CA2398136A1 - Immunotherapy using interleukin 13 receptor subunit alpha 2 - Google Patents

Abstract

A method for stimulating an 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 a subject. A compostion for stimulating an 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.

Description

CROSS REFERENCE TO RELATED APPLICATION
The present application claims the benefit of U.S. provisional application serial number 60/181,000 filed February 8, 2000.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made with Government support under grant number CA74154 awarded by the National Cancer Institute of the National Institutes of Health.
The Government may have certain rights in the invention.
FIELD OF THE INVENTION
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-13Ra2) as an immune system modulator and target for vaccines for the treatment and prevention of cancer.
BACKGROUND
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.
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 Immunolo~y: 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.
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-l, glycoprotein 100 (gp100), tyrosinase, BAGS, and GAGE. Reviewed by Maeurer et al., supra.
One of the most exciting recent findings in cancer immunology came after the SERER (for serological analysis of recombinant cDNA expression libraries) technique was developed.
Sahin et al., Proc. Natl. Acad. Sci. USA, 92: 11810-11813, 1995. The SERER
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, SERER 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.
In addition, SERER 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-l, SCPl and CT7) have unequivocally been shown to stimulate an immune response in a subject.

SUMMARY
The invention relates to the discovery that IL-13Ra2 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-13Ra2.
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-13Ra2 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-13Ra2, 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-13Ra2 serves as a selective target for HGG and other cancers that express IL-13Ra2 because, as described in more detail below, with the exception of testis, normal human tissue expresses little or no IL-13Ra2.
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-13Ra2 (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-13Ra1) and a 140 kDa component (termed IL-4Ra).
As indicated above, our tissue distributions studies showed that, among normal tissues, IL-13Ra2 is strongly expressed only in testis. This finding along with the showing that (a) IL-13Ra2 is preferentially over-expressed on HGG but not normal central nervous system (CNS) tissue and (b) that the IL-13Ra2 gene is localized to chromosome X, indicates that IL-13Ra2 is a CTA. Because other CTAs have proven to stimulate a strong immune response against cancer cells, the present invention provides methods and compositions useful for generating or increasing an anti-cancer immune response in a subject.
Accordingly, in one aspect the invention features a method for stimulating a immune response against IL-13Ra2 in a subject having or at risk for developing a disease having cells expressing IL-13Ra2. 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-13Ra2 when administered to an animal; and (b) administering the vaccine to the subject in an amount sufficient to stimulate an immune response against IL-13Ra2 in the subject.
In another aspect the invention features a composition for stimulating an immune response against IL-13Ra2 when administered to an animal. The composition includes:
(a) an isolated agent that can stimulate an immune response against IL-13Ra2 when administered to an animal; and (b) a pharmaceutically acceptable carrier.
In both of the foregoing method and composition, the agent that can stimulate an immune response against IL-13Ra2 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-13Ra2 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.
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.
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-13Ra2 i (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-13Ra2 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.
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.
S Definitions of molecular biology terms can be found, for example, in Rieger et al., Glossary of Genetics: Classical and Molecular, Sth 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.
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 1 S 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.
When referring to a nucleic acid molecule or polypeptide, the term "native"
refers to a 2S naturally-occurring (e.g., a "wild-type") nucleic acid or polypeptide. A
"homolog" of an IL-13Ra2 gene is a gene sequence encoding an IL-13Ra2 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.
By the terms "IL-13Ra2 gene" or "IL-13Ra2 polynucleotide" is meant a native IL-13Ra2 encoding nucleic acid sequence (e.g., the IL-13Ra2 cDNA sequence shown as SEQ
ID NO: 2 (Fig. 2)), genomic sequences from which IL-13Ra2 cDNA can be transcribed, and/or allelic variants and homologs of the foregoing.
S

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.
By "IL-13Ra2 protein" "IL-13Ra2 polypeptide," or simply "IL-13Ra2" is meant an expression product of an IL-13Ra2 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:l and cross-reacts with antibodies that specifically bind the protein of SEQ ID NO:1.
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, WI 53705).
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.
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.
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).
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 105 to 106 literslmole for that second molecule.
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).

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
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:
Fig. 1 is the amino acid sequence of the native H. Sapiens IL-13Ra2 protein.
Fig. 2 is the nucleic acid sequence of a cDNA corresponding to a native mRNA
encoding the native H. Sapiens IL-13Ra2 protein.
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 a-chain. B, 45-kDa IL13R al-chain; A and B
constitute the elements of the heterodimeric high affinity IL13/4R. C, a 42-kDa monomer of IL13Ra2.
Fig. 4 is a Northern blot analysis of human IL13Ra2 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.
Fig. 5 is a Northern blot analysis of human IL13Ra2 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.
Fig. 6 is a Northern blot analysis of human 140-kDa IL4R a-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.
Fig. 7 is a Northern blot analysis of human ~i-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.

Fig. 8 is a Northern blot analysis of transcripts of different IL 13 receptors in malignant glioma cells (G-48, A-172 MG, U-373 MG, and U-251 MG), normal human umbilical vein endothelial cells (HIJVEC) and in surgical specimens of GBM and normal human brain. The migration position of mRNA is shown in kilobases. Films were exposed for 2 weeks, except for actin (1 hr).
DETAILED DESCRIPTION
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-l3Ra,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 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-13Ra2 as a Cancer/Testis Antigen As its name implies, IL-13Ra2 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-13Ra1 (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-l3Ral, the shared receptor also includes a protein referred to as p140 (or IL-4Ra), the subcomponent responsible for IL-4 binding.
Idzerda et al., J. Exp. Med., 171: 861-873, 1990; Hilton et al., Proc. Natl.
Acad. Sci. USA, 93: 497-501, 1996; Debinski et al., Nature Biotech., 16: 449-453, 1995;
Zurawski et al., EMBO J., 12: 2663-2670, 1993; Minty et al., Nature, 36: 248-251, 1993.
Exposing cells to IL-13 results in responses very similar to those responses that occur after exposure to IL-4.
Zurawski, G., and J.E. de Vries, Stem Cells. 12: 169-174, 1994. Examples of cellular responses resulting from both IL-13 and IL-4 exposure include enhanced expression of CD72, IgM, and MHC class II antigen, as well as induced CD23 expression and IgE
heavy-chain gene production in B lymphocytes. Id.
In an interesting development, it was found that IL-13Ra1 was not the only IL-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-13Ra1/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-13Ra2, was subsequently cloned and sequenced. Id. This novel IL-13 binding protein, referred to herein as IL-13Ra2, 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 axe shown in Fig.

3.
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-13Ra2 in order to characterize the normal tissue expression pattern of this new receptor in detail. From these studies, we discovered that IL-13Ra2 expression is virtually absent in all normal adult tissue except testis. In earlier studies, the gene encoding IL-13Ra2 was localized to the X chromosome. Guo et al., Genomics, 42: 141-145, 1997.
Accordingly, our discovery allowed us to characterize the IL-13Ra2 protein as a member of the CTA group of tumor antigens. Moreover, because IL-13Ra2 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-13Ra2 on their surface. This discovery that IL-13Ra2 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 The invention provides vaccines that can stimulate an immune response against IL-13Ra2 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-13Ra2 when introduced into a subject. Typical immune responses include (a) the production of, or increase in titer of, antibodies that specifically bind IL-13Ra2 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 The antigenic agent for use in the vaccines of the invention can take the form of the native IL-13Ra2 (SEQ ID NO:1) or a peptide fragment of IL-13Ra2. 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 ~-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-13Ra2.
As indicated above, vaccines with in the invention can include an IL-13Ra2 protein as an antigenic agent. Preferred forms of IL-13Ra2 protein include a purified native IL-13Ra2 protein that has the amino acid sequence shown in Fig. 1 (SEQ ID NO:1 ).
Variants of the native IL-13Ra2 protein such as fragments, analogs and derivatives of native IL-13Ra2 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-13Ra2 gene, a polypeptide encoded by a homolog of the native IL-13Ra2 gene, and a polypeptide encoded by a non-naturally occurring variant of the native IL-13Ra2 gene.
Preferred versions of such variants are those that are able to stimulate an immune response to native IL-13Ra2 upon administration to a subject as part of a vaccine.
IL-13Ra2 protein variants have a peptide sequence that differs from the native IL-13Ra2 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-13Ra2 polypeptide. Amino acid insertions are preferably of about 1 to 4 contiguous amino acids, ZO and deletions are preferably of about 1 to 10 contiguous amino acids. In some applications, variant IL-13Ra2 proteins substantially maintain a native IL-13Ra2 protein functional activity (e.g., the ability to specifically bind IL-13). For other applications, variant IL-13Ra2 proteins lack or feature a significant reduction in an IL-13Ra2 protein functional activity.
Where it is desired to retain a functional activity of native IL-13Ra2 protein, preferred IL-?5 13Ra2 protein variants can be made by expressing nucleic acid molecules within the invention that feature silent or conservative changes. Variant IL-13Ra2 proteins with substantial changes in functional activity can be made by expressing nucleic acid molecules within the invention that feature less than conservative changes.
IL-13Ra2 protein fragments corresponding to one or more particular motifs (e.g., 30 ~ 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-13Ra2 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-13Ra2 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. Patent 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 subjects) for the stimulation of an immune response. Those peptide fragments that induce the desired response can then be selected.
Isolated peptidyl portions of IL-13Ra2 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-13Ra2 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-13Ra2 protein.
Another aspect of the present invention concerns recombinant forms of the IL-13Ra2 proteins. Recombinant polypeptides preferred for use in the present invention, in addition to native IL-13Ra2 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 N0:2. In a preferred embodiment, variant IL-13Ra2 have the ability to stimulate an immune response against the native IL-13Ra2 protein.
IL-13Ra2 protein variants can be generated through various techniques known in the art. For example, IL-13Ra2 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-13Ra2 protein variant having more, substantially the same, or merely a subset of the antigenic activity of the native IL-13Ra2 protein. Other variants of IL-13Ra2 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-13Ra2 protein variant having greater or lesser antigenic activity than native IL-13Ra2 protein can be readily determined by comparing the variant with the native IL-13Ra2 protein for the ability to stimulate an immune response against IL-13Ra2 in subjects vaccinated with the respective proteins.
, As another example, IL-13Ra2 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-13Ra2 protein sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al.
(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.
(1983) Nucleic Acid Res. 11:477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390;
Roberts et al. (1992) Proc. Natl. Acad. Sci. USA 89:2429-2433; Devlin et al.
(1990) Science 249: 404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382; as well as U.S: Pat. Nos. 5,223,409; 5,198,346; and 5,096,815).
Similarly, a library of coding sequence fragments can be provided for an IL-13Ra2 gene clone in order to generate a variegated population of IL-13Ra2 protein fragments for screening and subsequent selection of fragments having the ability to stimulate an immune response against IL-13Ra2 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-13Ra2 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 S 1 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-13Ra2 proteins to generate mimetics, e.g. peptide or non-peptide agents, that are able to stimulate an immune response against IL-13Ra2 in a subject. For instance, non-hydrolyzable peptide analogs of the amino acid residues of IL-13Ra2 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, III, 1985), beta-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647;
and Sato et al. '(1986) J. Chem. Soc. Perkin. Trans. 1:1231), and b-aminoalcohols (Gordon et al. (1985) Biochem. Biophys. Res. Commun. 126:419; and Dann et al. (1986) Biochem. Biophys. Res. Commun. 134:71). IL-13Ra2 proteins may also be chemically modified to create IL-13Ra2 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-13Ra2 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.
IL-13Ra2 proteins may also be fused to one or more other proteins. For example, an IL-13Ra2 protein or immunogenic portion thereof may be fused to another protein that serves as a targeting ligand to deliver the IL-13Ra2 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-13Ra2 protein or peptide can be fused to a mutant IL-13 molecule or anti-IL-13 receptor antibody to specifically target the IL-13Ra2 protein or peptide to a tumor, e.g., a HGG.
Numerous methods of fusing two or more proteins together are known in the art, e.g., making and expressing a recombinant fusion construct, or using a cross-linking agent to covalently bond the two or more proteins together to form one molecule. Any suitable for this application might be used in the invention.
The IL-13Ra2 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-13Ra2 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.
For example, after an IL-13Ra2 protein or peptide has been expressed in a cell, it can be isolated using immuno-affinity chromatography. For instance, an anti-IL-13Ra2 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-13Ra2 proteins or peptides utilized in the invention are expressed as a fusion protein containing an affinity tag (e.g., GST) that facilitates its purification.
In association with an antigenic agent (e.g., a IL-13Ra2 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 RibiTM adjuvant system (Corixa, Seattle, Washington) (3) aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc); (4) saponin-based adjuvants (StimulonTM from Aquila Biosciences, Framingham, Massachusetts); (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.

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 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 proteinlpeptide 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-13Ra2 when administered to a subject. Examples of such nucleic acids include those that encode the native IL-13Ra2 such as the nucleic acid shown herein as SEQ ID
N0:2 (Fig. 2), a variant of the native IL-13Ra2, or a peptide fragment of that native or variant IL-13Ra2.
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.
The form of the nucleic acid used in a vaccine of the invention can be any suitable for stimulating an immune response against IL-13Ra2 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 subjects) for the stimulation of an immune response. Those nucleic acids that induce the desired response can then be selected.
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-13Ra2 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 ).
Other nucleic acid molecules useful in the invention are variants of the native IL-13Ra2 gene such as those that encode fragments (e.g., post-translationally processed forms of), analogs and derivatives of a native IL-13Ra2 protein. Such variants may be, e.g., a naturally occurring allelic variant of the native IL-13Ra2 gene, a homolog of the native IL-13Ra2 gene, or a non-naturally occurring variant of the native IL-13Ra2 gene.
These variants have a nucleotide sequence that differs from the native IL-13Ra2 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-13Ra2 gene. Nucleic acid insertions are preferably of about 1 to 10 contiguous nucleotides, and deletions are preferably of about 1 to 30 contiguous nucleotides.
Naturally occurring allelic variants of the native IL-13Ra2 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-13Ra2 gene, and ZS encode polypeptides having structural similarity'to native IL-13Ra2 protein. Homologs of the native IL-13Ra2 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-13Ra2 gene, and encode polypeptides having structural similarity to native IL-13Ra2 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-13Ra2 gene.

Non-naturally occurring IL-13Ra2 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-13Ra2 gene, and encode polypeptides having structural similarity to native IL-13Ra2 protein. Examples of non-naturally occurring IL-13Ra2 gene variants are those that encode a fragment of a IL-13Ra2 protein, those that hybridize to the native IL-13Ra2 gene or a complement of to the native IL-13Ra2 gene under stringent conditions, those that share at least 65% sequence identity with the native IL-13Ra2 gene or a complement of the native IL-13Ra2 gene, and those that encode a IL-13Ra2 fusion protein.
Nucleic acids encoding fragments of native IL-13Ra2 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-13Ra2 protein. Shorter oligonucleotides (e.g., those of 6, 7, 8, 9, 10, 1 l, 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-13Ra2 protein can be used. Nucleic acids encoding fragments of native IL-13Ra2 protein can be made by enzymatic digestion (e.g., using a restriction enzyme) or chemical degradation of the full length native IL-13Ra2 gene or variants thereof.
Nucleic acid molecules encoding IL-13Ra2 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-13Ra2 fusion protein when introduced into a suitable host. For example, such a construct can be made by ligating a first polynucleotide encoding an IL-13Ra2 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-13Ra2 fusion proteins can be used, e.g., to enhance the immunogenicity of IL-13Ra2 peptides, to facilitate purification of IL-13Ra2 proteins/peptides, or to track the location of the IL-13Ra2 fusion protein after it has been administered to a subject.
Using the nucleotide sequence of the native IL-13Ra2 gene arid the amino acid sequence of a native IL-13Ra2 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-13Ra2 nucleic acid molecules can be expressed to produce variant IL-13Ra2 proteins.
Naked Nucleic Acid Vaccines The invention provides for the use of naked nucleic acid vaccines to stimulate an immune response against IL-13Ra2. Representative naked nucleic acid vaccines for use in this method include a DNA encoding one or more immunogenic portions of IL-13Ra2 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-13Ra2. Naked nucleic acid vaccines can be administered to a subject by any suitable technique. For example, naked DNA
encoding a peptide portion of IL-13Ra2 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 The invention also provides for the use of expression vector vaccines to stimulate an immune response against IL-13Ra2. In a typical application of this technique, a nucleic acid encoding one or more peptide or protein antigens of IL-13Ra2 is incorporated into a vector that allows expression of the antigens) in a host cell (e.g., a cell inside a subject or administered to a subject). The nucleic acid encoding the antigens) 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 antigens) encoded by the vector are expressed when the vector is introduced into a host cell in a subject. After expression, the antigens) 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).

Vectors for use in the invention can be any capable of expressing an encoded antigens) 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 Cell-based vaccines are provided in the invention to stimulate an immune response against IL-13Ra2. 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.
The invention further provides an isolated cell expressing IL-13Ra2 or a peptide fragment of IL-13Ra2. Cells expressing IL-13Ra2 can be isolated from a subject having such cells (e.g., from testis or HGG). Cells that do not express IL-13Ra2 can be made to express this protein in a number of different ways. As one example, cells can be cultured with IL-13Ra2 or peptide fragments thereof under conditions in which fragments of IL-13Ra2 become associated with MHC molecules on the cell surface. Alternatively, cells can be made to express IL-13Ra2 by introducing a nucleic acid encoding an IL-13Ra2 protein, a peptide fragment of IL-13Ra2, 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 pz'otein, peptide, or variant in lysates of a cell.

Cell types suitable for stimulating an immune response against IL-13Ra2 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.
Further examples of cells that can be used to stimulate an immune response against IL-13Ra2 include those that express a peptide comprising a least.? (e.g., 7, 8, 9, 10, 1 l, 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: l might also be used.
Although any cell that can express IL-13Ra2 protein, a peptide fragment of IL-13Ra2, 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.
Since DC are known to function as particularly strong APCs, their use in the cell-based vaccine of the invention is particularly preferred. See, e.g., Banchereau et al., Ann.
Rev. Immunology, 18:767, 2000. DC can be made to express an IL-13Ra2 protein, a peptide fragment of IL-13Ra2, or a variant thereof as described above. For example, DC
can be removed from a subject, contacted with the selected antigen, and then returned to the subject to stimulate an immune response. To enhance their antigen presentation capability, the DC
can also be treated with an activating substance such as a cytokines.
Those cell-based vaccines that are most effective in stimulating an immune response against IL-13Ra2 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 subjects) for the stimulation of an immune response. Those vaccines that induce the desired response can then be selected.
Anti-idiotypic Antibody Vaccines The invention also contemplates the use of anti-idiotypic antibody vaccines to stimulate an immune response against IL-13Ra2 in a subject. In this method, anti-idiotypic antibodies are prepared that feature an internal "image" of one or more immunogenic,portions of IL-13Ra2. See, e.g., U.S. Patent 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-13Ra2 which cross-reacts against actual immunogenic portions of IL-13Ra2. As one example, polyclonal anti-idiotypic antibodies can be generated by immunizing a host animal with monoclonal antibodies raised against an epitope of IL-13Ra2. Methods of preparing monoclonal and polyclonal antibodies as described in more detail below.
Antibody Production 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')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-13Ra2 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.
Human or humanoid.antibodies that specifically bind a TL-13Ra2 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-13Ra2. As another example, human antibodies against IL-13Ra2 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 US Patent 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-13Ra2 can be made from non-human antibodies by adapting known methods such as those described in U.S. Patent Nos. 5,530, 101;
5,585,089;
5,693,761; and 5,693,762.
Once produced, polyclonal or monoclonal antibodies can be tested for specific IL-13Ra2 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-13Ra2 are useful in the invention. For example, such antibodies can be used in an immunoassay to monitor the level of IL-13Ra2 in a sample (e.g., to determine the amount of cellular expression or subcellular location of IL-13Ra2, or the presence and amount of soluble forms of IL-13Ra2 in a liquid sample).
Preferably, IL-13Ra2 protein selective antibodies of the invention are produced using fragments of the IL-13Ra2 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-13Ra2 protein antibodies are produced using a fragment of a IL-13Ra2 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.
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-13Ra2 proteins or control proteins, such as glucocorticoid receptor, CAT, or luciferase.
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-13Ra2 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.
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')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')2 fragments.
Alternatively, Fab expression libraries can be constructed (Ruse 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-13Ra2 Immune Response in a Subject The invention provides methods for stimulating a immune response against IL-13Ra2 in a subject having or at risk for developing a cancer having cells expressing IL-13Ra2. 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-13Ra2 in the subj ect.

Subj ects 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-13Ra2 (e.g., a brain cancer such as HGG) is a particularly preferred subject.
IL-13Ra2 as a Component of a Polyvalent Vaccine The invention also provides polyvalent vaccines that incorporate one or more of the foregoing compositions that can stimulate an immune response against IL-13Ra2 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-13Ra2 (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-13Ra2 and (b) a different agent that can stimulate an immune response against a molecule other than IL-13Ra2 (e.g., another TSA or TAA).
Administering Vaccines to a Subject The vaccine compositions of the present invention can be used in a method for stimulating an immune response against IL-13Ra2 in a subject. In this method, an vaccine compositon of the invention can be administered to a subject by any method that stimulates the aforesaid immune response. The exact method selected is determined by the particular vaccine composition to administered. For parenteral administration by injection, the injection can be in situ (i.e., to a particular tissue or location on a tissue, e.g., into a tumor or lymph node), intramuscular, intravenous, intraperitoneal, or by another parenteral route. For example, for a protein/peptide based vaccine the vaccine may be administered by subcutaneous or intradermal injection. In some cases other routes can be used, e.g.
intravenous injection, intraperitoneal injection, or in situ injection into target tissue.
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.
Formulations for injection may be presented in unit dosage form, for example, in ampoules or in mufti-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.
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. LTSP grade carriers and excipients are particularly preferred for delivery of ,0 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 ~5 implantation) or by injection. Methods for making the foregoing formulations are well known and can be found in, for example, ReminQton's Pharmaceutical Sciences.
Dosing The vaccine compositions of the invention are preferably administered to a subject in an amount sufficient to stimulate an immune response against IL-13Ra2 in the subject, and not cause an overly toxic effect. Such a therapeutically effective amount can be determined as described below.
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 LDSO (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDSO/EDSO. 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 EDSO with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
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 70 administered to a subject at least 24 hours before a second (booster) dose is administered to the subject.
Kits The invention also provides kits for stimulating an immune response against IL-13Ra2 in a subject. Such kits can include a container holding one or more of the antigenic ,5 agents described above in a pharmaceutically acceptable form. The antigenic agents) 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 30 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-13Ra2 Mimics the Biological Features of an HGG-associated receptor for Normal Chinese hamster ovary (CHO) cells were transfected with a pcDNA 3.1 plasmid (Invitrogen) containing the full length open reading frame of IL-13Ra2 and positive clones were selected with geneticin. The expression of IL-13Ra2 in these clones was tested for their ability to bind Izsl_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-l3Ra 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-13Ra2 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-13Ra2 as a Cancer Testis Antigen Materials and Methods 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, CO).
2 ~,g of Poly (A)+ RNA was electrophoresed on a 1% agarose formaldehyde gel, transferred to 0.45 ~,m magna charge nylon (MSI, Westborough, MA) and UV-crosslinked (Stratagene, La Jolla, CA). RNA-blotted membranes were also purchased from Clontech (Palo Alto, CA).
Two Multiple Tissue Expression (MTETM) Blots (cat # 7770-1 and 7775-l;
www.clontech.com/mtn/index.html) were analyzed to determine the tissue distribution of the IL13 binding proteins. Two sets of Human Brain Multiple Tissue Northern (MTNTM) Blots l0 (cat # 7755-1 and 7769-1) were assayed to confirm the true presence of the transcripts. In addition, two Human Tissue Northern (MTNTM) Blots (cat # 7759-1 and 7760-1) were analyzed to verify the tissue distribution of the IL-13Ra2 transcript.
cDNA Probes. cDNA probes were generated either by PCR (IL-13Ra2 and ILl3Ral) or by restriction digest (IL-4Ra = p 140). cDNA containing human IL 13Ra2 was provided by L 5 Dr. Pascual Ferrara of Sanofi Recherche. cDNA containing human IL 13Ra 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 IL4Ra 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, CA).
?0 Actin cDNA was purchased from Clontech Labs.
The primers for human IL-13Ra2 were as follows:
forward 5' - AAGATTTGGAAGCTTATGGCTTTCGTTTGC - 3' (SEQ ID N0:3) reverse 5' - TCCCTCGAAGCTTCATGTATCACAGAAAAA - 3' (SEQ ID N0:4) The primers for human IL13Ra1 were as follows:
?5 forward 5' - ATTATTAAGCTTATGGAGTGGCCGGCG - 3' (SEQ ID NO:S) reverse 5' - TAACCGGAAGCTTCACTGAGAGGCTTT - 3' (SEQ ID N0:6) Northern Blot Analysis. Membranes were pre-hybridized overnight at 42°
C in a solution consisting of 50% formamide, Sx SSC, 50 mM sodium phosphate, Sx Denhardt's, 50 ~.g/ml sheared salmon sperm DNA, and 1% SDS. Membranes were subsequently hybridized overnight at 42° C in the same solution with the addition of full length cDNA probes labeled by random priming (Life Technologies, Rockville, MD) with 32P-dCTP using 1-2x cpm/ml. Following hybridization, the membranes were washed with 2x SSCl0.2%
SDS at 42° C for 20 minutes followed by two washes with lx SSC/0.1% SDS at 42° C for 20 minutes each. The membranes were exposed to autoradioraphic film X-GMAT AR (Eastman Kodak Co., Rochester, NY) and placed at -80° C for l, 3 and 14 days. The membranes were subsequently stripped and re-probed up to three more times. The membranes were probed first with IL-13Ra2, followed by ILl3Ral, IL-4Ra= p140, and actin. Films were scanned on a transparency scanner at a pixel size of 88x88 micron (Molecular Dynamics, Sunnyvale, CA). The images were compiled in Paint Shop Pro V 5.0 (Jasc software Inc., Eden Prairie, MN).
Results Northern blot analysis of transcripts for IL-13Ra2 in normal organs. To explore the expression of IL-13Ra2, 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-13Ra2, IL13Ra1, IL4a and ~i-actin. This assured that the levels of transcripts for IL-13Ra2 were not underestimated due to the usage of membranes with mRNA. Both the dot-blot analyses (not shown) and the Z0 electrophoretically separated transcripts for IL-13Ra2 (Fig. 4, panels I-IV) demonstrated mostly undetectable, or very weak signals in few cases, of IL-13Ra2 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-13Ra2 cDNA
probe to transcripts derived from testis. This was also found using another Northern blot membrane.
?5 A few other organs had transcripts that hybridized to the IL-13Ra2 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-13Ra2 expression in the CNS.
To confirm these findings made using dot blot analysis, additional blots were 30 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-13Ra2. No transcripts for IL-I3Ra2 were readily detected in the CNS.
Northern blot analysis of transcripts for IL13Ra1 in normal tissues. The expression of IL13Ra1, 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. S, panels I-IV).
LO The results unequivocally demonstrated that IL13Ra1 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 l 5 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 IL13Ra1 (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).
?0 Gene expression analysis of IL4Ra in normal tissues. In addition to ILl3Ral, IL4Ra 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 IL4Ra gene expression corresponded to that of IL13Ra1 was analyzed. All Northern blot analysis membranes used in this study demonstrated enriched content of the IL4Ra transcripts in a variety of tissues ?5 (Fig. 6, panels I, II, and IV). The presence of the transcripts within the CNS was most evident, as it was for IL13Ra1, 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 IL4Ra, which was generally similar to that seen with IL l3Ra 1 (not shown). Thus, discrete regions of normal human brain contain i0 transcripts for both ILl3Ral and IL4Ra, a complete heterodimer of the shared IL1314 receptor. Furthermore, several vital peripheral organs contained the two subunits of the IL13/4 receptor, including heart, liver, lung and intestinal tract.

Control hybridization of ~3-actin. All membranes used for Northern blot analysis of IL13 receptors transcripts were also hybridized with a cDNA probe for a house-keeping gene, ~3-actin (Fig. 7; dot' blots and panel III not shown). The intensity of the signals for ~3-actin was usually in accordance with the amount of mRNA present on the membranes, as estimated by the manufacturer.
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-13Ra2, ILl3Ral, IL4Ra and ~i-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 LO MG, U-373 MG, and U-251 MG) demonstrated intense signals for IL-13Ra2 (Fig.
8). On the other hand, the transcripts for the elements of the shared IL13/4 receptor, IL-l3Ral and IL4Ra, were found at lower levels when compared with that for IL-13Ra2 (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-13Ra2 l5 and IL-l3Ral were seen in cells (Fig. 8). In a control assay, human umbilical vein endothelial cells (HUVEC) showed the presence of transcripts for IL-l3Ra 1 and IL4Ra, but not those for IL-13Ra2 (Fig. 8). In summary, gene expression of IL-13Ra2 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-13Ra1 were found in all ?0 ofthese specimens. In other experiments, several additional HGG brain tumor specimens were determined to express IL-13Ra2.
Example 3- Representative Immunogenic Peptides of IL-13Ra2 Table I presents a list of IL-13Ra2 peptides that might be used to stimulate an immune response against IL-13Ra2 in a subject. The listed peptides were obtained using a ?5 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 30 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 I
Binding peptides prediction:
* = high stringency Allele Peptide Position Score t1/2 A1 IVDP-GYLGY 16 - 24 7.120 1236.45043346563 1 A I 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 - 4.020 55.7011058267956 A 0201 YLLCSWKPGI * 128 - 5.190 179.468552931832 A 0201 VLLD-TNYNL * 139 - 6.320 555.572992451403 A 0201 NLFY-WYEGL * 146 - 4.080 59.1454698498823 A 0201 GLDH-ALQCV * 153 - 4.160 64.0715225999366 A 0201 NIGC-RFPYL 170 -178 3.420 30.5694150210502 A 0201 FQLQNIVKPL * 206 - 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 - 4.250 70.1054123466879 A 0205 IVDP.GYLGYL 16 - 25 3.120 22.6463796431754 A 0205 YLYLQWQPPL * 24 - 33 4.140 62.8028214492017 3 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 - 4.670 106.697742432451 A 0205 VLLD-TNYNL * 138 - 3.740 42.0979901649969 Allele Peptide Position Score t1/2 A 0205 NLFY-WYEGL 146 - 154 3.040 20.9052432350928 A 0205 FQLQNIVKPL * 206 - 4.610 100.484149636389 A3 LLDTNYNLFY I40 - 149 3.190 24.2884274430946 A3 ALQC-VDYIIC 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-II1 4.090 59.7398917041452 1 A24 VYYN-WQYLL * 122 - 5.300 200.336809974792 ~ 130 A24 WYEG-LDHAL * 150 - 5.890 361.405284372286 A24 DYIKADGQNI * 162 - 4.500 90.0171313005218 A24 SYFTFQLQNI * 202 - 4.090 59.7398917041452 . 211 A DLSK-KTLLR 311-319 3.300 27.1126389206579 I A68.1 TVEY-ELKYR * 42 - 50 5.300 200.336809974792 S

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 - 4.790 120.301368663215 A68.1 FTFQLQNIVK * 204-213 4.090 59.7398917041452 0 A68.1 FTRESSCEIK 222 - 231 3.400 29.964100047397 A68.1 ESSC-EIKLK 225 - 233 3.300 27.1126389206579 A68.1 TVENETYTLK * 263 - 4.790 120.301368663215 A68.1 YTLKTTNETR * 269 - 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 21 I - 220 3.410 30.2652442594001 B7 EIRE-DDTTL 251 - 259 3.690 40.0448469572867 3 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 B 14 IRSSYFTFQL 199 - 208 3.000 20.0855369231877 3 B 2702 LQWQ-PPLSL 27 - 35 3.410 30.2652442594001 B 2702 WQPPLSLDHF 29 - 38 3.000 20.0855369231877 Allele Peptide Position Score t1/2 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 1 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 I B 2705 WQPPLSLDHF 29 -38 4.610 100.484149636389 S

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 3~ 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 35 B 2705 CRFP-YLEAS 173 - 181 6.910 1002.24724229025 B 2705 FPYLEASDYK 175 - 184 3.910 49.8989519734079 Allele Peptide Position Score t1/2 B 2705 IRSSYFTFQL 199 - 208 7.600 1998.19589510412 B 2705 RSSY - FTFQL Z00 - 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 1 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 I B 2705 GIWS-EWSDS 296 - 304 3.910 49.8989519734079 S

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 25 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 3 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-YLGYG 17 - 25 3.690 40.0448469572867 B 3701 KDGFDLNKGI 68 - 77 3.690 40.04484695272867 35 B 3701 IEAK-IHTLL 77 - 85 4.320 75.1886282920231 B 3701 LDTN-YNLFY 141 - 149 3.690 40.0448469572867 Allele Peptide Position Score t1/2 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 TRESSCEIKI, 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 1 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 I B 3902 VKPL-PPVYL 212 - 220 3.000 20.0855369231877 S

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 25 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 3 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 35 B 4403 GPIPARCFDY 239 - 248 3.810 45.1504388663187 B 4403 YEIEIREDDT 248 - 257 3.000 20.0855369231877 Allele Peptide Position Score t1/2 B 4403 IEIAEDDTTL 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 SIOI 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 I S B 5101 EGEDLSKKTL 308 - 317 4.190 66.0227909604099 B510I 8merNPPQDFEI 9 - 16 6.100 445.857770082517 B5101 8merPPQDFEIV 10-17 3.110 22.4210444007463 B5101 8merDPGYLGYL 18 - 25 5.300 200.336809974792 B5101 8merEAKIHTLL 78 - 85 4.700 109.947172452124 B5101 8merWAETTYWI 99 - 106 5.400 221.406416204187 B5101 8merQGIPETKV 109 - 116 3.800 44.7011844933008 B5101 8merKPGIGVLL 134-141 4.120 61.5592422644285 B5101 8merIGCRFPYL 171 - 178 3.260 26.0495371425183 B5101 8merKPLPPVYL 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 Allele Peptide Position Score t1/2 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 1 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 15 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 Smer 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 25 B5102 8mer KPLPPVYL 213 - 220 5.710 301.87106828279 BS 102 8merIPLGPIPA 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 35 B 5103 IPARCFDYEI 241 - 250 3.800 44.7011844933008 B 5201 NPPQ-DFEIV 9 - 17 4.700 109.947172452124 Allele Peptide Position Score t112 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 KTII'TKNLHY 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 1 ~ 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 1$ 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 1 77 - 85 5.870 354.248980267765 B60 RESS-CEIKL 224 - 232 6.560 706.271694595366 20 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 25 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 35 Cw 0301 VYYN-WQYLL , 122- 130 3.000 20.0855369231877 Cw 0301 VLLDTNYNLF 139 - 148 3.400 29.964100047397 Allele Peptide Position Score t1/2 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 1 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 15 Cw 0401 EYEL-KYRNI 44 - 52 3.220 25.0281201813378 Cw 0401 TWKKTIITKNL 56 - 65 3.690 40.0448469572867 Cw 0401 TYWI-SPQGI 103 - 1 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 3 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 Other Embodiments 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 LISTING
<l 10> The Penn State Research Foundation Waldemar, Debinski <120> llVIMUNOTHERAPY USING INTERLEUKIN 13 RECEPTOR SUBUNIT ALPHA
<130> 6460-32 <160> 6 <170> PatentIn version 3.0 <210> 1 <21I> 380 <212> PRT
<213> Homo Sapiens <400> 1 Met Ala Phe Val Cys Leu Ala Ile Gly Cys Leu Tyr Thr Phe Leu Ile Ser Thr Thr Phe Gly Cys Thr Ser Ser Ser Asp Thr Glu Ile Lys Val Asn Pro Pro Gln Asp Phe Glu Ile Val Asp Pro Gly Tyr Leu Gly Tyr Leu Tyr Leu Gln Trp Gln Pro Pro Leu Ser Leu Asp His Phe Lys Glu Cys Thr Val Glu Tyr Glu Leu Lys Tyr Arg Asn Ile Gly Ser Glu Thr Trp Lys Thr Ile Ile Thr Lys Asn Leu His Tyr Lys Asp Gly Phe Asp Leu Asn Lys GIy Ile Glu Ala Lys Ile His Thr Leu Leu Pro Trp Gln Cys Thr Asn Gly Ser Glu Val Gln Ser Ser Trp Ala Glu Thr Thr Tyr Page I

Trp Ile Ser Pro Gln Gly Ile Pro Glu Thr Lys Val Gln Asp Met Asp Cys Val Tyr Tyr Asn Trp Gln Tyr Leu Leu Cys Ser Trp Lys Pro Gly Ile Gly Val Leu Leu Asp Thr Asn Tyr Asn Leu Phe Tyr Tm Tyr Glu Gly Leu Asp His Ala Leu Gln Cys Val Asp Tyr Ile Lys Ala Asp Gly Gln Asn Ile Gly Cys Arg Phe Pro Tyr Leu Glu Ala Ser Asp Tyr Lys Asp Phe Tyr Ile Cys Val Asn Gly Ser Ser Glu Asn Lys Pro Ile Arg Ser Ser Tyr Phe Thr Phe Gln Leu Gln Asn Ile Val Lys Pro Leu Pro Pro Val Tyr Leu Thr Phe Thr Arg Glu. Ser Ser Cys Glu Ile Lys Leu Lys Trp Ser Ile Pro Leu Gly Pro Ile Pro Ala Arg Cys Phe Asp Tyr Glu Ile Glu Ile Arg Glu Asp Asp Thr Thr Leu Val Thr Ala Thr Val Glu Asn Glu Thr Tyr Thr Leu Lys Thr Thr Asn Glu Thr Arg Gln Leu .

Cys Phe Val Val Arg Ser Lys Val Asn Ile Tyr Cys Ser Asp Asp Gly Ile Trp Ser Glu Trp Ser Asp Lys Gln Cys Trp Glu Gly Glu Asp Leu Ser Lys Lys Thr Leu Leu Arg Phe Trp Leu Pro Phe Gly Phe Ile Leu Ile Leu Val Ile Phe Val Thr Gly Leu Leu Leu Arg Lys Pro Asn Thr Tyr Pro Lys Met Ile Pro Glu Phe Phe Cys Asp Thr <210> 2 <211> 1298 <212> DNA
<213> Homo sapiens <400> 2 ggtgcctgtc ggcggggaga gaggcaatat caaggtttta aatctcggag aaatggcttt 60 cgtttgcttg gctatcggat gcttatatac ctttctgata agcacaacat ttggctgtac 120 ttcatcttca gacaccgaga taaaagttaa ccctcctcag gattttgaga tagtggatcc 180 cggatactta ggttatctct atttgcaatg gcaaccccca ctgtctctgg atcattttaa 240 ggaatgcaca gtggaatatg aactaaaata ccgaaacatt ggtagtgaaa catggaagac 300 catcattact aagaatctac attacaaaga tgggtttgat cttaacaagg gcattgaagc 360 gaagatacac acgcttttac catggcaatg cacaaatgga tcagaagttc aaagttcctg 420 ggcagaaact acttattgga tatcaccaca aggaattcca gaaactaaag ttcaggatat 480 ggattgcgta tattacaatt ggcaatattt actctgttct tggaaacctg gcataggtgt 540 acttcttgat accaattaca acttgtttta ctggtatgag ggcttggatc atgcattaca 600 gtgtgttgat tacatcaagg ctgatggaca aaatatagga tgcagatttc cctatttgga 660 ggcatcagac tataaagatt tctatatttg tgttaatgga tcatcagaga acaagcctat 720 cagatccagt tatttcactt ttcagcttca aaatatagtt aaacctttgc cgccagtcta 780 tcttactttt actcgggaga gttcatgtga aattaagctg aaatggagca tacctttggg 840 acctattcca gcaaggtgtt ttgattatga aattgagatc agagaagatg atactacctt 900 ggtgactgct acagttgaaa atgaaacata caccttgaaa acaacaaatg aaacccgaca 960 attatgcttt gtagtaagaa gcaaagtgaa tatttattgc tcagatgacg gaatttggag 1020 tgagtggagt gataaacaat gctgggaagg tgaagaccta tcgaagaaaa ctttgctacg 1080 tttctggcta ccatttggtt tcatcttaat attagttata tttgtaaccg gtctgctttt 1140 gcgtaagcca aacacctacc caaaaatgat tccagaattt ttctgtgata catgaagact 1200 ttccatatca agagacatgg tattgactca acagtttcca gtcatggcca aatgttcaat 1260 atgagtctca ataaactgaa tttttcttgc gaatgttg 1298 <210> 3 <211> 30 <212> DNA
<213> Artificial Sequence <220>
<221> misc_feature <223> Forward PCR Primer for IL-l3Ralpha2 <400> 3 aagatttgga agcttatggc tttcgtttgc 30 <210> 4 <211> 30 <212> DNA
<213> Artificial Sequence <220>
<221> misc_feature <223> Reverse PCR Primer for IL-l3Ralpha2 <400> 4 tccctcgaag cttcatgtat cacagaaaaa 30 <210> 5 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<221> misc_feature <223> Forward PCR Primer for IL-l3Ralphal <400> 5 attattaagc ttatggagtg gccggcg 27 <210> 6 <211> 27 <212> DNA
<213> Artficial Sequence <220>
<221> misc_feature <223> Reverse PCR Primer For IL-l3Ralphal <400> 6 taaccggaag cttcactgag aggcttt 27

Claims (32)

What is claimed is:

1. A method for stimulating a immune response against IL-13R.alpha.2 in a subject having or at risk for developing a disease having cells expressing IL-13R.alpha.2, the method comprising the steps of:
(a) formulating an anti-cancer vaccine outside of the subject, the vaccine comprising an agent that can stimulate an immune response against IL-13R.alpha.2 when administered to an animal; and (b) administering the vaccine to the subject in an amount sufficient to stimulate an immune response against IL-13R.alpha.2 in the subject.

2. The method of claim l, wherein the agent that can stimulate an immune response against IL-13R.alpha.2 comprises a peptide comprising at least seven contiguous amino acids of SEQ ID NO:1.

3. The method of claim 1, wherein the agent that can stimulate an immune response against IL-13R.alpha.2 is a protein comprising the amino acid sequence of SEQ ID NO:1.

4. The method claim 1, wherein the vaccine further comprises an adjuvant.

5. The method of claim 4, wherein the adjuvant comprises a substance selected from the group consisting of an aluminum salt; an oil-in-water emulsion; a composition comprising saponin; a composition comprising a bacterial protein; and a cytokine.

6. The method of claim 4, wherein step (b) of administering the vaccine to the subject in an amount sufficient to stimulate an immune response against IL-13R.alpha.2 in the subject comprises administering the vaccine in at least a first dose and a second dose, wherein said first dose is administered to the subject at least 24 hours before said second dose is administered to the subject.

7. The method of claim 1, wherein the agent that can stimulate an immune response against IL-13R.alpha.2 comprises a nucleic acid that encodes a peptide comprising at least seven contiguous amino acids of SEQ ID NO:1.

8. The method of claim 7, wherein the nucleic acid is a naked DNA.

9. The method of claim 7, wherein the nucleic acid is incorporated into an expression vector.

10. The method of claim 1, wherein the agent that can stimulate an immune response against IL-13R.alpha.c2 comprises a cell expressing a peptide comprising at least seven contiguous amino acids of SEQ ID NO:1.

11. The method of claim 10, wherein the peptide comprising at least seven contiguous amino acids of SEQ ID NO:1 is a protein comprising the amino acid sequence of SEQ ID NO:1.

12. The method of claim 1, wherein the agent that can stimulate an immune response against IL-13R.alpha.2 comprises a cell into which has been introduced a purified nucleic acid that encodes a peptide comprising at least seven contiguous amino acids of SEQ ID
NO:1.

13. The method of claim 1, further comprising the step of providing a subject having or at risk for developing a cancer having cells expressing IL-13R.alpha.2.

14. The method of claim 13, wherein the cells expressing IL-13R.alpha.2 are glioma cells.

15. The method of claim 13, wherein the subject is a human being.

16. A composition for stimulating an immune response against IL-13R.alpha.2 when administered to an animal, the composition comprising:
(a) an isolated agent that can stimulate an immune response against IL-13R.alpha.2 when administered to an animal; and (b) a pharmaceutically acceptable carrier.

17. The composition of claim 16, wherein the agent that can stimulate an immune response against IL-13R.alpha.2 when administered to an animal comprises a peptide comprising at least seven contiguous amino acids of SEQ ID NO:1.

18. The composition of claim 17, wherein the peptide comprising at least seven contiguous amino acids of SEQ ID NO:1 is a protein comprising the amino acid sequence of SEQ ID NO:1.

19. The composition of claim 17, wherein the composition further comprises an adjuvant.

20. The composition of claim 19, wherein the adjuvant comprises a substance selected from the group consisting of an aluminum salt; an oil-in-water emulsion; a composition comprising saponin; a composition comprising a bacterial protein;
and a cytokine.

21. The composition of claim 20, wherein the agent that can stimulate an immune response against IL-13R.alpha.2 when administered to an animal comprises a nucleic acid that encodes a peptide comprising at least seven contiguous amino acids of SEQ ID
NO:1.

22. The composition of claim 21, wherein the nucleic acid is a naked DNA.

23. The composition of claim 21, wherein the nucleic acid is incorporated into an expression vector.

24. The composition of claim 20, wherein the agent that can stimulate an immune response against IL-13R.alpha.2 comprises a cell expressing a peptide comprising at least seven contiguous amino acids of SEQ ID NO:1.

25. The method of claim 24, wherein the peptide comprising at least seven contiguous amino acids of SEQ ID NO:1 is a protein comprising the amino acid sequence of SEQ ID NO:1.

26. The composition of claim 25, wherein the agent that can stimulate an immune response against IL-13R.alpha.2 comprises a cell into which has been introduced a purified nucleic acid that encodes a peptide comprising at least seven contiguous amino acids of SEQ ID
NO:1.

27. A method for directing an antibody to cells expressing IL-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; and (b) administering the pharmaceutical composition to the subject in an amount sufficient to allow the antibody to specifically bind to the cells expressing IL-13R.alpha.2 in the subject.

28. The method of claim 27, wherein the antibody is a monoclonal antibody.

29. The method of claim 27, wherein the antibody is a polyclonal antibody.

30. A pharmaceutical composition comprising an antibody that specifically binds IL-13R.alpha.2 and a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 30, wherein the antibody is a monoclonal antibody.

32. The pharmaceutical composition of claim 30, wherein the antibody is a polyclonal antibody.