 15% COZ), pH (generally about 7.0 to 8.0,
preferably
7.5), and culture fluid components that depend on host cell type. Ih vivo, the
conditions
that permit expression are primarily the health of the non-human transgenic
animal,
wluch depends on adequate nutrition, water, habitation, and environmental
conditions
(light-dark cycle, temperature, humidity, noise level). In either system,
expression may
depend on a repressor or inducer control system, as well known in the art.
The term "heterologous" refers to a combination of elements not naturally
occurring in a particular locus. For example, heterologous DNA refers to DNA
not
naturally located in the cell or in a particular chromosomal site of the cell.
A
heterologous expression regulatory element is such an element operatively
associated
with a different gene than the one it is operatively associated with in
nature. In the
context of the present invention, a construct coding sequence is heterologous
to the vector
DNA in which it is inserted for cloning or expression and it is heterologous
to a host cell
containing such a vector in which it is expressed, e.g., a CHO cell.
The term "gene delivery", "gene transfer", "transfection", and the like as
used herein mean the introduction of a ''foreign" (i. e., extrinsic or
extracellular) gene,
DNA or RNA sequence into a host cell, so that the host cell will express the
introduced
gene or sequence to produce a desired substance, typically a protein or enzyme
encoded
by the introduced gene or sequence. The introduced gene or sequence may also
be called
a "cloned" or "foreign" gene or sequence, may include regulatory or control
sequences,
such as start, stop, promoter, signal, secretion, or other sequences used by a
cell's genetic
machinery. The gene or sequence may include nonfunctional sequences or
sequences
with no l~nown function. A host cell that receives and expresses introduced
DNA or
RNA has been "transfected" and is a "transfectant" or a "ie." The DNA or RNA
introduced to a host cell can come from any source, including cells of the
same genus or
species as the host cell, or cells of a different genus or species.
A "gene delivery vehicle" is defined as any molecule that can carry
inserted polynucleotides into a host cell. Examples of gene delivery vehicles
are
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37
liposomes, biocompatible polymers, including natural polymers and synthetic
polymers;
lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial
viral
envelopes; metal particles; and bacteria, viruses, such as baculovirus,
adenovirus,
retrovirus, lentivirus, and adeno-associated virus, bacteriophage, cosrnid,
plasmid, fungal
vectors and other recombination vehicles typically used in the art, which have
been
described for expression in a variety of eukaryotic and prokaryotic hosts, and
may be
used for gene therapy as well as for simple protein expression.
The terms "vector", "cloning vector" and "expression vector" mean the
gene delivery vehicle by which a DNA or RNA sequence (e.g., a foreign gene)
can be
introduced into a host cell, so as to transform the host and promote
expression (e.g.,
transcription and translation) of the introduced sequence.
Vectors typically comprise the DNA of a transmissible agent, into which
foreign DNA is inseued. A common way to insert one segment of DNA into another
segment of DNA involves the use of enzymes called restriction enzymes that
cleave
DNA at specific sites (specific groups of nucleotides) called restriction
sites. A
"cassette" refers to a DNA segment that can be inserted into a vector or into
another piece
of DNA at a defined restriction site. Preferably, a cassette is an "expression
cassette" in
which the DNA is a coding sequence or segment of DNA that codes for an
expression
product that can be inserted into a vector at defined restriction sites. The
cassette
restriction sites generally are designed to ensure insertion of the cassette
in the proper
reading frame. Generally, foreign DNA is inserted at one or more restriction
sites of the
vector DNA, and then is carried by the vector into a host cell along with the
transmissible
vector DNA. A segment or sequence of DNA having inserted or added DNA, such as
an
expression vector, can also be called a "DNA construct." A common type of
vector is a
"plasmid" that generally is a self contained molecule of double-stranded DNA,
usually of
bacterial origin, that can readily accept additional (foreign) DNA and which
can be
readily introduced into a suitable host cell. A plas;~.zid vector often
contains boding DNA
and promoter DNA and has one or more r~~str~ction sites suitable for inserting
foreign
DNA. A large number of vectors, including plasmid and fungal vectors, have
been
described for replication and/or expression in a variety of eulcaryotic and
prokaryotic
hosts. Non-limiting examples include pKK plasmids (Amersham Pharmacia
Biotech),
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38
pUC plasmids, pET plasmids (Novagen, Inc., Madison, W~, pRSET or PREP plasmids
(Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly,
MA),
and many appropriate host cells, using methods disclosed or cited herein or
otherwise
known to those spilled in the relevant art. Recombinaxlt cloning vectors will
often
include one or more replication systems for cloning or expression, one or more
markers
for selection in the host, e.g. antibiotic resistance, and one or more
expression cassettes.
The term "host cell" or "recipient cell" means any cell of any organism
that is selected, modified, transformed, grown, or used or manipulated in any
way by
introduction of a nucleic acid or vector encoding a variant construct, for the
production of
a substance by the cell, for example the expression by the cell of a gene, a
DNA or RNA
sequence, or protein, i.e., the variant construct. The host cell may be found
in vit>~o, i.e.,
in tissue culture, or ifz vivo, i.e., in a microbe, plant or animal. These
terms are intended
to include progeny of a single cell, and 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 or introduction of
further variations.
The cells may be prokaryotic or eukaryotic, and include, but are not limited
to, bacterial
cells, yeast cells, animal cells, and mammalian cells, e.g., marine, rat,
hamster, simian, or
human cells.
The temp "expression system" means a host cell and compatible vector
under suitable conditions, e.g., for the expression of a protein coded for by
foreign DNA
carried by the vector and introduced to the host cell. Preferably the
introduced nucleic
acid is stably or transiently maintained in the host cell. Stable maintenance
typically
requires that the introduced nucleic acid either contains an origin of
replication
compatible with the host cell or integrates into a replicon of the host cell
such as an
extrachromosoml replicon (e.g., a plasmid) or a nuclear or mitochondrial
chromosome.
Common expression systems include E. coli host cells and plasmid vectors,
insect host
cells and Baculovi~°us vectors, and mammalian host cells and vectors.
In a specific
embodiment, the construct is expressed in COS-1 or CHO cells. Other suitable
cells
include NSO cells HeLa cells, ?93e (human kidney cells), mouse primary
myoblasts and
NIH 3T3 ells.
The term "culturing" refers to the ifz vitro propagation of host cells or
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39
organisms on or in media of various kinds. Preferably, culturing occurs under
conditions
that permit expression of the variant construct. By "expanded" is meant any
proliferation
or division of cells.
The terms "mutant" and "mutation" mean any detectable change in genetic
material, e.g., DNA, or any process, mechanism, or result of such a change.
This
includes gene mutations, in which the structure (e.g., DNA sequence) of a gene
is altered,
any gene or DNA arising from a~ly mutation process, and any expression product
(e.g.,
protein or enzyme) expressed by a modified gene or DNA sequence. The term
"variant"
may also be used to indicate a modified or altered gene, DNA sequence, enzyme,
cell,
etc., i. e., any kind of mutant.
As used herein, the term "oligonucleotide" refers to a nucleic acid,
generally of at least 10, preferably at least 15, and more preferably at least
20 nucleotides,
preferably no more than about 100 nucleotides, that is hybridizable to a
genomic DNA
molecule, a cDNA molecule, or an mRNA molecule having a sequence of interest.
Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to
which a
label, such as biotin, has been covalently conjugated. In one embodiment, a
labeled
oligonucleotide can be used as a probe to detect the presence of a nucleic
acid. In another
embodiment, oligonucleotides (one or both of which may be labeled) can be used
as PCR
primers, either for cloning full length or a fragment of the epitope string,
or to detect the
presence of nucleic acids encoding the epitope string. In a further
embodiment, an
oligonucleotide of the invention can form a triple helix with a epitope string-
encoding
DNA molecule, e.g., for purification purposes. Generally, oligonucleotides are
prepared
synthetically, preferably on a nucleic acid synthesizer. Accordingly,
oligonucleotides can
be prepared with non-naturally occurnng phosphoester analog bonds, such as
thioester
bonds, etc.
To induce an immune response in a subject, the peptide constructs of the
invention can be administered as polynucleotides encoding the polypeptides.
The
polynucleotides can be administered in a gene deliv,~ry vehicle or by
inserting into a host
cell which in turn recombinantly transcribes, translates and processes the
encoded
polypeptide. Isolated host cells containing the polynucleotides of this
invention in a
pharmaceutically acceptable canier can be therefore combined with appropriate
and
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effective amounts of an adjuvant, cytol~ine or co-stimulatory molecule for an
effective
vaccine regimen. In one embodiment, the host cell is an APC such as a
dendritic cell
(DC), a monocyte/macrophage, a B lymphocyte, or other cell types) expressing
the
necessary MHC/co-stimulatory molecules. The host cell can be further modified
by
inserting a polynucleotide coding for an effective amount of either or both a
cytolcine and
a co-stimulatory molecule.
APCs can be transduced in vitro with viral vectors encoding the peptide
constructs of the invention. The most common viral vectors include recombinant
poxviruses such as vaccinia and fowlpox virus (Bronte, et al. (1997) PNAS
94:3183-
3188; Kim, et al. (1997) J. Immunother. 20:276-286) and, preferentially,
adenovirus
(Arthur, et al. (1997) J. Immunol. 159:1393-1403; Wan, et al. (1997) Human
Gene
Therapy 8:1355-1363; Huang, et al. (1995) J. Virol. 69:2257-2263). Retrovirus
also may
be used for transduction (Marin, et al. (1996) J. Virol. 70:2957-2962).
Transduced APCs
can subsequently be administered to the host via an intravenous, subcutaneous,
intranasal,
intramuscular or intraperitoneal route of delivery. W 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.
Alternatively, iyi vivo transduction of DCs, or other APCs, can be
accomplished by administration of 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 1x101°-1x1012 i.u.
Although viral gene delivery is more efficient, DCs can also be transduced
ih vitrolex 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 4:17-25). Gene gun delivery or injection of naked plasmid
DNA
into the skin also leads to transduction of DCs (Condon et al. (1996) Nature
Med.
2:1122-1128; Raz et al (1994) PNAS 91:9519-9523). W tramuscular delivery of
plasmid
DNA may also be used for immunization (Rosato et al. (1997) Human Gene Therapy
8:1451-1458).
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41
Examples of Useful APC Receptors Targeted by the Constructs of the Invention
As specified above, the peptide constructs of the invention comprise APC-
targeting sequences.
Typically, antigen presenting cells (APCs) present exogenous antigen on
major histocompatibility complex class II (MHC) molecules, and endogenously
synthesized antigen on MHC I molecules. In exceptional circumstances exogenous
antigens can be presented on MHC I molecules; this phenomenon is central to
indirect
presentation of antigens, particularly as it relates to priming of naive CD8+
T
lymphocytes specific for tumors and other noninfectious agents (Bevan, J. Exp.
Med.
182, 639-641 (1995)).
To generate a primary CTL response to tumor-associated antigens
(TAAs), with the capacity to eliminate tumor cells, APCs have to present
peptides
complexed to MHC class I. It is now becoming apparent that professional APCs,
DCs,
monocytes and macrophages are capable of internalizing exogenous antigens for
processing and presentation on MHC class I molecules. Several mechanisms by
which
APCs may incorporate peptides and present peptides in association with MHC
class I
have been reposed. Firstly, peptides from degraded proteins may be produced in
high
concentrations externally and then exchanged with peptides on the surface MHC
class I
molecules (Carbone and Bevan (1990) J. Exp. Med. 171: 377-387; Pfeifer et al.
(1993)
Nature 361: 359-362). Alternatively, APCs may take up antigen by endocytosis
or
phagocytosis, process it by proteolysis in the endosomal vesicle where it
associates with
MHC class I and then it is transported to the surface without having ever
entered the
cytosol (Bachmann et al. (1995) Eur. J. T_m_m__unol. 25: 1739-1743; Liu et al.
(1997)
Stand. J. Tm_m__unol. 45: 527-533). Recently, a third mechanism whereby
exogenous
antigens were internalized by APCs, transferred to the cytosol, processed and
then
presented via the classical endogenous MHC class I pathway was demonstrated
for both
macrophages (Kovacsovics-Bankowski and Rock, (1995) Science 267: 243-246) and
DCs (Rodriguez et al., (1999) Nature Cell Biol. 1: 362-368).
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42
It has been demonstrated previously that exogenous antigens chaperoned
by heat shock proteins (HSP) such as gp96 can be taken up by APCs and
presented
through their MHC I molecules, leading to stimulation of antigen-specific CD8+
T cells
(Suto and Srivastava, Science 269, 1585-1588 (1995)). A wide array of peptides
are
chaperoned by gp96, depending upon the source from which they are isolated
(Srivastava
et al., Immunity 8, 657-665 (1998)). Tumor-derived gp96 carries tumor-
antigenic
peptides (Ishii et al., J. Immunol. 162, 1303-1309 (1999)), gp96 preparations
from virus-
infected cells carry viral epitopes (Suto and Srivastava, Science 269, 1585-
1588 (1995);
Nieland et al., Proc. Natl Acad. Sci. USA 95, 1800-1805 (1998)) and gp96
preparations
from cells transfected with model antigens such as ovalbumin or -galactosidase
are
associated with the corresponding epitopes (Arnold et al., J. Exp. Med. 182,
885-889
(1995); Breloer et al., Eur. J. Immunol. 28, 1016-1021 (1998)). The
association of gp96
with peptides occurs ifZ vivo (Menoret and Srivastava, Biochem. Biophys. Res.
Commun.
262, 813-818 (1999)). Complexes of gp96 with peptides, whether isolated from
cells
(Tamura et al., Science 278, 117-120 (1997)) or reconstituted ah vat3°o
(Blachere et al., J.
Exp. Med. 186, 1183-1406 (1997)), are excellent immunogens and have been used
extensively to elicit CD8+ T cell responses specific to the gp96-chaperoned
antigenic
peptides.
Binder et al. (Nature Immunol. (2000) 1:151-155) have shown that APCs
can take up exogenous antigenic peptides chaperoned by heat shock protein gp96
and re-
present them through the endogenous pathway on their MHC class I molecules.
The high
efficiency of this process has been attributed previously to a receptor for
gp96 on APCs.
The CD91 molecule (also called 2-macroglobulin receptor or the low density
lipoprotein-
related protein) was suggested by Binder et al. to be a cell surface receptor
for the heat
shock protein gp96. CD91 is present on cells of monocytic lineage as well as
hepatocytes, fibroblasts and lceratinocytes. Binder et al. have shown that HSP
gp96 is a
ligand for CD91. The p80 fragment of CD91 shown to bind gp96 directly is an N-
terminal degradation product of the CD91 a subunit. APC-gp96 interaction leads
to
interaction of gp96 with CD91 and consequent re-presentation of gp96-
chaperoned
peptides by MHC I molecules of the APC, followed by stimulation of antigen-
specific T
cells (Suto and Srivastava, Science 269, 1585-1588 (1995)) and secretion of
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43
proinflamrnatory cytol~ines such as TNF, granulocyte-macrophage colony-
stimulating
factor (GM-CSF) and interleukin 12 (Basu et al., Int. T_m_m__unol. (2000)
12:1539-1546).
Similarly, Suzie et al. (Proc. Natl. Acad. Sci. USA, 94:13146-13151
(1997)) have shown that a soluble hsp70 fusion protein having a large fragment
of
chicken ovalbumin as fusion partner could, in the absence of adjuvants,
stimulate H-2b
mice to produce ovalbumin-specific CD8+ CTL. Mice immunized with heat shock
proteins (hsps) isolated from mouse tumor cells (donor cells) produce CD8+
cytotoxic T
lymphocytes (CTL) that recognize donor cell peptides in association with the
major
histocompatibility complex (MHC) class I proteins of the responding mouse.
It follows that the hsp molecules are capable of delivering non-covalently
associated peptides to MHC class I proteins of other (recipient) cells,
including APCs.
Accordingly, in one of the embodiments of the present invention, the APC
targeting
sequence is derived from an hsp sequence, which mediates interaction with the
hsp
receptor (HSR) on the surface of APC (e.g., CD91 receptor).
DCs internalize exogenous antigens by fluid-phase pinocytosis or by
receptor-mediated endocytosis. DCs express several receptors that facilitate
the
internalization and presentation of antigens, including C-type lectin
receptors such as the
mannose receptor (Sallusto et al., (1995) J. Exp. Med. 182:389), DEC205 (Jiang
et al.,
(1995) Nature 375:151) and DC-SIGN (Engering et al., J Irnmunol (2002)
168:2118-
2126), as well as receptors for the Fc domain of Igs (Sallusto and
Lanzavecchia (1994) J.
Exp. Med. 179:1109).
FcRs are expressed on most cells of the hemopoietic lineages, including
DCs, and play a pivotal role in linking the humoral and cellular arms of the
immune
response. Antigen-IgG complexes (immune complexes (ICs)), which bind to and
cross-
link FcRs, mediate a variety of responses if2 vitro ranging from phagocytosis
to antibody-
mediated cellular cytotoxicity. FcR-mediated internalization of ICs by DCs is
associated
with enhanced presentation of both MHC class I- and MHC class n-binding
peptides
derived from the antigen present in the ICs and leads to activation of the DCs
and enables
DCs to prime peptide-specific CD8+ CTLs in vivo (Amigorena and Bonnerot (1999)
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44
Semin. Tmmunol. 11:385; Amigorena and Bonnerot (1999) Tm_m__unol. Rev.
172:279;
Regnault et al. (1999) J. Exp. Med. 189:371).
Schuurhuis et al. (J Immunol (2002) 168:2240-2246) have shown that
OVA/anti-OVA IC-treated D~Cs primed CTLs against the dominant CTL epitope
derived
from the OVA Ag present in the ICs.
Among the more than eight members of the IgG Fc receptor family, one
receptor, Fc~yRI (CD64), is constitutively expressed only on monocytes,
macrophages and
DCs (Pan et al. (1990) J. hnmunol. 145: 267-275; Fanger et al. (1997) J.
Immunol. 158:
3090-3098). Targeting antigen to Fc~yRI on DC in the form of ICs was shown to
result in
efficient class I-restricted presentation that required proteosomal
degradation and was
dependent on functional TAP (transporter associated with antigen processing)
(Regnault
et al. (1999) J. Exp. Med., 189: 371-380). Kaufinan et al. ((1996) Tumor
Target. 2: 17-
28) have shown in human clinical trials that a bispecific antibody specific
for Fc~yRI
efficiently targets monocytes and induces a host anti-tumor response in some
patients.
Furthermore, Wallace et al. ((2001) J. Immunol. Methods 248: 183-194) have
recently
demonstrated that a fusion protein based on a monoclonal antibody (mAb) that
targets
Fc~yRI, in which heavy chain CH2 and CH3 domains were removed and replaced
with the
prostate specific antigen (PSA), was internalized and processed by the human
myeloid
THP-1 cell line resulting in presentation of MHC class I-associated PSA
peptides and
lysis of THP-1 by PSA-specific human CTL.
It follows that the IgG-based complexes (e.g., immune complexes (ICs) or
chimeric IgG fusion polypeptides), which bind to FcRs are capable of
delivering non-
covalently associated peptides to MHC class I proteins of other (recipient)
cells, including
APCs. Accordingly, in one of the embodiments of the present invention, the APC
targeting sequence is derived from an IgG sequence, which mediates interaction
with the
FcR on the surface of APC (e.g., Fc~yRI).
Several receptors expressed by 'immature DCs belong to the C-type lectin
superfamily, including Langerin (CD207), the mannose receptor (MR; CD206), and
DEC-205 (CD205) (Mason, ed. In Leucocyte Typifag Yol. Yll: 2000 Oxford
University
Press, Oxford).
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Mucin (MUC1) is highly expressed in adenocarcinomas. In mice,
oxidized mannan linked to MUCl (M-FP), given in vivo, induces potent MHC-
restricted
CTL and tumor protection. Apostolopoulos et al. (Vaccine (2000) 18: 3174-84)
have
shown that murine mannose receptor (MR) bearing macrophages derived from
peritoneal
exudate cells (PEC) and cultured ex vivo with M-FP can, after adoptive
transfer,
efficiently present MUC1 to T cells, leading to the generation of high
frequency of CTL
and protection from tumor challenge. M-FP targets the MR and ensures rapid
passage of
peptides to MHC class I molecules, and can also directly stimulate ih vit~~o
IL-12
production by macrophages. In addition, targeting MR in other studies was
shown to
lead to efficient class II presentation, as after binding to the MR there is
internalization
with passage to lysosomes and phagosomes (Tan et al. (1997) Eur. J.
T_m_m__unol. 27: 2426-
2432; Engering et al. (1997) Eur. J. Immunol. 27: 2417-2425; Wileman (1985) J.
Biochem. 260: 7387-7393; Prigozy (1997) hnmunity 6: 187-193).
Mahnke et al. (J Cell Biol (2000) 151:673-84) have demonstrated in DCs
that the DEC-205 multilectin receptor targets late endosomes or lysosomes rich
in MHC
class II products, whereas the homologous macrophage mamiose receptor (MMR) is
found in more peripheral endosomes. It was concluded that the DEC-205
cytosolic
domain mediates a new pathway of receptor-mediated endocytosis that entails
efficient
recycling through late endosomes and a greatly enhanced efficiency of antigen
presentation to CD4+ T cells.
Geijtenbeek et al. ((2000) Cell 100:575) have recently identified a novel
C-type lectin, DC-specific ICAM-grabbing non-integrin (DC-SIGN; CD209), that
is
exclusively expressed on DCs, in contrast to the MR and DEC-205, which are
also
expressed on other cell types. DC-SIGN functions as cell adhesion receptor
mediating
both DC migration and T cell activation. DC-SIGN also functions as an HIV-1R
that
captures HIVgp120 and facilitates DC-induced HIV transmission of T cells.
Internalization motifs in the cytoplasmic tail of DC-SIGN hint to a function
of DC-SIGN
as endocytic receptor. Engering et al. (J Immunol (2002) 168:2118-2126) have
demonstrated that on DCs DC-SIGN is rapidly internalized upon binding of
soluble
ligand and are targeted to late endosomes/lysosomes. Moreover, ligands
internalized by
DC-SIGN are efficiently processed and presented to CD4+ T cells.
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46
It follows that at least some of C-type lectin receptors on the surface of
APCs (such as mannose receptor (MR; CD206), DEC-205 (CD205) and DC-SIGN
(CD209)) have the potency to be used for targeting antigens (e.g., TAAs)
specifically to
APCs (e.g., DCs) to induce antigen-specific immunity. Accordingly, in a
specific
embodiment, the APC targeting sequence of the invention comprises the sequence
targeting C-type lectin receptors.
Nucleic Acids and Gene Vaccines
Recombinant nucleic acids, particularly DNA molecules, provide for
efficient expression of the peptide constructs of the invention. In one
embodiment, the
invention provides a nucleic acid molecule encoding the APC-targeted peptide
construct
described above. The invention also provides an expression vector comprising
such
nucleic acid operably associated with an expression control sequences) and a
host cell
(e.g., APC transfected or transformed with the expression vector) and a
recombinant non-
human host comprising such nucleic acid. According to a specific embodiment,
the
expression vector comprising the nucleic acid encoding the epitope constructs
of the
invention may further comprise or may be combined with an APC targeting
mechanism
and may therefore be targeted directly to be expressed in APCs of the host.
The peptide
construct of the invention can be produced recombinantly by isolating it from
the host
cells grown under conditions that permit expression of the nucleic acid
encoding it.
In accordance with the present invention there may be employed
conventional molecular biology, microbiology, and recombinant DNA techniques
within
the skill of the art. Such techniques are explained fully in the literature.
See, e.g.,
Sambroolc, Fritsch & Maniatis, Molecular Clotti>zg: A Labor°atoty
Manual, Second
Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York
(herein "Sambrook et al., 1989"); DNA Cloniizg.~ A Pt°actical
Appt~oach, Volumes I and II
(Glover ed. 1985); Oligottucleotide Synthesis (Gait ed. 1984); Nucleic Acid
Hybt°idizatiott, Hames & Higgins eds. (1985); TYattscription And
Ti°anslation, Hames &
Higgins, eds. (1984); Artit~tal Cell CultuYe, Freshney, ed. (1986);
hntttobilized Cells Attd
Ettzy>Ttes, IRL Press, (1986); Perbal, A Practical Guide To Molecula>~
ClotZiftg (1984);
Ausubel et al. (eds.), CurYent Protocols irz Molecular Biology, John Wiley &
Sons, Inc.
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47
(1994); Goeddel et al., Gene ExpressiofZ Technology, Academic Press (1991);
Gacesa and
Rarnji, Irector~s: Essential Data Series, John Wiley & Sons (1994).
In a specific embodiment, vectors comprising the nucleotide sequence
encoding the peptide construct of the invention are administered to treat or
prevent a
disease or disorder associated with the expression or function of a molecule
to wluch the
construct elicits a specific immune response.
Any of the methods for gene therapy available in the art can be used
according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see, U. S. Patent Nos.
6,228,844; 5,693,622; 5,589,466; 5,580,859; 6,214,804; and 5,703,055,
International
Patent Publication Nos. WO 90/11092, WO 89/12458, WO 94/29469, EP 1026253,
Goldspiel et al., Clinical Pharmacy 1993, 12:488-505; Wu and Wu, Biotherapy
1991,
3:87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 1993, 32:573-596; Mulligan,
Science 1993, 260:926-932; and Morgan and Anderson, Ann. Rev. Biochem. 1993,
62:191-217; and May, TIBTECH 1993, 11:155-215. Methods commonly l~nown in the
art of recombinant DNA technology that can be used are described in Ausubel et
al.,
(eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY;
Kriegler,
1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;
and in
Chapters 12 and 13, Dracopoli et al., (eds.), 1994, Current Protocols in Human
Genetics,
John Wiley & Sons, NY. Vectors suitable for gene therapy are described above.
The present invention also provides delivery vehicles suitable for delivery
of a nucleic acid molecule of the invention into cells (whether in vivo, ex
vivo, or isz
vitro). The nucleic acid molecule of the invention can be contained within a
cloning or
expression vector. These vectors (especially expression vectors) can in turn
be
manipulated to assume any of a number of forms which may, for example,
facilitate
delivery to and/or entry into a cell.
When the vectors are used for gene . therapy iya vivo or ex vivo, a
pharmaceutically acceptable vector is preferred, such as a replication-
incompetent
retroviral or adenoviral vector. Pharmaceutically acceptable vectors
containing the
nucleic acids of this invention can be further modified for transient or
stable expression
of the inserted polynucleotide.
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Host cells containing the nucleic acids of the invention are useful for the
recombinant replication of the polynucleotides and for the recombinant
production of
peptides. Alternatively, the cells may be used to induce an immune response in
a subject
in the methods described herein. When the host cells are APCs, they can be
used to
expand a population of immune effector cells such as tumor infiltrating
lymphocytes
(TILs), which in turn are useful in adoptive inmnunotherapies.
In one aspect, the therapeutic vector comprises a nucleic acid molecule
that expresses the construct in a suitable host. In particular, such a vector
has a promoter
operably linked to the coding sequence for the peptide epitope construct. The
promoter
can be inducible or constitutive and, optionally, tissue-specific. In another
embodiment, a
nucleic acid molecule is used in which the antibody coding sequences and any
other
desired sequences are flanked by regions that promote homologous recombination
at a
desired site in the genome, thus providing for expression of the construct
from a nucleic
acid molecule that has integrated into the genome (Koller and Smithies, Proc.
Natl. Acad.
Sci. USA 1989, 86:8932-8935; Zijlstra et al., Nature 1989, 342:435-438).
Delivery of the vector into a patient may be either direct, in which case the
patient is directly exposed to the vector or a delivery complex, or indirect,
in which case,
cells are first transformed with the vector ifa vitf°~ then
transplanted into the patient.
These two approaches are lmown, respectively, as iya vivo and ex vivo gene
therapy.
In a specific embodiment, the vector is directly administered in vivo,
where it enters the cells of the organism and mediates expression of the
constructs. This
can be accomplished by any of numerous methods known in the art, e.g., by
constructing
it as part of an appropriate expression vector and administering it so that it
becomes
intracellular, e.g., by infection using a defective or attenuated retroviral
or other viral
vector (see, U.S. Patent No. 4,980,286), or by direct injection of naked DNA,
or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); or coating
with lipids or
cell-surface receptors or transfecting agents, encapsulation in biopolymers
(e.g., poly-
~31~4-N-acetylglucosamine polysaccharide; see, U.S. Patent No. 5,635,493),
encapsulation in liposomes, microparticles, or microcapsules; by administering
it in
linkage to a peptide or other ligand known to enter the nucleus; or by
administering it in
linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J.
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49
Biol. Chem. 1987, 62:4429-4432), etc. In another embodiment, a nucleic
acid/ligand
complex can be formed in which the ligand comprises a fusogenic viral peptide
to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet
another
embodiment, the nucleic acid can be targeted in vivo for cell specific uptake
and
expression, by targeting a specific receptor (see, e.g., PCT Publication Nos.
WO
92/06180, WO 92/22635, WO 92/20316 and WO 93/14188). These methods are in
addition to those discussed above in conjunction with "Viral and Non-viral
Vectors".
The form and amount of therapeutic nucleic acid envisioned for use
depends on the type of disease and the severity of the desired effect, patient
state, etc.,
and can be determined by one skilled in the art.
Therapeutic Use of the Constructs of the Invention
The invention also provides methods for treating a disease by
administration of a therapeutic of the invention. Such therapeutics include
the peptide
constructs of the invention and nucleic acids encoding the constructs of the
invention.
In the disclosed compositions, the peptide constructs of the invention or
the nucleic acids encoding the constructs of the invention are present in
immunogenically
effective amount. For each specific antigen, the immunogenically effective
amount is
readily determined experimentally (talcing into consideration specific
characteristics of a
given patient and/or type of treatment) using well-known methods. Generally,
this
amount is in the range of 0.1 ~g-100mg of an antigen per kg of the body
weight.
The subjects to which the present invention is applicable may be any
mammalian or vertebrate species that include, but are not limited to, cows,
horses, sheep,
pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice, rats, moneys,
rabbits,
chimpanzees, and humans. In a preferred embodiment, the subject is a human.
Tmmunogenicity enhancing methods of the invention can be used to
combat various cancers, which include without limitation fibrosarcoma,
myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio-sarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon
carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma,
leukemia,
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squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
hepatocarcinoma, sweat
gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic
carcinoma,
renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,
seminoma,
embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma,
small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma, among others.
Methods of the invention are also useful in treatment of infections, which
include, but are not limited to, parasitic infections (such as those caused by
plasmodia)
species, etc.), viral infections (such as those caused by influenza viruses,
leukemia
viruses, immunodeficiency viruses such as HIV, papilloma viruses, herpes
virus, hepatitis
viruses, measles virus, poxviruses, mumps virus, cytomegalovirus [CMV],
Epstein-Barr
virus, etc.), bacterial infections that involve MHC class I (such as those
caused by
staphylococcus, streptococcus, pneumococcus, Neisse~ia gonory~hea, Bornelia,
pseudomonas, mycobacteria, Salrrzoraella, etc.), and fungal infections (such
as those
caused by Can.dida, Ti~ieh~plzyton, Pty~sponzdm, etc.).
Methods of the invention are also useful in treatment of autoimmune
diseases, which include, but are not limited to, most forms of arthritis,
ulcerative colitis,
asthma, multiple sclerosis, lupus, and myasthenia gravis.
Tieatmerzt arid Pfeverztiorz of Cancers
In a preferred embodiment, the invention provides methods of treating or
preventing cancers. The method includes administering to a subject in need of
such
treatment or prevention a peptide construct of the invention or a nucleic
acids encoding it.
Cancers, including any disease or disorder characterized by uncontrolled
cell growth, in which the tumor cells express a tumor associated antigen as
described
herein having immunogenic properties relevant to human cancers, can be treated
or
prevented by administration of a construct of the invention. Whether a
particular
therapeutic is effective to treat or prevent a certain type of cancer can be
determined by
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any method known in the art.
In other embodiments of the invention, the subject being treated with the
construct may, optionally, be treated with other cancer treatments such as
surgery,
radiation therapy, or chemotherapy. In particular, the therapeutic of the
invention used to
treat or prevent cancer may be administered in conjunction with one or a
combination of
chemotherapeutic agents including, but not limited to, methotrexate, taxol,
taxotere,
mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,
ifosfamide,
nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine,
etoposides,
campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,
plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine,
paclitaxel,
docetaxel, etc.
Vaccine F~t~azulatio~zs asad Ady~air~ists~atiorz
The invention also provides vaccine formulations containing peptide
constructs of the invention or nucleic acids encoding them, which vaccine
formulations
are suitable for administration to elicit a protective immune (humoral and/or
cell
mediated) response against cancer cells bearing a ligand as described herein,
e.g., for the
treatment and prevention of diseases.
The peptide constructs of the invention or nucleic acids encoding them can
be used in a variety of formulations, which may vary depending on the intended
use.
The peptide constructs of the invention or nucleic acids encoding them can
be covalently or non-covalently linked (complexed) to various other molecules,
the
nature of which may vary depending on the particular purpose. For example, a
peptide of
the invention can be covalently or non-covalently complexed to a
macromolecular
carrier, including, but not limited to, natural and synthetic polymers,
proteins,
polysaccharides, poly(amino acid), polyvinyl alcohol, polyvinyl pyrrolidone,
and lipids.
A peptide can be conjugated to a fatty acid, for introduction into a liposome.
U.S. Pat.
No. 5,837,249. A synthetic peptide of the invention can be complexed
covalently or non-
covalently with a solid support, a variety of which are known in the art. A
synthetic
antigenic peptide epitope of the invention can be associated with an antigen-
presenting
matrix with or without co-stimulatory molecules, as described in more detail
below.
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Examples of protein carriers include, but are not limited to, superantigens,
serum albumin, tetanus toxoid, ovalburnin, thyroglobulin, myoglobulin, and
immmzoglobulin.
Peptide-protein carrier polymers may be formed using conventional
crosslinking agents such as carbodiimides. Examples of carbodiimides are 1-
cyclohexyl-
3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), 1-ethyl-3-(3-
dimethyaminopropyl)
carbodiimide (EDC) and 1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.
Examples of other suitable crosslinking agents are cya~logen bromide,
glutaraldehyde and succinic anhydride. In general, any of a number of
homobifunctional
agents including a homobifunctional aldehyde, a homobifunctional epoxide, a
homobifmctional imidoester, a homobifinctional N-hydroxysuccinimide ester, a
homobifinctional maleimide, a homobifunctional alkyl halide, a
homobifunctional
pyridyl disulfide, a homobifinctional aryl halide, a homobifimctional
hydrazide, a
homobi functional diazonium derivative and a homobifinctional photoreactive
compound
may be used. Also included are heterobifunctional compounds, for example,
compounds
having an amine-reactive and a sulfhydryl-reactive group, compounds with an
amine-
reactive and a photoreactive group and compounds with a carbonyl-reactive and
a
sulfhydryl-reactive group.
specific examples of such homobifunctional crosslinking agents include
the bifunctional N-hydroxysuccinimide esters
dithiobis(succinimidylpropionate),
disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional
imidoesters
dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the
bifunctional sulfliydryl-reactive crosslinkers 1,4-di-[3'-(2'-pyridyldithio)
propion-
amido]butane, bismaleimidohexane, and bis-N-maleimido-1,8-octane; the
bifunctional
aryl halides 1,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'-
dinitrophenylsulfone;
bifunctional photoreactive agents such as bis-[b-(4-
azidosalicylamido)ethyl]disulfide; the
bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde,
glutaraldehyde,
and adipaldehyde; a bifunctional epoxide such as 1,4-butaneodiol diglycidyl
ether, the
bifunctional hydrazides adipic acid dihydrazide, carbohydrazide, and succinic
acid
dihydrazide; the bifunctional diazoniums o-tolidine, diazotized and bis-
diazotized
benzidine; the bi:functional alkylhalides N1N'-ethylene-bis(iodoacetamide),
N1N'-
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53
hexamethylene-bis(iodoacetamide), N1N'-undecamethylene-bis(iodoacetamide), as
well
as benzylhalides and halomustards, such as ala'-diiodo-p-xylene sulfonic acid
and tri(2-
chloroethyl)amine, respectively.
Examples of other common heterobifun.ctional cross-linking agents that
may be used to effect the conjugation of proteins to peptides include, but are
not limited
to, SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MBS (m-
maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4-
iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate),
GMBS (N-(.gamma.-rnaleimidobutyryloxy)succinimide ester), MPBH (4-(4-N-
maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl)
cyclohexane-1-carboxyl-hydrazide), SMPT (succinimidyloxycarbonyl-a-methyl-a-(2-
pyridyldithio)toluene), and SPDP(N-succinimidyl 3-(2-
pyridyldithio)propionate).
Crosslinl~ing may be accomplished by coupling a carbonyl group to an
amine group or to a hydrazide group by reductive arnination.
Peptide constructs of the invention also may be formulated as non-
covalent attachment of monomers through ionic, adsorptive, or biospecific
interactions.
Complexes of peptides with highly positively or negatively charged molecules
may be
done through salt bridge formation under low ionic strength environments, such
as in
deionized water. Large complexes can be created using charged polymers such as
poly-
(L-glutamic acid) or poly-(L-lysine) which contain numerous negative and
positive
charges, respectively. Adsorption of peptides may be done to surfaces such as
microparticle latex beads or to other hydrophobic polymers, forming non-
covalently
associated peptide-superantigen complexes effectively mimicking crosslinlced
or
chemically polymerized protein. Finally, peptides may be non-covalently linked
through
the use of biospecific interactions between other molecules. For instance,
utilization of
the strong affinity of biotin for proteins such as avidin or streptavidin or
their derivatives
could be used to form peptide complexes. These biotin-binding proteins contain
four
binding sites that can interact with biotin in solution or be covalently
attached to another
molecule. Wilchek (1988) Anal Biochem. 171:1-32 F~~udes can be modified to
possess
biotin groups using common biotinylation reagents such as the N-
hydroxysuccinimidyl
ester of D-biotin (NHS-biotin) which reacts with available amine groups on the
protein.
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Biotinylated peptides then can be incubated with avidin or streptavidin to
create large
complexes. The molecular mass of such polymers can be regulated through
careful
control of the molar ratio of biotinylated peptide to avidin or streptavidin.
The peptide constructs of this invention also can be combined with various
liquid phase carriers, such as sterile or aqueous solutions, pharmaceutically
acceptable
carriers, suspensions and emulsions. Examples of non-aqueous solvents include
propyl
ethylene glycol, polyethylene glycol and vegetable oils. When used to prepare
antibodies,
the carriers also can include an adjuvant that is useful to non-specifically
augment a
specific immune response. A skilled artisan can easily determine whether an
adjuvant is
required and select one. However, for the purpose of illustration only,
suitable adjuvants
include, but are not limited to, Freund's Complete and Incomplete, mineral
salts and
polynucleotides.
Suitable preparations of such vaccines and therapeutics include
injectables, either as liquid solutions or suspensions; solid forms suitable
for solution in,
suspension in, liquid prior to injection, may also be prepared. The
preparation may also
be emulsified, or the constructs antibodies encapsulated in liposomes. The
active
immunogenic ingredients are often mixed with excipients which are
pharmaceutically
acceptable and compatible with the active ingredient. Suitable excipients are,
for
example, water, saline, buffered saline, dextrose, glycerol, ethanol, sterile
isotonic
aqueous buffer or the like and combinations thereof. In addition, if desired,
the vaccine
preparation may also include minor amounts of auxiliary substances such as
wetting or
emulsifying agents, pH buffering agents, and/or adjuvants that enhance the
effectiveness
of the vaccine. The construct when prepared as a vaccine can be introduced in
microspheres or microcapsules, e.g., prepared from PGLA (see, U.S. Patent Nos.
5,814,344, 5,100,669, and 4,849,222; PCT Publication Nos. WO 95/11010 and WO
93/07861).
The effectiveness of an adjuvant may be determined by measuring the
induction of an immune response directed against the targeted antigen.
The composition, vaccines, and therapeutics can be a liquid solution,
suspension, emulsion, tablet, pill, capsule, sustained release formulation, or
powder. For
oral administration, the therapeutics can take the form of, for example,
tablets or capsules
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prepared by conventional means with pharmaceutically acceptable excipients
such as
binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl
methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or
calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato
starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl
sulphate). The
tablets can be coated by methods well known in the art. Liquid preparations
for oral
administration can take the form of, for example, solutions, syrups, emulsions
or
suspensions, or they can be presented as a dry product for constitution with
water or other
suitable vehicle before use. Such liquid preparations can be prepared by
conventional
means with pharmaceutically acceptable additives such as suspending agents
(e.g.,
sorbitol syrup, cellulose derivatives or hydrogenated edible fats);
emulsifying agents
(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily
esters, ethyl alcohol
or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-
hydroxybenzoates or sorbic acid). The preparations can also contain buffer
salts,
flavoring, coloring and sweetening agents as appropriate.
Generally, the ingredients are supplied either separately or mixed together
in unit dosage form, for example, as a dry lyophilized powder or water-free
concentrate
in a sealed container such as a vial or sachette indicating the quantity of
active agent.
Where the composition is administered by injection, an ampoule of sterile
diluent can be
provided so that the ingredients may be mixed prior to administration.
In a specific embodiment, the lyophilized construct of the invention is
provided in a first container; a second container comprises diluent consisting
of an
aqueous solution of 50% glycerin, 0.25% phenol, and an antiseptic (e.g.,
0.005% brilliant
green).
The invention also provides a pharmaceutical pack or kit comprising one
or more containers filled with one or more of the ingredients of the vaccine
formulations
of the invention. Associated with such containers) can be a notice in the form
prescribed
by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or
biological products, which notice reflects approval by the agency of
manufacture, use or
sale for human administration.
Many methods may be used to introduce the vaccine formulations of the
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56
invention; these include but are not limited to oral, intracerebral;
intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal routes,
and via
scarification (scratching through the top layers of skin, e.g., using a
bifurcated needle) or
any other standard routes of immunization.
Effective Dose and Safety Evaluations
According to the methods of the present invention, the pharmaceutical and
vaccine compositions described herein are administered to a patient at
immunogenically
effective doses, preferably, with minimal toxicity. As recited in the Section
entitled
"Definitions", "immunogenically effective dose" or "therapeutically effective
dose" of
disclosed formulations refers to that amount of an antigen-containing
composition that is
sufficient to produce an effective immune response in the treated subject and
therefore
sufficient to result in a healthful benefit to said subject.
Following methodologies which are well-established in the art (see, e.g.,
reports on evaluation of several vaccine formulations in a collaborative
effort between the
Center for Biological Evaluation and Food and Drug Administration and the
National
Institute of Allergy and Infectious Diseases [Goldenthal et al., National
Cooperative
Vaccine Development Working Group. AIDS Res. Hum. Retroviruses, 1993, 9:545-
5490, effective doses and toxicity of the compounds and compositions of the
instant
invention are first determined in preclinical studies using small animal
models (e.g.,
mice) in which these compounds and compositions have been found to be
immunogenic
and that can be reproducibly immunized by the same route proposed for the
human
clinical trials.
In a specific embodiment, the efficiency of epitope-specific CD8+ T cell
responses to the pharmaceutical and vaccine compositions of the invention is
determined
by the enzyme-linked immunospot technique (ELISPOT). ELISPOT is a standard
method in the art originally developed by the present inventors a~zd their co-
workers
(Miyahira et al., J. Izn~nunol. Meth., 181: 45-54, 1995) and widely used by
others (see,
e.g., Guelly et al., Eur. J. Tmmunol., 32:182-192, 2002; Nilcitina and
Gabrilovich, Int. J.
Cancer, 94:825-833, 2001; Field et al., Immunol. Rev., 182:99-112, 2001;
Altfeld et al.,
J. Tm_m__unol., 167:2743-2752, 2001; Skoberne et al., J. Tm_m__unol., 167:2209-
2218, 2001).
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This method employs pairs of antibodies, directed against distinct epitopes of
a cytokine,
and allows the visualization of cytokine secretion by individual T cells
following in vitro
stimulation with an antigen. ELISPOT has the advantage of detecting only
activated/memory T cells and the cytolcine release can be detected at the
single cell
levels, allowing direct determination of T cell frequencies (Czerkinsky et
al., J. T_mmunol.
Methods, 25:29, 1988; Taguchi et al., J. Tm_m__unol. Methods, 128:65, 1990).
The cytol~ine
captured by the immobilized antibody in the ELISPOT assay is detected i~a situ
using an
insoluble peroxidase substrate. Thus, the cytokine secretion by individual
cells is clearly
visualized. The high sensitivity and easy performance, allowing a direct
enumeration of
peptide-reactive T cells without prior in vitro expansion, make the ELISPOT
assay
eminently well suited to moutor and measure T cell responses, particularly,
CD8+ T cell
responses of very low frequencies. According to alternative embodiments, the
efficiency
of epitope-specific CD8+ T cell responses to the pharmaceutical and vaccine
compositions of the invention can be determined using other art-recognized
irmnmiodetection methods such as, e.g., ELISA (Tanguay and Million,
Lympholcine
Cytolcine Res., 13:259, 1994) and intracellular staining (Carter and Swain,
Curr. Opin.
T_m_m__unol, 9:1977, 1997).
As disclosed herein, for any pharmaceutical composition or vaccine used
in the methods of the invention, the therapeutically effective dose can be
estimated
initially from animal models to achieve a circulating plasma concentration
range that
includes the ICSO (i.e., the concentration of the test compound which achieves
a half
maximal inhibition of symptoms). Dose-response curves derived from animal
systems
are then used to determine testing doses for the initial clinical studies in
humans. W
safety determinations for each composition, the dose and frequency of
immunization
should meet or exceed those anticipated for use in the clinical trial.
The dose of peptide constructs, nucleic acids encoding them and other
components in the compositions of the present invention is determined to
ensure that the
dose administered ,continuously or intermittently will not exceed a certain
amount in
consideration of the results in test animals and the individual conditions of
a patient. A
specific dose naturally varies depending on the dosage procedure, the
conditions of a
patient or a subject animal such as age, body weight, sex, sensitivity, feed,
dosage period,
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58
drugs used in combination, seriousness of the disease. The appropriate dose
and dosage
times under certain conditions can be determined by the test based on the
above-
described indices and should be decided according to the judgment of the
practitioner and
each patient's circumstances according to standard clinical techniques. In
this
connection, the dose of an antigen is generally in the range of 0.1 ~,g-100 mg
per kg of
the body weight.
Toxicity and therapeutic efficacy of immunogenic compositions of the
invention can be determined by standard pharmaceutical procedures in
experimental
animals, e.g., by determining the LDSO (the dose lethal to 50% of the
population) and the
EDSO (the dose therapeutically effective in 50% of the population). The dose
ratio
between toxic and therapeutic effects is the therapeutic index and it can be
expressed as
the ratio LDsn/EDSO. Compositions that exhibit large therapeutic indices are
preferred.
While therapeutics that exhibit toxic side effects can be used (e.g., when
treating severe
forms of cancer or life-threatening infections), care should be taken to
design a delivery
system that targets such immunogenic compositions to the specific site (e.g.,
a tumor or
an organ supporting replication of the infectious agent) in order to minimize
potential
damage to other tissues and organs and, thereby, reduce side effects. As
disclosed herein,
the constructs of the invention are not only highly irnlnunostimulating at
relatively low
doses (e.g., 0.1-100 ~,g per kg of the body weight) but also possess low
toxicity and do
not produce significant side effects.
As specified above, the data obtained from the animal studies can be used
in formulating a range of dosages for use in humans. The therapeutically
effective
dosage of compositions of the present invention in humans lies preferably
within a range
of circulating concentrations that include the EDSO with little or no
toxicity. The dosage
can vary within this range depending upon the dosage fore employed and the
route of
administration utilized. Ideally, a single dose should be used.
Ti'YAMPT.Ti
The following Example illustrates the invention without limiting its scope.
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59
Example 1: Construction of Epitope String Containing Amino Acid Sequences
from CEA
Carcinoembryonic antigen (CEA) is a tumor associated aaxtigen expressed
by many types of tumor cells (Thomas et al., Biochem. Biophys. Acta, 1032: 177-
79
(1990)). Amino acid sequence of CEA (SEQ JD NO:1; GenBank Accession No.
AAA62835) is provided below:
MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLP
QHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTG
FYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYL
WWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYG
PDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSG
SYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTY
LWWVIIRSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGTQNELSVDHSDPVILNVLY
GPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNS
GLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTT
YLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVL
YGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRTNGIPQQHTQVLLIAKIQPNN
NGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATAGIMIGVLVGVALI
An epitope string containing CTL epitopes of human CEA is constructed
with tlxe standard cloning steps and conditions.
The following CEA epitopes are incorporated into epitope string
constructs:
Seguence Seguence
Type
CTL CTL CEA1: YLSGANLNL (SEQ ID NO: 2), Zaremba S.,
et al. (1997)
Cancer Research 57(20), 4570-4577.
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CTL CEA2: HLFGYSWYK (SEQ ID NO: 3), Kawashima L,
et al.
(1999) Cancer Research 59(2), 431-435.
CTL CEA3: IPQQHTQVL (SEQ ID NO: 4), Lu J. and Celis
E. (2000)
Cancer Research 60(18), 5223-5227.
T-helper HIV gp120, as 421-444
epitope KQIINMWQEVGKAMYAPPISGQIR (SEQ ID NO: 5)
B-cell Amino acids 524-57 of the A3 domain; as 1-107 of
the N-domain of
epitope CEA (SEQ ~ NO: 1)
The CEA epitope sequences are then linked to the appropriate flanking
sequences and the resulting epitope string construct is linked at the C-
terminus to the
CD91 APC targeting sequence comprising the gp96 sequence which interacts with
the N-
terminal p80 fragment of the alpha subunit of CD91, receptor for heat shock
proteins (see
Binder et al., Nature hnmunol. 1: 151-55 (2000)).
The final epitope string construct may be represented by the following
general formula:
N-[Leu-Xaa-Xaa-Asp-Xaa-Xaa-Pro] [Xaa-Lys-Xaa-Lys-Phe]-[CEA epitope 1]- [Leu-
Xaa-Xaa-Asp-Xaa-Xaa-Pro] [Xaa-Lys-Xaa-Lys-Phe]-[CEA epitope 2]-...-[CEA
epitope n]-[Leu-Xaa-Xaa-Asp-Xaa-Xaa-Pro] [Xaa-Lys-Xaa-Lys-Phe]-[CD91
targeting sequence from gp96]-C
wherein n represents the number of epitopes and Xaa represents any amino acid.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the invention
in
addition to those described herein will become apparent to those skilled in
the art from
the foregoing description and the accompanying figures. Such modifications are
intended
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61
to fall within the scope of the appended claims.
All patents, applications, publications, test methods, literature, and other
materials cited herein are hereby incorporated by reference.
