AU774958B2 - Fibroblast growth factor-5 (FGF-5) is a tumor associated T-cell antigen - Google Patents

Description

WO 01/25271 PCT/US00/26689 FIBROBLAST GROWTH FACTOR-5 (FGF-5) IS A TUMOR ASSOCIATED T-CELL ANTIGEN

FIELD

This disclosure relates to the treatment of a neoplasm expressing FGF-5, for example by stimulation of the immune response to treat a neoplasm, more specifically to the use of FGF-5 to stimulate a cytotoxic T cell response against a neoplasm expressing

BACKGROUND

The use of immunologic agents has been proposed as an alternative for anti-neoplastic chemotherapy. For example, the administration of purified antigens, alone or in combination with various adjuvants, can be used to stimulate the B cell response. In addition, the regulation of circulating growth factors has been proposed as another way to treat tumor progression U.S.

Patent 5,919,459). Adjuvant formulations have also been proposed which are designed to generally stimulate cytotoxic T cells (see, PCT/US98/18495).

The process by which T cells recognize and interact with other cells is complex and involves cell surface complexes on the other cells of peptides and molecules referred to as human leukocyte antigens (HLA) or major histocompatibiltiy complexes (MHC). The interaction of T cells and complexes of MHC with an antigen is restricted, requiring a specific T cell for a specific complex of MHC and peptide antigen. For example, particular antigens have been identified which are expressed on cell surfaces, which can lead to lysis of the tumor cells by specific cytotoxic T cells. Genes that encode the antigens found on the surface of the cancer are called tumor rejection antigen precursors (TRAP) molecules, and the peptides derived from these genes are referred to as tumor rejection antigens (TRA) or tumor associated antigens (TAA). However, only a few TAA molecules have been recognized, and these TAAs are only known to be present on a limited number of tumor types (see, U.S. Patent No. 5.939,526).

Class I MHC molecules (referred to as HLAs, in humans) are expressed on the surface of almost all nucleated cells. A peptide fragment from the TAA is presented in the groove of the HLA, on the surface of the tumor. This allows cytotoxic T lymphocytes (CTLs) to recognize and destroy the tumor cells. In general, CTL responses are not directed against all possible epitopes of the TAA. Rather, they are restricted to a few immunodominant determinants. Additional factors, mostly linked to a processing event, can also play a role in dictating which of the many potential epitopes will be presented to the CTL cells.

A large number of CTL clones directed against melanoma have been obtained. In several instances, the antigens recognized by these CTLs have been characterized (for review see Parmiani, WO 01/25271 PCT/US00/26689 -2- Eur. J. Cancer. 34:S42-S47, 1998; Kirkin et al., APMIS. 106:665-79, 1998). In contrast, although several CTL clones directed against renal cell carcinoma (RCC) have been obtained, very little information is available on the TAAs recognized by these CTLs.

(initially termed FGF-3) is an oncogene-encoded glycoprotein bearing mitogenic activity for fibroblasts and endothelial cells. FGF-5 was previously isolated by transfecting a bladder carcinoma DNA into NIH-3T3 cells (Zhan et al., Mol Cell Biol. 8:3487-95, 1988 and corresponding U.S. Patent Nos. 5,155,217 (referred to herein as the '217 patent) and 5,238.916 (referred to herein as the '916 patent)). Both the nucleotide and protein sequences were submitted to GenBank under Accession Nos. NM 004464 and NP_004455 in 1990, which are incorporated by reference, as are the '217 and '916 patents.

Several publications describe the expression of FGF-5 in tumor cell lines and tissue samples. FGF-5 is expressed in RCC (Yoshimura et al., Cancer Lett. 103:91-7, 1996), as well as cancers of the bladder, liver and endometrium (Zhan et al., Mol Cell Biol. 8:3487-95, 1988; the '916 and '217 patents; Yoshimura et al., Cancer Lett. 103:91-7, 1996), pancreas (Kornmann et al., Oncogene. 15:1417-24, 1997), gastric and esophageal adenocarcinomas (Altorki et al., Cancer, 72:649-57, 1993), breast (Cullen et al., Cancer Res. 51:4978-85, 1991) and malignant melanomas (Albino et al., Cancer Res. 51:4815-20, 1991). However, whether FGF-5 expression in these tissues is correlated with the immunogenicity of FGF-5 was not determined. In addition, none of these publications disclose the use of FGF-5 as an immune target for cancer immunotherapies.

Expression of FGF-5 has been observed in several murine embryonic tissues (Goldfarb et al., Ann N YAcad Sci. 638:38-52, 1991; Haub and Goldfarb, Development. 112:397-406, 1991) and some normal adult tissues including pancreatic tissue (Kornmann et al., Oncogene, 15:1417- 24, 1997) human fibroblasts (Werner et al., Oncogene. 6:2137-44, 1991; Albino el al., 1991), skeletal muscle (Hannon et al., J. Cell Biol. 132:1151-9, 1996 and Hughes et al., Neuron, 10:369- 77, 1993); and adult neurotrophic cells (Goldfarb el al., Ann N YAcad Sci. 638:38-52, 1991; Kitaoka et al., Invest. Ophthalmol. Vis. Sci. 35:3189-98, 1994).

Renal cell carcinoma, although not a common malignancy, accounts for 3 of all adult cancers. Conventional therapies involving the systemic administration of both interleukin-2 (IL-2) and interferon a (IFN-a) result in the long-term remission in less than 20% of patients with metastatic RCC. In addition, this approach is associated with unwanted side effects. To date, no highly effective treatment for RCC is available, and the survival time of the patients is very short.

It is therefore important to develop new treatment strategies for these patients.

Osband Patent No. 5,192.537) discloses a method for treating RCC by in vitro incubation of a patient's T-cells with a tumor extract. The activated T-cells are then infused into WO 01/25271 PCT/US00/26689 -3the patient to reduce or eliminate the tumor burden. Cimetidine is concurrently administered to inhibit the patient's suppressor cells, and augment the anti-tumor response.

The '217 and the '916 patents demonstrate that FGF-5 is expressed in bladder, liver, and endometrial cancers. In addition, these patents disclose a method for treating FGF-5 expressing cancers by the administration of antibodies which recognize FGF-5. Such antibodies are also disclosed as diagnostic agents. However, there is no disclosure of FGF-5 expression in RCC, or how FGF-5 might be used to treat cancers using the CTL-mediated immune response.

Gospodarowicz (WO 97/30155) teaches the administration of an FGF-5 nucleic acid sequence, lacking the signal sequence, to promote angiogenesis in patients suffering from myocardial ischemia. The signal sequence was deleted to remove the oncogenic potential of FGF- Also disclosed are techniques for delivery of FGF-5 nucleic acid sequences to cells.

Gaugler et al. (Immunogenetics. 44:323-30, 1996) and corresponding U.S. Patent No.

5.939,526 describe the identification of RAGEL, a Renal tumor AntiGEn recognized by autologous CTLs. RAGEI was observed in 37% of RCC cell lines, but only in one of 57 renal cell carcinoma samples. RAGE1 was also expressed in other cancers including: sarcomas, infiltrating bladder carcinomas, and melanomas, but not expressed in normal tissues, except the retina. Gaugler et al.

propose that immunization of cancer patients against RAGE1 antigens may be an effective cancer immunotherapy, but also disclose that only a small percentage of patients with RCC might benefit, due to the low frequency of expression of RAGE1 in RCC tumor samples (1 of 57). Techniques for immunizing subjects against TAA are disclosed in U.S. Patent No. 5,939,526, which is incorporated by reference.

Several TAAs recognized by CTLs have been identified in melanomas (for a review see Kirkin et al., APMIS. 106:665-79, 1998). These antigens include: MAGE, BAGE, GAGE, PRAME, NY-ESO-1, gp 100, TRP-I. TRP- CDK4. MUM-1, and p-catenin, some of which have been tested for their ability to treat cancer in vivo. Although some antigens such as MAGE and PRAME should potentially be highly immunogenic, only a few patients have been identified who respond to these TAAs in vivo, indicating their genuinely low immunogenicity. These studies demonstrate that it is difficult to predict with any certainty which TAA will elicit a functional response in vivo. In addition, the need remains to identify alternative TAAs which might generate a greater response in vivo.

SUMMARY OF THE DISCLOSURE The disclosure includes a method of treating a subject having a neoplasm expressing or over-expressing FGF-5. In one embodiment, the method involves modulating an immune response, such as increasing or decreasing an immune response, or modulating FGF-5 expression or activity, WO 01/25271 PCT/US00/26689 -4such as increasing or decreasing FGF-5 expression or activity. In particular embodiments, the neoplasm expressing or over-expressing FGF-5 can be a carcinoma, such as an adenocarcinoma.

In other embodiments, the carcinoma is a carcinoma of the prostate, breast, bladder, or pancreas, or RCC, all of which express or overexposes FGF-5. In specific embodiments, the neoplasm is a

RCC.

In one embodiment, the method includes modulating an immune response sufficient to stimulate a cytotoxic T cell response to a cell of the FGF-5 expressing or over-expressing neoplasm. In a particular embodiment, the method includes administering a therapeutically effective amount of an agent that modulates an immune response, such as an FGF-5 polypeptide (including variants, polymorphisms, mutants, fusions, and fragments thereof), and immunoreactive sensitized T cells sensitized with Alternative embodiments of the method include administering a therapeutically effective amount of an FGF-5 polypeptide that modulates an immune response to treat a subject having a neoplasm expressing or over-expressing FGF-5. In some disclosed embodiments, the polypeptide protein has the amino acid sequence shown in either SEQ ID NOS: 4, 6, 8, 10, 12, 16, 18, or 19 or amino acid sequences that differ from those specified in SEQ ID NOS: 4, 6, 8, 10, 12, 16, 18 or 19 by one or more conservative amino acid substitutions, or amino acid sequences having at least 70% sequence identity to those sequences, for example sequences that are at least 90%, 95% or even 98% or 99% identical. In addition to such variants that retain the ability to modulate an immune response, fragments of the sequences that have or retain such activity may be used. Such fragments may, for example, include at least 50%, 70%, 75%, 90% or 95% of the amino acid residues of the FGF-5 peptide sequence. In other embodiments, the sequence is no more than 15 or 20 contiguous residues in length, however longer sequences (such as up to 60 or more amino acids in length) are also included. Also included are fragments and variants of the amino acid sequence of SEQ ID NO 19, which includes the HLA-A3 restricted epitope.

Although the polypeptide or fragment can be administered to a subject in a therapeutically effective amount sufficient to stimulate an immune response against cells of the tumor, alternative embodiments of the disclosure include administering a nucleic acid encoding the polypeptide, or the fragment, polymorphism, or variant thereof, sufficient to stimulate a cytotoxic T cell response. Alternatively, a vector can be administered to the subject which includes a nucleic acid sequence which encodes and expresses FGF-5, or a fragment, polymorphism, or variant thereof, sufficient to stimulate a cytotoxic T cell response to a cell of the neoplasm. In particular examples, the vector is a viral vector, such as a retroviral vector.

The FGF-5 polypeptide (including variants, polymorphisms, mutants, fusions, and fragments thereof) that modulates an immune response, can also be administered to the subject by WO 01/25271 PCT/US00/26689 administering an effective amount of a host cell expressing a recombinant nucleic acid encoding (including variants, polymorphisms, mutants, fusions, and fragments thereof), sufficient to stimulate a cytotoxic T cell response to a cell of the neoplasm, and inhibit neoplastic proliferation.

In some embodiments, the inhibition of neoplastic proliferation induces a regression of the tumor, for example as determined by radiographic or other laboratory evidence of disease (such as serologic tumor markers, such as FGF-5 specific binding agents). The T cell response is sufficient to stimulate the T cell to react with a cell of the tumor, such as RCC.

Alternatively, the subject can be treated by administering to the subject an effective, neoplasm-inhibiting amount of immunoreactive sensitized T cells, wherein the sensitized T cells are sensitized with FGF-5. The immunoreactive sensitized T cells may be autologous or heterologous.

In yet other embodiments, the method includes modulating FGF-5 expression or activity, for example by increasing or decreasing FGF-5 expression or activity. In a particular embodiment, the method includes administering a therapeutically effective amount of an agent that decreases expression or activity, such as an FGF-5 antisense molecule or FGF-5 specific binding agent. In another particular embodiment, the method includes administering a therapeutically effective amount of an agent that increases FGF-5 expression or activity, such as polypeptides and nucleic acids encoding FGF-5 polypeptides. FGF-5 antisense molecules hybridize to an RNA or a plus strand of an FGF-5 nucleic acid (including variants, polymorphisms, mutants, fusions, and fragments thereof) and inhibit FGF-5 expression and/or activity by a desired amount.

such as by at least 20%, 50%, 70%, 80% or 90%. In some embodiments, the FGF-5 specific binding agent is capable of specifically binding to an FGF-5 polypeptide (including variants, polymorphisms, mutants, fusions, and fragments thereof). In particular examples, the specific binding agent is an antibody, such as polyclonal antibodies, monoclonal antibodies, and fragments of monoclonal antibodies.

In view of the discovery that at least certain fragments of FGF-5 are HLA-A3 restricted.

the disclosure also includes administering the FGF-5 polypeptide, or a fragment or variant, to HLA-A3 individuals. Individuals can be pre-screened to select HLA-A3+ individuals to whom to administer the FGF-5 polypeptide or fragment.

In yet another embodiment, a method is disclosed for stimulating a cytotoxic T cell response against a RCC, by contacting the T cell with a therapeutically effective amount of an polypeptide or a cell expressing FGF-5 sufficient to stimulate the T cell to react with a cell of the RCC.

Also disclosed herein are methods for detecting an enhanced susceptibility of a subject to disease associated with abnormal FGF-5 expression, such as increased FGF-5 expression, by detecting the presence of multiple copies of an FGF-5 and/or transcription factor gene, and/or an WO 01/25271 PCT/US00/26689 -6increase in FGF-5 protein in cells of a subject, such as a human. The disease may be a tumor (such as a malignant tumor) in which FGF-5 is abnormally increased, such as RCC and carcinoma of the prostate, breast, bladder, and pancreas. In other embodiments, the disease is Hippel-Lindau disease, horseshoe kidneys, adult polycystic kidney disease, and acquired renal cystic disease. In certain embodiments, the presence of multiple copies of an FGF-5 and/or transcription factor(s) gene can be detected by incubating a nucleic acid, such as an oligonucleotide, with the nucleic acid of the cell under conditions such that the oligonucleotide will specifically hybridize to an and/or transcription factor(s) gene present in the nucleic acid to form an and/or transcription factor(s) gene complex, and then detecting an increase or decrease of oligonucleotide:FGF-5 and/or transcription factor(s) complexes, wherein the presence of said complexes indicates the presence of an FGF-5 and/or transcription factor(s) gene.

The present disclosure also provides methods for detecting the presence of FGF-5 protein in a cell by incubating a specific binding agent of the present disclosure with proteins of the cell under conditions such that the specific binding agent will specifically bind to an FGF-5 protein present in the cell to form a specific binding agent:FGF-5 protein complex, and detecting an increase or decrease (or quantity) of specific binding agent:FGF-5 protein complexes, including the presence of the FGF-5 protein, wherein an increase of the complexes relative to specific binding protein complexes in a non-neoplastic cell indicates expression or overexpression of and an enhanced susceptibility of the subject to a disease of abnormal FGF-5 expression.

Also disclosed herein are methods for lysing a cell of an FGF-5 expressing neoplasm by enhancing an immune response against FGF-5 in the subject, sufficient to induce regression of the neoplasm. In one embodiment, the cell is characterized by increased expression of an protein, or the cell has increased FGF-5 expression or copy number, relative to FGF-5 expression in a same tissue type that is non-neoplastic. In certain embodiments, enhancing the immune response can be achieved by exposing the cell to a therapeutically effective amount of an polypeptide (including fragments, polymorphisms, fusions, and variants) disclosed herein. In other embodiments, enhancing the immune response can be achieved by administering a therapeutically effective amount of any FGF-5-expressing nucleic acid (including fragments, polymorphisms, fusions, and variants). In yet other embodiments, enhancing the immune response is achieved by administering a therapeutically effective amount of immuno-reactive sensitized T-cells wherein the sensitized T cells are sensitized with The agents disclosed herein (such as those which modulate an immune response those which modulate FGF-5 expression or activity) can be administered prophylactically, prior to the development of a tumor, for example to persons at elevated risk of developing the tumor (such as persons with a family history of the tumor, and persons with Hippel-Lindau disease, horseshoe 7 kidneys, adult polycystic kidney disease, and acquired renal cystic disease). The agents can also be administered therapeutically, to a person who already has developed the tumor, for example a person who has undergone surgical resection ofa RCC primary or metastatic lesion, or a person who is undergoing chemotherapy for treatment of the tumor. Alternatively, the agents can be administered as the sole therapy for the tumor.

The agent(s) can be administered with pharmaceutically acceptable carrier. In addition, the agents can be administered in combination with other therapeutic treatments, such as other anti-neoplastic or anti-tumorigenic therapies. Such agents can be administered concurrently or sequentially, with the other therapies.

The foregoing features and advantages disclosed herein will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that 25 any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

S".BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a bar graph and table showing the analysis of the reactivity of Clone 2 CTL using a panel of tumor lines, and demonstrating HLA-A3 restriction of the CTL response to FGF-5. The HLA type of each tumor is shown on the right.

FIGS. 2A and 2B are bar graphs showing the effect of various anti-HLA monoclonal antibodies on the recognition of tumors by an HLA-A2 restricted CTL or Clone 2 CTL.

FIG. 3A is a bar graph illustrating the recognition of three cDNA clones S" in a HLA-A3 restricted manner by Clone 2 CTL.

\\melbfiles\home$\Simeona\Keep\Speci\78371 OO.doc 12/01/04 7A FIG. 3B is a schematic drawing showing the alignment of the three tumor-derived FGF-5 clones. The nucleotide changes (inside each bar), their positions (under 10E4-1), and any resulting amino acid changes (above each bar) are shown.

FIG. 4 is a graphical representation showing the normalization of FGFcopy number in normal adult tissues.

FIG. 5 is a graphical representation showing the normalization of copy number by p-actin copy number (filled bars) and the recognition by the CTL as represented by IFN-y concentration in each cell line. In the insert graph, the correlation between FGF-5 expression and recognition by the CTL (p<0.0001) is shown.

FIG. 6 is a sequence listing of an FGF-5 ORF-1 and ORF-2 from U.S.

Patent No. 5,238,916, which is incorporated by reference.

FIG. 7 is a graphical representation showing FGF-5 peptides that are immunogenic (unfilled bars) and are non-immunogenic (filled bars). The numbers below each bar represent the nucleotide number (upper numbers), and amino acid number (lower numbers) of the FGF-5 sequences shown in SEQ ID NO: 17.

\\melb_files\homeS\Simeona\Keep\Speci\78371 00.doc 12/01/04 WO 01/25271 PCT/US00/26689 -8- SEQUENCE LISTING The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NO I shows a DNA sequence for a first open reading frame of SEQ ID NO 2 shows an amino acid sequence for a first open reading frame of MUSFGF- SEQ ID NO 3 shows a DNA sequence for a second open reading frame of SEQ ID NO 4 shows an amino acid sequence for a second open reading frame of SEQ ID NO 5 shows a cDNA sequence for a variation of IA3-1.

SEQ ID NO 6 shows an amino acid sequence for a variation of IA3-1.

SEQ ID NO 7 shows a cDNA sequence for another variation of IA3-1.

SEQ ID NO 8 shows an amino acid sequence for another variation of IA3-1.

SEQ ID NO 9 shows a cDNA sequence for a variation of 6A4-1.

SEQ ID NO 10 shows an amino acid sequence for a variation of 6A4-1.

SEQ ID NO 11 shows a cDNA sequence for another variation of 6A4-1.

SEQ ID NO 12 shows the amino acid sequence for another variation of 6A4-1.

SEQ ID NO 13 shows a cDNA sequence for a variation of 10E4-1.

SEQ ID NO 14 shows a cDNA sequence for another variation of 10E4-1.

SEQ ID NO 15 shows a full-length cDNA sequence of construct 10E4-1 with ORF-1.

SEQ ID NO 16 shows an amino acid sequence of ORF-I from construct 10E4-1.

SEQ ID NO 17 shows a full-length cDNA sequence of construct 10E4-1 with ORF-2.

SEQ ID NO 18 shows an amino acid sequence of ORF-2 from construct 10E4-1.

SEQ ID NO 19 shows a 60 amino acid sequence which contains the FGF-5 epitope for HLA-A3+ individuals.

SEQ ID NO 20 and 21 show nucleic acid sequences of forward primers that can be used to RT-PCR SEQ ID NO 22 and 23 show nucleic acid sequences of reverse primers that can be used to RT-PCR SEQ ID NO 24 and 25 show nucleic acid sequences of forward and reverse primers, respectively, that can be used to clone the HLA-A3 gene from autologous 1764 RCC by RT-PCR.

WO 01/25271 PCT/US00/26689 -9- DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS The following definitions and methods are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. It must be noted that as used herein and in the appended claims, the singular forms or "an" or "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of such proteins and reference to "the antibody" includes reference to one or more antibodies and equivalents thereof known to those skilled in the an, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the an to which this disclosure belongs.

Abbreviations and Definitions CTL cytotoxic T lymphocyte FGF-5 fibroblast growth RCC renal cell carcinoma RT room temperature SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis TAA tumor-associated antigen TCR T-cell receptor TILs tumor infiltrating lymphocytes Antineoplastic agent: A drug or biologic that inhibits the proliferation of neoplastic cells.

for example arresting their growth or causing the regression of a tumor. Examples include alkylating agents (such a vincristine, vinblastine or taxol), anthracycline antibiotics such as daunorubicin and doxorubicin, hormonal therapies such as tamoxifen, and miscellaneous agents such as cis-diamminedichloroplatimun and hydroxyurea. Antineoplastic agents also include biologics, such as IL-2 and alpha-interferon, and immunotherapy, for example with bacille Calmette-Guerin (BCG). Protocols for administration of such agents are known in the an, and examples can be found in Goodman and Gilman, The Pharmacological Basis of Therapeutics, 17* edition, section XIII.

Antisense molecules or antisense oligonucleotides: Nucleic acid molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA (Weintraub, Scientific American 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids WO 01/25271 PCT/US00/26689 interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double stranded. In one embodiment, the antisense oligomer is about 15 nucleotides, which are easily synthesized. The use of antisense molecules to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem. 172:289, 1988).

Therapeutically effective antisense molecules are characterized by their ability to inhibit the expression of FGF-5. Complete inhibition is not necessary for therapeutic effectiveness, some oligonucleotides will be capable of inhibiting the expression of FGF-5 by at least 15%, 30%, 60%, 70%, 80% or Therapeutically effective antisense molecules are additionally characterized by being sufficiently complementary to FGF-5 encoding nucleic acid sequences. As described below, sufficient complementary means that the therapeutically effective oligonucleotide or oligonucleotide analog can specifically disrupt the expression of FGF-5, and not significantly alter the expression of genes other than Cancer: Malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and is capable of metastasis.

cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences which determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

Chemical synthesis: The artificial means by which one can make a protein or peptide, for example as described in EXAMPLE 17.

Deletion: The removal of a sequence of DNA, the regions on either side being joined together.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid, RNA).

The repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides, referred to as codons, in DNA molecules code for amino acid in a polypeptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

cDNA: A FGF-5 cDNA is functionally defined as cDNA molecule which encodes a protein having the ability to modulate an immune response, and includes fragments, variants, and polymorphisms of an FGF-5 cDNA that retain the ability to modulate an immune response. The FGF-5 cDNA can be derived by reverse transcription from the mRNA encoded by a FGF-5 gene WO 01/25271 PCTUS00/26689 I and lacks internal non-coding segments and transcription regulatory sequences present in the gene.

fusion protein: A fusion protein comprising a FGF-5 protein (or variants, polymorphisms, mutants, or fragments thereof) linked to other amino acid sequences that do not inhibit the desired activity of FGF-5, for example an immunogenic activity. The other amino acid sequences may be, for example, no more than 10, 20, 30, or 50 amino acid residues in length..

Gene: A gene which encodes an FGF-5 protein having the ability to modulate an immune response. The definition of a FGF-5 gene includes the various sequence polymorphisms and allelic variations that may exist within a population, or in other species.

FGF-5 Protein or FGF-5 Polypeptide: A protein encoded by a FGF-5 gene or cDNA, as well as fragments, variants, and polymorphisms that retain the ability to modulate an immune response. This protein may be functionally characterized by its ability to modulate an immune response as described herein. FGF-5 proteins include the full-length FGF-5 transcript (SEQ ID NOS: 4 and 18), as well as shorter peptides (for example SEQ ID NOS: 6, 8, 10, 12, and 19) and fusion proteins which retain the ability to modulate an immune response.

RNA: The RNA transcribed from a FGF-5 gene.

Isolated: An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been "isolated" thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. The method of the present disclosure can include administration of isolated FGF-5 to a subject.

Malignant: Cells which have the properties of anaplasia invasion and metastasis.

Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

Mimetic: A molecule (such as an organic chemical compound) that mimics the activity of a protein, such as the ability to modulate an immune response. Peptidomimetic and organomimetic embodiments are within the scope of this term, wherein the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid sidechains in the peptide, resulting in such peptido- and organomimetics of the peptides having the ability to stimulate a CTL response against a tumor that is expressing or overexpressing FGF-5. For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for WO 01/25271 PCT/US00/26689 12biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology (ed. Munson, 1995). chapter 102 for a description of techniques used in computer assisted drug design. EXAMPLE 16 describes other methods which can be used to generate mimetics.

Modulating an Immune Response: Includes the ability to increase or decrease an immune response in a subject, such as the ability to stimulate a CTL immune response, such as an HLA-A3-restricted CTL response, against FGF-5 expressing or over-expressing tumors, by a desired amount.

Agents that modulate an immune reponse include, but are not limited to: polypeptides (including fragments, variants, fusion proteins, and polymorphisms thereof), nucleic acid molecules encoding FGF-5- polpeptides, FGF-5 specific binding agent, antisense molecules, and immunoreactive sensitized T cells sensitized with Modulating FGF-5 expression or activity: Includes increasing or decreasing expression or activity. In one embodiment, modulating FGF-5 expression or activity includes administering a therapeutically effective amount of an agent that decreases FGF-5 expression or activity, such as an FGF-5 antisense molecule or FGF-5 specific binding agent. In yet another particular embodiment, modulating FGF-5 expression or activity includes administering a therapeutically effective amount of an agent that increases FGF-5 expression or activity, such as the polypeptides and nucleic acids encoding FGF-5 polypeptides disclosed herein.

Mutant FGF-5 gene: A mutant form of a FGF-5 gene which in some embodiments is associated with disease, for example a carcinoma, such as an adenocarcinoma, RCC, breast cancer, and prostate cancer. In other embodiments, the disease is an FGF-5-expressing tumor, for example RCC. breast, and prostate cancers.

Mutant FGF-5 protein: A protein encoded by a mutant FGF-5 gene.

Mutant FGF-5 RNA: An RNA transcribed from a mutant FGF-5 gene.

Neoplasm: Abnormal growth of cells.

Normal cells: Non-tumor, non-malignant cells.

Oligonucleotide: A linear polynucleotide sequence of up to about 200 nucleotide bases in length, for example a polynucleotide (such as DNA or RNA) which is at least 6 nucleotides, for example at least 15, 25, 50, 100 or even 200 nucleotides long.

Operably linked: 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 WO 01/25271 PCT/US00/26689 -13second 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 the same reading frame.

ORF (open reading frame): A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.

Ortholog: Two nucleotide sequences are orthologs of each other if they share a common ancestral sequence, and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences.

PCR (polymerase chain reaction): Describes a technique in which cycles of denaturation, annealing with primer, and then extension with DNA polymerase are used to amplify the number of copies of a target DNA sequence.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful herein are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the DNA. RNA, proteins, and antibodies herein disclosed.

Embodiments of the disclosure comprising medicaments can be prepared with conventional pharmaceutically acceptable carriers, adjuvants and counterions as would be known to those of skill in the art.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, ethanol, sesame oil, combinations thereof, or the like, as a vehicle. The medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. The carrier and composition can be sterile, and the formulation suits the mode of administration. For solid compositions powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol.

lactose, starch, sodium saccharine, cellulose, magnesium carbonate, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

WO 01/25271 PCT/USOO/26689 14- The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Further embodiments are provided in EXAMPLE 18.

Polynucdotide: A linear nucleic acid sequence of any length. Therefore, a polynucleotide includes molecules which are 15, 50, 100, 200 (oligonuclcotides) and also nucleotides as long as a full length cDNA. In particular embodiments, the polynucleotides are no longer than 15, 50, 100, 120, 180, or 200 nucleotides in length.

Probes and primers: Nucleic acid probes and primers may readily be prepared based on the amino acid sequences provided herein. A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Intersciences (1987).

Primers are short nucleic acids, such as DNA oligonucleotides 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, by PCR or other nucleic-acid amplification methods known in the art.

Methods for preparing and using probes and primers are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989), Ausubel et al., 1987, and Innis et al., PCR Protocols, A Guide to Methods and Applications,1990, Innis et al. 21-27, Academic Press, Inc., San Diego, California. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).

Promoter: An array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or WO 01/25271 PCT/US00/26689 protein is more enriched than the peptide or protein is in its natural environment within a cell.

Preferably, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. The method disclosed herein can include administration of purified FGF-5 to a subject to provoke a CTL.response against a tumor that is expressing or overexpressing Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, by genetic engineering techniques.

Sample: Includes biological samples containing genomic DNA, RNA, or protein obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.

Sequence Identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

Homologs or orthologs of nucleic acid or amino acid sequences will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or cDNAs are derived from species which are more closely related human and chimpanzee sequences), compared to species more distantly related human and C. elegans sequences). Typically, FGF-5 orthologs are at least identical at the nucleotide level and at least 50% identical at the amino acid level when comparing orthologus sequences.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith Waterman, Adv. Appl. Math. 2:482, 1981; Needleman Wunsch, J. Mol. Biol. 48:443, 1970; Pearson Lipman, Proc. Natl. Acad.

Sci. USA 85:2444, 1988; Higgins Sharp, Gene, 73:237-44, 1988; Higgins Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8. 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al,. J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.

215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda. MD WO 01/25271 PCT/US00/26689 -16- 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

Homologs of an FGF-5 protein are typically characterized by possession of at least 85%, 90%, 95%, 98% or 99%% sequence identity counted over full-length alignment with the amino acid sequence of FGF-5 using the NCBI Blast 2.0, gapped blastp set to default parameters. Queries searched with the blastn program are filtered with DUST (Hancock, and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs use SEG. Alternatively, one may manually align the sequences and count the number of identical amino acids. This number divided by the total number of amino acids in the reference sequence multiplied by 100 results in the percent identity.

For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9. extension gap I penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 70%, 80%, 85%, 90%, 98%, 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 20 amino acids, and may possess sequence identities of at least 85%, 90%, 95%, 98% or 99% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site.

One of ordinary skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. Provided herein are the peptide homologs described above, as well as nucleic acid molecules that encode such homologs.

An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequencedependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5 C to 20 C lower than the thermal melting point (TJ) for the specific sequence at a defined ionic strength and pH. The T, is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized to a perfectly matched probe or complementary strand. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1988) and Tijssen (Laboratory Techniques in Biochemistry and Molecular Biology-- WO 01125271 PCT/US00/26689 -17- Hybridization with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York, 1993). Nucleic acid molecules that hybridize under stringent conditions to an FGF-5 gene sequence will typically hybridize to a probe based on either an entire FGF-5 gene or selected portions of the gene under wash conditions of 2x SSC at 50 C. A more detailed discussion of hybridization conditions is presented in EXAMPLE 7.

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.

Such homologous peptides may, for example, possess at least 70%, 80%, 85%, 98%. or 99% sequence identity determined by this method. When less than the entire sequence is being compared for sequence identity, homologs may, for example, possess at least 75%, 85% 90%, 95%, 98% or 99% sequence identity over short windows of 10-20 amino acids. Methods for determining sequence identity over such short windows can be found at the NCBI web site. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs or other variants could be obtained that fall outside of the ranges provided.

The disclosure provides not only the peptide homologs that are described above, but also nucleic acid molecules that encode such homologs.

An alternative (and not necessarily cumulative) indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

Specific binding agent: An agent that binds substantially only to a defined target. As used herein, the term "FGF-5 specific binding agent" includes anti-FGF-5 peptide antibodies and other agents that bind substantially only to an FGF-5 peptide. The antibodies may be monoclonal or polyclonal antibodies that are specific for an FGF-5 peptide, as well as immunologically effective portions ("fragments") thereof. In one embodiment, the antibodies used herein are monoclonal antibodies (or immunologically effective portions thereof) and may also be humanized monoclonal antibodies (or immunologically effective portions thereof). Immunologically effective portions of monoclonal antibodies include Fab, Fab', Fabc and Fv portions (for a review, see Better and Horowitz, Methods. Enzymol. 178:476-96, 1989). Anti-inhibitory peptide antibodies WO 01/25271 PCT/US00/26689 -18may also be produced using standard procedures described in a number of texts, including Antibodies, A Laboratory Manual by Harlow and Lane, Cold Spring Harbor Laboratory (1988).

The determination that a particular agent binds substantially only to a FGF-5 peptide may readily be made by using or adapting routine procedures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Antibodies, A Laboratory Manual by Harlow and Lane). Western blotting may be used to determine that a given peptide binding agent, such as an anti-FGF-5 peptide monoclonal antibody, binds substantially only to a FGF-5 protein. The specific binding agents disclosed herein may be used to modulate FGF-5 activity, for example to cause regression of an FGF-5 expressing or overexpressing neoplasm. In addition, specific binding agents of disclosed herein may be used for diagnostic purposes, for example to determine if a subject has an enhanced susceptibility to develop a disease associated with FGF-5 expression or over-expression, or to monitor a subject's progress during therapy to treat an FGF-5 expressing or over-expressing neoplasm Specifically hybridizable and specifically complementary: Terms which indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide (or its analog) and the DNA or RNA target. The oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence to be specifically hybridizable. An oligonucleotide or analog is specifically hybridizable when binding of the oligonucleotide or analog to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non-target sequences under conditions in which specific binding is desired, for example under physiological conditions in the case of in vivo assays. Such binding is referred to as "specific hybridization." See EXAMPLE 7 for examples of hybridization conditions.

Subject: Living multicellular vertebrate organisms, a category which includes, both human and veterinary subjects for example, mammals, birds and primates.

Sufficient complementarity: When used, indicates that a sufficient number of base pairs exist between the oligonucleotide and the target sequence to achieve detectable binding, and disrupt expression of gene products (such as FGF-5). When expressed or measured by percentage of base pairs formed, the percentage complementarity that fulfills this goal can range from as little as about complementarity to full, (100%) complementary. In general, sufficient complementarity is at least about 50%. In one embodiment, sufficient complementarity is at least about complementarity. In another embodiment, sufficient complementarity is about 90% or about complementarity. In yet another embodiment, sufficient complementarity is about 98% or 100% complementarity.

WO 01/25271 PCT/US00/26689 -19- A thorough treatment of the qualitative and quantitative considerations involved in establishing binding conditions that allow one skilled in the art to design appropriate oligonucleotides for use under the desired conditions is provided by Beltz et al. Methods Enzymol 100:266-285, 1983, and by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

Target sequence: A portion of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) or RNA that upon hybridization to an therapeutically effective oligonucleotide or oligonucleotide analog results in the inhibition of VIAF expression. Either an antisense or a sense molecule can be used to target a portion of dsDNA, since both will interfere with the expression of that portion of the dsDNA. The antisense molecule can bind to the plus strand, and the sense molecule can bind to the minus strand. Thus, target sequences can be ssDNA, dsDNA, and RNA.

An oligonucleotide "binds" or "stably binds" to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties of the target:oligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including both functional and physical binding assays. Binding may be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation and the like.

Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures.

For example, one method that is widely used, because it is so simple and reliable, involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and target disassociate or melt.

The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature at which 50% of the oligomer is melted from its target. A higher means a stronger or more stable complex relative to a complex with a lower (Tm).

Therapeutically Effective Amount: The preparations disclosed herein are administered in therapeutically effective amounts. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, stimulates the desired response. In the case of treating cancer, the desired response is inhibiting or stopping the progression of the cancer, or inducing regression of the disease. Treatment may involve only slowing the progression of the WO 01/25271 PCT/US00/26689 disease temporarily, although more preferably, it involves halting or reversing the progression of the disease permanently.

The therapeutically effective amount also includes a quantity of FGF-5 protein, fusion protein, FGF-5 antisense molecule, FGF-5 specific binding agent, and/or autologous CTLs specific to FGF-5, sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to improve signs and/or symptoms a disease such as cancer, for example by modulating, for example increasing a CTL response against a tumor expressing or overexpressing An effective amount of FGF-5 protein, FGF-5 antisense molecule, FGF-5 specific binding agent, and/or autologous CTLs specific to FGF-5, may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of will be dependent on the source applied FGF-5 isolated from a cellular extract versus a chemically synthesized and purified FGF-5, or a variant or fragment that may not retain full activity), the subject being treated, the severity and type of the condition being treated, and the manner of administration. For example, a therapeutically effective amount of FGF-5 protein, FGFantisense molecule, FGF-5 specific binding agent, and/or autologous CTLs specific to FGF-5 can vary from about 0.01 mg/kg body weight to about 1 g/kg body weight.

The methods disclosed herein have equal application in medical and veterinary settings.

Therefore, the general term "subject being treated" is understood to include all animals (e.g.

humans, apes, dogs, cats, horses, and cows) that require modulation of a CTL response against a tumor that is expressing or overexpressing Therapeutically effective dose: A dose sufficient to stimulate a CTL response to a tumor expressing or over-expressing FGF-5, resulting in a regression of a pathological condition, or which is capable of relieving signs or symptoms caused by the condition, such as regression of the tumor and/or lysis of the cells of the tumor.

Transformed: A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

Transgenic Cell: Transformed cells which contain foreign, non-native DNA.

Tumor: A neoplasm Variants or fragments: The production of FGF-5 protein can be accomplished in a variety of ways (for example see EXAMPLE DNA sequences which encode for the protein, or a fragment or variant of the protein, can be engineered such that they allow the protein to be WO 01/25271 PCT/US00/26689 -21expressed in eukaryotic cells, bacteria, insects, and/or plants. In order to accomplish this expression, the DNA sequence can be altered and operably linked to other regulatory sequences.

The final product, which contains the regulatory sequences and the therapeutic protein, is referred to as a vector. This vector can then be introduced into the eukaryotic cells, bacteria, insect, and/or plant. Once inside the cell the vector allows the protein to be produced.

One of ordinary skill in the art will appreciate that the DNA can be altered in numerous ways without affecting the activity of the encoded protein. For example, PCR may be used to produce variations in the DNA sequence which encodes FGF-5. Such variants may be variants that are optimized for codon preference in a host cell that is to be used to express the protein, or other sequence changes that facilitate expression.

Two types of cDNA sequence variant may be produced. In the first type, the variation in the cDNA sequence is not manifested as a change in the amino acid sequence of the encoded polypeptide. These silent variations are simply a reflection of the degeneracy of the genetic code.

In the second type, the cDNA sequence variation does result in a change in the amino acid sequence of the encoded protein. In such cases, the variant cDNA sequence produces a variant polypeptide sequence. In order to optimize preservation of the functional and immunologic identity of the encoded polypeptide, any such amino acid substitutions may be conservative. Conservative substitutions replace one amino acid with another amino acid that is similar in size, hydrophobicity, etc.

Variations in the cDNA sequence that result in amino acid changes, whether conservative or not, are minimized to enhance preservation of the functional and immunologic identity of the encoded protein. The immunologic identity of the protein may be assessed by determining whether it is recognized by an antibody to FGF-5; a variant that is recognized by such an antibody is immunologically conserved. In particular embodiments, any cDNA sequence variant will introduce no more than 20, for example fewer than 10 amino acid substitutions into the encoded polypeptide.

Variant amino acid sequences can, for example, be 70%, 80%, 90% or even 95% identical to the native amino acid sequence.

Conserved residues in the same or similar proteins from different species can also provide guidance about possible locations for making substitutions in the sequence. A residue which is highly conserved across several species is more likely to be important to the function of the protein than a residue that is less conserved across several species.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.

WO 01/25271 PCT/US00/26689 -22- Additional definitions of terms commonly used in molecular genetics can be found in Benjamin Lewin, Genes V published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

To identify TAA, tumor infiltrating lymphocytes (TIL) were obtained from a metastatic pulmonary lesion from a renal cell carcinoma patient who demonstrated a mixed spontaneous regression of lesions following nephrectomy. These TIL were multiply restimulated with an autologous tumor cell line, expanded in IL-2, and cloned by limiting dilution. One CTL clone, Clone 2, demonstrated HLA-A3 restricted recognition of autologous tumor, but did not recognize an autologous EBV-B cell line or an autologous fibroblast cell line. Limiting dilution appeared to be an important step for the identification of auto-RCC specific CTL activity.

Clone 2 recognized a product of the fibroblast growth factor-5 (FGF-5) gene. Direct sequencing of genomic DNA from autologous EBVB cells showed no mutations in the putative amino acid sequence for FGF-5 in tumor-derived cDNA, indicating that a tumor-specific mutation is not the basis of immune recognition by Clone 2. FGF-5 was not expressed in normal adult organs, and was over-expressed in multiple renal cell carcinomas, prostate carcinomas, and a breast carcinoma. Expression of FGF-5 by renal and other tumors corresponded to the recognition of these tumor cells in an HLA-A3 restricted fashion.

EXAMPLE 1 Isolation of Tumor Infiltrating Lymphocytes (TILs) This example describes the isolation of TILs from a metastatic RCC lung lesion. Similar methods can be used to isolate TILs from other neoplasms.

The surgically resected remnant of a spontaneously regressing metastatic RCC lung lesion was enzymatically digested Collagenase type IV, 30u/ml deoxyribonuclease type IV, and 0.01% hyaluronidase type V [Sigma, St. Louis, MO]) at room temperature (RT) for three hours.

After digestion, the cell suspension was filtered through 100 pm nylon mesh and separated by density gradient using Lymphocyte Separation Medium (Organon Teknica, Durham, NC). The lymphocyte-containing interface was recovered, washed with Hanks' Balanced Salt Solution, and used for TIL culture in RPMI (Biofluids, Rockville, MD) 10% human AB serum (Biochemed Pharmacologicals. Winchester, VA) with 6000 IU/ml of recombinant human IL-2 (Chiron Corp., WO 01/25271 PCT/US00/26689 -23- Emoryville, CA) in 24 well plates at a cell density of 1 x 106 cells/well. The culture was stimulated every two weeks with an irradiated autologous RCC cell line established from a primary left nephrectomy sample (EXAMPLE 2).

An autologous EBV-B cell line was established by EBY infection using a culture supernatant of cell line B95-9 (American Type Culture Collection, ATCC, Manassas, VA). An autologous fibroblast line was established by infecting a one-week old cultured tumor sample with a retrovirus that encoded the papilloma virus E6/E7 proteins and performing limiting dilution. RCC cell lines UOK125, UOK127, UOK130, UOK131, UOK150, and UOK 171 were obtained from Dr. W. Marston Linehan (NCI, Bethesda, MD). Lung cancer cell lines SKGT2, SKGT4, and esophageal cancer cell line HCE-4 were obtained from Dr. David Schrump (NCI, Bethesda, MD). 1570 RCC, 1581 RCC, 1645 RCC, 1764 RCC, CY13, 501 mel, 526 mel, 586 mel. 624.38 mel, 888 mel, 1088 mel, 1199 mel, and 1479 mel were established from surgical samples. SW480, PC-3, and DU 145 were obtained from ATCC (Manassas, VA). TSU-PR1 was obtained from Dr.

Suzanne Topalian (NCI, Bethesda, MD).

EXAMPLE 2 Identification of a CTL Clone with Specific Recognition of Autologous RCC One of the regressing remnants of a metastatic lung lesion of a renal cell carcinoma produced a TIL line when cultured in media with IL-2. This bulk TIL line was stimulated periodically with an irradiated autologous tumor cell line established from a left nephrectomy operation sample. After three months of culture, the autologous tumor-specific T cell clone (Clone 2 CTL) was established by limiting dilution of the bulk TIL line. Clone 2 CTL was CD3*, CD8' and was found to utilize V312 by PCR-based TCR (T-cell receptor) analysis.

The reactivity of Clone 2 CTL was assessed using an autologous RCC cell line (1764 CTL), an autologous EBV-B cell line, an autologous fibroblast cell line, and other allogeneic RCC cell lines described in EXAMPLE 1. RCC cells (5 x 10 cells/well), fibroblasts (5 x 10' cells), or EBV-B cells (1 x 10' cells/well) were plated into 96 well flat bottom plates and 2 x 104 cells/well of Clone 2 CTL were added. After incubating for 20 hours, supernatants were harvested and IFN-y concentration was analyzed by ELISA (Endogen, Wobum, MA), where IFN-y concentration is considered proportional to CTL activation.

ELISA plates (96 well flat bottom, Costar, NY) were coated with anti-human IFN-y monoclonal antibody (2G1. ENDOGEN, MA) at 1 pg/ml, 100 pl/well overnight. After washing.

plates were blocked with PBS-5% FBS (fetal bovin serum) for one hour (200 /l/well), the samples added (100 il/well) and incubated for 90 minutes. Subsequently, the plates were washed and biotin-labeled anti-human IFN-y monoclonal antibody (B133.5, ENDOGEN, MA) was added at WO 01/25271 PCT/US00/26689 -24- /ug/ml, 100 pl/well, and incubated for one hour. The plates were subsequently washed and 2000times diluted HRP-streptavidin conjugate (Zymed Laboratories, CA) was added and incubated for minutes. Plates were washed and DAKO TMB One-Step Substrate System (DAKO Corporation, CA) was added (100 pl/well). The coloration reaction was stopped by adding 0.18 M sulfuric acid (100 pl/well) and the optical density at 450nm wave-length was measured.

Recombinant human IFN-y (ENDOGEN, MA) was used as a standard.

As shown in FIG. 1, the reactivity of Clone 2 CTL with a panel of HLA-typed tumors indicated that Clone 2 CTL recognized an antigen shared among renal cell carcinomas, and this recognition appeared to be restricted by HLA-A3. To confirm the restriction by HLA-A3, a blocking study using HLA-specific monoclonal antibodies (mAbs) was performed. W6/32, MA2.1, and GAPA3 hybridomas were obtained from ATCC and the antibodies purified by Lofstrand Labs (Gaithersburg, MD). B1.23.1 was obtained from NCI, Bethesda, MD. Irradiated tumor cells (5 x 10' cells in 100 pl) were incubated with mAbs (20 ug/ml) for 30 minutes at RT, and 2 x 10' CTL were added. Following 20 hours of culture at 37*C, the supernatant was assayed for IFN-y concentration by ELISA as described above. As a control (FIG. 2A), a CTL and autologous tumor target whose interaction is known to be restricted by HLA-A2 was used. The effect of a blocking mAb on the recognition of autologous tumor by Clone 2 CTL is shown in FIG.

2B (anti-class I MHC mAb=W6/32, anti-HLA-A2 mAb=MA2.1, anti-HLA-A3 mAb=GAPA3, anti-HLA-BC mAb=B1.23.1).

As shown in FIG. 2B, tumor recognition by Clone 2 CTL was maximally reduced by a pan-anti-class I MHC mAb (W6/32) and an anti-HLA-A3 mAb (GAPA3). At least 75% inhibition was observed, when compared to the CTL activity of the HLA-A2 restricted CTL. However, blocking by an anti-HLA-A2 mAb (MA2.1) and an anti-HLA-B/C mAb (Bl.23.1) was similar to the effect observed with the anti HLA-B/C antibody on an HLA-A2-restricted CTL. Therefore, Clone 2 CTL reactivity is restricted by HLA-A3.

EXAMPLE 3 Cloning the Antigen Recognized by Clone 2 CTL To identify the gene coding for the antigen recognized by Clone 2 CTL, expression cloning was performed utilizing a plasmid-based cDNA library. Poly RNA was prepared from the autologous RCC cell line using a mRNA isolation system (Fast Track kit 2.0; Invitrogen, Carlsbad, CA). cDNA was prepared with the Superscript plasmid system (Life Technologies, Rockville, MD) and ligated into the eukaryotic plasmid expression vector pME18S (Dr. Atsushi Miyajima, University of Tokyo). After electroporation and titration, a cDNA library was prepared in pools by inoculating approximately 100 bacterial clones/well in 1 ml LB/well and purifying WO 01/25271 PCT/US00/26689 plasmid using the Qiaprep 96 turbo system (Qiagen, Valencia, CA).

To serve as the antigen-presenting target cell line, COS-7 cells were retrovirally transduced with the HLA-A3 gene derived from the autologous tumor cell line. As a control target, the HLA-A0201 gene was retrovirally transduced into COS-7 cells (hereafter referred to as COS-A3 or COS-A2, respectively). To introduce HLA-A3 into non-HLA-A3-expressing cells, the HLA-A3 gene was cloned from autologous 1764 RCC by RT-PCR (forward primer TTGGGGAGGGAGCACAGGTCAGCGTGGGAAG-3', SEQ ID NO: 24; reverse primer GGACTCAGAATCTCCCCAGACGCCGAG-3'. SEQ ID NO: 25), sequenced, and subcloned into the retroviral vector pRx-IRES-Bsr (Wakimoto et al., Jpn. J. Cancer Res. 88:296-305, 1997).

Vesicular stomatitis virus G protein-pseudotyped retrovirus was prepared by transiently transfecting the 293 GP cell line using standard methods (see Wang et al., Cancer Res. 58:3519-25, 1998, incorporated by reference). Forty-eight hours after transfection, culture supernatant was harvested, filtered, and used for infection with 8 pg/ml of polybrene. Infection efficiencies ranged from 100% and the HLA-A3 positive population was further selected by 5 pg/ml of blasticidin S (Calbiochem, San Diego, CA).

Three independent clones: 1A3 (SEQ ID NOS: 7 and 6A4 (SEQ ID NOS: 11 and 12).

and 10E4 (SEQ ID NOS: 13-18) selected after subcloning reactive cDNA pools were transfected (100 ng of plasmid) into 5 x 10' COS-A2 and COS-A3 using 1 p of lipofectAMINE (Life Technologies, Rockville, MD) in 96-well plates according to the manufacturer's instructions. The next day, Clone 2 CTL (2 x 10' cells/well) were added and after 20 hours incubation, supernatants were assayed for IFN-y secretion by Clone 2 CTL as described in the above EXAMPLES. An gene from an independent source (FGF-5 MG, from Dr. Mitchell Goldfarb, Mount Sinai School of Medicine) was analyzed the same way.

As shown in FIG 3A, the measured IFN-y concentration is greater than 2500 pg/ml for the COS-A3 cells, but less than 1 pg/ml for the COS-A2 cells. The clones that conferred recognition of COS-A3 by the CTL (FIG. 3A) were identified and sequenced on an automated sequencer (ABI Prizm 310; Perkin-Elmer Corp., Foster City, CA).

All three clones encoded all or part of an FGF-5 sequence. As shown in FIG. 3B, the sequence of the clones is similar, but not identical to, the human FGF-5 sequence from GenBank Accession No M37825 (MUSFGF5A; SEQ ID NOS 1-4 and FIG. There were eight nucleotide mismatches at positions 79 287 732 810 876 895 974 and 975 four of which resulted in amino acid changes. The smallest cDNA clone (FIG. 3B) recognized by Clone 2 CTL (1A3-1) (SEQ ID NOS: 7 and 8) had six nucleotide mismatches and two of these changed the amino acid sequence. The longest clone (10E4-1) contained the full-length FGF-5 cDNA sequence (SEQ ID NOS 15 and 17).

WO 01/25271 PCT/US00/26689 -26- The genomic sequence for FGF-5 obtained from autologous EBV-B cells was identical to the autologous tumor-derived sequence for FGF-5. In addition, FGF-5 cDNA from an independent source (FGF-5 MG) was also recognized by Clone 2 CTL when transfected into COS-A3 (FIG.

3A). The DNA sequence of the FGF-5 MG plasmid was identical to clone 1A3-1 (SEQ ID NO: Therefore, the previously published FGF-5 sequence available on GenBank Accession No.

M37825 contains at least eight nucleotide and four amino acid differences.

These data demonstrate that Clone 2 CTL recognizes a non-mutated epitope within the 268 amino acid full-length FGF-5. Fragments of FGF-5 were also shown to activate CTL clonal expansion. Fragments of FGF-5 tested are shown in FIG. 7. Fragments which activated CTL clonal expansion are shown in FIG. 7 as open bars, while fragments which did not activate CTL clonal expansion are shown in FIG. 7 as filled bars. Fragment 610-822 did not activate an immune response, while a fragment as short as 60 amino acid residues, 643-822, (SEQ ID NO 19) are immunogenic in HLA-A3 individuals. The region of amino acids 643-679 may be modified in cells. The disclosure therefore provides a number of species of immunogenic petides, and permits one to easily screen for other immunogenic peptides with the assay of this example.

However, other epitopes within the FGF-5 peptide may be immunogenic for individuals who are not HLA-A3' (for example HLA-A2 subjects, who constitute about 50% of the Caucasian population). Hence amino acid sequences as short as eight contiguous amino acid residues, for example 8-12, or 20 or less residues of FGF-5 (SEQ ID NOS: 4, 6, 8, 10, 12, and 16-19) are included within the present disclosure. In other embodiments, sequences at least contiguous residues, for example at least 80, 100, 120, 140, 160, 180, 200, 220 or 240 contiguous amino acids of the sequence shown in FIG. 6, or SEQ ID NOS: 3 or 18 are suitable for use with this method.

EXAMPLE 4 Analysis of FGF-5 Expression in Normal and Tumor Cells To analyze FGF-5 expression, real-time-PCR (RT-PCR) analysis and INF-y release was measured in normal cells, tumor cells naturally expressing HLA-A3, and in non-HLA-A3 expressing tumors in which HLA-A3 was introduced by retroviral transduction (see EXAMPLE 3).

Similar methods can be used to analyze FGF-5 expression in any sample from any organism.

To determine the FGF-5 and P-actin copy number, real-time PCR was used. Total RNA of normal adult human tissues were purchased from Clontech Laboratories (Palo Alto, CA). Total RNA from tumor cell lines were prepared using the RNeasy mini kit according to the manufacturer's instructions (Qiagen, Valencia. CA). First strand cDNA was synthesized using the Superscript Preamplification System (Life Technologies. Rockville, MD) utilizing 5 pg of total WO 01/25271 PCT/US00/26689 -27- RNA from either normal tissue or tumor cell lines. For the analysis, 2 ul out of 20 pl of the first strand cDNA was used. Two forward primers (FGF-F1 (SEQ ID NO: 20) and FGF-F2 5'-TGCAGAGTGGGCATCGGTTTC (SEQ ID NO: 21)) were planned in exon 1 and two reverse primers (FGF-RI 5'-TATGCTCAATGCAGAGGTAC (SEQ ID NO: 22) and FGF-R2 5'-CGTAGTCCCTGTTATTTAAC (SEQ ID NO: 23)) were planned in exon 3.

Using the FI and RI pair and Taq polymerase (Life Technologies, Rockville, MD), the first PCR reaction was performed at 94*C for one minute followed by 16 cycles of 94*C for seconds, 61 C for 45 seconds, and 72 0 C for 60 seconds. As a template for the second nested PCR reaction, 2 tl out of 50 pl of the first PCR reaction was used with the primer pair of F2 and R2 using the same PCR program. The PCR product (10 pi) was subjected to agarose gel analysis. As a control, the expression level of P-actin was also measured.

To measure IFN-y concentration, tumor cells (5 x 10') were plated in flat bottom 96 well plates and 2 x 10' Clone 2 CTL were added. After incubating for 20 hours, the supernatants were assayed for IFN-y concentration by ELISA as described above in EXAMPLE 2.

copy number was normalized to P-actin copy number in each cell line and the copy number/10s P-actin copy number plotted (filled bars in FIGS. 4 and In contrast to autologous RCC cell lines (for example 1764 RCC, FIG. 5) which showed strong expression, in the normal tissues analyzed, FGF-5 expression was below the level of detection. As shown in FIG. 4, in normal tissues FGF-5 was only detectable in brain and kidney. However, the copy number in these tissues (about 20 FGF-5 copies/10 s P-actin copies) was lower than the calculated recognition threshold for CTL clone 2 (FIG. No detectable FGF-5 expression was observed in normal tissues using Northern blotting.

In contrast, as shown in FIG. 5, six of 10 RCC, two of three prostate carcinomas (PC3 and TSU-PRI) and 1 of 2 breast cancers (MDA231) showed significant recognition by CTL clone 2 (as judged by IFN-y>50 pg/ml, and at least twice that generated against an autologous EBVB control). In addition, these carcinomas showed a higher FGF-5 copy number (bars in FIG. compared with normal tissues (FIG. for example at least 50%, at least 75%, at least 500% or even at least 1000% greater FGF-5 copy number. None of eight malignant melanomas (526, 586, 624.38, 1479, 501, 888, 1088, and 1199 mel), three lung cancers (SKGT-2, 4, and one esophageal carcinoma (HCE-4), and two colon carcinomas (SW480 and CYI3) were recognized.

As shown in the inset graph of FIG. 5, the recognition by CTL clone 2 highly correlated with FGFcopy number (p<0.0001). Marginally recognized cells such as 1570 RCC and TSU-PR1 indicates that the FGF-5 expression threshold for recognition by CTL clone 2 was 50-100 mRNA copies/10' D actin copies. Therefore, cells having at least 75 FGF-5/10' P-actin copies, for WO 01/25271 PCT/US00/26689 -28example at least 100 FGF-5/10 P-actin copies, for example at least 500 FGF-5/105 P-actin copies, for example at least 1000 FGF-5/105 s -actin copies, are expected to generate an immune response.

EXAMPLE Immune Therapies This example describes how the expression of FGF-5 by tumor cells can be used to stimulate the immune system for cancer therapies. In particular embodiments, the immune therapy is preceded by determining whether FGF-5 expression is increased, for example using the method of EXAMPLE 4. The immune therapy can then be administered if FGF-5 expression is found to be increased in the tumor cell. The methods disclosed herein can also be combined with other antineoplastic treatments, such as radiotherapy or administration of anti-neoplastic drugs or biologics.

as a Tumor Antigen satisfies several criteria as a tumor antigen for cancer therapy. It is expressed substantially specifically in tumors but not in normal tissues in humans, particularly adults (FIGS. 4 and 5A and 5B). Although FGF-5 expression has been reported at a low level in brain (Goldfarb et al. Ann. NYAcad. Sci. 638:38-52, 1991), FGF-5 was not detected by RT-PCR in brain tissue, even though RT-PCR was sensitive enough to detect FGF-5 expression in tumors. Hence expression in adult human brain is low, if present at all. As an immunologically privileged site, the central nervous system is also unlikely to be an auto-immune target during FGF-5 targeted immunotherapy.

is a non-mutated antigen, over-expressed in some RCC, breast carcinomas, and prostate cancers (FIGS. 5A and 5B). According to the literature, FGF-5 is over-expressed in bladder cancers and pancreatic cancers (Yoshimura et al., Cancer Lett. 103:91-7, 1996; Kornman et al. Oncogene 15:1417-24, 1997). Therefore it represents an attractive tumor antigen for these cancers, and particularly such cancers which lack identified vaccine targets.

Most tumor-specific antigens are categorized in three groups; 1) tissue differentiation antigens MART-1, cyrosinase, gplOO); 2) tumor-specific mutations 0-catenin, CASP-8); and 3) proteins sharing expression on tumor and testis MAGE-1, BAGE). Recently antigens overexpressed on tumors but not expressed by testis have been described telomerase catalytic subunit (hTERT), p53). FGF-5 is believed to belong to this new class of tumor antigens.

Therapeutics directed against A therapeutic directed against FGF-5 is believed to have substantial therapeutic potential to interfere with important biological functions of tumors, since FGF-5 is believed to have an WO 01/25271 PCT/US00/26689 -29important biological function in maintaining the malignant phenotype of a tumor. Examples of such therapies include, but are not limited to immuno-therapeutics such as anti-FGF-5 antibodies (EXAMPLE 9) or the use of antisense molecules which disrupt FGF-5 expression (EXAMPLES 19 AND 20). FGF-5 was originally identified as an oncogene that transformed NIH-3T3 cells (Zhan et al., Mol. Cell. Biol. 8:3487-95, 1988). In pancreatic cancer, FGF-5 is believed to have autocrine and paracrine growth-promoting activities, and to function as an angiogenic factor such that it may therefore be suitable as an anti-angiogenic target.

Administration of Autologous CTLs, specific to Therapeutic approaches based upon the disclosure take advantage of a response by a subject's immune system, leading to treatment of cells that express or overexpress FGF-5 (for example lysis or death of the cells, or regression of the tumor), such as HLA-A3 restricted RCC cells. One such therapeutic approach is the administration of autologous CTLs, specific to to a subject with RCC, or other tumor characterized by overexpression of FGF-5. Techniques for obtaining such CTLs in vitro are known, as illustrated by the foregoing examples. Autologous peripheral blood mononuclear cells (PBMCs) such as lymphocytes can be used, or cells of dendritic autologous cells, which are stimulated with the FGF-5 antigen.

Generally, a sample of cells taken from a subject, such as blood cells, are contacted with a cell presenting the complex and capable of provoking CTLs to proliferate. In addition to the target cells listed above, the target cell can be a transfectant, such as a COS cell of the type described above. These transfectants present the desired HLA complex on their surface and, when combined with a CTL of interest, stimulate proliferation of the CTL of interest. COS cells, such as those used herein, are widely available, as are other suitable host cells. Specific production of a CTL clone has been described above. The clonally expanded autologous CTLs may then be administered to the subject.

In adoptive transfer, disclosed by Greenberg, J. Immunol. 136:1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et al, Eur. J. Immunol. 21:1403-10, 1991; and Kast el al., Cell 59:603-14, 1989, cells presenting the desired complex (FGF-5) are combined with CTLs, leading to proliferation of the specific CTLs. The proliferated CTLs arc administered to a subject suffering from an FGF-5 over-expressing tumor, such as RCC, carcinoma of the breast, prostate, bladder or pancreas. The CTLs then lyse the FGF-5 expressing tumor cells, and achieve the desired therapeutic goal of assisting in regression of the tumor.

CTLs can also be stimulated in vivo, using a number of approaches. One approach is the use of non-proliferative cells expressing the complex of FGF-5 and an HLA presenting molecule (such as HLA-A3), to stimulate an immune response. The cells used in this approach may be those WO 01/25271 PCT/US00/26689 that normally express the complex, such as irradiated tumor cells or cells transfected with one or both of the genes necessary for presentation of the complex. Chen et al., Proc. Natl. Acad. Sci.

USA 88:110-4, 1991 exemplifies this approach. Similarly, vectors carrying one or both of the genes of interest may be used, such as viral or bacterial vectors (EXAMPLE 14). For example, nucleic acids which encode FGF-5 may be operably linked to promoter and enhancer sequences which direct expression of the FGF-5 in certain tissues or cell types. The nucleic acid may be incorporated into an expression vector (EXAMPLE 8).

Expression vectors may be unmodified extrachromosomal nucleic acids, plasmids or viral genomes constructed or modified to enable insertion of exogenous nucleic acids, such as those encoding FGF-5. Nucleic acids encoding FGF-5 also may be inserted into a retroviral genome, thereby facilitating integration of the nucleic acid into the genome of the target tissue or cell type.

In these systems, the gene of interest is carried by a microorganism, a Vaccinia virus or retrovirus, which infect host cells. The cells which result present the complex of interest and are recognized by autologous CTLs, which then proliferate.

Administration of A similar effect can be achieved by combining the FGF-5, or a stimulatory fragment thereof, with an adjuvant to facilitate incorporation into cells (such as HLA-A3 presenting cells) in vivo. Generally, subjects receive an intradermal injection of an effective amount of the and/or immunogenic fragments or variants derived therefrom. Initial doses can be followed by booster doses, following immunization protocols standard in the art.

As part of the immunization protocols, substances which potentiate the immune response may be administered with nucleic acid or peptide components of a cancer vaccine. Such immune response potentiating compounds may be classified as either adjuvants or cytokines. Adjuvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages), activating macrophages and stimulating specific sets of lymphocytes.

Adjuvants of many kinds are well known in the art; specific examples include MPL (SmithKline Beecham), a congener obtained after purification and acid hydrolysis of Salmonella minnesota Re 595 lipopolysaccharide, QS21 (SmithKline Beecham), a pure QA-21 saponin purified from Quillja saponaria extract, and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol. Cytokines are also useful in vaccination protocols as a result of lymphocyte stimulatory properties. Many cytokines useful for such purposes will be known to one of ordinary skill in the art, including interleukin-12 (IL-12) which has been shown to enhance the protective effects of vaccines (Science 268: 1432-4, 1995).

WO 01/25271 PCT/US00/26689 -31- When administered, the therapeutic compositions of the present disclosure may be, for example, administered in pharmaceutically acceptable preparations (EXAMPLE 18). Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents (including other anti-neoplastic agents).

When it is desired to stimulate a cytotoxic lymphocyte immune response using a therapeutic composition disclosed herein, it is believed that doses of immunogens ranging from one nanogram/kilogram to 100 miligrams/kilogram, depending upon the mode of administration, would be effective. The range may, for example, be between 500 nanograms and 500 micrograms per kilogram. The absolute amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual subject parameters including age, physical condition, size, weight, and the stage of the disease.

These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

EXAMPLE 6 Probes, Primers and DNA Encoding Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided herein (SEQ ID NOS: 3, 5, 7, 9, 11, 13-15, 17 and FIG. A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992).

Primers are short nucleic acid molecules, such as DNA oligonucleotides 15 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.

Methods for preparing and using probes and primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York, 1989), Ausubel er al. (In Current Protocols in Molecular Biology. Greene Publ. Assoc. and Wiley- WO 01/25271 PCT/US00/26689 -32- Intersciences, 1992), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA). One of ordinary skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides of the gene will anneal to a target sequence. Thus, in order to obtain greater specificity, probes and primers can be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides of FGF-5 gene sequences.

The disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the disclosed gene sequences. Such molecules may comprise at least 20, 25. 30, 35. 40 or consecutive nucleotides of these sequences, and may be obtained from any region of the disclosed sequences. By way of example, the gene sequences may be apportioned into halves or quarters based on sequence length, and the isolated nucleic acid molecules may be derived from the first or second halves of the molecules, or any of the four quarters.

The probes and primers can be used for the detection of FGF-5 DNA and the detection of specific mutations or polymorphisms associated with tumor development or prognosis.

EXAMPLE 7 FGF-5 Sequence Variants The amino acid sequences of FGF-5 (SEQ ID NOS: 4, 6, 8, 10, 12, and 16-19 and FIG.

6) encoded by FGF-5 cDNAs (SEQ ID NOS: 3, 5, 7, 9, 11, 13-15, 17 and FIGS. 3C and 6) are disclosed. The distinctive functional characteristic of FGF-5 is its ability to modulate an immune response, for example the ability to stimulate an HLA-A3-restricted CTL immune response against FGF-5 expressing or over-expressing tumors, as measured by IFN-y concentration. This activity is reduced by about at least 50%, for example 75%, in the presence of pan-anti-class I MHC mAb (such as W6/32) or anti-HLA-A3 mAb (such as GAPA3). Another distinctive functional characteristic of FGF-5 is the observation that FGF-5 is expressed at undetectable levels in normal adult tissues, but is expressed or over-expressed in some cancers, such as RCC, prostate and breast carcinomas. These activities of FGF-5 proteins may readily be determined using the assays described above, for example those described in EXAMPLES 2-4.

Having presented the nucleotide sequence of FGF-5 cDNAs and the amino acid sequence of these proteins, this disclosure facilitates the creation of DNA molecules, and thereby proteins, which are derived from those disclosed but which vary in their precise nucleotide or amino acid WO 01/25271 PCT/US00/26689 33sequence from those disclosed. Such variants may be obtained through a combination of standard molecular biology laboratory techniques and the nucleotide sequence information disclosed herein.

variants, polymorphisms, mutants, and fragments will retain the ability to modulate an immune response, for example stimulate an immune response, for example, an HLA-A3restricted CTL immune response. Such an immune response in particular embodiments, induces a regression of the FGF-5 expressing tumor. In other embodiments, in the presence of pan-anti-class I MHC mAb (such as W6/32) or anti-HLA-A3 mAb (such as GAPA3) and FGF-5 variants, polymorphisms, mutants, and fragments, the stimulated immune response, such as an HLA-A3restricted CTL immune response, is blocked. This as observed by an at least 50% decrease in IFN-y concentration using the assay described in EXAMPLE 2.

Substitutions of FGF-5 amino acid sequences can be made either in regions that are highly conserved between species, or regions that share less conservation between species. In particular embodiments regions of FGF-5 which are highly conserved between species ideally do not substantially diverge from the wild-type sequence shown in SEQ ID NOS: 4, 6. 8, 10, 12, and 16- 19 and FIG. 6. Other important residues include the receptor binding domain and the heparin binding domain (both located in the center of the protein). Other important residues include amino acids 161-220 of FGF-5 (shown in SEQ ID NO: 19). In these regions, conservative substitutions will be better tolerated than non-conservative substitutions. Alterations in regions that are less conserved are predicted to have less of an effect on the function of the FGF-5 protein. Eight examples of differences among the FGF-5 sequences are at positions 79 287 732 810 876 895 974 and 975 Thus, the methods disclosed herein may be practiced with molecules that differ from the exact molecules disclosed, but which retain the requisite ability to modulate an immune response or FGF-5 expression or activity.

Variants and fragments may retain at least 60%, 70%, 75% 80%, 85%, 90%, 95%. 98%, or greater sequence identity to the FGF-5 amino acid sequences disclosed herein, and in particular embodiments at least this much identity to SEQ ID NOS: 4, 6, 8. 10, 12, and 16-19 and FIG. 6.

Less identity is allowed, as long as the variant FGF-5 sequence maintains the functional activity of the FGF-5 protein as defined herein. Such activity can be readily determined using the assays disclosed herein.

The simplest modifications involve the substitution of one or more amino acid residues (for example 2. 5 or 10 residues) for amino acid residues having similar biochemical properties.

These so-called conservative substitutions are likely to have minimal impact on the activity of the resultant protein. Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions WO 01/25271 PCT/US00/26689 -34generally are conservative when it is desired to finely modulate the characteristics of the protein.

Examples of amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gin or His for Asn; Glu for Asp; Ser for Cys; Asn for Gin; Asp for Glu; Pro for Gly; Asn or Gin for His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gin for Lys; Leu or lie for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and lie or Leu for Val.

Amino acid substitutions are typically of single residues, for example 1, 2, 3, 4, 5, 10 or more substitutions; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. Obviously, the mutations that are made in the DNA encoding the protein must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.

Changes in function or immunological identity (increase or decrease) are made by selecting substitutions that are less conservative than those listed above, selecting residues that differ more significantly in their effect on maintaining the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which a hydrophilic residue, seryl or threonyl, is substituted for (or by) a hydrophobic residue, leucyl, isoleucyl, phenylalanyl, valyl or alanyl: a cysteine or proline is substituted for (or by) any other residue; a residue having an electropositive side chain, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, glutamyl or aspartyl; or a residue having a bulky side chain, phenylalanine, is substituted for (or by) one not having a side chain, glycine. Such FGF-5 variants can be readily selected for additional testing by performing an assay (such as that shown in EXAMPLES 2-4) to determine if the FGF-5 variant retains the ability to stimulate an HLA-A3-restricted CTL immune responscjagainst expressing or over-expressing tumors, as measured by IFN-y concentration.

Variant DNA molecules include those created by standard DNA mutagenesis techniques, for example, M13 primer mutagenesis. Details of these techniques are provided in Sambrook et al.

(In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Chapter herein incorporated by reference). By the use of such techniques, variants may be created which differ in minor ways from those disclosed. DNA molecules and nucleotide sequences which are derivatives of those specifically disclosed herein and which differ from those disclosed by the WO 01/25271 PCT/US00/26689 deletion, addition or substitution of nucleotides while still encoding a protein which possesses the functional characteristics of FGF-5 proteins are comprehended by this disclosure.

Also disclosed are small DNA molecules which are derived from the disclosed DNA molecules. Such small DNA molecules include oligonucleotides suitable for use as hybridization probes or PCR primers. As such, these small DNA molecules will comprise at least a segment of cDNA molecules or FGF-5 genes. For example, the sequences will comprise at least 30, 40, or 50 contiguous nucleotides of nucleotide sequence of FGF-5 (SEQ ID NOS: 3, 5, 7, 9, 11, 13-15, 17 and FIG. 6, or their complementary strands) at least 20-50 consecutive nucleotides of the FGF-5 cDNA/gene sequences). It will be appreciated that such longer length nucleotide sequences will provide greater specificity in hybridization or PCR applications than shorter length sequences. Accordingly, superior results may be obtained using these longer stretches of consecutive nucleotides.

DNA molecules and nucleotide sequences which are derived from the disclosed DNA molecules as described above may also be defined as DNA sequences which hybridize under stringent conditions to the DNA sequences disclosed, or fragments thereof. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used.

Generally, the temperature of hybridization and the ionic strength (especially the Na' concentration) of the hybridization buffer will determine the stringency of hybridization.

Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989 ch. 9 and 11), herein incorporated by reference.

Specific hybridization refers to the binding, duplexing, or hybridizing of a molecule only or substantially only to a particular nucleotide sequence when that sequence is present in a complex mixture total cellular DNA or RNA). Specific hybridization may also occur under conditions of varying stringency.

Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially the Na concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989 chapters 9 and 11).

By way of illustration only, a hybridization experiment may be performed by hybridization of a DNA molecule (for example, a variant of a FGF-5 cDNA) to a target DNA molecule (for WO 01/25271 PCT/USOO/26689 -36example, a FGF-5 cDNA) which has been electrophoresed in an agarose gel and transferred to a nitrocellulose membrane by Southern blotting (Southern, J. Mol. Biol. 98:503, 1975), a technique well known in the art and described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

Hybridization with a target probe labeled, for example, with [P]-dCTP is generally carried out in a solution of high ionic strength such as 6xSSC at a temperature that is 20-25 0

C

below the melting temperature, Tm, described below. For such Southern hybridization experiments where the target DNA molecule on the Southern blot contains 10 ng of DNA or more, hybridization is typically carried out for 6-8 hours using 1-2 ng/ml radiolabeled probe (of specific activity equal to 109 CPM/jg or greater). Following hybridization, the nitrocellulose filter is washed to remove background hybridization. The washing conditions should be as stringent as possible to remove background hybridization but to retain a specific hybridization signal.

The term T, represents the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Because the target sequences are generally present in excess, at Tn, of the probes are occupied at equilibrium. The T.of such a hybrid molecule may be estimated from the following equation (Bolton and McCarthy, Proc. Natl. Acad. Sci. USA 48:1390, 1962): Tm 81.5*C 16.6(loglo[Na+]) 0.41(%G+C) formamide) (600/1); where I the length of the hybrid in base pairs.

This equation is valid for concentrations of Na* in the range of 0.01 M to 0.4 M, and it is less accurate for calculations of Tm in solutions of higher The equation is also primarily valid for DNAs whose G+C content is in the range of 30% to 75%, and it applies to hybrids greater than 100 nucleotides in length (the behavior of oligonucleotide probes is described in detail in Ch. 11 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

Thus, by way of example, for a 150 base pair DNA probe with a hypothetical GC content of 45 a calculation of hybridization conditions required to give particular stringencies may be made as follows. For this example, it is assumed that the filter will be washed in 0.3 xSSC solution following hybridization, thereby: 0.045 M; %GC 45%; Formamide concentration 0; 1 150 base pairs; Tm=81.5 16.6(logoNal*) (0.41 x 45) (600/150); and so T, 74.4 C.

The Tm of double-stranded DNA decreases by 1-1.5 C with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81:123, 1973). Therefore, for this given example, washing the filter in 0.3 xSSC at 59.4-64.4*C will produce a stringency of hybridization equivalent to 90%; that is, DNA molecules with more than 10% sequence variation relative to the target WO 01/25271 PCT/US00/26689 -37cDNA will not hybridize. Alternatively, washing the hybridized filter in 0.3 xSSC at a temperature of 65.4-68.4C will yield a hybridization stringency of 94%; that is, DNA molecules with more than 6% sequence variation relative to the target cDNA molecule will not hybridize. The above example is given entirely by way of theoretical illustration. One skilled in the art will appreciate that other hybridization techniques may be utilized and that variations in experimental conditions will necessitate alternative calculations for stringency.

Examples of stringent conditions are those under which DNA molecules with more than 15%, 10%, 6% or 2% sequence variation (also termed "mismatch") will not hybridize.

Stringent conditions are sequence dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An example of stringent conditions is no more than about 5"C lower than the thermal melting point T. for the specific sequence at a defined ionic strength and pH. An example of stringent conditions is a salt concentration of at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and a temperature of at least about 30"C for short probes 10 to 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For example, conditions of 5X SSPE (750 mM NaCI, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30 0 C are suitable for allele-specific probe hybridizations.

A perfectly matched probe has a sequence perfectly complementary to a particular target sequence. The test probe is typically perfectly complementary to a portion (subsequence) of the target sequence. The term "mismatch probe" refers to probes whose sequence is deliberately selected not to be perfectly complementary to a particular target sequence.

Transcription levels can be quantitated absolutely or relatively. Absolute quantitation can be accomplished by inclusion of known concentrations of one or more target nucleic acids (for example control nucleic acids such as Bio B or with a known amount the target nucleic acids themselves) and referencing the hybridization intensity of unknowns with the known target nucleic acids (for example by generation of a standard curve).

The degeneracy of the genetic code further widens the scope of the disclosure as it enables major variations in the nucleotide sequence of a DNA molecule while maintaining the amino acid sequence of the encoded protein. For example, the eighteenth amino acid residue of FGF-5 protein is alanine. This is encoded in the FGF-5 cDNA by the nucleotide codon triplet GCC. Because of the degeneracy of the genetic code, three other nucleotide codon triplets, GCT, GCA and GCG, also code for alanine. Thus, the nucleotide sequence of the FGF-5 cDNA could be changed at this position to any of these three codons without affecting the amino acid composition of the encoded protein or the characteristics of the protein. Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from the cDNA molecules disclosed herein using standard DNA WO 01/25271 PCT/USOO/26689 -38mutagenesis techniques as described above, or by synthesis of DNA sequences. DNA sequences which do not hybridize under stringent conditions to the cDNA sequences disclosed by virtue of sequence variation based on the degeneracy of the genetic code are herein also comprehended by this disclosure.

One skilled in the art will recognize that the DNA mutagenesis techniques described above may be used not only to produce variant DNA molecules, but will also facilitate the production of proteins which differ in certain structural aspects from FGF-5 proteins, yet which proteins are clearly derivative of this protein and which maintain the essential characteristics of a FGF-5 protein as defined above. Newly derived proteins may also be selected in order to obtain variations on the characteristic of a FGF-5 protein, as will be more fully described below. Such derivatives include those with variations in amino acid sequence including minor deletions, additions and substitutions.

While the site for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed protein variants screened for optimal activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence as described above are well known.

genes, cDNA, DNA molecules derived therefrom and the protein encoded by these cDNAs and derivative DNA molecules may be utilized in aspects of both the study of FGF-5 and for diagnostic and therapeutic applications related to FGF-5. Those skilled in the art will recognize that the utilities herein described are not limited to the specific experimental modes and materials presented and will appreciate the wider potential utility of this disclosure.

EXAMPLE 8 Recombinant Expression of With the provision of FGF-5 cDNAs (SEQ ID NOS: 3, 5, 7, 9, 11, 13-15, 17 and FIGS.

3C and 6) and amino acid sequences (SEQ ID NOS: 4, 6, 8, 10, 12, and 16-19 and FIG. the expression and purification of the corresponding proteins by standard laboratory techniques is now enabled. The purified protein may be used for functional analyses, antibody production and therapy in a subject as described herein. Furthermore, the DNA sequence of FGF-5 cDNAs and any variant or fragment thereof, can be manipulated in studies to understand the expression of the gene and the function of its product.

Partial or full-length cDNA sequences encoding an FGF-5 protein, may be ligated into bacterial expression vectors. Methods for expressing large amounts of protein from a cloned gene introduced into E. coli may be utilized for the purification, localization and functional analysis of WO 01/25271 PCT/US00/26689 -39proteins. For example, fusion proteins consisting of amino terminal peptides encoded by a portion of the E. coli lacZ or trpE gene linked to FGF-5 may be used to prepare polyclonal and monoclonal antibodies against FGF-5. Thereafter, these antibodies may be used to purify proteins by immunoaffinity chromatography, in diagnostic assays to quantitate the levels of protein, to localize FGF-5 in tissues and individual cells by immunofluorescence and as a therapeutic to treat expressing or over-expressing tumors.

Intact native protein, or variants or fragments thereof, may also be produced in E. coli in large amounts for functional studies. Methods and plasmid vectors for producing fusion proteins and intact native proteins in bacteria are described in Sambrook el al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Chapter 17, herein incorporated by reference). Such fusion proteins may be made in large amounts, are easy to purify, and can be used to elicit antibody response. Native proteins can be produced in bacteria by placing a strong, regulated promoter and an efficient ribosome binding site upstream of the cloned gene. If low levels of protein are produced, additional steps may be taken to increase protein production; if high levels of protein are produced, purification is relatively easy. Suitable methods are presented in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and are well known in the art. Often, proteins expressed at high levels are found in insoluble inclusion bodies. Methods for extracting proteins from these aggregates are described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Chapter 17).

Vector systems suitable for the expression of lacZ fusion genes include the pUR series of vectors (Ruther and Muller-Hill, 1983, EMBO J. 2:1791), pEX 1-3 (Stanley and Luzio, 1984, EMBO J. 3:1429) and pMR100 (Gray et al., 1982, Proc. Nail. Acad. Sci. USA 79:6598). Vectors suitable for the production of intact native proteins include pKC30 (Shimatake and Rosenberg, 1981, Nature 292:128), pKK177-3 (Amann and Brosius, 1985, Gene 40:183) and pET-3 (Studiar and Moffatt, 1986, J. Mol. Biol. 189:113). FGF-5 fusion proteins may be isolated from protein gels, lyophilized, ground into a powder and used as an antigen. The DNA sequence can also be transferred to other cloning vehicles, such as other plasmids, bacteriophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al., 1987, Science 236:806-12). These vectors may then be introduced into a variety of hosts including somatic cells, and simple or complex organisms, such as bacteria, fungi (Timberlake and Marshall, 1989, Science 244:1313-7), invertebrates, plants (Gasser and Fraley, 1989, Science 244:1293), and mammals (Pursel et al., 1989, Science 244:1281-8), which cell or organisms are rendered transgenic by the introduction of the heterologousFGF-5 cDNA.

WO 01/25271 PCT/US00/26689 For expression in mammalian cells, the cDNA sequence may be ligated to heterologous promoters, such as the simian virus SV40, promoter in the pSV2 vector (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6), and introduced into cells, such as monkey COS-1 cells (Gluzman, 1981, Cell 23:175-82), to achieve transient or long-term expression. The stable integration of the chimeric gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin (Southern and Berg, 1982, J. Mol. Appl. Genet. 1:327-41) and mycophoenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6).

DNA sequences can be manipulated with standard procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequencealteration via single-stranded bacteriophage intermediate or with the use of specific oligonucleotides in combination with PCR.

The cDNA sequence (or portions derived from it) or a mini gene (a cDNA with an intron and its own promoter) may be introduced into eukaryotic expression vectors by conventional techniques. These vectors are designed to permit the transcription of the cDNA eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation. Vectors containing the promoter and enhancer regions of the SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation and splicing signal from SV40 are readily available (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci.

USA 78:2072-6; Gorman et al., 1982, Proc. Natl. Acad. Sci USA 78:6777-81). The level of expression of the cDNA can be manipulated with this type of vector, either by using promoters that have different activities (for example, the baculovirus pAC373 can express cDNAs at high levels in S. frugiperda cells (Summers and Smith, 1985, Genetically Altered Viruses and the Environment, Fields et al. (Eds.) 22:319-328, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.) or by using vectors that contain promoters amenable to modulation, for example, the glucoconicoid-responsive promoter from the mouse mammary tumor virus (Lee et al., 1982, Nature 294:228). The expression of the cDNA can be monitored in the recipient cells 24 to 72 hours after introduction (transient expression).

In addition, some vectors contain selectable markers such as the gpt (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6) or neo (Southern and Berg, 1982, J. Mol. Appl.

Genet. 1:327-41) bacterial genes. These selectable markers permit selection of transfected cells that exhibit stable, long-term expression of the vectors (and therefore the cDNA). The vectors can be maintained in the cells as episomal, freely replicating entities by using regulatory elements of viruses such as papilloma (Sarver et al., 1981, Mol. Cell Biol. 1:486) or Epstein-Barr (Sugden et al.. 1985. Mol. Cell Biol. 5:410). Alternatively, one can also produce cell lines that have WO 01/25271 PCT/US00/26689 -41integrated the vector into genomic DNA. Both of these types of cell lines produce the gene product on a continuous basis. One can also produce cell lines that have amplified the number of copies of the vector (and therefore of the cDNA as well) to create cell lines that can produce high levels of the gene product (Alt et al., 1978, J. Biol. Chem. 253:1357).

The transfer of DNA into eukaryotic, in particular human or other mammalian cells, is now a conventional technique. The vectors are introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, 1973, Virology 52:466) or strontium phosphate (Brash et al., 1987, Mol. Cell Biol. 7:2013), electroporation (Neumann er al., 1982, EMBO J. 1:841), lipofection (Feigner et al., 1987, Proc.

Natl. Acad. Sci USA 84:7413), DEAE dextran (McCuthan et al., 1968. J. Natl. Cancer Inst.

41:351), microinjection (Mueller et al., 1978, Cell 15:579), protoplast fusion (Schafner, 1980, Proc. Natl. Acad. Sci. USA 77:2163-7), or pellet guns (Klein et al., 1987, Nature 327:70).

Alternatively, the cDNA can be introduced by infection with virus vectors. Systems are developed that use, for example, retroviruses (Bernstein et al., 1985, Gen. Engrg. 7:235), adenoviruses (Ahmad et al., 1986, J. Virol. 57:267), or Herpes virus (Spaete et al., 1982, Cell 30:295).

These eukaryotic expression systems can be used for studies of FGF-5 genes and mutant forms of these genes, FGF-5 proteins and mutant forms of these proteins. Such uses include, for example, the identification of regulatory elements located in the 5' region of FGF-5 genes on genomic clones that can be isolated from genomic DNA libraries, such as human or other nonhuman primate libraries, using the information contained in the present disclosure. The eukaryotic expression systems may also be used to study the function of the normal complete protein, specific portions of the protein, or of naturally occurring or artificially produced mutant proteins. Naturally occurring mutant proteins may exist in a variety of diseases in which apoptosis has become disregulated, while artificially produced mutant proteins can be designed by site directed mutagenesis as described above. These latter studies may probe the function of any desired amino acid residue in the protein by mutating the nucleotide coding for that amino acid.

Using the above techniques, the expression vectors containing FGF-5 genes or cDNA sequences or variants, fragments, or mutants thereof, can be introduced into human cells, mammalian cells from other species or non-mammalian cells as desired. The choice of cell is determined by the purpose of the treatment. For example, monkey COS cells (Gluzman, 1981, Cell 23:175-82) that produce high levels of the SV40 T antigen and permit the replication of vectors containing the SV40 origin of replication may be used. Similarly, Chinese hamster ovary (CHO), mouse NIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may be used.

One method that can be used to express FGF-5 polypeptides from cloned FGF-5 cDNA sequences in mammalian cells is to use the cloning vector, pXTI (Stratagene,), which contains the WO 01/25271 PCT/US00/26689 -42- Long Terminal Repeats (LTRs) and a portion of the GAG gene from Moloney Murine Leukemia Virus. The position of the viral LTRs allows highly efficient, stable transfection of the region within the LTRs. The vector also contains the Herpes Simplex Thymidine Kinase promoter (TK), active in embryonal cells and in a wide-variety of tissues in mice, and a selectable neomycin gene conferring G418 resistance. Two unique restriction sites BgII and XhoI are directly downstream from the TK promoter. FGF-5 cDNA, including the entire open reading frame for the proteins and the 3' untranslated region of the cDNAs are cloned into one of the two unique restriction sites downstream from the promoter.

The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc.) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600 pg/ml G418 (Sigma, St. Louis, MO). The protein is released into the supernatant and may be purified by standard immunoaffinity chromatography techniques using antibodies raised against FGF-5 proteins (see EXAMPLE 9).

Expression of FGF-5 proteins, variants, polymorphisms, fragments of variants thereof, in eukaryotic cells can be used as a source of proteins to raise antibodies. The proteins may be extracted following release of the protein into the supernatant as described above, or, the cDNA sequence may be incorporated into a eukaryotic expression vector and expressed as a chimeric protein with, for example, P-globin. Antibody to P-globin is thereafter used to purify the chimeric protein. Corresponding protease cleavage sites engineered between the P-globin gene and the cDNA are then used to separate the two polypeptide fragments from one another after translation.

One useful expression vector for generating P-globin chimeric proteins is pSG5 (Stratagene). This vector encodes rabbit P-globin.

The recombinant cloning vector then comprises the selected DNA of the DNA sequences disclosed herein for expression in a suitable host. The DNA is operatively linked in the vector to an expression control sequence in the recombinant DNA molecule so that the FGF-5 polypeptide can be expressed. The expression control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof. The expression control sequence may be specifically selected from the group consisting of the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus and simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors and combinations thereof.

WO 01/25271 PCT/US00/26689 -43 The host cell, which may be transfected with the vector disclosed herein, may be selected from the group consisting of bacteria, yeast, fungi, plant, insect, mouse or other animal subject; or human tissue cells.

It is appreciated that for mutant or variant DNA sequences, similar systems are employed to express and produce the mutant or variant product.

EXAMPLE 9 Production and Use of Antibodies This example describes several methods than can be used to produce antibodies that recognize FGF-5. Such antibodies can be used to purify proteins by immunoaffinity chromatography, in diagnostic assays to quantitate FGF-5 levels, to localize FGF-5 in tissues and individual cells for example by immunofluorescence, and as a therapeutic agent to treat expressing or over-expressing tumors.

Production of Antibodies Monoclonal or polyclonal antibodies may be produced to either normal FGF-5 proteins, or variants, polymorphisms, fragments and mutant forms thereof. Optimally, antibodies raised against the protein will specifically detect the protein. That is, antibodies raised against the protein (e.g.

would recognize and bind the protein and would not substantially recognize or bind to other cellular proteins (such as serum albumin). The determination that an antibody specifically detects an FGF-5 protein is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

To determine that a given antibody preparation (such as one produced in a mouse against the FGF-5 protein) specifically detects the FGF-5 protein by Western blotting, total cellular protein is extracted from cells (for example, lymphocytes) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. The proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane.

After washing the membrane to remove non-specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase; application of the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immunolocalized alkaline phosphatase.

Antibodies which specifically detect FGF-5 proteins will, by this technique, be shown to bind to the protein band. which will be localized at a given position on the gel determined by its WO 01/25271 PCT/US00/26689 -44molecular weight. Non-specific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-FGF-5 protein binding.

Antibodies that specifically bind to an FGF-5 protein (or any of the other novel proteins disclosed herein) belong to a class of molecules that are referred to herein as "specific binding agents." Specific binding agents that are capable of specifically binding to a FGF-5 protein may include polyclonal antibodies, monoclonal antibodies (including humanized monoclonal antibodies) and fragments of monoclonal antibodies such as Fab, F(ab')2 and Fv fragments, as well as any other agent capable of specifically binding to a FGF-5 protein (or the other disclosed proteins).

Substantially pure FGF-5 protein suitable for use as an immunogen is isolated from the transfected or transformed cells as described. Concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per milliliter. Monoclonal or polyclonal antibody to the protein can then be prepared as follows.

Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes of FGF-5 proteins, identified and isolated as described, can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495, 1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein (or epitope thereof) over a period of a few weeks.

The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).

The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Enzymol. 70:419, 1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies: A Laboratory Manual. 1988, Cold Spring Harbor Laboratory, New York). In addition, protocols for producing humanized forms of monoclonal antibodies (for therapeutic applications) and fragments of monoclonal antibodies are known in the art.

WO 01/25271 PCT/US00/26689 Polyclonal Antibody Production by Immunization Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis et al. Clin. Endocrinol. Metab.

33:988-91, 1971).

Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al. (In: Handbook of Experimental Immunology, Wier, D. Chapter 19. Blackwell.

1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 pM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Chapter 42.

1980).

Synthetic Peptides Another approach to raising antibodies against FGF-5 proteins is to use synthetic peptides synthesized on a commercially available peptide synthesizer based upon the predicted amino acid sequence of FGF-5 proteins.

Antibodies may be raised against FGF-5 proteins by subcutaneous injection of a DNA vector which expresses a FGF-5 protein into laboratory animals, such as mice. Delivery of the recombinant vector into the animals may be achieved using a hand-held form of the Biolistic system (Sanford et al., 1987, Pariculate Sci. Technol. 5:27-37) as described by Tang et al. (Nature, 356:152-4, 1992). Expression vectors suitable for this purpose may include those which express a cDNA under the transcriptional control of either the human actin promoter or the cytomegalovirus (CMV) promoter.

Antibody preparations prepared according to these protocols are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; WO 01/25271 PCT/US00/26689 -46they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.

Antibodies Raised by Injection of FGF-5 cDNA Antibodies may be raised against FGF-5 proteins by subcutaneous injection of a DNA vector which expresses FGF-5 protein (or variant, fragment, polymorphism, or mutant thereof) into laboratory animals, such as mice. Delivery of the recombinant vector into the animals may be achieved using a hand-held form of the Biolistic system (Sanford et al., Particulate Sci. Technol.

5:27-37, 1987) as described by Tang et al. (Nature 356:152-4, 1992). Expression vectors suitable for this purpose may include those which express the FGF-5 cDNA under the transcriptional control of either the human p-actin promoter or the CMV promoter.

Antibody preparations prepared according to these protocols are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.

Labeled Antibodies Antibodies disclosed herein can be conjugated with various labels for their direct detection (see Chapter 9, Harlow and Lane, Antibodies: A Laboratory Manual. 1988). The label, which may include, but is not limited to, a radiolabel, enzyme, fluorescent probe, or biotin, is chosen based on the method of detection available to the user.

Antibodies can be radiolabeled with iodine which yields low-energy gamma and Xray radiation. Briefly, 10 fg of protein in 25 Al of 0.5 M sodium phosphate (pH 7.50 is placed in a ml conical tube. To this. 500 pC of Na'"l, and 25 il of 2 mg/ml chloramine T is added and incubated for 60 seconds at RT. To stop the reaction, 50pl of chloramine T stop buffer is added (2.4 mg/ml sodium metabisulfite, 10 mg/ml tyrosine, 10% glycerol, 0.1% xylene cyanol in PBS).

The iodinated antibody is separated from the iodotyrosine on a gel filtration column. Antibodies disclosed herein can also be labeled with biotin, with enzymes such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) or with fluorescent dyes. The method of producing these conjugates is determined by the reactive group on the label added.

Therapeutic Uses for Antibodies The antibodies described above can be used to treat FGF-5 expressing or overexpressing tumors, for example by decreasing FGF-5 activity. Assays to determine whether an antibody modulates the immune response are described in EXAMPLES 2-4. Antibodies which recognize WO 01/25271 PCT/US00/26689 -47may cause regression of an FGF-5 expressing or overexpressing tumor. In addition, antibodies can be used as diagnostic agents, for example to monitor the progression or regression of an FGF-5 expressing or over expressing tumor in a subject, for example a subject undergoing therapy to treat the neoplasim.

EXAMPLE Diagnostic Methods One embodiment disclosed herein is a method of pre-screening cells, such as tumor cells to determine if the cells express or over-express FGF-5, and if the subject is HLA-A3 This can be determined by conventional methods for identifying cells which present a particular HLA molecule, and identifying cells expressing DNA of the pertinent sequences, in this case a sequence. In one embodiment, once cells presenting the relevant HLA complex are identified via the foregoing screening methodology, they can be combined with a sample from a subject, where the sample contains CTLs. If the complex-presenting cells are lysed by the mixed CTL sample, then it can be assumed that a FGF-5 TAA is being presented, and the subject is an appropriate candidate for the therapeutic approaches set forth herein.

A subject can be screened to determine if the cells of a subject carry an FGF-5 gene, for example a wild-type FGF-5 gene, polymorphic, mutant or variant FGF-5 genes having a heterozygous or homozygous nucleotide change, or insertions, partial deletion, or duplications of the FGF-5 gene. One major application of the FGF-5 sequence information presented herein is in the area of genetic testing for predisposition to disease, such as cancer, owing to the presence and expression or overexpression of an FGF-5 gene. Predisposition to other diseases, including, but not limited to Hippel-Lindau disease, horseshoe kidneys, adult polycystic kidney disease, and acquired renal cystic disease, owing to the presence and expression or overexpression of an gene, can also be determined using the methods disclosed herein.

The sequence of FGF-5 genes, including intron-exon boundaries, is also useful in such diagnostic methods. The method consists of providing a biological sample obtained from the subject, in which sample includes DNA or RNA, and providing an assay for detecting in the biological sample the presence, absence, variant, or mutation of a FGF-5 gene and FGF-5 RNA.

Suitable biological samples include samples obtained from body cells, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, fine needle aspirate specimen, amniocentesis samples and autopsy material. The detection in the biological sample may be performed by a number of methodologies, as outlined below.

The foregoing assay may be assembled in the form of a diagnostic kit and may include, for example, hybridization with oligonucleotides; PCR amplification of the gene or a part thereof using WO 01/25271 PCT/US00/26689 -48oligonucleotide primers; RT-PCR amplification of the RNA or a pan thereof using oligonucleotide primers; or direct sequencing of an FGF-5 gene of the subject's genome using oligonucleotide primers. The efficiency of these molecular genetic methods should permit a rapid classification of patients affected by the presence of one or more copies of an FGF-5 gene, or deletions, variants, polymorphisms or mutations of a FGF-5 gene.

One embodiment of such detection techniques is the PCR amplification of reverse transcribed RNA (RT-PCR) of RNA isolated from cells (for example cells from a tumor) followed by direct DNA sequence determination of the products. The presence of one or more nucleotide differences between the obtained sequence and the cDNA sequences, and especially, differences in the ORF portion of the nucleotide sequence are taken as indicative of a potential FGF-5 gene mutation.

Alternatively, DNA extracted from tumor or other cells may be used directly for amplification. The direct amplification from genomic DNA would be appropriate for analysis of the entire FGF-5 gene including regulatory sequences located upstream and downstream from the open reading frame. Reviews of direct DNA diagnosis have been presented by Caskey (Science 236:1223-8, 1989) and by Landegren et al. (Science 242:229-37, 1989).

Further studies of FGF-5 genes isolated from subjects may reveal particular mutations, duplications, variants, polymorphisms, or deletions, which occur at a high frequency within this population of individuals. In this case, rather than sequencing the entire FGF-5 gene, it is possible to design DNA diagnostic methods to specifically detect the most common FGF-5 mutations, variants, polymorphisms, or deletions.

The detection of specific DNA mutations may be achieved by methods such as hybridization using specific oligonucleotides (Wallace et al., 1986, Cold Spring Harbor Symp.

Quant. Biol. 51:257-61), direct DNA sequencing (Church and Gilbert, 1984, Proc. Natl. Acad.

Sci. USA. 81:1991-5), the use of restriction enzymes (Flavell et al., 1978, Cell 15:25; Geever et al., 1981, Proc. Natl. Acad. Sci USA 78:5081), discrimination on the basis of electrophoretic mobility in gels with denaturing reagent (Myers and Maniatis, 1986, Cold Spring Harbor Symp.

Quant. Biol. 51:275-84), RNase protection (Myers et al., 1985, Science 230:1242), chemical cleavage (Cotton et al., 1985, Proc. Natl. Acad. Sci. USA 85:4397-401), and the ligase-mediated detection procedure (Landegren et al., 1988, Science 241:1077).

Oligonucleotides specific to normal, variant, polymorphic, or mutant sequences are chemically synthesized using commercially available machines, labeled radioactively with isotopes (such as UP) or non-radioactively, with tags such as biotin (Ward and Langer et al., 1981, Proc.

Nail. Acad. Sci. USA 78:6633-57), and hybridized to individual DNA samples immobilized on membranes or other solid supports by dot-blot or transfer from gels after electrophoresis. The WO 01/25271 PCT/US00/26689 -49presence of these specific sequences are visualized by methods such as autoradiography or fluorometric (Landegren et al., 1989, Science 242:229-37) or colorimetric reactions (Gebeyehu et al., 1987, Nucleic Acids Res. 15:4513-34). The absence of hybridization would indicate a mutation in the particular region of the gene, or a deleted FGF-5 gene.

Sequence differences between normal, variant, polymorphic, and mutant forms of genes may also be revealed by the direct DNA sequencing method of Church and Gilbert (Proc.

Natl. Acad. Sci. USA 81:1991-5, 1988). Cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR (Wrichnik et al., 1987, Nucleic Acids Res. 15:529-42; Wong et al., 1987, Nature 330:384-6; Stoflet t al., 1988, Science 239:491-4). In this approach, a sequencing primer which lies within the amplified sequence is used with double-stranded PCR product or single-stranded template generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent tags.

Sequence alterations may occasionally generate fortuitous restriction enzyme recognition sites or may eliminate existing restriction sites. Changes in restriction sites are revealed by the use of appropriate enzyme digestion followed by conventional gel-blot hybridization (Southern, 1975, J. Mol. Biol. 98:503). DNA fragments carrying the site (either normal or mutant) are detected by their reduction in size or increase of corresponding restriction fragment numbers. Genomic DNA samples may also be amplified by PCR prior to treatment with the appropriate restriction enzyme; fragments of different sizes are then visualized under UV light in the presence of ethidium bromide after gel electrophoresis.

Genetic testing based on DNA sequence differences can be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing reagent.

Small sequence deletions and insertions can be visualized by high-resolution gel electrophoresis.

For example, a PCR product with small deletions is clearly distinguishable from a normal sequence on an 8% non-denaturing polyacrylamide gel (WO 91/10734; Nagamine et al., 1989, Am. J. Hum.

Genet. 45:337-9). DNA fragments of different sequence compositions may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific "partial-melting" temperatures (Myers et al., 1985, Science 230:1242). Alternatively, a method of detecting a mutation comprising a single base substitution or other small change could be based on differential primer length in a PCR. For example, an invariant primer could be used in addition to a primer specific for a mutation. The PCR products of the normal and mutant genes can then be differentially detected in acrylamide gels.

WO 01/25271 PCT/USOO/26689 In addition to conventional gel-electrophoresis and blot-hybridization methods, DNA fragments may also be visualized by methods where the individual DNA samples are not immobilized on membranes. The probe and target sequences may be both in solution, or the probe sequence may be immobilized (Saiki et al., 1989, Proc. Nat. Acad. Sci. USA 86:6230-4). A variety of detection methods, such as autoradiography involving radioisotopes, direct detection of radioactive decay (in the presence or absence of scintillant), spectrophotometry involving calorigenic reactions and fluorometry involved fluorogenic reactions, may be used to identify specific individual genotypes.

If more than one mutation is frequently encountered in FGF-5 genes, a system capable of detecting such multiple mutations is desirable. For example, a PCR with multiple, specific oligonucleotide primers and hybridization probes may be used to identify all possible mutations at the same time (Chamberlain et al., 1988, Nucl. Acids Res. 16:1141-55). The procedure may involve immobilized sequence-specific oligonucleotides probes (Saiki et al., 1989, Proc. Nat. Acad.

Sci. USA 86:6230-4).

One method particularly suitable for detecting the presence of FGF-5 genes disclosed herein is the use of high density oligonucleotide arrays (also known as "DNA chips") as described by Hacia et al. (Nat. Genet. 14:441-7, 1996).

EXAMPLE 11 Two Step Assay to Detect the Presence of FGF-5 gene in a Sample A tissue sample from a subject can be processed according to the method disclosed by Antonarakis el al. (New Eng. J. Med. 313:842-848, 1985), separated through a 1% agarose gel and transferred to a nylon membrane for Southern blot analysis. Membranes are UV cross linked at 150 mJ using a GS Gene Linker (Bio-Rad, Hercules, CA). An FGF-5 probe is subcloned into pTZ18U. The phagemids can be transformed into E. coli MV 1190 infected with M13K07 helper phage (Bio-Rad, Richmond, Calif.). Single stranded DNA is isolated according to standard procedures (Sambrook, er al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York. 1989).

Blots are prehybridized for 15-30 minutes at 65 0 C in 7% sodium dodecyl sulfate (SDS) in 0.5 M NaPO,. The methods follow those described by Nguyen, et al. (BioTechniques 13:116-123, 1992). The blots are hybridized overnight at 65*C in 7% SDS, 0.5 M NaPO, with 25-50 ng/ml single stranded probe DNA. Post-hybridization washes consist of two 30 minute washes in SDS, 40 mM NaPO 4 at 65"C, followed by two 30-minute washes in 1% SDS, 40 mM NaPO, at

C.

WO 01/25271 PCT/US00/26689 -51 The blots are subsequently rinsed with phosphate buffered saline (pH 6.8) for five minutes at RT and incubated with 0.2% casein in PBS for five minutes. The blots are then preincubated for 5-10 minutes in a shaking water bath at 45 C with hybridization buffer consisting of 6 M urea, 0.3 M NaCI, and 5X Denhardt's solution (see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989). The buffer is removed and replaced with 50-75 pl/cm' fresh hybridization buffer plus 2.5 nM of the covalently cross-linked oligonucleotide sequence complementary to the universal primer site (UP-AP, Bio-Rad). The blots are hybridized for 20-30 minutes at 45"C and post hybridization washes are incubated at 45 0 C as two 10 minute washes in 6 M urea, IX standard saline citrate (SSC), 0.1 SDS and one 10 minute wash in IXSSC, 0.1% Triton"X-100. The blots are rinsed for 10 minutes at RT with 1XSSC.

Blots are incubated for 10 minutes at RT with shaking in the substrate buffer consisting of 0.1 M diethanolarnine, 1 mM MgCI 2 0.02% sodium azide, pH 10.0. Individual blots are placed in heat sealable bags with substrate buffer and 0.2 mM AMPPD (3-(2'-spiroadamantane)-4-methoxy- 4-(3'-phosphoryloxy)phenyl-1,2-dioxetane, disodium salt, Bio-Rad). After a 20 minute incubation at RT with shaking, the excess AMPPD solution is removed. The blot is exposed to X-ray film overnight. Patient samples which show no hybridizing bands lack a FGF-5 gene. Patient samples which show positive bands indicate the presence of at least one copy of an FGF-5 gene, possibly more, which would indicate the possibility of ongoing disease such as cancer, such as RCC, prostate or breast cancer, or an enhanced susceptibility to developing a disease, such as cancer, in the future.

In other embodiments, the method involves assaying for the presence of transcription factors which may modulate expression of FGF-5. Patient samples which show positive bands for the transcription factor indicate the presence of at least one copy of the transcription factor gene, possibly more, which would indicate the possibility of ongoing disease such as cancer, such as RCC, prostate or breast cancer, or an enhanced susceptibility to developing a disease, such as cancer, in the future.

EXAMPLE 12 Quantitation of FGF-5 Proteins An alternative method of diagnosing the presence of an FGF-5 and/or transcription factor gene is to quantitate the level of FGF-5 protein in the cells of a subject. This diagnostic tool is useful for detecting elevated levels of FGF-5 protein which result from, for example, expression or overexpression of the FGF-5 gene, the presence of multiple copies of an FGF-5 and/or transcription factor(s) gene, or mutations (such as mutations within the gene, or mutations upstream or downstream from the gene which affect its expression) which result in increased expression of WO 01/25271 PCT/US00/26689 -52- These diagnostic methods, in addition to those described in EXAMPLES 10 and 11, provide an enhanced ability to diagnose susceptibility to diseases caused by FGF-5 expression or overexpression.

The determination of elevated FGF-5 protein levels is an alternative or supplemental approach to the direct determination of whether the FGF-5 gene (or transcription factor gene(s)) is present in multiple copies or if mutations are present (for example mutations up- or down-stream of an FGF-5 gene) that would result in increased FGF-5 expression, using the methods outlined above in EXAMPLES 10 and 11. The availability of antibodies specific to the FGF-5 protein (for example those described in EXAMPLE 9) will facilitate the quantitation of cellular FGF-5 protein by one of a number of immunoassay methods which are well known in the art and are presented in Harlow and Lane (Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory, New York.

1988).

Such assays permit both the detection of FGF-5 proteins in a biological sample and the quantitation of such proteins. Typical methods involve providing a biological sample of the subject in which the sample contains cellular proteins, and providing an immunoassay for quantitating the level of FGF-5 protein in the biological sample. This can be achieved by combining the biological sample with a FGF-5 specific binding agent, such as an anti-FGF-5 antibody (such as monoclonal or polyclonal antibodies), so that complexes form between the binding agent and the FGF-5 protein present in the sample, and then detecting or quantitating such complexes.

In particular forms, these assays may be performed with the FGF-5 specific binding agent immobilized on a support surface, such as in the wells of a microtiter plate or on a column. The biological sample is then introduced onto the support surface and allowed to interact with the specific binding agent so as to form complexes. Excess biological sample is then removed by washing, and the complexes are detected with a reagent, such as a second anti-FGF-5 protein antibody that is conjugated with a detectable marker.

In an alternative assay, the cellular proteins are isolated and subjected to SDS-PAGE followed by Western blotting. After resolving the proteins, the proteins are transferred to a membrane, which is probed with specific binding agents that recognize FGF-5. The proteins are detected, for example with HRP-conjugated secondary antibodies, and quantitated.

In yet another assay, the level of FGF-5 protein in cells is analyzed using microscopy.

Using specific binding agents which recognize FGF-5, samples can be analyzed for the presence of proteins. For example, frozen biopsied tissue sections are thawed at room temperature and fixed with acetone at -200 0 C for five minutes. Slides are washed twice in cold PBS for five minutes each, then air-dried. Sections are covered with 20-30 fl of antibody solution (15-45 Atg/ml) (diluted in PBS, 2% BSA at 15-50 /g/ml) and incubated at RT in a humidified chamber for WO 01/25271 PCT/US00/26689 -53minutes. Slides are washed three times with cold PBS five minutes each, allowed to air-dry briefly (5 minutes) before applying 20-30 pl of the second antibody solution (diluted in PBS, 2% BSA at 15-50 pg/ml) and incubated at RT in humidified chamber for 30 minutes. The label on the second antibody may contain a fluorescent probe, enzyme, radiolabel, biotin, or other detectable marker. The slides are washed three times with cold PBS five minutes each then quickly dipped in distilled water, air-dried, and mounted with PBS containing 30% glycerol. Slides can be stored at 4"C prior to viewing.

For samples prepared for electron microscopy (versus light microscopy), the second antibody is conjugated to gold particles. Tissue is fixed and embedded with epoxy plastics, then cut into very thin sections 1-2 pm). The specimen is then applied to a metal grid, which is then incubated in the primary anti-FGF-5 antibody, washed in a buffer containing BSA. then incubated in a secondary antibody conjugated to gold particles (usually 5-20 nm). These gold particles are visualized using electron microscopy methods.

For the purposes of quantitating FGF-5 proteins in a sample specimen, a "control" sample of the subject, which sample does not express FGF-5 or expresses FGF-5 at lower levels, can be used to compare FGF-5 expression in the sample to be analyzed. Such a "control" sample may be obtained from normal adult cells, such as those present in which expression of the protein is not detected. As shown in FIG. 4, for example, FGF-5 is not expressed in detectable levels in any normal tissue tested. However, peripheral blood leukocytes is clearly the most accessible and convenient source from which comparative samples can be obtained. Sample specimens can be obtained from peripheral blood, urine, saliva, tissue biopsy, amniocentesis samples, surgical specimens, fine needle aspirates, and autopsy material, particularly cancer cells. Quantitation of proteins can be made by immunoassay and compared to levels of the protein found in nonexpressing cells, such as normal adult tissues, or to the level of FGF-5 in healthy, normal cells (for example, cells of the same origin that are not neoplastic or are free of the disease of interest). A significant (for example 50% or greater) increase in the amount of FGF-5 protein in the cells of a subject compared to the amount of FGF-5 protein found in non-FGF-5 expressing cells or that found in normal cells, would be taken as an indication that the presence of the gene locus in the subject.

EXAMPLE 13 Gene Therapy A new gene therapy approach for subjects suffering from tumors which express or overexpress FGF-5 is now made possible by the present disclosure. Administration of FGF-5 to a subject suffering from a tumor that expresses or over-expresses FGF-5, may further enhance the WO 01/25271 PCT/US00/26689 -54modulation of the immune response, such as stimulating CTLs to lyse the FGF-5 expressing tumor cells, and achieve the desired therapeutic goal of assisting in regression of the tumor. In some subjects, expression or overexpression of FGF-5 by the tumor may be insufficient to stimulate the immune system, for example in non HLA-A3 subjects. Alternatively, modulation of the immune response may be augmented by additional FGF-5 expression. Essentially, cells may be removed from a subject, such as dendritic cells, suffering from a tumor that expresses or over-expresses and then transfected with an expression vector containing FGF-5 cDNA. These transfected cells will thereby produce functional FGF-5 protein and can be reintroduced into the subject to stimulate CTLs.

The scientific and medical procedures required for human cell transfection are now routine procedures. The provision herein of FGF-5 cDNAs now allows the development of human gene therapy based upon these procedures. Immunotherapy of melanoma patients using genetically engineered tumor-infiltrating lymphocytes (TILs) has been reported by Rosenberg et al. Engl.

J. Med. 323:570-8, 1990). In that study, a retrovirus vector was used to introduce a gene for neomycin resistance into TILs. A similar approach may be used to introduce the FGF-5 cDNA into patients affected by FGF-5 expression or overexpression.

In some embodiments, a method of treating tumors which express or overexpress or in which greater FGF-5 expression is desired, is disclosed. These methods can be accomplished by introducing a gene coding for FGF-5 into a subject. A general strategy for transferring genes into donor cells is disclosed in U.S. Patent No. 5,529,774. Generally, a gene encoding a protein having therapeutically desired effects is cloned into a viral expression vector, and that vector is then introduced into the target organism. The virus infects the cells, and produces the protein sequence in vivo, where it has its desired therapeutic effect. See, for example, Zabner et al. (Cell 75:207- 16, 1993).

In some of the foregoing examples, it may only be necessary to introduce the genetic or protein elements into certain cells or tissues. For example, in the case of benign nevi and psoriasis, introducing them into only the skin may be sufficient. However, in some instances tumors), it may be more therapeutically effective and simple to treat all of the patients cells, or more broadly disseminate the vector, for example by intravascular administration.

The nucleic acid sequence encoding at least one therapeutic agent is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, the gene's native promoter, retroviral LTR promoter, or adenoviral promoters, such as the adenoviral major late promoter; the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; inducible promoters, such as the MMTV promoter; the metallothionein promoter; heat shock promoters; the albumin promoter; the histone promoter: the a-actin promoter; TK WO 01/25271 PCT/US00/26689 promoters; B19 parvovirus promoters; and the ApoAl promoter. However the scope of the disclosure is not limited to specific foreign genes or promoters.

The recombinant nucleic acid can be administered to the subject by any method which allows the recombinant nucleic acid to reach the appropriate cells. These methods include injection, infusion, deposition, implantation, or topical administration. Injections can be intradermal or subcutaneous. The recombinant nucleic acid can be delivered as part of a viral vector, such as avipox viruses, recombinant vaccinia virus, replication-deficient adenovirus strains or poliovirus, or as a non-infectious form such as naked DNA or liposome encapsulated DNA.

EXAMPLE 14 Viral Vectors for Gene Therapy Adenoviral vectors may include essentially the complete adenoviral genome (Shenk et al., Curr. Top. Microbiol. Immunol. 111:1-39, 1984). Alternatively, the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted.

In one embodiment, the vector includes an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; a DNA sequence encoding a therapeutic agent; and a promoter for expressing the DNA sequence encoding a therapeutic agent. The vector is free of at least the majority of adenoviral El and E3 DNA sequences, but is not necessarily free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins transcribed by the adenoviral major late promoter. In another embodiment, the vector may be an adeno-associated virus (AAV) such as described in U.S. Patent No. 4,797,368 (Carter et al.) and in McLaughlin et al. Virol. 62:1963- 73, 1988) and AAV type 4 (Chiorini et al. J. Virol. 71:6823-33, 1997) and AAV type 5 (Chiorini et al. J. Virol. 73:1309-19, 1999).

Such a vector may be constructed according to standard techniques, using a shuttle plasmid which contains, beginning at the 5' end, an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter); a tripartite leader sequence, a multiple cloning site (which may be as herein described); a poly A signal; and a DNA segment which corresponds to a segment of the adenoviral genome.

The DNA segment serves as a substrate for homologous recombination with a modified or mutated adenovirus, and may encompass, for example, a segment of the adenovirus 5' genome no longer than from base 3329 to base 6246. The plasmid may also include a selectable marker and an origin of replication. The origin of replication may be a bacterial origin of replication. A desired DNA sequence encoding a therapeutic agent may be inserted into the multiple cloning site of the plasmid.

The plasmid may be used to produce an adenoviral vector by homologous recombination with a modified or mutated adenovirus in which at least the majority of the El and E3 adenoviral WO 01/25271 PCT/US00/26689 -56- DNA sequences have been deleted. Homologous recombination may be effected through cotransfection of the plasmid vector and the modified adenovirus into a helper cell line, such as 293 cells, by CaPO, precipitation. The homologous recombination produces a recombinant adenoviral vector which includes DNA sequences derived from the shuttle plasmid between the Not I site and the homologous recombination fragment, and DNA derived from the El and E3 deleted adenovirus between the homologous recombination fragment and the 3' ITR.

In one embodiment, the adenovirus may be constructed by using a yeast artificial chromosome (or YAC) containing an adenoviral genome according to the method described in Ketner et al. (Proc. Natl. Acad. Sci. USA, 91:6186-90, 1994), in conjunction with the teachings contained herein. In this embodiment, the adenovirus yeast artificial chromosome is produced by homologous recombination in vivo between adenoviral DNA and yeast artificial chromosome plasmid vectors carrying segments of the adenoviral left and right genomic termini. A DNA sequence encoding a therapeutic agent then may be cloned into the adenoviral DNA. The modified adenoviral genome then is excised from the adenovirus yeast artificial chromosome in order to be used to generate adenoviral vector particles as hereinabove described.

The adenoviral particles are administered in an amount effective to produce a therapeutic effect in a subject. The exact dosage of adenoviral panicles to be administered is dependent upon a variety of factors, including the age, weight, and sex of the subject to be treated, and the nature and extent of the disease or disorder to be treated. The adenoviral particles may be administered as part of a preparation having a titer of adenoviral particles of at least 1 x 10'0 pfu/ml, and in general not exceeding 2 x 10" pfu/ml. The adenoviral particles may be administered in combination with a pharmaceutically acceptable carrier in a volume up to 10 ml. The pharmaceutically acceptable carrier may be, for example, a liquid carrier such as a saline solution, protamine sulfate (Elkins- Sinn, Inc., Cherry Hill, NJ), or Polybrene (Sigma Chemical) as well as those described in EXAMPLE 18.

In another embodiment, the viral vector is a retroviral vector. Retroviruses have been considered for experiments in gene therapy because they have a high efficiency of infection and stable integration and expression (Orkin et al., 1988, Prog. Med. Genet.7:130-42). Partial or full length FGF-5 genes can be cloned into a retroviral vector and driven from either its endogenous promoter or from the retroviral LTR (long terminal repeat). Examples of retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. The vector is generally a replication defective retrovirus particle.

WO 01/25271 PCT/US00/26689 -57- Retroviral vectors are useful as agents to effect retroviral-mediated gene transfer into eukaryotic cells. Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest.

Most often, the structural genes gag, pol, and env) are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.

Other viral transfection systems may also be utilized for this type of approach, including Vaccinia virus (Moss et al.. 1987, Annu. Rev. Immunol. 5:305-24), Bovine Papilloma virus (Rasmussen et al., 1987, Methods Enzymol. 139:642-54) or members of the herpes virus group such as Epstein-Barr virus (Margolskee et al., 1988, Mol. Cell. Biol. 8:2837-47). Recent developments in gene therapy techniques include the use of RNA-DNA hybrid oligonucleotides, as described by Cole-Strauss et al. (Science 273:1386-9, 1996). This technique can allow for sitespecific integration of cloned sequences, permitting accurately targeted gene replacement.

New genes may be incorporated into proviral backbones in several general ways. In the most straightforward constructions, the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the long terminal repeat (LTR). Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter. Alternatively, two genes may be expressed from a single promoter by the use of an Internal Ribosome Entry Site.

EXAMPLE Peptide Modifications The present disclosure includes biologically active molecules that mimic the action (mimetics) of the FGF-5 proteins disclosed herein. The disclosure therefore includes synthetic embodiments of naturally-occurring peptides, as well as analogues (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences) and variants (homologs) of these peptides that stimulate an HLA-A3-restricted CTL immune response against FGF-5 expressing or over-expressing tumors, as measured by IFN-y concentration. Each peptide ligand disclosed is comprised of a sequence of amino acids, which may be either L- and/or D- amino acids, naturally occurring and otherwise.

Peptides may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other WO 01/25271 PCT/US00/26689 -58desirable properties. For example, carboxylic acid groups of the peptide, whether carboxylterminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a CI-C16 ester, or converted to an amide of formula NRIR2 wherein R1 and R2 are each independently H or CI-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCI, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to CI-C16 alkyl or dialkyl amino or further converted to an amide.

Hydroxyl groups of the peptide side chain may be converted to C -C16 alkoxy or to a Cl- C16 ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chain may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with CI-C16 alkyl, C1-C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C2- C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides disclosed herein to select and provide conformational constraints to the structure that result in enhanced stability. For example, a carboxyl-terminal or amino-terminal cysteine residue can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, thereby generating a cyclic peptide. Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.

To maintain an optimally functional peptide, particular peptide variants will differ by only a small number of amino acids from the peptides disclosed herein. Such variants may have deletions (for example of 1-3 or more amino acid residues), insertions (for example of 1-3 or more residues), or substitutions that do not interfere with the desired activity of the peptides.

Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. In particular embodiments, such variants will have amino acid substitutions of single residues, for example 1, 3, 5 or even 10 substitutions in the protein (SEQ ID NOS: 4, 6, 8, 10, 12, and 16-19).

Peptidomimetic and organomimetic embodiments are also disclosed, whereby the threedimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains in the peptide, resulting in such peptido- and organomimetics of the peptides having the ability to modulate an immune response against FGF-5 expressing or over-expressing tumors, as measured by IFN-y concentration (FGF-5 mimetics). For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for modulating an WO 01/25271 PCT/US00/26689 -59immune response activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology (ed. Munson, 1995), chapter 102 for a description of techniques used in CADD. Also disclosed are mimetics prepared using such techniques that produce either peptides or conventional organic pharmaceuticals that retain the ability to modulate an immune response or modulate FGF-5 expression or activity.

The above described mimetics are examined for their ability to stimulate an HLA-A3restricted CTL immune response against FGF-5 expressing or over-expressing tumors, as measured by IFN-y concentration. Such activities can be readily determined using the assays disclosed herein, for example using the methods described in EXAMPLES 24. Suitable mimetics would demonstrate the ability to modulate an immune response or modulate FGF-5 expression or activity as defined above.

EXAMPLE 16 Methods for Generating Mimetics Compounds or other molecules which mimic normal FGF-5 function, such as compounds which modulate an immune response or modulate FGF-5 expression or activity, for example modulate an immune response against FGF-5 expressing or over-expressing tumors, as measured by IFN-y concentration, can be identified and/or designed. These compounds or molecules are known as mimetics, because they mimic the biological activity of the normal protein.

Crystallography To identify the amino acids that interact between FGF-5 and the MHC of the HLA-A3restricted CTLs (the MHC), FGF-5 is co-crystallized in the presence of the MHC. One method that can be used is the hanging drop method. In this method, a concentrated salt, the protein and the MHC protein solution is applied to the underside of a lid of a multiwell dish. A range of concentrations may need to be tested. The lid is placed onto the dish, such that the droplet "hangs" from the lid. As the solvent evaporates, a protein crystal is formed, which can be visualized with a microscope. This crystallized structure is then subjected to X-ray diffraction or NMR analysis which allows for the identification of the amino acid residues that are in contact with one another. The amino acids that contact the transcription factors establish a pharmacophore that can then be used to identify drugs that interact at that same site.

WO 01/25271 PCT/US00/26689 Identification of drugs Once these amino acids have been identified, one can screen synthetic drug databases (which can be licensed from several different drug companies), to identify drugs that interact with the same amino acids of a FGF-5 protein that the MHC interacts with. Moreover, structure activity relationships and computer assisted drug design can be performed as described in Remington, The Science and Practice of Pharmacy, Chapter 28.

Designing synthetic peptides In addition, synthetic peptides can be designed from the sequence of the MHC that interacts with FGF-5. Several different peptides could be generated from this region(s). This could be done with or without the crystallography data. However, once crystallography data is available, peptides can also be designed that bind better than The chimeric peptides may be expressed recombinantly, for example in E. coli. One advantage of the synthetic peptides over the monoclonal antibodies is that they are smaller, and therefore diffuse easier, and are not as likely to be immunogenic. Standard mutagenesis of such peptides can also be performed to identify variant peptides having even greater ability to modulate an immune response or modulate FGF-5 expression or activity as defined herein.

After synthetic drugs or peptides that bind to FGF-5 have been identified, their ability to stimulate an HLA-A3-restricted CTL immune response against FGF-5 expressing or overexpressing tumors, as measured by IFN-y concentration, can be tested as described in EXAMPLES 2-4. Those that are positive would be good candidates for therapies, such as treatment of tumors in which FGF-5 is expressed or overexpressed.

EXAMPLE 17 Peptide Synthesis and Purification The disclosed peptides (and variants and fragments thereof) can be chemically synthesized by any of a number of manual or automated methods of synthesis known in the art. For example, solid phase peptide synthesis (SPPS) is carried out on a 0.25 millimole (mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizer and using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection, coupling with dicyclohexylcarbodiimide/ hydroxybenzotriazole or 2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/ hydroxybenzotriazole (HBTU/HOBT), and using p-hydroxymethylphenoxymethylpolystyrene (HMP) or Sasrin resin for carboxyl-terminus acids or Rink amide resin for carboxyl-terminus amides.

ii WO 01/25271 PCT/US00/26689 -61 Fmoc-derivatized amino acids are prepared from the appropriate precursor amino acids by tritylation and triphenylmethanol in trifluoroacetic acid, followed by Fmoc derivitization as described by Atherton et al. (Solid Phase Peptide Synthesis, IRL Press: Oxford, 1989).

Sasrin resin-bound peptides are cleaved using a solution of 1 TFA in dichloromethane to yield the protected peptide. Where appropriate, protected peptide precursors are cyclized between the amino- and carboxyl-termini by reaction of the amino-terminal free amine and carboxylterminal free acid using diphenylphosphorylazide in nascent peptides wherein the amino acid sidechains are protected.

HMP or Rink amide resin-bound products are routinely cleaved and protected sidechaincontaining cyclized peptides deprotected using a solution comprised of trifluoroacetic acid (TFA), optionally also comprising water, thioanisole, and ethanedithiol, in ratios of 100 5 5 2.5, for 3 hours at RT.

Crude peptides are purified by preparative high pressure liquid chromatography (HPLC), for example using a Waters Delta-Pak C18 column and gradient elution with 0.1% TFA in water modified with acetonitrile. After column elution, acetonitrile is evaporated from the eluted fractions, which are then lyophilized. The identity of each product so produced and purified may be confirmed by fast atom bombardment mass spectroscopy (FABMS) or electrospray mass spectroscopy (ESMS).

EXAMPLE 18 Pharmaceutical Compositions and Modes of Administration Various delivery systems for administering the combined therapy disclosed herein are known, and include encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (see Wu and Wu, J. Biol. Chem. 1987, 262:4429-32), and construction of a therapeutic nucleic acid as part of a retroviral or other vector.

Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, the pharmaceutical compositions may be introduced into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.

WO 01/25271 PCT/US00/26689 -62- In one embodiment, it may be desirable to administer the pharmaceutical compositions disclosed herein locally to the area in need of treatment, for example, by local infusion during surgery, topical application, in conjunction with a wound dressing after surgery, by injection, through a catheter, by a suppository or an implant, such as a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.

The use of liposomes as a delivery vehicle is one delivery method of interest. The liposomes fuse with the target site and deliver the contents of the lumen intracellularly. The liposomes are maintained in contact with the target cells for a sufficient time for fusion to occur, using various means to maintain contact, such as isolation and binding agents. Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus. The lipids may be any useful combination of known liposome forming lipids, including cationic lipids, such as phosphatidylcholine. Other potential lipids include neutral lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like. For preparing the liposomes, the procedure described by Kato et al. Biol. Chem. 1991, 266:3361) may be used.

The present disclosure also provides pharmaceutical compositions which include a therapeutically effective amount of autologous CTLs specific to FGF-5 and/or an FGF-5 protein, RNA, DNA, antisense molecule or specific binding agent (for example, antibodies), alone or with a pharmaceutically acceptable carrier. In one example, homogeneous compositions of the therapeutic molecules includes compositions that are comprised of at least 90% of the peptide, variant, analog, derivative or mimetic in the composition.

Delivery systems Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation suits the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.

The amount of the therapeutic agent that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined WO 01/25271 PCT/US00/26689 -63by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions.

Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Instructions for use of the composition can also be included.

The pharmaceutical compositions or methods of treatment may be administered in combination with other therapeutic treatments, such as other antineoplastic or antitumorigenic therapies.

Administration of Nucleic Acid Molecules In an embodiment in which an FGF-5 nucleic acid is employed for gene therapy, the analog is delivered intracellularly by expression from a nucleic acid vector or by receptormediated mechanisms). In an embodiment where the therapeutic molecule is a nucleic acid or antisense molecule, administration may be achieved by an appropriate nucleic acid expression vector which is administered so that it becomes intracellular, by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see Joliot et al., Proc. Natl. Acad. Sci. USA 1991, 88:1864-8). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within a cell's cellular DNA for expression, by homologous recombination.

The vector pcDNA, is an example of a method of introducing the foreign cDNA into a cell under the control of a strong viral promoter (CMV) to drive the expression. However, other vectors can be used (see EXAMPLES 8 and 14). Other retroviral vectors (such as pRETRO-ON, Clontech), also use this promoter but have the advantages of entering cells without any transfection aid, integrating into the genome of target cells only when the target cell is dividing (as cancer cells do, especially during first remissions after chemotherapy) and they are regulated. It is also possible to turn on the expression of the FGF-5 nucleic acid by administering tetracycline when these WO 01/25271 PCTFIS00/26689 -64plasmids are used. Hence these plasmids can be allowed to transfect the cells, then administer a course of tetracycline with a course of chemotherapy to achieve better cytotoxicity.

Other plasmid vectors, such as pMAM-neo (Clontech) or pMSG (Amersham Pharmacia Biotech) use the MMTV-LTR promoter (which can be regulated with steroids) or the SV10 late promoter (pSVL, Amersham Pharmacia Biotech) or metallothionein responsive promoter (pBPV, Amersham Pharmacia Biotech) and other viral vectors, including retroviruses. Examples of other viral vectors include adenovirus, AAV (adeno-associated virus), recombinant HSV, poxviruses (vaccinia) and recombinant lentivirus (such as HIV). These vectors achieve the basic goal of delivering into the target cell the cDNA sequence and control elements needed for transcription.

The present disclosure includes all forms of nucleic acid delivery, including synthetic oligos. naked DNA, plasmid and viral, integrated into the genome or not.

Administration of Antibodies In an embodiment where the therapeutic molecule is an antibody, specifically an antibody that recognizes FGF-5 proteins, administration may be achieved by direct injection, or by use of microparticle bombardment a gene gun, Biolistic, Dupont), or coating with lipids or cellsurface receptors or transfecting agents. Similar methods can be used to administer proteins, of fragments thereof.

The present disclosure also provides pharmaceutical compositions which include a therapeutically effective amount of the antibody, and a pharmaceutically acceptable carrier or excipient.

EXAMPLE 19 Antisense Disruption of FGF-5 Expression This example describes methods that can be used to decrease FGF-5 expression. Such methods are useful when treatment of tumors which express or over-express FGF-5 is desired, for example in RCC and carcinoma of the breast, prostate, bladder or pancreas. For example, some subjects may not respond to modulation of the immune response by an agent having FGF-5 activity.

Such subjects may include non-HLA-A3 subjects. As an alternative, or additional, therapy, subjects having an FGF-5 expressing or overexpressing neoplasm may respond to therapies which modulate FGF-5 expression or activity, for example by decreasing any neoplastic response to FGFexpression, for example angiogenesis. One approach to decreasing FGF-5 expression, activity, or function, is to use antisense oligonucleotides.

To design an antisense oligonucleotide, the mRNA sequence from the desired molecule, such as human FGF-5, is examined. Regions of the sequence containing multiple repeats, such as WO 01/25271 PCT/US00/26689 TTT IT'T are not as desirable because they will lack specificity. Several different regions can be chosen. Of those, oligos are selected by the following characteristics: ones having the best conformation in solution; ones optimized for hybridization characteristics; and one having less potential to form secondary structures. Antisense molecules having a propensity to generate secondary structures are less desirable.

Plasmids containing FGF-5 antisense sequences can also be genereated. For example, cDNA fragments coding for human FGF-5 are PCR amplified. The nucleotides are then amplified using Pfu DNA polymerase (Stratagene) and cloned in antisense orientation a vector, such as pcDNA vectors (InVitrogen). The nucleotide sequence and orientation of the insert can be confirmed by dideoxy sequencing using a Sequenase kit (Amersham Pharmacia Biotech).

Generally, the term "antisense" refers to a nucleic acid capable of hybridizing to a portion of an FGF-5 RNA (such as mRNA) by virtue of some sequence complementarity. The antisense nucleic acids disclosed herein can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.

The FGF-5 antisense nucleic acids are polynucleotides, and may be oligonucleotides (ranging from 6 to about 100 oligonucleotides). In specific aspects, the oligonucleotide is at least 15, 20. 62, 65, or 100 nucleotides, or a polynucleotide of at least 200 nucleotides. The antisense nucleic acids may be much longer constructs. The nucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.

The nucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, and may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86:6553-6; Lemaitre et al., Proc. Nal. Acad. Sci. USA 1987, 84:648-52; PCT Publication No. WO 88/09810) or bloodbrain barrier (see, PCT Publication No. WO 89/10134), hybridization triggered cleavage agents (see, Krol et al., BioTechniques 1988, 6:958-76) or intercalating agents (see, e.g., Zon, Phanr. Res. 1988, 5:539-49).

In one embodiment disclosed herein, an FGF-5 antisense polynucleotide (including oligonucleotides) is provided, for example of single-stranded DNA. The FGF-5 antisense polynucleotide may recognize any species of FGF-5. The antisense polynucleotide may be modified at any position on its structure with substituents generally known in the art. For example, a modified base moiety may be 5-fluorouracil, 5-bromouracil, 5-chlorouracil, hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta- D-galactosylqueosine, inosine, N-6-sopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2- WO 01/25271 PTU0168 PC r1USOO/26689 66 dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6adenine, 7-methylguanine, 5-methylaminornethyluracil, methoxyarninotnethyl-2-thiouraci, beta-Dmannosylqueosinc, S '-methoxyc-arboxymethyluracil. 5-methoxyuracil, 2-methytthio-N6isopentenyladenine, uracil-5-oxyacetic acid. pseudouracil, queosine, 2-thiocytosine. 5-methyl-2thiouracil, 2-thiouracil, 4-thiouracil, 5-metbyluracil. uracil-5-oxyacetic acid methylester, uracil-Soxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6diaminopurine.

In another embodiment, the polynucleotide includes at least one modified sugar moiety such as arabmnose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate. a phosphoramidate. a phosphordiamidate, a tuethyiphosphonate, an alkyl phosphotniester, or a formacetal or analog thereof.

In yet another embodiment, the polynucleotide is an a-anomeric oligonucleotide. An aanomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual 1-units, the strands run parallel to each other (Gautier et Nudl.

Acids Res. 1987, 15:6625-41). The oligonucleotide may be conjugated to another molecule, a peptide, hybridization triggered cross-linking agent, transport agent, or hybridizto-rgee cleavage agent. Oligonucleotides may include a targeting moiety that enhances uptake of the molecule by tumor cells. T1he targeting moiety can be a specific binding molecule, such as an antibody or fragment thereof that recognizes a molecule present on the surface of a diseased cell, such as a tumor cell.

As an alternative to antisense inhibitors, catalytic nucleic acid compounds, such as ribozymes or anti-sense conjugates, can be used to inhibit gene expression. Ribozymes may be synthesized and administered to the subject, or may be encoded on an expression vector, from which the ribozynie is synthesized in the targeted cell (as in PCT publication WO 9523225, and Beigelmnan et al. Nutc!. Acids Res. 1995, 23:4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of antisense with a metal complex, e.g.

terpyridylCu capable of mediating rtRNA hydrolysis, are described in Bashkin et al., 1995, App!. Biochem Biorechnol. 54:43-56.

Polynucleotides disclosed herein can be synthesized by standard methods known in the art, for example by use of an automated DNA synthesizer (Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligos; may be synthesized by the method of Stein et al. (Attc!. Acids Res. 1998, 16:3209), methylphosphonate oligos can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988. Proc. Nail. Acad. Sci. USA 85:7448-51). In a specific embodiment, the ILP-2 and ILP-3 antisense oligonucleotides comprise catalytic RNA, or a WO 01/25271 PCT/US00/26689 -67ribozyme (see PCT International Publication WO 90/11364, Sarver et al., Science 1990, 247:1222- In another embodiment, the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., Nucl.

Acids Res. 1987, 15:6131-48), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 1987, 215:327-330).

The antisense polynucleic acids disclosed herein comprise a sequence complementary to at least a portion of an RNA transcript of an FGF-5 gene, such as a human FGF-5 gene. However, absolute complementarity, although advantageous, is not required. A sequence can be complementary to at least a portion of an RNA, meaning a sequence having sufficient complementarily to be able to hybridize with the RNA, forming a stable duplex; in the case of double-strandedFGF-5antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a FGF-5 RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

The relative ability of polynucleotides (such as oligonucleotides) to bind to complementary strands is compared by determining the melting temperature of a hybridization complex of the poly/oligonucleotide and its complementary strand. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). A reduction in UV absorption indicates a higher Tm. The higher the Tm the greater the strength of the binding of the hybridized strands. As close to optimal fidelity of base pairing as possible achieves optimal hybridization of a poly/oligonucleotide to its target RNA.

The amount of FGF-5 antisense nucleic acid which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In one embodiment, pharmaceutical compositions comprising FGF-5 antisense nucleic acids are administered via liposomes, microparticles, or microcapsules. In other embodiments, it may be useful to use such compositions to achieve sustained release of the FGF-5 antisense nucleic acids. In yet other embodiments, it may be desirable to utilize liposomes targeted via antibodies to specific identifiable tumor antigens (Leonetti et al. Proc. Natl. Acad. Sci. USA 1990, 87:2448-51; Renneisen et al. J. Biol. Chem.

1990, 265:16337-42).

WO 01/25271 PCT/US00/26689 -68- EXAMPLE Methods of Treatment using Antisense Molecules When FGF-5 levels are prematurely downregulated by various antisense strategies, the FGF-5 expressing or over-expressing tumor may regress (see EXAMPLE 19). FGF-5 antisense oligonucleotides (EXAMPLE 19) can be used to disrupt cellular expression of FGF-5 proteins, which results in a decrease in FGF-5 expression or activity, such as by a factor of at least or 80%. The decrease in FGF-5 expression and/or activity may cause regression of expressing or non-expressing tumors.

The subject suffering from a disease in which FGF-5 is expressed or over-expressed can be treated with a therapeutically effective amount of FGF-5 antisense oligonucleotides. After the antisense has taken effect (FGF-5 levels are downregulated), for example after 24-48 hours.

the subject can be monitored for regression of the tumor and/or lysis of the cells of the tumor.

Prophylactic Treatments The treatments disclosed herein can also be used prophylactially, for example to inhibit or prevent progression to of a disease in which FGF-5 is expressed or over-expressed. Such administration is indicated where the treatment is shown to have utility for treatment or prevention of the disorder. The prophylactic use is indicated in conditions known or suspected of preceding progression to diseases associated with an undesired amount of FGF-5 expression. Such diseases may include tumors in which FGF-5 expression is elevated, such as RCC, breast cancer and prostate cancer.

EXAMPLE 21 Generation and Expression of FGF-5 Fusion Proteins Methods for making fusion proteins are well known to those skilled in the art. For example U.S. Patent No. 6,057,133 to Bauer et al. (herein incorporated by reference) discloses methods for making fusion molecules composed of human interleukin-3 (hlL-3) variant or mutant proteins functionally joined to a second colony stimulating factor, cytokine, lymphokine.

interleukin, hematopoietic growth factor or IL-3 variant. U.S. Patent No. 6,072,041 to Davis et al. (herein incorporated by reference) discloses the generation of fusion proteins comprising a single chain Fv molecule directed against a transcytotic receptor covalently linked to a therapeutic protein.

Similar methods can be used to generate fusion proteins comprising FGF-5 (or variants, mutants, polymorphisms, or fragments thereof) linked to other amino acid sequences. Linker WO 01/25271 PCT/US00/26689 -69regions can be used to space the two portions of the protein from each other and to provide flexibility between them. The linker region is generally a polypeptide of between 1 and 500 amino acids in length, for example less than 30 amino acid in length. The linker joining the two molecules can be designed to allow the two molecules to fold and act independently of each other, not have a propensity for developing an ordered secondary structure which could interfere with the functional domains of the two proteins, have minimal hydrophobic or charged characteristic which could interact with the functional protein domains and provide steric separation of the two regions. Typically surface amino acids in flexible protein regions include Gly, Asn and Ser. Other neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Additional amino acids may also be included in the linker due to the addition of unique restriction sites in the linker sequence to facilitate construction of the fusions. Other moieties may also be included, as desired. These may include a binding region, such as avidin or an epitope, such as a polyhistadine tag, which may be useful for purification and processing of the fusion protein. In addition, detectable markers can be attached to the fusion protein, so that the traffic of the fusion protein through a body or cell may be monitored conveniently. Such markers may include radionuclides, enzymes, fluors, and the like.

Fusing of the nucleic acid sequences of FGF-5 (or variants, mutants, polymorphisms, or fragment thereof), with the nucleic acid sequence of another protein (or variants, mutants, polymorphisms, or fragment thereof), can be accomplished by the use of intermediate vectors.

Alternatively, one gene can be cloned directly into a vector containing the other gene. Linkers and adapters can be used for joining the nucleic acid sequences, as well as replacing lost sequences, where a restriction site was internal to the region of interest. Genetic material (DNA) encoding one polypeptide, peptide linker, and the other polypeptide is inserted into a suitable expression vector which is used to transform prokaryotic or eukaryotic cells, for example bacteria, yeast, insect cells or mammalian cells (see EXAMPLE The transformed organism is grown and the protein isolated by standard techniques, for example by using a detectable marker such as nickel-chelate affinity chromatography, if a polyhistadine tag is used. The resulting product is therefore a new protein, a fusion protein, which has FGF-5 joined by a linker region to a second protein. To confirm that the fusion protein was expressed, the purified protein is subjected to electrophoresis in SDS-polyacrylamide gels, and transferred onto nitrocellulose membrane filters using established methods. The protein products can be identified by Western blot analysis using antibodies directed against the individual components, polyhistadine tag and FGF-5 (see EXAMPLE 9).

WO 01/25271 PCT/US00/26689 EXAMPLE 28 Transgenic Plants and Animals The creation of transgenic plants and animals which express FGF-5 can be made by techniques known in the art, for example those disclosed in U.S. Patent Nos. 5,574,206; 5,723,719; 5,175,383; 5,824,838; 5,811,633; 5.620,881; and 5,767,337, which are incorporated by reference. Methods for generating transgenic mice are described in Gene Targeting, Joyuner ed., Oxford University Press, 1995 and Watson et al., Recombinant DNA 2nd Ed., W.H. Freeman and Co., New York, 1992, Chapter 14.

EXAMPLE 29 Methods for Measuring an Immune Response Several methods known to those skilled in the art can be used to monitor a modulation in an immune response in a subject. Although this example provides specific examples of assays which can be used for this purpose, other methods can be used to measure an immune response.

For example, modulations in an immune response, for example an increase or decrease in an immune response, can be measured by a observing a change in the activity or number of T-cells in the peripheral blood of a subject. In another embodiment, the immune response can be determined by measuring IFN-y concentration, using the assays described in EXAMPLES 2 and 3.

For example, when an immune response is increased, the concentration of IFN-y may increase by a desired amount, for example by at least 50%, at least 75% or even at least 1000%. This immune response may reduced by a desired amount, for example by at least 50%, for example at least in the presence of pan-anti-class I MHC mAb (such as W6/32) or anti-HLA-A3 mAb (such as GAPA3).

The immune response can be modulated by increasing or decreasing the immune response.

For example, FGF-5 or autologous CTLs specific to FGF-5, can be used provoke a CTL response against a tumor that is expressing or overexpressing FGF-5 in a subject to whom it is administered.

In another embodiment, it is an amount of FGF-5 or autologous CTLs, specific to FGF-5, required to increase by more than a desired amount, for example by at least 50%, at least 75% or at least 1000% using the assay described in EXAMPLES 2 and 3.

Having illustrated and described the principles of methods of treating FGF-5 expressing or overexpressing tumors, it should be apparent to one skilled in the art that the disclosure can be modified in arrangement and detail without departing from such principles. In view of the many possible embodiments to which the principles of our disclosure may be applied, it should be WO 01/25271 PCT/US00/26689 -71 recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as a limitation on the scope of the disclosure. Rather, the scope of the disclosure is in accord with the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

EDITORIAL NOTE APPLICATION NUMBER 78371/00 The following Sequence Listing pages 1 to 17 are part of the description. The claims pages follow on pages 72 to 76.

WO 01/25271 W00115271PCI'/USOO/26689 SEQUENCE LISTING <110> The Government of the United States of America <120> FIBROBLAST GROWTH FACTOR-5 (FGF-5) IS A TUMOR ASSOCIATED T-CELL ANTIGEN <130> 55911 <140> <141> <150> 60/157,103 <151> 1999-10-02 <160> <170> Patentln Ver. 2.1 <210> 1 <211> 143 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (27)..(140) <400> 1 cctctcccct tctcttcccc gaggct atg tcc acc cgg tgc ggc gag gcg ggc 53 Met Ser Thr Arg Cya Gly Glu Ala Gly 1 aga gcc aga ggc acg cay ccg cac agg ggc tac aga gcc cag aat cay 101 Arg Ala Arg Gly Thr Gin Pro His Arg Gly Tyr Arg Ala Gin Asn Gin 15 20 ccc tac aag atg cac tia gga ccc ccg cgg ctg gaa gaa tga 143 Pro Tyr Lys Met His Leu Gly Pro Pro Arg Leu Glu Glu <210> 2 <211> 38 <212> PRT <213> Homo sapiens <400> 2 Met Ser Thr Arg Cys Gly Glu Ala Gly Arg Ala Arg Gly Thr Gin Pro 1 5 10 His Arg Gly Tyr Arg Ala Gin Asn Gin Pro Tyr Lys met His Leu Gly 25 Pro Pro Arg Leu Glu Glu <210> 3 <211> 1123 WO 01/25271 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (140)..(946) <400> 3 cctCtCCCCt ictettecec gaggctatgt gaggcaegea gccgcacagg ggctacagag gaceccgcg gctggaaga atg agc ttg PCTIUSOO/26689 ccaceggtg cggcgaggcg ggeagagcca eccagaatca gccctaeaag atgcaettag tee tte etc ctc etc ctc ttC ttc Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe age Ser cc Pro tee Ser tee Ser eag Gin tac Tyr aaa Lys ttt Phe aaa Lys 140 ttc Phe aa t Asn tgg cac His aaa Lys age Ser tee Ser age Ser tge Cys gte: Val get Ala 125 ttt Phe aea Thr ace Thr tat etg Leu ggg Gly age Ser tee Ser agt Ser aga Arg aa t Asn 110 gtg Val tta Leu gat Asp tat Tyr gtt ate Ile caa Gin aga Arg ec Pro tte Phe gtg Vai gga Giy t Ct Ser geg Al a gac Asp gee Ala 175 gee etc Leu ce Pro eag Gin gea Al a cag Gin gge Gly tee Ser eag Gin atg Met tge Cys 160 tea Ser ctg age gee tgg get eac ggg Ser gga Gly age Ser get Ala 65 tgg Trp ate Ile eac His ggg Gly tea Ser 145 aag Lys gca Ala aat Ala ec Pro age Ser so tet Ser age Ser ggt cay gaa Giu att Ile 130 aaa Lys tte Phe ata Ile aaa Trp get Ala 35 agt Ser ctg Leu eee Pro tte Phe gee Al a 115 gta Val1 aaa Lys agg Arg eat His aga Ala 20 gee Ala age Ser gge Gly teg Ser eat His 100 aat Asn gga Gly gga Gly gag Glu aga Arg 180 gga His aect Thr get Ala age Ser ggg Gly 85 ctg Leu atg Met ata Ile aaa Lys cgt Arg 165 act rhr aaa 2 Gly gat Asp atg Met caa Gin 70 ege Arg cag Gin tta Leu ega Arg etc Leu 150 t tt Phe gaa Giu gee gag Glu agg Arg tet Ser gga Gly egg Arg ate Ile agt Ser gga Gly 135 eat His caa Gin aag egt etc gc Lys aae Asn tee Ser agt Se r ace Thr ta e Tyr gtt Val 120 gtt Val gea Ala gaa Glu Arg cet Pro tet Ser ggc Gly gge Gly ceg Pro 105 ttg Leu ttC Phe agt Ser aat Asn Leu ata Ile tet Ser ttg Leu age Ser gat Asp gaa Glu age Ser gc Ala age Se r Al a gge Gly gee Al a gag Giu ete Leu ggc Gly ata Ile aac Asn aag Lys 155 tat Tyr 220 268 316 364 412 460 508 556 604 652 700 748 170 aaa aea ggg egg gag Lys Thr Gly Arg Giu 185 aaa ega ggg tgc age WO 01/25271 PTU0168 PCTIUSOO/26689 Trp, Tyr Val 190 Ala Leu Asn Lys Gly Lys Ala Lys Arg Gly Cys Ser 200 cit cca aga tic Leu Pro Arg Phe ccc cgg Pro Arg 205 gtt aaa ccc cag Val Lys Pro Gin aic ici acc cat Ile Ser Thr His cag tcg gag Gin Ser Glu aag aaa aai cca Lys Lys Asn Pro cag cca Gin Pro 225 cct agc Pro Ser 240 acc aac Thr Asn gaa ctt tct tic Glu Leu Ser Phe cci atc aag tca Pro Ile Lys Ser 245 ica gtg aaa tac Ser Val Lys Tyr gtt act git cct Val Thr Val Pro aag ait ccc ctt iCt gca Lys Ile Pro Leu Ser Ala 250 aga ctc aag tic cgc ttt Arg Leu Lys Phe Arg Phe 265 cct cgg aaa Pro Arg Lys gga taa iattaatctt ggccitgtga gaaaccattc tticccctca ggagtttcta Gly taggigicit cagagttctg aagaaaaatt aciggacaca gcitcagcta tacttacact giattgaagt cacgtcattt gttcagigi gactgaaaca aaatgttttt tgataggaag gaaac ig <210> 4 <211> 268 <212> PRT <213> Homo sapiens <400> 4 Mei Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu Ile Leu 996 1056 1116 1123 1 5 Ala Al a Ser Gly Ser His 100 Asn Gly Gly Giu Arg 180 Gly His Gly Giu Thr Asp Arg Ala Met Ser Ser Gin Gly Gly Arg Arg Leu Gin Ile Met Leu Ser Ile Arg Gly 135 Lys Leu His 150 Arg Phe Gin 165 Thr Giu Lys Lys Ala Lys Lys Asn Se r Se r Thr Tyr Val 120 Val Ala Clu Thr Arg 200 Lys Ser Ser Ser Cys Val Ala 125 Phe Thr Thr Tyr Arg 205 Gly Gin Pro Ser Arg Gin Ser Pro Ala Ser Phe Gin so Arg Val Gly Asn Gly Ser 110 Val Ser Gin Leu Ala Met Asp Asp Cys 160 Tyr Ala ser 175 Val Ala Leu 190 Val Lys Pro Asn Lys Arg 195 Gly Cys Ser Pro WVO 01/25271 PTU0168 PCT/USOO/26689 Gin His Ile Ser Thr His Phe 210 215 Pro Giu Leu Ser Phe Thr Val Leu Pro Arg Phe Gin Ser Giu Gin Lys Asn Pro Pro Thr Val Pro Pro Ile Lys Lys Ile Pro Leu Arg Leu Lys Phe 265 Ser Ala Pro Arg Lys 250 Arg Phe Gly Asn Ser Val <210> <211> 531 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (531) <400> tgc aga gtg ggc ate ggt ttc eat ctg eag atc tac ceg gat Cys Arg Val Gly Ile Gly Phe His Leu Gin Ile Tyr Pro Asp ggc aaa Gly Lys gtc aat gga Val Asn Gly gct gtg tct Ala Val Ser cac gaa gee aat His Giu Ala Asn tta agt gtt ttg Leu Ser Vai Leu gaa ata ttt Giu Ile Phe cag ggg att gta Gin Gly Ile Val ata ega gga gtt ttc agc aae aaa Ile Arg Gly Vai Phe Ser Asn Lys ttt tta Phe Leu gcg atg tca aaa Ala Met Ser Lys gga aaa ctc cat Gly Lys Leu His agt gee aag tte Ser Ala Lys Phe aca gat gac tge aag Thr Asp Asp Cys Lys acc tat gcc tea gca Thr Tyr Ala Ser Ala as agg gag egt ttt Arg Glu Arg Phe gaa aat agc tat Glu Asn Ser Tyr ata cat aga act Ile His Arg Thr aaa aca ggg cgg Lys Thr Gly Arg gag tgg Giu Trp tat gtt gcc ctg aat aaa aga gga aaa Tyr Val Ala Leu Asn Lys Arg Gly Lys 100 105 gec aaa ega ggg Ala Lys Arg Gly tgc age ec Cys Scr Pro 110 336 egg gtt aaa Arg Val Lys 115 eec eag cat ate Pro Gin His Ile ace eat ttt ctt eea aga tte aag Thr His Phe Leu Pro Arg Phe Lys 125 cag teg Gin Scr 130 gag eag cea gaa Giu Gin Pro Glu tct ttc aeg gtt Ser Phe Thr Val gtt ect gaa aag Vai Pro Giu Lys 432 480 aaa aat eea ec age c ate aag tea aag Lys Asn Pro Pro Ser Pro Ile Lys Ser Lys ccc ett tet gea Pro Leu Ser Ala WO 01/25271 PTUOI68 PCTIUSOOf26689 cgg aaa aat acc aac tca gtg aaa tac aga ctc aag ttt cgc ttt gga Arg Lys Asn Thr Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 165 170 175 taa <210> 6 <211> 176 <212> PRT <213> Homo sapiens 531 <400> 6 Cys Arg 1 Val Asn Ala Vai Phe Leu so Thr Asp Thr Tyr Tyr Val Arg Val Gin Ser 130 Lys Asn 145 Arg Lys Gly Phe His Leu Gin Ile Tyr Pro Asp Giy Lys Ile Asm Lys Tyr Giu Ser Phe Glu Ala Phe 175 <210> 7 <211> 531 <212> DNA <213> Homno sapiens <220> <221> CDS <222> <400> 7 tgc aga gtg ggc atc Cys Arg Val Gly Ile ggt ttc cat ctg Giy Phe His Leu cag atc tac ccg gat ggc aaa 48 Gin Ile Tyr Pro Asp Gly Lys gte aat gga tcc cac gaa gcc aat Val Asn Gly Ser His Glu Ala Asn gct gtg tet eag ggg att gta gga Ala Vai Ser Gin Gly Ile Vai Giy 40 tta agt gtt Leu Ser Val ttg gaa ata ttt 96 Leu Giu Ile Phe ttc age aac aaa 144 Phe Ser Asn Lys ata cga gga gtt Ile Arg Gly Val WO 01/25271 PTU0168 PCTIUSOO/26689 ttt tta Phe Leu gcg atg tca aaa Ala Met Ser Lys aaa gga aaa ctc Lys Gly Lys Leu 55 agg gag Cgt ttt A-rg Giu Arg Phe cat His gca agt gcc aag ttc Ala Ser Ala Lys Phe gaa aat agc tat aat Giu Asn Ser Tyr Asn gat gac tgc aag Asp Asp Cys Lys acc tat gcc Thr Tyr Ala tca gca Ser Ala ata cat aga act Ile His Arg Thr aaa aca ggg cgg Lys Thr Gly Arg gag tgg Giu Trp tat gtg gcc ctg aat aaa aga gga aaa gcc aaa cga ggg tgc agc ccc Tyr Val Ala Leu Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro 100 105 110 cgg gtt aaa Arg Val Lys 115 ccc cag cat atc Pro Gin His Ile acc cat ttt ctg Thr His Phe Leu aga. ttc aag Arg Phe Lys cag tcg Gin Ser 130 gag cag cca gaa Giu Gin Pro Glu tct ttc acg gtt Ser Phe Thr Val act gtt cct gaa aag Thr Val Pro Giu Lys 140 ccc ctt tct gca cct Pro Leu Ser Ala Pro aaa aag cca cct agc Lys Lys Pro Pro Ser 145 cgg aaa aat acc aac Arg Lys Asn Thr Asn 165 atc aag cca aag Ile Lys Pro Lys tca gtg aaa tac Ser Val Lys Tyr ctc aag ttt cgc Leu Lys Phe Arg ttt gga Phe Gly 175 taa <210> 8 <211> 176 <212> PRT <213> Homo sapiens <400> 8 Cys Arg Val 1 Val Asn Gly Ala Vai Ser Gly Ile Gly Phe His Leu Gin Ile Tyr Pro Asp Gly Lys Ile Phe His Giu Ala Asn Gly Ile Val Gly Ser Val Leu Arg Gly Val Phe Leu Ala Met Ser Lys Lys Gly Lys Leu His Ala Arg Giu Arg Phe Gin Giu Ser Asn Lys Ala Lys Phe Asp Asp Cys Lys Phe Asn Ser Tyr Tyr Ala Ser Ile His Lys Arg Arg Thr Glu Gly Lys Ala Lys Thr Gly Lys Arg Gly Tyr Val Ala Leu Arg Giu Trp Cys Ser Pro 110 Arg Phe Lys Pro Glu Lys Arg Val Lye 115 Gin Ser Glu 130 Lys Lys Pro Gin His Ile His Phe Leu Gin Pro Glu Leu 135 Pro Ser Pro Ile Phe Thr Val Thr Lys Pro Lys Ile 6 Leu Ser Ala Pro WO 01/25271 WO 01l5271PCT/US00126689 145 150 155 160 Arg Lys Asn Thr Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 165 170 175 <210> 9 <211> 741 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <400> 9 gag aag cgt cte gci Glu Lys Arg Leu Al, 1 agg aac cct ata ggi Arg Ann Pro Ile Gl' ici ice tct tct gci ser Ser Ser Ser Al.

gga agt ggc ttg gai Gly Ser Gly Leu Gli s0 cgg acc ggc agc cti Arg Thr Gly Ser Lei ate tac ccg gat ggi Ile Tyr Pro Asp Gl, 8' agt gtt itg gaa at.

Ser Val Leu Glu Ili 100 gga gtt ttc agc aa Gly Val Phe Ser Asi 115 cat gca agt gcc aac.

His Ala Ser Ala Ly: 130 caa gaa aat agc tal Gin Glu Ann Ser Tyi 145 aaa aca ggg cgg ga( Lys Thr Gly Arg G11 16! aaa cga ggg tgc agc ccc aaa ggg caa ccc gga ccc gct gcc act gat Pro Lys Gly Gin Pro Gly Pro Ala Ala Thr Asp agc Ser tee Ser agc Ser 55 tge Cys gtc Val.

gct Ala iii Phe aca Thr 135 acc Thr tat Tyr egg aga Arg ccc Pro tiC Phe gig Val gga Gly ici Ser 105 gcg Ala gac Asp gcc Al a gce Ala aaa age agi Ser Ser tet ctg Ser Leu age ec Ser Pro ggt tte Gly Phe gaa gee Giu Ala att gta Ile Val aaa aaa Lys Lys 125 tt agg Phe Arg 140 ata cat Ile His aaa aga Lys Arg cat ate atg Met caa Gln ege Arg eag Gin tta Leu cga Arg etc Leu ttt Phe gaa Glu 160 gee Ala cat 96 144 192 240 288 336 384 432 480 528 576 WO 01/25271 Lys Arg Gly ttt ctt cca Phe Leu Pro 195 PCTIUSOO/26689 Ser Pro Arg aga ttc aag cag Arg Phe Lys Gin Val Lys 185 tcg gag Ser Giu 200 aat cca Asn Pro Pro Gin His Ile cag cca gaa Gin Pro Giu cct agc cct Pro Ser Pro 220 Ser Thr His 190 tct ttc acg Ser Phe Thr aag tca aag Lys Ser Lys gtt act Val Thr 210 att ccc Ile Pro gtt cct gaa aag Val Pro Glu Lys ctt tct gca Leu Ser Ala cct cgg Pro Arg 230 gga taa Gly aaa aat acc Lys Asn Thr aac tca Asn Ser 235 gtg aaa tac Val Lys Tyr aag ttt cgc Lys Phe Arg <210> <211> 246 <212> PRT <213> Hoino sapiens <400> Glu Lys 1 Arg Asn Ser Ser Gly Ser Arg Thr Ile TyrI Ser Val I Giy ValI His Ala 130 Gin Giu 145 Lys Thr Lys Arg( Phe Leu I Val Thr 1 210 Ile Pro 1 225 Leu Lys I Pro Ser Se r Gin Tyr 70 Lys Phe Lys Phe Asn 150 Trp Pro Lys Lys Pro 230 Gly Gly Gin Ser Arg Ser Pro Ser Phe Arg Val Asn Gly Val Ser 105 Leu Ala 120 Asp Asp Tyr Ala Val Ala Val Lys 185 Ser Giu 200 Asn Pro Lys Asn Gly Ser Ala Trp Ile 75 His Gly Ser Lys Al a 155 Asn Gin Pro Ser Pro Ser Ser Se r Gly Giu Ile Lys Phe 140 Ile Lys His Glu Pro 220 Ala Ser Leu Pro Phe Ala Val Lys 125 Arg His Arg Ile Leu 205 Ile Thr Asp Ala Met Ser Gin Gly Arg Leu Gin Met Leu Ile Arg Lys Leu Arg Phe Thr Giu 160 Lys Ala 175 Thr His Phe Thr Lys Ser Lys Asn Ser Val Lys Tyr WO 01/25271 PCTtUS00126689 <210> 11 <211> 741 <212> DNA <213> Homo sapiens <220> <221> CDS <222> <400> 11 gag aag cgt ctc gcc ccc aaa ggg caa ccc gga ccc gct gcc act gat 48 Giu Lys Arg Leu Ala Pro Lys Gly Gin Pro Giy Pro Ala Ala Thr Asp 1 5 10 agg aac cct aga ggc tcc agc agc aga cag agc agc agt agc gct atg 96 Arg Asn Pro Arg Gly Ser Ser Ser Arg Gin Ser Ser Ser Ser Ala Met 25 tct tcc tct tct gcc tcc tcc tcc ccc gca gct tct ctg ggc agc caa 144 Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala Ala Ser Leu Gly Ser Gin 40 gga agt ggc ttg gag cag agc agt ttc cag tgg agc ccc tcg ggg cgc 192 Gly Ser Gly Leu Giu Gin Ser Ser Phe Gin Trp Ser Pro 5cr Gly Arg 55 cgg acc ggc agc ctc tac tgc aga. gtg ggc atc ggt ttc cat ctg cag 240 Arg Thr Gly 5cr Leu Tyr Cys Arg Val Gly Ile Gly Phe His Leu Gin 70 75 atc tac ccg gat ggc aaa gtc aat gga tcc cac gaa gcc aat atg tta 288 Ile Tyr Pro Asp Gly Lys Val Asa Gly Ser His Giu Ala Asa Met Leu as 90 agt gtt ttg gaa ata ttt gct gtg tct cag ggg att gta gga ata cga 336 Ser Vai Leu Giu Ile Phe Ala Val Ser Gin Gly Ile Val Gly Ile Arg 100 105 110 gga gtt ttc agc aac aaa ttt tta gcg atg tca aaa aaa gga aaa ctc 384 Gly Val Phe Ser Asn Lys Phe Leu Ala Met Ser Lys Lys Gly Lys Leu 115 120 125 cat gca agt gcc aag ttc aca gat gac tgc aag ttc agg gag cgt ttt 432 His Ala Ser Ala Lys Phe Thr Asp Asp Cys Lys Phe Arg Giu Arg Phe 130 135 140 caa gaa aat agc tat aat acc tat gcc tca gca ata cat aga act gaa 480 Gin Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala Ile His Arg Thr Giu 145 i5o 155 160 aaa aca ggg cgg gag tgg tat gtg gcc ctg aat aaa aga gga aaa gcc 528 Lys Thr Giy Arg Glu Trp Tyr Vai Ala Leu Asn Lys Arg Giy Lys Aia 165 170 175 aaa cga ggg tgc agc ccc cgg gtt aaa ccc cag cat atc tct acc cat 576 Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gin His Ile Ser Thr His 180 185 190 ttt ctg cca aga ttc aag cag tcg gag cag cca. gaa ctt tct ttc acg 624 Phe Leu Pro Arg Phe Lys Gin Ser Giu Gin Pro Glu Leu Ser Phe Thr 195 200 205 WO 01/25271 WO 0125271PCT(USOO/26689 gtt act gtt Val Thr Val 210 att ccc ett Ile Pro Leu 225 etc aag ttt Leu Lys Phe cct gaa aag aaa aag cca cct agc ccc ate aag cca aag Pro Giu Lys Lys Lys Pro Pro Ser Pro Ile Lys Pro Lys 215 220 tet gca cct egg aaa aat acc aac tea gtg aaa tac aga Ser Ala Pro Arg Lys Asn Thr Asn Ser Val Lys Tyr Arg 230 235 240 cgc ttt gga taa Arg Phe Gly 245 <210> 12 <211> 246 <212> PRT <213> Homo sapiens <400> 12 Giu Lys Arg Leu Ala 1 5 Arg Asn Pro Arg Gly Ser Ser Ser Ser Ala Gly Ser Gly Leu Glu Arg Thr Gly Ser Leu Ile Tyr Pro Asp Giy Ser Val Leu Glu Ile 100 Gly Val Phe Ser Asn 115 His Ala Ser Ala Lys 130 Gin Giu Asn Ser Tyr 145 Lys Thr Gly Arg Giu 165 Lys Arg Gly Cys Ser 180 Phe Leu Pro Arg Phe 195 Val Thr Val Pro Glu 210 Ile Pro Leu Ser Ala 225 Leu Lys Plie Arg Phe 245 <210> 13 <211> 920 <212> DNA <213> Homo sapiens <400> 13 Pro Gin Ala Gin Gly Ser Gin Met Cys Ser Leu 170 Pro Gin Pro Gly Se r Aia Trp Ile 75 His Giy Ser Lys Ala 155 Asn Gin Pro Ser Pro Ser Ser Ser Gly Giu Ile Lys Phe 140 Ile Lys His Giu Pro 220 Ala Ser Leu Pro Phe Aila Val1 Lys 125 Arg His Arg Ile Leu 205 Ile Asp Met Gin Arg Gin Leu Arg Leu Phe Glu 160 Al a His Thr Lys Arg 240 Arg Lys Asn Thr Asn 5cr Vai Lys Tyr WO 01/25271 atgtccaccc agagcccaga tgtccttcct agaagcgtct gctccagcag ccgcagcttc cctcggggcg tctacccgga tatttgctgt cgatgtcaaa gggagcgttt aaacagggcg gcCcccgggt agcagccaga tcaagtcaaa tcaagtttcg PCT/US00126689 ggtgcggcga atcagcccta cctcctcctc cgcccccaaa cagacagagc tctgggcagc ccggaccggc tggcaaagtc gtctcagggg aaaaggaaaa t caagaaaat ggagtggtat taaaccccag actttctttc gattcccctt ctttggataa ggcgggcaga caagatgcac ttcttcagcc gggcaacccg agcagtagcg caaggaagtg agcctctact aatggatccc attgtaggaa ctccatgcaa agctataata gttgccctga catatctcta acggttactg tctgcacctc gccagaggca t taggacc cc acctgatcct gacccgctgc ctatgtcttc gcttggagca gcagagtggg acgaagccaa tacgaggagt gtgccaagtt cctatgcctc ataaaagagg cccattttct ttcctgaaaa ggaaaaatac cgcagccgca cgcggctgga cagcgcctgg cactgatagg ctcttctgcc gagcagtttc catcggtttc tatgttaagt tttcagcaac cacagatgac agcaatacat aaaagccaaa tccaagattc gaaaaat cca caactcagtg caggggctac agaatgagct gctcacgggg aaccctatag tcctcctccc cagtggagcc catctgcaga gttttggaaa aaatttttag tgcaagttca agaactgaaa cgagggtgca aagcagtcgg cctagcccta aaatacagac <210> 14 <211> 920 <212> DNA <213> HOMO sapiens <400> 14 atgtccaccc agagcccaga tgtccttcCt agaagcgtc t gctccagcag ccgcagcttc cctcggggcg t ctacccgga tatttgctgt cgatgtcaaa gggagcgttt aaacagggcg gcccccgggt agcagccaga tcaagccaaa tcaagtttcg ggtgcggcga atcagcccta cctcctcctc cgcccccaaa cagacagagc tctgggcagc ccggaccggc tggcaaagtc gtctcagggg aaaaggaaaa tcaagaaaat ggagtggtat taaaccccag actttctttc gattcccctt ctttggataa ggcgggcagc caagatgcac ttcttcagcc gggcaacccg agcagtageg caaggaagtg agcctctact aatggatccc attgtaggaa ctccatgcaa agct ataata gtggccctga catatctcta acggttactg tctgcacctc gcc agaggca ttaggacccc acctgatcct gacccgctgc ctatgtcttc gcttggagca gcagagtggg acgaagccaa tacgaggagt gtgccaagtt cctatgcctc at aaaagagg cccattttct ttcctgaaaa ggaaaaatac cgcagccgca cgcggctgga cagcgcctgg cactgatagg ctcttctgcC gagcagtttc catcggtttc tatgttaagt tttcagcaac cacagatgac agcaatacat aaaagccaaa gccaagattc gaaaaagcca caactcagtg caggggctac agaatgagct gctcacgggg aaccctagag tcCtCctccc cagtggagcc cat ctgcaga gttttggaaa aaatttttag tgcaagttca agaac tgaaa cgagggtgca aagcagtcgg cctagcccta aaatacagac <210> <211> 1653 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (50)..(166) <400> cggacgcgtg ggctctctct tcccctctcc ccttctcttc cccgaggct atg tcc acc 58 Met Ser Thr cgg tgc ggc gag gcg ggc agc gcc aga Arg Cys Gly Glu Ala Gly Ser Ala Arg 10 ggc acg cag ccg cac agg ggc Gly Thr Gin Pro His Arg Gly is tac aga gcc cag aat cag ccc tac aag atg cac 11 tta gga ccc ccg cgg 154 WO 01t25271 PCTUSOOI26689 Tyr Arg Ala Gin Asn Gin Pro Tyr Lys Met His Leu Gly Pro Pro Arg 25 30 ctg gaa gaa tga. gcttgtcctt cctcctcctc ctcttcttca gccacctgat 206 Leu Glu Glu cctcagcgcc tgccactgat ttcCtcttct gcagagcagt gggcatcggt caatatgtta agttttcagc gttcacagat ctcagcaata aggaaaagcc tctgccaaga aaagaaaaag taccaactca tgagaaacca at tac tggac tgtgactgaa gggagcacac t t tagaact t acagattaac ttgatgcaga cctctgcatt attttatgct ttacattttt aa tatgt tta aaaaaaa tgggctcacg aggaacccta gcctctcct ttccagtgga ttccatctgc agtgttttgg aacaaatttt gactgcaagt catagaactg aaacgagggt ttcaagcagt ccacct agcc gtgaaataca ttctttcccc acagcttcag acaaaatgtt tcc ttcagt t tgtattttcg ctgaaagaac taaaatattt gagcatattt gtttatgaat cctattcact gtttacatt gggagaagcg gaggc t ccag cccccgcagc gcccctcggg agatctaccc aaatatttgc tagcgatgtc tcagggagcg aaaaaacagg gcagcccccg cggagcagcc ctatcaagcc gactcaagtt tcaggagtt ctatacttac ttttgatagg cagcaagaca.

gaaagttaaa atacatgata tgttaacttt tcttactttt tataaatgtg gcactttttt ttaaaatgtt tctcgccccc cagcagacag ttctctgggc gcgccggacc ggatggcaaa tgtgtct cag aaaaaaagga ttttcaagaa gcgggagtgg ggttaaaccc agaactttct aaagattccc tcgctttgga ctataggtgt actgtattga aaggaaac ig taaagc ci i taacagggac catttttatt tgtttttttt attattttaa tttatagctc attgttttta taaattctci aaagggcaac agcagcagta agccaaggaa ggcagcctci gtcaatggai gggattgtag aaactccatg aatagctata tatgtggccc cagcatatct ttcacggt ta ctttctgcac taatattcct cttcagagit agtcacgi ca gaattctttg tgctttatgc tacgtattti tttggtttcc tgtttgtttt tiaatatgac atttgtaata tttciagcca t icacagcaa ccggacccgc 266 gcgctatgtc 326 gtggcitgga 386 acigcagagt 446 cccacgaagc 506 gaatacgagg 566 caagtgccaa 626 atacctatgc 686 tgaataaaag 746 ctacccattt 806 ctgttcctga 866 ctcggaaaaa 926 cttggccttg 986 ctgaagaaaa 1046 ttgttcag 1106 iactaataca 1166 ttgagggata 1226 tctgactttt 1286 aaagaatatt 1346 cttaaaagta 1406 ataagcaaic 1466 tggaaatctt 1526 tacctcagat 1586 aaaaaaaaaa 1646 1653 <210> 16 <211> 38 <212> PRT <213> Homo sapiens WO 01/25271 WO 0125271PCT/USOO/26689 <400> 16 Met Ser Thr Arg cys Gly Glu Ala Gly Ser Ala Arg Gly Thr Gin Pro 1 5 His Arg Gly Tyr Arg Ala Gin Asn Gin Pro Tyr Lys Met His 25 Pro Pro Arg Leu Giu Glu <210> 17 <211> 1653 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (163)..(969) <400> 17 cggacgcgtg ggctctctct tcecctetce ccttctcttc ccgaggeta tgtccacccg gtgcggcgag gcgggcagcg ccagaggcac gcagccgcac aggggctaca gagceagaa 120 tcagceetac aagatgcact taggaccce gcggctggaa ga atg age ttg tee Met Ser Leu Ser 1 etc age gee tgg get Leu Ser Ala Trp Ala tte etc ctc etc etc Pile Leu Leu Leu Leu tte ttc Pile Phe age eac ctg Ser His Leu eac ggg gag aag His Gly Glu Lys etc gee eec aaa.

Leu Ala Pro Lys caa eec gga ec Gin Pro Gly Pro get gee Ala Ala act gat agg Thr Asp Arg cet aga ggc tee Pro Arg Gly Ser age aga eag age age agt age Ser Arg Gin Ser Ser Ser Ser 318 get atg tet tee tet tet gee Ala Met Ser Ser Ser Ser Ala tee tee eee gea Ser Ser Pro Ala tet etg gge Ser Leu Gly age caa Ser Gin gga agt gge ttg Gly Scr Giy Leu eag age agt ttc Gin Ser Ser Pile tgg age ce teg Trp Ser Pro Ser ege egg ace ggc Arg Arg Thr Gly etc tac tge aga Leu Tyr Cya Arg ggc ate ggt ttc Gly Ile Gly Phe etg eag ate tac Leu Gin Ile Tyr gat ggc aaa. gte aat gga tee eac gaa Asp Gly Lys Val Asn Gly Ser His Giu 110 gee aat Ala Asn 115 atg tta agt Met Leu Ser ttg gaa ata ttt Leu Glu Ile Pile gig tet cag ggg Val Ser Gin Gly att gta gga Ile Val Gly 130 558 WO 01 /25271 ata cga gga Ile Arg Gly PCTIUSOO026689 gtt tte agc aac aaa tt tta. geg atg tca. aaa aaa gga Val Phe Ser Asn Phe Leu Ala Met Ser Lys Lys Gly aaa ctc Lys Leu 150 cgt ttt Arg Phe caa.

Gin gca agt gce Ala Ser Ala gaa aat agc Glu Asn Ser 170 aca ggg cgg Thr Gly Axg aag ttc Lys Phe 155 tat aat Tyr Asn gag tgg Glu Trp, aca gat gac tgc aag Thr Asp Asp Cys Lys 160 ace tat gcc tea gca Thr Tyr Ala Ser Ala 175 tat gtg gcc ctg aat Tyr Val Ala Leu Asn 190 cgg gtt aaa ccc cag Arg Val Lys Pro Gin ttc agg gag Phe Arg Glu ata cat Ile His gaa aaa Glu Lys aaa gcc aaa.

Lys Ala Lys ace cat ttt Thr His Phe 215 tte acg gtt Phe Thr Val 230 tgc agc ccc Cys Ser Pro aaa aga gga Lys Arg Gly 195 eat ate tet His Ile Ser 210 gaa. ctt tct Giu Leu Ser cet ate aag Pro Ile Lys cca aga ttc Pro Arg Phe aag Lys 220 aag Lys tcg gag cag Ser Glu Gin act gtt cct Thr Val Pro aaa aag cca Lys Lys Pro aag att ccc ett Lys Ile Pro Leu ect egg aaa Pro Arg Lys aat ace Ass Thr 255 aac tea gtg Asn Ser Val 606 654 702 750 798 846 894 942 989 1049 1109 1169 1229 1289 1349 1409 1469 1529 1589 1649 1653 tac aga etc aag ttt ege ttt gga taa Tyr Arg Leu Lys Phe Arg Phe Gly 265 tattcctctt ggecttgtga.

gaaaceattc actggacaca gactgaaaca agcacactcc agaactttgt gattaacctg atgcagataa ctgcattgag ttatgctgtt catttttcct atgtttagtt tttccectca gcttcageta aaatgttttt ttcagttcag attttcggaa aaagaacata aatattttgt catattttct tatgaattat attcactgca ttacatttta ggagtttcta tacttaeact tgat aggaag caagacataa agttaaataa catgatacat taacttttgt tacttttatt aaatgtgttt ct ttt ttat t aaatgtttaa taggtgtctt gtattgaagt gaaactggaa agccttttgc cagggactac ttttattttt ttttttttgt attttaatta atagctcatt gtttttattt cagagttctg eacgtcatt t ttetttgtac tttatgettg gtatttttct ggtt teeaaa ttgttttctt atatgacata.

tgtaatatgg ctagccatac aagaaaaatt gttteagtgt taatacaggg agggatattt gaettttaea gaatattttg aaaagtaeet agcaateatt aaatctttta etcagataat attctetttc acagcaaaaa aaaaaaaaaa aaaa WO 01/25271 PTUO/68 PCTIUSOO/26699 <210> 18 <211> 268 <212> PRT <213> Homo sapiens <400> 18 Ser Ala Al a Ser Gly Ser His 100 Asn Gly Gly Glu Arg 180 Gly Ser Ser Lys Lys 260 Phe Leu His Gly Thr Asp Ala Met Ser Gin Gly Arg Leu Gin Met Leu Ile Arg Lys Leu 150 Arg Phe 165 Thr Giu Lys Ala Thr His Phe Thr 230 Pro Lys 245 Tyr Arg Leu Leu Phe Lys Arg Leu 25 Asn Pro Arg Ser Ser Ser Ser Gly Leu Thr Gly Ser Tyr Pro Asp 105 Val Leu Glu 120 Val Phe Ser Ala Ser Ala Glu Asa Ser 170 Tbr Gly Arg 185 Arg Gly Cys 200 Leu Pro Arg Thr Val Pro Pro Leu Ser 250 Lys Phe Arg 265 Leu Ile Gly Gin Ser Arg Scr Pro Ser Phe Arg Val Asn Gly 110 Val Ser Leu Ala Asp Asp Tyr Ala 175 Val Ala 190 Val Lys Ser Giu Lys Pro Lys Asn 255 <210> 19 <211> <212> PRT <213> Homo sapiens <400> 19 Lys Phe Arg Giu Arg Phe 1 5 Ala Ile His Arg Thr Giu Asn Lys Arg Gly Lys Ala Gin Giu Asa Ser 10 Lys Thr Gly Arg 25 Lys Arg Gly Cys 40 Tyr Asn Thr Tyr Al1a Ser Glu Trp Tyr Val Ala Leu Ser Pro Arg Val Lys Pro Gin His Ile Ser Thr His Phe Leu Pro Arg Phe WO 01/25271 PCT/US00/26689 <210> <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Forward Primer <400> cttcttcagc cacctgatcc tc 22 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Forward Primer <400> 21 tgcagagtgg gcatcggttt c 21 <210> 22 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Reverse Primer <400> 22 tatgctcaat gcagaggtac <210> 23 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Reverse Primer <400> 23 cgtagtccct gttatttaac <210> 24 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer that can be used to clone HLA-A3 <400> 24 WO 01/25271 PCT/USOOIZ6689 ttggggaggg agcacaggtc agcgtgggaa g 31 <210> <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Prim-er that can be used to clone HLA-A3.

<400> ggactcagaa tctccccaga cgccgag 27

Claims (43)

1. A method of treating a subject having a neoplasm expressing fibroblast growth factor-5 (FGF-5) comprising: a) stimulating an immune response to FGF-5 in the subject sufficient to stimulate a cytotoxic T-cell response to a cell of the neoplasm; or b) administering a therapeutically effective amount of an agent which decreases FGF-5 expression or activity.

2. The method of claim 1, wherein the neoplasm expressing FGF-5 is selected from the group consisting of a prostate carcinoma, a breast carcinoma, a bladder carcinoma, a pancreas carcinoma, and a renal cell carcinoma (RCC).

3. The method of claim 2, wherein the neoplasm is a RCC.

4. The method of any one of claims 1 to 3, wherein the cytotoxic T cell response is stimulated by administering a therapeutically effective amount of an agent that stimulates the immune response. The method of claim 4, wherein the agent that stimulates an immune response is selected from the group consisting of an FGF-5 polypeptide and an immunoreactive sensitized T cell sensitized with

6. The method of claim 5, wherein the FGF-5 polypeptide that stimulates an immune response comprises an amino acid sequence selected from the group consisting of: the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 16, 18 or 19; amino acid sequences that differ from those specified in by one 25 or more conservative amino acid substitutions that retain the ability to stimulate an immune response; fragments of the amino acid sequence of or that retain the ability to stimulate an immune response; and amino acid sequences having at least 70% sequence identity to the sequences specified in and that retain the ability to stimulate an immune response.

7. The method of claim 6, wherein the FGF-5 polypeptide that stimulates an immune response comprises an amino acid sequence selected from the group consisting of: 35 the sequence shown in SEQ ID NO 8, 12, 18 or 19; an immunogenic fragment thereof that retains the ability to Sstimulate an immune response; and \\melb_files\home$\Simeona\Keep\Speci\78371 OO.doc 12/01/04 73 a sequence having at least 70% sequence identity to or that retains the ability to stimulate an immune response.

8. The method of any one of claims 5 to 7, wherein administering the therapeutically effective amount of FGF-5 polypeptide comprises administering a nucleic acid encoding the FGF-5 polypeptide sufficient to stimulate a cytotoxic T cell response.

9. The method of claim 8, wherein administering the nucleic acid comprises administering an effective amount of a vector comprising a nucleic acid encoding the polypeptide sufficient to stimulate a cytotoxic T cell response to a cell of the neoplasm. The method of claim 9, wherein the vector is a viral vector.

11. The method of claim 10, wherein the viral vector is a retroviral vector.

12. The method of any one of claims 5 to 7, wherein administering the therapeutically effective amount of FGF-5 polypeptide comprises administering an effective amount of a host cell expressing a recombinant nucleic acid encoding sufficient to stimulate a cytotoxic T cell response to a cell of the neoplasm.

13. The method of any one of claims 5 to 7, wherein administering the therapeutically effective amount of FGF-5 polypeptide comprises administering a purified FGF-5 polypeptide sufficient to stimulate a cytotoxic T cell response.

14. The method of claim 5, wherein the immunoreactive sensitized T cell sensitized with FGF-5 is autologous. The method of claim 5, wherein the immunoreactive sensitized T cell sensitized with FGF-5 is heterologous.

16. The method of claim 1, wherein the agent that decreases 25 expression is an FGF-5 antisense molecule.

17. The method of claim 16, wherein the FGF-5 antisense molecule hybridizes to an RNA or a plus strand of an FGF-5 nucleic acid.

18. The method of claim 1, wherein the agent that decreases FGF-5 activity is an FGF-5 specific binding agent.

19. The method of claim 18, wherein the FGF-5 specific binding agent is capable of specifically binding to an FGF-5 polypeptide.

20. The method of claim 19, wherein the FGF-5 specific binding agent is selected from the group consisting of: polyclonal antibodies; monoclonal antibodies; and fragments of monoclonal antibodies. 35 21. The method of claim 1, wherein the agent that stimulates an immune response is therapeutically immunogenic in HLA-A3+ individuals.

22. The method of claim 21, further comprising the step of selecting HLA- \\melb_filea\homeS\Simeona\Keep\Speci\78371 00.doc 12/01/04 74 A3+ individuals to whom to administer the agent that stimulates an immune response.

23. A method of stimulating a cytotoxic T cell response against a RCC, comprising: contacting the T cell with an effective amount of an FGF-5 polypeptide or a cell expressing the FGF-5 polypeptide sufficient to stimulate the T cell to react with a cell of the RCC.

24. The method of claim 4, wherein the agent that stimulates an immune response is present in a pharmaceutically acceptable carrier. The method of claim 1, wherein the agent that decreases expression or activity is present in a pharmaceutically acceptable carrier.

26. The method of claim 1 or claim 4, further comprising administering one or more other anti-neoplastic compounds.

27. The method of claim 1, wherein the agent that decreases or increases expression or activity is present in a pharmaceutically acceptable carrier.

28. A method for detecting an enhanced susceptibility of a subject to a disease of abnormal FGF-5 expression, the method comprising detecting an increase in protein in a cell of the subject.

29. The method of claim 28, wherein the disease is selected from the group consisting of Hippel-Lindau disease, horseshoe kidneys, adult polycystic kidney disease, acquired renal cystic disease, a prostate carcinoma, a breast carcinoma, a bladder carcinoma, a pancreas carcinoma, and a RCC. The method of claim 28, wherein the disease is RCC.

31. The method of any one of claims 28 to 30, wherein the method comprises detecting FGF-5 protein in the cell of the subject, the method comprising: incubating an FGF-5 specific binding agent with proteins of the cell under conditions such that the specific binding agent will specifically bind to a protein present in the cell to form a specific binding agent:FGF-5 protein complex; and detecting an increase or decrease of specific binding agent:FGF-5 protein complexes, wherein an increase of the complexes relative to specific binding agent:FGF-5 protein complexes in a non-neoplastic cell indicates expression or overexpression of FGF-5, and an enhanced susceptibility of the subject to a disease of abnormal FGF-5 expression.

32. A method of lysing a cell of an FGF-5 expressing neoplasm in a subject, Scomprising enhancing an immune response against FGF-5 in the subject, sufficient to induce regression of the neoplasm.

33. The method of claim 32, wherein the cell is characterized by increased expression of a FGF-5 protein compared to FGF-5 expression in a same tissue type that \\melb files\home$\Simeona\Keep\Speci\78371 00.doc 12/01/04 is non-neoplastic.

34. The method of claim 33, wherein enhancing the immune response comprises exposing the cell to a therapeutically effective amount of an polypeptide, sufficient to provoke an immune response against

35. The method of claim 33, wherein enhancing the immune response comprises administering a therapeutically effective amount of a nucleic acid which can express the FGF-5 polypeptide.

36. The method of claim 33, wherein enhancing the immune response comprises administering a therapeutically effective amount of immuno-reactive sensitized T-cells wherein the sensitized T cells are sensitized with

37. Use of an agent which stimulates an immune response to FGF-5 in a subject in the preparation of a medicament for use in the treatment of an expressing neoplasm, wherein the agent is able to stimulate a cytotoxic T-cell response to the FGF-5 expressing neoplasm.

38. Use of an agent which decreases FGF-5 expression or activity in the preparation of a medicament for use in the treatment of an FGF-5-expressing neoplasm

39. A method of treating a subject having a neoplasm which expresses fibroblast growth factor-5 (FGF-5), the method comprising: administering a therapeutically-effective amount of an FGF-5 polypeptide to the subject sufficient to stimulate an immune response to the FGF-5 polypeptide, wherein the immune response stimulates a cytotoxic T-cell response to the neoplasm. The method of claim 39, wherein the FGF-5 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOS: 4, 6, 8, 10, 12, 16, 17, 18, 19 and the amino acid sequence disclosed in Figure 6.

41. A method of treating a subject having a neoplasm which expresses fibroblast growth factor-5 (FGF-5), the method comprising: administering a therapeutically-effective amount of a polynucleotide which encodes an polypeptide to the subject sufficient to stimulate an immune response to the polypeptide, wherein the immune response stimulates a cytotoxic T-cell response to the neoplasm.

42. The method of claim 41, wherein the polynucleotide encodes an polypeptide which comprises a sequence selected from the group consisting of SEQ ID NOS: 4, 6, 8, 10, 12, 16, 17, 18, 19 and the amino acid sequence disclosed in Figure 6.

43. The method of claim 41, wherein the polynucleotide comprises a 35 sequence selected from the group consisting ofSEQ ID NOS: 3, 5, 7, 9, 11, 13, 14, 17, and the nucleic acid sequence disclosed in Figure 6.

44. A method of treating a subject having a neoplasm which expresses fibroblast growth factor-5 (FGF-5), the method comprising: administering FGF-5-sensitized cytotoxic T-cells to the subject in an amount sufficient 40 to produce a therapeutic response to the neoplasm.

45. A method of treating a subject having a neoplasm which expresses fibroblast growth factor-5 (FGF-5), the method comprising: administering a therapeutically-effective amount of a polynucleotide which is antisense to a gene which encodes an FGF-5 polypeptide, in an amount sufficient to decrease the 45 expression of the FGF-5 polypeptide by the neoplasm. Hi\joannem\keep\78371-00 US govt 2.doc 26/05/04 -76-

46. A method of treating a subject having a neoplasm which expresses fibroblast growth factor-5 (FGF-5), the method comprising: administering a therapeutically effective amount of a specific binding agent to an FGF- polypeptide sufficient to decrease FGF-5 activity or expression by the neoplasm, and wherein the specific binding agent is selected from the group consisting of any one or more of polyclonal antibodies, monoclonal antibodies, Fab fragments of monoclonal antibodies, F(ab') 2 fragments of monoclonal antibodies and Fv fragments of monoclonal antibodies.

47. Use of an agent which decreases FGF-5 expression or activity in the preparation of a medicament for use in the treatment of an FGF-5-expressing neoplasm.

48. A method according to claim 1, substantially as herein described with reference to the description or any one of the examples or figures.

49. A method according to claim 28, substantially as herein described with reference to the description or any one of the examples or figures.

50. A method according to claim 39, substantially as herein described with reference to the accompanying drawings. Dated this 2 6 th day of May 2004 THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia 0 H:\joannem\keep\78371-00 US govt 2.doc 26/05/04