U.S. patent number 7,867,977 [Application Number 12/092,449] was granted by the patent office on 2011-01-11 for immunogenic peptides and methods of use for treating and preventing cancer.
This patent grant is currently assigned to N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Jay A. Berzofsky, Lee J. Helman, Crystal MacKall, Leon T. Van Den Broeke.
United States Patent |
7,867,977 |
Berzofsky , et al. |
January 11, 2011 |
Immunogenic peptides and methods of use for treating and preventing
cancer
Abstract
Disclosed are immunogenic peptides, related fusion proteins,
nucleic acids encoding the peptides or fusion proteins, conjugates,
expression vectors, host cells, and antibodies. Also, disclosed are
pharmaceutical compositions, vaccines for use in the treatment or
prevention of cancer, e.g., alveolar rhabodomyosarcoma, methods of
stimulating a T cell to kill a tumor cell, methods of stimulating
CD4.sup.+ and CD8.sup.+ T cells, and methods of treating or
preventing cancer are further provided herein.
Inventors: |
Berzofsky; Jay A. (Bethesda,
MD), Van Den Broeke; Leon T. (Zoutelande, NL),
MacKall; Crystal (Silver Spring, MD), Helman; Lee J.
(Bethesda, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (N/A)
|
Family
ID: |
37708288 |
Appl.
No.: |
12/092,449 |
Filed: |
October 24, 2006 |
PCT
Filed: |
October 24, 2006 |
PCT No.: |
PCT/US2006/041462 |
371(c)(1),(2),(4) Date: |
October 06, 2008 |
PCT
Pub. No.: |
WO2007/055902 |
PCT
Pub. Date: |
May 18, 2007 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090117116 A1 |
May 7, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60733319 |
Nov 3, 2005 |
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Current U.S.
Class: |
514/19.3;
530/328; 514/19.6 |
Current CPC
Class: |
A61P
35/00 (20180101); C07K 14/70539 (20130101); C07K
14/4748 (20130101); C07K 16/2833 (20130101); C07K
7/06 (20130101); A61K 39/0011 (20130101) |
Current International
Class: |
A61K
38/00 (20060101) |
References Cited
[Referenced By]
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WO 93/20185 |
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Oct 1993 |
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WO |
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WO 97/10269 |
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Mar 1997 |
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WO |
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WO02069900 |
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Sep 2002 |
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WO |
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generation vaccines," Nat. Rev. Immunol., 1 (3), 209-219 (2001).
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subunit-rotavirus VP7 fusion protein in transgenic potato," Mol.
Biotechnol., 31 (3), 193-202 (2005). cited by other .
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298, 209-223 (2005). cited by other .
Kirin et al., "Amino acid and peptide bioconjugates of copper(II)
and zinc(II) complexes with a modified N,N-bis(2-picolyl)amine
ligand," Inorg. Chem., 44 (15), 5405-5415 (2005). cited by other
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Sarobe et al., "Enhanced in vitro potency and in vivo
immunogenicity of a CTL epitope from hepatitis C virus core protein
following amino acid replacement at secondary HLA-A2.1 binding
positions," J. Clin. Invest., 102 (6), 1239-1248 (1998). cited by
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peptides but affect their extracellular antigen processing," Int.
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Acad. Sci. USA, 86 (7), 2361-2364 (1999). cited by other.
|
Primary Examiner: Huff; Sheela J
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Claims
The invention claimed is:
1. An isolated immunogenic peptide comprising the amino acid
sequence SPQNSIRHNL (SEQ ID NO: 3), a functional portion or
functional variant thereof, or a pharmaceutically acceptable salt
thereof, wherein the peptide, or functional portion or functional
variant thereof, binds to an MHC Class I molecule, provided that
the peptide does not consist of the amino acid sequence
TIGNGLSPQNSIRHNLSL (SEQ ID NO: 4) or NPTGTIGNGLSPQNSIRHNLSLH (SEQ
ID NO: 5), wherein the functional portion comprises 50% or more of
the immunogenic peptide comprising SEQ ID NO: 3, wherein the
functional variant comprises one conservative amino acid
substitution, and wherein the peptide consists of 8 to 10 amino
acids.
2. An immunogenic peptide comprising the amino acid sequence
SPX.sub.1NX.sub.2X.sub.3RHNL (SEQ ID NO: 6), wherein X.sub.1 is any
amino acid except for Gln, X.sub.2 is any amino acid except for
Ser, and X.sub.3 is any amino acid except for Ile, a functional
portion, or a pharmaceutically acceptable salt thereof, wherein the
peptide or functional portion thereof binds to an MHC Class I
molecule, wherein the functional portion comprises 50% or more of
the immunogenic peptide comprising SEQ ID NO: 3.
3. The immunogenic peptide of claim 1, wherein the functional
variant has enhanced ability to bind to the MHC Class I molecule as
compared to a peptide that lacks one conservative amino acid
substitution.
4. The immunogenic peptide of claim 1, wherein the MHC Class I
molecule is HLA-B7.
5. The immunogenic peptide of claim 1, wherein the immunogenic
peptide stimulates cytotoxic T lymphocytes.
6. The immunogenic peptide of claim 5, wherein the immunogenic
peptide stimulates cytotoxic T lymphocytes to kill tumor cells.
7. The immunogenic peptide of claim 6, wherein the tumor cells are
alveolar rhabdomyosarcoma cells.
8. The immunogenic peptide of claim 2, wherein each of X.sub.1,
X.sub.2 and X.sub.3 is independently any small, aliphatic amino
acid.
9. The immunogenic peptide of claim 8, wherein X.sub.1 is Ser, Thr,
or Ala; X.sub.2 is Thr or Ala; and X.sub.3 is Ser, Thr, or Ala.
10. The immunogenic peptide of claim 2, wherein the peptide is
selected from the group consisting of: SPANSIRHNL (SEQ ID NO: 7);
SPQNAIRHNL (SEQ ID NO: 8); and SPQNSARHNL (SEQ ID NO: 9).
11. The immunogenic peptide of claim 1, wherein the peptide is
SPQNSIRHNL (SEQ ID NO: 3).
12. A fusion protein comprising the immunogenic peptide of claim 1
and an MHC Class I molecule, or a functional portion thereof.
13. The fusion protein of claim 12, wherein the MHC Class I
molecule is HLA-B7.
14. A conjugate comprising the immunogenic peptide of claim 1
conjugated to a targeting moiety.
15. A pharmaceutical composition comprising the peptide of claim 1
and a pharmaceutically acceptable carrier.
16. A pharmaceutical composition comprising the fusion protein of
claim 12 and a pharmaceutically acceptable carrier.
17. A pharmaceutical composition comprising the conjugate of claim
14 and a pharmaceutically acceptable carrier.
Description
BACKGROUND OF THE INVENTION
Most tumors express mutated or inappropriately expressed,
nonmutated tumor-associated antigens (TAAs) that often contain
cytotoxic T lymphocyte (CTL) epitopes. Yet, the immune system often
remains incapable of overtaking the growth potential of the
malignant cells. Many approaches have been attempted to obtain
protective and therapeutic anti-tumor immunity. However, for some
of these approaches, limited success was observed (Dagher et al.,
Med Pediatr Oncol 38: 158-164 (2002); and Rodeberg et al., Cancer
Immuno Immunother 54: 526-534 (2005)).
The present invention seeks to overcome the aforementioned problems
by providing immunogenic peptides, dendritic cells presenting the
immunogenic peptides, and methods of treating and preventing
cancer. These and other advantages of the invention, as well as
additional inventive features, will be apparent from the
description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides immunogenic peptides which bind to a
Major Histocompatibility Complex (MHC) Class I molecule, e.g.,
HLA-B7. Fusion proteins and conjugates comprising at least one of
the inventive immunogenic peptides described herein are also
provided by the present invention.
The present invention further provides nucleic acids encoding any
of the inventive immunogenic peptides or fusion proteins described
herein, expression vectors comprising the nucleic acids, and host
cells comprising the vectors. Isolated antibodies, or antigen
binding portions thereof, that bind to any of the inventive
immunogenic peptides described herein are furthermore provided by
the present invention.
Pharmaceutical compositions comprising any of the inventive
immunogenic peptides, fusion proteins, conjugates, nucleic acids,
expression vectors, host cells, or antibodies, and a
pharmaceutically acceptable carrier, are provided herein. Also,
vaccines comprising any of the inventive immunogenic peptides,
fusion proteins, conjugates, nucleic acids, expression vectors, or
host cells are provided.
Methods of stimulating a T cell to kill a tumor cell, methods of
stimulating CD4.sup.+ and CD8.sup.+ T cells, as well as methods of
treating or preventing cancer, are further provided by the present
invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
FIG. 1 depicts the CTL lytic activity at different effector
cell:target cell (E:T) ratios. The effector cells are the CTL
generated in Example 2 described herein, while the target cells are
C1R-B7 cells pulsed with RS10 peptide (squares) or control peptide
(triangles).
FIG. 2 depicts the CTL lytic activity at different E:T ratios. The
effector cells are the CTL generated in Example 2 described herein,
while the target cells are C1R-B7 cells pulsed with RS10 peptide
(squares) or control peptide (triangles). Anti-HLA-B7 antibody used
at 10% v/v (open circles) or 20% v/v (closed circles).
FIG. 3 depicts the CTL lytic activity at different E:T ratios. The
effector cells are the CTL generated in Example 2 described herein,
while the target cells are rhabdomyosarcoma cells expressing HLA-B7
(Rh5; squares), or two control cell lines, RD (triangles) or CTR
(circles), which do not express HLA-B7.
FIG. 4 depicts the fluorescence index at different concentrations
of RS10 peptide (squares) or RS10-3A mutant peptide
(triangles).
FIG. 5 depicts the CTL lytic activity at different E:T ratios,
wherein the effector cells are the CTL generated in Example 2
described herein, and the target cells are C1R-B7 cells pulsed with
either RS10-3A peptide (squares) or control peptide (SS1;
triangles).
FIG. 6 depicts the fluorescence index at different concentrations
of RS10 peptide (squares), RS10-3A mutant peptide (triangles),
RS10-5A mutant peptide (circles), or RA10-6A mutant peptide
(diamonds).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to immunogenic peptides. In one
embodiment, each of the peptides has an amino acid sequence based
on the PAX3-FKHR fusion protein breakpoint region. The amino acid
and nucleotide sequences of the PAX3-FKHR fusion protein are known
in the art (SEQ ID NOs: 1 and 2, respectively) (See, for instance,
Galili et al., Nature Genetics 5: 230-235 (1993), and Shapiro et
al., Cancer Research 6: 5108-5112 (1993)).
In a preferred embodiment, the present inventive immunogenic
peptide comprises the amino acid sequence SPQNSIRHNL (SEQ ID NO:
3), but does not consist of the amino acid sequence
TIGNGLSPQNSIRHNLSL (SEQ ID NO: 4) or NPTGTIGNGLSPQNSIRHNLSLH (SEQ
ID NO: 5).
In another embodiment, the present inventive immunogenic peptide
comprises the amino acid sequence SPX.sub.1NX.sub.2X.sub.3RHNL (SEQ
ID NO: 6), wherein X.sub.1 is any amino acid except for Gln,
X.sub.2 is any amino acid except for Ser, and X.sub.3 is any amino
acid except for Ile.
The present inventive immunogenic peptides have one or more
attractive properties. The peptides bind to an MHC Class I
molecule. The MHC Class I molecule to which the peptide binds can
be any MHC Class I molecule known in the art (see, for example,
Janeway et al., Immunobiology: The Immune System in Health and
Disease, 4.sup.th ed., Current Biology Publications: Garland
Publishing, New York, N.Y., 1999). The MHC Class I molecule can,
for example, be an MHC Class I molecule of any mammal, e.g., mouse,
rat, human rabbit, etc. Suitable MHC Class I molecules include, for
example, HLA-A, -B, and --C molecules, such as HLA-B7, -B8, B44,
-A2, -A3, -A11, -A31, and -C1. Preferably, the MHC Class I molecule
is HLA-B7. As one of ordinary skill in the art appreciates, it is
possible for the inventive immunogenic peptides to bind to more
than one MHC Class I molecule or to both an MHC Class I molecule
and an MHC Class II molecule. Immunogenic peptides having such
dual- or multi-specificities for MHC molecules are included within
the scope of the invention.
Methods of determining whether a given peptide binds to an MHC
Class I molecule are known in the art, and include, for instance,
binding assays, such as Far Western binding assays, surface plasmon
resonance binding assays, and the binding assay as illustrated in
Example 1.
Desirably, the present inventive immunogenic peptides or portions
thereof not only bind to an MHC Class I molecule, but also
stimulate CD8.sup.+ T cells, e.g., cytotoxic T lymphocytes (CTL).
By "stimulate" in the context of T cells is meant activating
intracellular signaling pathways in a T cell through the
antigen-specific T cell receptor (TCR) expressed on that T cell,
which activation leads to one or more T cell responses, such as T
cell proliferation, cytolytic activity, and cytokine production,
e.g., IFN-.gamma.. Preferably, the T cells are stimulated by the
present inventive peptides to kill or lyse a target cell, which
presents the peptide recognized by the TCR of the T cell.
Desirably, the target cell is a tumor cell. Also, in some
instances, it is preferable for the peptides to stimulate CD4.sup.+
T cells, in addition to CD8.sup.+ T cells. Stimulation of CD4.sup.+
T cells by the immunogenic peptide desirably aids a B cell mediated
immune response, which includes the production of antibodies.
The immunogenic peptides of the present invention can be of any
length, i.e., can comprise any number of amino acids, provided that
the peptides are able to bind to an MHC Class I molecule. For
example, the peptide can be 5 to 654 amino acids long, such as 5,
6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19, 20, 25, 50, 75, 100 or more
amino acids in length. In a preferred embodiment, the peptide
consists of 8 to 10 amino acids.
The immunogenic peptides of the present invention can comprise
synthetic amino acids in place of one or more naturally-occurring
amino acids. Such synthetic amino acids are known in the art, and
include, for example, aminocyclohexane carboxylic acid, norleucine,
.alpha.-amino n-decanoic acid, homoserine,
S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,
4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,
4-carboxyphenylalanine, .beta.-phenylserine
.beta.-hydroxyphenylalanine, phenylglycine,
.alpha.-naphthylalanine, cyclohexylalanine, cyclohexylglycine,
indoline-2-carboxylic acid,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic
acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine,
N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine,
.alpha.-aminocyclopentane carboxylic acid, .alpha.-aminocyclohexane
carboxylic acid, .alpha.-aminocycloheptane carboxylic acid,
.alpha.-(2-amino-2-norbornane)-carboxylic acid,
.alpha.,.gamma.-diaminobutyric acid,
.alpha.,.beta.-diaminopropionic acid, homophenylalanine, and
.alpha.-tert-butylglycine.
The present inventive immunogenic peptides can be glycosylated,
amidated, carboxylated, phosphorylated, esterified, N-acylated,
cyclized via, e.g., a disulfide bridge, or converted into an acid
addition salt and/or optionally dimerized or polymerized, or
conjugated.
When the immunogenic peptides of the present invention are in the
form of a salt, preferably, the peptides are in the form of a
pharmaceutically acceptable salt. Suitable pharmaceutically
acceptable acid addition salts include those derived from mineral
acids, such as hydrochloric, hydrobromic, phosphoric,
metaphosphoric, nitric, and sulphuric acids, and organic acids,
such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic,
glycolic, gluconic, succinic, and arylsulphonic acids, for example,
p-toluenesulphonic acid.
The present invention also provides functional portions of the
immunogenic peptides described herein. The term "functional
portion" when used in reference to an immunogenic peptide refers to
any part or fragment of the immunogenic peptide of the present
invention, which part or fragment retains the biological (e.g.,
immunogenic) activity of the immunogenic peptide of which it is a
part. Functional portions encompass, for example, those parts of an
immunogenic peptide (the parent peptide) that retain the ability to
bind to an MHC Class I molecule to a similar extent, the same
extent, or to a higher extent, as the parent peptide. In reference
to the parent peptide, the functional portion can comprise, for
instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of
the parent peptide. The functional portion can comprise additional
amino acids at the amino or carboxy terminus of the portion, or at
both termini, which additional amino acids are not found in the
amino acid sequence of the parent peptide. Desirably, the
additional amino acids do not interfere with the biological
function of the functional portion, e.g., binding to an MHC Class I
molecule, stimulation of CD8.sup.+ and CD4.sup.+ T cells,
stimulation of T cells to kill tumor cells, and treatment and/or
prevention of cancer.
The present invention also provides functional variants of the
immunogenic peptides described herein. The term "functional
variant" as used herein refers to an immunogenic peptide having
substantial or significant sequence identity or similarity to a
parent immunogenic peptide, which functional variant retains the
biological activity of the immunogenic peptide of which it is a
variant. Functional variants encompass, for example, those variants
of the immunogenic peptide (the parent peptide) that retain the
ability to bind to an MHC Class I molecule to a similar extent, the
same extent, or to a higher extent, as the parent peptide. In
reference to the parent peptide, the functional variant can, for
instance, be at least 30%, 50%, 75%, 80%, 90%, 98% or more
identical to the parent peptide.
The functional variant can, for example, comprise the amino acid
sequence of the parent immunogenic peptide with at least one
conservative amino acid substitution. Conservative amino acid
substitutions are known in the art, and include amino acid
substitutions in which one amino acid having certain physical
and/or chemical properties is exchanged for another amino acid that
has the same chemical or physical properties. For instance, the
conservative amino acid substitution can be an acidic amino acid
substituted for another acidic amino acid (e.g., Asp or Glu), an
amino acid with a nonpolar side chain substituted for another amino
acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu,
Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for
another basic amino acid (Lys, Arg, etc.), an amino acid with a
polar side chain substituted for another amino acid with a polar
side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.
Alternatively or additionally, the functional variants can comprise
the amino acid sequence of the parent immunogenic peptide with at
least one non-conservative amino acid substitution. In this case,
it is preferable for the non-conservative amino acid substitution
to not interfere with or inhibit the biological activity of the
peptide. Preferably, the non-conservative amino acid substitution
enhances the biological activity of the peptide.
The immunogenic peptides can consist essentially of the specified
amino acid sequence, such other components of the peptide, e.g.,
other amino acids, do not materially change the biological, e.g.,
immunogenic, activity of the peptide. In this regard, the present
inventive immunogenic peptide can, for example, consist essentially
of the amino acid sequence SPQNSIRHNL (SEQ ID NO: 3). Also, for
instance, the inventive peptide can consist essentially of the
amino acid sequence of SEQ ID NO: 5.
With respect to the immunogenic peptides comprising an amino acid
sequence of SEQ ID NO: 5, it is preferred that each of X.sub.1,
X.sub.2 and X.sub.3 is independently any small, aliphatic amino
acid. Small aliphatic amino acids are known in the art and include,
for example, Ser, Thr, or Ala. In a more preferred embodiment,
X.sub.1 is Ser, Thr, or Ala; X.sub.2 is Thr or Ala; and X.sub.3 is
Ser, Thr, or Ala. In an even more preferred embodiment, the peptide
comprises an amino acid sequence selected from the group consisting
of: SPANSIRHNL (SEQ ID NO: 7); SPQNAIRHNL (SEQ ID NO: 8); and
SPQNSARHNL (SEQ ID NO: 9).
With respect to the immunogenic peptide comprising the amino acid
sequence of SEQ ID NO: 3, it is preferred that the peptide consists
or consists essentially of the amino acid sequence SPQNSIRHNL (SEQ
ID NO: 3). Also, with respect to the immunogenic peptides of SEQ ID
NO: 3, it is preferred that the peptides are isolated and/or
purified.
The immunogenic peptides of the present invention can be obtained
by methods known in the art. Suitable methods of de novo
synthesizing peptides are described herein and in references, such
as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford
University Press, Oxford, United Kingdom, 2005; Peptide and Protein
Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope
Mapping, ed. Westwoood et al., Oxford University Press, Oxford,
United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Also, peptides
can be recombinantly produced using the nucleic acids described
herein using standard recombinant methods. See, for instance,
Sambrook et al., Molecular Cloning: A Laboratory Manual, 3.sup.rd
ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and
Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates and John Wiley & Sons, NY, 1994. Further,
some of the immunogenic peptide can be isolated and/or purified
from a source, such as a plant, a bacterium, a mammal, e.g., a rat,
a human, etc. Methods of isolation and purification are well-known
in the art. Alternatively, the peptides described herein can be
commercially synthesized by companies, such as Synpep (Dublin,
Calif.), Peptide Technologies Corp. (Gaithersburg, Md.), and
Multiple Peptide Systems (San Diego, Calif.). In this respect, the
immunogenic peptides of the present invention can be synthetic,
recombinant, isolated, and/or purified.
The present invention further provides a fusion protein comprising
at least one of the immunogenic peptides (including functional
portions and variants thereof) described herein and an MHC Class I
molecule, or a portion thereof. The MHC Class I molecule can be any
of the MHC Class I molecules known in the art, such as any of those
described herein. Preferably, the MHC Class I molecule is HLA-B7.
The portion of the MHC Class I molecule can be any part of the MHC
Class I molecule. Preferably, the portion comprises the peptide
binding portion of the MHC Class I molecule. More preferably, the
portion comprises the peptide binding portion and the T cell
receptor binding portion of the MHC Class I molecule. Such portions
of MHC Class I molecules are known in the art.
The fusion protein can comprise one or more copies of the
immunogenic peptide and/or one or more copies of the MHC Class I
molecule or part thereof. For instance, the fusion protein can
comprise 1, 2, 3, 4, 5, or more copies of the immunogenic peptide
and/or of the MHC Class I molecule or part thereof. Suitable
methods of making fusion proteins are known in the art, and
include, for example, recombinant methods. See, for instance, Choi
et al., Mol Biotechnol 31: 193-202 (2005).
The present invention further provides conjugates, e.g.,
bioconjugates, comprising any of the immunogenic peptides
(including any of the functional portions or variants thereof).
Conjugates, as well as methods of synthesizing conjugates of
peptides in general, are known in the art (See, for instance,
Hudecz, F., Methods Mol Biol 298: 209-223 (2005) and Kirin et al.,
Inorg Chem 44(15): 5405-5415 (2005)).
The present invention provides a nucleic acid comprising a
nucleotide sequence encoding any of the immunogenic peptides,
functional portions or variants thereof, or fusion proteins
thereof, described herein. By "nucleic acid" as used herein
includes "polynucleotide," "oligonucleotide," and "nucleic acid
molecule," and generally means a polymer of DNA or RNA, which can
be single-stranded or double-stranded, synthesized or obtained
(e.g., isolated and/or purified) from natural sources, which can
contain natural, non-natural or altered nucleotides, and which can
contain a natural, non-natural or altered internucleotide linkage,
such as a phosphoroamidate linkage or a phosphorothioate linkage,
instead of the phosphodiester found between the nucleotides of an
unmodified oligonucleotide. It is generally preferred that the
nucleic acid does not comprise any insertions, deletions,
inversions, and/or substitutions. However, it may be suitable in
some instances, as discussed herein, for the nucleic acid to
comprise one or more insertions, deletions, inversions, and/or
substitutions.
Preferably, the nucleic acids of the present invention are
recombinant. As used herein, the term "recombinant" refers to (i)
molecules that are constructed outside living cells by joining
natural or synthetic nucleic acid segments to nucleic acid
molecules that can replicate in a living cell, or (ii) molecules
that result from the replication of those described in (i) above.
For purposes herein, the replication can be in vitro replication or
in vivo replication.
The nucleic acids can be constructed based on chemical synthesis
and/or enzymatic ligation reactions using procedures known in the
art. See, for example, Sambrook et al., supra, and Ausubel et al.,
supra. For example, a nucleic acid can be chemically synthesized
using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex
formed upon hybridization (e.g., phosphorothioate derivatives and
acridine substituted nucleotides). Examples of modified nucleotides
that can be used to generate the nucleic acids include, but are not
limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxymethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N.sup.6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N.sup.6-substituted adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N.sup.6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
3-(3-amino-3-N.sup.2-carboxypropyl) uracil, (acp3) w, and
2,6-diaminopurine. Alternatively, one or more of the nucleic acids
of the present invention can be purchased from companies, such as
Macromolecular Resources (Fort Collins, Colo.) and Synthegen
(Houston, Tex.).
The nucleic acids of the present invention can be incorporated into
an expression vector. In this regard, the present invention
provides expression vectors comprising any of the nucleic acids of
the present invention. For purposes herein, the term "expression
vector" means a genetically-modified oligonucleotide (i.e.,
polynucleotide) construct that permits the expression of a protein
or a peptide by a host cell, when the construct comprises a
nucleotide sequence encoding the protein or peptide, and the vector
is contacted with the cell under conditions sufficient to have the
protein expressed within the cell. The vectors of the present
invention are not naturally-occurring as a whole. However, parts of
the vectors can be naturally-occurring. The present inventive
expression vectors can comprise any type of nucleotides, including,
but not limited to DNA and RNA, which can be single-stranded or
double-stranded, synthesized or obtained in part from natural
sources, and which can contain natural, non-natural or altered
nucleotides. The expression vectors can comprise
naturally-occurring, non-naturally-occurring internucleotide
linkages, or both types of linkages. Preferably, the non-naturally
occurring or altered nucleotides or internucleotide linkages does
not hinder in any way the transcription or replication of the
vector.
The expression vector of the present invention can be any suitable
expression vector, and can be used to transform or transfect any
suitable host. Suitable vectors include those designed for
propagation and expansion or for expression or both, such as
plasmids and viruses. The vector can be selected from the group
consisting of the pUC series (Fermentas Life Sciences), the
pBluescript series (Stratagene, LaJolla, Calif.), the pET series
(Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech,
Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.).
Bacteriophage vectors, such as .lamda.GT10, .lamda.GT11,
.lamda.ZapII (Stratagene), .lamda.EMBL4, and .lamda.NM1149, also
can be used. Examples of plant expression vectors include pBI01,
pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of
animal expression vectors include pEUK-C1, pMAM and pMAMneo
(Clontech).
The expression vectors of the present invention can be prepared
using standard recombinant DNA techniques described in, for
example, Sambrook et al., supra, and Ausubel et al., supra.
Constructs of expression vectors, which are circular or linear, can
be prepared to contain a replication system functional in a
prokaryotic or eukaryotic host cell. Replication systems can be
derived, e.g., from ColE1, 2.mu. plasmid, .lamda., SV40, bovine
papilloma virus, and the like.
Desirably, the expression vector comprises regulatory sequences,
such as transcription and translation initiation and termination
codons, which are specific to the type of host (e.g., bacterium,
fungus, plant, or animal) into which the vector is to be
introduced, as appropriate and taking into consideration whether
the vector is DNA- or RNA-based.
The expression vector can include one or more marker genes, which
allow for selection of transformed or transfected hosts. Marker
genes include biocide resistance, e.g., resistance to antibiotics,
heavy metals, etc., complementation in an auxotrophic host to
provide prototrophy, and the like. Suitable marker genes for the
present inventive expression vectors include, for instance,
neomycin/G418 resistance genes, hygromycin resistance genes,
histidinol resistance genes, tetracycline resistance genes, and
ampicillin resistance genes.
The expression vector can comprise a native or normative promoter
operably linked to the nucleic acid encoding the protein. The
selection of promoters, e.g., strong, weak, inducible,
tissue-specific and developmental-specific, is within the ordinary
skill of the artisan. Similarly, the combining of a nucleic acid
with a promoter is also within the skill of the artisan. The
promoter can be a non-viral promoter or a viral promoter, e.g., a
cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter,
and a promoter found in the long-terminal repeat of the murine stem
cell virus.
The present inventive expression vectors can be designed for either
transient expression, for stable expression, or for both. Also, the
expression vectors can be made for constitutive expression or for
inducible expression. Further, the expression vectors can be made
to include a suicide gene.
As used herein, the term "suicide gene" refers to a gene that
causes the cell expressing the suicide gene to die. The suicide
gene can be a gene that confers sensitivity to an agent, e.g., a
drug, upon the cell in which the gene is expressed, and causes the
cell to die when the cell is contacted with or exposed to the
agent. Suicide genes are known in the art (see, for example,
Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J.
(Cancer Research UK Centre for Cancer Therapeutics at the Institute
of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004) and
include, for example, the Herpes Simplex Virus (HSV) thymidine
kinase (TK) gene, cytosine daminase, purine nucleoside
phosphorylase, and nitroreductase.
The present invention further provides a host cell comprising any
of the expression vectors described herein. As used herein, the
term "host cell" refers to any type of cell that can contain the
present inventive expression vector. The host cell can be a
eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a
prokaryotic cell, e.g., bacteria or protozoa. The cell can be a
cultured cell or a primary cell, i.e., isolated directly from an
organism, e.g., a human. The cell can be an adherent cell or a
suspended cell, i.e., a cell that grows ill suspension. Suitable
host cells are known in the art and include, for instance,
DH5.alpha. E. coli cells, Chinese hamster ovarian cells, monkey
VERO cells, COS cells, HEK293 cells, and the like. For purposes of
amplifying or replicating the recombinant expression vector, the
host cell is preferably a prokaryotic cell, e.g., a DH5.alpha.
cell. For purposes of producing a recombinant protein, the host
cell is preferably a mammalian cell. Most preferably, the host cell
is a human cell. While the host cell can be of any cell type, can
originate from any type of tissue, and can be of any developmental
stage, the host cell preferably is an antigen presenting cell, such
as, for instance, a dendritic cell, a macrophage, or a B cell.
Preferably, the host cell is a dendritic cell.
The present invention further provides an antibody, or an antigen
binding portion thereof, that binds to any of the immunogenic
peptides described herein. The antibody can be any type of
immunoglobulin that is known in the art. For instance, the antibody
can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The
antibody can be monoclonal or polyclonal. The antibody can be a
naturally-occurring antibody, e.g., an antibody isolated and/or
purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,
hamster, human, etc. Alternatively, the antibody can be a
genetically-engineered antibody, e.g., a humanized antibody or a
chimeric antibody. The antibody can be in monomeric or polymeric
form. Also, the antibody can have any level of affinity or avidity
for the peptide of the present invention. Desirably, the antibody
is specific for the peptide, such that there is minimal
cross-reaction with other peptides or proteins.
Methods of testing antibodies for the ability to bind to any of the
immunogenic peptides are known in the art an include any
antibody-antigen binding assay, such as, for example,
radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation,
and competitive inhibition assays (see, e.g., Janeway et al.,
supra, and U.S. Patent Application Publication No. 2002/0197266
A1).
Suitable methods of making antibodies are known in the art. For
instance, standard hybridoma methods are described in, e.g., Kohler
and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane
(eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C.
A. Janeway et al. (eds.), Immunobiology, 5.sup.th Ed., Garland
Publishing, New York, N.Y. (2001)). Alternatively, other methods,
such as EBV-hybridoma methods (Haskard and Archer, J. Immunol.
Methods, 74(2), 361-67 (1984), and Roder et al., Methods Enzymol.,
121, 140-67 (1986)), and bacteriophage vector expression systems
(see, e.g., Huse et al., Science, 246, 1275-81 (1989)) are known in
the art. Further, methods of producing antibodies in non-human
animals are described in, e.g., U.S. Pat. Nos. 5,545,806,
5,569,825, and 5,714,352, and U.S. Patent Application Publication
No. 2002/0197266 A1).
Phage display furthermore can be used to generate the antibody of
the present invention. In this regard, phage libraries encoding
antigen-binding variable (V) domains of antibodies can be generated
using standard molecular biology and recombinant DNA techniques
(see, e.g., Sambrook et al. (eds.), Molecular Cloning, A Laboratory
Manual, 3.sup.rd Edition, Cold Spring Harbor Laboratory Press, New
York (2001)). Phage encoding a variable region with the desired
specificity are selected for specific binding to the desired
antigen, and a complete or partial antibody is reconstituted
comprising the selected variable domain. Nucleic acid sequences
encoding the reconstituted antibody are introduced into a suitable
cell line, such as a myeloma cell used for hybridoma production,
such that antibodies having the characteristics of monoclonal
antibodies are secreted by the cell (see, e.g., Janeway et al.,
supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).
Antibodies can be produced by transgenic mice that are transgenic
for specific heavy and light chain immunoglobulin genes. Such
methods are known in the art and described in, for example U.S.
Pat. Nos. 5,545,806 and 5,569,825, and Janeway et al., supra.
Methods for generating humanized antibodies are well known in the
art and are described in detail in, for example, Janeway et al.,
supra, U.S. Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European
Patent No. 0239400 B1, and United Kingdom Patent No. 2188638.
Humanized antibodies can also be generated using the antibody
resurfacing technology described in U.S. Pat. No. 5,639,641 and
Pedersen et al., J. Mol. Biol., 235, 959-973 (1994).
The present inventive invention also provides antigen binding
portions of any of the antibodies described herein. The antigen
binding portion can be any portion that has at least one antigen
binding site, such as Fab, F(ab').sub.2, dsFv, sFv, diabodies, and
triabodies.
A single-chain variable region fragment (sFv) antibody fragment,
which consists of a truncated Fab fragment comprising the variable
(V) domain of an antibody heavy chain linked to a V domain of a
light antibody chain via a synthetic peptide, can be generated
using routine recombinant DNA technology techniques (see, e.g.,
Janeway et al., supra). Similarly, disulfide-stabilized variable
region fragments (dsFv) can be prepared by recombinant DNA
technology (see, e.g., Reiter et al., Protein Engineering, 7,
697-704 (1994)). Antibody fragments of the present invention,
however, are not limited to these exemplary types of antibody
fragments.
The present inventive immunogenic peptides, fusion proteins,
conjugates, nucleic acids, expression vectors, host cells, and
antibodies, can be isolated and/or purified. The term "isolated" as
used herein means having been removed from its natural environment.
The term "purified" as used herein means having been increased in
purity, wherein "purity" is a relative term, and not to be
necessarily construed as absolute purity. For example, the purity
can be at least 50%, can be greater than 60%, 70% or 80%, or can be
100%.
The present inventive immunogenic peptides (including functional
portions and variants thereof), fusion proteins, conjugates,
nucleic acids, expression vectors, host cells, and antibodies
(including antigen binding portions thereof), all of which are
collectively referred to as "immunogenic materials" hereinafter,
can be formulated into a composition, such as a pharmaceutical
composition. In this regard, the present invention provides a
pharmaceutical composition comprising any of the immunogenic
peptides, fusion proteins, conjugates, nucleic acids, expression
vectors, host cells, and antibodies, and a pharmaceutically
acceptable carrier. The present inventive pharmaceutical
compositions containing any of the immunogenic materials can
comprise more than one immunogenic material, e.g., a peptide and a
nucleic acid, or two or more different peptides. Alternatively, the
pharmaceutical composition can comprise an immunogenic material in
combination with another pharmaceutically active agents or drugs,
such as a chemotherapeutic agents e.g., asparaginase, busulfan,
carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,
gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,
vinblastine, vincristine, etc.
Preferably, the carrier is a pharmaceutically acceptable carrier.
With respect to pharmaceutical compositions, the carrier can be any
of those conventionally used and is limited only by
chemico-physical considerations, such as solubility and lack of
reactivity with the active compound(s), and by the route of
administration. The pharmaceutically acceptable carriers described
herein, for example, vehicles, adjuvants, excipients, and diluents,
are well-known to those skilled in the art and are readily
available to the public. It is preferred that the pharmaceutically
acceptable carrier be one which is chemically inert to the active
agent(s) and one which has no detrimental side effects or toxicity
under the conditions of use.
The choice of carrier will be determined in part by the particular
immunogenic material, as well as by the particular method used to
administer the immunogenic material. Accordingly, there are a
variety of suitable formulations of the pharmaceutical composition
of the present invention. The following formulations for oral,
aerosol, parenteral, subcutaneous, intravenous, intramuscular,
intraarterial, intrathecal, interperitoneal, rectal, and vaginal
administration are exemplary and are in no way limiting. More than
one route can be used to administer the immunogenic materials, and
in certain instances, a particular route can provide a more
immediate and more effective response than another route.
Topical formulations are well-known to those of skill in the art.
Such formulations are particularly suitable in the context of the
present invention for application to the skin.
Formulations suitable for oral administration can consist of (a)
liquid solutions, such as an effective amount of the immunogenic
material dissolved in diluents, such as water, saline, or orange
juice; (b) capsules, sachets, tablets, lozenges, and troches, each
containing a predetermined amount of the active ingredient, as
solids or granules; (c) powders; (d) suspensions in an appropriate
liquid; and (e) suitable emulsions. Liquid formulations may include
diluents, such as water and alcohols, for example, ethanol, benzyl
alcohol, and the polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant. Capsule forms
can be of the ordinary hard- or soft-shelled gelatin type
containing, for example, surfactants, lubricants, and inert
fillers, such as lactose, sucrose, calcium phosphate, and corn
starch. Tablet forms can include one or more of lactose, sucrose,
mannitol, corn starch, potato starch, alginic acid,
microcrystalline cellulose, acacia, gelatin, guar gum, colloidal
silicon dioxide, croscarmellose sodium, talc, magnesium stearate,
calcium stearate, zinc stearate, stearic acid, and other
excipients, colorants, diluents, buffering agents, disintegrating
agents, moistening agents, preservatives, flavoring agents, and
other pharmacologically compatible excipients. Lozenge forms can
comprise the immunogenic material in a flavor, usually sucrose and
acacia or tragacanth, as well as pastilles comprising the
immunogenic material in an inert base, such as gelatin and
glycerin, or sucrose and acacia, emulsions, gels, and the like
containing, in addition to, such excipients as are known in the
art.
The immunogenic material, alone or in combination with other
suitable components, can be made into aerosol formulations to be
administered via inhalation. These aerosol formulations can be
placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. They also
may be formulated as pharmaceuticals for non-pressured
preparations, such as in a nebulizer or an atomizer. Such spray
formulations also may be used to spray mucosa.
Formulations suitable for parenteral administration include aqueous
and non-aqueous, isotonic sterile injection solutions, which can
contain anti-oxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. The immunogenic material can be
administered in a physiologically acceptable diluent in a
pharmaceutical carrier, such as a sterile liquid or mixture of
liquids, including water, saline, aqueous dextrose and related
sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol,
a glycol, such as propylene glycol or polyethylene glycol,
dimethylsulfoxide, glycerol, ketals such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol)
400, oils, fatty acids, fatty acid esters or glycerides, or
acetylated fatty acid glycerides with or without the addition of a
pharmaceutically acceptable surfactant, such as a soap or a
detergent, suspending agent, such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters.
Suitable soaps for use in parenteral formulations include fatty
alkali metal, ammonium, and triethanolamine salts, and suitable
detergents include (a) cationic detergents such as, for example,
dimethyl dialkyl ammonium halides, and alkyl pyridinium halides,
(b) anionic detergents such as, for example, alkyl, aryl, and
olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl-.beta.-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
The parenteral formulations will typically contain from about 0.5%
to about 25% by weight of the immunogenic material in solution.
Preservatives and buffers may be used. In order to minimize or
eliminate irritation at the site of injection, such compositions
may contain one or more nonionic surfactants having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17.
The quantity of surfactant in such formulations will typically
range from about 5% to about 15% by weight. Suitable surfactants
include polyethylene glycol sorbitan fatty acid esters, such as
sorbitan monooleate and the high molecular weight adducts of
ethylene oxide with a hydrophobic base, formed by the condensation
of propylene oxide with propylene glycol. The parenteral
formulations can be presented in unit-dose or multi-dose sealed
containers, such as ampoules and vials, and can be stored in a
freeze-dried (lyophilized) condition requiring only the addition of
the sterile liquid excipient, for example, water, for injections,
immediately prior to use. Extemporaneous injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
Injectable formulations are in accordance with the present
invention. The requirements for effective pharmaceutical carriers
for injectable compositions are well-known to those of ordinary
skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice,
J.B. Lippincott Company, Philadelphia, Pa., Banker and Chalmers,
eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs,
Toissel, 4th ed., pages 622-630 (1986)). Preferably, when
administering cells, e.g., dendritic cells, the cells are
administered via injection.
Additionally, the immunogenic materials, or compositions comprising
such immunogenic materials, can be made into suppositories by
mixing with a variety of bases, such as emulsifying bases or
water-soluble bases. Formulations suitable for vaginal
administration can be presented as pessaries, tampons, creams,
gels, pastes, foams, or spray formulas containing, in addition to
the active ingredient, such carriers as are known in the art to be
appropriate.
It will be appreciated by one of skill in the art that, in addition
to the above-described pharmaceutical compositions, the immunogenic
materials of the present invention can be formulated as inclusion
complexes, such as cyclodextrin inclusion complexes, or
liposomes.
For purposes of the present invention, the amount or dose of the
immunogenic material administered should be sufficient to effect,
e.g., a therapeutic or prophylactic response, in the subject or
animal over a reasonable time frame. For example, the dose of the
immunogenic material should be sufficient to bind to an MHC Class I
molecule, stimulate tumor cell killing, stimulate CD4.sup.+ and
CD8.sup.+ T cells, or treat or prevent cancer in a period of from
about 2 hours or longer, e.g., 12 to 24 or more hours, from the
time of administration. In certain embodiments, the time period
could be even longer. The dose will be determined by the efficacy
of the particular immunogenic material and the condition of the
animal (e.g., human), as well as the body weight of the animal
(e.g., human) to be treated.
Many assays for determining an administered dose are known in the
art. For purposes of the present invention, an assay, which
comprises comparing the extent to which CD8.sup.+ T cells are
stimulated to kill tumor cells upon administration of a given dose
of a immunogenic material to a mammal among a set of mammals of
which is each given a different dose of the immunogenic material,
could be used to determine a starting dose to be administered to a
mammal. The extent to which the tumor cells are killed upon
administration of a certain dose can be assayed by methods known in
the art, including, for instance, the method described herein as
Example 3.
The dose of the immunogenic material also will be determined by the
existence, nature and extent of any adverse side effects that might
accompany the administration of a particular immunogenic material.
Typically, the attending physician will decide the dosage of the
immunogenic material with which to treat each individual patient,
taking into consideration a variety of factors, such as age, body
weight, general health, diet, sex, immunogenic material to be
administered, route of administration, and the severity of the
condition being treated. By way of example and not intending to
limit the present invention, the dose of the immunogenic material
can be about 0.001 to about 1000 mg/kg body weight of the subject
being treated/day, from about 0.01 to about 10 mg/kg body
weight/day, about 0.01 mg to about 1 mg/kg body weight/day.
One of ordinary skill in the art will readily appreciate that the
immunogenic materials of the present invention can be modified in
any number of ways, such that the therapeutic or prophylactic
efficacy of the immunogenic materials is increased through the
modification. For instance, the immunogenic materials can be
conjugated either directly or indirectly through a linker to a
targeting moiety. The practice of conjugating compounds, e.g.,
immunogenic materials, to targeting moieties is known in the art.
See, for instance, Wadwa et al., J. Drug Targeting 3: 111 (1995)
and U.S. Pat. No. 5,087,616. The term "targeting moiety" as used
herein, refers to any molecule or agent that specifically
recognizes and binds to a cell-surface receptor, such that the
targeting moiety directs the delivery of the immunogenic materials
to a population of cells on which surface the receptor is
expressed. Targeting moieties include, but are riot limited to,
antibodies, or fragments thereof, peptides, hormones, growth
factors, cytokines, and any other natural or non-natural ligands,
which bind to cell surface receptors (e.g., Epithelial Growth
Factor Receptor (EGFR), T-cell receptor (TCR), B-cell receptor
(BCR), CD28, Platelet-derived Growth Factor Receptor (PDGF),
nicotinic acetylcholine receptor (nAChR), etc.). The term "linker"
as used herein, refers to any agent or molecule that bridges the
immunogenic materials to the targeting moiety. One of ordinary
skill in the art recognizes that sites on the immunogenic
materials, which are not necessary for the function of the
immunogenic materials, are ideal sites for attaching a linker
and/or a targeting moiety, provided that the linker and/or
targeting moiety, once attached to the immunogenic materials,
do(es) not interfere with the function of the immunogenic
materials, i.e., the ability to bind to an MHC Class I molecule, to
stimulate CD4.sup.+ and CD8.sup.+ T cells, to stimulate the killing
of tumor cells, or to treat or prevent cancer.
Alternatively, the immunogenic materials can be modified into a
depot form, such that the manner in which the immunogenic materials
is released into the body to which it is administered is controlled
with respect to time and location within the body (see, for
example, U.S. Pat. No. 4,450,150). Depot forms of immunogenic
materials can be, for example, an implantable composition
comprising the immunogenic materials and a porous or non-porous
material, such as a polymer, wherein the immunogenic materials is
encapsulated by or diffused throughout the material and/or
degradation of the non-porous material. The depot is then implanted
into the desired location within the body and the immunogenic
materials are released from the implant at a predetermined
rate.
The present inventive immunogenic peptides (including functional
portions and variants thereof), fusion proteins, conjugates,
nucleic acids, expression vectors, and host cells can be made as
part of a vaccine. As such, the present invention also provides a
vaccine comprising any of the immunogenic peptides, fusion
proteins, nucleic acids, expression vectors, and host cells
described herein. The term "vaccine" as used herein means any
substance that causes activation of an animal's immune system
without causing actual disease. The vaccines of the present
invention comprise an immunogen that induces an immune response
directed against a tumor antigen, e.g., PAX3-FKHR. The immunogen of
the present inventive vaccines is any of the immunogenic peptides,
fusion proteins, nucleic acids, expression vectors, and host cells
described herein.
In one embodiment, the vaccine is an expression vector encoding a
PAX3-FKHR peptide. To provide a vaccine to an individual, a
nucleotide sequence which encodes for the PAX3-FKHR peptide is
inserted into a expression vector, as described above, and
introduced into the mammal to be immunized. Examples of vectors
that may be used in the aforementioned vaccines include, but are
not limited to, defective retroviral vectors, adenoviral vectors
vaccinia viral vectors, fowl pox viral vectors, or other viral
vectors (Mulligan, R. C., (1993) Science 260:926-932). The viral
vectors carrying the PAX3-FKHR nucleic acid can be introduced into
a mammal either prior to any evidence of the disease, e.g., cancer,
or to mediate regression of the disease in a mammal afflicted with
the disease. Examples of methods for administering the viral vector
into the mammals include, but are not limited to, exposure of cells
to the virus ex vivo, or injection of the retrovirus or a producer
cell line of the virus into the affected tissue or intravenous
administration of the virus. Alternatively, the viral vector
carrying all or part of the PAX3-FKHR nucleic acid sequence may be
administered locally by direct injection or topical application in
a pharmaceutically acceptable carrier.
The quantity of viral vector carrying the PAX3-FKHR nucleic acid
sequence to be administered is based on the titer of virus
particles. A preferred range of the immunogen to be administered
may be about 10.sup.6 to about 10.sup.11 virus particles per
mammal, preferably a human. After immunization, the efficacy of the
vaccine can be assessed by production of antibodies or immune cells
that recognize the antigen (PAX3-FKHR), as assessed by specific
lytic activity or specific cytokine production or by regression of
the disease. One skilled in the art knows the conventional methods
to assess the aforementioned parameters. If the mammal to be
immunized is already afflicted with the disease, the vaccine can be
administered in conjunction with other therapeutic treatments.
Examples of other therapeutic treatments includes, but are not
limited to, adoptive T cell immunotherapy, coadministration of
cytokines or other therapeutic drugs for the disease.
Alternatively, the PAX3-FKHR peptides may be administered as a
vaccine in a pharmaceutically acceptable carrier. Ranges of
PAX3-FKHR peptide that may be administered are about 0.001 to about
100 mg per patient, preferred doses are about 0.01 to about 100 mg
per patient. Immunization is repeated as necessary, until a
sufficient titer of anti-immunogen antibody or immune cells has
been obtained.
In yet another alternative embodiment, mammalian cells expressing
the PAX3-FKHR antigen can be administered to mammals and serve as a
vaccine. Examples of mammalian cells include, but are not limited
to, primary manmalian cultures or continuous mammalian cultures,
COS cells, NIH3T3, or 293 cells (ATCC #CRL 1573). Examples of how
the cells expressing PAX3-FKHR antigens can be administered
include, but not limited to, intravenous, intraperitoneal or
intralesional administration.
In yet another embodiment of this invention, PAX3-FKHR peptides,
may be exposed to dendritic cells cultured in vitro. The cultured
dendritic cells provide a means of producing T-cell dependent
antigens comprised of dendritic cell modified antigen or dendritic
cells pulsed with antigen, in which the antigen is processed and
expressed on the antigen activated dendritic cell. The PAX3-FKHR
antigen-activated dendritic cells or processed dendritic cell
antigens may be used as immunogens for vaccines or for the
treatment of cancer. The dendritic cells should be exposed to
antigen for sufficient time to allow the antigens to be
internalized and presented on the dendritic cells surface. The
resulting dendritic cells or the dendritic cell process antigens
can than be administered to an individual in need of therapy. Such
methods are described in Steinman et al. (WO 93/208185) and in
Banchereau et al. (EP Application 0563485A1) which are incorporated
herein by reference.
Vaccination can be conducted by conventional methods. For example,
the immunogen can be used in a suitable diluent, such as saline or
water, or a complete or incomplete adjuvant. Further, the immunogen
can be bound or unbound to a carrier to make the peptide
immunogenic. Examples of such carrier molecules include but are not
limited to bovine serum albumin (BSA), keyhole limpet hemocyanin
(KLH), tetanus toxoid, and the like. The immunogen also can be
coupled with a lipoprotein or administered in liposomal form or
with adjuvants. The immunogen can be administered by any route
appropriate for antibody production such as intravenous,
intraperitoneal, intramuscular, subcutaneous, and the like. The
immunogen may be administered once or at periodic intervals until a
significant titer of anti-PAX3-FKHR immune cells or anti-PAX3-FKHR
antibody is produced. The presence of anti-PAX3-FKHR immune cells
may be assessed by measuring the frequency of precursor CTL against
PAX3-FKHR antigen prior to and after immunization by a CTL
precursor analysis assay (Coulie, P. et al., (1992) Internat J Can
50:289-297). The antibody may be detected in the serum using
standard immunoassays known in the art.
The vaccine formulations may be evaluated first in animal models,
initially rodents, and in non-human primates and finally in humans.
The safety of the immunization procedures is determined by looking
for the effect of immunization on the general health of the
immunized animal (weight change, fever, appetite behavior etc.) and
looking for pathological changes on autopsies. After initial
testing in animals, diseased patients can be tested. Conventional
methods would be used to evaluate the immune response of the
patient to determine the efficiency of the vaccine.
The vaccine may be used either prophylactically or therapeutically.
When provided prophylactically, the vaccine is provided in advance
of any evidence of disease, e.g., cancer. The prophylactic
administration of the vaccine should serve to prevent or attenuate
the disease in a mammal. In a preferred embodiment, mammals,
preferably humans, at high risk for the disease are
prophylactically treated with the vaccines of the present
invention. Examples of such mammals include, but are not limited
to, humans with a family history of the disease or humans
previously afflicted with the disease and therefore at risk for
re-occurrence. When provided therapeutically, the vaccine is
provided to enhance the patient's own immune response to the
antigen, e.g., tumor antigen, present in the patient.
It is contemplated that the present inventive pharmaceutical
compositions and vaccines can be used in methods of treating or
preventing cancer. Without being bound to any particular theory, it
is believed that the present inventive peptides bind to a MHC Class
I molecule, e.g., HLA-B7, and to a T cell receptor, such that the
corresponding T cell is stimulated to create an immune response,
e.g., a cellular immune response. More particularly, the T cells
having the TCR, which binds to the peptides of the present
invention, once bound to the peptide are stimulated to lyse and/or
kill the target cell, e.g., a tumor cell. In this respect, the
present invention provides a method of stimulating a T cell to kill
a tumor cell. The method comprises contacting a T cell with a cell
presenting on its surface any of the immunogenic peptides described
herein and with a tumor cell in a manner effective for the T cell
to kill the tumor cell.
The tumor cell lysed by the T cell can be any type of tumor cell,
such as, for instance, a tumor cell from a benign tumor or a
cancerous tumor. The tumor cell can be a tumor cell from any of the
following cancers: alveolar rhabdomyosarcoma, breast cancer,
prostate cancer, lung cancer, colon cancer, rectal cancer, urinary
bladder cancer, non-Hodgkin lymphoma, melanoma, renal cancer,
pancreatic cancer, cancer of the oral cavity, pharynx cancer,
ovarian cancer, thyroid cancer, stomach cancer, brain cancer,
multiple myeloma, esophageal cancer, liver cancer, cervical cancer,
larynx cancer, cancer of the intrahepatic bile duct, acute myeloid
leukemia, soft tissue cancer, small intestine cancer, testicular
cancer, chronic lymphocytic leukemia, Hodgkin lymphoma, chronic
myeloid cancer, acute lymphocytic cancer, cancer of the anus, anal
canal, or anorectum, cancer of the vulva, cancer of the neck,
gallbladder, or pleura, malignant mesothelioma, bone cancer, cancer
of the joints, hypopharynx cancer, cancer of the eye, cancer of the
nose, nasal cavity, or middle ear, nasopharynx cancer, ureter
cancer, peritoneum, omentum, and mesentery cancer, or
gastrointestinal carcinoid tumor. Preferably, the tumor cell is an
alveolar rhabodomyosarcoma cell.
With respect to the present inventive method of stimulating a T
cell to kill a tumor cell, the T cell can be contacted with the
tumor cell and the peptide-presenting cell simultaneously or
sequentially. For instance, the T cell can be contacted with the
peptide-presenting cell before being contacted with the tumor cell.
Alternatively, the T cell can be contacted with the tumor cell and
the peptide-presenting cell at the same time.
Also, the T cell can be contacted with the peptide-presenting cell
in vitro, in vivo or ex vivo. For example, the T cell can be
contacted with the peptide-presenting cell in vitro or ex vivo and
then subsequently contacted with the tumor cell in vivo.
Alternatively, the T cell can be contacted with the
peptide-presenting cell in vivo and then subsequently contacted
with the tumor cell in vivo, or can be contacted with the
peptide-presenting cell and the tumor cell in vivo and
simultaneously.
In a preferred embodiment, the method provides for the killing of
multiple tumor cells in a manner effective to treat cancer in a
mammal. In this regard, the present invention provides a method of
treating cancer. Preferably, the T cells are autologous to the
mammal being treated.
Further, without being bound to any particular theory, some of the
present inventive peptides can also be able to stimulate CD4.sup.+
T cells, such that the T cells help B cells to produce antibodies
against the peptides. In this regard, the peptides desirably
stimulate a humoral immune response, in addition to the cellular
immune response mediated through the CD8.sup.+ T cells.
Accordingly, the present invention also provides methods of
stimulating CD4.sup.+ and CD8.sup.+ T cells. The method comprises
contacting a CD4.sup.+ and a CD8.sup.+ T cell with any of the
dendritic cells described herein.
With respect to the present inventive method of stimulating a
CD4.sup.+ T cell and a CD8.sup.+ T cell, the T cell can be
contacted with the CD4.sup.+ T cell and a CD8.sup.+ T cell
simultaneously or sequentially. For instance, the CD4.sup.+ T cell
can be contacted with the dendritic cell at the same or different
time as the time that the CD8.sup.+ T cell is contacted with the
dendritic cell.
Also, either of the CD4.sup.+ T cell and the CD8.sup.+ T cell can
be contacted with the dendritic cell in vitro, in vivo or ex vivo.
For example, the CD8.sup.+ T cell can be contacted with the
dendritic cell in vitro or ex vivo. Alternatively, the T cell can
be contacted with the dendritic cell in vivo.
In a preferred embodiment, the T cells, e.g., the CD8.sup.+ T
cells, are contacted with a tumor cell after being contacted with
the dendritic cell, such that the T cell lyses and kills the tumor
cell.
In a more preferred embodiment, the method provides for the killing
of multiple tumor cells in a manner effective to treat cancer in a
mammal. In this regard, the present invention provides a method of
treating cancer. Preferably, the T cells are autologous to the
mammal being treated.
A method of treating or preventing cancer in a mammal is further
provided herein. The method comprises administering to the mammal
any of the pharmaceutical compositions or vaccines described herein
in an amount effective to treat or prevent cancer in the
mammal.
With respect to the methods of treating or preventing cancer, the
cancer can be any cancer, such as any of those described herein.
Preferably, the cancer is alveolar rhabdomyosarcoma.
With respect to the methods of treating or preventing cancer, the
pharmaceutical compositions or vaccines can be administered by any
method known in the art, including any of the routes described
herein. As one of ordinary skill in the art recognizes, some
pharmaceutical compositions and vaccines are more amenable to
certain routes than others. For example, it is preferable for the
pharmaceutical compositions and vaccines comprising host cells to
be administered through injection, as opposed to orally or
transdermally. The route appropriate for the particular
pharmaceutical composition or vaccine can easily be determined by
one of ordinary skill in the art.
The terms "treat," and "prevent" as well as words stemming
therefrom, as used herein, do not necessarily imply 100% or
complete treatment or prevention. Rather, there are varying degrees
of treatment or prevention of which one of ordinary skill in the
art recognizes as having a potential benefit or therapeutic effect.
In this respect, the present inventive methods can provide any
amount of any level of treatment or prevention of cancer in a
mammal.
As used herein, the term "mammal" refers to any mammal, including,
but not limited to, mammals of the order Rodentia, such as mice and
hamsters, and mammals of the order Logomorpha, such as rabbits. It
is preferred that the mammals are from the order Carnivora,
including Felines (cats) and Canines (dogs). It is more preferred
that the mammals are from the order Artiodactyla, including Bovines
(cows) and Swines (pigs) or of the order Perssodactyla, including
Equines (horses). It is most preferred that the mammals are of the
order Primates, Ceboids, or Simoids (monkeys) or of the order
Anthropoids (humans and apes). An especially preferred mammal is
the human.
The following examples further illustrate the invention but, of
course, should not be construed as in any way limiting its
scope.
EXAMPLES
The following cell lines and peptides are used in the examples
described herein below.
C1R.B7, a specific transfectant of the B lymphoblastoid C1R cell
line, which, in native form, expresses no endogenous HLA-A or HLA-B
gene products (Storkus et al., Proc. Natl. Acad. Sci., 86,
2361-2364 (1999)), is a gift of William Biddison (National
Institute of Neurological Disorders and Stroke, NIH, Bethesda,
Md.). T2.B7 is a specific transfectant of the hybrid B and T
lymphoblastoid T2 cell line, which is deficient in TAP1 and TAP2
gene expression (Salter et al., Immunogenetics 21: 235-246 (1985);
and Spies and DeMars, Nature 351: 323-324 (1991)), and which is a
gift of Peter Cresswell (Yale University, New Haven, Conn.). The
Rh5 alveolar rhabdomyosarcoma cell line is kindly provided by Dr.
P. Houghton (St Jude's Children's Research Hospital). The RD and
CTR embryonal rhabdomyosarcoma cell lines are obtained from the
American Type Culture Collection and Dr. M. Tsokos (National Cancer
Institute), respectively. Cell lines are maintained in culture
medium with heat inactivated fetal calf serum (FCS) (10% v/v).
Culture medium consists of RPMI1640 medium (Cellgro, Bethesda, Md.)
containing L-glutamine (2 mM), penicillin (100 IU/ml), streptomycin
(100 .mu.g/ml), nonessential amino acids (10 .mu.l/ml),
sodium-pyruvate (1.0 mM), gentamicm (25 .mu.g/ml), and
2-mercaptoethanol (50 .mu.M).
Full-length peptides are purchased from Peptide Technologies Corp.
(Gaithersburg, Md.) and from Multiple Peptide Systems (San Diego,
Calif.) at >95% purity and are single peaks by reverse-phase
high-performance liquid chromatography. Optimal epitopes are
synthesized on an automated peptide synthesizer (Symphony
Multiplex; Protein Technologies, Phoenix, Ariz.) using
9-fluoroenyImethyloxycarbonyl chemistry (Stewart et al., Solid
Phase Peptide Synthesis, 2.sup.nd ed., Rockford, Ill., Pierce
Chemical Company, 1984). The peptides are cleaved from the resin
with trifluoroacetic acid. Purification to single peaks is achieved
using reverse-phase high-performance liquid chromatography on
bondapack reverse-phase CIS columns (Waters Associates, Milford,
Mass.).
Example 1
This example demonstrates the binding of a PAX3-FKHR peptide
epitope to a MHC Class I molecule.
A chromosomal translocation-generated fusion protein breakpoint
peptide, RS10 (SPQNSIRHNL), is selected on the basis of predicted
potential binding to HLA-B7 (Rammensee et al., Immunogenetics 41:
178-228 (1995)). Peptide binding to HLA-B7 is assessed by using
stabilization of HLA-B7 molecules on the surface of T2-B7 cells
that lack the TAP transporter and therefore express only
short-lived empty HLA-B7 molecules unless a peptide is present that
can bind and stabilize them. These assays are described in (Stuber
et al., Eur. J. Immunol., 22, 2697-2703 (1992), Nijman et al., Eur.
J. Immunol., 23, 1215-1219 (1993), Zeh et al., Hum. Immunol., 39,
79-86 (1994), and Smith et al., Internat. Immunol., 9, 1085-1093
(1997)). Cells of the TAP1/TAP2-deficient T2 cell line (Salter et
al., Immunogenetics, 21, 235-246 (1985), and Spies et al., Nature,
351, 323-324 (1991)) transfected with the HLA-B7 gene are suspended
in culture medium containing heat inactivated FCS (2.5% v/v) and
added to 96-well round-bottomed plates at 2.times.10.sup.5
cells/well. Human .beta.2-microglobulin (Sigma Chemical Co.) is
also added at 20 .mu.g/well. Where appropriate, peptide is added to
the desired concentration. The cells are then incubated overnight
at 37.degree. C. in 5% CO.sub.2, followed by washing with PBS
containing NaN.sub.3 (0.5% w/v and FCS 2% v/v). Next, cells are
incubated on ice for 30 min in the presence of primary anti-HLA-B7
specific antibody (BB7.1 hybridoma culture supernatant; ATCC),
followed by washing and incubation for 30 min in goat anti-mouse
immunoglobulin FITC (Becton Dickinson). Analysis is performed by
flow cytometry.
Conventional mAb staining is conducted in PBS containing 0.01%
sodium azide on ice. Cells are labeled with FITC- or PE-conjugated
nAbs obtained from BD PharMingen (San Diego, Calif.). For each
staining of interest, the appropriate isotype-matched control is
included. All reagents are used at optimal concentration as
determined experimentally. Flow cytometric analysis is performed
with a FACScan (BD Biosciences, Mountain View, Calif.). Data are
collected on 5000-10,000 viable cell events and analyzed with
CellQuest software.
Based on these binding assays, the RS10 peptide is found to bind
HLA-B7 in a binding assay (data not shown).
This example demonstrated that the RS10 peptide binds to an MHC
Class I molecule.
Example 2
This example demonstrates a method of making dendritic cells pulsed
with the present inventive peptides and T cells specific for the
peptides of the present invention.
To determine whether this peptide could elicit human CTL, dendritic
cells presenting the RS10 peptide are generated. Specifically,
leukopheresed human mononuclear cells are elutriated from an
HLA-B7.sup.+ normal healthy blood donor to separate a monocyte and
a lymphocyte fraction. The monocyte fraction is converted into
dendritic cells by growth in GM-CSF and IL-4 and maturation with
CD40L. Specifically, elutriated monocytes and lymphocytes are
obtained from apheresed HLA-B7-positive subjects from the NIH
normal donor pool. Monocytes are cultured for 7 days in 75 cm.sup.2
culture flasks (Costar Corporation) at 3.5.times.10.sup.6 cells/ml
in culture medium containing heat inactivated autologous plasma
(10% v/v), h-IL-4 (800 U/ml; R&D) and h-GMCSF (50 ng/ml;
Immunex) at 37.degree. C. in an atmosphere of 5% CO.sub.2 in air.
At day 3, the cells are re-fed by removing 5 ml of the medium from
the culture flasks and adding back 5 ml of fresh culture medium
supplemented with cytokines (h-IL4: 400 U/ml; h-GMCSF: 25 ng/ml).
At day 4, CD40 ligand trimer (Immunex Corp., now Amgen, Seattle) is
added at 1 .mu.g/ml (Cella et al., J. Exp. Med., 184, 747-752,
(1996)). Cells are harvested on day 6 and stained for CD14, CD19,
CD56, CD80, CD83, CD86, and HLA-DR antigens. The staining is then
quantified by flow cytometry as described above.
The DCs are found to be strongly positive for CD80 and CD86
costimulatory molecules, HLA-DR, and the maturation marker CD83,
but are negative for CD14, CD19, and CD56 (data not shown).
The DCs are then pulsed with the RS10 peptide and used to stimulate
autologous lymphocytes from the same apheresis to generate a
specific CTL line. Specifically, lymphocytes are suspended in
culture medium containing heat inactivated autologous plasma (10%
v/v)) and plated in a 24-well plate at 4.times.10.sup.6 cells/well.
Autologous dendritic cells are pulsed with RS10 peptide (10 .mu.M)
in culture medium for 4 hours. Next, the dendritic cells are
irradiated with 3000 rads and added to the lymphocytes at
4.times.10.sup.5 cells/well. The next day (day 1), cultures are
supplemented with hIL-2 (12.5 U/ml), hIL-7 (2400 U/ml), hIL-1.beta.
(150 U/ml) and hIL-12 (.+-.1 ng/ml). At day 7, the cells are
harvested, washed, plated in a 24-well plate at 1.5.times.10.sup.6
cells/well, and restimulated with irradiated RS10 pulsed autologous
DCs (1.5.times.10.sup.5 cells/well). At day 8, the cells are
supplemented with IL-2 (12.5 U/ml) and hIL-7 (2400 U/ml).
Restimulations are done weekly using the same conditions. Cultures
are checked every week for relative CD4, CD8, and CD56 expression
and, if necessary, depleted of CD4.sup.+ or CD56+ cells by magnetic
cell sorting using Magnetic Micro Beads (Miltenyi Biotec
Midi-MACS).
The line is found to express CD8, but not CD4 and CD56 (data not
shown).
This example demonstrated the generation and characterization of
DCs pulsed with PAX3-FKHR peptide and the generation and
characterization of a CTL line specific for the PAX3-FKHR peptide
and MHC molecule expressed on the DCs.
Example 3
This example demonstrates that the peptides of the present
invention stimulate T cells to kill tumor cells.
The human CTL line generated and characterized in Example 2 is
tested for the ability to kill human C1R-B7 target cells pulsed
with the specific RS10 peptide or a control SSI peptide from
synovial sarcoma that also binds HLA-B7 (Worley et al., Cancer
Res., 61, 6868-6875 (2001)) (FIG. 1). In particular, specific
cytotoxic activities are determined in a standard 4 h .sup.51Cr
release assay at various effector:target (E:T) ratios. Briefly,
graded doses of viable effector cells are plated in triplicate in
96-well U-bottom culture plates (Corning Glass, Corning, N.Y.) and
co-cultured for 4 h with sodium chromate-labeled (100 .mu.Ci; NEN,
Boston, Mass.) peptide pulsed (10 .mu.M) C1R-B7 target cells. In
some experiments, the Rh5, RD and CTR tumor cell lines are used as
a target. Supernatants are collected, radioactivity measured, and
specific lysis is calculated according to the equation: percentage
of specific cytotoxicity=(experimental cpm-spontaneous
cpm)/(maximum cpm-spontaneous cpm).times.100.
Maximum .sup.51Cr release is determined from supernatants of lysed
target cells incubated with Triton X-100 (5% v/v). Spontaneous
release is determined from target cells incubated without added
effector cells.
The lysis is clearly specific for the PAX-FKHR-derived RS10
peptide. Furthermore, the killing is restricted by the human HLA-B7
class I MHC molecule as demonstrated by blockade of killing with
antibody to HLA-B7 (FIG. 2).
This example demonstrated that the generated CTL can lyse tumor
cells.
Example 4
This example demonstrates that human CTL specific for the RS10
PAX-FKHR fusion peptide kill rhabdomyosarcoma cells expressing
HLA-B7.
To determine whether this RS10 epitope is naturally processed and
presented by HLA-B7 in human tumor cells that express endogenous
PAX-FKHR fusion protein, the lytic ability of the RS10-specific CTL
line is tested against a rhabdomyosarcoma tumor cell expressing
HLA-B7 (Rh5), and two control lines RD and CTR not expressing
HLA-B7 (FIG. 3). CTL activity is assessed as described in Example
3.
Clear specific lysis of the Kh5 cells compared to the other control
tumor cells indicates 1) that the RS10 epitope is indeed naturally
endogenously processed and presented in unmanipulated human tumor
cells, and 2) that CTL raised against this epitope could kill human
tumor cells.
This example demonstrated that human CTL specific for the RS10
peptide kill tumor cells expressing an MHC Class I molecule.
Example 5
This example demonstrates the generation and testing of variant
peptides of RS10.
To maximize immunogenicity, it is often helpful to modify the
sequence of an epitope to increase the affinity for the relevant
MHC molecule, in a process that is called epitope enhancement
(Berzofsky et al., Ann. N.Y. Acad. Sci., 690, 256-264 (1993),
Berzofsky et al., Ann. N.Y. Acad. Sci., 754, 161-168 (1995), Ahlers
et al., Proc. Natl. Acad. Sci. USA, 94, 10856-10861 (1997), Sarobe
et al., J. Clin. Invest., 102, 1239-1248 (1998), Ahlers et al., J.
Clin. Invest., 108, 1677-1685 (2001), Berzofsky et al., Nature
Reviews Immunology, 1, 209-219 (2001), Okazaki et al., J. Immunol.,
171, 2548-2555 (2003), Oh et al., Cancer Research, 64, 2610-2618
(2004), and Berzofsky et al., J. Clin. Invest., 113, 1515-1525
(2004)). Such improvements in binding affinity can sometimes be
achieved by replacement of an amino acid residue causing an adverse
interaction with one that has a small, neutral side chain, such as
alanine (Berzofsky et al., Ann. N.Y. Acad. Sci., 690, 256-264
(1993), Berzofsky et al., Ann. N.Y. Acad. Sci., 754, 161-168
(1995), Ahlers et al., Proc. Natl. Acad. Sci. USA, 94, 10856-10861
(1997), Ahlers et al., J. Clin. Invest., 108, 1677-1685 (2001), and
Boehncke et al., J. Immunol., 150, 331-341 (1993)). To screen for
such a possibility, a series of peptides with Ala substitutions at
each position in RS10 is synthesized and tested for binding to
HLA-B7 in a T2-B7 binding assay. The variants with the Ala
substitution at positions 3, 5, and 6 (RS10-3A, RS10-5A, and
RS10-6A, respectively) show higher binding affinity for HLA-B7 than
the natural RS10 peptide (FIGS. 4 and 6). The concentration
required for a 50% increase in HLA-B7 expression is decreased from
approximately 0.3 .mu.M for RS10 to approximately 0.05 .mu.M for
the RS10-3A variant.
To be sure that the epitope enhanced peptide with increased binding
affinity for the HLA molecule has not simultaneously lost
recognition by the T cell, the ability of the RS40-specific human
CTL to kill C1R-B7 targets pulsed with the RS10-3A peptide is
tested as described above. As shown in FIG. 5, the specific killing
observed confirms that the enhanced epitope is not so altered in
the surface presented to the T cell receptor that it had lost
recognition by the human CTL. This enhanced peptide may therefore
serve as a candidate vaccine to elicit specific CTL immunity in
HLA-B7.sup.+ patients with alveolar rhabdomyosarcoma, without the
concern that self tolerance could dampen the response, or that the
tumor could escape by losing the fusion protein.
All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the
same extent as if each reference were individually and specifically
indicated to be incorporated by reference and were set forth in its
entirety herein.
The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention (especially in the
context of the following claims) are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. The terms "comprising," "having,"
"including," and "containing" are to be construed as open-ended
terms (i.e., meaning "including, but not limited to,") unless
otherwise noted. Recitation of ranges of values herein are merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range, unless otherwise
indicated herein, and each separate value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments may become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
SEQUENCE LISTINGS
1
91836PRTHomo sapiens 1Met Thr Thr Leu Ala Gly Ala Val Pro Arg Met
Met Arg Pro Gly Pro1 5 10 15Gly Gln Asn Tyr Pro Arg Ser Gly Phe Pro
Leu Glu Val Ser Thr Pro 20 25 30Leu Gly Gln Gly Arg Val Asn Gln Leu
Gly Gly Val Phe Ile Asn Gly 35 40 45Arg Pro Leu Pro Asn His Ile Arg
His Lys Leu Val Glu Met Ala His 50 55 60His Gly Ile Arg Pro Cys Val
Ile Ser Arg Gln Leu Arg Val Ser His65 70 75 80Gly Cys Val Ser Lys
Ile Leu Cys Arg Tyr Gln Glu Thr Gly Ser Ile 85 90 95Arg Pro Gly Ala
Ile Gly Gly Ser Lys Pro Lys Gln Val Thr Thr Pro 100 105 110Asp Val
Glu Lys Lys Ile Glu Glu Tyr Lys Arg Glu Asn Pro Gly Met 115 120
125Phe Ser Trp Glu Ile Arg Asp Lys Leu Leu Lys Asp Ala Val Cys Asp
130 135 140Arg Asn Thr Val Pro Ser Val Ser Ser Ile Ser Arg Ile Leu
Arg Ser145 150 155 160Lys Phe Gly Lys Gly Glu Glu Glu Glu Ala Asp
Leu Glu Arg Lys Glu 165 170 175Ala Glu Glu Ser Glu Lys Lys Ala Lys
His Ser Ile Asp Gly Ile Leu 180 185 190Ser Glu Arg Ala Ser Ala Pro
Gln Ser Asp Glu Gly Ser Asp Ile Asp 195 200 205Ser Glu Pro Asp Leu
Pro Leu Lys Arg Lys Gln Arg Arg Ser Arg Thr 210 215 220Thr Phe Thr
Ala Glu Gln Leu Glu Glu Leu Glu Arg Ala Phe Glu Arg225 230 235
240Thr His Tyr Pro Asp Ile Tyr Thr Arg Glu Glu Leu Ala Gln Arg Ala
245 250 255Lys Leu Thr Glu Ala Arg Val Gln Val Trp Phe Ser Asn Arg
Arg Ala 260 265 270Arg Trp Arg Lys Gln Ala Gly Ala Asn Gln Leu Met
Ala Phe Asn His 275 280 285Leu Ile Pro Gly Gly Phe Pro Pro Thr Ala
Met Pro Thr Leu Pro Thr 290 295 300Tyr Gln Leu Ser Glu Thr Ser Tyr
Gln Pro Thr Ser Ile Pro Gln Ala305 310 315 320Val Ser Asp Pro Ser
Ser Thr Val His Arg Pro Gln Pro Leu Pro Pro 325 330 335Ser Thr Val
His Gln Ser Thr Ile Pro Ser Asn Pro Asp Ser Ser Ser 340 345 350Ala
Tyr Cys Leu Pro Ser Thr Arg His Gly Phe Ser Ser Tyr Thr Asp 355 360
365Ser Phe Val Pro Pro Ser Gly Pro Ser Asn Pro Met Asn Pro Thr Ile
370 375 380Gly Asn Gly Leu Ser Pro Gln Asn Ser Ile Arg His Asn Leu
Ser Leu385 390 395 400His Ser Lys Phe Ile Arg Val Gln Asn Glu Gly
Thr Gly Lys Ser Ser 405 410 415Trp Trp Met Leu Asn Pro Glu Gly Gly
Lys Ser Gly Lys Ser Pro Arg 420 425 430Arg Arg Ala Ala Ser Met Asp
Asn Asn Ser Lys Phe Ala Lys Ser Arg 435 440 445Ser Arg Ala Ala Lys
Lys Lys Ala Ser Leu Gln Ser Gly Gln Glu Gly 450 455 460Ala Gly Asp
Ser Pro Gly Ser Gln Phe Ser Lys Trp Pro Ala Ser Pro465 470 475
480Gly Ser His Ser Asn Asp Asp Phe Asp Asn Trp Ser Thr Phe Arg Pro
485 490 495Arg Thr Ser Ser Asn Ala Ser Thr Ile Ser Gly Arg Leu Ser
Pro Ile 500 505 510Met Thr Glu Gln Asp Asp Leu Gly Glu Gly Asp Val
His Ser Met Val 515 520 525Tyr Pro Pro Ser Ala Ala Lys Met Ala Ser
Thr Leu Pro Ser Leu Ser 530 535 540Glu Ile Ser Asn Pro Glu Asn Met
Glu Asn Leu Leu Asp Asn Leu Asn545 550 555 560Leu Leu Ser Ser Pro
Thr Ser Leu Thr Val Ser Thr Gln Ser Ser Pro 565 570 575Gly Thr Met
Met Gln Gln Thr Pro Cys Tyr Ser Phe Ala Pro Pro Asn 580 585 590Thr
Ser Leu Asn Ser Pro Ser Pro Asn Tyr Gln Lys Tyr Thr Tyr Gly 595 600
605Gln Ser Ser Met Ser Pro Leu Pro Gln Met Pro Ile Gln Thr Leu Gln
610 615 620Asp Asn Lys Ser Ser Tyr Gly Gly Met Ser Gln Tyr Asn Cys
Ala Pro625 630 635 640Gly Leu Leu Lys Glu Leu Leu Thr Ser Asp Ser
Pro Pro His Asn Asp 645 650 655Ile Met Thr Pro Val Asp Pro Gly Val
Ala Gln Pro Asn Ser Arg Val 660 665 670Leu Gly Gln Asn Val Met Met
Gly Pro Asn Ser Val Met Ser Thr Tyr 675 680 685Gly Ser Gln Ala Ser
His Asn Lys Met Met Asn Pro Ser Ser His Thr 690 695 700His Pro Gly
His Ala Gln Gln Thr Ser Ala Val Asn Gly Arg Pro Leu705 710 715
720Pro His Thr Val Ser Thr Met Pro His Thr Ser Gly Met Asn Arg Leu
725 730 735Thr Gln Val Lys Thr Pro Val Gln Val Pro Leu Pro His Pro
Met Gln 740 745 750Met Ser Ala Leu Gly Gly Tyr Ser Ser Val Ser Ser
Cys Asn Gly Tyr 755 760 765Gly Arg Met Gly Leu Leu His Gln Glu Lys
Leu Pro Ser Asp Leu Asp 770 775 780Gly Met Phe Ile Glu Arg Leu Asp
Cys Asp Met Glu Ser Ile Ile Arg785 790 795 800Asn Asp Leu Met Asp
Gly Asp Thr Leu Asp Phe Asn Phe Asp Asn Val 805 810 815Leu Pro Asn
Gln Ser Phe Pro His Ser Val Lys Thr Thr Thr His Ser 820 825 830Trp
Val Ser Gly 83523200DNAHomo sapiens 2ccgtttcgcc ttcacctgga
tataatttcc gagcgaagtg cccccaggat gaccacgctg 60gccggcgctg tgcccaggat
gatgcggccg ggcccggggc agaactaccc gcgtagcggg 120ttcccgctgg
aagtgtccac tcccctcggc cagggccgcg tcaaccagct cggcggcgtt
180tttatcaacg gcaggccgct gcccaaccac atccgccaca agatcgtgga
gatggcccac 240cacggcatcc ggccctgcgt catctcgcgc cagctgcgcg
tgtcccacgg ctgcgtctcc 300aagatcctgt gcaggtacca ggagactggc
tccatacgtc ctggtgccat cggcggcagc 360aagcccaagc aggtgacaac
gcctgacgtg gagaagaaaa ttgaggaata caaaagagag 420aacccgggca
tgttcagctg ggaaatccga gacaaattac tcaaggacgc ggtctgtgat
480cgaaacaccg tgccgtcagt gagttccatc agccgcatcc tgagaagtaa
attcgggaaa 540ggtgaagagg aggaggccga cttggagagg aaggaggcag
aggaaagcga gaagaaggcc 600aaacacagca tcgacggcat cctgagcgag
cgagcctcag caccccaatc agatgaaggc 660tctgatattg actctgaacc
agatttacca ctaaagagga aacagcgcag aagccgaacc 720accttcacag
cagaacagct ggaggaactg gagcgtgctt ttgagagaac tcattaccct
780gacatttata ctagggagga actggcccag agggcgaagc tcaccgaggc
ccgagtacag 840gtctggttta gcaaccgccg tgcaagatgg aggaagcaag
ctggggccaa tcaactgatg 900gctttcaacc atctcattcc cggggggttc
cctcccactg ccatgccgac cttgccaacg 960taccagctgt cggagacctc
ttaccagccc acatctattc cacaagctgt gtcagatccc 1020agcagcaccg
ttcacagacc tcaaccgctt cctccaagca ctgtacacca aagcacgatt
1080ccttccaacc cagacagcag ctctgcctac tgcctcccca gcaccaggca
tggattttcc 1140agctatacag acagctttgt gcctccgtcg gggccctcca
accccatgaa ccccaccatt 1200ggcaatggcc tctcacctca gaattcaatt
cgtcataatc tgtccctaca cagcaagttc 1260attcgtgtgc agaatgaagg
aactggaaaa agttcttggt ggatgctcaa tccagagggt 1320ggcaagagcg
ggaaatctcc taggagaaga gctgcatcca tggacaacaa cagtaaattt
1380gctaagagcc gaagccgagc tgccaagaag aaagcatctc tccagtctgg
ccaggagggt 1440gctggggaca gccctggatc acagttttcc aaatggcctg
caagccctgg ctctcacagc 1500aatgatgact ttgataactg gagtacattt
cgccctcgaa ctagctcaaa tgctagtact 1560attagtggga gactctcacc
cattatgacc gaacaggatg atcttggaga aggggatgtg 1620cattctatgg
tgtacccgcc atctgccgca aagatggcct ctactttacc cagtctgtct
1680gagataagca atcccgaaaa catggaaaat cttttggata atctcaacct
tctctcatca 1740ccaacatcat taactgtttc gacccagtcc tcacctggca
ccatgatgca gcagacgccg 1800tgctactcgt ttgcgccacc aaacaccagt
ttgaattcac ccagcccaaa ctaccaaaaa 1860tatacatatg gccaatccag
catgagccct ttgccccaga tgcctataca aacacttcag 1920gacaataagt
cgagttatgg aggtatgagt cagtataact gtgcgcctgg actcttgaag
1980gagttgctga cttctgactc tcctccccat aatgacatta tgacaccagt
tgatcctggg 2040gtagcccagc ccaacagccg ggttctgggc cagaacgtca
tgatgggccc taattcggtc 2100atgtcaacct atggcagcca ggcatctcat
aacaaaatga tgaatcccag ctcccatacc 2160caccctggac atgctcagca
gacatctgca gttaacgggc gtcccctgcc ccacacggta 2220agcaccatgc
cccacacctc gggtatgaac cgcctgaccc aagtgaagac acctgtacaa
2280gtgcctctgc cccaccccat gcagatgagt gccctggggg gctactcctc
cgtgagcagc 2340tgcaatggct atggcagaat gggccttctc caccaggaga
agctcccaag tgacttggat 2400ggcatgttca ttgagcgctt agactgtgac
atggaatcca tcattcggaa tgacctcatg 2460gatggagata cattggattt
taactttgac aatgtgttgc ccaaccaaag cttcccacac 2520agtgtcaaga
caacgacaca tagctgggtg tcaggctgag ggttagtgag caggttacac
2580ttaaaagtac ttcagattgt ctgacagcag gaactgagag aagcagtcca
aagatgtctt 2640tcaccaactc ccttttagtt ttcttggtta aaaaaaaaaa
acaaaaaaaa aaaccctcct 2700tttttccttt cgtcagactt ggcagcaaag
acatttttcc tgtacaggat gtttgcccaa 2760tgtgtgcagg ttatgtgctg
ctgtagataa ggactgtgcc attggaaatt tcattacaat 2820gaagtgccaa
actcactaca ccatataatt gcagaaaaga ttttcagatc ctggtgtgct
2880ttcaagtttt gtatataagc agtagataca gattgtattt gtgtgtgttt
ttggtttttc 2940taaatatcca attggtccaa ggaaagttta tactcttttt
gtaatactgt gatgggcctc 3000atgtcttgat aagttaaact tttgtttgta
ctacctgttt tctgcggaac tgacggatca 3060caaagaactg aatctccatt
ctgcatctcc attgaacagc cttggacctg ttcacgttgc 3120cacagaattc
acatgagaac caagtagcct gttatcaatc tgctaaatta atggacttgt
3180taaacttttg gaaaaaaaag 3200310PRTArtificial SequenceSynthetic
3Ser Pro Gln Asn Ser Ile Arg His Asn Leu1 5 10418PRTArtificial
SequenceSynthetic 4Thr Ile Gly Asn Gly Leu Ser Pro Gln Asn Ser Ile
Arg His Asn Leu1 5 10 15Ser Leu523PRTArtificial SequenceSynthetic
5Asn Pro Thr Gly Thr Ile Gly Asn Gly Leu Ser Pro Gln Asn Ser Ile1 5
10 15Arg His Asn Leu Ser Leu His 20610PRTArtificial
SequenceSynthetic 6Ser Pro Xaa Asn Xaa Xaa Arg His Asn Leu1 5
10710PRTArtificial SequenceSynthetic 7Ser Pro Ala Asn Ser Ile Arg
His Asn Leu1 5 10810PRTArtificial SequenceSynthetic 8Ser Pro Gln
Asn Ala Ile Arg His Asn Leu1 5 10910PRTArtificial SequenceSynthetic
9Ser Pro Gln Asn Ser Ala Arg His Asn Leu1 5 10
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