WO1999037665A1 - Isolated, polypeptides which bind to hla-a29 molecules, nucleic acid, the molecules encoding these, and uses thereof - Google Patents

Abstract

Peptides which bind to HLA-A29 molecules are disclosed. These molecules satisfy motifs defined by SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25. Also described are minigenes which encode the peptides of the invention as well as their use.

Description

ISOLATED, POLYPEPTIDES WHICH BUND TO HLA-A29

MOLECULES, NUCLEIC ACID, THE MOLECULES

ENCODING THESE, AND USES THEREOF

RELATED APPLICATION

This application is a continuation-in-part of Serial No. 08/531 ,662 filed September 21 ,

1995, which is a continuation-in-part of copending application Serial No. 08/370,648, filed

January 10, 1995, which is a continuation in part of copending patent application Serial No.

08/250,162 filed on May 27, 1994, which is a continuation-in-part of Serial No. 08/096,039 filed July 22, 1993. All of these applications are incorporated by reference.

FDELD OF THE INVENTION

This invention relates to a nucleic acid molecule which codes for a tumor rejection

antigen precursor. More particularly, the invention concerns genes, whose tumor rejection

antigen precursor is processed, inter aha, into at least one tumor rejection antigen that is

presented by HLA-Cw6 molecules. The genes in question do not appear to be related to other known tumor rejection antigen precursor coding sequences. The invention also relates to

peptides presented by the HLA-Cw6 molecules, and uses thereof. Also a part of the inventions are peptides presented by HLA-A29 molecules, and uses thereof.

BACKGROUND AND PRIOR ART

The process by which the mammalian immune system recognizes and reacts to foreign

or alien materials is a complex one. An important facet of the system is the T lymphocyte, or "T cell" response. This response requires that T cells recognize and interact with

complexes of cell surface molecules, referred to as human leukocyte antigens ("HLAs"), or major histocompatibility complexes ("MHCs"), and peptides. The peptides are derived from

larger molecules which are processed by the cells which also present the HLA/MHC molecule.

See in this regard Male et al. , Advanced Immunology (J.P. Lipincott Company, 1987),

especially chapters 6-10. The interaction of T cells and HLA/peptide complexes is restricted,

requiring a T cell specific for a particular combination 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. This mechanism is involved in the immune system's response to foreign materials,

in autoimmune pathologies, and in responses to cellular abnormalities. Much work has focused on the mechanisms by which proteins are processed into the HLA binding peptides.

See, in this regard, Barinaga, Science 257: 880 (1992); Fremont et al., Science 257: 919

(1992); Matsumura et al. , Science 257: 927 (1992); Latron et al., Science 257: 964 (1992).

Also see Engelhard, Ann. Rev. Immunol. 12: 181-207 (1994).

The mechanism by which T cells recognize cellular abnormalities has also been

implicated in cancer. For example, in PCT application PCT/US92/04354, filed May 22,

1992, published on November 26, 1992, and incorporated by reference, a family of genes is

disclosed, which are processed into peptides which, in turn, are expressed on cell surfaces,

which can lead to lysis of the tumor cells by specific CTLs cytolytic T lymphocytes, or

"CTLs" hereafter. 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 Application Serial Number 807,043, filed December 12, 1991, now U.S. Patent

No. 5,342,774.

In U.S. Patent Application Serial Number 938,334, now U.S. Patent No. 5,405,940,

the disclosure of which is incorporated by reference, it is explained that the MAGE-1 gene

codes for a tumor rejection antigen precursor which is processed to nonapeptides which are

presented by the HLA-A1 molecule. The reference teaches that given the known specificity

of particular peptides for particular HLA molecules, one should expect a particular peptide to

bind to one HLA molecule, but not to 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.

In U.S. Patent Application Serial Number 008,446, filed January 22, 1993 and

incorporated by reference, the fact that the MAGE-1 expression product is processed to a

second TR is disclosed. This second TRA is presented by HLA-C clone 10 molecules. The

disclosure shows that a given TRAP can yield a plurality of TRAs.

U.S. Patent Application Serial Number 994,928, filed December 22, 1992, and

incorporated by reference herein teaches that tyrosinase, a molecule which is produced by

some normal cells (e.g. , melanocytes) , is processed in tumor cells to yield peptides presented

by HLA-A2 molecules. In U.S. Patent Application Serial Number 08/032,978, filed March 18, 1993, and

incorporated by reference in its entirety, 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 U.S. Patent Application Serial Number 08/079, 110, filed June 17, 1993 and

incorporated by reference herein, an unrelated tumor rejection antigen precursor, the so-called

"BAGE" precursor, is described. The BAGE precursor is not related to the MAGE family.

The work which is presented by the papers, patents, and patent applications cited supra

deals, in large part, with the MAGE family of genes, and the unrelated BAGE gene. It has

not been found, however, that additional tumor rejection antigen precursors are expressed by

cells. These tumor rejection antigen precursors are referred to as "GAGE" tumor rejection

antigen precursors. They do not show homology to either the MAGE family of genes or the

BAGE gene. Thus the present invention relates to genes encoding such TRAPs, the tumor

rejection antigen precursors themselves as well as applications of both.

Thus, another feature of the invention are peptides which are anywhere from 9 to 16

amino acids long, and comprise the sequence:

Xaa Tip Pro Xaa Xaa Xaa Xaa Tyr

(SEQ ID NO: 23)

where Xaa is any amino acid and Xaa ₂₎ means that 1 or 2 amino acids may be N-terminal

to the Tip residue. These peptides bind to, and/or are processed to peptides which bind to

HLA-A29 molecules.

The invention is elaborated upon further in the disclosure which follows. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 sets forth lysis studies using CTL clone 76/6.

Figure 2 shows tumor necrosis factor ("TNF") release assays obtained with various

transfectants and controls.

Figure 3 compares lysis induced by cytolytic T lymphocytes of clone CTL 76/6. Peptides of

varying length were tested, including SEQ ID NO: 4.

Figure 4 presents an alignment of the cDNAs of the six GAGE genes discussed herein. In the

figure, identical regions are surrounded by boxes. Translation initiation sites and stop codons

are also indicated. Primers, used in polymerase chain reaction as described in the examples,

are indicated by arrows.

Figure 5 sets forth the alignment of deduced amino acid sequences for the members of the

GAGE family. Identical regions are shown by boxes, and the antigenic peptide of SEQ ID

NO: 4, is shown.

Figure 6 shows the results obtained when each ofthe GAGE cDNAs was transfected into COS

cells, together with HLA-Cw6 cDNA. Twenty-four hours later, samples of CTL 76/6 were

added, and TNF release was measured after twenty-four hours. Figure 7 compares the stimulation of CTL 22/23 by COS-7 cells, transfected with HLA-A29 cDNA, a MAGE, BAGE or GAGE sequence, as shown. Control values are provided by

MZ2-MEL.43 and COS cells, as stimulators.

Figure 8 presents results obtained by ⁵¹Cr release studies, using various peptides including

SEQ ID NO: 22 and various peptides derived therefrom.

DETAILED DESCRTPTION OF PREFERRED EMBODIMENTS

Example 1 A melanoma cell line, MZ2-MEL was established from melanoma cells taken from

patent MZ2, using standard methodologies. This cell line is described, e.g., in PCT Application PCT/US92/04354, filed May 22, 1992, published November 26, 1992, and

incoφorated by reference in its entirety. Once the cell line was established, 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 patent

MZ2, and contacted to the irradiated melanoma cells. The mixture was observed for lysis of

the melanoma cells, which indicated that CTLs specific for a complex of peptide and HLA

molecule presented by the melanoma cells were present in the sample.

The lysis assay employed was a chromium release assay following Herin et a , Int.

J. Cancer 39: 390-396 (1987), the disclosure of which is incoφorated by reference. The

assay, however, is described herein. The target melanoma cells were grown in vitro, and then

resuspended at IO⁷ cells/ml in DMEM, supplemented with 10 mM HEPES and 30% FCS, and incubated for 45 minutes at 37°C with 200 μCi/ml of Na(⁵¹Cr)O₄. Labelled cells were washed

three times with DMEM, supplemented with 10 mM Hepes. These were then resuspended

in DMEM supplemented with lOmM Hepes and 10% FCS, after which 100 ul aliquots

containing 10³ cells, were distributed into 96 well microplates. Samples of PBLs were added

in 100 ul of the same medium, and assays were carried out in duplicate. Plates were

centrifuged for 4 minutes at lOOg, and incubated for four hours at 37°C in an 8 % CO₂

atmosphere.

Plates were centrifuged again, and 100 ul aliquots of supernatant were collected and

counted. Percentage of ⁵¹Cr release was calculated as follows:

% ⁵¹Cr release = (ER-SR1 x 100

(MR-SR)

where ER is observed, experimental ⁵¹Cr release, SR is spontaneous release measured by

incubating IO³ labeled cells in 200 ul of medium alone, and MR is maximum release, obtained

by adding 100 ul 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. The CTL

clone MZ2-CTL 76/6 was thus isolated. The clone is referred to as "76/6" hereafter.

The same method was used to test target K562 cells, as well as the melanoma cell line.

Figure 1 shows that this CTL clone recognizes and lyses the melanoma cell line, i.e. , MZ2-

MEL but not K562. The clone was then tested against other melanoma cell lines and

autologous EBV-transformed B cells in the same manner described supra. Figure 1 shows that

autologous B cells, transformed by Epstein Barr Virus ("EBV") were not lysed, and that while

MZ2-MEL 3.0 was lysed by CTL clone 76/6, the cell line MZ2-MEL.4F, a variant which

does not express antigen F, was not. Hence, the clone appears to be specific for this antigen. The results presented supra are inconclusive as to which HLA molecule presents the

TRA. The lysed cell line, i.e., MZ2-MEL, is known to express HLA-A1, HLA-A29, HLA-

B37, HLA-B44, HLA-Cw6, and HLA-C clone 10. In experiments not reported here but

which followed the protocol of this example, a subline of MZ2-MEL was tested, which had

lost expression of HLA molecules A29, B44, and C clone 10. The subline was lysed, thus

indicating that the presenting molecule should be one of Al , B37 or Cw6.

Example 2

Further studies were carried out to determine if 76/6 also produced tumor necrosis

factor ("TNF") when contacted with target cells. The method used was that described by

Traversari et a , Immunogenetics 35: 145-152 (1992), the disclosure of which is incoφorated

by reference. Briefly, samples of the CTL line were combined with samples of a target cell

of interest in culture medium. After 24 hours, supernatant from the cultures was removed,

and then tested on TNF-sensitive WEHI cells. Cell line MZ2-MEL.43, a subclone of the

MZ2-MEL cell line discussed supra as well as in the cited references, gave an extremely

strong response, and was used in the following experiments.

Example 3

The results from Example 2 indicated that MZ2-MEL.43 presented the target antigen

of interest. As such, it was used as a source of total mRNA to prepare a cDNA library.

Total RNA was isolated from the cell line. The mRNA was isolated using an oligo-dT

binding kit, following well recognized techniques. Once the mRNA was secured, it was

transcribed into cDNA, via reverse transcription, using an oligo dT primer containing a NotI site, followed by second strand synthesis. The cDNA was then ligated to a BstXI adaptor, digested with NotI, size fractionated by a Sephacryl S-500 HR column, and then cloned, undirectionaUy, into the BstXI and NotI sites of pcDNA I/Amp. The recombinant plasmid

was then electroporated into DH5α E. coli bacteria. A total of 1500 pools of 100 recombinant

bacteria were seeded in microwells. Each contained about 100 cDNAs, because nearly all

bacteria contained an insert.

Each pool was amplified to saturation and plasmid DNA was extracted by alkaline lysis

and potassium acetate precipitation, without phenol extraction.

Example 4

Following preparation of the library described in Example 3, the cDNA was

transfected into eukaryotic cells. The transfections, described herein, were carried out in

dupUcate. Samples of COS-7 ceUs were seeded, at 15,000 ceUs/weU into tissue culture flat

bottom microwells, in Dulbecco's modified Eagles Medium ("DMEM") supplemented with

10 % fetal calf serum . The ceUs were incubated overnight at 37 ° C , medium was removed and then replaced by 50 μl/weU of DMEM medium containing 10 % Nu serum, 400 μg/ml DEAE-

dextran, and 100 μM chloroquine, plus 100 ng of the plasmids. As was indicated supra, the

lysis studies did not establish which HLA molecule presented the antigen. As a result, cDNA

for each of the HLA molecules which could present the antigen (Al, B37, Cw6) was used,

separately, to cotransfect the cells. Specifically, one of 28 ng of the gene encoding HLA-A1 ,

cloned into pCD-SRα, 50 ng of cDNA for HLA-B37 in pcDNA I/Amp, or 75 ng of cDNA

for HLA-Cw6 in pcDNAI-Amp, using the same protocol as were used for transfection with

the Ubrary. Transfection was carried out in duplicate wells, but only 500 pools of the HLA-Cw6

transfectants could be tested in single weUs. Following four hours of incubation at 37° C, the

medium was removed, and replaced by 50 μl of PBS containing 10% DMSO. This medium

was removed after two minutes and replaced by 200 μl of DMEM supplemented with 10%

FCS.

Following this change in medium, COS cells were incubated for 24-48 hours at 37°C.

Medium was then discarded, and 1000-3000 cells of CTL clone 76/6 were added, in 100 μl

of Iscove's medium containing 10% pooled human serum supplemented with 20-30 U/ml of

recombinant IL-2. Supernatant was removed after 24 hours, and TNF content was determined

in an assay on WEHI ceUs, as described by Traversari et a , Immunogenetics 35: 145-152

(1992), the disclosure of which is incoφorated by reference.

The 1500 pools transfected with HLA-A1 , and the 1500 pools transfected with HLA-

B37 stimulated TNF release to a concentration of 15-20 pg/ml, or 2-6 pg/ml, respectively.

Most of the HLA-Cw6 transfectants yielded 3-20 pg/ml, except for one pool, which yielded

more than 60 pg/ml. This pool was selected for further work.

Example 5

The bacteria of the selected pool were cloned, and 600 clones were tested. Plasmid

DNA was extracted therefrom, transfected into a new sample of COS ceUs in the same manner

as described supra, and the ceUs were again tested for stimulation of CTL clone 76/6. Ninety-

four positive clones were found. One of these, referred to as cDNA clone 2D6, was tested

further. In a comparative test COS cells were transfected with cDNA clone 2D6 and the

HLA-Cw6 cDNA, HLA-Cw6 cDNA alone, or cDNA 2D6 alone. Control ceU lines MZ2-

10 MEL F and MZ2-MEL F⁺ were also used. TNF release into CTL supernatant was measured

by testing it on WEHI ceUs, as referred to supra. The number of surviving WEHI cells was

measured by optical density after incubation of the ceUs with MTT. Figure 2 shows that the

COS ceUs transfected with HLA-Cw6 and cDNA-2D6, and the cell line MZ2-MEL F⁺

stimulated TNF release from CTL clone 76/6, indicating that HLA-Cw6 presented the subject

TRA.

Example 6

The cDNA 2D6 was sequenced following art known techniques. A sequence search

revealed that the plasmid insert showed no homology to known genes or proteins. SEQ ID

NO: 1 presents cDNA nucleotide information for the identified gene, referred to hereafter as

" GAGE" . A putative open reading frame is located at bases 51 -467 of the molecule. The first

two bases of this sequence are from the vector carrying the cDNA sequence, and are thus not

part of the cDNA itself.

Example 7

Following sequencing of the cDNA, as per Example 6, experiments were carried out

to determine if ceUs of normal tissues expressed the gene. To determine this, Northern

blotting was carried out on tissues and tumor cell lines, as indicated below. The blotting

experiments used cDNA for the complete sequence of SEQ ID NO: 1. PCT was then used

to confirm the results.

11 Table 1. Expression of gene GAGE

Normal tissues

PHA activated T ceUs

CTL clone 82/30

Liver

Muscle

Lung Brain

Kidney

Placenta

Heart

Skin Testis +

Tumor cell lines

Melanoma 7/16 Lung carcinoma 1/6

Sarcoma 0/1

Thyroid medullary carcinoma 0/1

Tumor samples

Melanoma 1/1

Example 8

DetaUed analysis of normal tissues and tumors was carried out by applying polymerase chain reaction ("PCR") and the GAGE gene information described supra.

First, total RNA was taken from the particular sample, using art recognized techniques.

This was used to prepare cDNA. The protocol used to make the cDNA involved combining

4 ul of reverse transcriptase buffer 5x, 1 ul of each dNTP, (10 mM), 2 ul of dithiothreitol

(100 mM), 2 ul of dT-15 primer (20 um), 0.5 ul of RNasin (40 units/ul), and 1 ul of MoMLV

reverse transcriptase (200 units/ul). Next, 6.5 ul of template RNA (1 ug/3.25 ul water, or 2

ug total template RNA) was added. The total volume of the mixture was 20 ul. This was

12 mixed and incubated an*2°C for 60 minutes, after which it was chiUeu ori ice. A total Of 80

ul of water was then added, to 100 ul total. This mixture was stored at -20°C until used in

PCR.

To carry out PCR, the primers

5 -AGA CGC TAC GTA GAG CCT-3 '

(sense)

and

5 ' CCA TCA GGA CCA TCT TCA-3'

(antisense)

SEQ ID NOS: 2 and 3, respectively, were used. The reagents included 30.5 ul water, 5 ul

of PCR buffer lOx, 1 ul of each dNTP (10 uM), 2.5 ul of each primer (20 uM), and 0.5 ul

of polymerizing enzyme Dynazyme (2 units/ul). The total volume was 45 ul. A total of 5 ul of cDNA was added (this corresponded to 100 ng total RNA). The mixture was combined,

and layered with one drop of mineral oU. The mixture was transferred to a thermocycler

block, preheated to 94°C, and amplification was carried out for 30 cycles, each cycle

consisting of the foUowing:

first denaturation: 94° C, 4 min.

denaturation: 94° C, 1 min.

annealing: 55 °C, 2 min.

extension: 72 °C, 3 min.

final extension: 72°C, 15 min.

FoUowing the cycling, 10 ul aliquots were run on a 1.5 % agarose gel, stained with ethidium

bromide.

cDNA ampUfied using the primers set forth supra yields a 238 base pair fragment.

There is no ampUfication of contaminating genomic DNA, if present.

13 The results are presented in Table 2, which follows. They confirm that the only normal tissue which expresses GAGE is testis, whereas a number of tumors, including

melanoma, lung, breast, larynx, pharynx, sarcoma, testicular seminoma, bladder and colon

express the gene. Thus, any one of these tumors can be assayed for expression of the GAGE

gene.

14 NORMAL TISSUES

Heart -

Brain -

Liver -

Lung -

Kidney -

Spleen

Lymphocytes -

Bone marrow -

Skin -

Naevus -

Melanocytes -

Fibroblasts -

Prostate -

Testis +

Ovary -

Breast -

Adrenals -

Muscle -

Placenta -

UmbiUcal cord -

TUMORS

CeU Lines Tumor Samples

Melanoma 40/63 46/146 (32%)

Lung cancer

Epidermoid carcinoma 10/41 (24%)

Adenocarcinoma 4/18

Small Cell Lung Cancer 6/23 0/2

Breast cancer 15/146 (10%)

Head and neck tumor

Larynx 6/15 (40%)

Pharynx 3/13

Sarcoma 1/4 6/18 (33 %)

Testicular seminoma 6/6 (100%)

Bladder cancer 5/37 (14%)

Prostate cancer 2/20

Colon cancer 5/13 0/38

Renal cancer 0/6 0/45

Leukemia 3/6 0/19

15 Example 9

The identification of the nucleic acid molecule referred to in the prior examples led to

further work directed to determination of tumor rejection antigens presented by HLA-Cw6

molecules, and derived from the GAGE gene. The complete cDNA of GAGE in expression vector pcDNA/Amp was digested with

restriction endonucleases NotI and SpEH, and then with exonuclease JH foUowing supplier's

instruction (Erase-a-base System, Promega). This treatment generated a series of progressive

deletions, staring at the 3' end.

The deletion products were Ugated back into pcDNAI/AMP, and then electroporated

into E. coU strain DH5 'IQ, using well known techniques. The transformants were selected with ampicillin (50 micrograms/ml).

Plasmid DNA was extracted from each recombinant clone and was then transfected into

COS-7 cells, together with a vector which coded for HLA-Cw6. The protocols used foUow

the protocols described above.

The transfectants were then tested in the TNF release assay. This permitted separation

of positive and negative clones. AU the negative clones showed a deletion ofthe entire GAGE

sequence. The smallest positive clone contained the first 170 nucleotides of SEQ ID NO: 1.

The analysis of this sequence, supra, notes that the open reading frame starts at nucleotide 51.

Thus, this fragment contains a sequence which encodes the first 40 amino acids of the GAGE

TRAP.

16 Example 10

Additional experiments were then carried out to define the region encoding the TRA

peptide more precisely. Polymerase chain reaction ("PCR") amplification was used to do this.

Two primers were synthesized. The first primer was a 22-mer complementary to a

sequence within the plasmid vector pcDNAI/Amp located upstream of a BamHI site. The

second primer was a 29-mer containing at the 3' end nucleotides 102-119 of SEQ ID NO: 1 ,

and at the 5' end an extension of 11 nucleotides containing an Xbal restriction site.

Following amplification, the PCR product was digested by BamHI and Xbal, and

cloned into the BamHI-Xbal sites of plasmid pcDNA-3. The recombinant colonies were

cotransfected into COS-7 cells with cDNA encoding HLA-Cw6, in accordance with Example

4, and a TNF release assay, also as described supra, was carried out, using CTL 76/6.

TNF release was observed, indicating that the "minigene" was processed to a TRA.

The minigene, i.e. , nucleotides 1-119 of SEQ ID NO: 1 , the coding region of which runs

from nucleotides 51-119, encoded the first 23 amino acids of the cDNA of SEQ ID NO: 1.

This information served as the basis for the next set of experiments.

Example 11

Two peptides were synthesized, based upon the first 23 amino acids of SEQ ID NO:

1. These were:

Met Ser Tφ Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg

(SEQ ID NO: 12)

and

Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val Glu Pro Pro Glu Met He

(SEQ ID NO: 13)

17 Each peptide was pulsed into COS-7 ceUs previously transfected with HLA-Cw6 cDNA, and

combined with CTL 76/6 to determine if TNF release would be induced. Peptides (20 ug/ml)

were added to COS-7 cells which had been transfected with the HLA-Cw6 cDNA twenty-four

hours previously After incubation at 37°C for 90 minutes, medium was discarded, and 3000

CTLs were added in 100 microliters of medium, containing 25 units/ml of IL-2. Eighteen

hours later, TNF content of supernatant was tested via determining toxicity on WEHI- 164- 13

ceUs. The second peptide (SEQ ID NO: 13) was found to induce more than 30 pg/ml of TNF,

whUe the first peptide (SEQ ID NO: 12) was found to induce less than 10 pg/ml of TNF. The

second peptide was used for further experiments.

Example 12

Various peptides based upon SEQ ID NO: 13 were synthesized, and tested, some of

which are presented below. To carry out these tests ⁵¹Cr labeUed LB33-EBV ceUs, which are

HLA-Cw6 positive, were incubated with one of the foUowing peptides:

Tyr Arg Pro Arg Pro Arg Arg Tyr

(SEQ ID NO: 4)

Thr Tyr Arg Pro Arg Pro Arg Arg Tyr

(SEQ ID NO: 5)

Thr Arg Pro Arg Pro Arg Arg Tyr Val

(SEQ ID NO: 6)

Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val

(SEQ ID NO: 7)

Arg Pro Arg Pro Arg Arg Tyr Val Glu

(SEQ ID NO: 8)

Met Ser Tφ Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg

(SEQ ID NO: 12)

18 The peptide concentration varied, as indicated in figure 3, and the ratio of CTL: LB33-EBV

("effector: target ratio"), was 10:1. ⁵¹Cr release was determined after four hours of incubation

at 37°C. Levels of lysis for positive ("F⁺", MZ2-MEL.3.1), and negative ("F"; MZ2-

MEL.2.2.5) control ceUs are indicated, in figure 3.

It was found, quite suφrisingly that the octamer of SEQ ID NO: 4 was the best

peptide, and appeared to be the tumor rejection antigen. This is the first time an octamer has

been reported as being involved in presentation by a human MHC molecule. There is some

precedent for a murine system, as reported by Engelhard, supra, at 199, for H-2K^b and H-2K^K

molecules. The nonamers of SEQ ID NO: 5 and SEQ ID NO: 6 also induced CTL lysis albeit

to a lesser extent than the octamer of SEQ ID NO: 4.

In results not reported here, a second CTL was tested (CTL 82/31). This CTL was

known to lyse ceUs presenting

MZ2-F. It, too, lysed HLA-Cw6 positive ceUs following pulsing with the peptide of SEQ ID

NO: 4.

Example 13

To find out whether the GAGE DNA set forth supra was unique, a cDNA Ubrary made

with RNA from MZ2-MEL.43 (the same Ubrary that was used for the cloning of GAGE) was

hybridized with a probe derived from the GAGE cDNA. The probe was a PCR fragment of

308 base pairs between positions 20 and 328 of SEQ ID NO: 1. Twenty positive cDNAs were

obtained. Six of them were entirely sequenced. They were aU highly related to the GAGE

sequence, but they were slightly different from it. Two of the six clones were identical to

each other, but aU the others differed from each other. Thus, five new sequences different

19 from but highly related to GAGE were identified. They are caUed GAGE-2, 3, 4, 5 and 6

(Figure 4) and are presented as SEQ ID NOS: 14-18, respectively. The fourteen other clones

were partiaUy sequenced at the 5' end and their sequence corresponded to one of the six

GAGE cDNAs.

The major difference between these cDNAs and GAGE-1 is the absence of a stretch

of 143 bases located at position 379 to 521 of the GAGE sequence of SEQ ID NO: 1. The

rest of the sequences shows mismatches only at 19 different positions, with the exception of

GAGE-3 whose 5 ' end is totally different from the other GAGE for the first 112 bases. This

region of the GAGE-3 cDNA contains a long repeat and a haiφin structure.

The deduced GAGE-1 protein corresponding to a tumor rejection antigen precursor is

about 20 amino acids longer than the 5 other proteins, whose last seven residues also differ

from the homologous residues of GAGE-1 (Figure 5). The rest ofthe protein sequences show

only 10 mismatches. One of these is in the region corresponding to the antigenic peptide of

SEQ ID NO: 4. The sequence of the peptide is modified in GAGE-3, 4 5 and 6 so that

position 2 is now W instead of R.

Example 14

To assess whether the change at position 2 affected the antigenicity of the peptide,

cDNA of the 6 GAGE cDNAs were individuaUy transfected into COS ceUs together with the

cDNA of HLA-Cw6, and the transfectants were tested for recognition by CTL 76/6 as

described, supra. Only GAGE-1 and GAGE-2 transfected ceUs were recognized, showing that

the modified peptide encoded by GAGE-3, 4, 5 and 6 was not antigenic in the context of this

experiment. Sequence analysis of the 5' end of the 14 other clones mentioned supra showed

20 that 7 of them contained the sequence encoding the antigenic peptide, and thus probably corresponded to either GAGE-1 or GAGE-2.

Example 15

The PCR primers used supra to test the expression of GAGE in tumor samples do not discriminate between GAGE-1 or 2 and the four other GAGE cDNAs that do not encode

antigen MZ2F. A new set of primers was prepared which specificaUy amplifies GAGE-1 and

2, and not GAGE-3, 4, 5 and 6. These primers are:

VDE44 5 '-GAC CAA GAC GCT ACG TAG-3 ' (SEQ ID NO: 9) VDE24 5'-CCA TCA GGA CCA TCT TCA-3' (SEQ ID NO: 10)

These primers were used as described, supra, in a RT-PCR reaction using a polymerase

enzyme in the foUowing temperature conditions:

40 min at 94 °C 30 cycles with 1 min at 94° C

2 min at 56°C

3 min at 72°C 15 min at 72°C

The results of this analysis are set forth in Table 3.

21 Table 3

Expression of GAGE genes by tumor samples and tumor cell lines

Histological type Number of GAGE positive tumors

All GAGE genes* GAGE-1 and 2**

Tumor samples

Melanomas primary lesions 5/39 5/39 (13%) metastases 47/132 36/131 (27%)

Sarcomas 6/20 6/20 (30%)

Lung carcinoma NSCLC 14/65 12/64 (19%)

Head and neck squamous cell carcinomas 13/55 10/54 (19%)

Prostatic carcinomas 2/20 2/20

Mammary carcinomas 18/162 14/162 (9%)

Bladder carcinomas superficial 1/20 1/20 infiltrating 5/26 3/26

Testicular seminomas 6/6

5/6

Colorectal carcinomas 0/43

Leukemias and lymphonas 0/25

Renal carcinomas 0/46

Tumor cell lines

Melanomas 45/74 40/74 (54%) Sarcomas 1/4 1/4 Lung carcinomas SCLC 7/24 7/24 (29%) NSCLC 1/2 1/2 Mesotheliomas 5/19 5/19 (26%) Head and neck squamous cell carcinomas 0/2 Mammary carcinomas 1/4 0/4 Bladder carcinomas 0/3 Colon carcinomas 5/13 5/13 Leukemias 3/6 1/6 Lymphomas 0/6 Renal carcinomas

0/6

^♦Expression of GAGE was tested by RT-PCR on total RNA with primers VDE-18 and VDE-24, detecting all GAGE genes. No PCR product was observed when these primers were assayed on DNA from MZ2-MEL.

**Expression of GAGE-1 and 2 was tested by RT-PCR on total RNA with primers VDE-44 and VDE-24, which distinguish GAGE-1 and 2 from the four other GAGE genes. No PCR product was observed when these primers were assayed on DNA from MZ2-MEL.

22 In further work, new primers were designed which amplified aU GAGE genes, to make sure that there was no expression of any of them in normal tissues. These primers are

VDE43 5'-GCG GCC CGA GCA GTT CA-3' (SEQ ID NO: 11)

VDE24 5'-CCA TCA GGA CCA TCT TCA-3 (SEQ ID NO: 10)

These were used exactly as for the PCR using the VDE44 and VDE24 primers. The results

are shown in Table 4. They confirm that the normal tissues are negative, except for testis.

23 Table 4

Expression of GAGE genes in normal adult and fetal tissues

GAGE Adult tissues expression*

Adrenal gland Benign naevus Bone marrow Brain Breast

Cerebellum Colon Heart Kidney Liver

Lung

Melanocytes Muscle Ovary Prostate

Skin

Splenocytes Stomach Testis Thymocytes

Urinal bladder Uterus Placenta Umbilical cord

Fetal tissues*

Fibroblasts Brain

Liver Spleen Thymus Testis

^♦Expression of GAGE was tested by RT-PCR amplification on total

RNA with primers VDE43 and VDE24 detecting all GAGE genes (Figure 7) . Absence of PCR product is indicated by - and presence by +.

No PCR product was observed when these primers were assayed on DNA from MZ2-MEL.

*Fetal tissues derive from fetuses older than 20 weeks.

24 Example 16

In work not reported here, it had been ascertained that cytolytic T ceU clone CTL

22/23 (Van den Eynde, et al. , Int. J. Cancer 44: 634-640 (1989), incoφorated by reference)

did not recognize melanoma cell MZ2-MEL.3.1. This melanoma cell line was reported by

Van der Bruggen, et al. , Eur. J. Immunol. 24: 2134-2140 (1994), to have lost expression of

MHC molecules HLA-A29, HLA-B24, and HLA-Cw^*1601. Studies were undertaken to

determine if transfection with one of these MHC molecules could render the line sensitive to

CTL 22/23. HLA-A29 was the first molecule tested. To do so, poly A⁺ RNA was extracted

from HLA-A29⁺ cell Une MZ2-MEL.43, using a commercially available extraction kit, and

foUowing the manufacturer's instructions. The mRNA was then converted to cDNA, using

standard methodologies, size fractionated, and then inserted unidirectionally, into the BstXI

and NotI sites of plasmid pcDNA-I/Amp. The plasmids were electroplated into E. coU strain

DH5α5'IQ, and selected with ampiciUin (50 μg/ml). The bacteria were plated onto

nitroceUulose filters, and dupUcated. The filters were prepared, and hybridized overnight in

6xSSC/0.1 % SDS/lx Denhardt's solution at 40°C, using ³²P labelled probe:

5 '-ACTCCATGAGGTATTTC-3 '

(SEQ ID NO: 19)

The probe is a sequence which surrounds the start codon of most HLA sequences.

The filters were washed twice, at room temperature for 5 minutes each time in 6xSSC,

and twice in 6xSSC at 43 °C. Positive sequences were then screened with probe:

5 '-TTTCACCACATCCGTGT-3 '

(SEQ ID NO: 20)

25 which had been labelled with ³²P. This sequence is specific for HLA-A29, as determined by

reference to the Kabat Database of sequences and proteins of immunological interest,

incoφorated by reference. This database is avaUable at the NCBI (USA), or on Web Solte

(Internet) WWW . NCBI . NLM . NJH . GO V . The filters were washed twice at room temperature

for 5 minutes each time, at 6xSSC, followed by two washes, at 6xSSC (5 minutes per wash),

at 42°C.

Example 17

Once positive HLA-A29 clones were isolated, these were transfected into COS-7 using

the DEAE-dextran chloroquine method out supra. In brief, 1.5 x IO⁴ COS-7 ceUs were treated

with 50ng of plasmid pcDNA-I/Amp containing HLA-A29, and 100 ng of cDNA containing

cDNA for one of the GAGE sequences mentioned supra, or one of the prior art MAGE or

BAGE sequences in plasmid pcDNAα-I/Amp or pcDSR-α, respectively. The transfectants

were then incubated for 24 hours at 37° C.

The transfectants were then tested for their abiUty to stimulate TNF production by

CTLs, using the assay explained at the end of example, 4, supra.

Figure 7, which presents the results of this study, shows that high levels of TNF

production were achieved using any of GAGE-3, 4, 5 or 6 and HLA-A29 as transfectants.

GAGE-1 and GAGE-2, in contrast, do not stimulate CTL clone 22/23, thus leading to the

conclusion that GAGE 3, 4, 5 and 6 are processed to an antigen or antigens presented by

HLA-A29 molecules and recognized by CTL 22/23.

26 Example 18

The fact that GAGE-3 , 4, 5 and 6 were processed to peptides presented by HLA- A29 +

ceUs, while GAGE-1 and GAGE-2 were not, suggested examination ofthe deduced amino acid

sequences for those common to GAGE 3, 4, 5 and 6 and absent from GAGE-1 and GAGE-2.

The sequence:

Arg Ser Thr Tyr Tyr Tφ Pro Arg Pro Arg Arg Tyr Val Gin

(SEQ ID NO: 21)

was identified. The peptide was synthesized, lyophilized, and then dissolved in 1 volume

DMSO, 9 volumes of 10 mM acetic acid in water. This methodology was used for the other

peptides synthesized, discussed infra.

The peptide (SEQ ID NO: 21) was tested in a ⁵¹Cr release experiment, foUowing the

method described supra.

It was found that this peptide did provoke lysis. Successive deletions were prepared,

and tested for their abiUty to provoke lysis, again using the ⁵¹Cr lytic assay. This work is

depicted in Figure 8. It was found that the shortest peptide to provoke lysis was

Tyr Tyr Tφ Pro Arg Pro Arg Arg Tyr

(SEQ ID NO: 22), which is common to all of GAGE-3 through 6. SpecificaUy, amino acids

10-18 of GAGE-3, and amino acids 9-17 of GAGE-4, 5 and 6 correspond to this peptide.

The members of the peptide famUy shown in Figure 8, and represented, e.g. , by SEQ

ID NOS: 21 and 22, do not accord with the data presented by Toubert, et al. , "HLA-A29

Peptide Binding Motif", Abstract No. 4183, Ninth International Congress of Immunology,

July 23-29, 1995, San Francisco, CA, incoφorated by reference. According to Toubert, et

27 al. , at the least a Phe residue is required at the third position of any peptide which binds to

HLA-A29. As is shown herein, such is not the case.

The foregoing examples show the isolation of nucleic acid molecules which code for

tumor rejection antigen precursors and tumor rejection antigens. These molecules, however, are not homologous with any of the previously disclosed MAGE and BAGE coding sequences described in the references set forth supra. Hence, one aspect of the invention is an isolated

nucleic acid molecule which comprises the nucleotide sequences set forth in any of SEQ ID

NOS: 1-6 as weU as fragments thereof, such as nucleotides 1-170, and 51-170 of SEQ ID NO:

1 , or any other fragment which is processed to a tumor rejection antigen. The sequences of

SEQ ID NOS: 1-6 are neither MAGE nor BAGE coding sequences, as will be seen by

comparing those to the sequence of any of these genes as described in the cited references.

Also a part ofthe invention are those nucleic acid molecules which also code for a non-MAGE

and non-BAGE tumor rejection antigen precursor but which hybridize to a nucleic acid

molecule containing the described nucleotide sequence of SEQ ID NO: 1 , under stringent

conditions. The term "stringent conditions" as used herein refers to parameters with which

the art is famihar. More specifically, stringent conditions, as used herein, refers to

hybridization in 1M NaCl, 1 % SDS, and 10% dextran sulfate for 18 hours at 65 °C. This is

foUowed by two washes of the filter at room temperature for 5 minutes, in 2xSSC, and one

wash for 30 minutes in 2xSSC, 0.1 % SDS, at 65°C. There are other conditions, reagents,

and so forth which can be used, which result in the same or higher degree of stringency. The

skiUed artisan wUl be famiUar with such conditions and, thus, they are not given here.

It will also be seen from the examples that the invention embraces the use of the

sequences in expression vectors, as well as to transform or transfect host ceUs and ceU lines,

28 be these prokaryotic (e.g. , R_coli), or eukaryotic (e.g. , CHO or COS ceUs). The expression vectors require that the pertinent sequence, i.e., those described supra, be operably linked to a promoter. As it has been found that both of human leukocyte antigens HLA-Cw6 and HLA-

A29 present tumor rejection antigens derived from these genes, the expression vector may also

include a nucleic acid molecule coding for one of HLA-Cw6 or HLA-A29. In a situation

where the vector contains both coding sequences, it can be used to transfect a ceU which does

not normally express either one. The tumor rejection antigen precursor coding sequence may

be used alone, when, e.g. , the host cell already expresses one or both of HLA-Cw6 and HLA-

A29. Of course, there is no Umit on the particular host cell which can be used. As the

vectors which contain the two coding sequences may be used in HLA-A29 or HLA-Cw6 presenting cells if desired, and the gene for tumor rejection antigen precursor can be used in

host cells which do not express HLA-A29 or HLA-Cw6.

The invention also embraces so caUed expression kits, which aUow the artisan to

prepare a desired expression vector or vectors. Such expression kits include at least separate

portions of each of the previously discussed coding sequences. Other components may be

added, as desired, as long as the previously mentioned sequences, which are required, are

included.

To distinguish the nucleic acid molecules and the TRAPs of the invention from the

previously described MAGE and BAGE materials, the invention shaU be referred to as the

GAGE family of genes and TRAPs. Hence, whenever "GAGE" is used herein, it refers to

the tumor rejection antigen precursors coded for by the previously described sequences.

"GAGE coding molecule" and simUar terms are used to describe the nucleic acid molecules

themselves.

29 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 such as melanoma,

characterized by expression of the TRAP, or presentation of the tumor rejection antigen.

These methods involve determining expression of the TRAP gene, and/or TRAs derived

therefrom , such as a TRA presented by HLA-Cw6 or HLA- A29. In the former situation , such

determinations can be carried out via any standard nucleic acid determination assay, including the polymerase chain reaction, or assaying with labelled 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. To carry out the assay, it is preferred to make sure that testis ceUs are

not present, as these normaUy express GAGE. This is not essential, however, as one can

routinely differentiate between testis and other ceU types. Also, it is practicaUy impossible

to have testis cells present in non-testicular sample.

The isolation of the TRAP gene also makes it possible to isolate the TRAP molecule

itself, especially TRAP molecules containing the amino acid sequence coded for by any of

SEQ ID NOs: 2-6. These isolated molecules when presented as the TRA, or as complexes of

TRA and HLA, such as HLA-Cw6 or HLA-A29, may be combined with materials such as

adjuvants to produce vaccines useful in treating disorders characterized by expression of the TRAP molecule.

Exemplary adjuvants include Freund's complete and incomplete adjuvant, kiUed R

pertussis organisms, "BCG", or BacUle Calmente-Guerin, Al(OH)₃, muramyl dipeptide and

its derivatives, which may be emulsified in metabolizable oUs, such as squalene,

monophosphoryl lipid A (MPL), keyhole limpet hemocyanin (KLH), saponin extracts such

30 as QA-7, QA-19, and QA-21 (also referred to as QS-21), these having been described in U.S.

Patent No. 5,057,540 to Kensil, et al. , incoφorated by reference, MTP-MF59, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), the cationic

amphiphile DOTMA, the neutral phospholipids such as DOPE, and combinations of these. This listing is by no means comprehensive, and the artisan of ordinary skill wUl be able to augment this Usting. AU additional adjuvants are encompassed herein.

In addition, vaccines can be prepared from ceUs which present the TRA/HLA

complexes on their surface, such as non-proliferative cancer ceUs, non-proUferative

transfectants, et cetera. In all cases where ceUs are used as a vaccine, these can be ceUs

transfected with coding sequences for one or both of the components necessary to provide a

CTL response, or be ceUs which express both molecules without transfection. Further, the

TRAP molecule, its associated TRAs, as weU as complexes of TRA and HLA, may be used

to produce antibodies, using standard techniques weU known to the art.

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, melanoma

in particular. Melanoma is well known as a cancer of pigment producing ceUs.

As indicate, supra, tumor rejection antigens, such as the one presented in SEQ ID NO:

4, are also part of the invention. Also a part of the invention are polypeptides, such as

molecules containing from 8 to 16 amino acids, where the polypeptides contain the amino acid

sequence set forth in SEQ ID NO: 4. As the examples indicate, those peptides which are

longer than the octamer of SEQ ID NO: 4 are processed into the tumor rejection antigen of

SEQ ID NO: 4 by the HLA-Cw6 presenting cancer ceUs, and presented thereby. The

presentation leads to lysis by cytolytic T lymphocytes present in a body fluid sample contacted

31 to the ceUs presenting the complex. Similarly, the peptides longer than SEQ ID NO: 22, such

as SEQ ID NO: 21 , are processed to the appropriate TRA, and are presented by cancer cells,

such as HLA-A29 positive ceUs.

Thus, another feature of the invention are peptides which are anywhere from 9 to 16

amino acids long, and comprise the sequence:

Xaa Xaa Tφ Pro Xaa Xaa Xaa Xaa Tyr

(SEQ ID NO: 23)

or

Xaa Xaa Tφ Xaa Arg Xaa Xaa Xaa Tyr

(SEQ ID NO: 24)

or

Xaa Xaa Tφ Xaa Xaa Xaa Xaa Arg Tyr

(SEQ ID NO: 25)

where Xaa in each case is any amino acid.

Especially preferred are peptides which, in accordance with the formula of SEQ ID

NOS: 23, 24 and 25 also satisfy one or more of the foUowing criteria: the N-terminal amino

acid position is Tyr, second position is Tyr, fourth position is Pro, fifth position is Arg, sixth

position is Pro, seventh position is Arg, and eighth position is Arg. Of course, the fourth

position is already fixed in SEQ ID NO: 23 , the fifth is already fixed in SEQ ID NO: 24, and

the eighth in SEQ ID NO: 25. When all of these criteria are satisfied and this peptide consists

of 9 amino acids, one has SEQ ID NO: 22. Any or aU of the foreign specific alternatives may

be combined in the peptides of the claimed invention, subject to the motif of SEQ ID NO: 23 ,

32 24 or 25 and the size of 9-16 amino acids. Especially preferred are peptides which are 9-14

amino acids long, and which include SEQ ID NO: 23, 24 or 25 subject to the above preferred

alternatives.

Also a part of the invention are so-caUed "minigenes", which are isolated nucleic acid molecules which encode any of SEQ ID NOS: 21 , 22, 23, 24 or 25 all of the especiaUy

preferred embodiments of SEQ ID NO: 23, 24 or 25 being included. There are only a limited

number of nucleic acid molecules which can encode, e.g., SEQ ID NO: 21 or 22, and they

can aU be deduced from the known rules of degeneracy for codons without any difficulty. The

use of these minigenes in encoding the peptides of the invention, in both standard in vivo and

in vitro methodologies is also encompassed by the invention. These peptides bind to, and/or

are processed to peptides which bind to HLA-A29 molecules. The fact that these peptides are

processed to the tumor rejection antigen in indicated by the examples.

This property may be exploited in the context of other parameters in confirming

diagnosis of pathological conditions, such as cancer, melanoma in particular. For example,

the investigator may study antigens shed into blood or urine, observe physiological changes,

and then confirm a diagnosis of melanoma using the CTL proliferation methodologies

described herein.

Also a part of the invention are complexes of HLA-A29 molecules, and one of the

peptides listed supra, preferably in soluble form. Such soluble complexes can be used, e.g. ,

to determine presence of CTLs in a sample, such as a body fluid sample, by adding the soluble

complexes to the sample, and then determining reaction with CTLs. Panning for CTLs using

soluble complexes of MHC molecule and peptide is taught by, e.g., Bousso et al., Immunol.

Lett. 59(2): 85-91 (1997), incoφorated by reference. The complexes are preferably

33 immobilized to faciUtate the enrichment ofthe CTLs. Attention is also drawn to Sakita, et al. ,

J. Immunol. Meth. 192: 105-115 (1996), also incoφorated by reference, which shows that such complexes can be used to stimulate CTLs in vivo. This is another feature of the invention. In an especiaUy preferred embodiment, the soluble complexes referred to supra are

multimeric, most preferably tetrameric. Altman et al. , Science 274: 94-96 (1996),

incoφorated by reference, describe how such structures can be made.

On their own, peptides in accordance with the invention may be used to carry out

HLA-typing assays. It is weU known that when a skin graft, organ transplant, etc., is

necessary one must perform HLA typing so as to minimize the possibiUty of graft rejection.

The peptides of the invention may be used to determine whether or not an individual is HLA-

Cw6 or HLA-A29 positive, so that appropriate donors may be selected. This type of assay

is simple to carry out. The peptides ofthe invention are contacted to a sample of interest, and

binding to ceUs in that sample indicates whether or not the individual from which the sample

is taken is HLA-Cw6 or HLA-A29 positive. One may label the peptides themselves,

conjugate or otherwise bind them to linkers which are labeled, immobilize them to sohd

phases, and so forth, so as to optimize such an assay. Other standard methodologies wiU be

clear to the skiUed artisan, and need not be presented herein.

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-A29 orHLA-

Cw6 ceUs. One such approach is the administration of CTLs specific to the complex to a

subject with abnormal ceUs of the phenotype at issue. It is within the skill of the artisan to

develop such CTLs in vitro. Specifically, a sample of ceUs, such as blood ceUs, are contacted

to a cell presenting the complex and capable of provoking a specific CTL to proliferate. The

34 target ceU can be a transfectant, such as COS cell of the type described supra. These transfectants present the desired complex on their surface and, when combined with a CTL of interest, stimulate its proliferation. COS ceUs, such as those used herein, are widely

ava able, as are other suitable host cells.

To detaU the 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 ceUular abnormality

which is characterized by certain of the abnormal ceUs presenting the particular complex,

where the complex contains the pertinent HLA molecule. 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 ceUs

present the relevant HLA/TRA complex. This can be determined very easUy, as the art is

very famiUar with methods for identifying ceUs which present a particular HLA molecule, as

well as how to identify cells expressing RNA ofthe pertinent sequences, in this case a GAGE

sequence. Once ceUs 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 ceUs are lysed by the mixed CTL sample, then it

can be assumed that a GAGE derived, tumor rejection antigen 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 avaUable in accordance with

the invention. CTLs can also be provoked jn vivo, using a number of approaches. One

35 approach, i.e. , the use of non-proUferative ceUs expressing the complex, has been elaborated

upon supra. The ceUs used in this approach may be those that normally express the complex,

such as irradiated melanoma ceUs 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 ceUs expressing

HPV E7 peptides in a therapeutic regime. Various ceU types may be used. SimUarly, vectors

carrying one or both of the genes of interest may be used. Viral or bacterial vectors are especiaUy preferred. In these systems, the gene of interest is carried by, e.g. , a Vaccina virus

or the bacteria BCG, and the materials de facto "infect" host ceUs. The ceUs 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 tumor rejection antigen or the

precursor itself with an adjuvant to facilitate incoφoration into HLAL-Cw6 presenting ceUs

which then present the HLA/peptide complex of interest. The TRAP is processed to yield the

peptide partner of the HLA molecule while the TRA is presented without the need for further

processing.

Other aspects of the invention wUl be clear to the skiUed artisan and need not be

repeated here.

The terms and expressions which have been employed are used as terms of description

and not of Umitation, and there is no intention in the use of such terms and expressions of

excluding any equivalents of the features shown and described or portions thereof, it being

recognized that various modifications are possible within the scope of the invention.

36 (1) GENERAL INFORMATION:

(i) APPLICANTS: Van der Bruggen, Pierre; Van den Eynde, Benoit; DeBacker, OUvier; Boon-FaUeur, Thierry

(ii) TITLE OF INVENTION: Isolated, Polypeptides Which Bind to HLA-A29 Molecules, Nucleic Acid, The Molecules Encoding These, and Uses Thereof

(iii) NUMBER OF SEQUENCES: 25

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fulbright & Jaworski L.L.P.

(B) STREET: 666 Fifth Avenue

(C) CITY: New York City

(D) STATE: New York

(E) COUNTRY: USA (F) ZIP: 10103-3198

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 kb storage

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: PC

(D) SOFTWARE: Wordperfect

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: 435

(vh) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 09/012,818

(B) FILING DATE: 23-January-1998

(C) CLASSIFICATION: 435

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/531,662

(B) HUNG DATE: 21 -September- 1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/370,648

(B) HUNG DATE: 10-January-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/250,162

(B) FILING DATE: 27-May-1994

37 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/096,039

(B) FILING DATE: 22-July-1993

(viii) ATTORNEY/ AGENT INFORMATION:

(A) NAME: Hanson, Norman D.

(B) REGISTRATION NUMBER: 30,946

(C) REFERENCE/DOCKET NUMBER: LUD 5531 PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 318-3168

(B) TELEFAX: (212) 752-5958

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 646 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

CTGCCGTCCG GACTCTTTTT CCTCTACTGA GATTCATCTG TGTGAAATAT 50

GAGTTGGCGA GGAAGATCGA CCTATCGGCC TAGACCAAGA CGCTACGTAG 100

AGCCTCCTGA AATGATTGGG CCTATGCGGC CCGAGCAGTT CAGTGATGAA 150

GTGGAACCAG CAACACCTGA AGAAGGGGAA CCAGCAACTC AACGTCAGGA 200

TCCTGCAGCT GCTCAGGAGG GAGAGGATGA GGGAGCATCT GCAGGTCAAG 250

GGCCGAAGCC TGAAGCTGAT AGCCAGGAAC AGGGTCACCC ACAGACTGGG 300

TGTGAGTGTG AAGATGGTCC TGATGGGCAG GAGATGGACC CGCCAAATCC 350

AGAGGAGGTG AAAACGCCTG AAGAAGAGAT GAGGTCTCAC TATGTTGCCC 400

AGACTGGGAT TCTCTGGCTT TTAATGAACA ATTGCTTCTT AAATCTTTCC 450

CCACGGAAAC CTTGAGTGAC TGAAATATCA AATGGCGAGA GACCGTTTAG 500

TTCCTATCAT CTGTGGCATG TGAAGGGCAA TCACAGTGTT AAAAGAAGAC 550

ATGCTGAAAT GTTGCAGGCT GCTCCTATGT TGGAAAATTC TTCATTGAAG 600

TTCTCCCAAT AAAGCTTTAC AGCCTTCTGC AAAGAAAAAA AAAAAA 646

38 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

AGACGCTACG TAGAGCCT 18

(2) INFORMATION FOR SEQ ID NO: 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Unear

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 3:

CCATCAGGAC CATCTTCA 18

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:

Tyr Arg Pro Arg Pro Arg Arg Tyr 5

(2) INFORMATION FOR SEQ ID NO: 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Thr Tyr Arg Pro Arg Pro Arg Arg Tyr 5

39 (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Tyr Arg Pro Arg Pro Arg Arg Tyr Val

5

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val 5 10

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 8:

Arg Pro Arg Pro Arg Arg Tyr Val Glu

5

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GACCAAGACG CTACGTAG 18

40 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 10:

CCATCAGGAC CATCTTCA 18

(2) INFORMATION FOR SEQ ID NO: 11 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 :

GCGGCCCGAG CAGTTCA 17

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Ser Tφ Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg 5 10 15

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val Glu Pro Pro Glu Met He 5 10 15

41 (2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 538 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Unear

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 14:

ACGCCAGGGA GCTGTGAGGC AGTGCTGTGT GGTTCCTGCC GTCCGGACTC 50

TTTTTCCTCT ACTGAGATTC ATCTGTGTGA AATATGAGTT GGCGAGGAAG 100

ATCGACCTAT CGGCCTAGAC CAAGACGCTA CGTAGAGCCT CCTGAAATGA 150

TTGGGCCTAT GCGGCCCGAG CAGTTCAGTG ATGAAGTGGA ACCAGCAACA 200

CCTGAAGAAG GGGAACCAGC AACTCAACGT CAGGATCCTG CAGCTGCTCA 250

GGAGGGAGAG GATGAGGGAG CATCTGCAGG TCAAGGGCCG AAGCCTGAAG 300

CTCATAGCCA GGAACAGGGT CACCCACAGA CTGGGTGTGA GTGTGAAGAT 350

GGTCCTGATG GGCAGGAGAT GGACCCGCCA AATCCAGAGG AGGTGAAAAC 400

GCCTGAAGAA GGTGAAAAGC AATCACAGTG TTAAAAGAAG ACACGTTGAA 450

ATGATGCAGG CTGCTCCTAT GTTGGAAATT TGTTCATTAA AATTCTCCCA 500

ATAAAGCTTT ACAGCCTTCT GCAAAGAAAA AAAAAAAA 538

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 560 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

CTCATATTTC ACACAGATGA GTTGGCGAGG AAGATCGACC TATTATTGGT 50

CTAGGCCAAT AATAGGTCGA TCTTCCTCGC CAACTCATAT TTCACACAGA 100

TGAATCTCAG TAGAGGAAAA TCGACCTATT ATTGGCCTAG ACCAAGGCGC 150

TATGTACAGC CTCCTGAAGT GATTGGGCCT ATGCGGCCCG AGCAGTTCAG 200

TGATGAAGTG GAACCAGCAA CACCTGAAGA AGGGGAACCA GCAACTCAAC 250

42 GTCAGGATCC TGCAGCTGCT CAGGAGGGAG AGGATGAGGG AGCATCTGCA 300

GGTCAAGGGC CGAAGCCTGA AGCTGATAGC CAGGAACAGG GTCACCCACA 350

GACTGGGTGT GAGTGTGAAG ATGGTCCTGA TGGGCAGGAG ATGGACCCGC 400

CAAATCCAGA GGAGGTGAAA ACGCCTGAAG AAGGTGAAAA GCAATCACAG 450

TGTTAAAAGA AGGCACGTTG AAATGATGCA GGCTGCTCCT ATGTTGGAAA 500

TTTGTTCATT AAAATTCTCC CAATAAAGCT TTACAGCCTT CTGCAAAGAA 550

AAAAAAAAAA 560

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 540 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Unear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CGCCAGGGAG CTGTGAGGCA GTGCTGTGTG GTTCCTGCCG TCCGGACTCT 50

TTTTCCTCTA CTGAGATTCA TCTGTGTGAA ATATGAGTTG GCGAGGAAGA 100

TCGACCTATT ATTGGCCTAG ACCAAGGCGC TATGTACAGC CTCCTGAAAT 150

GATTGGGCCT ATGCGGCCCG AGCAGTTCAG TGATGAAGTG GAACCAGCAA 200

CACCTGAAGA AGGGGAACCA GCAACTCAAC GTCAGGATCC TGCAGCTGCT 250

CAGGAGGGAG AGGATGAGGG AGCATCTGCA GGTCAAGGGC CGAAGCCTGA 300

AGCTGATAGC CAGGAACAGG GTCACCCACA GACTGGGTGT GAGTGTGAAG 350

ATGGTCCTGA TGGGCAGGAG ATGGACCCGC CAAATCCAGA GGAGGTGAAA 400

ACGCCTGAAG AAGGTGAAAA GCAATCACAG TGTTAAAAGA AGGCACGTTG 450

AAATGATGCA GGCTGCTCCT ATGTTGGAAA TTTGTTCATT AAAATTCTCC 500

CAATAAAGCT TTACAGCCTT CTGCAAAAAA AAAAAAAAAA 540

43 (2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISΉCS:

(A) LENGTH: 532 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

AGCTGTGAGG CAGTGCTGTG TGGTTCCTGC CGTCCGGACT CTTTTTCCTC 50

TACTGAGATT CATCTGTGTG AAATATGAGT TGGCGAGGAA GATCGACCTA 100

TTATTGGCCT AGACCAAGGC GCTATGTACA GCCTCCTGAA GTGATTGGGC 150

CTATGCGGCC CGAGCAGTTC AGTGATGAAG TGGAACCAGC AACACCTGAA 200

GAAGGGGAAC CAGCAACTCA ACGTCAGGAT CCTGCAGCTG CTCAGGAGGG 250

AGAGGATGAG GGAGCATCTG CAGGTCAAGG GCCGAAGCCT GAAGCTGATA 300

GCCAGGAACA GGGTCACCCA CAGACTGGGT GTGAGTGTGA AGATGGTCCT 350

GATGGGCAGG AGATGGACCC GCCAAATCCA GAGGAGGTGA AAACGCCTGA 400

AGAAGGTGAA AAGCAATCAC AGTGTTAAAA GAAGGCACGT TGAAATGATG 450

CAGGCTGCTC CTATGTTGGA AATTTGTTCA TTAAAATTCT CCCAATAAAG 500

CTTTACAGCC TTCTGCAAAG AAAAAAAAAA AA 532

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 539 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Unear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GCCAGGGAGC TGTGAGGCAG TGCTGTGTGG TTCCTGCCGT CCGGACTCTT 50

TTTCCTCTAC TGAGATTCAT CTGTGTGAAA TATGAGTTGG CGAGGAAGAT 100

CGACCTATTA TTGGCCTAGA CCAAGGCGCT ATGTACAGCC TCCTGAAGTG 150

ATTGGGCCTA TGCGGCCCGA GCAGTTCAGT GATGAAGTGG AACCAGCAAC 200

ACCTGAAGAA GGGGAACCAG CAACTCAACG TCAGGATCCT GCAGCTGCTC 250

44 AGGAGGGAGA GGATGAGGGA GCATCTGCAG GTCAAGGGCC GAAGCCTGAA 300

GCTGATAGCC AGGAACAGGG TCACCCACAG ACTGGGTGTG AGTGTGAAGA 350

TGGTCCTGAT GGGCAGGAGG TGGACCCGCC AAATCCAGAG GAGGTGAAAA 400

CGCCTGAAGA AGGTGAAAAG CAATCACAGT GTTAAAAGAA GACACGTTGA 450

AATGATGCAG GCTGCTCCTA TGTTGGAAAT TTGTTCATTA AAATTCTCCC 500

AATAAAGCTT TACAGCCTTC TGCAAAAAAA AAAAAAAAA 539

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

ACTCCATGAG GTATTTC 17

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: Unear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

TTTCACCACA TGCGTGT 17

(2) INFORMATION FOR SEQ ID NO: 21 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 :

Arg Ser Thr Tyr Tyr Tφ Pro Arg Pro Arg Arg Tyr Val Gin 5 10

45 (2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Tyr Tyr Tφ Pro Arg Pro Arg Arg Tyr 5

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ix) FEATURE:

(D) OTHER INFORMATION: Each Xaa may be any amino acid (xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 23:

Xaa Xaa Tφ Pro Xaa Xaa Xaa Xaa Tyr

5

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: Unear

(ix) FEATURE:

(D) OTHER INFORMATION: Each Xaa may be any amino acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Xaa Xaa Tφ Xaa Arg Xaa Xaa Xaa Tyr

5

46 2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: Unear

(ix) FEATURE:

(D) OTHER INFORMATION: Each Xaa may be any amino acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

Xaa Xaa Tφ Xaa Xaa Xaa Xaa Arg Tyr 5

47

Claims

We claim:

1. An isolated peptide consisting of from 9 to 16 amino acids and containing SEQ

ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

2. The isolated peptide of claim 1 , consisting of SEQ ID NO: 23, SEQ ID NO:

24, or SEQ ID NO: 25.

3. Method for determining presence of cytolytic T lymphocytes in a body fluid

sample which are specific for complexes of HLA-A29 molecules and SEQ ID NO: 23, SEQ

ID NO: 24, or SEQ ID NO: 25 comprising contacting a sample of ceUs which present HLA-

A29 on their surface with a polypeptide comprising SEQ ID NO: 23, SEQ ID NO: 24, or

SEQ ID NO: 25 under conditions favoring processing of said polypeptide to the polypeptide

SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 and binding of SEQ ID NO: 23, SEQ

ID NO: 24 or SEQ ID NO: 25 to said HLA-A29 molecules, contacting a body fluid sample

beUeved to contain said cytolytic T lymphocytes to said ceUs presenting complexes of SEQ ID

NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 and HLA-A29 on their surface, and determining

at least one of (i) tumor necrosis factor released by cytolytic T lymphocytes or (ii) lysis of said

ceUs presenting said complexes, as a determination of presence of said cytolytic T lymphocytes

in said sample.

4. The method of claim 3, comprising determining release of tumor necrosis

factor.

48

5. The method of claim 3, comprising determining lysis by determining release

of radiolabelled chromium.

6. The isolated peptide of claim 1 , wherein at least the N-terminus of SEQ ID NO:

23 is Tyr, or the second amino acid is Tyr, or the fifth amino acid is Arg, or the sixth amino

acid is Pro, or the seventh amino acid is Arg, or the eighth amino acid is Arg.

7. Isolated nucleic acid molecule consisting of a nucleotide sequence which encodes

the peptide of SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

8. The isolated nucleic acid molecule of claim 7, consisting of a nucleotide sequence

which encodes the peptide of SEQ ID NO: 21.

9. The isolated nucleic acid molecule of claim 8, consisting of a nucleotide sequence

which encodes the peptide of SEQ ID NO: 22.

10. The isolated peptide of claim 1 , wherein at least the N-terminus of SEQ ID NO:

24 is Tyr, or the second amino acid is Tyr, or the fourth amino acid is Pro, or the sixth amino

acid is Pro, or the seventh amino acid is Arg, or the eighth amino acid is Arg.

11. The isolated peptide of claim 1 , wherein at least the N-terminus of SEQ ID NO:

25 is Tyr, or the second amino acid is Tyr, or the fourth amino acid is Tyr, or the fourth

49 amino acid is Pro, or the fifth amino acid is Arg, or the sixth amino acid is Pro, or the seventh

amino acid is Arg.

12. A method of making a polypeptide consisting of the amino acid sequence of SEQ

ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 comprising transforming or transfecting a cell

with a nucleic acid molecule sequence which encodes said peptide to produce said peptide.

13. The method of claim 12, wherein said peptide consists of SEQ ID NO: 21.

14. The method of claim 12, wherein said peptide consists of SEQ ID NO: 22.

15. The method of claim 12, wherein said nucleic acid molecule is transformed or

transfected into said ceU in vivo.

16. The method of claim 12, wherein said nucleic acid molecule is transformed or

transfected into said cell in vitro.

17. Isolated complex of an HLA-A29 molecule and the peptide of claim 1.

18. The isolated complex of claim 17 in a solution.

19. Method for determining presence of cytolytic T ceUs in a sample, wherein said

cytolytic T cells have a receptor specific for complexes of HLA-A29 and a peptide consisting

50 of the amino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25 with the isolated complex of claim 18, and determining binding of a cytolytic T ceU to said

isolated complex as a determination of said cytolytic T ceUs in said sample.

20. A method for stimulating cytolytic T ceUs in vivo, comprising administering the isolated complex of claim 18 to a subject, in an amount sufficient to stimulate cytolytic T ceUs specific for said complex.

21. The isolated complex of claim 17, wherein said complex is multimeric.

22. The isolated complex of claim 21 , wherein said multimeric complex is a tetramer.

51