U.S. patent application number 09/780926 was filed with the patent office on 2002-12-26 for immunotherapy using interleukin 13 receptor subunit alpha 2.
Invention is credited to Debinski, Waldemar.
Application Number | 20020197266 09/780926 |
Document ID | / |
Family ID | 22662463 |
Filed Date | 2002-12-26 |
United States Patent
Application |
20020197266 |
Kind Code |
A1 |
Debinski, Waldemar |
December 26, 2002 |
Immunotherapy using interleukin 13 receptor subunit alpha 2
Abstract
A method for stimulating a immune response against
IL-13R.alpha.2 in a subject having or at risk for developing a
disease having cells expressing IL-13R.alpha.2 includes the steps
of formulating the anti-cancer vaccine outside of the subject and
administering the vaccine to the subject in an amount sufficient to
stimulate an immune response against IL-13R.alpha.2 in the subject.
A composition for stimulating a immune response against
IL-13R.alpha.2 in a subject having or at risk for developing a
disease having cells expressing IL-13R.alpha.2 includes an isolated
agent that can stimulate immune response against IL-13.alpha.2.
Inventors: |
Debinski, Waldemar;
(Hershey, PA) |
Correspondence
Address: |
Stanley A. Kim
Akerman, Senterfitt & Eidson, P.A.
222 Lakeview Avenue, Suite 400
P.O. Box 3188
West Palm Beach
FL
33402-3188
US
|
Family ID: |
22662463 |
Appl. No.: |
09/780926 |
Filed: |
February 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60181000 |
Feb 8, 2000 |
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Current U.S.
Class: |
424/185.1 ;
424/85.1 |
Current CPC
Class: |
A61P 35/04 20180101;
A61P 25/00 20180101; A61P 43/00 20180101; A61K 39/001184 20180801;
A61P 35/00 20180101; C07K 16/2866 20130101; C07K 14/7155 20130101;
A61K 2039/545 20130101; A61K 2039/53 20130101; A61K 2039/505
20130101; A61K 2039/5156 20130101 |
Class at
Publication: |
424/185.1 ;
424/85.1 |
International
Class: |
A61K 039/00; A61K
038/19 |
Goverment Interests
[0002] This invention was made with Government support under grant
number CA74154 awarded by the National Cancer Institute of the
National Institutes of Health. The Government may have certain
rights in the invention.
Claims
What is claimed is:
1. A method for stimulating a immune response against
IL-13R.alpha.2 in a subject having or at risk for developing a
disease having cells expressing IL-13R.alpha.2, the method
comprising the steps of: (a) formulating an anti-cancer vaccine
outside of the subject, the vaccine comprising an agent that can
stimulate an immune response against IL-13R.alpha.2 when
administered to an animal; and (b) administering the vaccine to the
subject in an amount sufficient to stimulate an immune response
against IL-13R.alpha.2 in the subject.
2. The method of claim 1, wherein the agent that can stimulate an
immune response against IL-13Rox2 comprises a peptide comprising at
least seven contiguous amino acids of SEQ ID NO:1.
3. The method of claim 1, wherein the agent that can stimulate an
immune response against IL-13R..alpha.2 is a protein comprising the
amino acid sequence of SEQ ID NO:1.
4. The method claim 1, wherein the vaccine further comprises an
adjuvant.
5. The method of claim 4, wherein the adjuvant comprises a
substance selected from the group consisting of: an aluminum salt;
an oil-in-water emulsion; a composition comprising saponin; a
composition comprising a bacterial protein; and a cytokine.
6. The method of claim 4, wherein step (b) of administering the
vaccine to the subject in an amount sufficient to stimulate an
immune response against IL-13R.alpha.2 in the subject comprises
administering the vaccine in at least a first dose and a second
dose, wherein said first dose is administered to the subject at
least 24 hours before said second dose is administered to the
subject.
7. The method of claim 1, wherein the agent that can stimulate an
immune response against IL-13R.alpha.2 comprises a nucleic acid
that encodes a peptide comprising at least seven contiguous amino
acids of SEQ ID NO:1.
8. The method of claim 7, wherein the nucleic acid is a naked
DNA.
9. The method of claim 7, wherein the nucleic acid is incorporated
into an expression vector.
10. The method of claim 1, wherein the agent that can stimulate an
immune response against IL-13R.alpha.2 comprises a cell expressing
a peptide comprising at least seven contiguous amino acids of SEQ
ID NO:1.
11. The method of claim 10, wherein the peptide comprising at least
seven contiguous amino acids of SEQ ID NO:1 is a protein comprising
the amino acid sequence of SEQ ID NO:1.
12. The method of claim 1, wherein the agent that can stimulate an
immune response against IL-13R.alpha.2 comprises a cell into which
has been introduced a purified nucleic acid that encodes a peptide
comprising at least seven contiguous amino acids of SEQ ID
NO:1.
13. The method of claim 1, further comprising the step of providing
a subject having or at risk for developing a cancer having cells
expressing IL-13R.alpha.2.
14. The method of claim 13, wherein the cells expressing
IL-13R.beta..alpha.2 are glioma cells.
15. The method of claim 13, wherein the subject is a human
being.
16. A composition for stimulating an immune response against
IL-13R.alpha.2 when administered to an animal, the composition
comprising: (a) an isolated agent that can stimulate an immune
response against IL-13R.alpha.2 when administered to an animal; and
(b) a pharmaceutically acceptable carrier.
17. The composition of claim 16, wherein the agent that can
stimulate an immune response against IL-13R.alpha.2 when
administered to an animal comprises a peptide comprising at least
seven contiguous amino acids of SEQ ID NO:1.
18. The composition of claim 17, wherein the peptide comprising at
least seven contiguous amino acids of SEQ ID NO:1 is a protein
comprising the amino acid sequence of SEQ ID NO:1.
19. The composition of claim 17, wherein the composition further
comprises an adjuvant.
20. The composition of claim 19, wherein the adjuvant comprises a
substance selected from the group consisting of: an aluminum salt;
an oil-in-water emulsion; a composition comprising saponin; a
composition comprising a bacterial protein; and a cytokine.
21. The composition of claim 20, wherein the agent that can
stimulate an immune response against IL-13R.alpha.2 when
administered to an animal comprises a nucleic acid that encodes a
peptide comprising at least seven contiguous amino acids of SEQ ID
NO:1.
22. The composition of claim 21, wherein the nucleic acid is a
naked DNA.
23. The composition of claim 21, wherein the nucleic acid is
incorporated into an expression vector.
24. The composition of claim 20, wherein the agent that can
stimulate an immune response against IL-13R.alpha.2 comprises a
cell expressing a peptide comprising at least seven contiguous
amino acids of SEQ ID NO:1.
25. The method of claim 24, wherein the peptide comprising at least
seven contiguous amino acids of SEQ ID NO:1 is a protein comprising
the amino acid sequence of SEQ ID NO:1.
26. The composition of claim 25, wherein the agent that can
stimulate an immune response against IL-13R.alpha.2 comprises a
cell into which has been introduced a purified nucleic acid that
encodes a peptide comprising at least seven contiguous amino acids
of SEQ ID NO:1.
27. A method for directing an antibody to cells expressing
IL-13RU,2 in a subject, the method comprising the steps of: (a)
formulating a pharmaceutical composition outside of a subject, the
pharmaceutical composition comprising an antibody that specifically
binds IL-13R.alpha.2 and a pharmaceutically acceptable carrier; and
(b) administering the pharmaceutical composition to the subject in
an amount sufficient to allow the antibody to specifically bind to
the cells expressing IL-13R.alpha.2 in the subject.
28. The method of claim 27, wherein the antibody is a monoclonal
antibody.
29. The method of claim 27, wherein the antibody is a polyclonal
antibody.
30. A pharmaceutical composition comprising an antibody that
specifically binds IL-13R.alpha.2 and a pharmaceutically acceptable
carrier.
31. The pharmaceutical composition of claim 30, wherein the
antibody is a monoclonal antibody.
32. The pharmaceutical composition of claim 30, wherein the
antibody is a polyclonal antibody.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of U.S.
provisional application serial No. 60/181,000 filed Feb. 8,
2000.
FIELD OF THE INVENTION
[0003] The invention relates generally to the fields of biology,
immunology, medicine, and oncology. More particularly, the
invention relates to the use of the interleukin 13 (IL-13) receptor
subunit alpha 2 (IL-13R.alpha.2) as an immune system modulator and
target for vaccines for the treatment and prevention of cancer.
BACKGROUND
[0004] Cancer is presently the second leading cause of death in
developed nations. Wingo et al., J. Reg. Management, 25:43-51
(1998). Despite recent research that has revealed many of the
molecular mechanisms of tumorigenesis, few new treatments have
achieved widespread clinical success in treating solid tumors. The
mainstay treatments for most malignancies thus remain gross
resection, chemotherapy, and radiotherapy. While increasingly
successful, each of these treatments still causes numerous
undesired side effects. The primary cause of this is that none of
these conventional methods specifically targets only diseased
cells. For example, surgery results in pain, traumatic injury to
healthy tissue, and scarring. Radiotherapy and chemotherapy cause
nausea, immune suppression, gastric ulceration and secondary
tumorigenesis.
[0005] In an effort to develop techniques to more specifically
target diseased cells, progress in tumor immunology has led to the
discovery of antigens that are preferentially or specifically
expressed on cancer cells. These tumor-associated antigens (TAA) or
tumor-specific antigens (TSA) have been used as antigenic agents in
cancer vaccines designed to stimulate an immune response
selectively directed against cancer cells expressing such antigens.
See, Tumor Immunology: Immunotherapy and Cancer Vaccines, A. G.
Dalgleish and M. J. Browning, eds., Cambridge University Press,
1996; Immunotherapy in Cancer, M. Gore and P. Riches, eds., John
Wiley & Son Ltd., 1996; Maeurer et al., Melanoma Res., 6:11-24
(1996). Among the most widely studied of these antigens are
melanoma associated antigens, prostate specific antigen (PSA), E6
and E7, carcinoembryonic antigen (CEA), p53, and gangliosides
(e.g., GM2). More recent studies have shown that certain TAAs and
TSAs are particularly effective at stimulating specific immune
responses.
[0006] For example, pioneering research with melanoma associated
antigens led to the identification of MAGE-1 (Melanoma Antigen 1)
as a T-cell activating TSA. Traversari et al., Immunogenetics, 35:
145-152, 1992. Subsequently other groups using similar techniques
identified other T-cell activating melanoma antigens including
other MAGEs, MART-1, glycoprotein 100 (gp100), tyrosinase, BAGE,
and GAGE. Reviewed by Maeurer et al., supra. One of the most
exciting recent findings in cancer immunology came after the SEREX
(for serological analysis of recombinant cDNA expression libraries)
technique was developed. Sahin et al., Proc. Natl. Acad. Sci. USA,
92: 11810-11813, 1995. The SEREX technique involves screening a
cDNA expression library of an autologous tumor by exposing the
library to antibodies contained in a patient's sera. Several active
cancer antigens have been identified using this technique. See,
Old, L. J. and T. C. Chen, J. Exp. Med., 187: 1163-1167, 1998.
Moreover, SEREX analysis showed that patients produce a high titer
of IgG antibodies against cancer antigens- a finding that indicated
that helper T cells (e.g., CD4+ T cells) and B cells cooperate in
stimulating an immune response against the cancer.
[0007] In addition, SEREX analyses led to the identification of a
group of cancer antigens termed "cancer/testis" antigens (CTAs).
CTAs share several common features including (a) among normal
organs, almost exclusive expression in the testis, (b) expression
in a wide variety of tumors, (c) presence of multiple members in
each identified family, and (d) localization of their genes to the
X chromosome (with the notable exception of SCP 1). Chen et al., J.
Biol. Chem., 273: 17618-17625, 1998. Based on the foregoing
criteria, several previously identified TAAs or TSAs (e.g., MAGE,
BAGE and GAGE) were re-discovered as CTAs. Notably, unlike many
non-CTA antigens, most of these previously identified CTAs as well
as newly identified CTAs (e.g., SSX2, NY-ESO-1, SCP1 and CT7) have
unequivocally been shown to stimulate an immune response in a
subject.
SUMMARY
[0008] The invention relates to the discovery that IL-13R.alpha.2
is a cancer/testis antigen. This discovery is important because, in
contrast to most other cancer-associated agents, most of the
cancer/testis antigens so far tested as active immunotherapy agents
against cancer have proven very effective in stimulating
anti-cancer immune responses in subjects. Thus, the present
discovery provides methods and compositions for preventing and/or
treating cancers that express IL-13R.alpha.2.
[0009] In particular, the invention relates to the treatment and/or
prevention of high-grade gliomas (HGG) in a subject as HGG cells
have been shown to express high levels of IL-13R.alpha.2 on their
surfaces. Human HGG are rapidly progressing heterogeneous brain
tumors of astroglial origin. The present invention is especially
important because no effective modalities for treating HGG are yet
accepted for clinical use. Previously, it was shown that the vast
majority of HGG patients over-express a more restrictive receptor
for IL-13, that is a receptor that binds IL-13 in an IL-4
independent manner. Recently, a new IL-13 binding protein, termed
IL-13R.alpha.2, was cloned. This protein was shown to have affinity
for IL-13 but not IL-4. In a rough comparison, this characteristic
relates to the more restrictive receptor for IL-13 expressed on
HGG. Here we demonstrate that, IL-13R.alpha.2 serves as a selective
target for HGG and other cancers that express IL-13R.alpha.2
because, as described in more detail below, with the exception of
testis, normal human tissue expresses little or no IL-13R.alpha.2.
And although many normal tissues express a receptor that binds
IL-13, this receptor (sometimes termed the "shared" receptor
because it binds both IL-13 and IL-4) differs functionally from
IL-13R.alpha.2 (believed to be the "restrictive" receptor) in that
the shared receptor binds both IL-13 and IL-4, while the
restrictive receptor binds only IL-13. The two receptors also
differ structurally, with the restrictive receptor being a 42 kDa
monomer and the shared receptor being a heterodimer composed of a
45 kDa component (termed IL-13R.alpha.1) and a 140 kDa component
(termed IL-4R.alpha.).
[0010] As indicated above, our tissue distributions studies showed
that, among normal tissues, IL-13R.alpha.2 is strongly expressed
only in testis. This finding along with the showing that (a)
IL-13R.alpha.2 is preferentially over-expressed on HGG but not
normal central nervous system (CNS) tissue and (b) that the
IL-13R.alpha.2 gene is localized to chromosome X, indicates that
IL-13R.alpha.2 is a CTA. Because other CTAs have proven to
stimulate a strong immune response against cancer cells, the
present invention provides methods and compositions useful for
generating or increasing an anti-cancer immune response in a
subject.
[0011] Accordingly, in one aspect the invention features a method
for stimulating a immune response against IL-13R.alpha.2 in a
subject having or at risk for developing a disease having cells
expressing IL-13R.alpha.2. The method includes the steps of: (a)
formulating an anti-cancer vaccine outside of the subject, the
vaccine including an agent that can stimulate an immune response
against IL-13R.alpha.2 when administered to an animal; and (b)
administering the vaccine to the subject in an amount sufficient to
stimulate an immune response against IL-13R.alpha.2 in the
subject.
[0012] In another aspect the invention features a composition for
stimulating an immune response against IL-13R.alpha.2 when
administered to an animal. The composition includes: (a) an
isolated agent that can stimulate an immune response against
IL-13R.alpha.2 when administered to an animal; and (b) a
pharmaceutically acceptable carrier.
[0013] In both of the foregoing method and composition, the agent
that can stimulate an id immune response against IL-13R.alpha.2 can
include a peptide including at least seven contiguous amino acids
of SEQ ID NO: 1. For example, the agent can be a protein including
the amino acid sequence of SEQ ID NO: 1. The agent can also take
the form of a nucleic acid that encodes a peptide including at
least seven contiguous amino acids of SEQ ID NO: 1. Such a nucleic
acid can be used as a naked DNA or in an expression vector
construct including the nucleic acid. The agent that can stimulate
an immune response against IL-13R.alpha.2 can also be a cell. This
cell can be one that expresses a peptide including at least seven
contiguous amino acids of SEQ ID NO: 1, or one into which a
purified nucleic acid that encodes a peptide including at least
seven contiguous amino acids of SEQ ID NO: 1 has been
introduced.
[0014] The vaccines and compositions within the invention can
further include an adjuvant such as an aluminum salt; an
oil-in-water emulsion; a composition including saponin; a
composition including a bacterial protein; or a cytokine.
[0015] The method of the invention can further include a step of
providing a subject (e.g., a human being) having or at risk for
developing a cancer having cells expressing IL-13R.alpha.2 (e.g.,
glioma cells). Also in the method, the step of administering the
vaccine to the subject in an amount sufficient to stimulate an
immune response against IL-13R.alpha.2 in the subject can include
administering the vaccine in at least a first dose and a second
dose, wherein the first dose is administered to the subject at
least 24 hours before the second dose is administered to the
subject.
[0016] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Definitions of
molecular biology terms can be found, for example, in Rieger et
al., Glossary of Genetics: Classical and Molecular, 5th edition,
Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford
University Press: New York, 1994. Standard one-letter nomenclature
for nucleotide bases, and one- and three-letter nomenclature for
amino acid residues are used.
[0017] As used herein, a "nucleic acid" means a chain of two or
more nucleotides. For example, RNA (ribonucleic acid) and DNA
(deoxyribonucleic acid) are nucleic acids. An "isolated" nucleic
acid is one that has been substantially separated or purified away
from other nucleic acid sequences in the cell of the organism in
which the nucleic acid naturally occurs, i.e., other chromosomal
and extrachromosomal DNA and RNA, e.g., by conventional nucleic
acid purification methods. The term therefore includes a
recombinant nucleic acid molecule incorporated into a vector, into
an autonomously replicating plasmid or virus, or into the genomic
DNA of a prokaryote or eukaryote. It includes a separate molecule
such as a cDNA, a genomic fragment, a fragment produced by
polymerase chain reaction (PCR), or a restriction fragment. It also
includes recombinant nucleic acid molecules and chemically
synthesized nucleic acid molecules. A "recombinant" nucleic acid
molecule is one made by an artificial combination of two otherwise
separated segments of sequence, e.g., by chemical synthesis or by
the manipulation of isolated segments of nucleic acids by genetic
engineering techniques.
[0018] When referring to a nucleic acid molecule or polypeptide,
the term "native" refers to a naturally-occurring (e.g., a
"wild-type") nucleic acid or polypeptide. A "homolog" of an
IL-13R.alpha.2 gene is a gene sequence encoding an IL-13R.alpha.2
polypeptide isolated from a species other than Homo sapiens. By the
phrase "naked nucleic acid" is meant an isolated nucleic acid not
incorporated in an expression vector.
[0019] By the terms "IL-13R.alpha.2 gene" or "IL-13R.alpha.2
polynucleotide" is meant a native IL-13R.alpha.2 encoding nucleic
acid sequence (e.g., the IL-13R.alpha.2 cDNA sequence shown as SEQ
ID NO: 2 (FIG. 2)), genomic sequences from which IL-13R.alpha.2
cDNA can be transcribed, and/or allelic variants and homologs of
the foregoing.
[0020] As used herein, "protein," "peptide," or "polypeptide" means
any peptide-linked chain of amino acids, regardless of length or
post-translational modification, e.g., glycosylation or
phosphorylation. Generally, the term "peptide" is used herein to
refer to amino acid chains less than about 25 amino acid residues
in length, while the terms "protein" and "polypeptide" are used to
refer to larger amino acid chains. When referring to a protein or
peptide, the term "isolated" means proteins or peptides that are
isolated from other cellular proteins or are made synthetically.
The term thus encompasses both purified and recombinant
polypeptides. The term "recombinant protein" or "recombinant
peptide" refers to a protein or peptide that is produced by
recombinant nucleic acid techniques, wherein generally, a nucleic
acid encoding the peptide or protein is inserted into a suitable
expression vector which is in turn used to transform a host cell
such that, when cultured under appropriate conditions, the cell
produces the peptide or protein.
[0021] By "IL-13R.alpha.2 protein" "IL-13R.alpha.2 polypeptide," or
simply "IL-13R.alpha.2" is meant an expression product of an
IL-13R.alpha.2 gene such as the protein of SEQ ID NO: 1 (FIG. 1);
or a ffi protein that shares at least 65% (but preferably 75, 80,
85, 90, 95, 96, 97 ,98, or 99%) amino acid sequence identity with
SEQ ID NO: 1 and cross-reacts with antibodies that specifically
bind the protein of SEQ ID NO: 1.
[0022] As used herein, "sequence identity" means the percentage of
identical subunits at corresponding positions in two sequences when
the two sequences are aligned to maximize subunit matching, i.e.,
taking into account gaps and insertions. When a subunit position in
both of the two sequences is occupied by the same monomeric
subunit, e.g., if a given position is occupied by an adenine in
each of two DNA molecules, then the molecules are identical at that
position. For example, if 7 positions in a sequence 10 nucleotides
in length are identical to the corresponding positions in a second
10-nucleotide sequence, then the two sequences have 70% sequence
identity. Preferably, the length of the compared sequences is at
least 60 nucleotides, more preferably at least 75 nucleotides, and
most preferably 100 nucleotides. Sequence identity is typically
measured using sequence analysis software (e.g., Sequence Analysis
Software Package of the Genetics Computer Group, University of
Wisconsin Biotechnology Center, 1710 University Avenue, Madison,
Wis. 53705).
[0023] A first nucleic-acid sequence is "operably" linked with a
second nucleic-acid sequence when the first nucleic-acid sequence
is placed in a functional relationship with the second nucleic-acid
sequence. For instance, a promoter is operably linked to a coding
sequence if the promoter affects the transcription or expression of
the coding sequence. Generally, operably linked DNA sequences are
contiguous and, where necessary to join two protein coding regions,
in reading frame.
[0024] As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. A vector capable of directing the expression of a
gene to which it is operatively linked is referred to herein as an
"expression vector." As used herein, the term "promoter" means a
nucleic acid sequence that regulates expression of a selected
nucleic acid sequence operably linked to the promoter, and which
effects expression of the selected nucleic acid sequence in cells.
The term encompasses "tissue specific" promoters, i.e. promoters,
which effect expression of the selected nucleic acid sequence only
in specific cells (e.g. cells of a specific tissue). The term also
covers so-called "leaky" promoters, which regulate expression of a
selected nucleic acid primarily in one tissue, but cause expression
in other tissues as well. The term also encompasses both non-tissue
specific promoters and promoters that are constitutively active and
inducible.
[0025] By the phrase "stimulating an immune response" is meant
eliciting or increasing the activation of a lymphocyte (e.g., a B
cell or T cell) or other immune system component. The stimulation
of an immune response against a specific antigen can be measured as
an increase in antibody titer against that antigen or the
activation of one or more lymphocytes having a surface receptor
specific for the antigen. Activation of lymphocytes can be
determined by conventional assays, e.g., the induction of mitosis,
secretion of cytokines, modulation of cell surface molecule
expression, secretion of immunoglobulin (B cells), and increased
killing of target cells (cytotoxic T cells).
[0026] As used herein, "bind," "binds," or "interacts with" means
that one molecule recognizes and adheres to a particular second
molecule in a sample, but does not substantially recognize or
adhere to other structurally unrelated molecules in the sample.
Generally, a first molecule that "specifically binds" a second
molecule has a binding affinity greater than about 10.sup.5 to
10.sup.6 liters/mole for that second molecule.
[0027] By the term "antibody" is meant any antigen-binding peptide
derived from an immunoglobulin. The term includes polyclonal
antisera, monoclonal antibodies, fragments of immunoglobulins
produced by enzymatic digestion (e.g., Fab fragments) or genetic
engineering (e.g., sFv fragments).
[0028] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions will control. In addition, the particular
embodiments discussed below are illustrative only and not intended
to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The invention is pointed out with particularity in the
appended claims. The above and further advantages of this invention
may be better understood by referring to the following description
taken in conjunction with the accompanying drawings, in which:
[0030] FIG. 1 is the amino acid sequence of the native H. sapiens
IL-13R.alpha.2 protein.
[0031] FIG. 2 is the nucleic acid sequence of a cDNA corresponding
to a native mRNA encoding the native H. sapiens IL-13R.alpha.2
protein.
[0032] FIG. 3 is a schematic representation of two types of IL13
receptors: the shared with IL4 physiological, heterodimeric
IL13/4R, and an IL4-independent monomeric, HGG-associated IL13R. A,
140-kDa IL4R .alpha.-chain. B, 45-kDa IL13R .alpha.1-chain; A and B
constitute the elements of the heterodimeric high affinity IL13/4R.
C, a 42-kDa monomer of IL13R.alpha.2.
[0033] FIG. 4 is a Northern blot analysis of human IL13R.alpha.2
transcripts (closed figure) in series of CNS (panels I and II) and
peripheral tissues (panels III and IV). The migration position of
MRNA is shown in kilobases. Films were exposed for 2 weeks.
[0034] FIG. 5 is a Northern blot analysis of human IL13R.alpha.2
transcripts (closed figure) in series of CNS (panels I and II) and
peripheral tissues (panels III and IV). The migration position of
mRNA is shown in kilobases. Films were exposed for 2 weeks except
for membranes shown in panels III and IV, which were exposed for 3
days.
[0035] FIG. 6 is a Northern blot analysis of human 140-kDa IL4R
.alpha.-chain transcripts (closed figure) in series of CNS (panels
I and II) and peripheral tissues (panel IV). The migration position
of mRNA is shown in kilobases. Films were exposed for 2 weeks.
[0036] FIG. 7 is a Northern blot analysis of human P-actin
transcripts in CNS (panels I and II) and peripheral tissues (panel
IV). The migration position of rnRNA is shown in kilobases. Films
were exposed for 1-3 hours.
[0037] FIG. 8 is a Northern blot analysis of transcripts of
different IL 13 receptors in malignant glioma cells (G-48, A-172
MG, U-373 MG, and U-251 MG), normal human umbilical vein
endothelial cells (HUVEC) and in surgical specimens of GBM and
normal human brain. The migration position of mRNA is shown in
kilobases. Films were exposed for 2 weeks, except for actin (1
hr).
DETAILED DESCRIPTION
[0038] The invention encompasses compositions and methods relating
to stimulating an immune response against IL-13R.alpha.2 in a
subject having or being at risk for developing a cancer or other
disease having cells expressing IL-13R.alpha.2. The below described
preferred LO embodiments illustrate adaptations of these
compositions and methods. Nonetheless, from the description of
these embodiments, other aspects of the invention can be made
and/or practiced based on the description provided below.
[0039] Biological Methods
[0040] Methods involving conventional molecular biology techniques
are described herein. Such techniques are generally known in the
art and are described in detail in methodology treatises such as
Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed.
Sambrook el al., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates). Methods for chemical synthesis of
nucleic acids are discussed, for example, in Beaucage and
Carruthers, Tetra. Letts. 22:1859-1862, 1981,and Matteuccietal., J.
Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleic acids
can be performed, for example, on commercial automated
oligonucleotide synthesizers. Immunological methods (e.g.,
preparation of antigen-specific antibodies, immunoprecipitation,
and immunoblotting) are described, e.g., in Current Protocols in
Immunology, ed. Coligan et al., John Wiley & Sons, New York,
1991; and Methods of Immunological Analysis, ed. Masseyeff et al.,
John Wiley & Sons, New York, 1992. Conventional methods of gene
transfer and gene therapy can also be adapted for use in the
present invention. See, e.g., Gene Therapy: Principles and
Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene
Therapy Protocols (Methods in Molecular Medicine), ed. P. D.
Robbins, Humana Press, 1997; and Retro-vectors for Human Gene
Therapy, ed. C. P. Hodgson, Springer Verlag, 1996.
[0041] Identification of IL-13R.alpha.2 as a Cancer/Testis
Antigen
[0042] As its name implies, IL-13R.alpha.2 is a receptor for the
lymphokine IL-13. IL-13 has been identified as a homologue of IL-4
that is secreted by both B and T cells. Minty et al., Nature, 36:
248-251, 1993; McKenzie et al., Proc. Natl. Acad. Sci. USA, 90:
3735-3739, 1993. Several types of normal cells contain an IL-13
receptor termed the shared IL-13/IL-4 receptor, which is a
heterodimer that includes an IL-13 binding subcomponent named
IL-13R.alpha.1 (Interleukin 13 receptor alpha one). Hilton et al.,
Proc. Natl. Acad. Sci. USA, 93: 497-501, 1996; Aman et al., J.
Biol. Chem., 271: 29265-29270, 1996; Miloux et al., FEBS Letters,
40: 163-166, 1997. In addition to IL-13R.alpha.1, the shared
receptor also includes a - protein referred to as p140 (or IL-4Ra),
the subcomponent responsible for IL-4 binding. Idzerda et al., J.
Exp. Med., 171: 861-873, 1990; Hilton et al., Proc. Natl. Acad.
Sci. USA, 93: 497-501, 1996; Debinski et al., Nature Biotech., 16:
449-453, 1995; Zurawski et al., EMBO J., 12: 2663-2670, 1993; Minty
et al., Nature, 36: 248-251, 1993. Exposing cells to IL-13 results
in responses very similar to those responses that occur after
exposure to IL-4. Zurawski, G., and J.E. de Vries, Stem Cells. 12:
169-174, 1994. Examples of cellular responses resulting from both
IL-13 and IL-4 exposure include enhanced expression of CD72, IgM,
and MHC class II antigen, as well as induced CD23 expression and
IgE heavy-chain gene production in B lymphocytes. Id.
[0043] In an interesting development, it was found that
IL-13R.alpha.1 was not the only IL-13 binding site that existed on
cells. In previous studies, it was demonstrated that many cancers,
most notably HGG, are capable of binding IL-13. Debinski et al.,
Clin. Cancer Res., 1:1253-1258, 1995; Debinski et al., J. Biol.
Chem., 271: 22428-22433, 1996; Debinski et al., Nature Biotech.,
16: 449-453, 1998; Debinski et al., Critic Rev. Oncogen., 9:
256-268, 1998; Debinski et al., Clin. Cancer Res., 5: 985-990,
1999. Through these studies, it became increasingly clear that the
IL-13 binding capacity of many of these tumors was not mediated
through the shared IL-13/IL-4 receptor (i.e., the receptor now
known to be a heterodimer composed of IL-13R.alpha.1/p140).
Notably, in lymphoid cells that contain the shared receptor,
saturating the receptors with IL-4 blocked IL-13 binding. Zurawski
et al., EMBO J., 12: 2663-2670, 1993. This was not the case using
HGG cells, where IL-13 binding was unaltered even where a large
excess of IL-4 used in neutralization assays. Debinski et al.,
Clin. Research Res., 1: 1253-1258, 1995; Debinski et al., J. Biol.
Chem., 271: 22428-22433, 1996; Debinski et al., Nature Biotech.,
16: 449-453, 1998. In further experiments, rationally designed
IL-13 mutants were generated that maintained their ability to bind
glioblastoma (HGG) cells but lost their ability to interact and
cause signaling in cells expressing only the IL-4/IL-13 shared
receptor. Debinski et al., Nature Biotech., 16: 449-453, 1998;
Thompson, J. P. and W. Debinski, J. Biol. Chem., 274: 29944-29950,
1999; Debinski, W., and J. P. Thompson, Clin. Cancer Res., 5:
3143s-3147s, 1999. This evidence supported the existence of an
additional IL-13 binding protein, unrelated to known IL-4 binding
proteins. Additional evidence was derived when a novel IL-13
binding protein on cells of renal cell carcinoma metastases (Caki-1
cells) was isolated and the gene encoding the protein cloned. Caput
et al., J. Biol. Chem., 271:16921, 1996. The gene encoding this
protein, termed IL-13R.alpha.2, was subsequently cloned and
sequenced. Id. This novel IL-13 binding protein, referred to herein
as IL-13R.alpha.2, was shown not to specifically bind IL-4. The
proposed structures of the shared -15- IL-13/4 receptor and the
IL-4-independent receptor for IL-13 are shown in FIG. 3.
[0044] To investigate whether this newly discovered receptor is
present in HGG, we evaluated its gene expression in HGG established
cell lines, and HGG explant cells and tumor specimens. In addition
to these studies on HGG, we screened a plethora of normal central
nervous system (CNS) tissues and peripheral organs for the MRNA
transcripts of IL-13R.alpha.2 in order to characterize the normal
tissue expression pattern of this new receptor in detail. From
these studies, we discovered that IL-13R.alpha.2 expression is
virtually absent in all normal adult tissue except testis. In
earlier studies, the gene encoding IL-13R.alpha.2 was localized to
the X chromosome. Guo et al., Genomics, 42: 141-145, 1997.
[0045] Accordingly, our discovery allowed us to characterize the
IL-13R.alpha.2 protein as a member of the CTA group of tumor
antigens. Moreover, because IL-13R.alpha.2 is a transmembrane
receptor, it is exposed to the extracellular environment
independently of MHC presentation. Thus, in contrast to
intracellular antigens that must be displayed as a peptide fragment
in complex with an MHC molecule on the cell surface to be
recognized by immune system components, cytotoxic agents or
antibodies can be directly targeted to cancer cells bearing
IL-13R.alpha.2 on their surface. This discovery that IL-13R.alpha.2
is a CTA associated with HGG is significant because no other
HGG-associated antigens of this prevalence are known that could
serve as a basis for a rational design of anti-glioma vaccines.
[0046] Vaccines
[0047] The invention provides vaccines that can stimulate an immune
response against IL-13R.alpha.2 in a subject when administered to
the subject. Vaccines within the invention include an antigenic
agent which can take the form of any substance that can evoke or
increase an immune response against IL-13R.alpha.2 when introduced
into a subject. Typical immune responses include (a) the production
of, or increase in titer of, antibodies that specifically bind
IL-13R.alpha.2 and (b) the activation of T lymphocytes (e.g., to
kill a target cell or provide help in the activation of antibody
production in B lymphocytes). A number of different antigenic
agents have been shown to be effective in stimulating an immune
response against a protein antigen, including, for example,
protein- and peptide-based vaccines, tumor-cell vaccines, dendritic
cell/gene therapy vaccines and DNA/viral vaccines. See, e.g.,
Greten, T. F. and E. M. Jaffee, J. Clin. Oncol., 17: 1047-1060,
1999. In addition to the foregoing, various substances such as
adjuvants and excipients/carriers can be included in the vaccine
compositions of the invention to non-specifically enhance the
antigen-specific immune response stimulated by the antigenic agent
and to facilitate delivery of the other components of the vaccine
to a subject.
[0048] Protein/Peptide Based Vaccines
[0049] The antigenic agent for use in the vaccines of the invention
can take the form of the native IL-13R.alpha.2 (SEQ ID NO:1) or a
peptide fragment of IL-13R.alpha.2. Vaccines made with the whole
protein antigen are advantageous because they have the capability
of stimulating an immune response against all of the potential
antigenic sites expressed by the protein. Vaccines made with
peptide antigens (e.g., 7-15 or 8-12 contiguous amino acids of the
whole protein), on the other hand, will generally stimulate an
immune response against fewer than all of the potential antigenic
sites expressed by the protein. Peptide-based vaccines are
sometimes advantageous over whole protein-based vaccines where it
is desired to more specifically target the stimulated immune
response, e.g., to avoid undesired cross reactions. For example,
peptides for use in the vaccine can be selected to correspond to
(1) specific epitopes of the antigens that are known to be
presented by MHC class I or MHC class II molecules, or (2) a
modified form of an epitope that either exhibits an increased
stability in vivo or a higher binding affinity for an MHC molecule
than the native epitope, while still being capable of specific
activation of T-cells. See, Ayyoub et al., J. Biol. Chem., 274:
10227-10234, 1999; Parkhurst et al., Immunol., 157: 2539-2548,
1996. Peptide-based vaccines have been shown to circumvent immune
tolerance to the intact proteins. Disis et al., J. Immunol., 156:
3151-3158, 1996. In addition to vaccines composed of only one type
of peptide fragment, other vaccines within the invention also
include those made up of a cocktail of several different peptides
derived from IL-13R.alpha.2.
[0050] As indicated above, vaccines with in the invention can
include an IL-13R.alpha.2 protein as an antigenic agent. Preferred
forms of IL-13R.alpha.2 protein include a purified native
IL-13R.alpha.2 protein that has the amino acid sequence shown in
FIG. 1 (SEQ ID NO: 1). Variants of the native IL-13R.alpha.2
protein such as fragments, analogs and derivatives of native
IL-13R.alpha.2 are also contemplated for use as an antigenic agent
in the vaccines of the invention. Such variants include, e.g., a
polypeptide encoded by a naturally occurring allelic variant of the
native IL-13R.alpha.2 gene, a polypeptide encoded by a homolog of
the native IL-13R.alpha.2 gene, and a polypeptide encoded by a
non-naturally occurring variant of the native IL-13R.alpha.2 gene.
Preferred versions of such variants are those that are able to
stimulate an immune response to native IL-13R.alpha.2 upon
administration to a subject as part of a vaccine.
[0051] IL-13R.alpha.2 protein variants have a peptide sequence that
differs from the native IL-13R.alpha.2 protein in one or more amino
acids. The peptide sequence of such variants can feature a
deletion, addition, or substitution of one or more amino acids of
the native IL-13R.alpha.2 polypeptide. Amino acid insertions are
preferably of about 1 to 4 contiguous amino acids, and deletions
are preferably of about 1 to 10 contiguous amino acids. In some
applications, variant IL-13R.alpha.2 proteins substantially
maintain a native IL-13R.alpha.2 protein functional activity (e.g.,
the ability to specifically bind IL-13). For other applications,
variant IL-13R.alpha.2 proteins lack or feature a significant
reduction in an IL-13R.alpha.2 protein functional activity. Where
it is desired to retain a functional activity of native
IL-13R.alpha.2 protein, preferred IL-13R.alpha.2 protein variants
can be made by expressing nucleic acid molecules within the
invention that feature silent or conservative changes. Variant
IL-13R.alpha.2 proteins with substantial changes in functional
activity can be made by expressing nucleic acid molecules within
the invention that feature less than conservative changes.
[0052] IL-13R.alpha.2 protein fragments corresponding to one or
more particular motifs (e.g., those likely to bind with high
affinity to MHC molecules) and/or domains are within the invention
as are those of arbitrary sizes. For example, peptide fragments of
IL-13R.alpha.2 protein consisting of at least 5, 10, 25, 30, 40,
50, 50, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 300 or more
contiguous amino acids of the IL-13R.alpha.2 protein are within the
scope of the present invention. Fragments of between 7 and 15 amino
acids (preferably 8-12 amino acids) in length (e.g., those sized to
fit in the grooves of MHC molecules) are preferred as peptides of
such size have been shown to serve as efficient immunogenic agents.
Methods for identifying efficiently immunogenic peptides of a whole
protein are known in the art, e.g., using amphipathicity
algorithms. See, e.g., Berzofsky, J. A., Ann. N. Y. Acad. Sci.,
12:256, 1993; U.S. Pat. Nos. 5,976,541 and 5,980,899. Peptides that
are most immunogenic in a subject can also be determined by
preparing a series of overlapping peptide fragments (e.g., 7-30
amino contiguous amino acids long) of the whole antigen,
administering the subject (or a series of genetically similar such
subjects) such fragments in a vaccine composition, and analyzing
the subject(s) for the stimulation of an immune response. Those
peptide fragments that induce the desired response can then be
selected.
[0053] Isolated peptidyl portions of IL-13R.alpha.2 proteins can be
obtained by screening peptides recombinantly produced from the
corresponding fragment of the nucleic acid encoding such peptides.
In addition, fragments can be chemically synthesized using
techniques known in the art such as conventional Merrifield solid
phase f-Moc or t-Boc chemistry. For example, similar to the
technique described above, an IL-13R.alpha.2 protein of the present
invention may be arbitrarily divided into fragments of desired
length with no overlap of the fragments, or preferably divided into
overlapping fragments of a desired length. The fragments can be
produced (recombinantly or by chemical synthesis) and tested to
identify those peptidyl fragments which can function antigenic
agents that stimulate an immune response against an IL-13R.alpha.2
protein.
[0054] Another aspect of the present invention concerns recombinant
forms of the IL-13R.alpha.2 proteins. Recombinant polypeptides
preferred for use in the present invention, in addition to native
IL-13R.alpha.2 protein, are encoded by a nucleic acid that has at
least 85% sequence identity (e.g., 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, 100%) with the nucleic acid sequence of
SEQ ID NO:2. In a preferred embodiment, variant IL-13R.alpha.2 have
the ability to stimulate an immune response against the native
IL-13R.alpha.2 protein.
[0055] IL-13R.alpha.2 protein variants can be generated through
various techniques known in the art. For example, IL-13R.alpha.2
protein variants can be made by mutagenesis, such as by introducing
discrete point mutation(s), or by truncation. Mutation can give
rise to an IL-13R.alpha.2 protein variant having more,
substantially the same, or merely a subset of the antigenic
activity of the native IL-13R.alpha.2 protein. Other variants of
IL-13R.alpha.2 that can be generated include those that are
resistant or more susceptible to proteolytic cleavage, as for
example, due to mutations which alter protease target sequences.
Whether a change in the amino acid sequence of a peptide results in
a IL-13R.alpha.2 protein variant having greater or lesser antigenic
activity than native IL-13R.alpha.2 protein can be readily
determined by comparing the variant with the native IL-13R.alpha.2
protein for the ability to stimulate an immune response against
IL-13R.alpha.2 in subjects vaccinated with the respective
proteins.
[0056] As another example, IL-13R.alpha.2 protein variants can be
generated from a degenerate oligonucleotide sequence. Chemical
synthesis of a degenerate gene sequence can be carried out in an
automatic DNA synthesizer, and the synthetic genes then ligated
into an appropriate expression vector. The purpose of a degenerate
set of genes is to provide, in one mixture, all of the sequences
encoding the desired set of potential IL-13R.alpha.2 protein
sequences. The synthesis of degenerate oligonucleotides is well
known in the art (see for example, Narang, SA (1983) Tetrahedron
39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland
Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp
273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura
et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.
11:477. Such techniques have been employed in the directed
evolution of other proteins (see, for example, Scott et al. (1990)
Science 249:386-390; Roberts et al. (1992) Proc. Natl. Acad. Sci.
USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla
et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382; as well as
U.S. Pat. Nos. 5,223,409; 5,198,346; and 5,096,815).
[0057] Similarly, a library of coding sequence fragments can be
provided for an IL-13R.alpha.2 gene clone in order to generate a
variegated population of IL-13R.alpha.2 protein fragments for
screening and subsequent selection of fragments having the ability
to stimulate an immune response against IL-13R.alpha.2 in a
subject. A variety of techniques are known in the art for
generating such libraries, including chemical synthesis. In one
embodiment, a library of coding sequence fragments can be generated
by (i) treating a double-stranded PCR fragment of an IL-13R.alpha.2
gene coding sequence with a nuclease under conditions wherein
nicking occurs only about once per molecule; (ii) denaturing the
double-stranded DNA; (iii) renaturing the DNA to form
double-stranded DNA which can include sense/antisense pairs from
different nicked products; (iv) removing single-stranded portions
from reformed duplexes by treatment with S1 nuclease; and (v)
ligating the resulting fragment library into an expression vector.
By this exemplary method, an expression library can be derived
which codes for N-terminal, C-terminal and internal fragments of
various sizes.
[0058] The invention also provides for reduction of IL-13R.alpha.2
proteins to generate mimetics, e.g. peptide or non-peptide agents,
that are able to stimulate an immune response against
IL-13R.alpha.2 in a subject. For instance, non-hydrolyzable peptide
analogs of the amino acid residues of IL-13R.alpha.2 proteins and
peptides thereof can be generated using benzodiazepine (e.g., see
Freidinger et al. in Peptides: Chemistry and Biology, G. R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine
(e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
substituted gamma lactam rings (Garvey et al. in Peptides:
Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al.
(1986) J. Med. Chem. 29:295; and Ewenson et al. in Peptides:
Structure and Function (Proceedings of the 9th American Peptide
Symposium) Pierce Chemical Co. Rockland, Ill., 1985), beta-turn
dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and
Sato et al. (1986) J. Chem. Soc. Perkin. Trans. 1:1231), and
b-aminoalcohols (Gordon et al. (1985) Biochem. Biophys. Res.
Commun. 126:419; and Dann et al. (1986) Biochem. Biophys. Res.
Commun. 134:71). IL-13Ru2 proteins may also be chemically ZU 2E
modified to create IL-13R.alpha.2 derivatives by forming covalent
or aggregate conjugates with other chemical moieties, such as
glycosyl groups, lipids, phosphate, acetyl groups and the like.
Covalent derivatives of IL-13R.alpha.2 proteins or peptides can be
prepared by linking the chemical moieties to functional groups on
amino acid side chains of the protein/peptide or at the N-terminus
or at the C-terminus of the protein/peptide.
[0059] IL-13R.alpha.2 proteins may also be fused to one or more
other proteins. For example, an IL-13R.alpha.2 protein or
immunogenic portion thereof may be fused to another protein that
serves as a targeting ligand to deliver the IL-13R.alpha.2 protein
or portion to a particular target site in a subject (e.g., in order
to stimulate a local immune response at that site). For instance,
an IL-13R.alpha.2 protein or peptide can be fused to a mutant IL-13
molecule or anti-IL-13 receptor antibody to specifically target the
IL-13R.alpha.2 protein or peptide to a tumor, e.g., a HGG. Numerous
methods of fusing two or more proteins together are known in the
art, e.g., making and expressing a recombinant fusion construct, or
using a cross-linking agent to covalently bond the two or more
proteins together to form one molecule. Any suitable for this
application might be used in the invention.
[0060] The IL-13R.alpha.2 proteins and peptides of the invention
can be made by known methods. For example, a host cell transfected
with a nucleic acid vector directing expression of a nucleotide
sequence encoding the subject proteins or peptides can be cultured
under appropriate conditions to allow expression of the peptide to
occur. The cells may be harvested, lysed, and the protein isolated.
A recombinant IL-13R.alpha.2 protein or peptide can be isolated
from host cells using techniques known in the art for purifying
proteins including ion-exchange chromatography, gel filtration
chromatography, ultrafiltration, electrophoresis, and
immunoaffinity purification with antibodies specific for such
protein or peptide.
[0061] For example, after an IL-13R.alpha.2 protein or peptide has
been expressed in a cell, it can be isolated using immuno-affinity
chromatography. For instance, an anti-IL-13R.alpha.2 antibody that
specifically binds the subject proteins or peptides can be
immobilized on a column chromatography matrix, and the matrix can
be used for immuno-affinity chromatography to purify the proteins
or peptides from cell lysates by standard methods (see, e.g.,
Ausubel et al., supra). After immuno-affinity chromatography, the
proteins or peptides can be further purified by other standard
techniques, e.g., high performance liquid chromatography (see,
e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular
Biology, Work and Burdon, eds., Elsevier, 1980). In another
embodiment, the IL-13R.alpha.2 proteins or peptides utilized in the
invention are expressed as a fusion protein containing an affinity
tag (e.g., GST) that facilitates its purification.
[0062] In association with an antigenic agent (e.g., a
IL-13R.alpha.2 protein or peptide fragment thereof) of a vaccine of
the invention, an adjuvant can be used to boost the immune
response. Suitable adjuvants for use in the invention can include
any substance that can non-specifically enhance an antigen-specific
immune response stimulated by an antigenic agent. Many such
adjuvants are known, including for example: (1) Freund's adjuvant
(complete and incomplete) (2) oil-in-water emulsion formulations
such as the Ribi.TM. adjuvant system (Corixa, Seattle, Wash.) (3)
aluminum salts (e.g., aluminum hydroxide, aluminum phosphate,
aluminum sulfate, etc); (4) saponin-based adjuvants (Stimulon.TM.
from Aquila Biosciences, Framingham, Mass.); (5) cytokines such as
IL-1, IL-2, macrophage colony stimulating factor, and tumor
necrosis factor; and (6) other substances that act as
immunostimulating agents such as muramyl peptides or bacterial cell
wall components, toxins, and toxoids.
[0063] To facilitate their formulation for administration to a
subject, the vaccine compositions of the invention (e.g., the
protein/peptide antigen and adjuvant) can further contain a
pharmaceutically acceptable carrier or excipient. For example the
protein/peptide antigen and adjuvant can be mixed with a diluent
such as water, saline, glycerol, ethanol, etc. Other substances,
such as preservatives, surfactants, emulsifying agents, buffers,
etc. can also be included. Typically, the protein/peptide-based
vaccine compositions of the invention are prepared for parenteral
injection as liquid solutions or suspensions. The vaccine
compositions can also be prepared as solids (e.g., a lyophilized
powder) that can be reconstituted in a liquid (e.g., saline) prior
to injection into a subject. The vaccine compositions can also be
emulsified or encapsulated in liposomes.
[0064] Nucleic Acid-Based Vaccines
[0065] Nucleic acid-based vaccines are known to elicit a prominent
cell-mediated immune response. See, e.g., Donnely et al., 1997;
Rosenberg, S. A., Immunity 10:281, 1999. Thus, in addition to
protein/peptide based vaccines, the antigenic agent for use in the
vaccines of the invention can take the form of a nucleic acid that
can stimulate an immune response against IL-13R.alpha.2 when
administered to a subject. Examples of such nucleic acids include
those that encode the native IL-13R.alpha.2 such as the nucleic
acid shown herein as SEQ ID NO:2 (FIG. 2), a variant of the native
IL-13R.alpha.2, or a peptide fragment of that native or variant
IL-13R.alpha.2. Vaccines made with a nucleic acid that encodes the
whole protein antigen are advantageous because they have the
potential for stimulating an immune response against all of the
different antigenic sites expressed by the protein. Vaccines made
with a nucleic acid that encodes a peptide antigen (e.g., 7-15
amino acids of the whole protein), on the other hand, will
generally stimulate an immune response against fewer than all of
the potential antigenic sites expressed by the protein.
[0066] The form of the nucleic acid used in a vaccine of the
invention can be any suitable for stimulating an immune response
against IL-13R.alpha.2 when administered to a subject. For example,
the nucleic acid can be in the form of "naked DNA" or it can be
incorporated in an expression vector. A description of suitable
nucleic acids is presented below. Nucleic acids that are most
immunogenic in a subject can be determined by preparing several of
the below listed nucleic acids (e.g., those that encode the whole
antigen or peptide fragments thereof), administering the subject
(or a series of genetically similar such subjects) such nucleic
acids in a vaccine composition (e.g., as naked nucleic acid or in
an expression vector in a suitable carrier), and analyzing the
subject(s) for the stimulation of an immune response. Those nucleic
acids that induce the desired response can then be selected.
[0067] Nucleic acid molecules utilized in the present invention as
an antigenic agent may be in the form of RNA or in the form of DNA
(e.g., cDNA, genomic DNA, and synthetic DNA). The DNA may be
double-stranded or single-stranded, and if single-stranded may be
the coding (sense) strand or non-coding (anti-sense) strand. The
coding sequence which encodes the native IL-13R.alpha.2 protein may
be identical to the nucleotide sequence shown in FIG. 2. It may
also be a different coding sequence which, as a result of the
redundancy or degeneracy of the genetic code, encodes the same
polypeptide as shown in SEQ ID NO:1 (FIG. 1).
[0068] Other nucleic acid molecules useful in the invention are
variants of the native IL-13R.alpha.2 gene such as those that
encode fragments (e.g., post-translationally processed forms of),
analogs and derivatives of a native IL-13R.alpha.2 protein. Such
variants may be, e.g., a naturally occurring allelic variant of the
native IL-13R.alpha.2 gene, a homolog of the native IL-13R.alpha.2
gene, or a non-naturally occurring variant of the native
IL-13R.alpha.2 gene. These variants have a nucleotide sequence that
differs from the native IL-13R.alpha.2 gene in one or more bases.
For example, the nucleotide sequence of such variants can feature a
deletion, addition, or substitution of one or more nucleotides of
the native IL-13R.alpha.2 gene. Nucleic acid insertions are
preferably of about 1 to 10 contiguous nucleotides, and deletions
are preferably of about 1 to 30 contiguous nucleotides.
[0069] Naturally occurring allelic variants of the native
IL-13R.alpha.2 gene within the invention are nucleic acids isolated
from human tissue that have at least 75% (e.g., 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with the
native IL-13R.alpha.2 gene, and encode polypeptides having
structural similarity to native IL-13R.alpha.2 protein. Homologs of
the native IL-13R.alpha.2 gene within the invention are nucleic
acids isolated from other species that have at least 75% (e.g.,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence
identity with the native IL-13R.alpha.2 gene, and encode
polypeptides having structural similarity to native IL-13R.alpha.2
protein. Public and/or proprietary nucleic acid databases can be
searched in an attempt to identify other nucleic acid molecules
having a high percent (e.g., 70, 80, 90% or more) sequence identity
to the native IL-13R.alpha.2 gene.
[0070] Non-naturally occurring IL-13R.alpha.2 gene variants are
nucleic acids that do not occur in nature (e.g., are made by the
hand of man), have at least 75% (e.g., 76%, 77%, 78%, 79%,
80%,81%,82%,83%,84%,85%,86%,- 87%,88%,89%,90%,91%,92%,93%,94%,95%,
96%, 97%, 98%, and 99%) sequence identity with the native
IL-13R.alpha.2 gene, and encode polypeptides having structural
similarity to native IL-13R.alpha.2 protein. Examples of
non-naturally occurring IL-13R.alpha.2 gene variants are those that
encode a fragment of a IL-13R.alpha.2 protein, those that hybridize
to the native IL-13R.alpha.2 gene or a complement of to the native
IL-13R.alpha.2 gene under stringent conditions, those that share at
least 65% sequence identity with the native IL-13R.alpha.2 gene or
a complement of the native IL-13R.alpha.2 gene, and those that
encode a IL-13R.alpha.2 fusion protein.
[0071] Nucleic acids encoding fragments of native IL-13R.alpha.2
protein within the invention are those that encode, e.g.,
2,5,6,7,8,9,10,11,12, 13,14,15,16,17,18, 19,20,25,30,40, 50, 60,
70, 80, 90, 100, 150, 200, 250, 300 or more amino acid residues of
the native IL-13R.alpha.2 protein. Shorter oligonucleotides (e.g.,
those of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 30,
50, 100, 125, 150, or 200 base pairs in length) that encode
fragments of the native IL-13R.alpha.2 protein can be used. Nucleic
acids encoding fragments of native IL-13R.alpha.2 protein can be
made by enzymatic digestion (e.g., using a restriction enzyme) or
chemical degradation of the full length native IL-13R.alpha.2 gene
or variants thereof.
[0072] Nucleic acid molecules encoding IL-13R.alpha.2 fusion
proteins are also within the invention. Such nucleic acids can be
made by preparing a construct (e.g., an expression vector) that
expresses a IL-13R.alpha.2 fusion protein when introduced into a
suitable host. For example, such a construct can be made by
ligating a first polynucleotide encoding an IL-13R.alpha.2 protein
fused in frame with a second polynucleotide encoding another
protein (e.g., a detectable label or carrier protein) such that
expression of the construct in a suitable expression system yields
a fusion protein. IL-13R.alpha.2 fusion proteins can be used, e.g.,
to enhance the immunogenicity of IL-13R.alpha.2 peptides, to
facilitate purification of IL-13R.alpha.2 proteins/peptides, or to
track the location of the IL-13R.alpha.2 fusion protein after it
has been administered to a subject.
[0073] Using the nucleotide sequence of the native IL-13R.alpha.2
gene and the amino acid sequence of a native IL-13R.alpha.2
protein, those skilled in the art can create nucleic acid molecules
that have minor variations in their nucleotide sequences, by, for
example, standard nucleic acid mutagenesis techniques or by
chemical synthesis. Variant IL-13R.alpha.2 nucleic acid molecules
can be expressed to produce variant IL-13R.alpha.2 proteins.
[0074] Naked Nucleic Acid Vaccines
[0075] The invention provides for the use of naked nucleic acid
vaccines to stimulate an immune response against IL-13R.alpha.2.
Representative naked nucleic acid vaccines for use in this method
include a DNA encoding one or more immunogenic portions of
IL-13R.alpha.2 along with sufficient other 5' and 3' elements to
direct expression of the foregoing. The use of naked nucleic acids
for stimulating both class I and class II restricted immune
responses against a particular protein is known in the art. See,
e.g., Rosenberg, S. A., Immunity 10:281, 1999; Ulmer et al.,
Science, 259:1745, 1993; Donnelly et al., Ann. NY Acad. Sci.,
772:40, 1995; Scheurs et al., Cancer res. 58:2509, 1998; Hurpin et
al., Vaccine 16:208, 1998; Lekutis et al., J. Immunol. 158:4471,
1997; Manickan et al., J. Leukoc. Biol. 61:125, 1997. These methods
can be adapted for use in the present invention by using a nucleic
acid encoding one or more immunogenic portions of IL-13R.alpha.2.
Naked nucleic acid vaccines can be administered to a subject by any
suitable technique. For example, naked DNA encoding a peptide
portion of IL-13R.alpha.2 can be injected into muscle cells of a
subject or naked DNA-coated gold particles can be introduced into
skin cells (to be taken up by dendritic cells) of a subject using a
gene gun.
[0076] Expression Vector Vaccines
[0077] The invention also provides for the use of expression vector
vaccines to stimulate an immune response against IL-13R.alpha.2. In
a typical application of this technique, a nucleic acid encoding
one or more peptide or protein antigens of IL-13R.alpha.2 is
incorporated into a vector that allows expression of the antigen(s)
in a host cell (e.g., a cell inside a subject or administered to a
subject). The nucleic acid encoding the antigen(s) is generally be
under the operational control of other sequences contained within
the vector such as a promoter sequences (e.g., tissue specific,
constitutively active, or inducible) or enhancer sequences. The
antigen(s) encoded by the vector are expressed when the vector is
introduced into a host cell in a subject. After expression, the
antigen(s) can associate with an MHC molecule for presentation to
immune system cells such as T lymphocytes, thus stimulating an
immune response. See, e.g., Corr et al., J. Exp. Med. 184:1555
(1996).
[0078] Vectors for use in the invention can be any capable of
expressing an encoded antigen(s) in a subject. For example, vectors
derived from bacterial plasmids and viruses may be used.
Representative viral vectors include retroviral, adenoviral, and
adeno-associated viral vectors. See, e.g., Gene Therapy: Principles
and Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene
Therapy Protocols (Methods in Molecular Medicine), ed. P. D.
Robbins, Humana Press, 1997; and Retro-vectors for Human Gene
Therapy, ed. C. P. Hodgson, Springer Verlag, 1996.
[0079] Cell-Based Vaccines
[0080] Cell-based vaccines are provided in the invention to
stimulate an immune response against IL-13R.alpha.2. In similar
approaches using different cancer-associated antigen, cancer cells
isolated from a patient have been harbored in vitro and transfected
with DNA encoding for immune stimulants, such as cytokines, MHC
molecules or co-stimulatory molecules. The transfected cancer cells
were then re-injected to the patient in order to activate the
immune system in order to generate an anti-cancer response. Greten,
T. F., and E. M. Jaffee, J. Clin. Oncol., 17: 1047-1060, 1999;
Simons et al., Cancer Res., 57: 1537-1546, 1997.
[0081] The invention further provides an isolated cell expressing
IL-13R.alpha.2 or a peptide fragment of IL-13R.alpha.2. Cells
expressing IL-13R.alpha.2 can be isolated from a subject having
such cells (e.g., from testis or HGG). Cells that do not express
IL-13R.alpha.2 can be made to express this protein in a number of
different ways. As one example, cells can be cultured with
IL-13R.alpha.2 or peptide fragments thereof under conditions in
which fragments of IL-13R.alpha.2 become associated with MHC
molecules on the cell surface. Alternatively, cells can be made to
express IL-13R.alpha.2 by introducing a nucleic acid encoding an
IL-13R.alpha.2 protein, a peptide fragment of IL-13R.alpha.2, or a
variant of the foregoing into the cells, and culturing such cells
under conditions that cause the cells to express the protein or
peptide. Cellular expression of the protein, peptide, or variant
can be monitored by any conventional technique. For example,
fluorescently labeled antibodies that specifically bind the
protein, peptide, or variant can be used to detect expression of
the protein, peptide, or variant on a cell. See, e.g., Kim et al.,
J. Immunother. 20:276, 1997. In addition, Western blotting using
antibodies that specifically bind the protein, peptide, or variant
can be used to detect expression of the protein, peptide, or
variant in lysates of a cell.
[0082] Cell types suitable for stimulating an immune response
against IL-13R.alpha.2 can be prokaryotic or eukaryotic. A number
of such cells are known in the art, so an exhaustive list is not
provided herein. Examples of suitable prokaryotic cells include
bacterial cells such as E. coli, B. subtilis, and mycobacteria.
Examples of suitable eukaryotic cells include plant, yeast, insect,
avian, nematode (e.g., C. elegans), and mammalian cells (e.g.,
autologous cells from a human patient that are to be later
reintroduced into the patient). These cells can be cultured in
conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0083] Further examples of cells that can be used to stimulate an
immune response against IL-13R.alpha.2 include those that express a
peptide comprising a least 7 (e.g., 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20 or more) contiguous amino acids of SEQ ID
NO:1. For instance, an isolated cell expressing a protein having
the sequence of SEQ ID NO: 1 can be used. Cells into which have
been introduced a purified nucleic acid that encodes a peptide
comprising a least 7 contiguous amino acids of SEQ ID NO: 1 might
also be used.
[0084] Although any cell that can express IL-13R.alpha.2 protein, a
peptide fragment of IL-13R.alpha.2, or a variant of the foregoing
can be used to stimulate an immune response in a subject, some are
preferred because of their particular antigen presentation
capabilities. Examples of such cells include antigen-presenting
cells (APCs) such as B lymphocytes, monocytes/macrophages,
dendritic cells (DC), and other cells expressing major
histocompatability complex (MHC) and/or costimulatory
molecules.
[0085] Since DC are known to function as particularly strong APCs,
their use in the cell-based vaccine of the invention is
particularly preferred. See, e.g., Banchereau et al., Ann. Rev.
Immunology, 18:767, 2000. DC can be made to express an
IL-13R.alpha.2 protein, a peptide fragment of IL-13R.alpha.2, or a
variant thereof as described above. For example, DC can be removed
from a subject, contacted with the selected antigen, and then
returned to the subject to stimulate an immune response. To enhance
their antigen presentation capability, the DC can also be treated
with an activating substance such as a cytokines.
[0086] Those cell-based vaccines that are most effective in
stimulating an immune response against IL-13R.alpha.2 in a subject
can be determined by preparing a series of different cell-based
vaccine (e.g. those expressing whole antigen or specific peptide
fragments of the antigen), administering a subject (or a series of
genetically similar subjects) such different vaccines, and
analyzing the subject(s) for the stimulation of an immune response.
Those vaccines that induce the desired response can then be
selected.
[0087] Anti-Idiotypic Antibody Vaccines
[0088] The invention also contemplates the use of anti-idiotypic
antibody vaccines to stimulate an immune response against
IL-13R.alpha.2 in a subject. In this method, anti-idiotypic
antibodies are prepared that feature an internal "image" of one or
more immunogenic portions of IL-13R.alpha.2. See, e.g., U.S. Pat.
Nos. 5,053,224; 5,208,146; 5,612,030; and 5,925,362. Administration
of these anti-idiotypic antibodies in a vaccine composition to a
subject can stimulate an immune response against the "image" of an
immunogenic portion of IL-13R.alpha.2 which cross-reacts against
actual immunogenic portions of IL-13R.alpha.2. As one example,
polyclonal anti-idiotypic antibodies can be generated by immunizing
a host animal with monoclonal antibodies raised against an epitope
of IL-13R.alpha.2. Methods of preparing monoclonal and polyclonal
antibodies as described in more detail below.
[0089] Antibody Production
[0090] The vaccines/antigenic agents featured in the invention can
be used to raise antibodies useful in the invention. Polyclonal
antibodies are heterogeneous populations of antibody molecules that
are contained in the sera of the immunized animals. Antibodies
within the invention therefore include polyclonal antibodies and,
in addition, monoclonal antibodies, single chain antibodies, Fab
fragments, F(ab').sub.2 fragments, and molecules produced using a
Fab expression library. Monoclonal antibodies, which are
homogeneous populations of antibodies to a particular antigen, can
be prepared using the IL-13R.alpha.2 proteins and peptides
described above and standard hybridoma technology (see, for
example, Kohler et al., Nature 256:495, 1975; Kohler et al., Eur.
J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292,
1976; Hammerling et al., "Monoclonal Antibodies and T Cell
Hybridomas," Elsevier, N.Y., 1981; Ausubel et al., supra). In
particular, monoclonal antibodies can be obtained by any technique
that provides for the production of antibody molecules by
continuous cell lines in culture such as described in Kohler et
al., Nature 256:495, 1975, and U.S. Pat. No. 4,376,110; the human
B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72,
1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026, 1983), and
the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies
and Cancer Therapy," Alan R. Liss, Inc., pp. 77-96, 1983). Such
antibodies can be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. A hybridoma producing a
'nAb of the invention may be cultivated in vitro or in vivo. The
ability to produce high titers of rmabs in vivo makes this a
particularly useful method of production.
[0091] Human or humanoid antibodies that specifically bind a
IL-13R.alpha.2 protein can also be produced using known methods.
For example, polyclonal antibodies can also be collected from human
subjects having such antibodies in their sera, e.g., subjects
administered vaccines that stimulate antibody production against
IL-13R.alpha.2. As another example, human antibodies against
IL-13R.alpha.2 protein can be made by adapting known techniques for
producing human antibodies in animals such as mice. See, e.g.,
Fishwild, D. M. et al., Nature Biotechnology 14 (1996): 845-851;
Heijnen, I. et al., Journal of Clinical Investigation 97 (1996):
331-338; Lonberg, N. et al., Nature 368 (1994): 856-859; Morrison,
S. L., Nature 368 (1994): 812-813; Neuberger, M., Nature
Biotechnology 14 (1996): 826; and U.S. Pat. Nos. 5,545,806;
5,569,825; 5,877,397; 5,939,598; 6,075,181; 6,091,001; 6,114,598;
and 6,130,314. Humanoid antibodies against IL-13R.alpha.2 can be
made from non-human antibodies by adapting known methods such as
those described in U.S. Pat. Nos. 5,530, 101; 5,585,089; 5,693,761;
and 5,693,762.
[0092] Once produced, polyclonal or monoclonal antibodies can be
tested for specific IL-13R.alpha.2 recognition by Western blot or
immunoprecipitation analysis by standard methods, for example, as
described in Ausubel et al., supra. Antibodies that specifically
recognize and bind to IL-13R.alpha.2 are useful in the invention.
For example, such antibodies can be used in an immunoassay to
monitor the level of IL-13R.alpha.2 in a sample (e.g., to determine
the amount of cellular expression or subcellular location of
IL-13R.alpha.2, or the presence and amount of soluble forms of
IL-13R.alpha.2 in a liquid sample).
[0093] Preferably, IL-13R.alpha.2 protein selective antibodies of
the invention are produced using fragments of the IL-13R.alpha.2
protein that lie outside highly conserved regions and appear likely
to be antigenic by criteria such as high frequency of charged
residues. Cross-reactive anti-IL-13R.alpha.2 protein antibodies are
produced using a fragment of a IL-13R.alpha.2 protein that is
conserved among members of this family of proteins. In one specific
example, such fragments are generated by standard techniques of
PCR, and are then cloned into the pGEX expression vector (Ausubel
et al., supra). Fusion proteins are expressed in E. coli and
purified using a glutathione agarose affinity matrix as described
in Ausubel, et al., supra.
[0094] In some cases it may be desirable to minimize the potential
problems of low affinity or specificity of antisera. In such
circumstances, two or three fusions can be generated for each
protein, and each fusion can be injected into at least two rabbits.
Antisera can be raised by injections in a series, preferably
including at least three booster injections. Antiserum is also
checked for its ability to immunoprecipitate recombinant
IL-13R.alpha.2 proteins or control proteins, such as glucocorticoid
receptor, CAT, or luciferase.
[0095] Techniques described for the production of single chain
antibodies (e.g., U.S. Pat. Nos. 4,946,778, 4,946,778, and
4,704,692) can be adapted to produce single chain antibodies
against a IL-13R.alpha.2 protein, or a fragment thereof. Single
chain antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide.
[0096] Antibody fragments that recognize and bind to specific
epitopes can be generated by known techniques. For example, such
fragments include but are not limited to F(ab').sub.2 fragments
that can be produced by pepsin digestion of the antibody molecule,
and Fab fragments that can be generated by reducing the disulfide
bridges of F(ab').sub.2 fragments. Alternatively, Fab expression
libraries can be constructed (Huse et al., Science 246:1275, 1989)
to allow rapid and easy identification of monoclonal Fab fragments
with the desired specificity.
[0097] Method of Inducing an Anti-IL-13R.alpha.2 Immune Response in
a Subject
[0098] The invention provides methods for stimulating a immune
response against IL-13R.alpha.2 in a subject having or at risk for
developing a cancer having cells expressing IL-13R.alpha.2. Such
methods can be performed by (a) formulating as anti-cancer vaccine
composition (as described above) outside of the subject and (b)
administering the vaccine to the subject in an amount sufficient to
stimulate an immune response against IL-13R.alpha.2 in the
subject.
[0099] Subjects
[0100] The compositions and methods of the invention can be
utilized with any suitable subject, e.g., an animal such as a
mammal (e.g., human beings, dogs, cats, goats, sheep, cows, horses,
etc.). A human patient suffering or at risk for developing a cancer
or other disease that has cells that overexpress IL-13R.alpha.2
(e.g., a brain cancer such as HGG) is a particularly preferred
subject.
[0101] IL-13R.alpha.2 as a Component of a Polyvalent Vaccine
[0102] The invention also provides polyvalent vaccines that
incorporate one or more of the foregoing compositions that can
stimulate an immune response against IL-13R.alpha.2 in a subject.
Two general types of polyvalent vaccines are within the invention.
First, a vaccine that contains more than one agent that can
stimulate and immune response against IL-13R.alpha.2 (e.g., a
composition that contains 2, 3, 4, 5, 6 , 7, 8, or more different
peptides listed in Table 1 below). Second, a vaccine that contains
both (a) an agent that can stimulate and immune response against
IL-13R.alpha.2 and (b) a different agent that can stimulate an
immune response against a molecule other than IL-13R.alpha.2 (e.g.,
another TSA or TAA).
[0103] Administering Vaccines to a Subject
[0104] The vaccine compositions of the present invention can be
used in a method for stimulating an immune response against
IL-13R.alpha.2 in a subject. In this method, an vaccine compositon
of the invention can be administered to a subject by any method
that stimulates the aforesaid immune response. The exact method
selected is determined by the particular vaccine composition to
administered. For parenteral administration by injection, the
injection can be in situ (i.e., to a particular tissue or location
on a tissue, e.g., into a tumor or lymph node), intramuscular,
intravenous, intraperitoneal, or by another parenteral route. For
example, for a protein/peptide based vaccine the vaccine may be
administered by subcutaneous or intradermal injection. In some
cases other routes can be used, e.g. intravenous injection,
intraperitoneal injection, or in situ injection into target
tissue.
[0105] Naked nucleic acid vaccines or expression vector vaccines
may be administered by intramuscular injection. Cell-based vaccines
can be introduced into an animal by any suitable method, e.g.,
subcutaneous injection. In addition to parenteral routes, the
vaccines of the invention can also be administered by a
non-parenteral route, e.g, by oral, buccal, urethral, vaginal, or
rectal administration.
[0106] Formulations for injection may be presented in unit dosage
form, for example, in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the vaccine compositions
may be in powder form (e.g., lyophilized) for constitution with a
suitable vehicle, for example, sterile pyrogen-free water, before
use.
[0107] To facilitate delivery of the antigenic compositions (e.g.,
antigenic agent plus adjuvant) of the invention to an animal, the
antigenic compositions can be mixed with a pharmaceutically
acceptable carrier or excipient. Examples of such pharmaceutically
acceptable carriers and excipients include diluents such as water,
saline, citrate buffered saline, phosphate buffered saline, acetate
buffered saline, and bicarbonate buffered saline; and stabilizing
agents such as amino acids, alcohols, proteins (for example, serum
albumin), EDTA, mannitol, sorbitol, and glycerol. To minimize the
chance of infection or adverse reaction when administered to a
subject, carriers and excipients are preferably sterile and
pyrogen-free. USP grade carriers and excipients are particularly
preferred for delivery of vaccine compositions to human subjects.
The vaccine compositions can also be formulated for long-term
release as a depot preparation by adding the antigenic agent to
suitable polymeric or hydrophobic materials or ion exchange resins.
They can also be made by preparing the vaccine composition as a
sparingly soluble derivative. Depot preparations can be
administered to a subject by implantation (e.g., subcutaneous or
intramuscular surgical implantation) or by injection. Methods for
making the foregoing formulations are well known and can be found
in, for example, Remington's Pharmaceutical Sciences.
[0108] Dosing
[0109] The vaccine compositions of the invention are preferably
administered to a subject in an amount sufficient to stimulate an
immune response against IL-13R.alpha.2 in the subject, and not
cause an overly toxic effect. Such a therapeutically effective
amount can be determined as described below.
[0110] Toxicity and therapeutic efficacy of the vaccines utilized
in the invention can be determined by standard pharmaceutical
procedures, using either cells in culture or experimental animals
to determine the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Vaccines that exhibit
large therapeutic indices are preferred. While those that exhibit
toxic side effects may be used, care should be taken to design a
delivery system that minimizes the potential damage of such side
effects. Data obtained from animal studies can be used in
formulating a range of dosage for use in humans. The dosage of such
vaccines lies preferably within a range that include an ED.sub.50
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized.
[0111] The vaccines of the invention can be administered to a
subject using various different vaccination schedules. For example,
a nucleic acid vaccine might be administered to a subject only
once, while a protein/peptide- based vaccine might be administered
to the subject on multiple occasions (1, 2, 3, 4, 5 or more times).
For example, in an effort to stimulate a strong immune response, a
first dose of a vaccine compositions of the invention may be
administered to a subject at least 24 hours before a second
(booster) dose is administered to the subject.
[0112] Kits
[0113] The invention also provides kits for stimulating an immune
response against IL-13R.alpha.2 in a subject. Such kits can include
a container holding one or more of the antigenic agents described
above in a pharmaceutically acceptable form. The antigenic agent(s)
in the container can be in liquid form (e.g., as a solution) or in
solid form (e.g., as a lyophilized or desiccated powder). Where,
for example, the antigenic agent is a solid, the kits within the
invention can further include a container holding a
pharmaceutically acceptable solution (e.g., sterile saline with or
without dextrose) for reconstituting the solid into a liquid
suitable for injection. The kits of the invention can further
include (a) one or more devices to administer the antigenic agent,
e.g., a needle or syringe, a packaged alcohol pad, etc.; and/or (b)
printed instructions for using the kit.
EXAMPLES
Example 1
IL-13R.alpha.2 Mimics the Biological Features of an HGG-associated
receptor for IL-13
[0114] Normal Chinese hamster ovary (CHO) cells were transfected
with a pcDNA 3.1 plasmid (Invitrogen) containing the full length
open reading frame of IL-13R.alpha.2 and positive clones were
selected with geneticin. The expression of IL-13R.alpha.2 in these
clones was tested for their ability to bind .sup.125I-labeled
IL-13. Selected clones were shown to bind labeled IL-13
independently of IL-4. In addition, labeled IL-13 was displaced by
IL-13.E13K, a mutant of IL-13 shown to have a greater affinity for
the IL-13 binding protein on HGG than for the shared IL-13/IL-4
receptor found in a plethora of tissues under a physiological
state. Furthermore, these IL-13R.alpha. transfected CHO cells were
exposed to an IL-13.E13K-PE38QQR cytotoxin, a fusion protein
showing potent dose dependent cytotoxicity on HGG cells. The clones
expressing the receptor were killed in direct proportion to their
affinity for IL-13, but not CHO cells alone or CHO cells
transfected with an empty plasmid. In neutralization experiments,
an excess of IL-13 prevented the cytotoxic effect of IL-13.E
13K-PE38QQR. Therefore the only way the toxin, PE38QQR, could have
entered and killed the cells was through receptor-mediated
endocytosis, a process directed through the IL-13 portion of the
cytotoxin. Use of an IL13.E13K/enhanced green flourescent protein
(EGFP) fusion protein confirmed that this process occurred. Thus,
IL-13R.alpha.2 was demonstrated to share properties ascribed to
more restrictive, IL-4 independent, IL-13 binding sites found on
HGGs in situ and in vitro.
Example 2
Identification of IL-13R.alpha.2 as a Cancer Testis Antigen
[0115] Materials and Methods
[0116] Sources of RNA.
[0117] High-grade glioma cell lines A-172 MG, U-373 MG, U-251 MG
and human glioblastoma multiforme explant cells (G-48) were grown
in culture in appropriate media. Total RNA was extracted from the
cells using the acid-guanidium isothiocyanate-phenol-chloroform
method. Poly(A)+ RNA was further isolated using the Mini-oligo(dT)
Cellulose Spin Column Kit (5 prime-3 prime Inc., Boulder, Colo.). 2
.mu.g of Poly (A)+ RNA was electrophoresed on a 1% agarose
formaldehyde gel, transferred to 0.45 .mu.m magna charge nylon
(MSI, Westborough, Mass.) and UV-crosslinked (Stratagene, La Jolla,
Calif.). RNA-blotted membranes were also purchased from Clontech
(Palo Alto, Calif.). Two Multiple Tissue Expression (MTETM) Blots
(cat #7770-1 and 7775-1; www.clontech.com/mtn/index.html) were
analyzed to determine the tissue distribution of the IL13 binding
proteins. Two sets of Human Brain Multiple Tissue Northern (MTNTM)
Blots (cat # 7755-1 and 7769-1) were assayed to confirm the true
presence of the transcripts. In addition, two Human Tissue Northern
(MTNTM) Blots (cat #7759-1 and 7760-1) were analyzed to verify the
tissue distribution of the IL-13 Ra2 transcript.
[0118] cDNA Probes.
[0119] cDNA probes were generated either by PCR (IL-13R.alpha.2 and
IL13R.alpha.1) or by restriction digest (IL-4R.alpha.=p140). cDNA
containing human IL13R.alpha.2 was provided by Dr. Pascual Ferrara
of Sanofi Recherche. cDNA containing human IL-13R.alpha.1 (and also
93 bases of murine IL-13) was provided by Dr. Douglas J. Hilton of
The Walter and Eliza Hall Institute of Medical Research. Plasmid
pHuIL4R/ID was used to obtain a fragment of IL4R.alpha. by the
restriction digest. The fragments were electrophoresed on a 1%
agarose gel, excised from the gel and purified using QlAquick Gel
Extraction Kit (Qiagen Inc., Valencia, Calif.). Actin cDNA was
purchased from Clontech Labs.
[0120] The primers for human IL-13R.alpha.2 were as follows:
1 forward 5'-AAGATTTGGAAGCTTATGGCTTTCGTTTGC-3' (SEQ ID NO:3)
reverse 5'-TCCCTCGAAGCTTCATGTATCACAGAAAAA-3' (SEQ ID NO:4)
[0121] The primers for human IL13R.alpha.1 were as follows:
2 forward 5'-ATTATTAAGCTTATGGAGTGGCCGGCG-3' (SEQ ID NO:5) reverse
5'-TAACCGGAAGCTTCACTGAGAGGCTTT-3' (SEQ ID NO:6)
[0122] Northern Blot Analysis.
[0123] Membranes were pre-hybridized overnight at 42.degree. C. in
a solution consisting of 50% formamide, 5.times. SSC, 50 mM sodium
phosphate, 5.times. Denhardt's, 50 .mu.g/ml sheared salmon sperm
DNA, and 1% SDS. Membranes were subsequently hybridized overnight
at 42.degree. C. in the same solution with the addition of full
length cDNA probes labeled by random priming (Life Technologies,
Rockville, Md.) with .sup.32P-dCTP using 1-2.times.10.sup.6 cpm/ml.
Following hybridization, the membranes were washed with
2.times.SSC/0.2% SDS at 42.degree. C. for 20 minutes followed by
two washes with 1.times. SSC/0.1% SDS at 42.degree. C. for 20
minutes each. The membranes were exposed to autoradioraphic film
X-OMAT AR (Eastman Kodak Co., Rochester, N.Y.) and placed at
-80.degree. C. for 1, 3 and 14 days. The membranes were
subsequently stripped and re-probed up to three more times. The
membranes were probed first with IL-13R.alpha.2, followed by
IL13R.alpha.1, IL-4R.alpha.=p140, and actin. Films were scanned on
a transparency scanner at a pixel size of 88.times.88 micron
(Molecular Dynamics, Sunnyvale, Calif.). The images were compiled
in Paint Shop Pro V 5.0 (Jasc software Inc., Eden Prairie,
Minn.).
[0124] Results
[0125] Northern blot analysis of transcripts for IL-13R.alpha.2 in
normal organs. To explore the expression of IL-13R.alpha.2, an
extensive examination of the presence of transcripts for this
protein among multiple normal tissues, including 20 discrete
regions of the CNS and a variety of normal peripheral organs was
performed. All Northern blots using same membranes were performed
with respective labeled cDNAs in the following order:
IL-13R.alpha.2, ILI3R.alpha.1, IL4.alpha. and .beta.-actin. This
assured that the levels of transcripts for IL-13R.alpha.2 were not
underestimated due to the usage of membranes with mRNA. Both the
dot-blot analyses (not shown) and the electrophoretically separated
transcripts for IL-13R.alpha.2 (FIG. 4, panels I-IV) demonstrated
mostly undetectable, or very weak signals in few cases, of
IL-13R.alpha.2 transcripts in the organs studied, even after 2-week
of film exposure. The first dot blot performed, however,
surprisingly showed an unusually high density of labeling with
IL-13R.alpha.2 cDNA probe to to transcripts derived from testis.
This was also found using another Northern blot membrane. A few
other organs had transcripts that hybridized to the IL-13R.alpha.2
cDNA (aorta, liver, and pituitary gland). The density of labeling
in the dot blots was much lower than in the testis blot. Of
importance, there was no evidence for the presence of significant
IL-13R.alpha.2 expression in the CNS.
[0126] To confirm these findings made using dot blot analysis,
additional blots were performed using electrophoretically separated
mRNAs. Again, the discrete regions of normal human brain did not
produce clear-cut hybridization signals (FIG. 4, panels I and II).
On the other hand, the only organ with prominent hybridization band
corresponding to the mRNA of 1.5 kb was seen in testis (FIG. 4,
panel III). Poorly detectable signals were seen in placenta, liver,
and kidney (FIG. 4, panel IV). Thus, among normal tissues, testes
was the only one that prominently expressed IL-13R.alpha.2. No
transcripts for IL-13R.alpha.2 were readily detected in the
CNS.
[0127] Northern blot analysis of transcripts for IL13R.alpha.1 in
normal tissues. The expression of IL13R.alpha.1, a component of a
heterodimeric form of IL13 receptor that is shared with IL4, IL
13/4 receptor was examined in a variety of normal human tissues
(FIG. 5) by either dot-blot analyses (not shown) or blots of
electrophoretically separated transcripts (FIG. 5, panels I-IV).
The results unequivocally demonstrated that IL 13R.alpha.1 was
expressed in a variety of the organs, including CNS tissue from
medulla, spinal cord, substantia nigra, thalamus, and corpus
callosum. Size fractionated mRNAs confirmed the many positive
signals seen in dot blots with the strongest signals observed in
ovary, heart, liver and lung (FIG. 5, panels III and IV,
respectively). Of interest, liver showed two hybridized species of
MRNA: one of 4.5 kb and the other of 2.0 kb, as an example of a
normal organ with doublet of positive signals of different sizes.
In summary, discrete regions of normal human brain did produce
clear-cut positive hybridization signals for IL 13R.alpha.1 (FIG.
5, panels I and II). In addition, many vital peripheral organs
exhibited hybridization bands corresponding to the mRNA of 4.5-4.65
kb (FIG. 5, panels III and IV).
[0128] Gene expression analysis of IL4R.alpha. in normal tissues.
In addition to IL13R.alpha.1, IL4R.alpha. is another component of a
heterodimeric form of IL13 receptor that is shared with IL4, i.e.,
the shared IL13/4 receptor. Thus, whether the distribution of IL4RU
gene expression corresponded to that of IL13R.alpha.1 was analyzed.
All Northern blot analysis membranes used in this study
demonstrated enriched content of the IL4R.alpha. transcripts in a
variety of tissues (FIG. 6, panels I, II, and IV). The presence of
the transcripts within the CNS was most evident, as it was for
IL13R.alpha.1, in medulla, spinal cord, substantia nigra and
thalamus (FIG. 6, panels I and II). Among normal peripheral organs,
liver, lung, kidney, intestinal tract, spleen, stomach, and testis
demonstrated gene expression of IL4R.alpha., which was generally
similar to that seen with IL13R.alpha.1 (not shown). Thus, discrete
regions of normal human brain contain transcripts for both
IL13R.alpha.1 and IL4R.alpha., a complete heterodimer of the shared
IL13/4 receptor. Furthermore, several vital peripheral organs
contained the two subunits of the IL13/4 receptor, including heart,
liver, lung and intestinal tract.
[0129] Control Hybridization of .beta.-Actin.
[0130] All membranes used for Northern blot analysis of IL13
receptors transcripts were also hybridized with a cDNA probe for a
house-keeping gene, .beta.-actin (FIG. 7; dot blots and panel III
not shown). The intensity of the signals for .beta.-actin was
usually in accordance with the amount of mRNA present on the
membranes, as estimated by the manufacturer.
[0131] Gene Expression of IL 13Receptors in Cells.
[0132] Gene expression of the two IL13 receptors was also examined
in malignant and normal cells (FIG. 8). Transcripts for
IL13R.alpha.2, IL13R.alpha.1, IL4R.alpha. and .beta.-actin were
examined in serial hybridization assays. Isolated explant cells of
HGG (G-48) as well as human malignant glioma established cell lines
(A-172 MG, U-373 MG, and U-251 MG) demonstrated intense signals for
IL-13R.alpha.2 (FIG. 8). On the other hand, the transcripts for the
elements of the shared IL 13/4 receptor, IL-13R.alpha.1 and
IL4R.alpha., were found at lower levels when compared with that for
IL-13R.alpha.2 (FIG. 8). A-172 MG cells appeared to be the most
enriched in the components of the IL 13/4 receptor heterodimer. Of
interest, two species of different sizes of the transcripts for
both IL-13R.alpha.2 and IL-13R.alpha.1 were seen in cells (FIG. 8).
In a control assay, human umbilical vein endothelial cells (HUVEC)
showed the presence of transcripts for IL-13R.alpha.1 and
IL4R.alpha., but not those for IL-13R.alpha.2 (FIG. 8). In summary,
gene expression of IL-13R.alpha.2 was detected in two specimens of
HGG (FIG. 8, HGG 13 and HGG 52), but not in two normal brain
specimens (FIG. 8, NB 3 and NB 6). However, the transcripts for
IL-13R.alpha.1 were found in all of these specimens. In other
experiments, several additional HGG brain tumor specimens were
determined to express IL-13R.alpha.2.
Example 3
Representative Immunogenic Peptides of IL-13R.alpha.2
[0133] Table I presents a list of IL-13R.alpha.2 peptides that
might be used to stimulate an immune response against
IL-13R.alpha.2 in a subject. The listed peptides were obtained
using a computer program provided by the Ludwig Institute For
Cancer Research (Lausanne, Switzerland) on the Internet at
http://www-ludwig.unil.ch.SEREX.html. This program provided the
best (at high stingency) fit of predicted immunogenic peptides that
bind specific classes of MHC molecules (i.e., the various alleles
of human MHC Class I indicated in Table I). The peptides indicated
with the "*" are those that should bind under high stringency. The
skilled artisan could produce these peptides as described herein
(e.g., by automated peptide synthesis) and use each in a vaccine
preparation that would be administered to a variety of test
subjects (e.g. those with different MHC types) as also described
herein. The immune response stimulated by each of these peptides in
the subjects could then be assessed, so that those that stimulate
the desired immune responses in particular test subjects could be
identified.
3TABLE I Binding peptides prediction: Allele Peptide Position Score
t1/2 A1 IVDP-GYLGY 16-24 7.120 1236.45043346563 A1 LLDTNYNLFY
140-149 4.820 123.965090779824 A_0201 YLYLQWQPPL * 24-33 5.760
317.34832891785 A_0201 YLQWQPPLSL * 26-35 4.600 99.4843156419338
A_0201 LQWQ-PPLSL 27-35 3.430 30.876642749677 A_0201 SLDHFKECTV
34-43 3.330 27.9383417032365 A_0201 NLHYKDGFDL * 64-73 4.830
125.210960654765 A_0201 WQCT-NGSEV 87-95 3.490 32.7859477062319
A_0201 CVYY-NWQYL * 121-129 4.020 55.7011058267956 A_0201
YLLCSWKPGI * 128-137 5.190 179.468552931832 A_0201 VLLD-TNYNL *
139-147 6.320 555.572992451403 A_0201 NLFY-WYEGL * 146-154 4.080
59.1454698498823 A_0201 GLDH-ALQCV * 153-161 4.160 64.0715225999366
A_0201 NIGC-RFPYL 170-178 3.420 30.5694150210502 A_0201 FQLQNIVKPL
* 206-215 4.450 85.6269440022006 A_0201 QLQN-IVKPL * 207-215 3.900
49.4024491055302 A_0201 NIVK-PLPPV 210-218 3.090 21.9770779757634
A_0201 YLTFTRESSC 219-228 3.140 23.1038668587222 A_0201 QLCFVVRSKV
* 279-288 4.250 70.1054123466879 A_0205 IVDPGYLGYL 16-25 3.120
22.6463796431754 A_0205 YLYLQWQPPL * 24-33 4.140 62.8028214492017
A_0205 LQWQ-PPLSL 27-35 3.350 28.5027336437673 A_0205 LQWQ-PPLSL
26-35 3.040 20.9052432350928 A_0205 CVYY-NWQYL * 121-129 4.430
83.9314169102688 A_0205 VLLD-TNYNL * 139-147 4.670 106.697742432451
A_0205 VLLD-TNYNL * 138-147 3.740 42.0979901649969 A_0205
NLFY-WYEGL 146-154 3.040 20.9052432350928 A_0205 FQLQNIVKPL *
206-215 4.610 100.484149636389 A3 LLDTNYNLFY 140-149 3.190
24.2884274430946 A3 ALQC-VDYIK 157-165 4.520 91.8355979781567 A3
GIWS-EWSDK 296-304 3.410 30.2652442594001 A24 DFEIVDPGYL 13-22
3.410 30.2652442594001 A24 LYLQ-WQPPL * 25-33 5.710 301.87106828279
A24 EYEL-KYRNI * 44-52 4.320 75.1886282920231 A24 TYWI-SPQGI *
103-111 4.090 59.7398917041452 A24 VYYN-WQYLL * 122-130 5.300
200.336809974792 A24 WYEG-LDHAL * 150-158 5.890 361.405284372286
A24 DYIKADGQNI * 162-171 4.500 90.0171313005218 A24 SYFTFQLQNI *
202-211 4.090 59.7398917041452 A DLSK-KTLLR 311-319 3.300
27.1126389206579 A68.1 TVEY-ELKYR * 42-50 5.300 200.336809974792
A68.1 TVEY-ELKYR * 41-50 4.600 99.4843156419338 A68.1 ETWK-TILTK *
55-63 4.500 90.0171313005218 A68.1 CVNG-SSENK * 189-197 4.790
120.301368663215 A68.1 FTFQLQN1VK * 204-213 4.090 59.7398917041452
A68.1 FTRESSCEIK 222-231 3.400 29.964100047397 A68.1 ESSC-EIKLK
225-233 3.300 27.1126389206579 A68.1 TVENETYTLK * 263-272 4.790
120.301368663215 A68.1 YTLKTPNETR * 269-278 4.600 99.4843156419338
A68.1 ETRQLCFVVR * 276-285 5.010 149.904736149047 B7 DPGYLGYLYL
18-27 4.390 80.640418980477 B7 CVYY-NWQYL 121-129 3.000
20.0855369231877 B7 GVLLDTNYNL 138-147 3.000 20.0855369231877 B7
IVKPLPPVYL 211-220 3.410 30.2652442594001 B7 EIRE-DDTTL 251-259
3.690 40.0448469572867 B8_8mer EAKIHTLL 78-85 3.470
32.1367424447532 B8_8mer EIKLKWSI 229-236 3.690 40.0448469572867
B8_8mer VVRSKVNI 283-290 3.000 20.0855369231877 B14 QNIGCRFPYL
169-178 3.400 29.964100047397 B14 IRSSYFTFQL 199-208 3.000
20.0855369231877 B_2702 LQWQ-PPLSL 27-35 3.410 30.2652442594001
B_2702 WQPPLSLDHF 29-38 3.000 20.0855369231877 B_2702 YRNI-GSETW
49-57 4.610 100.484149636389 B_2702 VQSSWAETTY 95-104 3.000
20.0855369231877 B_2702 VQDM-DCVYY 116-124 3.000 20.0855369231877
B_2702 GQNIGCRFPY 168-177 3.000 20.085536923 1877 B_2702 CRPP-YLEAS
173-181 3.920 50.4004447780655 B_2702 IRSSYFTFQL 199-208 4.100
60.340287597362 B_2702 TRESSCEIKL 223-232 4.100 60.340287597362
B_2702 ARCFDYEIEI 243-252 4.100 60.340287597362 B_2702 IRED-DJTLV
252-260 3.000 20.085536923 1877 B_2702 VRSK-VNIYC 284-292 3.000
20.085536923 1877 B_2705 FEIV-DPGYL 14-22 3.400 29.964 100047397
B_2705 YLYLQWQPPL 24-33 5.010 149.904736149047 B_2705 LQWQ-PPLSL
27-35 6.910 1002.24724229025 B_2705 LQWQ-PPLSL 26-35 3.400
29.964100047397 B_2705 WQPPLSLDHF 29-38 4.610 100.484149636389
B_2705 KECT-VEYEL 39-47 4.500 90.0171313005218 B_2705 YRNIGSETWK
49-58 7.600 1998.19589510412 B_2705 RNIG-SETWK 50-58 4.090
59.7398917041452 B_2705 SETWKTIITK 54-63 3.400 29.964100047397
B_2705 KNLH-YKDGF 63-71 3.400 29.964100047397 B_2705 NLHYKDGFDL
64-73 3.400 29.964100047397 B_2705 IEAK-IHTLL 77-85 3.400
29.964100047397 B_2705 WQCT-NGSBV 87-95 4.100 60.340287597362
B_2705 VQSSWAETTY 95-104 4.610 100.484149636389 B_2705 VQDM-DCVYY
116-124 4.610 100.484149636389 B_2705 CVYY-NWQYL 121-129 3.910
49.8989519734079 B_2705 WQYL-LCSWK 126-134 6.910 1002.24724229025
B_2705 CSWKPGIGVL 131-140 3.910 49.8989519734079 B_2705 VLLD-TNYNL
139-147 3.400 29.964100047397 B_2705 TNYN-LFYWY 143-151 3.910
49.8989519734079 B_2705 NLFY-WYEGL 146-154 5.010 149.904736149047
B_2705 ALQC-VDYIK 157-165 3.400 29.964100047397 B_2705 LQCV-DYIKA
158-166 3.000 20.0855369231877 B_2705 GQNIGCRFPY 168-177 4.610
100.484149636389 B_2705 CRFP-YLEAS 173-181 6.910 1002.24724229025
B_2705 FPYLEASDYK 175-184 3.910 49.8989519734079 B_2705 IRSSYFTFQL
199-208 7.600 1998.19589510412 B_2705 RSSY-FTFQL 200-208 3.400
29.964100047397 B_2705 FTFQLQNIVK 204-213 3.910 49.8989519734079
B_2705 FQLQNIVKPL 206-215 4.100 60.340287597362 B_2705 TRES-SCEIK
223-231 7.600 1998.19589510412 B_2705 RESS-CEIKI 224-232 4.500
90.0171313005218 B_2705 ARCFDYEIEI 243-252 6.400 601.845037872082
B_2705 RCFDYEIEIR 244-253 4.320 75.1886282920231 B_2705 IRED-DTTLV
252-260 6.400 601.845037872082 B_2705 IEIREDDTTL 250-259 3.400
29.964 100047397 B_2705 VENE-TYTLK 264-272 3.400 29.964 100047397
B_2705 TRQL-CFVVR 277-285 6.910 1002.24724229025 B_2705 RQLCFVVRSK
278-287 5.200 181.272241875151 B_2705 VRSK-VNIYC 284-292 5.300
200.336809974792 B_2705 GIWS-EWSDS 296-304 3.910 49.8989519734079
B_2705 KQCW-EGEDL 304-312 6.400 601.845037872082 B_2705 QCWEGEDLSK
305-314 3.910 49.8989519734079 B_2705 WEGE-DLSKK 307-315 3.400
29.964100047397 B_2705 GEDLSKKTLL 309-318 3.400 29.964100047397
B_3501 DPGY-LGYLY 18-26 3.700 40.4473043600674 B_3501 QPPL-SLDHF
30-38 3.000 20.0855369231877 B_3501 FPYL-EASDY 175-183 4.110
60.9467175696222 B_3501 KPIRSSYFTF 197-206 3.690 40.0448469572867
B_3501 KPLPPVYLTF 213-222 3.690 40.0448469572867 B_3501 GPIPARCFDY
239-248 3.700 40.4473043600674 B3501_8mer DPGYLGYL 18-25 3.000
20.0855369231877 B3501_8mer KPGIGVLL 134-141 3.690 40.0448469572867
B3501_8mer KPIRSSYF 197-204 3.690 40.0448469572867 B3501_8mer
KPLPPVYL 213-220 3.690 40.0448469572867 B3501_8mer LPPVYLTF 215-222
3.000 20.0855369231877 B3501_8mer GPIPARCF 239-246 3.000
20.0855369231877 B3501_8mer IPARCEDY 241-248 3.700 40.4473043600674
B_3701 VDPG-YLGYL 17-25 3.690 40.0448469572867 B_3701 KDGFDLNKGI
68-77 3.690 40.04484695272867 B_3701 IEAK-IHTLL 77-85 4.320
75.1886282920231 B_3701 LDTN-YNLFY 141-149 3.690 40.0448469572867
B_3701 EDLS-KKTLL 310-318 5.300 200.336809974792 B_3701 EDLS-KKTLL
309-318 3.910 49.8989519734079 B LHYK-DGFDL 65-73 3.400
29.964100047397 B_3901 LHYK-DGFDL 65-73 5.190 179.468552931832
B_3901 DHALQCVDYI 155-164 3.810 45.1504388663187 B_3901 TRESSCEIKL
223-232 3.120 22.6463796431754 B_3901 IRED-DTTLV 252-260 3.400
29.964100047397 B3901_8mer DHFKECTV 36-43 4.090 59.7398917041452
B3901_8mer IREDDTTL 252-259 4.500 90.0171313005218 B_3902
LQWQ-PPLSL 27-35 3.000 20.0855369231877 B_3902 FKECTVEYEL 38-47
3.180 24.046753520645 B_3902 WKTI-ITKNEL 57-65 3.180
24.0467535520645 B_3902 WKPG-IGVLL 133-141 3.180 24.0467535520645
B_3902 FQLQNIVKPL 206-215 3.180 24.0467535520645 B_3902 VKPL-PPVYL
212-220 3.000 20.0855369231877 B_3902 IKLK-WSIPL 230-238 3.180
24.0467535520645 B_3902 LKTTNETRQL 271-280 3.000 20.0855369231877
B_3902 KQCW-EGEDL 304-312 3.000 20.0855369231877 B_3902 DKQCWBGEDLY
303-312 3.000 20.0855369231877 B40 FEIV-DPGYL 14-22 4.390
80.640418980477 B40 KECT-VEYEL 39-47 3.000 20.0855369231877 B40
IEAK-IHTLL 77-85 3.690 40.0448469572867 B40 RESS-CEIKIL 224-232
3.000 20.085536923 1877 B40 IEIREDDTTL 250-259 4.390
80.640418980477 B40 SEWS-DKQCW 299-307 3.690 40.0448469572867 B40
GEDL-SKKTL 309-317 3.000 20.0855369231877 B_4403 QDFEIVDPGY 12-21
3.120 22.6463796431754 B_4403 FEIV-DPGYI 14-22 3.000
20.0855369231877 B_4403 VDPGYLGYLY 17-26 3.210 22.6463796431754
B_4403 KTIITKNLHY 58-67 3.530 34.1239676147544 B_4403 QNIG-CRFPY
169-177 3.530 34.1239676147544 B_4403 LEASDYKDFY 178-187 5.480
239.846707374255 B_4403 SENKPIRSSY 194-203 5.480 23 9.846707374255
B_4403 CEIK-LKWSI 228-236 3.000 20.0855369231877 B_4403 GPIPARCFDY
239-248 3.810 45.1504388663187 B_4403 YEIEIREDDT 248-257 3.000
20.0855369231877 B_4403 IEIREDDTTL 250-259 3.410 30.2652442594001
B_4403 SEWS-DKQCW 299-307 3.180 24.0467535520645 B_5101 NPPQ-DFEIV
9-17 5.410 223.631587680546 B_5101 DPGYLGYLYL 18-27 5.400
221.406416204187 B_5101 IGSE-TWKTI 52-60 5.050 156.022464486395
B_5101 DGFD-LNKGI 69-77 6.070 432.680681574476 B_5101 SPQGIPETKV
107-116 5.410 223.631587680546 B_5101 IPET-KVQDM 111-119 3.770
43.3800648358516 B_5101 EGLDHALQCV 152-161 4.790 120.3013686632215
B_5101 HALQ-CVDYI 156-164 5.300 200.336809974792 B_5101 EASDYKDFYI
179-188 6.090 441.421411145971 B_5101 NGSS-ENKPI 191-199 4.590
98.4944301619463 B_5101 IPARCFDYEI 241-250 6.260 523.218940108001
B_5101 PARC-FDYEI 242-250 3.000 20.0855369231877 B_5101 EGEDLSKKTL
308-317 4.190 66.0227909604099 B5101_8mer NPPQDFEI 9-16 6.100
445.857770082517 B5101_8mer PPQDFEIV 10-17 3.110 22.4210444007463
B5101_8mer DPGYLGYL 18-25 5.300 200.336809974792 B5101_8mer
EAKIHTLL 78-85 4.700 109.947172452124 B5101_8mer WAETTYWI 99-106
5.400 221.406416204187 B5101_8mer QGIPETKV 109-116 3.800
44.7011844933008 B5101_8mer KPGIGVLL 134-141 4.120 61.5592422644285
BS101_8mer IGCRFPYL 171-178 3.260 26.0495371425183 B5101_8mer
KPLPPVYL 213-220 3.920 50.4004447780655 B_5102 NPPQ-DFEIV 9-7 5.510
247.151127067624 B_5102 DPGYLGYLYL 18-27 4.810 122.731617517265
B_5102 IGSE-TWKTI 52-60 4.790 120.301368663215 B_5102 DGFD-LNKGI
69-77 6.200 592.749041093256 B_5102 KGIEAKIHTL 75-84 4.400
81.4508686649681 B_5102 LPWQ-CTNGS 85-93 3.430 30.876642749677
B_5102 SSWAETIYWI 97-106 3.200 24.5325301971094 B_5102 TYWI-SPQGI
103-111 3.100 22.1979512814416 B_5102 TTYWISPOGI 102-111 3.100
22.1979512814416 B_5102 SPQGIPETKV 107-116 6.100 445.857770082517
B_5102 YLLCSWKPGI 128-137 3.180 24.0467535520645 B_5102 EGLDHALQCV
152-161 4.900 134.289779684936 B_5102 HALQ-CVDYI 156-164 6.600
735.095189241973 B_5102 FPYL-EASDY 175-183 3.510 33.4482677839449
B_5102 EASDYKDFYI 179-188 5.400 221.406416204187 B_5102 NGSS-ENKPI
191-199 4.590 98.4944301619463 B_5102 KPIR-SSYFT 197-205 3.510
33.4482677839449 B_5102 SYFTFQLQNI 202-211 3.300 27.1126389206579
B_5102 FTFQ-LQNIV 204-212 3.200 24.5325301971094 B_5102 KPLP-PVYLT
213-221 3.410 30.2652442594001 B_5102 IPLGPIPARC 236-245 4.200
66.6863310409252 B_5102 IPARCFDYEI 241-250 6.100 445.857770082517
B_5102 RCFD-YEIEI 244-252 3.000 20.0855369231877 B_5102 FVVR-SKVNI
282-290 3.280 26.575772699874 B_5102 LCF-VRSKV 280-288 3.100
22.1979512814416 B_5102 NIYC-SDDGI 289-297 3.000 20.0855369231877
B5102_8mer NPPQDFEI 9-16 6.200 492.749041093256 B5102_8mer PPQDFEIV
10-17 3.010 20.2873999252409 B5102_8mer DPGYLGYL 18-25 4.610
100.484149636389 B5102_8mer EAKIHTLL 78-85 3.320 27.6603505585 167
B5102_8mer WAETTYWI 99-106 4.810 122.731617517265 B5102_8mer
YWISPQGI 104-111 3.280 26.575772699874 B5102_8mer QGIPETKV 109-116
5.000 148.413159102577 B5102_8mer KPGIGVLL 134-141 4.710
111.052159905699 B5102_8mer IGCRIFPYL 171-178 3.100
22.1979512814416 B5102_8mer FTFQLQNI 204-211 3.890 48.9108865237319
B5102_8mer KPLPPVYL 213-220 5.710 301.87106828279 B5102_8mer
IPLGPIPA 236-243 3.610 36.9660528148225 B_5103 NPPQ-DFEIV 9-17
3.800 44.7011844933008 B_5103 IGSETWKTII 52-61 3.900
49.4024491055302 B_5103 DGFD-LNKGI 69-77 3.980 53.5170342274912
B_5103 SPQGIPETKV 107-116 3.800 44.7011844933008 B_5103 EGLDHALQCV
152-161 3.980 53.5170342274912 B_5103 HALQ-CVDYI 156-164 4.890
132.953574051283 B_5103 EASDYKDFYI 179-188 4.610 100.484149636389
B_5103 NGSS-ENKPI 191-199 3.700 40.4473043600674 B_5103 IPARCFDYEI
241-250 3.800 44.7011844933008 B_5201 NPPQ-DFEIV 9-17 4.700
109.947172452124 B_5201 NPPQ-DFEIV 8-17 3.680 39.6463940725726
B_5201 IGSETWKTII 52-61 4.600 99.4843156419338 B_5201 DGFD-LNKGI
69-77 4.110 60.9467175696222 B_5201 FTFQ-LQNIV 204-212 4.600
99.4843156419338 B_5801 KTIITKNLHY 58-67 3.000 20.0855369231877
B_5801 SSWA-ETTYW 97-105 4.390 80.640418980477 B_5801 QSSWAETTYW
96-105 4.390 80.640418980477 B_5801 DTNY-NLFYW 142-150 3.370
29.0785270577971 B_5801 KPLPPVYLTF 213-222 3.100 22.1979512814416
B_5801 SSCE-IKLKW 226-234 5.690 295.893620640484 B_5801 SSCE-IKLKW
225-234 3.800 44.7011844933008 B_5801 TTNETRQLCF 273-282 4.490
89.1214458786587 B_5801 CSDDGIWSEW 292-301 4.900 134.289779684936
B_5801 WSEWSDKQCW 298-307 4.390 80.640418980477 B60 FEIV-DPGYL
14-22 5.770 320.537732647356 B60 VDPG-YLGYL 17-25 3.000
20.085536923 1877 B60 KECT-VEYEL 39-47 5.870 3 54.248980267765 B60
IEAK-IHTLL 77-85 5.870 354.248980267765 B60 RESS-CELKL 224-232
6.560 706.271694595366 B60 IEIREDDTTL 250-259 5.770
320.537732647356 B60 GEDL-SKKTL 309-317 5.080 160.774055928607 B60
EDLS-KKTLL 310-318 3.690 40.0448469572867 B61 REDDTTLVTA 253-262
3.100 22.1979512814416 B61 NETR-QLCFV 275-283 4.380
79.8380334050845 B61_8mer SEVQSSWA 93-100 3.690 40.0448469572867
B61_8mer REDDTTLV 253-260 3.790 44.2564002759834 Cw_0301 FEIV-DPGYL
14-22 3.000 20.0855369231877 Cw_0301 LYLQ-WQPPL 25-33 3.000
20.0855369231877 Cw_0301 YLYLQWQPPL 24-33 3.000 20.0855369231877
Cw_0301 VEYELKYRNI 43-52 3.630 37.7128166171817 Cw_0301 LHYK-DGFDL
65-73 3.000 20.0855369231877 Cw_0301 KGIEAKIHTL 75-84 3.590
36.2340759264765 Cw_0301 CVYY-NWQYL 121-129 3.360 28.7891908792427
Cw_0301 DCVYYNWQYL 120-129 3.360 28.7891908792427 Cw_0301
VYYN-WQYLL 122-130 3.000 20.0855369231877 Cw_0301 VLLDTNYNLF
139-148 3.400 29.964100047397 Cw_0301 GVLLDTNYNL 138-147 3.000
20.0855369231877 Cw_0301 YNLFYWYEGL 145-154 3.610 100.484149636389
Cw_0301 NLFY-WYEGL 146-154 3.410 30.2652442594001 Cw_0301
QNIGCRFPYL 169-178 3.610 100.484149636389 Cw_0301 KPIRSSYFTF
197-206 3.810 45.1504388663187 Cw_0301 FQLQNIVKPL 206-215 3.180
24.0467535520645 Cw_0301 KPLPPVYLTF 213-222 5.010 149.904736149047
Cw_0301 IKLK-WSIPL 230-238 3.000 20.0855369231877 Cw_0301
ATVENETYTL 262-271 3.590 36.2340759264765 Cw_0401 DFEIVDPGYL 13-22
5.300 200.336809974792 Cw_0401 DPGYLGYLYL 18-27 4.390
80.640418980477 Cw_0401 LYLQ-WQPPL 25-33 5.300 200.336809974792
Cw_0401 QPPL-SLDHF 30-38 4.490 89.1214458786587 Cw_0401 HFKE-CTVEY
37-45 3.400 29.964100047397 Cw_0401 EYEL-KYRNI 44-52 3.220
25.0281201813378 Cw_0401 TWKKTIITKNL 56-65 3.690 40.0448469572867
Cw_0401 TYWI-SPQGI 103-111 3.220 25.0281201813378 Cw_0401
IPET-KVQDM 111-199 4.390 80.640418980477 Cw_0401 VYYN-WQYLL 122-130
5.300 200.336809974792 Cw_0401 SWKP-GIGVL 132-140 4.560
95.5834798300662 Cw_0401 WYEG-LDHAL 150-158 5.300 200.336809974792
Cw_0401 WYEG-LDHAL 149-158 3.870 47.9423860808193 Cw_0401
DYIKADGQNI 162-171 3.220 25.0281201813378 Cw_0401 RFPYLEASDY
174-183 3.220 25.0281201813378 Cw_0401 DYKD-FYICV 182-190 3.400
29.964100047397 Cw_0401 KPIRSSYFTF 197-206 3.700 40.4473043600674
Cw_0401 YFTF-QLQNI 203-211 3.910 49.8989519734079 Cw_0401
SYFTFQLQNI 202-211 3.910 49.8989519734079 Cw_0401 KPLPPVYLTF
213-222 3.880 48.4242150713452 Cw_0401 TFTRESSCEI 221-230 3.220
25.0281201813378 Cw_0401 CFVVRSKVNI 281-290 3.220 25.0281201813378
Cw_0702 DPGY-LGYLY 18-26 3.870 47.9423860808193 Cw_0702 DPGY-LGYLY
17-26
3.460 31.8169765146677 * = high stringency
[0134] Other Embodiments
[0135] This description has been by way of example of how the
compositions and methods of invention can be made and carried out.
Those of ordinary skill in the art will recognize that various
details may be modified in arriving at the other detailed
embodiments, and that many of these embodiments will come within
the scope of the invention. Therefore, to apprise the public of the
scope of the invention and the embodiments covered by the
invention, the following claims are made.
Sequence CWU 1
1
6 1 380 PRT Homo sapiens 1 Met Ala Phe Val Cys Leu Ala Ile Gly Cys
Leu Tyr Thr Phe Leu Ile 1 5 10 15 Ser Thr Thr Phe Gly Cys Thr Ser
Ser Ser Asp Thr Glu Ile Lys Val 20 25 30 Asn Pro Pro Gln Asp Phe
Glu Ile Val Asp Pro Gly Tyr Leu Gly Tyr 35 40 45 Leu Tyr Leu Gln
Trp Gln Pro Pro Leu Ser Leu Asp His Phe Lys Glu 50 55 60 Cys Thr
Val Glu Tyr Glu Leu Lys Tyr Arg Asn Ile Gly Ser Glu Thr 65 70 75 80
Trp Lys Thr Ile Ile Thr Lys Asn Leu His Tyr Lys Asp Gly Phe Asp 85
90 95 Leu Asn Lys Gly Ile Glu Ala Lys Ile His Thr Leu Leu Pro Trp
Gln 100 105 110 Cys Thr Asn Gly Ser Glu Val Gln Ser Ser Trp Ala Glu
Thr Thr Tyr 115 120 125 Trp Ile Ser Pro Gln Gly Ile Pro Glu Thr Lys
Val Gln Asp Met Asp 130 135 140 Cys Val Tyr Tyr Asn Trp Gln Tyr Leu
Leu Cys Ser Trp Lys Pro Gly 145 150 155 160 Ile Gly Val Leu Leu Asp
Thr Asn Tyr Asn Leu Phe Tyr Trp Tyr Glu 165 170 175 Gly Leu Asp His
Ala Leu Gln Cys Val Asp Tyr Ile Lys Ala Asp Gly 180 185 190 Gln Asn
Ile Gly Cys Arg Phe Pro Tyr Leu Glu Ala Ser Asp Tyr Lys 195 200 205
Asp Phe Tyr Ile Cys Val Asn Gly Ser Ser Glu Asn Lys Pro Ile Arg 210
215 220 Ser Ser Tyr Phe Thr Phe Gln Leu Gln Asn Ile Val Lys Pro Leu
Pro 225 230 235 240 Pro Val Tyr Leu Thr Phe Thr Arg Glu Ser Ser Cys
Glu Ile Lys Leu 245 250 255 Lys Trp Ser Ile Pro Leu Gly Pro Ile Pro
Ala Arg Cys Phe Asp Tyr 260 265 270 Glu Ile Glu Ile Arg Glu Asp Asp
Thr Thr Leu Val Thr Ala Thr Val 275 280 285 Glu Asn Glu Thr Tyr Thr
Leu Lys Thr Thr Asn Glu Thr Arg Gln Leu 290 295 300 Cys Phe Val Val
Arg Ser Lys Val Asn Ile Tyr Cys Ser Asp Asp Gly 305 310 315 320 Ile
Trp Ser Glu Trp Ser Asp Lys Gln Cys Trp Glu Gly Glu Asp Leu 325 330
335 Ser Lys Lys Thr Leu Leu Arg Phe Trp Leu Pro Phe Gly Phe Ile Leu
340 345 350 Ile Leu Val Ile Phe Val Thr Gly Leu Leu Leu Arg Lys Pro
Asn Thr 355 360 365 Tyr Pro Lys Met Ile Pro Glu Phe Phe Cys Asp Thr
370 375 380 2 1298 DNA Homo sapiens 2 ggtgcctgtc ggcggggaga
gaggcaatat caaggtttta aatctcggag aaatggcttt 60 cgtttgcttg
gctatcggat gcttatatac ctttctgata agcacaacat ttggctgtac 120
ttcatcttca gacaccgaga taaaagttaa ccctcctcag gattttgaga tagtggatcc
180 cggatactta ggttatctct atttgcaatg gcaaccccca ctgtctctgg
atcattttaa 240 ggaatgcaca gtggaatatg aactaaaata ccgaaacatt
ggtagtgaaa catggaagac 300 catcattact aagaatctac attacaaaga
tgggtttgat cttaacaagg gcattgaagc 360 gaagatacac acgcttttac
catggcaatg cacaaatgga tcagaagttc aaagttcctg 420 ggcagaaact
acttattgga tatcaccaca aggaattcca gaaactaaag ttcaggatat 480
ggattgcgta tattacaatt ggcaatattt actctgttct tggaaacctg gcataggtgt
540 acttcttgat accaattaca acttgtttta ctggtatgag ggcttggatc
atgcattaca 600 gtgtgttgat tacatcaagg ctgatggaca aaatatagga
tgcagatttc cctatttgga 660 ggcatcagac tataaagatt tctatatttg
tgttaatgga tcatcagaga acaagcctat 720 cagatccagt tatttcactt
ttcagcttca aaatatagtt aaacctttgc cgccagtcta 780 tcttactttt
actcgggaga gttcatgtga aattaagctg aaatggagca tacctttggg 840
acctattcca gcaaggtgtt ttgattatga aattgagatc agagaagatg atactacctt
900 ggtgactgct acagttgaaa atgaaacata caccttgaaa acaacaaatg
aaacccgaca 960 attatgcttt gtagtaagaa gcaaagtgaa tatttattgc
tcagatgacg gaatttggag 1020 tgagtggagt gataaacaat gctgggaagg
tgaagaccta tcgaagaaaa ctttgctacg 1080 tttctggcta ccatttggtt
tcatcttaat attagttata tttgtaaccg gtctgctttt 1140 gcgtaagcca
aacacctacc caaaaatgat tccagaattt ttctgtgata catgaagact 1200
ttccatatca agagacatgg tattgactca acagtttcca gtcatggcca aatgttcaat
1260 atgagtctca ataaactgaa tttttcttgc gaatgttg 1298 3 30 DNA
Artificial Sequence misc_feature Forward PCR Primer for
IL-13Ralpha2 3 aagatttgga agcttatggc tttcgtttgc 30 4 30 DNA
Artificial Sequence misc_feature Reverse PCR Primer for
IL-13Ralpha2 4 tccctcgaag cttcatgtat cacagaaaaa 30 5 27 DNA
Artificial Sequence misc_feature Forward PCR Primer for
IL-13Ralpha1 5 attattaagc ttatggagtg gccggcg 27 6 27 DNA Artificial
Sequence misc_feature Reverse PCR Primer For IL-13Ralpha1 6
taaccggaag cttcactgag aggcttt 27
* * * * *
References