AU705768B2 - Rage tumor rejection antigens - Google Patents

Description

WO 96/29409 PCTISO ~/IlnAi7i -1- RAGE TUMOR REJECTION ANTIGENS Field of the Invention This invention relates to nucleic acid molecules which code for tumor rejection antigens and precursors thereof. The tumor rejection antigen precursors are processed, inter alia, into at least one tumor rejection antigen that is presented by HLA molecules. The nucleic acid molecules, proteins coded for by such molecules and peptides derived therefrom, as well as related antibodies and cytotoxic lymphocytes, are useful, inter alia, in diagnostic and therapeutic contexts.

Background of the Invention The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is complex. An important facet of the system is the T cell response. T cells can recognize and interact with other cells via cell surface complexes on the other cells of peptides and molecules referred to as human leukocyte antigens or major histocompatibility complexes The peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecule.

See Male et al., Advanced Immunology Lipincott Company, 1987), especially chapters 6-10. The interaction ofT cells and complexes of HLA/peptide is restricted, requiring a specific T cell for a specific complex of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present Similarly, there is no response if the specific complex is absent, but the T cell is present The mechanism is involved in the immune system's response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities.

The mechanism by which T cells recognize alien materials also has been implicated in cancer. A number of cytolytic T lymphocyte (CTL) clones directed against autologous melanoma have been described.

In some instances, the antigens recognized by these clones have been characterized. In PCT application PCT/US92/04354, published on November 26, 1992, the "MAGE" family, a tumor specific family of genes, is disclosed. The expression products of these genes are processed into peptides which, in turn, are expressed on cell surfaces. This can lead to lysis of the tumor cells by specific CTLs. The genes are said to code for "tumor rejection antigen precursors" or "TRAP" molecules, and the peptides derived therefrom are referred to as "tumor rejection antigens" or "TRAs". See Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et al., Science 254:1643 (1991), for further information on this family of genes. Also, see U.S.

Patent No. 5,342,774.

WO Qi/l09QM TDrUT9Cf^C% f 2 J.I UO WU* UJ/ -2- In U.S. Patent 5,405,940, MAGE nonapeptides are taught which are presented by the HLA-A1 molecule. Given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to bind one HLA molecule, but not others. This is important, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area.

because cellular abnormalities are not restricted to one particular HLA phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.

It also was discovered that a MAGE expression product is processed to a second TRA. This second TRA is presented by HLA-C clone 10 molecules. Therefore, a given TRAP can yield a plurality of TRAs.

In PCT W094/14459, published July 7, 1994, tyrosinase is described as a tumor rejection antigen precursor. This reference discloses that a molecule which is produced by some normal cells melanocytes), is processed in tumor cells to yield a tumor rejection antigen that is presented by HLA-A2 molecules.

In PCT W094/21126, published September 29, 1994, a second TRA, not derived from tyrosinase is taught to be presented by HLA-A2 molecules. The TRA is derived from a TRAP. but is coded for by a non-MAGE gene. This disclosure shows that a particular HLA molecule may present TRAs derived from different sources.

In PCT W095/00159, published January 5. 1995, an unrelated tumor rejection antigen precursor, the so-called "BAGE" precursor, is described. TRAs are derived from the TRAP and also are described. They form complexes with MHC molecule HLA-C-Clone In PCT W095/03422, published February 2. 1995, another unrelated tumor rejection antigen precursor, the so-called "GAGE" precursor, is described. The GAGE precursor is not related to the BAGE or the MAGE family.

The work which is presented by the papers, patents and patent applications described above deal. for the most part, with the MAGE family of genes, the BAGE gene and the GAGE gene. These genes are expressed in a number of tumors but are completely silent in normal tissues except testis. None is expressed in renal carcinoma.

It now has been discovered that another gene family, the "RAGE" genes. encodes additional tumor rejection antigens and precursors thereof. The RAGE genes do not show homology to the MAGE family of genes, to the BAGE gene or the GAGE gene. The RAGE genes are expressed in renal tumor cells, but not in normal renal cells. The RAGE genes are also expressed in certain other tumor cell types.

WO 96/29409 PCT/US96/04037 The invention is elaborated upon in the disclosure which follows.

Summary of the Invention The invention provides isolated nucleic acid molecules, expression vectors containing those molecules and host cells transfected with those molecules. The invention also provides isolated proteins and peptides, and antibodies to those proteins and peptides. Kits containing the foregoing molecules additionally are provided. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of a RAGE TRA or TRAP.

According to one aspect of the invention, an isolated polypeptide is provided. It includes at least the amino acid sequence of SEQ.ID.NO.40 and is a RAGE TRA. In preferred embodiments, the isolated polypeptide includes at least the amino acid sequence of SEQ.ID.NO. 43. In other embodiments the isolated polypeptide may consist essentially of or may even be only the amino acid sequence of SEQ.ID.NO.40 or SEQ.ID.NO.43.

According to another aspect of the invention, an isolated nucleic acid molecule is provided. The molecule encodes a polypeptide selected from the group consisting of SEQ.ID.NO.40 and SEQ.ID.NO.43.

The isolated nucleic acid can include SEQ.ID.NO.44 and preferably includes SEQ.ID.NO.45. In other embodiments the isolated nucleic acid may consist essentially of or may even be only SEQ.ID.NOs.44 or According to another aspect of the invention, an isolated nucleic acid molecule is provided which hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence of SEQ.ID.NO.1, SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.10. SEQ.ID.NO.12. SEQ.ID. NO.13. SEQ.ID.NO.14, SEQ.ID.NO. 17, SEQ.ID.NO.23, and/or SEQ.ID.NO.35. and which codes for RAGE TRAs or TRAPs, with the proviso that the isolated nucleic acid molecule does not code for a MAGE. GAGE or BAGE TRA or TRAP. In preferred embodiments, the isolated nucleic acid molecule is an mRNA molecule or a cDNA molecule. In one embodiment, the isolated nucleic acid molecule is complementary to nucleotides selected from the group consisting of 204 to 326 of SEQ.ID.NO.1, 313 to 399 of SEQ.ID.NO.1.

444 to 665 of SEQ.ID.NO.1, 273 to 449 of SEQ.ID.NO.12. 217 to 276 of SEQ.ID.NO.12, 185 to 247 of SEQ.ID.NO.13 and 269 to 832 of SEQ.ID.NO.14. In another embodiment, the isolated nucleic acid consists essentially of SEQ.ID.NO.1, SEQ.ID.NO.4. SEQ.ID. NO.6. SEQ.ID.NO.10. SEQ.ID.NO.12. SEQ.ID.

NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15. SEQ.ID.NO.17. SEQ.ID.NO.23. SEQ.ID.NO.35. SEQ.ID.NO.44 and/or According to another aspect of the invention, expression vectors and host cells containing those expression vectors are provided. The expression vectors include any one or more of the isolated nucleic acid WO 96/29409 DF/T Inl Aki 4 I j molecules described above. In one embodiment, the expression vector comprises the isolated nucleic acid of SEQ.ID.NOs.44 or 45. Other expression vectors according to the invention include the isolated nucleic acids described above and a nucleic acid which codes for an HLA molecule which can present the TRAs of the invention to cytolytic T cells. One example is HLA-B7. The host cells may endogenously express the HLA molecule such as HLA-B7.

According to another aspect of the invention, isolated nucleic acid molecules that are unique fragments of SEQ.ID.NO.1, SEQ.ID.NO.12, SEQ.ID.NO.13 or SEQ.ID.NO. 14 or their complements are provided. Such unique fragments are used to identify or to selectively amplify the nucleic acids described above. When the unique fragments are used for identifying expression of the above nucleic acids, the unique fragments preferably are between 200 and 1310 nucleotides in length, 200 and 1234 nucleotides in length.

200 and 2050 nucleotides in length or 200 and 1167 nucleotides in length. When the unique fragments are used to amplify the above-described nucleic acid molecules, the unique fragments are between 12 and 32 nucleotides in length. It will be recognized that amplification procedures are not exclusive of procedures that might be used to identify a nucleic acid molecule.

According to another aspect of the invention, kits for detecting the presence of expression of a TRA or TRAP are provided. Such kits employ two or more of the above-described molecules isolated in separate containers and packaged in a single package. In one such kit a pair of amplification primers are provided.

each of the pair consisting essentially of a 12-32 in length nucleotide contiguous segment of SEQ.ID.NO. or the complement thereof, SEQ.ID.NO. 12 or the complement thereof, SEQ.ID.NO. 13 or the complement thereof, or SEQ.ID.NO.14 or the complement thereof, and wherein the contiguous segments are nonoverlapping. Preferably the amplification primers are PCR primers, wherein one of the primers is a contiguous segment of the Watson strand and another of the primers is the complement of a contiguous segment of Crick strand. In certain embodiments, primers are constructed and arranged to selectively amplify and/or identify only one of the RAGE family, such as only RAGE 1 or a portion of only RAGE 1. etc. For example, one of the pair can be contiguous in RAGE 1 genes and allelic variants thereof but not contiguous in RAGE 2, 3 or 4 genes. More specifically as an example, a first primer can be a nucleic acid consisting essentially of any one of SEQ.ID.NOs.50-57. and a second primer can consist essentially of a 12-32 in length nucleotide contiguous segment of SEQ.ID.NO. 1, or the complement thereof depending upon the choice of the first primer.

Another kit according to the invention is an expression kit comprising a separate portion of the isolated nucleic acid molecule which codes for a RAGE TRAP. or a molecule including a RAGE TRA. and an HLA presenting molecule that forms a complex with that TRA and that stimulates a cytolytic T cell WO 96/29409 DPTTeO/IA'7 response. One such kit includes a nucleic acid which codes for the peptide of SEQ.ID.NO.40 or SEQ.ID.NO.43 and a nucleic acid molecule which codes for HLA-B7. Another kit according to the invention is an expression kit comprising a separate portion of the isolated nucleic acid molecule which hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence of SEQ.ID.NO.1.

SEQ.ID.NO.4, SEQ.ID.NO.6, SEQ.ID.NO.10, SEQ.ID.12, SEQ.ID.13, SEQ.ID.14, SEQ.ID.15. SEQ.ID.17, SEQ.ID.23 and/or SEQ.ID.35 and which codes for a RAGE TRAP. and a nucleic acid molecule which codes for HLA-B7.

According to another aspect of the invention isolated TRAPs coded for by the above molecules and useful fragments thereof also are provided. Antibodies to such molecules and to complexes of HLA and RAGE TRAs also are provided.

According to another aspect of the invention, methods for diagnosing a disorder characterized by expression of a RAGE TRAP are provided. One method involves a RAGE TRAP which is processed to a RAGE derived TRA that forms a complex with HLA molecules. The method involves contacting a biological sample isolated from a subject with an agent that binds the complex and then determining binding between the complex and the agent as a determination of the disorder. In one embodiment, the method determines binding of the agent to a complex of RAGE TRA and HLA-B7. In this embodiment, the RAGE TRA can be selected from the group consisting of the peptide of SEQ.ID.NO.40 and the peptide of SEQ.ID.NO.43. Another method involves contacting a biological sample isolated from a subject with an agent that is specific for a RAGE nucleic acid or an expression product thereof Interaction between the agent and the nucleic acid or expression product thereof then is determined, interaction being indicative of the disorder. The agent may be a nucleic acid which hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence selected from the group consisting of SEQ.ID.NO. 1. SEQ.ID.NO.4.

SEQ.ID.NO.6. SEQ.ID.NO.10, SEQ.ID.NO.12. SEQ.ID. NO.13, SEQ.ID.NO.14. SEQ.ID.NO. 17, SEQ.ID.NO.23 and SEQ.ID.NO.35. and which codes for a TRAP. with the proviso that the isolated nucleic acid molecule does not code for a MAGE. GAGE. or BAGE TRAP. Another method involves contacting a biological sample isolated from a subject with an agent that is specific for a RAGE tumor rejection antigen peptide and then determining interaction between the peptide and the agent as a determination of the disorder. In one embodiment, the peptide is selected from the group consisting of and SEQ.ID.NO.43.

According to another aspect of the invention, an isolated biological preparation is provided. The preparation consists essentially ofcytolytic T cells specific for complexes of an HLA molecule and a RAGE TRA. In one embodiment, the cytolytic T cells are specific for complexes of an HLA-B7 molecule and the Wn 9 Qn'lQ prrTUSTQ6;/nAn3'7 -6- 6 TRA. In this embodiment, the antigen can be a peptide selected from the group consisting of the peptide of and the peptide of SEQ.ID.NO.43.

Another aspect of the invention thus involves a method for enriching selectively a population of T cells with cytolytic T cells specific for complexes of an HLA molecule and a RAGE TRA. The method involves contacting an isolated population ofT cells containing cytolytic T cell precursors with an agent resulting in presentation of a complex of a RAGE TRA and HLA presenting molecule, in an amount sufficient to selectively enrich the isolated population ofT cells with said cytolytic T cells. In one preferred embodiment, the HLA molecule is HLA-B7 and the RAGE TRA is selected from the group consisting of a peptide consisting of the amino acids of SEQ.ID.NO.40 and a peptide consisting of the amino acids of SEQ.ID.NO.43.

Still another aspect of the invention involves methods for treating a subject with a disorder characterized by expression of a RAGE TRA or TRAP. One such method involves administering to a subject in need of such treatment an effective amount of an agent which enriches selectively in the subject the presence of complexes of HLA and RAGE TRA. resulting in cytolytic T cell response reactive with such complexes, sufficient to ameliorate the disorder. Such agents include the RAGE TRAPs and recombinant cells expressing complexes of the HLA and RAGE TRA. In one embodiment, such agents include cells expressing a complex of HLA-B7 and a peptide consisting of the peptide of SEQ.ID.NO.40 or the peptide of SEQ.ID.NO.43. Another method involves administering to a subject in need of such treatment an amount of autologous cytolytic T cells sufficient to ameliorate the disorder, wherein the autologous cytolytic T cells are specific for complexes of an HLA molecule and a RAGE TRA.

In connection with any isolated nucleic acid encoding a TRAP or TRA as described above, the invention also embraces degenerate nucleic acids that differ from the isolated nucleic acid in codon sequence only due to the degeneracy of the genetic code or complements of any of the foregoing nucleic acids.

The invention also embraces functional variants and equivalents of all of the molecules described above.

The invention also involves the discovery and isolation of TRAPs and TRAs which are expressed in tumor cells, particularly in renal tumor cells, and not expressed in normal renal cells. Prior to the present invention. TRAPs and/or TRAs of the type described herein were not known and identified for renal carcinomas, despite the knowledge and identity of other TRAPs and TRAs for numerous other cell types.

Surprisingly, the RAGE-1 gene of the present invention was expressed in only 1 of 57 renal carcinomas, and the best antigenic peptide for a particular cytotoxic T cell clone was discovered to be a decamer, not the usual nonamer peptide. Thus, according to another aspect of the invention, the invention provides TRAPs. TRAs WO 961/9409 PCW'T/Uc/040An7 WO 96I 9Q409 ff'TQAQc UIV'.U, -7and nucleic acids coding for TRAPs and TRAs, which are not MAGE, BAGE AND GAGE TRAPs. TRAs and nucleic acids, which are expressed in tumor cells, particularly in renal tumor cells but not in normal renal cells, and are obtainable by a process comprising isolating renal tumor cells from a patient isolating lymphocytes from the patient, contacting the renal tumor cells with the lymphocytes in vitro, isolating a cytotoxic T cell clone among the lymphocytes reactive with the renal tumor cells.

preparing an expression library from mRNA of the renal tumor cells, screening the expression library with the cytotoxic T cell clone for a library member reactive with the cytotoxic T cell clone, isolating said library member reactive with the cytotoxic T cell clone, and sequencing the renal cell nucleic acid in said library member, said renal cell nucleic acid encoding the TRAP (or portion thereof) including the TRA. The invention also provides sequences having homology to such nucleic acids and coding for renal associated TRAPs and TRAs. sequences which hybridize under stringent conditions to such nucleic acids and coding for renal associated TRAPs and TRAs, complements, unique fragments and 'degenerates' of the foregoing, the TRAPs and TRAs themselves, as well as functional variants and equivalents of any of the foregoing, all of which can be considered to be RAGE nucleic acids.

TRAPs and TRAs.

The invention also provides agents that selectively enrich in a subject the presence of complexes of HLA/RAGE TRAs for use as a medicament. Such agents include, but are not limited to. RAGE TRAs and/or RAGE TRAPs; recombinant cells expressing RAGE TRAs and/or RAGE TRAPs and also expressing appropriate HLA molecules, recombinant or not; and functional variants and equivalents of the foregoing. Specific examples include the RAGE TRA of SEQ.ID.NO. 43: any fragment of the RAGE TRAP of SEQ.ID.NO. 5 including the TRA of SEQ.ID.NO. 43: the RAGE TRAP of SEQ.ID.NO. recombinant cells expressing the TRA of SEQ.ID.NO. 43 and HLA-B7: and/or any other RAGE TRA.

RAGE TRAP or functional fragment thereof and/or cells expressing such molecules.

The invention also provides agents that selectively enrich in a subject the presence of complexes of HLA/RAGE TRAs in the manufacture of a medicament for treating cancer. Such agents include, but are not limited to, RAGE TRAs and/or RAGE TRAPs; recombinant cells expressing RAGE TRAs and/or RAGE TRAPs and also expressing appropriate HLA molecules, recombinant or not; and functional variants and equivalents of the foregoing. Specific examples include the RAGE TRA of SEQ.ID.NO. 43; any fragment of the RAGE TRAP of SEQ.ID.NO. 5 including the TRA of SEQ.ID.NO. 43; the RAGE TRAP of WO 96/29409 Pr'T/TTO/nAn7 WO 96/29409 "PCf -l VtT~ IAZ7 -8- SEQ.ID.NO. 5; recombinant cells expressing the TRA of SEQ.ID.NO. 43 and HLA-B7; and/or any other RAGE TRA, RAGE TRAP or functional fragment thereof and/or cells expressing such molecules.

The invention also provides cytotoxic T cells specific for complexes of HLA and RAGE TRA for use as a medicament. One nonlimiting example is autologous cytotoxic T cells specific for tumor cells expressing complexes of HLA-B7 and RAGE TRA.

The invention also provides cytotoxic T cells specific for complexes of HLA and RAGE TRA in the manufacture of a medicament for treating cancer. One nonlimiting example is autologous cytotoxic T cells specific for tumor cells expressing complexes of HLA-B7 and RAGE TRA.

The invention also provides pharmaceutical preparations containing any one or more of the medicaments described above or throughout the specification. Such pharmaceutical preparations can include pharmaceutically acceptable diluent carriers or excipients.

These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.

Brief Description of the Drawings Figure 1 is a graph showing the levels of tumor necrosis factor produced when CTL Clone 263/17 is combined with COS cells transfected with HLA-B7 cDNA and a cDNA encoding a RAGE TRAP.

Figure 2 is a schematic representation of the RAGE-1. 2, 3 and 4 cDNAs. Closed black boxes indicate the different ORF in each of the three reading frames. Shaded areas in the RAGE-2, 3 and 4 cDNAs represent sequences that are unrelated to the RAGE-1 sequence, including two insertions. The 5' terminal sequence obtained by PCR is indicated with dashed boxes. The 3' end of this PCR sequence is identical to the overlapping 5' end sequences of the RAGE-2, 3 and 4 cDNAs. The antigenic peptide encoded by RAGE-1 is indicated.

Figure 3 is a graph showing the levels of tumor necrosis factor produced when CTL Clone 263/17 is combined with COS cells transfected with HLA-B7 cDNA and a cDNA encoding a RAGE TRAP or a minigene encoding ORF2 of a RAGE TRAP.

Figure 4 is a graph detailing the levels of tumor necrosis factor produced when CTL Clone 263/17 is combined with peptide fragments of the TRAP encoded by ORF2 of the RAGE gene and COS cells transfected with HLA-B7.

Figure 5 is a graph depicting the lytic activity of CTL clone 267/17 against HLA-B7- LB23-EBV B cells pulsed with increasing concentrations of the peptides including a RAGE TRA.

Dd'Ir rFTQ QC /A A f%'2pT WOl 96/29409D'IT~ f~l'

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Brief Description of the Sequences SEQ.ID.NO. I is the nucleotide sequence of the RAGE- I cDNA.

SEQ.JD.NO.2 is open reading fr-ame 1 (ORFi1) of the cDNA of SEQ.ID.NO. 1.

SEQ.JD.NO.3 is the translated amino acid sequence of SEQ.ID.NO.2.

SEQ.JD.NO.4 is open reading frame 2 (ORF2) of the cDNA of SEQ.TD.NO. 1.

is the translated amino acid sequence of SEQ.JDNO.4.

SEQ.LD.NO.6 is open reading frame 3 (ORF3) of the cDNA of SEQ.ID.NO. 1.

SEQ.ID.NO.7 is the translated amino acid sequence of SEQ.ID.NO.6.

SEQ.ID.NO.8 is open reading frame 4 (ORF4) of the cDNA of SEQ.ID.NO. 1.

SEQ.ID.NO.9 is the translated amino acid sequence of SEQ.TD.NO.8.

SEQ.ID.NO. 10 is open reading frame 5 (ORF5) of the cDNA of SEQ.ID.NO. 1.

SEQ.JD.NO. 11 is the translated amino acid sequence of SEQ.ID.NO. SEQ.ID.NO. 12 is the nucleotide sequence of the RAGE-2 cDNA.

SEQ.ID.NO. 13 is the nucleotide sequence of the RAGE-3 cDNA.

SEQ.ID.NO. 14 is the nucleotide sequence of the RAGE-4 eDNA.

SEQ.ID.NO. 15 is open reading f-rme 2' (ORF2') of the cDNA of SEQ.JD.NO. 12.

SEQ.JD.NO. 16 is the trnslated amino acid sequence of SEQ.ID.NO. SEQ.ID.NO. 17 is open reading fr-ame 3'(ORF3 of the cDNA of SEQ.JD.NO. 12.

SEQ.JD.NO. 18 is the translated amino acid sequence of SEQJID.NO. 17.

SEQ.ID.NO. 19 is open reading frame 4 (ORF4) of the cDNA of SEQ.JD.NO. 12.

is the translated amino acid sequence of SEQ.JD.NO. 19.

SEQ.ID.NO.21 is open reading frame 5 (ORF5 of the cDNA of SEQ.JD.NO. 12.

SEQ.ID.NO.22 is the translated amino acid sequence of SEQ.ID.NO.2 1.

SEQ.ID.NO.23 is open reading frame 6 (ORF6) of the cDNA of SEQ.ID.NO. 13.

SEQ.ID.NO.24 is the translated amino acid sequence of SEQ.ID.NO.23.

is open reading frame 2' (OR.F2') of the cDNA of SEQ.ID.NO. 13 SEQ.ID.NO.26 is the translated amino acid sequence of SEQ.ID.NO.27 is open reading fr-ame -3 '(ORF3 of the cDNA of SEQ.ID.NO. 13.

SEQ.ID.NO.28 is the translated amino acid sequence of SEQ.ID.NO.27.

SEQ.ID.NO.29 is open reading frame 4 (ORF4) of thie cDNA of SEQ.ID.NO. 13 SEQ.ID.NO.3 0 is the translated amino acid sequence of SEQ.ID.NO.29.

SEQ.ID.NO.31I is open reading fr-ame 5 (ORES) of the cDNA of SEQ.ID.NO. 13

I

W~O OL/OAn0 PFr'T Tn tfA AUl9w' SI T- 10 JrL. 11 UYVIU 10 SEQ.ID.NO.32 is the translated amino acid sequence of SEQ.ID.NO.3 1.

SEQ.ID.NO.33 is open reading fiame 2' (ORF2') of the cDNA of SEQ.ID.NO.14.

SEQ.ID.NO.34 is the translated amino acid sequence of SEQ.ID.NO.33.

is open reading fame 3"(ORF3") of the cDNA of SEQ.ID.NO.14.

SEQ.ID.NO.36 is the translated amino acid sequence of SEQ.ID.NO.37 is open reading frame 4' (ORF4') of the cDNA of SEQ.ID.NO.14.

SEQ.ID.NO.38 is the translated amino acid sequence of SEQ.ID.NO.37.

SEQ.ID.NO.39 is the dodecamer peptide containing the RAGE tumor rejection antigen mentioned in connection with Figure 4.

SEQ.ID.NO.40 is a nonamer fragment (amino acids 1-9) of the peptide described in SEQ.ID.NO.39.

SEQ.ID.NO.41 is a nonamer fragment (amino acids 2-10) of the peptide described in SEQ.ID.NO.39.

SEQ.ID.NO.42 is a nonamer fiagment (amino acids 3-11) of the peptide described in SEQ.ID.NO.39.

SEQ.ID.NO.43 is a decamer fragment (amino acids 1-10) of the peptide described in SEQ.ID.NO.39.

SEQ.ID.NO.44 is the nucleotide sequence of a DNA which encodes the peptide of is the nucleotide sequence of a DNA which encodes the peptide of SEQ.ID.NO.43.

SEQ.ID.NO.46 is a sense primer used in PCR tests for expression of the RAGE TRAP.

SEQ.ID.NO.47 is an antisense primer used in PCR tests for expression of the RAGE TRAP.

common to all RAGE genes tested.

SEQ.ID.NO.48 is an antisense primer. specific for RAGE-1, used in PCR tests for expression of the RAGE-1 TRAP gene.

SEQ.ID.NO.49 represents the region of RAGE genes flanking the insertion point of ORF2. with the insertion designated by N.

SEQ.ID.NOs.50-57 are PCR primers useful in identification of RAGE 1.

Detailed Description of the Invention An antigen recognized on a renal cell carcinoma by autologous CTL restricted by HLA-B7 is encoded by a previously unknown gene. This gene is silent in all normal tissues (including testis). except for retina, and it is expressed in several tumor samples.

WO 96/29409 PCT/US96/04037 -11- EXAMPLE 1: Description of an anti-renal cell carcinoma CTL clone of patient LE9211 Tumor line LE9211-RCC is a renal cell carcinoma line derived from a tumor sample of a female patient named LE9211. A sample thereof was irradiated, so as to render it non-proliferative. These irradiated cells were then used to isolate cytolytic T cell clones ("CTLs") specific thereto.

A sample of peripheral blood mononuclear cells ("PBMCs") was taken from patient LE 9211. and contacted to the irradiated carcinoma cells. After 1 4 days, the mixture was observed for lysis of the carcinoma cells, which indicated that CTLs specific for a complex of peptide and HLA molecule presented by the carcinoma cells were present in the sample.

The lysis assay employed was a chromium release assay following Herin et al., Int. J. Cancer 39:390- 396 (1987). The assay, however, is briefly described herein. The target carcinoma cells were grown in vitro.

and then resuspended at 10 7 cells/ml in Dulbecco's Modified Eagles Medium (DMEM). supplemented with FCS. and incubated for 45 minutes at 37 0 C with 200 itCi/ml ofNa( 51 Cr)O 4 Labeled cells were washed three times with DMEM. These were then resuspended in DMEM supplemented with 10 mM Hepes and fetal calf serum (FCS), after which 100 ul aliquots containing 103 cells were distributed into 96 well microplates. Samples oflymphocytes were added in 100 ul of the same medium, and assays were carried out in duplicate. Plates were centrifuged for 4 minutes at 100g and incubated for four hours at 37°C in a 8% CO, atmosphere.

Plates were centrifuged again, and 100 ul aliquots of supematant were collected and counted.

Percentage of "Cr release was calculated as follows: 5 "Cr release (ER-SR) x 100

(MR-SR)

where ER is observed, experimental 5 'Cr release. SR is spontaneous release measured by incubating 103 labeled cells in 200 tl of medium alone, and MR is maximum release. obtained by adding 100 pl 0.3% Triton X-100 to target cells.

Those mononuclear blood samples which showed high CTL activity were expanded and cloned via limiting dilution, and were screened again, using the same methodology. A first CTL clone was then isolated.

The clone is referred to as 263/17 hereafter. A second CTL clone. 361A/17, was obtained similarly from the same experiment and was used as described further below when CTL 263/17 failed to grow indefinitley.

CTL clones 263/17 and 361A/17 were capable oflysing specifically the autologous tumor cells and not NK-target K562 cells. NK target K562 cells are available from the ATCC, Rockville. Maryland.

Wr 091f4oAnO PrTTSO nAn4n7 -12- CTL clone 263/17 produced TNF when stimulated with the autologous tumor cells. To identify the HLA molecule that presented the antigen to CTL clone 263/17, inhibition experiments were carried out where the production of TNF was tested in the presence of monoclonal antibodies directed against HLA molecules or against CD4/CD8 accessory molecules. Four monoclonal antibodies were found to inhibit the production of TNF by CTL 263/17: monoclonal antibody W6/32, which is directed against all HLA class I molecules (Parham et al., 1979, J. Immunol., 123:342); antibody B1.23.2 which recognizes HLA-B and C molecules (Rebai et al., 1983, Tissue Antigens, 22:107); antibody ME-1 which specifically recognizes HLA-B7 (Ellis et al., 1982.

Hum. Immunol., 5:49); and antibody B9.4.1 against CD8. No inhibition was found with antibodies directed against HLA Class II DR molecules (L243: Lampson et al.. 1980. J. Immunol.. 125:293), against HLA-A3 (GAPA 3: Berger et al.. 1982 Hybridoma, 1:87) or against CD4 (13B.8.82). The conclusion was that CTL 263/17 was of the CD8 type, and recognized an antigen presented by HLA-B7.

To define the tumor specificity of this CTL clone, normal kidney cells derived from another patient which are also HLA-B7 (PTEC-HLA-B7 cells) were tested. These cells derive from the proximal tubular epithelium which is the site of origin of renal cell carcinoma. PTEC-HLA-B7 cells were not lysed by the CTL, suggesting that the antigen is specifically expressed on tumors.

Renal cell carcinoma line MZ-1851, which is derived from another HLA-B7 patient, was also lysed by the CTL, showing that the antigen is shared by independent tumors.

EXAMPLE 2: Isolation of a cDNA clone that directs the expression of the antigen recognized by CTL 263/17 A. cDNA library RNA was isolated from LE-9211-RCC, and poly-A RNA was purified by oligo-dT binding. cDNA was prepared by reverse transcription with an oligo-dT primer containing a Not I site. followed by second strand synthesis (Superscript Choice System. BRL. Life Technologies). The cDNA was then ligated to a BstXI adaptor, digested with Not I, size-fractionated (Sephacryl S-500 HR columns. BRL, Life Technologies) and cloned unidirectionally into the BstXI and Not I sites ofpcDNA-I-Amp (Invitrogen). The recombinant plasmid was then electroporated into DH5a E coli bacteria. 1500 pools of 100 recombinant bacteria were amplified and plasmid DNA of each pool was extracted by alkaline lysis, potassium acetate precipitation and phenol extraction.

w o/114A0n PTr/oCo04AiAN" 13 B. Transfection of COS cells Plasmid DNA from the different pools was co-transfected into COS cells with 60 ng of the HLA-B7 cDNA (cloned by PCR from the cDNA of another HLA-B7 patient and inserted into plasmid vector pcDSRalpha). The transfection was made in duplicate wells. Briefly, samples of COS-7 cells were seeded, at 15,000 cells/well into tissue culture flat bottom microwells, in DMEM supplemented with 10% fetal calf serum. The cells were incubated overnight at 37 0 C. medium was removed and then replaced by 50 pl/well of DMEM medium containing 10% Nu-Serum (Collaborative Research, Bedford, MA). 400pg/ml DEAEdextran, and 100 pM chloroquine, plus 100 ng of the plasmids. Following four hours of incubation at 37 0

C,

the medium was removed, and replaced by 50 pl of PBS containing 10% dimethyl sulfoxide (DMSO). This medium was removed after two minutes and replaced by 200 pl of DMEM supplemented with 10% FCS.

Following this change in medium, COS cells were incubated for 24-48 hours at 37 0 C. The transfectants then were screened with CTL 263/17. After first removing the medium. 3000 CTL 263/17 cells were added to each well in 100pl of medium containing 25 U/ml IL-2. The amount of TNF present in the supematant was then measured by testing its cytotoxicity for WEHI 164.13 cells. Most pools gave a TNF concentration below 10 pg/ml. Two pools (1157 and 1319) gave higher concentrations in both of the duplicate wells (24 to 37 pg/ml). The bacteria of pool 1319 were cloned and 1200 clones were obtained.

Their plasmid DNA was extracted and transfected into COS cells with HLA-B7. The transfectants were screened with CTL 263/17. One cDNA clone (9H3) gave a high TNF production by CTL 263/17. Figure 1 shows the result obtained when this cDNA (60 ng) was transfected into COS cells with the HLA-B7 cDNA (60 ng) and screened with CTL 263/17.

This cDNA also was stably transfected into LB23-SAR cells, an HLA-B7 sarcoma line. The lysis test then was performed with CTL clone 361A/17. which recognizes the same antigen as CTL clone 263/17.

These stably transfected cells were recognized in the same manner as the COS-HLA-B7-cDNA 9H3 cells.

EXAMPLE 3: Sequence ofcDNA 9H3 cDNA clone 9H3 is 1130 bp long. This cDNA was not complete because its size was smaller than that of an mRNA observed on a Northern blot (1.6kb). The 5' end of the cDNA was cloned by RACE-PCR and the entire sequence was confirmed. This entire sequence is shown as SEQ.ID.NO.1. A comparison with the sequences reported in databanks showed at the 3' end a high homology with a short sequence of 235 bp called "expressed sequence tag". whose function is unknown and at the 5' end a limited homology in a stretch of 95 bases) with the antisense strand of two human endogenous retroviruses called RTVL-H2 and RGH2 3).

WO 96/29409 PCTIS96/04037 -14- The gene was called RAGE, for Renal tumor AntiGEn.

The sequence contains five open reading frames. ORF1: 99 base pairs encoding a protein of 32 residues; ORF2: 123 base pairs encoding a protein of 40 residues; ORF3: 87 base pairs encoding a protein of 28 residues; ORF4: 288 base pairs encoding a protein of 95 residues; and ORF5: 222 base pairs encoding a protein of 73 residues. (SEQ.ID.NOs.2, 4, 6, 8 and 10. respectively). SEQ.ID.NO.4 codes for the TRAP from which the antigenic peptide reactive with CTL 263/17 (as an HLA-B7/peptide complex) is derived.

EXAMPLE 4: Identification of Additional RAGE Genes This example describes the identification of three additional RAGE genes and the determination that only the RAGE gene identified in the above examples, now designated RAGE-1. encodes a RAGE TRA reactive with CTL 263/17.

A probe was prepared from RAGE-1 cDNA and used to screen a LE9211-RCC cDNA library for additional RAGE genes. Three cDNAs homologous to RAGE. labeled RAGE-2 (SEQ.ID.NO.12). RAGE-3 (SEQ.ID.NO.13) and RAGE-4 (SEQ.ID.NO.14), were isolated. The RAGE-2, 3 and 4 genes were sequenced by standard methods. Comparison of the nucleotide sequences of these RAGE cDNAs with the RAGE-1 cDNA showed that truncated and novel open reading frames (ORFs) were present in the newly identified RAGE cDNAs. RAGE-2, RAGE-3 and RAGE-4 contained an insert of 37 bp at position 249 ofRAGE1 (within the sequence corresponding to ORF2 (SEQ.ID.NO.4) of RAGE-1). For the RAGE-2 cDNA. comparison with the cosmid sequence indicated that this insertion corresponds to the beginning of an exon. Its absence from the RAGE-1 cDNA might result from the use of an alternative downstream acceptor site. In addition. RAGE-2. 3 and 4 differ from RAGE-1 in lacking a nucleotide at position 192 of RAGE-1. These changes significantly modify the ORFs of RAGE-2, 3 and 4 that are homologous to ORF2 and 3 of RAGE-1. In addition. RAGE-3 has another insertion of 47 bp at the 5' end. Except for these differences, the RAGE-1. 2 and 3 sequences are identical.

RAGE-4 is about 800 bp longer than the other RAGE cDNAs. Its 5' sequence is identical to that of RAGE-2. but from position 434 to the poly-A tail. the RAGE-4 sequence differs totally from the other RAGE cDNAs. The RAGE-4 cDNA was shown not to be chimeric. The starting position of the 3' unrelated sequence corresponds to an exon-intron boundry in the RAGE genomic sequence, and the 3' unrelated sequence was present in the 3' end of the RAGE gene. Therefore. the RAGE-4 cDNA appears to result from differential splicing of the RAGE-2 gene. The schematic alignment of the four cDNAs is shown in Fig. 2. There are 17 ORFs in the four RAGE cDNAs. Of these 17, 10 are different. The ORFs are as follows: WO 96/29409 PCTrITC Q Af/7 Gene ORF Nucleotide No. SEQ.ID.NO.

RAGE-1 ORF1 173-271 2 ORF2 204-326 4 ORF3 313-399 6 ORF4 323-610 8 444-665 RAGE-2 ORF2' 217-276 ORF3' 273449 17 ORF4 373-660 19 494-715 21 RAGE-3 ORF6 185-247 23 ORF2' 274-333 ORF3' 330-506 27 ORF4 430-717 29 551-772 31 RAGE-4 ORF2' 213-272 33 ORF3" 269-832 ORF4' 369-557 37 The RAGE-2, RAGE-3 and RAGE-4 cDNAs were cloned into expression plasmids by art-standard procedures and transfected as described with HLA-B7 into COS-7 cells to determine if these cDNAs also encoded the antigen recognized by CTL 263/17. Parallel control experiments with the RAGE cDNA (now referred to as RAGE-1) and with LE9211-RCC cells were also performed.

Incubation of LE9211-RCC cells or COS-7 cells cotransfected with RAGE-1 and HLA-B7 with CTL 263/17 strongly induced release of TNF by CTL 263/17. Cotransfection of RAGE-2. RAGE-3 or RAGE4 and HLA-B7 did not elicit TNF release. Therefore, only RAGE-1 was able to transfer expression of the antigen recognized by CTL 263/17.

EXAMPLE 5: Identification of ORF containing RAGE tumor rejection antigen The 37 bp insertion in RAGE-2. 3 and 4 caused premature temaination of ORF2 in these three genes.

It was reasoned, therefore, that the antigenic peptide recognized by CTL 263/17 was encoded by the 3' end of ORF2. To test this hypothesis, the DNA sequences corresponding to ORF2 of RAGE 1 and ORF2' of RAGE-2 and RAGE-3 were cloned into an expression vector and transfected into COS-7 cells with HLA-B7 as described above. As positive controls, the RAGE-1 cDNA was cotransfected with HLA-B7 into COS-7 cells or LE2911-RCC cells were used. These transfectants or LE2911-RCC cells were used to provoke release of TNF from CTL 263/17 cells. Among the ORF transfectants, only the ORF2 from WO Q96/od4f9 PCOIT/ST9Q IT -16- RAGE-1 successfully stimulated TNF release from CTL 263/17 cells (Fig. This experiment confirmed that the RAGE antigenic peptide recognized by CTL 263/17 cells was encoded by the 3' end of ORF2 of RAGE-1.

EXAMPLE 6: Identification of RAGE tumor rejection antigen peptide Synthetic peptides corresponding to the 3' end of RAGE-1 ORF2 were synthesized and tested for stimulation of TNF release from CTL 263/17 cells. COS-7 cells were transfected with HLA-B7 as described above and a synthetic peptide corresponding to a 3' portion of ORF2 was added to the culture. CTL 263/17 cells were added and the production of TNF was measured after 18 hours (Fig. Peptide SPSSNRIRNTST (SEQ.ID.NO.39) efficiently stimulated the release of TNF from CTL 263/17. Since peptides presented by HLA class I molecules are usually 9 amino acids in length, we tested nonameric peptides (SEQ.ID.NOs. 40,41 and 42) derived from the dodecameric peptide (SEQ.ID.NO.39) previously used to stimulate TNF release from CTL 263/17 cells. The results of these experiments are shown in Fig. 4.

One of these peptides (SPSSNRIRN, SEQ.ID.NO.40) was recognized by CTL 263/17, but to a far lesser extent than the dodecameric peptide, which suggested that the nonamer (SEQ.ID.NO.40) was not the optimal peptide. The decameric peptide (SPSSNRIRNT, SEQ.ID.NO.43) was very efficiently recognized by CTL 263/17.

EXAMPLE 7: Activity of RAGE tumor rejection antigen nonamer and decamer peptides This example shows the ability of the RAGE TRA peptide to induce lysis of HLA-B7-expressing cells and the relative efficiencies of the nonamer and decamer peptides.

Nonameric and decameric RAGE peptides (SEQ.ID.NOs. 40 and 43. respectively) were tested for the ability to induce cell lysis of HLA-B7- LB23-EBV B cells by CTL 263/17 cells in a dose response assay.

Lyophilized peptides were dissolved at 20 mg/ml in DMSO, then diluted to 2 mg/ml in 10mM acetic acid and stored at -80 0 C. Target cells, HLA-B7 EBV-transformed lymphoblastoid cells (LB23-EBV cells), were labeled with "Cr. as described above, for 1 hour at 37 0 C followed by extensive washing to remove unincorporated label. LB23-EBV cells were then incubated in 96-well microplates in the presence of various concentrations of peptides for 30 minutes at 37°C. CTL263/17 were then added in an equal volume of medium at an effector:target ratio of 10:1. Chromium-51 release was measured after 4 hours. Fig. 5 shows the results of the dose response assay. Half maximal lysis of LB23-EBV cells was induced at a concentration of 30 ng/ml SPSSNRIRNT peptide (SEQ.ID.NO.43).

WO o/240dnQ 'nT CnLj IAS9T -17- r EXAMPLE 8: Expression of RAGE-1 gene The expression of RAGE was tested by PCR using the following primers: SEQ.ID.NO.46 GTG TCT CCT TCG TCT CTA CTA (sense primer) SEQ.ID.NO.47 GGT GTG CCG ATG ACA TCG (antisense primer common to all RAGE genes) SEQ.ID.NO.48 GAG GTA TTC CTG ATC CTG (antisense primer specific for RAGE-1) First, total RNA was taken from the particular sample, using art recognized techniques. This RNA was used to prepare cDNA. The protocol used to make the cDNA involved combining 4 ul of 5x reverse transcriptase buffer, 1 ul of each dNTP (10mM), 2 ul of dithiothreitol (100mM), 2 pl of dT-15 primer pM), 0.5 ul of RNasin (40 units/pl), and 1 il of M-MLV reverse transcriptase (200 units/ Next. 6.5 ul of template RNA (1 ig/3.25 il water, or 2 pg total template RNA) was added. The total volume of the mixture was 20 il. This was mixed and incubated at 42 0 C for 60 minutes, after which it was chilled on ice. A total of 80 pl of water was then added, to 100 pl total. This mixture was stored at -20 0 C until used in PCR.

The reagents for PCR included: 5 microliters of 1 Ox DynaZyme buffer 20 pmoles of each primer 5 nanomoles of each dNTP 1 unit of polymerizing enzyme "Dynazyme" (2 units/pl) 5 pl of cDNA (corresponding to 100 ng total RNA) water to a final volume of 50 il The mixture was combined, and layered with one drop of mineral oil. The mixture was transferred to a thermocycler block, preheated to 94°C, and amplification was carried out for one cycle of 15 min at 94 0

C.

followed by 33 cycles of: 1 min. at 94 0

C

2 min. at 56°C or 60 0 C (see below) 3 min. at 72 0

C

A final extension step of 15 min. was then performed at 72°C. Expression of all RAGE genes was tested by PCR amplification with pan-RAGE sense (SEQ.ID.NO.46) and antisense (SEQ.ID.NO.47) primers using an annealing step of 60 0 C for 2 minutes. Expression of only RAGE-1 gene was tested by PCR amplification with pan-RAGE sense (SEQ.ID.NO.46) and RAGE-l-specific antisense (SEQ.ID.NO.48) primers using an WO ofi/0no Tr"r/r o i A rn WO 96I9409Q pf- eiT~ irk0407 -18-

IWO

annealing step of 56'C for 2 minutes. The PCR product of 194 base pairs (general to all RAGE genes tested) and 239 base pairs (specific for RAGE-1 genes) were visualized on an agarose gel containing ethidium bromide.

The gene was found to be tumor-specific. The gene was silent in all normal tissues tested, except for retina. In particular, the gene was silent in adrenals, bladder, bone marrow, brain, breast, cerebellum, colon, heart, kidney, liver, lung, melanocytes, muscle, nevus, ovary, placenta, prostate, skin, splenocytes, stomach.

testis, thymocytes, uterus and healing wounds. The gene, however, was found to be expressed in a variety of tumor cell lines and tumor tissue samples (Table It is also expressed in some other tumors which are not listed here, although not frequently.

Table 1. Expression of RAGE-1 Gene in Tumor Samples Histological Type Number of Tumors Expressing ALL RAGE RAGE-1 Tumor Samples Renal carcinoma 2/57 1/57 Sarcomas 5/25 3/25 Bladder tumors superficial -0/29 0/29 infiltrating -3/37 3/37 Melanomas primary lesions -2/60 2/60 metastases -8/177 6/177 Head and neck tumors 2/50 1/50 Mammary carcinomas 3/128 1/128 Prostatic carcinomas 0/22 0/22 Colorectal carcinomas 0/48 0/48 Leukemias 0/19 0/19 Lung carcinomas (NSCLC') 0/59 0/59

(SCLC)

Mesotheliomas 1/3 0/3 Brain tumors 0/11 0/11 Oesophage tumors 0/7 0/7 Ovarian tumors 0/3 0/3 Tumor Cell Lines Renal carcinoma 8/19 7/19 Bladder tumors 3/3 3/3 Mesotheliomas 11/19 8/19 WO 96/29409 -19- PCTfUS96/04037 Head and neck tumors 3/7 1/7 Sarcomas 2/6 1/6 Melanomas 11/78 7/78 Colorectal carcinomas 1/17 1/17 Lung carcinomas (NSCLC') 0/2 0/2

(SCLC)

0/26 0/26 Leukemias/Lymphomas 0/11 0/11 Brain tumors 0/1 0/1 Gastric tumors 0/2 0/2 SNSCLC: non-small cell lung carcinoma.

The foregoing examples show the isolation of a nucleic acid molecule which codes for a TRAP. This TRAP coding molecule, however, is not homologous with any of the previously disclosed coding sequences described in the references set forth supra. Hence, one aspect of the invention is an isolated nucleic acid molecule which includes all or a unique portion of the nucleotide sequence set forth in SEQ.ID.NO.1, SEQ.ID.NO.4, SEQ.ID.NO.6 or SEQ.ID.NO.10. It is also expected that antigens derived from other RAGE ORFs encoded by SEQ.ID.NOs. 12, 13 and 14 may be recognized cytolytic T lymphocyte clones other than CTL263/17. Thus, the invention in another aspect involves any one or more of the RAGE family of genes, including isolated unique portions thereof such as portions encoding TRAPs and TRAs, RAGE TRAPs and TRAs derived therefrom and all of the diagnostic and therapeutic modalities relating thereto. The foregoing sequences are not MAGE, BAGE or GAGE sequences, as will be seen by comparing them to the MAGE.

BAGE or GAGE sequences described in the references.

Also a part of the invention are those nucleic acid sequences which also code for a non-MAGE. non- BAGE and non-GAGE tumor rejection antigen precursor but which hybridize to a nucleic acid molecule consisting of the above described nucleotide sequences, under stringent conditions. The term "stringent conditions" as used herein refers to parameters with which the art is familiar. More specifically, stringent conditions, as used herein. refers to hybridization at 65 0 C in hybridization buffer (3.5 x SSC. 0.02% Ficoll.

0.02% Polyvinyl pyrolidone, 0.02% Bovine Serum Albumin. 25mM NaHPO, (pH7). 0.5% SDS. 2mM EDTA). SSC is 0.15M Sodium Chloride/0.15M Sodium Citrate. pH 7: SDS is Sodium dodecyl Sulphate: and EDTA is Ethylene diamine tetra acetic acid. After hybridization, the membrane upon which the DNA is transferred is washed at 2xSSC at room temperature and then at 0.1 xSSC/0.1 xSDS at 650C.

There are other conditions, reagents. and so forth which can be used, which result in the same degree of stringency. The skilled artisan will be familiar with such conditions. and thus they are not given here. The WO 96/29409 IIl/'^nn/llTlln' f^ L 20- r1/uayo/Ut43/ skilled artisan also is familiar with the methodology for screening cells, preferably cancer cells, for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid.

In screening for RAGE family members, a Southern blot may be performed using the foregoing conditions, together with a 3 2 P probe. After washing the membrane to which the DNA was finally transferred, the membrane can be placed against X-ray film to detect the radioactive signal.

The invention thus provides isolated unique fragments of SEQ.ID.NO. 1 or its complement. A unique fragment is one that is a 'signature' for RAGE genes. It for example, is long enough to assure that its precise sequence is not found in molecules outside of the RAGE family as defined by claim 23. Preferred unique fragments are those found only in ORF2 or its complement. Unique fragments can be used as probes in Southern blot assays to identify RAGE family members including those expressing ORF2 or can be used in amplification assays such as those employing PCR. As known to those skilled in the art. large probes such as 200 bp or more are preferred for certain uses such as Southern blots, while smaller fragments will be preferred for uses such as PCR. As will be recognized by those skilled in the art, the size of a unique fragent will depend upon its conservency in the genetic code. Thus, some regions of SEQ.ID.NO.1. SEQ.ID.NO.12, SEQ.ID.NO.13 and SEQ.ID.NO.14 will require longer segments to be unique while others will require only short segments, typically between 12 and 32 bp 12, 13, 14, 15, 16, 17. 18, 19, 20, 21, 22, 23,24,25,26, 27, 28, 29, 30, 31 and/or 32 bases long). Virtually any segment of SEQ.ID.NO. 1 that is 18 or more nucleotides in length will be unique. Those skilled in the art are well versed in methods for selecting such sequences, typically on the basis of the ability of the unique fragment to selectively distinguish the sequence of interest from nonfamily members. A comparison of the sequence of the fragment to those on known data bases typically is all that is necessary, although in vitro confirmatory hybridization and sequencing analysis may be performed.

For any pair of PCR primers constructed and arranged to selectively amplify RAGE-1, a RAGE-1 specific primer may be used. Such a primer is a contiguous stretch of RAGE-1 which hybridizes to both sides of the insertion point in ORF2 which is altered by the insertion of additional nucleotides in other RAGE genes. Such a specific primer would fully hybridize to a contiguous stretch of nucleotides only in RAGE-1.

but would hybridize only in part to RAGE genes that do not share ORF 2. For efficient PCR priming and RAGE 1 identification, the RAGE 1 specific primer should be constructed and arranged so it does not hybridize efficiently at its 3' end to RAGE genes other than RAGE 1. To accomplish this, the primer can be described as having two ends: a 5' end that is contiguous with and complementary to one side of the insertion point joined directly to a 3' end that is contiguous with and complementary to the opposite side of the insertion point. By making the 5' end of the primer substantially longer than the 3' end. and by making the 3' end short WO 96/29409 IDPT TCo/Lt io3'7 21 1-4 nucleotides), then the kinetics of hybridization will strongly favor hybridization at the 5' end. In this instance, 3' initiated PCR extension will occur only when both the 5' and 3' ends hybridize to the nucleic acid.

i.e. only when ORF 2 is present without an insert.

RAGE-1 specific primers. as described above, may be designed to prime DNA synthesis on either strand of the DNA helix, described herein as the Watson or the Crick strands. The sequence in RAGE 1 which flanks the insertion point, is 5'-CAAACANGGATCA-3' (SEQ.ID.NO.49; Watson strand. N is a nucleotide insert). A RAGE-1 specific primer designed to preferentially amplify the Watson strand of RAGE-1 typically would comprise 12 and preferably 15 or more nucleotides ary to the nucleotides of the Watson strand 3' to the insertion point. The remaining portion of the primer would be one to four nucleotides long and would be complementary to the sequence 5' to the insertion point. Such a primer would be perfectly complementary and contiguous with its complement in RAGE-1. The 3' end of the primer would hybridize to its complement in the Watson strand and initiate extension. In RAGE genes other than RAGE-1, the insertion ofnoncomplementary nucleotides at the insertion point of ORF2 would substantially eliminate hybridization of the 3' end of the RAGE-1 specific primer to the Watson strand 5' of the insert. The mismatch generated at the 3' end of the primer when hybridized to RAGE genes, other than RAGE-1. would preclude efficient amplification of those genes. Exemplary primers consist essentially of the following sequences.

wherein N is zero, one or more contiguous nucleotides on the appropriate Watson or Crick strands: 5'-NTATTCCTGATCCT-3'(SEQ.ID.NO. 5'-NTATTCCTGATCCTG-3'(SEQ.ID.NO. 51); 5'-NTATTCCTGATCCTGT-3'(SEQ.ID.NO. 52); 5'-NTATTCCTGATCCTGTT-3'(SEQ.ID.NO. 53); 5'-NCAAGTTCAAACAG-3'(SEQ.ID.NO. 54); 5'-NCAAGTTCAAACAGG-3'(SEQ.ID.NO. 5'-NCAAGTTCAAACAGGA-3'(SEQ.ID.NO. 56); and 5'-NCAAGTTCAAACAGGAT-3'(SEQ.ID.NO. 57).

The expression of RAGE-1 may also be detected by PCR using primers which initiate extension on opposite sides of the insertion point. Analysis of amplification products can distinguish RAGE-1 amplification products from non-RAGE-1 amplification products by the length of the amplification products.

Because the RAGE-1 gene does not contain the insert present in other RAGE genes, amplification products derived from RAGE-1 will be shorter than amplification products derived from other RAGE genes (by about wn O/4ono lT/US T Sv- 22 rK IUU3JI 37 base pairs). This difference may be distinguished readily using standard methods in the art. Additional methods which can distinguish nucleotide sequences of substantial homology, such as ligase chain rection and other methods, will be apparent to skilled artisans. RAGE 2. 3 and 4 specific primers may be prepared in a like manner.

The invention also includes the use of nucleic acid sequences which include alternative codons that encode the same amino acid residues as encoded by the RAGE genes. For example, as disclosed above in Example 7. a decameric peptide SPSSNRIRNT (SEQ.ID.NO.43) is a RAGE tumor rejection antigen. The serine residues (amino acids No. 1, 3 and 4 of SEQ.ID.NO.40) for example, are encoded by the codons TCA.

AGT and TCA, respectively. In addition to TCA and AGT, serine amino acid residues may also be encoded by the codons TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo. to incorporate a serine residue. Similarly, nucleotide sequence triplets which encode other amino acid residues comprising a RAGE tumor rejection antigen include: CCA. CCC. CCG and CCT (proline codons); CGA. CGC. CGG.

CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologcally isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.

The examples above also show the isolation ofpeptides which are RAGE TRAs. These exemplary peptides are processed translation products of the nucleic acids of SEQ.ID.NO.1. As such. it will be appreciated by one of ordinary skill in the art that the translation products from which a RAGE TRA is processed to a final form for presentation may be of any length or sequence so long as they encompass the RAGE TRA. As demonstrated in the examples above. peptides or proteins as small as 9, 10. or 12 amino acids and as large as the amino acid sequence encoded by ORFI are appropriately processed. presented by HLA-B7 and recognized by CTL263/17. The peptide of SEQ.ID.NO.23 may have one, two. three. four. five.

six. seven, eight nine. ten. or more amino acids added to either or both ends. The antigenic portion of such a peptide is cleaved out under physiological conditions for presentation by HLA class I molecules.

The amino acid sequence of proteins and peptides from which RAGE TRAs are derived may be of natural or non-natural origin, that is. they may comprise a natural RAGE TRAP molecule or may comprise a modified sequence as long as the amino acid sequence retains the tumor rejection antigen sequence recognized by the CTL when presented on the surface of a cell. For example, RAGE tumor rejection WO Q/?Q4fQ DPTIfTr I A ni4T7 23 antigens in this context may be fusion proteins of a RAGE tumor rejection antigen and unrelated amino acid sequences, the translated polypeptide of ORF2 of the RAGE-1 gene, synthetic peptides of amino acid sequences shown in SEQ.ID.NOs.39, 40 and 43, labeled peptides, peptides isolated from patients with renal cell carcinoma, peptides isolated from cultured cells which express RAGE-1, peptides coupled to nonpeptide molecules for example in certain drug delivery systems and other molecules which include the amino acid sequence of It will also be seen from the examples that the invention embraces the use of the sequences in expression vectors, as well as to transfect host cells and cell lines, be these prokaryotic E coli), or eukaryotic CHO cells. COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). The expression vectors require that the pertinent sequence, those described supra, be operably linked to a promoter. As it has been found that human HLA-B7 presents a TRA derived from these genes, the expression vector may also include a nucleic acid sequence coding for HLA-B7. In a situation where the vector contains both coding sequences, it can be used to transfect a cell which does not normally express either one. The TRAP or TRA coding sequence may be used alone, when. e.g. the host cell already expresses HLA-B7. Of course, there is no limit on the particular host cell which can be used. As the vectors which contain the two coding sequences may be used in HLA-B7 presenting cells if desired, and the nucleic acid coding for the TRAP or TRA can be used in host cells which do not express HLA-B7.

The invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors. Such expression kits include at least separate portions of at least two of the previously discussed materials. Other components may be added, as desired.

To distinguish the nucleic acid molecules and the TRAPs of the invention from the previously described MAGE family, BAGE gene and GAGE gene, the invention shall be referred to as the RAGE family of genes and TRAPs. Hence, whenever "RAGE" is used herein, specifically excluded are MAGE.

BAGE and GAGE genes, gene products. TRAPs and TRAs.

The invention as described herein has a number of uses. some of which are described herein. First. the invention permits the artisan to diagnose a disorder characterized by expression of the TRAP. These methods involve determining expression of the TRAP gene. and/or TRAs derived therefrom. such as a TRA presented by HLA-B7. ha the former situation. such determinations can be carried out via any standard nucleic acid determination assay, including the polymerase chain reaction. or assaying with labeled hybridization probes.

In the latter situation. assaying with binding partners for complexes of TRA and HLA. such as antibodies. is especially preferred. An alternate method for determination is a TNF release assay, of the type described supra.

WO 96/29409 P~rTS/04TToi;A7/- 24 The isolation of the TRAP gene also makes it possible to isolate the TRAP molecule itself, and/or TRAs derived therefrom, especially TRAP and/or TRA molecules containing the amino acid sequences coded for by SEQ.ID.NO. 1 or 4. Other TRAPs or TRAs encoded by SEQ.ID.NOs. 1, 12, 13 and 14 and recognized by other CTL clones and/or presented by other HLA molecules may be isolated by the procedures detailed herein. (There are numerous HLA molecules known to those skilled in the art, including but not limited to. those encoded by HLA-A, HLA-B, HLA-C, HLA-E. HLA-F and HLA-G genes.) A variety of methodologies well-known to the skilled practitioner can be utilized to obtain isolated TRAP molecules.

and/or TRAs derived therefrom. The protein may be purified from cells which naturally produce the protein.

Alternatively, an expression vector may be introduced into cells to cause production of the protein. In another method, mRNA transcripts may be microinjected or otherwise introduced into cells to cause production of the encoded protein. Translation ofmRNA in cell-free extracts such as the reticulocyte lysate system also may be used to produce protein. Peptides comprising TRAs of the invention may also be synthesized in vitro. Those skilled in the art also can readily follow known methods for isolating proteins in order to obtain isolated TRAP and/or TRAs derived therefrom. These include, but are not limited to. immunochromotography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.

These isolated molecules when processed and presented as the TRA. or as complexes of TRA and HLA, such as HLA-B7, may be combined with materials such as adjuvants to produce vaccines useful in treating disorders characterized by expression of the TRAP molecule. In addition. vaccines can be prepared from cells which present the TRA/HLA complexes on their surface, such as non-proliferative cancer cells, nonproliferative transfectants, etcetera. In all cases where cells are used as a vaccine, these can be cells transfected with coding sequences for one or both of the components necessary to provoke a CTL response. or be cells which already express both molecules without the need for transfection. Vaccines also encompass naked DNA or RNA, encoding a RAGE TRA or precursor thereof, which may be produced in vitro and administered via injection, particle bombardment nasal aspiration and other methods. Vaccines of the "naked nucleic acid" type have been demonstrated to provoke an immunological response including generation of CTLs specific for the peptide encoded by the naked nucleic acid (Science 259:1745-1748. 1993).

The TRAP molecule, its associated TRAs. as well as complexes of TRA and HLA. may be used to produce antibodies. using standard techniques well known to the art. Standard reference works setting forth the general principles of antibody production include Catty. Antibodies. A Practical Approach. Vol. 1.

IRL Press, Washington DC (1988); Klein. Immunolovg: The Science of Cell-Non-Cell Discrimination.

John Wiley and Sons, New York (1982); Kennett, et al., Monoclonal Antibodies. Hvbridoma. A New Dimension In Biological Analyses, Plenum Press. New York (1980); Campbell, Monoclonal Antibody WO 96/294Qdf UT/TCnjr n 25 U .7,UI UJi Technology, in Laboratory Techniques and Biochemistry and Molecular Biology, Vol. 13 (Burdon, R. et al.

EDS.), Elsevier Amsterdam (1984); and Eisen. Microbiology, third edition, Davis, B.D. et al. EDS.

(Harper Rowe, Philadelphia (1980).

The antibodies of the present invention thus are prepared by any of a variety of methods, including administering protein, fragments of protein, cells expressing the protein or fragments thereof and the like to an animal to induce polyclonal antibodies. The production of monoclonal antibodies is according to techniques well known in the art. As detailed herein, such antibodies may be used for example to identify tissues expressing protein or to purify protein. Antibodies also may be coupled to specific labeling agents for imaging or to antitumor agents, including, but not limited to, methotrexate, radioiodinated compounds, toxins such as ricin, other cytostatic or cytolytic drugs, and so forth. Antibodies prepared according to the invention also preferably are specific for the TRA/HLA complexes described herein.

When "disorder" is used herein, it refers to any pathological condition where the tumor rejection antigen precursor is expressed. An example of such a disorder is cancer, renal cell carcinoma in particular.

Some therapeutic approaches based upon the disclosure are premised on a response by a subject's immune system, leading to lysis of TRA presenting cells, such as HLA-B7 cells. One such approach is the administration of autologous CTLs specific to the complex to a subject with abnormal cells of the phenotype at issue. It is within the skill of the artisan to develop such CTLs in vitro. 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. The target cell can be a transfectant, such as a COS cell of the type described supra.

These transfectants present the desired complex of their surface and, when combined with a CTL of interest.

stimulate its proliferation. COS cells, such as those used herein are widely available, as are other suitable host cells. Specific production ofa CTL clone has been described above. The clonally expanded autologous CTLs then are administered to the subject. Other CTLs specific to RAGE-1 and CTLs specific to RAGE TRAs encoded by RAGE-2, 3, or 4 may be isolated and administered by similar methods.

To detail a therapeutic methodology, referred to as adoptive transfer (Greenberg. J. Immunol. 136(5): 1917 (1986); Riddel et al., Science 257:238 (7-10-92): Lynch et al. Eur. J. Immunol. 21:1403-1410 (1991); Kast et al.. Cell 59: 603-614 (11-17-89)). cells presenting the desired complex are combined with CTLs leading to proliferation of the CTLs specific thereto. The proliferated CTLs are then administered to a subject with a cellular abnormality which is characterized by certain of the abnormal cells presenting the particular complex. The CTLs then lyse the abnormal cells, thereby achieving the desired therapeutic goal.

The foregoing therapy assumes that at least some of the subject's abnormal cells present the relevant HLA/TRA complex. This can be determined very easily, as the art is very familiar with methods for WO 96/29409Q 26 rliUYbU4/vS7 identifying cells which present a particular HLA molecule, as well as how to identify cells expressing DNA of the pertinent sequences, in this case a RAGE sequence. Once cells presenting the relevant complex are identified via the foregoing screening methodology, they can be combined with a sample from a patient, where the sample contains CTLs. If the complex presenting cells are lysed by the mixed CTL sample, then it can be assumed that a RAGE derived, TRA is being presented, and the subject is an appropriate candidate for the therapeutic approaches set forth supra.

Adoptive transfer is not the only form of therapy that is available in accordance with the invention.

CTLs can also be provoked in vivo, using a number of approaches. One approach, the use of nonproliferative cells expressing the complex, has been elaborated upon supra. The cells used in this approach may be those 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- 114 (January, 1991) exemplifies this approach. showing the use of transfected cells expressing HPVE7 peptides in a therapeutic regime. Various cell types may be used. Similarly, vectors carrying one or both of the genes of interest may be used. Viral or bacterial vectors are especially preferred. For example, nucleic acids which encode a RAGE TRA may be operably linked to promoter and enhancer sequences which direct expresion of the RAGE TRA in certain tissues or cell types. The nucleic acid may be incorporated into an expression vector. 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 RAGE TRAs. Nucleic acids encoding a RAGE TRA 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. retrovirus or the bacteria BCG. and the materials de facto "infect" host cells. The cells which result present the complex of interest, and are recognized by autologous CTLs, which then proliferate.

A similar effect can be achieved by combining the TRAP or a stimulatory fragment thereof with an adjuvant to facilitate incorporation into HLA-B7 presenting cells in vivo. The TRAP is processed to yield the peptide partner of the HLA molecule while the TRA is presented without the need for further processing.

Generally. subjects can receive an intradermal injection of an effective amount of the RAGE TRAP. and/or TRAs 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 compound may be classified as either adjuvants or cytokines. Adjuvants may enhance the Wf Q~0A9Q0n T'n/^T/TTC'f tf\ ^1\'9 -27- r 1U U3 immunological response by providing a reservoir of antigen extracellularly or within macrophages), activating macrophages and stimulating specific sets oflymphocytes. 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 Quilla 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: 1432-1434, 1995).

When administered, the therapeutic compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible cariers. supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

The preparations of the invention are administered in 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 the progression of the cancer. This may involve only slowing the progression of the disease temporarily. although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed herein.

Where it is desired to stimulate an immune response using a therapeutic composition of the invention, this may involve the stimulation of a humoral antibody response resulting in an increase in antibody titer in serum, a clonal expansion ofcytotoxic lymphocytes, or some other desirable immunologic response. 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 preferred range is believed to 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 patient 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.

Other aspects of the invention will be clear to the skilled artisan and need not be repeated here.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of P.\OPER\EJMfl4298-96.CLM I1/2MM 28 the features show~n and described or portons thereof it being recognized that vaious modifications are possible within the scope of the invention Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

R-eferees 1) Gleser, and Swamop, A. 1992. Expressed sequence tags and chromosomal localization of cDNA clones from a subtaced retinal pigm~ent epithelium libraiY. Genomics 13. 873-876.

2) Mager, and Freeman, J.D. 1987. Human endogenous retrovirus-like genone with Type C pol sequences and gag sequences related to human T-cell lyrnPhotroPic viruses. J. Virol. 61. 4060-4066.

3) Hirose. Takarnatsu Harada, F. 1993. Presence of env genes in members Of the RWVL-H fimily of human endogenous retrovirus-like elements. Virology 192, 52-61.

wn on/ GoAn Pi CT/US96/04037 29 Sequence Listing GENERAL INFORMATION:

APPLICANT:

NAME: LUDWIG INSTITUTE FOR CANCER RESEARCH STREET:1345 Avenue of the Americas CITY: New York STATE: NEW YORK COUNTRY: UNITED STATES OF AMERICA ZIP: 10105

APPLICANT:

NAME: LEIDEN UNIVERSITY STREET: Stationsweg 46 CITY: Leiden COUNTRY: THE NETHERLANDS ZIP: 2312 AV (ii) TITLE OF INVENTION: RAGE TUMOR REJECTION ANTIGENS (iii) NUMBER OF SEQUENCES: 57 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: WOLF, GREENFIELD SACKS, P.C.

STREET: 600 ATLANTIC AVENUE CITY: BOSTON STATE: MASSACHUSETTS COUNTRY: UNITED STATES OF AMERICA ZIP: 02210 Wn O/I')QAnO Pr T/US96/04037 30 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/401,015 FILING DATE: 21-MAR-1995 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/530,569 FILING DATE: 20-SEP-1995 (viii) ATTORNEY/AGENT INFORMATION: NAME: GATES, EDWARD R.

REGISTRATION NUMBER: 31,616 REFERENCE/DOCKET NUMBER: L0461/7002WO (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 617-720-3500 TELEFAX: 617-720-2441 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: WO 96/29409 -3 LENGTH: 1311 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCT1US96/04037 (ii) MOLECULE TYPE: cDNA (iii) HYPTHTICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGTAGCAGC CAAAGCAGGC ATCCCCGCAG 'ITGACrTGCC ACCAAGGGAA TGTGGGTGAA TGACCAAGGC AGGCATCCTC GCGGTGATCA GACACCAATG GAGTGTGGGT GAATAATCAG GCAGGCATCC CCGCAGTGAT TAAACACCAA GAGAAGACTA TTCCI'GAGTC TGTGACTGGT GCGGAGT1 TGAGTCCACA GATAAAATGT GTCTCCICG TCI'CTACI'AG AGAGGAAAAA.

GAACTGGAAT TGGAAGAACA GGGAGACI'GA. AGGGTAGCAA GAGAGGCTGG AGAAGAGAGT GAAAAGACCG CITACCI'GAT TTGtAAATTGT CTGCAGCCCC TCI=CCTGG AGTAAATGAA CTGGACCAAA TCTCAAAAAA TCCACGATGT CATCGGCACA CCCGCTCAGA AGATCCTCAC CAAGTTCAAA CAGGATCAGG AATACCTCTA CTAACAACCA AT=GTCCCC ACAATGCCTC 120 180 240 300 360 420 480 PCTfUS96/040T7 WO 96/2940l9 P~nQI~ -32- TCCCTCCI'GC ACGCAATGGT GGCCTATGAT CCCGATGAGA GAATCGCCGC CCACCAGGCC CI'GCAGCACC CCTACTTCCA AGAACAGAGA AACAGTCCCT AAAGCAAGAG GAGGACCGTC CCAAGAGACG AGGACCCC TATGTCATGG AACTGCCCAA ACT'AAAGCIT TCGGGAGTGG TCAGACTGTC OTCI'TACTCC AGCCCCACGC TGCAGTCCGT GCTTCGA.TCT OGAACAAATO GAAGAGTGCC OGTGCTGAGA CCCTTGAAGT GCATCCOTGC GAGCAAGAAG AC7AGATCCGC AGAAGGACCT TAAGCCTGCC CCGCAGCAGT GTCGCCTGCC CACCATAGTG CGGAAAGGCG GAAGATAACT GAGCAGCACC GTCGTCTCGA CTTCCGAGGC AACACCAAGC CCGACCGGGC CAGGCCTGGG TGATCTGCJ7G CTGAGACGCC ACGGAGGGCT? GGGGATGCGC CI'GCGTCCGT TTCGCGCTGG CCGGGGC27CT GGGTGCTGCC CTGCGCCCJ7G CCGCACCOGC GGCCCGCGCA GCTGCCI'ACG ATGTTCTGGG CTAATATACI' TGTAAAACCA CCGCATTCTA GOGTTTTCTT TCATTTCGT TAAGAAJ ETG GGGCAGGAAA TACTTTGTAA CTTTGTATAT C-LATCAAAAC AACGAGCAG GCATTT CTGT GATGTOTTGG GCGTGGTTGG AAGGTGGGTT CTGCGTGTCC CTTCCCAGCG CTGCTGGTCA GTCGTGGAGO GCCATCATGT CT'TACCAGTG ACGCT'GCTGA CACCCCTGAC T=TTAAA GAATAAGCTG TCG'rTAAAAA AAAA AAAAAA A INFORMVATION FOR SEQ ID NO:2: 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1311 SEQUENCE CHIARACTERISTICS: WO 96/29409 PCT/US96/f4n7 3, LENGTH: 99 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..99 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ATG AAC TGG ACC AAA TCT CAA AAA ATC CAC GAT GTC ATC GGC ACA CCC 48 Met Asn Trp Thr Lys Ser Gin Lys Ile His Asp Val Ile Gly Thr Pro 1 5 10 GCT CAG AAG ATC CTC ACC AAG TTC AAA CAG GAT CAG GAA TAC CTC TAC 96 Ala Gin Lys Ile Leu Thr Lys Phe Lys Gin Asp Gin Glu Tyr Leu Tyr 20 25

TAA

WO 96/29409 WO 9629409PCTIUS96/04037 -34- INFORMVATION FOR SEQ ID NO:3: SEQUENCE CHRAC'ER.ISTICS: LENGTH: 32 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Asn Trp Thr Lys Ser Gin Lys Ile His Asp Val Ile Gly Thr Pro 1 5 10 Ala Gin Lys Ile Leu Thr Lys Phe Lys Gin Asp Gin Giu Tyr Leu-Tyr 25 INFORMATION FOR SEQ ID NO:4: SEQUENCE CTLARACTERISTICS: LENGTH: 123 base pairs TYPE: nucleic acid STRADEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 96/29409 WO 9629409PCTIUS96/04037 35 FRAGMv= TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .123 (xi) SEQ UENCE DESCRIPTION: SEQ ID NO:4:

ATG

met 1 TCA TCG Ser Ser GCA CAC CCG CIC AGA AGA Ala His Pro Leu Arg Arg

TCC

Ser 10 TCA OCA ACT TCA AAC AGG Ser Pro Ser Ser Asn Arg ATC AGG AAT Ile Arg Asn

ACC

Thr TCT ACT AAC AAC CAA Ser Thr Asn Asn Gin 25 GTC CCC ACA ATG CCT CIC Phe Val Pro Thr Met Pro Leu CCI CCI GCA Pro Pro Ala CCC AAT CGT CCC CIA TCA 123 Arg Asn Gly Cly Leu INFORMATION FOR SEQ ID SEQUENCE CAACTERISTICS: LENGTH: 40 amino acids (B3) TYPE: amino acid TOPOLOGY: linear WO 96/29409 -36- (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID PCTfUS96/04037 Met Ser Ser Ala His Pro Leu Arg Arg Ser 10 Ile Arg Asm Thr Ser Thr Asn Asn Gin Phe 25 I0 Pro Pro Ala Arg Asn Gly Gly Leu INFORMATION FOR SEQ ID NO:G: SEQUENCE CHARACTERISTICS: LENGTH: 87 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO F'RAGIV1ET TYPE: internal Ser Pro Ser Ser Asn Arg Val Pro Thr Met Pro Leu (vi) ORIGINAL SOURCE: ORGANISM: Homro sapiens WO 96/29409 PCT/US96/04037 -37 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..87 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATG GTG GCC TAT GAT CCC GAT GAG AGA Met Val Ala Tyr Asp Pro Asp Glu Arg 1 5 CAG CAC CCC TAC TTC CAA GAA CAG AGA Gin His Pro Tyr Phe Gin Glu Gin Arg ATC GCC GCC CAC CAG Ile Ala Ala His Gin 10 GCC CTG Ala Leu AAC AGT CCC TAA Asn Ser Pro INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Val Ala Tyr Asp Pro Asp Glu Arg Ile Ala Ala His Gin Ala Leu 1 5 10 Gin His Pro Tyr Phe Gin Glu Gin Arg Asn Ser Pro WO 96/29409 PCT/US96/04037 -38- INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 288 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..288 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATG ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro 1 5 10 ACT TCC AAG AAC AGA GAA ACA GTC CCT AAA GCA AGA GGA GGA CCG TCC WO 96/29409 Tbr Ser Lys 39 PCTfUS96/04037 Asn Arg Giu Thr Val Pro Lys Ala Arg Gly Gly Pro Ser 25 CAA GAG Gln Glu

ACG

Thr AOG ACC 000 CIA Arg Thr Gly Leu TG CAT OGA ACT GCC Cys His Gly Thr Ala 40 GTC 'ITA CTC CAG CCC Val Leu Leu Gin Pro

CAA

Gin

CAC

His ACT AAA GCT Thr Lys Ala 144

TTC

Phe

G

Gly AGT GOT CAG ACT Ser Gly Gin Thr

GTC

Val GCT GCA GTC Ala Ala Val 192 Arg GCT TOG ATC TG Ala Trp Ile Trp

AAC

Asn 70

GAG

Giu AAA TOG AAG AGT GCC Lys Trp Lys Ser Ala 75 GOT OCT GAG ACC Gly Ala Giu Thr Leu 240 OAA OTO CAT CCC TG Giu Val His Pro Gys CAA GAA GAC AGA TCC OCA OAA OGA CCT TAA Gin Giu Asp Arg Ser Ala Giu Gly Pro 90 288 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTEISTICS: LENGTH: 95 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: WO 96/29409 WO 9629409PCTIUS96/04037 40 Met Ile Pro Met Arg Glu Ser Pro Pro 1 5 Thr Ser Lys Asn Arg Glu Thr Val Pro 25 Thr Arg Pro Cys Ser Thr Pro 10 Lys Ala Arg Gly Gly Pro Ser Gin Glu Thr Arg Thr Gly Leu Cys His Giy Thx Ala Gin Thr Lys Ala 40 Phe Gly Ser Gly Gin Thr Val Val Leu Leu Gin Pro His Ala Ala Val 55 Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Giu Thr Leu 65 70 75 Glu Val His Pro Cys Glu Gin Giu Asp Arg Ser Ala Giu Giy Pro 90 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 222 base pairs TYPE: nucleic acid STRANDEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO WO 96/29409 WO 9629409PCTIUS96/04037 41 (iv) AN'TI-SNSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Hlomo sapiens (ix) FEATURFE: NAME/KEY: CDS LOCATION: 1. .222 (xi) SEQUENCE DESCRIPTION: SEQ ID

ATG

met 1

GAA

Glu CIG CCC AAA CTA AAG OTT TCG Leu Pro Lys Leu Lys Leu Ser 5 GGA GTG GTC AGA CTG TCG TOT Gly Val Val Arg Leu Ser Ser 10 TAC TCC AGC Tyr Ser Ser CCC ACG CTG CAG TCC GTG Pro Thr Leu Gin Ser Val 25 OTT OCA TOT OCA Leu Gly Ser Gly ACA AAT GCA Thr Asn Gly AGA GTG Arg Val Val OTG AGA CCC Leu Arg Pro

TIG.

Leu AAG TGC ATC COT GCG Lys Cys Ile Pro Ala

AGC

Ser AAG AAG Lys Lys 144 ACA CAT Thr Asp CCG GAG AAG GAC CTT Pro Gin Lys Asp Leu 55 AAG COT GCC CCG CAG GAG TGT CGC CIG Lys Pro Ala Pro Gin Gin Cys Arg Leu 192 CCC ACC ATA =T CCC AAA CCC CCA AGA TAA WO 96/29409 WO 9629409PCTIUS96/04037 42 Pro Thr Ile Val Arg Lys Gly Gly Arg INFORMATION FOR SEQ ID NO:ii: SEQUENCE CHARACTERISTICS: LEGTH: 73 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENE DESCRIPTION: SEQ ID NO:11: Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser Tyr Ser Ser Pro Thr Leu Gin Ser Val Leu Gly Ser Gly Thr Asn Gly Arg Val Pro Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys Tlir Asp Pro Gin Lys Asp Leu Lys Pro Ala Pro Gin Gln Cys Arg Leu Pro Thr Ie Val Arg Lys Gly Gly Arg INFORMAITION FOR SEQ ID N0:12: WO 96/29409 43 SEQUENCE CHARACTERISTICS: LENGTH: 1168 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO PCTIUS96/04037 1 5 (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CTGTGACTGG TGCTGGAGTT TGAGTCCAC AGATAAAATG TGTCT'CCTTC GTC TCTACTA GAGAGGAAAA AGAACTGGAA. TTGGAAGAAC AGGGAGACTG AAGGGTAGCA AGAGAGGCTG GAGAAGAGAG TGAAAAGACC GCTTACCTGA TTGAAATTG TCI'GC.AGCCC CTCTTTCCTG GAGTAAATGA ACTGGACCAA. ATCTCAAAAA TCCACGATGT CATCGGCACA CCCGCTCAGA AGATCCTCAC CAAGTTCAAA. CAGTCGAGAG CTATGAATI TGAWTI'CCT TTTAAAAAGG GATCAGGAAT ACCTCTACTA ACAACCAATT TGTCCCCAC.A ATGCCTCTCC CTCCTGCACG 120 180 240 300 360 Wn Ofl')GAAG VC-r QGKIAJA" Umf O~)O~lO CT1TQ~IAfl2 TVS- 44 t* LFJW CAATGGTGGC CTATGATCCC GA.TGAGAGAA TCGCCGCCCA CCAGGCCCTG CAGCACCCCT ACTTCCAAGA ACAGAGAAAC AGTCCCTAAA GCAAGAGGAG GACCGTCCCA AGAGACGAGG ACCGGCCTAT GTCATG7GAAC TGCCCAAACT AAAGCITTCG GGAGTGGTCA GACTGTCGTC TTACTCCAGC CCCACGCTGC AGTCCGTGCT TGGATCI'GGA ACAAATGGAA GAGTGCCGGT GCT GAGACCC TTGAAGTGCA TCCCTGCGAG CAAGAAGACA GATCCGCAGA AGGAC CTT AA GCCTGCCCCG CAGCAGTGTC GCCTGCCCAC CATAGTGCGG AAAGGCGGAA GATAAOTGAG CAGCACCGTC GTCTrCGACTT CGGAGGCAAC ACOAAGCCCO ACCGGGCCAG GCCTGGGTGA TCI'GCTGCTG AGACGCCACG GAGGGCI'OCC GATGCGCCTG CCTCCGTTTC GCGCTGCCG CGGCTCTGGG TGCTGCCCTG OGCCCTOCOG CACCCGCCGC CCGCGCAGC T GCCTAGGATG TCJ7GGGCTA ATATACTTGT AAAACOACCG CATTCTAGGG TT'ITCT'ITCA TTTTCGTTAA GAATFT'GGG CAGGAAATAC TTTGTAACTT TGTATATGAA TCAAAACAAA CGAGCAGGCA TTTC TGTGAT GTGTTGGGCG TGGTTGGAAG GTGGGTTCTG CGTGTCCCIT CCCAGCGCTG CIGGTCAGTC GTGGAGCGCC ATCATGTCTT ACCAGTGACG CTGCTGACAC CCCTGACTT TATTAAAGAA TAAGCTGTCG TTAAAAAA INFORMIVTON FOR SEQ ID NO:13: 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1168 SEQUENCE OHAFACTER.ISTICS: WO 96/29409 45 LENGTH: 1235 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCTIUS96/04037 (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATTCCTGAGT CTGTGACTGG TGCGAGTT TI'GAGTCCAC AGATAAAATG TGCT'CC GTCTCTACTA GAGAGGAAAA AGAACTGGAA TTGGAAGAAC AGGGAATG AAGGGTAGCA AGAGAGGCTG GAGAAGAGAG TGAAAAGACC GCTTACCTGA TITGAATTG ATGGCG GGGAATGAAG AATGTGATAT ACATCTTTGG AGTCTGTTCT GCA.GCCCCTC TTTCCTGGA.G TAAATGAACT GGACCAAATC TCAAAAATCC ACGATGTCAT CGGCACACCC GCTCAGAAGA TCCI'CACCAA GTTCAAACAG TCGAGAGCTA TGAATTITGA TTTCCITI AAAAAGGGAT CAGGAATACC TCTACTAACA ACCAATTTGT CCCCACAATG CCT'CT'CCCTC CCCGCAA 120 180 240 300 360 420 Wn 04110AM lDf'qrf]rTCn.C /A AA'27 46 -L J7UIU TGGTGGCCI'A TGATCCCGAT GAGAGAATCG CCGCCCACCA GGCCCTCAG CACCCCTACT TCCAAGAACA GAGAAACAGT CCCTAAAGCA AGAGGAGGAC CGTCCCAAGA GACGAGGACC GGCCI'ATGTC ATGGAAOTGC CCMAACTAAA GC CGGGA GTGGTCAGAC T=T=GT=TA CTCCAGCCCC ACGC TGCAGT CCGTGJIGG ATCTGGAACA AATGGAAGAG TGCCCGTGCT GAGACCC=T AAGTGCATCC CTGCGAGCAA GAAGACAGAT CCGCAGAAGG ACCTTAAGCC TGOCCCGCAG CAGTGTCGCC TGCCCACCAT AGTGCGGAAA GGCGGAAGAT AACTGAGCA G CACCGTCGTC TCGA =TCGG AGGCAACACC AAGCCCGACC GGGCCAGGCC TGGGTGATCT ==GCGAGA CGCCACGGAG GGCTGGGGAT GCGCCTGCG CCGTTCGCG CTGGCCGCGGG CTCTGGGTGC TGCCC TGCGC CC27CCGCAC CCGCGCCCO CGCAGCTCC TAOGATGTTC TGGGCTAATA TACTGTAAA ACCACCGCAT TCTAGGJTTT TCITCAT= TCGTTAAGAA 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1235 TTTGGGGCAG GAAATACIT GTAACITGT ATATGAATCA AAACAAACGA GCAGGCATTT CTGTGATGTG TTGGGCGTGG TTGGAAGGTG GGTFCTGCG GTCCCTCCC AGCGCTCT GTCAGTCGTG GAGCGCCATC ATGTCTACC ACTGACGOTG CTGACACCCC TGACrI'AT TAAAGAATAA GCTGTCGTTA CAGTA'TTGCA AAAAA INFOPMA.TION FOR SEQ ID NO:14: (i2 SEQUENCE OHARACrEISTICS: WO 96/29409 PCTIUS96/04037 47- LEGTH: 2051 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: No FRAGME~NT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GACI'GGTGCT GGAGTT'GA GTCCACAGAT AAAATGTGTC TC==ICGTr CI'ACTAGAGA GGAAGA CTGGAATTGG AAGAACAGGG AGArn'GAAGG GTAGCAAGAG AGGC7TGGAGA AGAGAGTGAA AAGACCGCI ACCTGATTG AAATI'CTCTG CAGCOCTOT TTCCTGGAGT AAATGAACTG GACCAAATCT CAAAAATCCA CGATGTCATC GGCACACCCG CTCAGAAGAT CCTCACCAAG TTCAAA.CAGT CGAGAGCIA GAATITGAT TTCCTTTTA AAAAGGGATC AGGAATACCI' C]7ACTACAA CCAATIGTC CCCACAATGC CTCT CCCTCC TGCACGCAAT GGTGGCCI'AT GATCCCGATG AGAGAATCGC CGCCCACCAkG GCCCTGCAGC ACCCCTACI 120 180 240 300 360 420 WO 961294(19 48 PC I /USYbU4t CCAAGAACAG AGAACCCAGA ACGAGCGA GGATGAAOGC CTCAGCCGTC CTCCTCCCCA ITCAAACACG TTCATCCCTC AACCCTO=GC TGAGCACCTG CATGCTGCCC CGCCGOAGTG TCACCC= CT TGTGTGAGCC TACCCTCATC CACCOA.CCTC ACCCTCCTGA C AAAGAA GACACCGCCC AC&AAGCACAG GOGAGCCCAG TOACACCOGA CACrOCGOG GGCAGGCC= GCAGGGAGAA GCAGTAAGCA OCCATOTOGA TCAGCCATT CCATCTGCA CT'CAGACGTG CACGTC=TCG TGTGACAGGC GGCAGCAGTG CGACCGTGAC CTCCCATCTG CT'CTGCTGTC OCCACACCIG CGGTGCAGCC AGCCTGCCAC AAGGCAGCTA GAGTCCAGCT AGACCCACCC CTGGCACGGC CGACCT'CrC CTGr=c CTGGGCCTAA TCCCCGTGCA TTCTCCAACG CCAGAAGTGT AAGAAAGTGC AAGGCAACAA GTGAGAAGAG CAAACCCAAA TCGTACCAGG GAAGCI'AGTC TICCAGGGC ACCTGAGTGA GGGCATGACC AGCCTGACG CTGCCTCOCT ACCATCTGCC CAGGGCC=GC TGAATGC=TG AGTCCATGGT GACAGTGGTG GGAACAGTTA CGAGGCAGTT AGAT17TGGA AGTCATGTTG GCCCACTGG CTACAGAGCA GTCTIAGG-AA CAGCACCATA AAAATAAAGA CIT'TCCTG ACACACATGC ATCTAGAGTA AACTGGGGCG TATCTGACAG CGTTAGTACA GTGATGGCCA AATGCAAACT GCATCCAGA ACCAGcGAAG GGTGACAGAC TGGGCTGAGG CAGAGCTAGG ACTAACCATC TCGAGTGATG CCA.TCTCGGG GCCAACAAAA GTTTT"GGACA CGGCTGGATC ATCTGACCAA ACTGCTCAAA TLCTITACACA 37 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 WO 96/29409 'PCTfFj4ZQf.1d1jf1'ZP7 49

V

ATTAITGTCC TGGTATTAAA CT ITICACG CCA CTTCCAA CAACAGGAG ACGAATAAG GAGATGACCA GGAAGATGGC TGGATTAAGA ATTCTAGACT TGGCCGGGTO CGGTOGOTCA CACCTGTAAT CCTAGCATCT TGGGAGGCTG AGGCAGGAGG ATCG CTT GAG CCCAGAG TTT GAGACCAGCC TAGGCAACAT AACGAGACCC CATCTCTACA AAATATCAAA AAMTTACCCA GGTATGGTGG CACACACGTG TGATTCCAGC TACTCGGGACG GCTGAGATOG GAGGATCAZT TGAACCCAGO AGGTTGGGGC TACAATGAGC TATGA.TCGCA CCA CTT CATT CCAGCCTGGA TGACAGAAGA CTOACTOCAT AGTTCATGGC CCCGTGATCC AGAGTCCCTG CTGGCGCCT CGAGTGGGGC AGGCGAGAA CTCAAGCTGT AAOTAATGTC TCCTCC!GAAG AAAACTAAAC CGAGGGCTGA GCTGATGTGA AGTTTTCCGT GGCTGCATTC ATACAAATGG TGAAAATGTA GCATACCTCC CCTCAAAAGC CTGAAAGTAA AGACATGCCC CCAATTAAT GTGATGAATT AGAGAAATAG GTTTCAGACA CITCATGGT TAAAGTJTCA CAAAATAAAG UITTCGAAGG AAAAAAAAAA A 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2051 INFORMVATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LEGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 96/29409 PTU9/43 50 PCTfUS96/04037 (i i) MOLECULE TYPE: cDNA.

(iii) HYPOTETICAL: NO (iv) ANTI-SENSE: NO FPRAGvENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURPE: NAMvE/KY: CDS LOCATION: (xi) SEQUENCE DESCRIPTION: SEQ ID

ATG

Met 1

CGA

Arg TCA TCG GCA. CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA AAC AGT Ser Ser Ala His Pro Leu .Arg Arg Ser Ser Pro Ser Ser Asn Ser 5 10 GAG OTA TGA Glu Leu INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid WO 96/29409 PCT/US96/04037 -51 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 1 5 10 Arg Glu Leu INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 177 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens WO 96/29409 WO 9629409PCTIUS96/04037 52 (ix) FEATURE: NAME/KEY: ODS LOCATION: 1. .177 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

ATG

Met 1 AAT TT Asn Phe GAT TI? CCT TI? AAA Asp Phe Pro Phe Lys 5

AAG

LYS

GGA TCA OGA ATA COT Gly Ser Gly Ile Pro 10 CTA CTA Leu Leu ATG GTG Met Val.

ACA

Thr ACC AAT Thr Asn

TIG

Leu

CCC

Pro TOC CCA CAA TGC Ser Pro Gin Cys

OI'C

Leu 25 TCC CC OTO CAC Ser Leu Leu His

GCA

Ala GCC TAT GAT Ala Tyr Asp GAT GAG AGA Asp Glu Arg

ATC

Ile 40 GCC GCC CAC CAG GCC Ala Ala His Gin Ala CTG GAG CAC Leu Gin His 144 CCC TAC TIC Pro Tyr Phe CAA GAA GAG AGA Gin Giu Gin Arg AAC AGT CCC TAA Asn Ser Pro 177 INFORMvATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGH: 58 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 96/29409 -53- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: PCT/US96/04037 Met Asn Phe Asp Phe 1 5 Thr Thr Asn Leu Ser Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu 10 Ser Leu Leu His Ala Met Val Pro Gin Cys Leu 25 Ala Tyr Asp Pro Asp Glu Arg Ile 40 Ala Ala His Gin Ala Leu Gin His Pro Tyr Phe Gin Glu Gin Arg Asn Ser Pro INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 288 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: WO 96/29409 54 ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .288 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i9: PCTIUS96/04037

ATG

met

I

ACT

Thr ATC CCG Ile Pro ATG AGA GAA TCG CCG CCC Met Arg Giu Ser Pro Pro 5

ACC

Thr 10 AGG CCC TGC AGC Arg Pro Cys Ser ACC COT Thr Pro CCG TCC Pro Ser TOC AAG Ser Lys

AAC

Asn

AGG

Arg AGA GAA ACA GTC Arg Giu Thr Val

CCT

Pro 25 AAA GCA AGA GGA GGA Lys Ala Arg Gly Gly CAA GAG Gin Giu

ACG

Thr ACC GGC CTA Thr Gly Leu TGT CAT Cys His GGA ACT GCC Giy Thr Ala CAA ACT Gin Thr AAA GCT Lys Ala 144

TTC

Phe GOG AGT GGT CAG, ACT Gly Ser Gly Gin Tlir GCT TGG ATC TGG AAC Ala Trp Ile Trp Asn 70

GTC

Val 55

AAA

Lys GTC TTA CTC CAG Vai Leu Leu Gin

CCC

Pro CAC GCT GCA GTC His Ala Ala Vai 192

CGT

Arg TGG AAG AGT Trp Lys Ser

GCC

Ala 75 GGT GCT GAG ACC CTT Gly Ala Giu Thr Leu 240 3 0 GAA Glu GTG CAT CCC TGC GAG CAA GAA GAC AGA Vai His Pro Cys Giu Gin Giu Asp Arg 90 TCC GCA. GAA GGA COT TAA Ser Ala Glu Gly Pro 288 WO 96/29409 WO 9629409PCTJUS96/04037 55 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 95 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro Thr Ser Lys Asn Axg Giu Thr Val Pro Lys Aia Arg Giy Gly Pro Ser Gin Giu Thr Arg Thr Gly Leu Cys His Gly Tlir Ala Gin Thr Lys Ala Phe Gly Ser Gly Gin Thr Val Val Leu Leu Gin Pro His Ala Ala Val Arg Ala Trp Ile Trp Asn Lys Trp Lys Ser Ala Gly Ala Glu Thr Leu Glu Val His Pro Cys Glu Gin Glu Asp Arg Ser Ala Glu Giy Pro INFORMATION FOR SEQ ID NO:21: WO 96/129409 VrCTlTQOCIflAfl2' -56- SEQUENMCE CHARACT'ERISTICS: LENGTH: 222 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .222 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: ATG GAA CI'G CCC AAA CIA AAG CI TCG OGA GTG GTC AGA CIGTCG TC' 48 Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu Ser Ser 1 5 10 TAC T1CC AGC CCC ACG CIG CAG TCC GTG CI GGA TCT GGA. ACA AAT GGA. 96 Tyr Ser Ser Pro Thr Leu Gin Ser Val Leu Gly Ser Gly Thr Asn Gly 25 WO 96/29409 WO 9629409PCTIUS96/04037 -57-

AGA

Arg

GTG

Val COG GTG CTG AGA CCC Pro Val Leu Arg Pro COG CAG AAG GACCI Pro Gin Lys Asp Leu 55 TTG AAG TGC ATC COT GO AGC AAG AAG Leu Lys Cys Ile Pro Ala Ser Lys Lys 40 144

ACA

Thr

OAT

Asp so AAG COT GCC CCG Lys Pro Ala Pro CAG CAG TOT CGC OTG Gin Gin Cys Arg Leu 192

CCC

Pro ACC ATA GTG COO Thr Ile Val Arg AAA GOC Lys Gly OGA AGA TAA Gly Arg 222 INFORMATION FOR SEQ ID NO:22: Wi SEQUENCE CHARACTERISTICS: LEGTH: 73 amino acids (W TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Met Giu Leu Pro Lys 1 5 Tyr Ser Ser Pro Thx Leu Lys Leu Ser Oly Val Val Arg Leu Ser Ser 10 Leu Gin Ser Val Leu Gly Ser Gly Thr Asn Gly 25 Arg Val Pro Vai Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys 40 WO 96/29409 Pr'T/fSO/0LfAn'7 -58- Thr Asp Pro Gin Lys Asp Leu Lys Pro Ala Pro Gin Gin Cys Arg Leu 55 v~vur Pro 65 Thr Ile Val Arg Lys Gly Gly Arg INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 63 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: WO 9612940f9 1DJ"r1rfFj4ZQK1AAAV7

ATG

Met 1

TOO

Ser 59 A 1AAG AAT OTO ATA TAO ATO ITT GGA OTO TGT TCT GOA GCC OCT CT Lys Asn Val Ile Tyr Ile Phe Gly Val Oys Ser Ala Ala Pro Leu 5 10 TGG AGT AAA TGA Trp Ser Lys INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE. protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: met 1 Lys Asn Val Ile Tyr Ile Phe Gly Val Cys Ser Ala Ala Pro Leu 5 10 Ser Trp Ser Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid WO 96/29409 60 PCTIUS96/04037 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHTICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. (xi) SEQUENCE DESCRIPTION: SEQ ID

ATG

met 1

CGA

Arg TCA TOG GCA CAC CCG CTC AGA AGA Ser Ser Ala His Pro Leu Arg Arg 5 TCC TCA CCA AGT TCA AAC ACT Ser Ser Pro Ser Ser Asn Ser 10 GAG CIA TGA Glii Leu INFORMVATION FOR SEQ ID NO:26: WO 96/29409 -6 Wi SEQUENCE CHAATERISTICS: LENGTH: 19 amino acids TYPE: amino acid TOPOLOGY: linear PCTfUS96/04037 (ii) MOLECULE TYPE: protein (xi) SEQ UEN CE DESCRIPTION: SEQ ID NO:26: Met Ser Ser Ala His 1 5 Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 10 Arg Glu Leu INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACT'ERISTICS: LENGTH: 177 base pairs TYPE: nucleic acid STRANDEDNBSS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal WO 96/29409 62 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: ODS LOCATION: 1. .177 (xi) SEQUJENCE DESCRIPTION: SEQ ID NO:27: PCTIUS96/04037 ATG AAT FIT GAT Met Asn Phe Asp TT1' OCT TIT AAA Phe Pro Phe Lys

AAG

LjYS ACA ACC AAT Thr Thr Asn

TTG

Leu TCC CCA CAA TGC Ser Pro Gin Cys

CTC

Leu 25

GC

Ala GGA TOA OGA ATA OCT Gly Ser Gly Ile Pro TOO CTO CTG CAC GCA Ser Leu Leu His Ala GOC CAC CAG GOC CTG Ala His Gin Ala Leu CTA CTA.

Leu Leu ATG GTG Met Val GOC TAT Ala Tyr

GAT

Asp

TTO

Phe COO GAT GAG AGA Pro Asp Giu Arg

ATO

Ile 40 CAG CAC Gin His 144 CCC TAO Pro Tr CAA GAA CAG AGA Gin Giu Gin Arg AAC AGT COO TAA Asn Ser Pro 177 INFORMVATION FOR SEQ ID NO:28: SEQUENCE OHARA CTE RISTICS: LENGTH: 58 amino acids TYPE: amino acid WO 96/29409 PCT/US96/04037 63 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Met Asn Phe Asp Phe 1 5 Pro Phe Lys Lys Gly 10 Ser Gly Ile Pro Leu Leu Thr Thr Asn Leu Ser Pro Gin Cys Ala Tyr Asp Pro Asp Glu Arg Ile 40 Leu Ser Leu Leu His Ala Met Val 25 Ala Ala His Gin Ala Leu Gin His Pro Tyr Phe Gin Glu Gin Arg Asn Ser Pro INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 288 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 96/29409 64 FRAT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens PCTfUS96/04037 (ix) FEATURE: NAIVE/KY: CDS LOCATION: 1. .288 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:

ATG

met 1

ACT

Tlir ATC CCG Ile Pro TCC AAG Ser Lys ATG AGA GAA TCG CCG Met Arg Giu Ser Pro 5 AAC AGA GAA ACA. GTC Asn Arg Glu Thr Val

CCC

Pro ACC AGG CCC TGC AGC Thx Arg Pro Cys Ser ACC CCT Thr Pro CCG TCC Pro Ser

CCT

Pro 25 AAA GCA AGA GGA Lys Ala Arg Gly

GGA

Gly CAA GAG ACG Gin Giu Thr

AGG

Arg ACC GGC CIA Thr Giy Leu

TGT

Cys 40 CAT GGA ACT GCC His Giy Thr Ala

CAA

Gin

CAC

His ACT AAA GCI1 Thr Lys Ala 144 TIC GGG AGT GGT CAG ACT Phe Gly Ser Giy Gin Thr CGT GCI TGG ATC TGG AAC Arg Ala Trp Ile Trp Asn 70

GTC

Val S5 GTC TIA CIC GAG CCC Val Leu Leu Gin Pro GCI GCA GTC Ala Ala Val 192 AAA TGG AAG AST GCC GGT Lys Trp Lys Ser Ala Gly 75 GCI GAG ACC CTT Ala Giu Thr Leu 240 WO 96/29409 GAA GTG CAT CCC TGC GAG CAA GAA GAC AGA TCC GCA GAA GGA Glu Val His Pro Cys Glu Gin Glu Asp Arg Ser Ala Glu Gly 90 PCTfUS96/04037 CCT TAA Pro 288 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 95 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ile Pro Met Arg 1 5 Glu Ser Pro Pro Thr Arg Pro Cys Ser 10 Thr Pro Thr Ser Lys Asn Arg Glu Thr Gin Glu Thr Arg Thr Gly Leu Phe Gly Ser Gly Gin Thr Val 55 Arg Ala Trp Ile Trp Asn Lys 65 70 Pro 25 Lys Ala Arg Gly Gly Pro Ser Cys His Gly Thr Ala Gin Thr Lys Ala 40 Val Leu Leu Gin Pro His Ala Ala Val Trp Lys Ser Ala Gly Ala Glu Thr Leu 75 WO 96/294 09 rF S9143 66 Glu Val His Pro Cys Glu Gln Glu Asp Arg Ser Ala Glu Gly Pro 90 INFORMATION FOR SEQ ID NO:31: fUS96/04037 SEQUENCE CHARACTERISTICS: LENGTH: 222 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA.

(iii) HYPOTHETICAL: No (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Horno sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .222 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: WO 96/29409 PTU9143 67 PCTIUS96/04037 ATO GAA CTG 000 Met Glu Leu Pro AAA CIA AAG CIT TOG LYS Leu Lys Leu Ser OGA GTG GTO AGA CI'G Gly Val Val Arg Leu TOG TOT Ser Ser TAO TOO AGO Tyr Ser Ser 000 Pro

GTG

Val

AOG

Thr CTG CAG TOO Leu Gin Ser

GTG

Val 25

AAG

Lys CIT GGA TOT GGA AOA AAT GGA Leu Gly Ser Gly Thx Asn Gly AGA GTG Arg Val

OOG

Pro

OOG

Pro OTG AGA 000 Leu Arg Pro

TTG

Leu 40 TGO ATO OOT GOG Oys Ile Pro Ala

AGO

Ser AAG AAG Lys Lys 144

ACA

Thr

GAT

Asp CAG AAG GAO CT Gin Lys Asp Leu 55 AAG OOT GOO OOG Lys Pro Ala Pro CAG CAG Gin Gin TGT OGO OTG Oys Arg Leu 192 000 Pro ACC ATA GTG OGG Thr Ile Val Arg AAA GGO Lys Gly GGA AGA TAA Giy Arg 222 INFORMATION FOR SEQ ID NO:32: SEQUENCE CH~AATERISTICS: LEGTH: 73 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECUL TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: WO 96/29409 -8 Met Glu Leu Pro Lys Leu Lys Leu Ser Gly Val Val Arg Leu 10 PCTfUS96/04037 Ser Ser Ty'r Ser Ser Arg Val Pro Pro Thr Leu Gin Ser Val Leu Gly Ser Gly Thr Asn Gly 20 25 Val Leu Arg Pro Leu Lys Cys Ile Pro Ala Ser Lys Lys 40 Gin Lys Asp Leu Lys Pro Ala Pro Gin Gin Cys Arg Leu 55 Thr Asp Pro Pro Thr Ile Val Arg Lys Gly Gly Arg INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOG: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTPETICAL: NO (iv) ANTI-SENSE: NO FRAGMNT TYPE: internal WO 96/29409 69 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAIVE/KEY: CDS LOCATION: 1. (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: PCTIUS96/04037

ATO

Met 1 TCA TCG GCA CAC CCG CTC AGA AGA TCC TCA CCA AGT TCA AAC AGT Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 5 10 CGA GAG CIA TGA Arg Glu Leu INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTRISTICS: LENGH: 19 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

I

WO 96/29409 PCTQUS96/04037 Met 1 Ser Ser Ala His Pro Leu Arg Arg Ser Ser Pro Ser Ser Asn Ser 5 10

V"

Arg Glu Leu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 564 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..564 (xi) SEQUENCE DESCRIPTION: SEQ ID WO 96/29409 WO 9629409PCTIUS96/04037 71

ATG

Met 1

ACA

Thr AAT =v GAT =I OCT =T AAA AAG Asn Phe Asp Phe Pro Phe Lys Lys

S

GGA

Gly 10 TGA GGA ATA OCT Ser Gly Ile Pro CIA CTA Leu Leu

ATGGT

Met Val ACC AAT Thr Asn

TTG

Leu

COO

Pro TCC OCA CAA TGO Ser Pro Gin Cys rCT Leu 25 TCO CTC =T CAC Ser Leu Leu His

GOA

Ala GCC TAT Ala Tyr

GAT

Asp GAT GAG AGA ATO Asp Giu Arg Ile 40 GCO GCC GAO GAG Ala Ala His Gin

GCC

Ala OTG GAG GAO Leu Gin His 144 CCC TAO Pro Tyr TTO GAA GAA GAG Phe Gin Giu Gin

AGA

Arg 55 ACC GAG AAO OGA Thr Gin Asn Gly

AGO

Ser GAG GAT GAA GGO Giu Asp Glu Giy 192 CTo Leu

GCT

Ala AGO =G OCT OCT Ser Arg Pro Pro 000 Pro 70 GAT TGA AAO AOG TTb His Ser Asn Thr Phe 75 ATO OCT GAA 000 Ile Pro Gin Pro

TCT

Ser 240 GAG GAO =T Giu His Leu

GAT

His GCT GOO CC 000 Ala Ala Arg Pro

GAG

Gin 90

CTO

Leu TGT GAO OCT TCT Oys His Pro Ser TGT GTG Oys Vai 288 AGO CTA 000 Ser Leu Pro OOG 000 AGA Pro Gly Arg 115

TGA

Ser 100 TOO ACC GAO CTO Ser Thr His Leu

ACC

Thr 105 =T ACC TTA Leu Thr Leu AAG AAG ACA Lys Lys Thr 336 AGO AGA GGG GAG 000 Ser Thr Giy Ciu Pro 120 ACT GAO ACC OGA GAO Ser His Thr Pro His TOG CG 00 Trp Arg Gly 384 WO 96/29409 WO 9629409PCTIUS96/04037 72

GAG

Gin

OCA

Pro 145 GOC TIG GAG GGA GAA GOA Ala Leu Gin Gly Glu Ala 130 135 GTA AGO AGO OAT Val Ser Ser His OTO OAT CAG OGA Leu His Gin Pro 140 GAO AGG CGG GAG Asp Arg Arg Gin

TI?

Phe

GAG

Gin 160 432 TOT GO ACT GAG Ser Gly Thr Gin

AOG

Thr 150 TGO AOG TOT TOG Oys Thr Ser Ser

TGT

Cys 155 480 TGO GAO OGT GAO Cys Asp Arg Asp

CTO

Leu 165 OGA TOT GOT OTG Pro Ser Ala Leu

COG

Leu 170 TOO OGA GAO OTG OGG TGO Ser Pro His Leu Arg Oys 175 528 AGO GAG OOT GC Ser Gin Pro Ala 180 AGA AGO GAG OTA GAG TOO AGO TAG Tbr Arg Gin Leu Glu Ser Ser 185 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LEGTH: 187 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Met Asn Phe Asp Phe Pro Phe Lys Lys Gly Ser Gly Ile Pro Leu Leu 1 5 10 WO 96/29409 WO 9629409PCTIUS96/04037 73 Thr Thr Asn Leu Ser Pro Gin Cys Leu Ser Leu Leu His Ala Met Val Ala Tyr Asp Pro Asp Glu Arg Ie Ala Ala His Gin Ala Leu Gin His Pro Tyr Phe Gin Giu Gin Arg Thr Gin Asn Gly Ser Glu Asp Glu Gly Leu Ser Arg Pro Pro Pro His Ser Asn Thx Phe Ile Pro Gin Pro Ser Ala Glu His Leu His Ala Ala Arg Pro Gin Cys His Pro Ser Cys Val Ser Leu Pro Ser Ser Thr His Leu Thr Leu Leu Thr Leu Lys Lys Thr 100 105 Pro Gly Arg Ser Thr Gly Glu Pro Ser His Thr Pro His Trp Arg Gly 115 120 125 Gin Ala Leu Gin Gly Glu Ala Val Ser Ser His Leu His Gin Pro Phe 130 135 140 Pro Ser Gly Thr Gin Thr Cys Thr Ser Ser Cys Asp Arg Arg Gin Gin 145 150 Cys Asp Arg Asp Leu Pro Ser Ala Leu Leu Ser Pro His Leu Arg Cys 175 Ser Gin Pro Ala Thr Arg Gin Leu Giu Ser Ser 180 WO 96/29409 PCT/US96/04037 -74- INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 189 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..189 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: ATC CCG ATG AGA GAA TCG CCG CCC ACC AGG CCC TGC AGC ACC CCT Ile Pro Met Arg Glu Ser Pro Pro Thr Arg Pro Cys Ser Thr Pro 5 10

ATG

Met 1 WO 96/29409 WO 9629409PCTJUS96/04037 75 ACT TCC Thr Ser AAG AAC AGA GAA CCC AGA Lys Asn Arg Glu Pro Arg

ACG

Thr 25 GAA GCG AGG ATG AAG GCC TCAk Giu Ala Arg Met Lys Ala Ser GCC GTC Ala Val AGC ACC Ser Thr

CTC

Leu CTC CCC ATI CAA Leu Pro Ile Gin Thr 40 CGT TCA TCC CTC AAC CCT CTG CI'G Arg Ser Ser Leu Asn Pro Leu Leu 144 TGC ATO CIG CCC GCC Cys Met Leu Pro Gly 55 CGC AGT GTC ACC CIT CIT GTG TGA Arg Ser Val Thr Leu Leu Val 189 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHRACT'ERISTICS: LENGTH: 62 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOHTICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Met Ile Pro Met Arg Glu Ser Pro Pro Thy Ar; Pro Cys Ser Thr Pro 1 5 10 Thr Ser Lys Asn Arg Glu Pro Arg Thr Glu Ala Ar; Met Lys Ala Ser 25 Bf-rA QQC1Ajft'2-F WOf 96IQ~l 0409~OJ~~,,,.9.

76 L&U Ala Val Leu Leu Pro Ile Gin Thr Arg Ser Ser Leu Asn Pro Leu Leu 40 Ser Thr Cys Met Leu Pro Gly Arg Ser Val Tlir Leu Leu Val 55 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARA CTE RISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTEETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Ser Pro Ser Ser Asn Arg Ile Arg Asn Thr Ser Thx INFORMATION FOR SEQ ID WO 96/29409 77 Wi SEQUENCE CHARACTEISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAMNT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Pro Ser Ser Asn Arg Ile Arg Asn 1 INFORMATION FOR SEQ ID NO:41: SEQUENCE CH~AACERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide PCTIUS96/04037 (iii) HYPOTHETICAL: NO WO 96/29409 78 FRAGT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens PCTIUS96/04037 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Pro Ser Ser Asn Arg Ile ZArg Asn Thr INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTI CS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOT14ETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: Ser Ser Asn Arg Ile Arg Asn Thr Ser 1 WO 96/29409 WO 9629409PCTIUS96/04037 79 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHiARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNBSS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO FRAGET TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Ser Pro Ser Ser Asn Arg Ile Arg Asn Tbr INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA WO 96129409 WO 9629409PCTfUS96/04037 80 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: TCACCAAGT' OMACAGGAT CAGGAAT INFORMATION FOR SEQ ID Wi SEQUENCE CHRAACI7EISTICS: LEGTH-: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA.

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGM'ENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens WO 96/29409 -8 (xi) SEQUENCE DESCRIPTION: SEQ ID TCACCAAGTT CAAACAGGAT CAGGAATACC INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs PCTfUS96/04037 TYPE: nucleic acid STANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGME~NT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homno sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GTGTCTCCTT CGTCI'CTACT A INFORMATION FOR SEQ ID NO:47: SEQUENCE CH-ARACTERISTICS: WO 96/29409 PCT/US96/04037 82 LENGTH: 18 base pairs TYE: nucleic acid STRATNDEDNESS: single TOPOLODGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHTICAL: NO (iv) ANTI-SENSE: YES FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GGTGTGCCGA. TGACATCG INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTEISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOETICAL: NO WO 96/29409 WO 9629409PCT/US96/04037 83 (iv) ANTI-SENSE: YES FRAMENT TYPE: internal (vi) ORIGINA~L SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GAGGTATTCC TGATCCTG INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 13 base pairs TYPE: nucleic acid SWRANDEDNESS: single TOPOLOG: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homno sapiens WO 96/29409 -84- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: CAAACANGGA TCA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal PCT/US9604037 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NTATTCCTGA TCCT INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs WO 96/29409 WO 9629409PCT1US96/04037 85 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA.

(iii) HTYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: NTATTCCTGA. TCCI'G INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDN7\ (iii) HYPOTHETICAL: NO WO 96/29409 WO 9629409PCTJUS96/04037 86 (iv) ANTI-SENSE: NO FRAG1V,1NT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: NTATI'CCIGA TCCI'GT INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACITERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECUJLE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens WO 96/29409 87 (xi) SEQUNCE DESCRIPTION: SEQ ID NO:53: NTATJ7CCTGA TCCTGFI' INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHTICAL: NO (iv) ANTI-SENSE: YES FRAGMVENT TYPE: internal PCT1US96/04037 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: NCAAGTTCAA ACAG INFORMVATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs WO 96/29409 88 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES FRAGMEN.T TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens PCTIUS96/04037 (xi) SEQUENCE DESCIPTION: SEQ ID NCAAGTTCAA ACAGG INFORMATION FOR SEQ ID NO:56: SEQUENCE CARACTERISTICS: LENGTH: 16 base pairs TYPE nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO ~111 WO 96/29409 PCT/US96/04037 -89- (iv) ANTI-SENSE: YES FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: NCAAGTTCAA ACAGGA INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: NCAAGTTCAA ACAGGAT

Claims (2)

1. An isolated polypeptide comprising the amino acid sequencc of SEQ.LO.NO.40 or a functional variant thereof.

52. The isolated polypeptide of claim 1 wherein the isolated polypeptide comprises the amino acid sequence of SEQ.ID.NO.43. 3. The isolated polypeptide of claim 1 whezein the isolated polypeptide comprises an amino acid sequence selccted from the group consisting of SEQ.ID.NO.40 and SEQ.ID.NO.43. 4. The isolated polypecride of claim 1 wherein the isolated polypeptide consists of an amino acid sequence selected from the group consisting of SEQ.ID.INO.40 and SEQ.ID.NO.43. 5. An isolated nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide of claim 1, the polypeptide of claim 2, the polypeptide of claim 3 and the polypeptide of claim 4. 6. The isolated nucleic acid of claim 5 wherein the nucleic acid comprises SEQ.ID.NO.44. 7. An expression vector comprising the isolated nucleic acid of claim 5 operably linked to a promoter. S. The expression vector of claim 7 wherein the nucleic, acid comprises SEQ.[D.NO.44. 9. The expression vector of claims 7 or 8 further comprising a nucleic acid which codes for 1-LA-137. AMENDED ShEET -91 A host cell transfected or transformed with an expression vector selected from the group consisting of the expression vector of chlim 7, the expression vector of claim 8 and the expression vector of claim 9. 11. A host cell transfected or transformed with an expression vector selected from the group of the expression vector of claim 7 and the expression vector of claim 8, and wherein the host cell expresses HLA-B7. 12. A kit for detecting the presence of the expression of a tumor rejection antigen precursor comprising: a first primer selected from the group consisting of a nucleic acid consisting of any one of SEQ.ID.NOs.50-57, and a second primer constructed and arranged to selectively amplify together with the first primer a portion of ORF2 characteristic only of RAGE I genes. 13. A method for enriching selectively a population of T cells with cytolytic T cells specific for a RAGE tumor rejection antigen comprising: contacting an isolated population ofT cells with an agent presenting a complex of a RAGE tumor rejection antigen and HLA presenting molecule in an amount sufficient to selectively enrich said isolated population of T cells with said cytolytic T cells. 14. The method of claim 13 wherein the HLA presenting molecule is HLA-B7 and wherein the RAGE tumor rejection antigen is selected from the group consisting of: a peptide consisting of the amino acid sequence of SEQ.ID.NO.40 and a peptide consisting of the amino acid sequence of SEQ.ID.NO.43. A method for diagnosing a disorder characterized by expression of a RAGE tumor rejection antigen peptide, comprising: contacting a biological sample isolated from a subject with an agent that is specific for the RAGE tumor rejection antigen peptide, and determining the interaction between the agent and the RAGE tumor rejection antigen peptide as a determination of the disorder. AMENDED SHEET P:\OPER\FJH\54298-96.CLM 18/2/99 -92- 16. The method of claim 15 wherein the peptide is selected from the group consisting of: a peptide consisting of the amino acid sequence of SEQ. ID. NO. 40 and a peptide consisting of the amino acid sequence of SEQ. ID. NO.43. 17. A method for diagnosing a disorder characterized by expression of a RAGE tumor rejection antigen peptide which forms a complex with HLA-B7 molecules, comprising: contacting a biological sample isolated from a subject with an agent that binds the complex; and determining binding between the complex and the agent as a determination of the disorder. 18. The method of claim 17 wherein the peptide is selected from the group consisting of: a peptide consisting of the amino acid sequence of SEQ. ID. NO.40 and a peptide consisting of the amino acid sequence of SEQ. ID. NO.43. 0 19. The use of a polypeptide comprising the amino acid sequence of SEQ ID or a functional variant thereof for the manufacture of a medicament in the treatment of conditions requiring the selective enrichment in a subject of complexes of HLA-B7 and RAGE tumour rejection antigen. The use of claim 19 wherein the RAGE tumour rejection antigen is selected from the group consisting of: a peptide consisting of the amino acid sequence of SEQ. ID. NO.40 and a peptide 0* consisting of the amino acid sequence of SEQ. ID. NO.43. 21. The use of a composition comprising autologous cytolytic T cells specific for complexes of an HLA-B7 molecule and a RAGE tumour rejection antigen in the manufacture of a medicament. of a medicament. P:\OPER\EJH\54298-96.CLM 18/2/99 -93- 22. The use of claim 21 wherein the RAGE tumour rejection antigen is selected from the group consisting of: a peptide consisting of the amino acids of SEQ. ID. NO.40 and a peptide consisting of the amino acids of SEQ. ID. NO.43. 23. An isolated nucleic acid molecule selected from the group consisting of (a) nucleic acid molecules which hybridize under stringent conditions, to a molecule consisting of a nucleic acid sequence selected from the group consisting of: SEQ. ID. NO. 1; SEQ. ID. NO.4; SEQ. ID. NO.6; SEQ. ID. NO.10; SEQ. ID. NO.12; SEQ. ID. NO.13; SEQ. ID. NO.14; SEQ. ID. NO.15; SEQ. ID. NO.17; SEQ. ID. NO.23; and/or SEQ. ID. NO.35, and which codes for a tumor rejection antigen precursor, with the proviso that the isolated nucleic acid molecule does not code for a MAGE, GAGE, BAGE tumour rejection antigen precursor; nucleic acid molecules that differ from the nucleic acid molecules of in codon sequence due to the degeneracy of the genetic code; and complements of and 24. The isolated nucleic acid molecule of claim 23 wherein the isolated nucleic acid molecular is a cDNA molecule or an mRNA molecule. g* 25. The isolated nucleic acid molecule of claim 23 wherein the isolated nucleic acid molecule codes for a tumour rejection antigen precursor coded for by a molecule having a sequence selected from the group consisting of SEQ. ID. NO.1; SEQ. ID. NO.12; SEQ. ID. NO.13; and/or SEQ. ID. NO.14;. 26. The isolated nucleic acid molecule of claim 23 wherein the isolated nucleic acid molecule comprises a nucleic acid molecule which is a complement of a nucleic acid molecule selected from the group consisting of: nucleotides 204 to 326 of SEQ. ID. NO.1; nucleotides 313 to 399 of SEQ. ID. NO.1; nucleotides 444 to 665 of SEQ. ID. NO.1; nucleotides 273 to 449 of SEQ. ID. NO.12; nucleotides 217 to 276 of SEQ. ID. NO.12; nucleotides 185 to 247 of SEQ. ID. NO.13; and/or nucleotides 269 to 832 of SEQ. ID. NO.14. 27. An isolated nucleic acid molecule consisting of a nucleotide sequence selected P:\OPFER\EJH\54298-96.CLM WIM9~ -93A- from the group consisting of: SEQ. ID. NO.1; SEQ. ID. NO.4; SEQ. ID. NO.6; SEQ. ID. SEQ. ID. NO.12; SEQ. ID. NO.13; SEQ. ID. NO.14; SEQ. ID. NO.15; SEQ. ID. NO.17; SEQ. ID. NO.23; SEQ. ID. NO.35; and/or SEQ. ID. 4 4. S 4* S 4. 4 4* 4* S S *4e 4. S. 4.4y 9S 4* 4 04 44 5 4 :0 0 0oo -94- 28. An expression vector comprising the isolated nucleic acid molecule of claims 23, 24, 25, 26 or 27, operably linked to a promoter. 29. A host cell transfected or transformed with the expression vector of claim 28. An isolated nucleic acid consisting of a unique fragment of a sequence selected from the group consisting of: SEQ.ID.NO. 1; SEQ.ID.NO. 12; SEQ.ID.NO. 13; SEQ.ID.NO. 14; the complement of SEQ.ID.NO. 1; the complement of SEQ.ID.NO. 12; the complement of SEQ.ID.NO. 13; and/or the complement of SEQ.ID.NO. 14, wherein the unique fragment is 12, 13, 14, 15, 16, 17, 18, 19,20,21,22,23,24,25,26,27,28,29,30,31 and/or 32 nucleotides in length, provided that the isolated nucleic acid excludes sequences consisting of EMBL database accession number M91400. 31. An isolated nucleic acid consisting of a unique fragment of a sequence selected from the group consisting of: SEQ.ID.NO. 1; SEQ.ID.NO. 12; SEQ.ID.NO. 13; SEQ.ID.NO. 14; the complement of SEQ.ID.NO. 1; the complement of SEQ.ID.NO. 12; the complement of SEQ.ID.NO. 13; and/or the complement of SEQ.ID.NO. 14, wherein the fragment is between 200 and 2050 nucleotides in length. 32. The isolated nucleic acid of claim 30 wherein the unique fragment is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and/or 32 nucleotides in length. 33. A kit for detecting the presence of the expression of a tumor rejection antigen precursor comprising a pair of isolated nucleic acid molecules constructed and arranged to selectively amplify the isolated nucleic acid molecule of claim 23. 34. The kit of claim 33 wherein the pair of isolated nucleic acid molecules are PCR primers. 35. An isolated tumor rejection antigen precursor coded for by the nucleic acid molecule of claims 23, 24, 25, 26 or 27. AMENDED SHEET 36. A method for diagnosing a disorder characterized by expression of a RAGE tumor rejection antigen precursor which is processed to a RAGE derived tumor rejection antigen which forms a complex with HLA molecules, comprising: contacting a biological sample isolated from a subject with an agent that binds said complex, and determining binding between said complex and said agent as a determinant of said disorder. 37. A method for diagnosing a disorder characterized by expression of a RAGE tumor rejection antigen precursor coded for by a nucleic acid, comprising: contacting a biological sample isolated from a subject with an agent that is specific for said nucleic acid or an expression product thereof, wherein the nucleic acid is a nucleic acid as recited in claim 23, and determining the interaction between said agent and said nucleic acid or said expression product as a determination of said disorder. 38. The method of claim 37 wherein the agent comprises a nucleic acid molecule selected from the group ofmolecules consisting of: SEQ.ID.NO. 1; SEQ.1D.NO, 12; SEQ.ID.NO. 13; SEQ.ID.NO. 14; a unique fragment of SEQ.ID.NO. I; a unique fragment of SEQ.ID.NO. 12; a unique fragment of SEQ.ID.NO. 13; and a unique fragment of SEQ.ID.NO. 14, wherein the unique fragment is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and/or 32 nucleotides in length. 39. The use of an agent which enriches selectively in the subject the presence of complexes of HLA and tumor rejection antigen that is derived from a tumor rejection antigen precursor coded for by a molecule as described in claims 23, 24, 25, 26 or 27, in the manufacture of a medicament. The use of a composition comprising autologous cytolytic T cells wherein the cytolyticT cells are specific for complexes of an HLA molecule and a tumor rejection antigen that is derived from a tumor rejection antigen precursor coded for by a molecule as described in claims 23, 24, 25, 26 or 27, in the manufacture of a medicament. AMENDED SHET P:\OPER\EJff\54298-96.CLM 25/2/99 -96- 44. An isolated polypeptide according to any one of claims 1 to 6 or an expression vector according to any one of claims 7 to 9 or a host cell according to claim 10 or 11 or a kit according to claim 12 or a method according to any one of claims 13 to 18 or 36 to 40 or a use according to any one of claims 19 to 22 or an isolated nucleic acid molecule according to any one of claims 23 to 27 or 30 to 33 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 25th day of February, 1999 Ludwig Institute for Cancer Research AND Leiden University By DAVIES COLLISON CAVE Patent Attorneys for the Applicants O *S 4