U.S. patent application number 12/886355 was filed with the patent office on 2011-05-12 for antibody cancer immunotherapy.
This patent application is currently assigned to THE PENN STATE RESEARCH FOUNDATION. Invention is credited to Neil Christensen, Waldemar Debinski, Akiva Mintz.
Application Number | 20110110955 12/886355 |
Document ID | / |
Family ID | 22662463 |
Filed Date | 2011-05-12 |
United States Patent
Application |
20110110955 |
Kind Code |
A1 |
Debinski; Waldemar ; et
al. |
May 12, 2011 |
ANTIBODY CANCER IMMUNOTHERAPY
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;
(Winston-Salem, NC) ; Christensen; Neil;
(Harrisburg, PA) ; Mintz; Akiva; (Brooklyn,
NY) |
Assignee: |
THE PENN STATE RESEARCH
FOUNDATION
University Park
PA
|
Family ID: |
22662463 |
Appl. No.: |
12/886355 |
Filed: |
September 20, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12020409 |
Jan 25, 2008 |
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12886355 |
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10104408 |
Mar 22, 2002 |
7338929 |
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12020409 |
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09780926 |
Feb 8, 2001 |
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10104408 |
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60181000 |
Feb 8, 2000 |
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Current U.S.
Class: |
424/156.1 ;
424/174.1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/04 20180101; A61K 39/001184 20180801; C07K 14/7155
20130101; C07K 16/2866 20130101; A61K 2039/545 20130101; A61K
2039/5156 20130101; A61P 25/00 20180101; A61K 2039/505 20130101;
A61K 2039/53 20130101; A61K 39/0011 20130101; A61P 43/00
20180101 |
Class at
Publication: |
424/156.1 ;
424/174.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under grant
number CA74145 awarded by the National Cancer Institute of the
National Institutes of Health. The Government may have certain
rights in the invention.
Claims
1. A method for directing an antibody to cancer cells expressing
IL-13R.alpha.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; (b) administering the pharmaceutical composition to the
subject in an amount sufficient to allow the antibody to
specifically bind to the cancer cells expressing IL-13R.alpha.2 in
the subject; and (c) measuring stimulation of an immune response
against cancer cells expressing IL-13R.alpha.2 in the subject after
administration of the pharmaceutical composition to the
subject.
2. The method of claim 1, wherein the antibody is a monoclonal
antibody.
3. The method of claim 1, wherein the antibody is a polyclonal
antibody.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation application of
U.S. patent application Ser. No. 12/020,409 filed Jan. 25, 2008,
which is a divisional application of U.S. patent application Ser.
No. 10/104,408 filed Mar. 22, 2002 which is a continuation-in-part
of U.S. patent application Ser. No. 09/780,926 filed Feb. 8, 2001
which claims the benefit of U.S. provisional application Ser. 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-R 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-R.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, such as MAGE and BAGE,
have proven to stimulate a strong immune response against cancer
cells (see Mintz and Debinski in Crit. Rev. Oncogen 11:77-95;
2000), the present invention provides methods and compositions
useful for generating or increasing an anti-cancer immune response
in a subject.
[0011] For the purpose of anti-cancer immunotherapy, IL-13R.alpha.2
has the following distinct advantages over other cancer-related
antigens. Firstly, IL-13R.alpha.2 is a cell-surface receptor,
affording it exposure to the humoral arm of the immune system.
Secondly, IL-13R.alpha.2 is expressed on the vast majority of HGGs
tested, indicating its critical role in HGG progression and its
potential as a target for immunotherapy. Thirdly, the physiological
distribution of IL-13R.alpha.2 is limited to cancer cells and the
testes, limiting the potential for autoimmune side affects that are
observed when the target is also expressed in healthy tissue.
Furthermore, autoimmune side affects are unlikely because the
testes are an immune-privileged organ that expresses little MHC
class I molecules. Fourthly, hIL-13R.alpha.2 is an ideal target for
anti-cancer immunotherapy because of its size (380 amino acids in
full length IL-13R.alpha.2 and 343 amino acids in the extracellular
domain), providing the immune system with multiple epitopes to
recognize and target.
[0012] 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.2in the
subject.
[0013] 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.
[0014] In both of the foregoing method and composition, the agent
that can stimulate an 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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 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.
[0023] 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).
[0024] 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.
[0025] 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.
[0026] 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).
[0027] 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.
[0028] 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).
[0029] 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
[0030] 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:
[0031] FIG. 1 is the amino acid sequence of the native H. sapiens
IL-13R.alpha.2 protein. 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 .beta.-actin
transcripts in CNS (panels I and II) and peripheral tissues (panel
IV). The migration position of mRNA is shown in kilobases. Films
were exposed for 1-3 hours.
[0037] FIG. 8 is a Northern blot analysis of transcripts of
different IL13 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).
[0038] FIG. 9 is two graphs showing the effectiveness of an
hIL13R.alpha.2 recombinant protein vaccine (A) and a nucleic acid
vaccine (B) in preventing tumor formation in an animal model.
DETAILED DESCRIPTION
[0039] The invention encompasses compositions and methods relating
to stimulating an immune response against IL-13RA2 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 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.
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 et 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 Matteucci et al.,
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.
Identification of IL-13R.alpha.2 as a Cancer/Testis Antigen
[0041] 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-4R.alpha.), 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.
[0042] 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 IL-13/4 receptor and the
IL-4-independent receptor for IL-13 are shown in FIG. 3.
[0043] 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.
[0044] 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.
Vaccines
[0045] 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.
Protein/Peptide Based Vaccines
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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. A G 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). 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. 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), eta-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-13R.alpha.2
proteins may also be chemically 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.
[0054] 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.
[0055] 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. 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.
[0056] 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.
[0057] 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.
[0058] 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.
Nucleic Acid-Based Vaccines
[0059] 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.
[0060] 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.
[0061] 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).
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
Naked Nucleic Acid Vaccines
[0068] 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.
Expression Vector Vaccines
[0069] 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).
[0070] 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.
Cell-Based Vaccines
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] As DC are known to function as particularly strong APCs able
to efficiently take up, process, and present various forms of
antigens to immunologically naive T cells, 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
primed with a specific tumor antigen (e.g., IL-13R.alpha.2 or
peptide fragments thereof) can thus activate an anti-tumor
cytotoxic T lymphocyte (CTL) response that can provide protection
against and cause regression of a tumor. Several tumor-associated
antigens represent tissue differentiation antigens that are poorly
immunogenic due to an immune tolerance to self-antigens.
Stimulation with antigen-loaded DC, however, can break tolerance to
tumor-associated antigens and induce anti-tumor cytotoxic immune
responses.
[0077] 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. Ex vivo protocols for DC
priming with tumor-associated antigen are known in the art. See,
e.g., Kumamoto et al., J Dermatol. 28:658, 2001 and Fong et al. J.
Immunol. 167:7150-2001. Generally, DC are isolated from peripheral
blood by, for example, density gradient separation,
fluorescence-activated cell sorting and immunological cell
separation methods. See, e.g., U.S. Pat. No. 6,194,204. The
isolated DC are then cultured in media supplemented with purified
antigen (e.g., IL-13R.alpha.2) so that the DC can process the
antigen for presentation to T cells. The antigen-loaded DC can be
administered to a patient (e.g., injection) in a therapeutically
effective amount (e.g., an amount that causes tumor regression). To
enhance this response, the DC may be exposed to a cytokine (e.g.,
GM-CSF/IL-4) prior to administration. Tanigawa et al., J.
Immunother. 26:493, 2001. In addition, specific antigen can be
targeted to DC according to known methods. See, e.g., Nature
Biotech. 17:253, 1999.
[0078] 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.
Anti-Idiotypic Antibody Vaccines
[0079] 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.
Antibody Production
[0080] 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 mAb
of the invention may be cultivated in vitro or in vivo. The ability
to produce high titers of mAbs in vivo makes this a particularly
useful method of production.
[0081] 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.
[0082] 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).
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
Method of Inducing an Anti-IL-13R.alpha.2 Immune Response in a
Subject
[0087] 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.
Subjects
[0088] 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.
IL-13R.alpha.2 as a Component of a Polyvalent Vaccine
[0089] 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).
Administering Vaccines to a Subject
[0090] 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 composition
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.
[0091] 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.
[0092] 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.
[0093] 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.
Dosing
[0094] 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.
[0095] 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.
[0096] 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.
Kits
[0097] 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
[0098] 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 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.E13K-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
Materials and Methods
[0099] Sources of RNA. 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 (MTE.TM.) 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 (MTN.TM.) Blots (cat #7755-1
and 7769-1) were assayed to confirm the true presence of the
transcripts. In addition, two Human Tissue Northern (MTN.TM.) Blots
(cat #7759-1 and 7760-1) were analyzed to verify the tissue
distribution of the IL-13R.alpha.2 transcript.
[0100] cDNA Probes. 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 IL13R.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 QIAquick Gel Extraction Kit (Qiagen
Inc., Valencia, Calif.). Actin cDNA was purchased from Clontech
Labs.
The primers for human IL-13R.alpha.2 were as follows:
TABLE-US-00001 forward (SEQ ID NO: 3)
5'-AAGATTTGGAAGCTTATGGCTTTCGTTTGC-3' reverse (SEQ ID NO: 4)
5'-TCCCTCGAAGCTTCATGTATCACAGAAAAA-3'
The primers for human IL13R.alpha.1 were as follows:
TABLE-US-00002 forward 5'-ATTATTAAGCTTATGGAGTGGCCGGCG-3' (SEQ ID
NO: 5) reverse 5'-TAACCGGAAGCTTCACTGAGAGGCTTT-3' (SEQ ID NO: 6)
[0101] Northern Blot Analysis. 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.).
Results
[0102] 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, IL13R.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 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.
[0103] 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.
[0104] 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, IL13/4
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 IL13R.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 IL13R.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).
[0105] 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
IL4R.alpha. 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.
[0106] Control hybridization of .beta.-actin. 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.
[0107] Gene expression of IL13 receptors in cells. Gene expression
of the two IL13 receptors was also examined in malignant and normal
cells (FIG. 8). Transcripts for IL-13R.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
IL13/4 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 IL13/4 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
[0108] 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.
TABLE-US-00003 TABLE 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-TIITK * 55-63 4.500 90.0171313005218 A68.1 CVNG-SSENK *
189-197 4.790 120.301368663215 A68.1 FTFQLQNIVK * 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 YTLKTTNETR * 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 20.0855369231877 B_2702 CRFP-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-DTTLV
252-260 3.000 20.0855369231877 B_2702 VRSK-VNIYC 284-292 3.000
20.0855369231877 B_2705 FEIV-DPGYL 14-22 3.400 29.964100047397
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-NGSEV 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-CEIKL 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
29.964100047397 B_2705 VENE-TYTLK 264-272 3.400 29.964100047397
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
IPARCFDY 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 DKQCWEGEDLY 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-CEIKL 224-232 3.000 20.0855369231877 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-DPGYL 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 5.480 239.846707374255 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 B5101_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 SSWAETTYWI 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 27.6603505585167 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
IGCRFPYL 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 20.0855369231877 B60 KECT-VEYEL 39-47 5.870 354.248980267765
B60 IEAK-IHTLL 77-85 5.870 354.248980267765 B60 RESS-CEIKL 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
Example 4
Protein and Nucleic Acid Vaccines Prevent The Development of
Tumors
[0109] The effect of an antibody-based immune response against
cells expressing IL-13R.alpha.2 was examined. An immunocompetent
syngeneic murine glioma model that expresses IL-13R.alpha.2 was
established. G-26 murine glioma cells were stably transfected with
hIL-13R.alpha.2 and were shown to contain the IL-13 binding
characteristics of human HGGs. Furthermore, tumors grown from these
IL-13R.alpha.2(+) cells immunocompetent C57BL/J6 mice maintained
the HGG restricted IL-13 binding properties, validating this model.
Immunocompetent C57BL/J6 mice were injected with affinity-purified
extracellular domain of IL-13R.alpha.2 recombinant protein domain
[6.times.(His)-(factor X restriction site)-IL13-R.alpha.2 (amino
acids 27-343)] produced in E. coli. together with Freund's Complete
adjuvant or Freund's adjuvant alone (10 male mice per/group, age 10
weeks). Mice were vaccinated every 2 weeks for a total of 3 times.
Three weeks after the last vaccination, a substantial load of
G-26-hIL-13R.alpha.2(+) tumor cells (5.times.10.sup.6 cells) were
implanted subcutaneously into the vaccinated mice and the controls.
Tumors appeared 16 days post tumor cells injection in the control
groups but not in the IL-13R.alpha.2 vaccinated group (FIG. 1A).
Additionally, mice vaccinated with recombinant IL-13R.alpha.2
manifested a strong specific antibody response against
IL-13R.alpha.2 as demonstrated by enzyme-linked immunosorbent assay
(ELISA).
[0110] Anti-tumor responses by the cell-mediated branch of the
immune system were also examined. A plasmid containing
IL-13R.alpha.2 under the CMV promoter, pcDNA3.1/IL13R.alpha.2, or
pcDNA3.1 alone was attached to gold particles and used to vaccinate
mice via gene gun (10 mice/group) (Vaccine 18:2937-2944; 2000).
Mice were immunized every two weeks for a total of 3 times. Three
weeks after the last immunization, mice were injected
subcutaneously with 5.times.10.sup.6 G-26-IL-13R.alpha.2(+) murine
glioma cells. Tumors appeared 16 days after tumor cell injection
only in mice vaccinated with pcDNA 3.1 vector alone but no tumors
were visible in mice vaccinated with pcDNA 3.1/R.alpha.2 (FIG.
1B).
Other Embodiments
[0111] 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
3171380PRTHomo sapiens 1Met Ala Phe Val Cys Leu Ala Ile Gly Cys Leu
Tyr Thr Phe Leu Ile1 5 10 15Ser Thr Thr Phe Gly Cys Thr Ser Ser Ser
Asp Thr Glu Ile Lys Val 20 25 30Asn Pro Pro Gln Asp Phe Glu Ile Val
Asp Pro Gly Tyr Leu Gly Tyr 35 40 45Leu Tyr Leu Gln Trp Gln Pro Pro
Leu Ser Leu Asp His Phe Lys Glu 50 55 60Cys Thr Val Glu Tyr Glu Leu
Lys Tyr Arg Asn Ile Gly Ser Glu Thr65 70 75 80Trp Lys Thr Ile Ile
Thr Lys Asn Leu His Tyr Lys Asp Gly Phe Asp 85 90 95Leu Asn Lys Gly
Ile Glu Ala Lys Ile His Thr Leu Leu Pro Trp Gln 100 105 110Cys Thr
Asn Gly Ser Glu Val Gln Ser Ser Trp Ala Glu Thr Thr Tyr 115 120
125Trp Ile Ser Pro Gln Gly Ile Pro Glu Thr Lys Val Gln Asp Met Asp
130 135 140Cys Val Tyr Tyr Asn Trp Gln Tyr Leu Leu Cys Ser Trp Lys
Pro Gly145 150 155 160Ile Gly Val Leu Leu Asp Thr Asn Tyr Asn Leu
Phe Tyr Trp Tyr Glu 165 170 175Gly Leu Asp His Ala Leu Gln Cys Val
Asp Tyr Ile Lys Ala Asp Gly 180 185 190Gln Asn Ile Gly Cys Arg Phe
Pro Tyr Leu Glu Ala Ser Asp Tyr Lys 195 200 205Asp Phe Tyr Ile Cys
Val Asn Gly Ser Ser Glu Asn Lys Pro Ile Arg 210 215 220Ser Ser Tyr
Phe Thr Phe Gln Leu Gln Asn Ile Val Lys Pro Leu Pro225 230 235
240Pro Val Tyr Leu Thr Phe Thr Arg Glu Ser Ser Cys Glu Ile Lys Leu
245 250 255Lys Trp Ser Ile Pro Leu Gly Pro Ile Pro Ala Arg Cys Phe
Asp Tyr 260 265 270Glu Ile Glu Ile Arg Glu Asp Asp Thr Thr Leu Val
Thr Ala Thr Val 275 280 285Glu Asn Glu Thr Tyr Thr Leu Lys Thr Thr
Asn Glu Thr Arg Gln Leu 290 295 300Cys Phe Val Val Arg Ser Lys Val
Asn Ile Tyr Cys Ser Asp Asp Gly305 310 315 320Ile Trp Ser Glu Trp
Ser Asp Lys Gln Cys Trp Glu Gly Glu Asp Leu 325 330 335Ser Lys Lys
Thr Leu Leu Arg Phe Trp Leu Pro Phe Gly Phe Ile Leu 340 345 350Ile
Leu Val Ile Phe Val Thr Gly Leu Leu Leu Arg Lys Pro Asn Thr 355 360
365Tyr Pro Lys Met Ile Pro Glu Phe Phe Cys Asp Thr 370 375
38021298PRTHomo sapiens 2Gly Gly Thr Gly Cys Cys Thr Gly Thr Cys
Gly Gly Cys Gly Gly Gly1 5 10 15Gly Ala Gly Ala Gly Ala Gly Gly Cys
Ala Ala Thr Ala Thr Cys Ala 20 25 30Ala Gly Gly Thr Thr Thr Thr Ala
Ala Ala Thr Cys Thr Cys Gly Gly 35 40 45Ala Gly Ala Ala Ala Thr Gly
Gly Cys Thr Thr Thr Cys Gly Thr Thr 50 55 60Thr Gly Cys Thr Thr Gly
Gly Cys Thr Ala Thr Cys Gly Gly Ala Thr65 70 75 80Gly Cys Thr Thr
Ala Thr Ala Thr Ala Cys Cys Thr Thr Thr Cys Thr 85 90 95Gly Ala Thr
Ala Ala Gly Cys Ala Cys Ala Ala Cys Ala Thr Thr Thr 100 105 110Gly
Gly Cys Thr Gly Thr Ala Cys Thr Thr Cys Ala Thr Cys Thr Thr 115 120
125Cys Ala Gly Ala Cys Ala Cys Cys Gly Ala Gly Ala Thr Ala Ala Ala
130 135 140Ala Gly Thr Thr Ala Ala Cys Cys Cys Thr Cys Cys Thr Cys
Ala Gly145 150 155 160Gly Ala Thr Thr Thr Thr Gly Ala Gly Ala Thr
Ala Gly Thr Gly Gly 165 170 175Ala Thr Cys Cys Cys Gly Gly Ala Thr
Ala Cys Thr Thr Ala Gly Gly 180 185 190Thr Thr Ala Thr Cys Thr Cys
Thr Ala Thr Thr Thr Gly Cys Ala Ala 195 200 205Thr Gly Gly Cys Ala
Ala Cys Cys Cys Cys Cys Ala Cys Thr Gly Thr 210 215 220Cys Thr Cys
Thr Gly Gly Ala Thr Cys Ala Thr Thr Thr Thr Ala Ala225 230 235
240Gly Gly Ala Ala Thr Gly Cys Ala Cys Ala Gly Thr Gly Gly Ala Ala
245 250 255Thr Ala Thr Gly Ala Ala Cys Thr Ala Ala Ala Ala Thr Ala
Cys Cys 260 265 270Gly Ala Ala Ala Cys Ala Thr Thr Gly Gly Thr Ala
Gly Thr Gly Ala 275 280 285Ala Ala Cys Ala Thr Gly Gly Ala Ala Gly
Ala Cys Cys Ala Thr Cys 290 295 300Ala Thr Thr Ala Cys Thr Ala Ala
Gly Ala Ala Thr Cys Thr Ala Cys305 310 315 320Ala Thr Thr Ala Cys
Ala Ala Ala Gly Ala Thr Gly Gly Gly Thr Thr 325 330 335Thr Gly Ala
Thr Cys Thr Thr Ala Ala Cys Ala Ala Gly Gly Gly Cys 340 345 350Ala
Thr Thr Gly Ala Ala Gly Cys Gly Ala Ala Gly Ala Thr Ala Cys 355 360
365Ala Cys Ala Cys Gly Cys Thr Thr Thr Thr Ala Cys Cys Ala Thr Gly
370 375 380Gly Cys Ala Ala Thr Gly Cys Ala Cys Ala Ala Ala Thr Gly
Gly Ala385 390 395 400Thr Cys Ala Gly Ala Ala Gly Thr Thr Cys Ala
Ala Ala Gly Thr Thr 405 410 415Cys Cys Thr Gly Gly Gly Cys Ala Gly
Ala Ala Ala Cys Thr Ala Cys 420 425 430Thr Thr Ala Thr Thr Gly Gly
Ala Thr Ala Thr Cys Ala Cys Cys Ala 435 440 445Cys Ala Ala Gly Gly
Ala Ala Thr Thr Cys Cys Ala Gly Ala Ala Ala 450 455 460Cys Thr Ala
Ala Ala Gly Thr Thr Cys Ala Gly Gly Ala Thr Ala Thr465 470 475
480Gly Gly Ala Thr Thr Gly Cys Gly Thr Ala Thr Ala Thr Thr Ala Cys
485 490 495Ala Ala Thr Thr Gly Gly Cys Ala Ala Thr Ala Thr Thr Thr
Ala Cys 500 505 510Thr Cys Thr Gly Thr Thr Cys Thr Thr Gly Gly Ala
Ala Ala Cys Cys 515 520 525Thr Gly Gly Cys Ala Thr Ala Gly Gly Thr
Gly Thr Ala Cys Thr Thr 530 535 540Cys Thr Thr Gly Ala Thr Ala Cys
Cys Ala Ala Thr Thr Ala Cys Ala545 550 555 560Ala Cys Thr Thr Gly
Thr Thr Thr Thr Ala Cys Thr Gly Gly Thr Ala 565 570 575Thr Gly Ala
Gly Gly Gly Cys Thr Thr Gly Gly Ala Thr Cys Ala Thr 580 585 590Gly
Cys Ala Thr Thr Ala Cys Ala Gly Thr Gly Thr Gly Thr Thr Gly 595 600
605Ala Thr Thr Ala Cys Ala Thr Cys Ala Ala Gly Gly Cys Thr Gly Ala
610 615 620Thr Gly Gly Ala Cys Ala Ala Ala Ala Thr Ala Thr Ala Gly
Gly Ala625 630 635 640Thr Gly Cys Ala Gly Ala Thr Thr Thr Cys Cys
Cys Thr Ala Thr Thr 645 650 655Thr Gly Gly Ala Gly Gly Cys Ala Thr
Cys Ala Gly Ala Cys Thr Ala 660 665 670Thr Ala Ala Ala Gly Ala Thr
Thr Thr Cys Thr Ala Thr Ala Thr Thr 675 680 685Thr Gly Thr Gly Thr
Thr Ala Ala Thr Gly Gly Ala Thr Cys Ala Thr 690 695 700Cys Ala Gly
Ala Gly Ala Ala Cys Ala Ala Gly Cys Cys Thr Ala Thr705 710 715
720Cys Ala Gly Ala Thr Cys Cys Ala Gly Thr Thr Ala Thr Thr Thr Cys
725 730 735Ala Cys Thr Thr Thr Thr Cys Ala Gly Cys Thr Thr Cys Ala
Ala Ala 740 745 750Ala Thr Ala Thr Ala Gly Thr Thr Ala Ala Ala Cys
Cys Thr Thr Thr 755 760 765Gly Cys Cys Gly Cys Cys Ala Gly Thr Cys
Thr Ala Thr Cys Thr Thr 770 775 780Ala Cys Thr Thr Thr Thr Ala Cys
Thr Cys Gly Gly Gly Ala Gly Ala785 790 795 800Gly Thr Thr Cys Ala
Thr Gly Thr Gly Ala Ala Ala Thr Thr Ala Ala 805 810 815Gly Cys Thr
Gly Ala Ala Ala Thr Gly Gly Ala Gly Cys Ala Thr Ala 820 825 830Cys
Cys Thr Thr Thr Gly Gly Gly Ala Cys Cys Thr Ala Thr Thr Cys 835 840
845Cys Ala Gly Cys Ala Ala Gly Gly Thr Gly Thr Thr Thr Thr Gly Ala
850 855 860Thr Thr Ala Thr Gly Ala Ala Ala Thr Thr Gly Ala Gly Ala
Thr Cys865 870 875 880Ala Gly Ala Gly Ala Ala Gly Ala Thr Gly Ala
Thr Ala Cys Thr Ala 885 890 895Cys Cys Thr Thr Gly Gly Thr Gly Ala
Cys Thr Gly Cys Thr Ala Cys 900 905 910Ala Gly Thr Thr Gly Ala Ala
Ala Ala Thr Gly Ala Ala Ala Cys Ala 915 920 925Thr Ala Cys Ala Cys
Cys Thr Thr Gly Ala Ala Ala Ala Cys Ala Ala 930 935 940Cys Ala Ala
Ala Thr Gly Ala Ala Ala Cys Cys Cys Gly Ala Cys Ala945 950 955
960Ala Thr Thr Ala Thr Gly Cys Thr Thr Thr Gly Thr Ala Gly Thr Ala
965 970 975Ala Gly Ala Ala Gly Cys Ala Ala Ala Gly Thr Gly Ala Ala
Thr Ala 980 985 990Thr Thr Thr Ala Thr Thr Gly Cys Thr Cys Ala Gly
Ala Thr Gly Ala 995 1000 1005Cys Gly Gly Ala Ala Thr Thr Thr Gly
Gly Ala Gly Thr Gly Ala 1010 1015 1020Gly Thr Gly Gly Ala Gly Thr
Gly Ala Thr Ala Ala Ala Cys Ala 1025 1030 1035Ala Thr Gly Cys Thr
Gly Gly Gly Ala Ala Gly Gly Thr Gly Ala 1040 1045 1050Ala Gly Ala
Cys Cys Thr Ala Thr Cys Gly Ala Ala Gly Ala Ala 1055 1060 1065Ala
Ala Cys Thr Thr Thr Gly Cys Thr Ala Cys Gly Thr Thr Thr 1070 1075
1080Cys Thr Gly Gly Cys Thr Ala Cys Cys Ala Thr Thr Thr Gly Gly
1085 1090 1095Thr Thr Thr Cys Ala Thr Cys Thr Thr Ala Ala Thr Ala
Thr Thr 1100 1105 1110Ala Gly Thr Thr Ala Thr Ala Thr Thr Thr Gly
Thr Ala Ala Cys 1115 1120 1125Cys Gly Gly Thr Cys Thr Gly Cys Thr
Thr Thr Thr Gly Cys Gly 1130 1135 1140Thr Ala Ala Gly Cys Cys Ala
Ala Ala Cys Ala Cys Cys Thr Ala 1145 1150 1155Cys Cys Cys Ala Ala
Ala Ala Ala Thr Gly Ala Thr Thr Cys Cys 1160 1165 1170Ala Gly Ala
Ala Thr Thr Thr Thr Thr Cys Thr Gly Thr Gly Ala 1175 1180 1185Thr
Ala Cys Ala Thr Gly Ala Ala Gly Ala Cys Thr Thr Thr Cys 1190 1195
1200Cys Ala Thr Ala Thr Cys Ala Ala Gly Ala Gly Ala Cys Ala Thr
1205 1210 1215Gly Gly Thr Ala Thr Thr Gly Ala Cys Thr Cys Ala Ala
Cys Ala 1220 1225 1230Gly Thr Thr Thr Cys Cys Ala Gly Thr Cys Ala
Thr Gly Gly Cys 1235 1240 1245Cys Ala Ala Ala Thr Gly Thr Thr Cys
Ala Ala Thr Ala Thr Gly 1250 1255 1260Ala Gly Thr Cys Thr Cys Ala
Ala Thr Ala Ala Ala Cys Thr Gly 1265 1270 1275Ala Ala Thr Thr Thr
Thr Thr Cys Thr Thr Gly Cys Gly Ala Ala 1280 1285 1290Thr Gly Thr
Thr Gly 1295330DNAHomo sapiens 3aagatttgga agcttatggc tttcgtttgc
30430DNAHomo sapiens 4tccctcgaag cttcatgtat cacagaaaaa 30527DNAHomo
sapiens 5attattaagc ttatggagtg gccggcg 27627DNAHomo sapiens
6taaccggaag cttcactgag aggcttt 2779PRTHomo sapiens 7Ile Val Asp Pro
Gly Tyr Leu Gly Tyr1 5810PRTHomo sapiens 8Leu Leu Asp Thr Asn Tyr
Asn Leu Phe Tyr1 5 10910PRTHomo sapiens 9Tyr Leu Tyr Leu Gln Trp
Gln Pro Pro Leu1 5 101010PRTHomo sapiens 10Tyr Leu Gln Trp Gln Pro
Pro Leu Ser Leu1 5 10119PRTHomo sapiens 11Leu Gln Trp Gln Pro Pro
Leu Ser Leu1 51210PRTHomo sapiens 12Ser Leu Asp His Phe Lys Glu Cys
Thr Val1 5 101310PRTHomo sapiens 13Asn Leu His Tyr Lys Asp Gly Phe
Asp Leu1 5 10149PRTHomo sapiens 14Trp Gln Cys Thr Asn Gly Ser Glu
Val1 5159PRTHomo sapiens 15Cys Val Tyr Tyr Asn Trp Gln Tyr Leu1
51610PRTHomo sapiens 16Tyr Leu Leu Cys Ser Trp Lys Pro Gly Ile1 5
10179PRTHomo sapiens 17Val Leu Leu Asp Thr Asn Tyr Asn Leu1
5189PRTHomo sapiens 18Asn Leu Phe Tyr Trp Tyr Glu Gly Leu1
5199PRTHomo sapiens 19Gly Leu Asp His Ala Leu Gln Cys Val1
5209PRTHomo sapiens 20Asn Ile Gly Cys Arg Phe Pro Tyr Leu1
52110PRTHomo sapiens 21Phe Gln Leu Gln Asn Ile Val Lys Pro Leu1 5
10229PRTHomo sapiens 22Gln Leu Gln Asn Ile Val Lys Pro Leu1
5239PRTHomo sapiens 23Asn Ile Val Lys Pro Leu Pro Pro Val1
52410PRTHomo sapiens 24Tyr Leu Thr Phe Thr Arg Glu Ser Ser Cys1 5
102510PRTHomo sapiens 25Gln Leu Cys Phe Val Val Arg Ser Lys Val1 5
102610PRTHomo sapiens 26Ile Val Asp Pro Gly Tyr Leu Gly Tyr Leu1 5
102710PRTHomo sapiens 27Tyr Leu Tyr Leu Gln Trp Gln Pro Pro Leu1 5
10289PRTHomo sapiens 28Leu Gln Trp Gln Pro Pro Leu Ser Leu1
5299PRTHomo sapiens 29Leu Gln Trp Gln Pro Pro Leu Ser Leu1
5309PRTHomo sapiens 30Cys Val Tyr Tyr Asn Trp Gln Tyr Leu1
5319PRTHomo sapiens 31Val Leu Leu Asp Thr Asn Tyr Asn Leu1
5329PRTHomo sapiens 32Val Leu Leu Asp Thr Asn Tyr Asn Leu1
5339PRTHomo sapiens 33Asn Leu Phe Tyr Trp Tyr Glu Gly Leu1
53410PRTHomo sapiens 34Phe Gln Leu Gln Asn Ile Val Lys Pro Leu1 5
103510PRTHomo sapiens 35Leu Leu Asp Thr Asn Tyr Asn Leu Phe Tyr1 5
10369PRTHomo sapiens 36Ala Leu Gln Cys Val Asp Tyr Ile Lys1
5379PRTHomo sapiens 37Gly Ile Trp Ser Glu Trp Ser Asp Lys1
53810PRTHomo sapiens 38Asp Phe Glu Ile Val Asp Pro Gly Tyr Leu1 5
10399PRTHomo sapiens 39Leu Tyr Leu Gln Trp Gln Pro Pro Leu1
5409PRTHomo sapiens 40Glu Tyr Glu Leu Lys Tyr Arg Asn Ile1
5419PRTHomo sapiens 41Thr Tyr Trp Ile Ser Pro Gln Gly Ile1
5429PRTHomo sapiens 42Val Tyr Tyr Asn Trp Gln Tyr Leu Leu1
5439PRTHomo sapiens 43Trp Tyr Glu Gly Leu Asp His Ala Leu1
54410PRTHomo sapiens 44Asp Tyr Ile Lys Ala Asp Gly Gln Asn Ile1 5
104510PRTHomo sapiens 45Ser Tyr Phe Thr Phe Gln Leu Gln Asn Ile1 5
10469PRTHomo sapiens 46Asp Leu Ser Lys Lys Thr Leu Leu Arg1
5479PRTHomo sapiens 47Thr Val Glu Tyr Glu Leu Lys Tyr Arg1
5489PRTHomo sapiens 48Thr Val Glu Tyr Glu Leu Lys Tyr Arg1
5499PRTHomo sapiens 49Glu Thr Trp Lys Thr Ile Ile Thr Lys1
5509PRTHomo sapiens 50Cys Val Asn Gly Ser Ser Glu Asn Lys1
55110PRTHomo sapiens 51Phe Thr Phe Gln Leu Gln Asn Ile Val Lys1 5
105210PRTHomo sapiens 52Phe Thr Arg Glu Ser Ser Cys Glu Ile Lys1 5
10539PRTHomo sapiens 53Glu Ser Ser Cys Glu Ile Lys Leu Lys1
55410PRTHomo sapiens 54Thr Val Glu Asn Glu Thr Tyr Thr Leu Lys1 5
105510PRTHomo sapiens 55Tyr Thr Leu Lys Thr Thr Asn Glu Thr Arg1 5
105610PRTHomo sapiens 56Glu Thr Arg Gln Leu Cys Phe Val Val Arg1 5
105710PRTHomo sapiens 57Asp Pro Gly Tyr Leu Gly Tyr Leu Tyr Leu1 5
10589PRTHomo sapiens 58Cys Val Tyr Tyr Asn Trp Gln Tyr Leu1
55910PRTHomo sapiens 59Gly Val Leu Leu Asp Thr Asn Tyr Asn Leu1 5
106010PRTHomo sapiens 60Ile Val Lys Pro Leu Pro Pro Val Tyr Leu1 5
10619PRTHomo sapiens 61Glu Ile Arg Glu Asp Asp Thr Thr Leu1
5628PRTHomo sapiens 62Glu Ala Lys Ile His Thr Leu Leu1 5638PRTHomo
sapiens 63Glu Ile Lys Leu Lys Trp Ser Ile1 5648PRTHomo sapiens
64Val Val Arg Ser Lys Val Asn Ile1 56510PRTHomo sapiens 65Gln Asn
Ile Gly Cys Arg Phe Pro Tyr Leu1 5 106610PRTHomo sapiens 66Ile Arg
Ser Ser Tyr Phe Thr Phe Gln Leu1 5 10679PRTHomo sapiens 67Leu Gln
Trp Gln Pro Pro Leu Ser Leu1 56810PRTHomo sapiens 68Trp Gln Pro Pro
Leu Ser Leu Asp His Phe1 5 10699PRTHomo sapiens 69Tyr Arg Asn Ile
Gly Ser Glu Thr Trp1 57010PRTHomo sapiens 70Val Gln Ser Ser Trp Ala
Glu Thr Thr Tyr1 5 10719PRTHomo sapiens 71Val Gln Asp Met Asp
Cys
Val Tyr Tyr1 57210PRTHomo sapiens 72Gly Gln Asn Ile Gly Cys Arg Phe
Pro Tyr1 5 10739PRTHomo sapiens 73Cys Arg Phe Pro Tyr Leu Glu Ala
Ser1 57410PRTHomo sapiens 74Ile Arg Ser Ser Tyr Phe Thr Phe Gln
Leu1 5 107510PRTHomo sapiens 75Thr Arg Glu Ser Ser Cys Glu Ile Lys
Leu1 5 107610PRTHomo sapiens 76Ala Arg Cys Phe Asp Tyr Glu Ile Glu
Ile1 5 10779PRTHomo sapiens 77Ile Arg Glu Asp Asp Thr Thr Leu Val1
5789PRTHomo sapiens 78Val Arg Ser Lys Val Asn Ile Tyr Cys1
5799PRTHomo sapiens 79Phe Glu Ile Val Asp Pro Gly Tyr Leu1
58010PRTHomo sapiens 80Tyr Leu Tyr Leu Gln Trp Gln Pro Pro Leu1 5
10819PRTHomo sapiens 81Leu Gln Trp Gln Pro Pro Leu Ser Leu1
5829PRTHomo sapiens 82Leu Gln Trp Gln Pro Pro Leu Ser Leu1
58310PRTHomo sapiens 83Trp Gln Pro Pro Leu Ser Leu Asp His Phe1 5
10849PRTHomo sapiens 84Lys Glu Cys Thr Val Glu Tyr Glu Leu1
58510PRTHomo sapiens 85Tyr Arg Asn Ile Gly Ser Glu Thr Trp Lys1 5
10869PRTHomo sapiens 86Arg Asn Ile Gly Ser Glu Thr Trp Lys1
58710PRTHomo sapiens 87Ser Glu Thr Trp Lys Thr Ile Ile Thr Lys1 5
10889PRTHomo sapiens 88Lys Asn Leu His Tyr Lys Asp Gly Phe1
58910PRTHomo sapiens 89Asn Leu His Tyr Lys Asp Gly Phe Asp Leu1 5
10909PRTHomo sapiens 90Ile Glu Ala Lys Ile His Thr Leu Leu1
5919PRTHomo sapiens 91Trp Gln Cys Thr Asn Gly Ser Glu Val1
59210PRTHomo sapiens 92Val Gln Ser Ser Trp Ala Glu Thr Thr Tyr1 5
10939PRTHomo sapiens 93Val Gln Asp Met Asp Cys Val Tyr Tyr1
5949PRTHomo sapiens 94Cys Val Tyr Tyr Asn Trp Gln Tyr Leu1
5959PRTHomo sapiens 95Trp Gln Tyr Leu Leu Cys Ser Trp Lys1
59610PRTHomo sapiens 96Cys Ser Trp Lys Pro Gly Ile Gly Val Leu1 5
10979PRTHomo sapiens 97Val Leu Leu Asp Thr Asn Tyr Asn Leu1
5989PRTHomo sapiens 98Thr Asn Tyr Asn Leu Phe Tyr Trp Tyr1
5999PRTHomo sapiens 99Asn Leu Phe Tyr Trp Tyr Glu Gly Leu1
51009PRTHomo sapiens 100Ala Leu Gln Cys Val Asp Tyr Ile Lys1
51019PRTHomo sapiens 101Leu Gln Cys Val Asp Tyr Ile Lys Ala1
510210PRTHomo sapiens 102Gly Gln Asn Ile Gly Cys Arg Phe Pro Tyr1 5
101039PRTHomo sapiens 103Cys Arg Phe Pro Tyr Leu Glu Ala Ser1
510410PRTHomo sapiens 104Phe Pro Tyr Leu Glu Ala Ser Asp Tyr Lys1 5
1010510PRTHomo sapiens 105Ile Arg Ser Ser Tyr Phe Thr Phe Gln Leu1
5 101069PRTHomo sapiens 106Arg Ser Ser Tyr Phe Thr Phe Gln Leu1
510710PRTHomo sapiens 107Phe Thr Phe Gln Leu Gln Asn Ile Val Lys1 5
1010810PRTHomo sapiens 108Phe Gln Leu Gln Asn Ile Val Lys Pro Leu1
5 101099PRTHomo sapiens 109Thr Arg Glu Ser Ser Cys Glu Ile Lys1
51109PRTHomo sapiens 110Arg Glu Ser Ser Cys Glu Ile Lys Leu1
511110PRTHomo sapiens 111Ala Arg Cys Phe Asp Tyr Glu Ile Glu Ile1 5
1011210PRTHomo sapiens 112Arg Cys Phe Asp Tyr Glu Ile Glu Ile Arg1
5 101139PRTHomo sapiens 113Ile Arg Glu Asp Asp Thr Thr Leu Val1
511410PRTHomo sapiens 114Ile Glu Ile Arg Glu Asp Asp Thr Thr Leu1 5
101159PRTHomo sapiens 115Val Glu Asn Glu Thr Tyr Thr Leu Lys1
51169PRTHomo sapiens 116Thr Arg Gln Leu Cys Phe Val Val Arg1
511710PRTHomo sapiens 117Arg Gln Leu Cys Phe Val Val Arg Ser Lys1 5
101189PRTHomo sapiens 118Val Arg Ser Lys Val Asn Ile Tyr Cys1
51199PRTHomo sapiens 119Gly Ile Trp Ser Glu Trp Ser Asp Ser1
51209PRTHomo sapiens 120Lys Gln Cys Trp Glu Gly Glu Asp Leu1
512110PRTHomo sapiens 121Gln Cys Trp Glu Gly Glu Asp Leu Ser Lys1 5
101229PRTHomo sapiens 122Trp Glu Gly Glu Asp Leu Ser Lys Lys1
512310PRTHomo sapiens 123Gly Glu Asp Leu Ser Lys Lys Thr Leu Leu1 5
101249PRTHomo sapiens 124Asp Pro Gly Tyr Leu Gly Tyr Leu Tyr1
51259PRTHomo sapiens 125Gln Pro Pro Leu Ser Leu Asp His Phe1
51269PRTHomo sapiens 126Phe Pro Tyr Leu Glu Ala Ser Asp Tyr1
512710PRTHomo sapiens 127Lys Pro Ile Arg Ser Ser Tyr Phe Thr Phe1 5
1012810PRTHomo sapiens 128Lys Pro Leu Pro Pro Val Tyr Leu Thr Phe1
5 1012910PRTHomo sapiens 129Gly Pro Ile Pro Ala Arg Cys Phe Asp
Tyr1 5 101308PRTHomo sapiens 130Asp Pro Gly Tyr Leu Gly Tyr Leu1
51318PRTHomo sapiens 131Lys Pro Gly Ile Gly Val Leu Leu1
51328PRTHomo sapiens 132Lys Pro Ile Arg Ser Ser Tyr Phe1
51338PRTHomo sapiens 133Lys Pro Leu Pro Pro Val Tyr Leu1
51348PRTHomo sapiens 134Leu Pro Pro Val Tyr Leu Thr Phe1
51358PRTHomo sapiens 135Gly Pro Ile Pro Ala Arg Cys Phe1
51368PRTHomo sapiens 136Ile Pro Ala Arg Cys Phe Asp Tyr1
51379PRTHomo sapiens 137Val Asp Pro Gly Tyr Leu Gly Tyr Leu1
513810PRTHomo sapiens 138Lys Asp Gly Phe Asp Leu Asn Lys Gly Ile1 5
101399PRTHomo sapiens 139Ile Glu Ala Lys Ile His Thr Leu Leu1
51409PRTHomo sapiens 140Leu Asp Thr Asn Tyr Asn Leu Phe Tyr1
51419PRTHomo sapiens 141Glu Asp Leu Ser Lys Lys Thr Leu Leu1
51429PRTHomo sapiens 142Glu Asp Leu Ser Lys Lys Thr Leu Leu1
51439PRTHomo sapiens 143Leu His Tyr Lys Asp Gly Phe Asp Leu1
51449PRTHomo sapiens 144Leu His Tyr Lys Asp Gly Phe Asp Leu1
514510PRTHomo sapiens 145Asp His Ala Leu Gln Cys Val Asp Tyr Ile1 5
1014610PRTHomo sapiens 146Thr Arg Glu Ser Ser Cys Glu Ile Lys Leu1
5 101479PRTHomo sapiens 147Ile Arg Glu Asp Asp Thr Thr Leu Val1
51488PRTHomo sapiens 148Asp His Phe Lys Glu Cys Thr Val1
51498PRTHomo sapiens 149Ile Arg Glu Asp Asp Thr Thr Leu1
51509PRTHomo sapiens 150Leu Gln Trp Gln Pro Pro Leu Ser Leu1
515110PRTHomo sapiens 151Phe Lys Glu Cys Thr Val Glu Tyr Glu Leu1 5
1015210PRTHomo sapiens 152Trp Lys Thr Ile Ile Thr Lys Asn Glu Leu1
5 101539PRTHomo sapiens 153Trp Lys Pro Gly Ile Gly Val Leu Leu1
515410PRTHomo sapiens 154Phe Gln Leu Gln Asn Ile Val Lys Pro Leu1 5
101559PRTHomo sapiens 155Val Lys Pro Leu Pro Pro Val Tyr Leu1
51569PRTHomo sapiens 156Ile Lys Leu Lys Trp Ser Ile Pro Leu1
515710PRTHomo sapiens 157Leu Lys Thr Thr Asn Glu Thr Arg Gln Leu1 5
101589PRTHomo sapiens 158Lys Gln Cys Trp Glu Gly Glu Asp Leu1
515911PRTHomo sapiens 159Asp Lys Gln Cys Trp Glu Gly Glu Asp Leu
Tyr1 5 101609PRTHomo sapiens 160Phe Glu Ile Val Asp Pro Gly Tyr
Leu1 51619PRTHomo sapiens 161Lys Glu Cys Thr Val Glu Tyr Glu Leu1
51629PRTHomo sapiens 162Ile Glu Ala Lys Ile His Thr Leu Leu1
51639PRTHomo sapiens 163Arg Glu Ser Ser Cys Glu Ile Lys Leu1
516410PRTHomo sapiens 164Ile Glu Ile Arg Glu Asp Asp Thr Thr Leu1 5
101659PRTHomo sapiens 165Ser Glu Trp Ser Asp Lys Gln Cys Trp1
51669PRTHomo sapiens 166Gly Glu Asp Leu Ser Lys Lys Thr Leu1
516710PRTHomo sapiens 167Gln Asp Phe Glu Ile Val Asp Pro Gly Tyr1 5
101689PRTHomo sapiens 168Phe Glu Ile Val Asp Pro Gly Tyr Leu1
516910PRTHomo sapiens 169Val Asp Pro Gly Tyr Leu Gly Tyr Leu Tyr1 5
1017010PRTHomo sapiens 170Lys Thr Ile Ile Thr Lys Asn Leu His Tyr1
5 101719PRTHomo sapiens 171Gln Asn Ile Gly Cys Arg Phe Pro Tyr1
517210PRTHomo sapiens 172Leu Glu Ala Ser Asp Tyr Lys Asp Phe Tyr1 5
1017310PRTHomo sapiens 173Ser Glu Asn Lys Pro Ile Arg Ser Ser Tyr1
5 101749PRTHomo sapiens 174Cys Glu Ile Lys Leu Lys Trp Ser Ile1
517510PRTHomo sapiens 175Gly Pro Ile Pro Ala Arg Cys Phe Asp Tyr1 5
1017610PRTHomo sapiens 176Tyr Glu Ile Glu Ile Arg Glu Asp Asp Thr1
5 1017710PRTHomo sapiens 177Ile Glu Ile Arg Glu Asp Asp Thr Thr
Leu1 5 101789PRTHomo sapiens 178Ser Glu Trp Ser Asp Lys Gln Cys
Trp1 51799PRTHomo sapiens 179Asn Pro Pro Gln Asp Phe Glu Ile Val1
518010PRTHomo sapiens 180Asp Pro Gly Tyr Leu Gly Tyr Leu Tyr Leu1 5
101819PRTHomo sapiens 181Ile Gly Ser Glu Thr Trp Lys Thr Ile1
51829PRTHomo sapiens 182Asp Gly Phe Asp Leu Asn Lys Gly Ile1
518310PRTHomo sapiens 183Ser Pro Gln Gly Ile Pro Glu Thr Lys Val1 5
101849PRTHomo sapiens 184Ile Pro Glu Thr Lys Val Gln Asp Met1
518510PRTHomo sapiens 185Glu Gly Leu Asp His Ala Leu Gln Cys Val1 5
101869PRTHomo sapiens 186His Ala Leu Gln Cys Val Asp Tyr Ile1
518710PRTHomo sapiens 187Glu Ala Ser Asp Tyr Lys Asp Phe Tyr Ile1 5
101889PRTHomo sapiens 188Asn Gly Ser Ser Glu Asn Lys Pro Ile1
518910PRTHomo sapiens 189Ile Pro Ala Arg Cys Phe Asp Tyr Glu Ile1 5
101909PRTHomo sapiens 190Pro Ala Arg Cys Phe Asp Tyr Glu Ile1
519110PRTHomo sapiens 191Glu Gly Glu Asp Leu Ser Lys Lys Thr Leu1 5
101928PRTHomo sapiens 192Asn Pro Pro Gln Asp Phe Glu Ile1
51938PRTHomo sapiens 193Pro Pro Gln Asp Phe Glu Ile Val1
51948PRTHomo sapiens 194Asp Pro Gly Tyr Leu Gly Tyr Leu1
51958PRTHomo sapiens 195Glu Ala Lys Ile His Thr Leu Leu1
51968PRTHomo sapiens 196Trp Ala Glu Thr Thr Tyr Trp Ile1
51978PRTHomo sapiens 197Gln Gly Ile Pro Glu Thr Lys Val1
51988PRTHomo sapiens 198Lys Pro Gly Ile Gly Val Leu Leu1
51998PRTHomo sapiens 199Ile Gly Cys Arg Phe Pro Tyr Leu1
52008PRTHomo sapiens 200Lys Pro Leu Pro Pro Val Tyr Leu1
52019PRTHomo sapiens 201Asn Pro Pro Gln Asp Phe Glu Ile Val1
520210PRTHomo sapiens 202Asp Pro Gly Tyr Leu Gly Tyr Leu Tyr Leu1 5
102039PRTHomo sapiens 203Ile Gly Ser Glu Thr Trp Lys Thr Ile1
52049PRTHomo sapiens 204Asp Gly Phe Asp Leu Asn Lys Gly Ile1
520510PRTHomo sapiens 205Lys Gly Ile Glu Ala Lys Ile His Thr Leu1 5
102069PRTHomo sapiens 206Leu Pro Trp Gln Cys Thr Asn Gly Ser1
520710PRTHomo sapiens 207Ser Ser Trp Ala Glu Thr Thr Tyr Trp Ile1 5
102089PRTHomo sapiens 208Thr Tyr Trp Ile Ser Pro Gln Gly Ile1
520910PRTHomo sapiens 209Thr Thr Tyr Trp Ile Ser Pro Gln Gly Ile1 5
1021010PRTHomo sapiens 210Ser Pro Gln Gly Ile Pro Glu Thr Lys Val1
5 1021110PRTHomo sapiens 211Tyr Leu Leu Cys Ser Trp Lys Pro Gly
Ile1 5 1021210PRTHomo sapiens 212Glu Gly Leu Asp His Ala Leu Gln
Cys Val1 5 102139PRTHomo sapiens 213His Ala Leu Gln Cys Val Asp Tyr
Ile1 52149PRTHomo sapiens 214Phe Pro Tyr Leu Glu Ala Ser Asp Tyr1
521510PRTHomo sapiens 215Glu Ala Ser Asp Tyr Lys Asp Phe Tyr Ile1 5
102169PRTHomo sapiens 216Asn Gly Ser Ser Glu Asn Lys Pro Ile1
52179PRTHomo sapiens 217Lys Pro Ile Arg Ser Ser Tyr Phe Thr1
521810PRTHomo sapiens 218Ser Tyr Phe Thr Phe Gln Leu Gln Asn Ile1 5
102199PRTHomo sapiens 219Phe Thr Phe Gln Leu Gln Asn Ile Val1
52209PRTHomo sapiens 220Lys Pro Leu Pro Pro Val Tyr Leu Thr1
522110PRTHomo sapiens 221Ile Pro Leu Gly Pro Ile Pro Ala Arg Cys1 5
1022210PRTHomo sapiens 222Ile Pro Ala Arg Cys Phe Asp Tyr Glu Ile1
5 102239PRTHomo sapiens 223Arg Cys Phe Asp Tyr Glu Ile Glu Ile1
52249PRTHomo sapiens 224Phe Val Val Arg Ser Lys Val Asn Ile1
52258PRTHomo sapiens 225Leu Cys Phe Val Arg Ser Lys Val1
52269PRTHomo sapiens 226Asn Ile Tyr Cys Ser Asp Asp Gly Ile1
52278PRTHomo sapiens 227Asn Pro Pro Gln Asp Phe Glu Ile1
52288PRTHomo sapiens 228Pro Pro Gln Asp Phe Glu Ile Val1
52298PRTHomo sapiens 229Asp Pro Gly Tyr Leu Gly Tyr Leu1
52308PRTHomo sapiens 230Glu Ala Lys Ile His Thr Leu Leu1
52318PRTHomo sapiens 231Trp Ala Glu Thr Thr Tyr Trp Ile1
52328PRTHomo sapiens 232Tyr Trp Ile Ser Pro Gln Gly Ile1
52338PRTHomo sapiens 233Gln Gly Ile Pro Glu Thr Lys Val1
52348PRTHomo sapiens 234Lys Pro Gly Ile Gly Val Leu Leu1
52358PRTHomo sapiens 235Ile Gly Cys Arg Phe Pro Tyr Leu1
52368PRTHomo sapiens 236Phe Thr Phe Gln Leu Gln Asn Ile1
52378PRTHomo sapiens 237Lys Pro Leu Pro Pro Val Tyr Leu1
52388PRTHomo sapiens 238Ile Pro Leu Gly Pro Ile Pro Ala1
52399PRTHomo sapiens 239Asn Pro Pro Gln Asp Phe Glu Ile Val1
524010PRTHomo sapiens 240Ile Gly Ser Glu Thr Trp Lys Thr Ile Ile1 5
102419PRTHomo sapiens 241Asp Gly Phe Asp Leu Asn Lys Gly Ile1
524210PRTHomo sapiens 242Ser Pro Gln Gly Ile Pro Glu Thr Lys Val1 5
1024310PRTHomo sapiens 243Glu Gly Leu Asp His Ala Leu Gln Cys Val1
5 102449PRTHomo sapiens 244His Ala Leu Gln Cys Val Asp Tyr Ile1
524510PRTHomo sapiens 245Glu Ala Ser Asp Tyr Lys Asp Phe Tyr Ile1 5
102469PRTHomo sapiens 246Asn Gly Ser Ser Glu Asn Lys Pro Ile1
524710PRTHomo sapiens 247Ile Pro Ala Arg Cys Phe Asp Tyr Glu Ile1 5
102489PRTHomo sapiens 248Asn Pro Pro Gln Asp Phe Glu Ile Val1
52499PRTHomo sapiens 249Asn Pro Pro Gln Asp Phe Glu Ile Val1
525010PRTHomo sapiens 250Ile Gly Ser Glu Thr Trp Lys Thr Ile Ile1 5
102519PRTHomo sapiens 251Asp Gly Phe Asp Leu Asn Lys Gly Ile1
52529PRTHomo sapiens 252Phe Thr Phe Gln Leu Gln Asn Ile Val1
525310PRTHomo sapiens 253Lys Thr Ile Ile Thr Lys Asn Leu His Tyr1 5
102549PRTHomo sapiens 254Ser Ser Trp Ala Glu Thr Thr Tyr Trp1
525510PRTHomo sapiens 255Gln Ser Ser Trp Ala Glu Thr Thr Tyr Trp1 5
102569PRTHomo sapiens 256Asp Thr Asn Tyr Asn Leu Phe Tyr Trp1
525710PRTHomo sapiens 257Lys Pro Leu Pro Pro Val Tyr Leu Thr Phe1 5
102589PRTHomo sapiens 258Ser Ser Cys Glu Ile Lys Leu Lys Trp1
52599PRTHomo sapiens 259Ser Ser Cys Glu Ile Lys Leu Lys Trp1
526010PRTHomo sapiens 260Thr Thr Asn Glu Thr Arg Gln Leu Cys Phe1 5
1026110PRTHomo sapiens 261Cys Ser Asp Asp Gly Ile Trp Ser Glu Trp1
5 1026210PRTHomo sapiens 262Trp Ser Glu Trp Ser Asp Lys Gln Cys
Trp1 5 102639PRTHomo sapiens 263Phe Glu Ile Val Asp Pro Gly Tyr
Leu1 52649PRTHomo sapiens 264Val Asp Pro Gly Tyr Leu Gly Tyr Leu1
52659PRTHomo sapiens 265Lys Glu Cys Thr Val Glu Tyr Glu Leu1
52669PRTHomo sapiens 266Ile Glu Ala Lys Ile His Thr Leu Leu1
52679PRTHomo sapiens 267Arg Glu Ser Ser Cys Glu Ile Lys Leu1
526810PRTHomo sapiens 268Ile Glu Ile Arg Glu Asp Asp Thr Thr Leu1 5
102699PRTHomo sapiens 269Gly Glu Asp Leu Ser Lys Lys Thr Leu1
52709PRTHomo sapiens 270Glu Asp Leu Ser Lys Lys Thr Leu Leu1
527110PRTHomo sapiens 271Arg Glu Asp Asp Thr Thr Leu Val Thr Ala1 5
102729PRTHomo sapiens 272Asn Glu Thr Arg Gln Leu Cys Phe Val1
52738PRTHomo sapiens 273Ser Glu Val Gln Ser Ser Trp Ala1
52748PRTHomo sapiens 274Arg Glu Asp Asp Thr Thr Leu Val1
52759PRTHomo sapiens 275Phe Glu Ile Val Asp Pro Gly Tyr Leu1
52769PRTHomo sapiens 276Leu Tyr Leu Gln Trp Gln Pro Pro Leu1
527710PRTHomo sapiens 277Tyr Leu Tyr Leu Gln Trp Gln Pro Pro Leu1 5
1027810PRTHomo sapiens 278Val Glu Tyr Glu Leu Lys Tyr Arg Asn Ile1
5 102799PRTHomo sapiens 279Leu His Tyr Lys Asp Gly Phe Asp Leu1
528010PRTHomo sapiens 280Lys Gly Ile Glu Ala Lys Ile His Thr Leu1 5
102819PRTHomo sapiens 281Cys Val Tyr Tyr Asn Trp Gln Tyr Leu1
528210PRTHomo sapiens 282Asp Cys Val Tyr Tyr Asn Trp Gln Tyr Leu1 5
102839PRTHomo sapiens 283Val Tyr Tyr Asn Trp Gln Tyr Leu Leu1
528410PRTHomo sapiens 284Val Leu Leu Asp Thr Asn Tyr Asn Leu Phe1 5
1028510PRTHomo sapiens 285Gly Val Leu Leu Asp Thr Asn Tyr Asn Leu1
5
1028610PRTHomo sapiens 286Tyr Asn Leu Phe Tyr Trp Tyr Glu Gly Leu1
5 102879PRTHomo sapiens 287Asn Leu Phe Tyr Trp Tyr Glu Gly Leu1
528810PRTHomo sapiens 288Gln Asn Ile Gly Cys Arg Phe Pro Tyr Leu1 5
1028910PRTHomo sapiens 289Lys Pro Ile Arg Ser Ser Tyr Phe Thr Phe1
5 1029010PRTHomo sapiens 290Phe Gln Leu Gln Asn Ile Val Lys Pro
Leu1 5 1029110PRTHomo sapiens 291Lys Pro Leu Pro Pro Val Tyr Leu
Thr Phe1 5 102929PRTHomo sapiens 292Ile Lys Leu Lys Trp Ser Ile Pro
Leu1 529310PRTHomo sapiens 293Ala Thr Val Glu Asn Glu Thr Tyr Thr
Leu1 5 1029410PRTHomo sapiens 294Asp Phe Glu Ile Val Asp Pro Gly
Tyr Leu1 5 1029510PRTHomo sapiens 295Asp Pro Gly Tyr Leu Gly Tyr
Leu Tyr Leu1 5 102969PRTHomo sapiens 296Leu Tyr Leu Gln Trp Gln Pro
Pro Leu1 52979PRTHomo sapiens 297Gln Pro Pro Leu Ser Leu Asp His
Phe1 52989PRTHomo sapiens 298His Phe Lys Glu Cys Thr Val Glu Tyr1
52999PRTHomo sapiens 299Glu Tyr Glu Leu Lys Tyr Arg Asn Ile1
530011PRTHomo sapiens 300Thr Trp Lys Lys Thr Ile Ile Thr Lys Asn
Leu1 5 103019PRTHomo sapiens 301Thr Tyr Trp Ile Ser Pro Gln Gly
Ile1 53029PRTHomo sapiens 302Ile Pro Glu Thr Lys Val Gln Asp Met1
53039PRTHomo sapiens 303Val Tyr Tyr Asn Trp Gln Tyr Leu Leu1
53049PRTHomo sapiens 304Ser Trp Lys Pro Gly Ile Gly Val Leu1
53059PRTHomo sapiens 305Trp Tyr Glu Gly Leu Asp His Ala Leu1
53069PRTHomo sapiens 306Trp Tyr Glu Gly Leu Asp His Ala Leu1
530710PRTHomo sapiens 307Asp Tyr Ile Lys Ala Asp Gly Gln Asn Ile1 5
1030810PRTHomo sapiens 308Arg Phe Pro Tyr Leu Glu Ala Ser Asp Tyr1
5 103099PRTHomo sapiens 309Asp Tyr Lys Asp Phe Tyr Ile Cys Val1
531010PRTHomo sapiens 310Lys Pro Ile Arg Ser Ser Tyr Phe Thr Phe1 5
103119PRTHomo sapiens 311Tyr Phe Thr Phe Gln Leu Gln Asn Ile1
531210PRTHomo sapiens 312Ser Tyr Phe Thr Phe Gln Leu Gln Asn Ile1 5
1031310PRTHomo sapiens 313Lys Pro Leu Pro Pro Val Tyr Leu Thr Phe1
5 1031410PRTHomo sapiens 314Thr Phe Thr Arg Glu Ser Ser Cys Glu
Ile1 5 1031510PRTHomo sapiens 315Cys Phe Val Val Arg Ser Lys Val
Asn Ile1 5 103169PRTHomo sapiens 316Asp Pro Gly Tyr Leu Gly Tyr Leu
Tyr1 53179PRTHomo sapiens 317Asp Pro Gly Tyr Leu Gly Tyr Leu Tyr1
5
* * * * *
References