CA2165435C - Isolated peptides which form complexes with mhc molecule hla-c-clone 10 and uses thereof - Google Patents

Abstract

A new family of tumor rejection antigen precursors, and the nucleic acid molecules which code for them, are disclosed. These tumor rejection antigen precursors are referred to as BAGE tumor rejection antigen precursors, and the nucleic acid molecules which code for them are referred to as BAGE coding molecules. Various diagnostic and therapeutic uses of the coding sequences and the tumorrejection antigen precursor molecules are described.

Description

r j'IELD OF THE INVENTION
This invention relates to peptides which are useful in connection with the diagnosis and treatment of pathological conditions. More particularly, it relates to peptides which are processed to a peptide presented by the MHC molecule HLA-C-clone 10, and the presented peptide itself. These peptides are useful in diagnosis and therapeutic contexts.
~~C1CGRODND 1~~!JD PRIOR ART
The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is a complex one. An important fx~cet of the system is the T cell response. This response requires that T cells recognize and interact with complexes of cell surface molecules, referred to as human leukocyte antigens ("HLA"), 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 cell and complexes of HLA/peptide 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 (1992j.
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, 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.
The genes are said to code for "tumor rejection antigen l0 precursors" or "T;RAP" 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.
In U.S. patent No. S,G05,940~
nonapeptides are taught 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 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 H?~A molecule has diagnostic and therapeutic ramifications, these axe 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 No. 5,5.58,995 .-the fact that the MAGE-1 expression product is processed to a second TRA 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.
In U.~~. Patent Number '_>, r87, X374, 1=yrosinase is described as a tumor rE=jecr:ion ~:mtigen pre~;ursor. This reference discloses that a molecule which. i.~; produ:,ed by some normal cells (e.g., melanocytes) ,, is processed ire tumor cells t:o yield a tumor rejection ant=igen that i.s presert:e<1 )v~y HLA--A2 molecules.
In L1.S. Paten':. I'lurnber 5, 620, 886, a second TRA, not derived from tyrosin<nse is taught to be presented by HLA-A2 molecules. The TRA is derived from a TRAP, but is coded f=or by a non MAGE gene. This di~c:losur:~ shows that a particular HLA
molecule may present 'I"RAs der_iver_1 from different sources.
In U.S. patent No. _'i,571.,'711, <3 new family of genes, referred to therein a; the BAt~F; :family, was disclosed. It was observed that these gE.me~> also ~_°.ode for r.umo~ rejection antigen precursors. It was of>served in that: application that the MHC
molecule known as HL~-~wi~'-clone 10 presented a tumor rejection antigen derived from ..~ BAC~E tumor rejection antigen precursor;
however, the tumor rejection <3ntig~ru was not disclosed. The present application dE:eals with. this peptide, referred to herein as SEQ ID 1VO:10, as ~,aeLa. as other ramificatvons stemming from the identifi<:ation of this peptide.
The invention i.s elaborated upon further in the disclosure which follo~as.
BRIEF DESCRIPTIOI\1 OF THE FIGURES
Figures 1A to LL~ set forth the results of a chromium release assay using C'1'u c;-Lone 82/8% .against M'Z2-KIEL cell line in Fig. lA, K56<'? cell lime ~n Fig. 1B, and MZ2-MEL 2.2.5 cell line for Figs. 1C and 1D, where f'ig. 1C is the transfected variation.
Figure 2 shows the results of <~ TNF release assay when CTL clone 82/82 was used against a panel of different cell lines.

Figure 3 depicts the results of a TNF release assay using transfected COS cells.
Figure 4 presents the results of a test of various transfectants using CTL clone 82/82, and measuring TNF
release.
Figure 5 shows results obtained when studies were undertaken to determine half maximal lysis of cells, following stimulation of cytolytic T cell clone CTL 82/82 with the peptide of SEQ ID NO: 10.
DETAILED pE8CRIPTION OF PREFERRED EMBODII~iENTB
example 1 A melanoma cell line, MZ2-MEL was established from melanoma cells taken from patient MZ2, using standard methodologies. This cell line is described, e.g., in PCT
Application PCT/US92/04354, filed May 22, 1992, published November 26, 199..
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 patient 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 al., Int. J. Cancer 39:390-396 (1987), The assay, however, is described herein. The target melanoma cells were grown ~ Y'~, and then resuspended at 10' cells/ml in DMEM, supplemented with 10 mM NEPES and 30~ FCS, and incubated for minutes at 37°C with 200 ~Ci/ml of Na(S~Cr)O'. Labelled cells were washed three times with DMEM, supplemented with 10 , mM Iiepes. These were then resuspended in DMEM supplemented 40 with 10 mM Hepes and 10~ FCS, after which 100 ul aliquots containing 103 cells, were distributed into 96 well 5 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 1008, and incubated for four hours at 37°C in a 8% Co2 atmosphere.
Plates were centrifuged again, and 100 ul aliquots of :10 supernatant were collected and counted. Percentage of S~Cr release was calculated as follows:
% ~~Cr release = jER-SRS x 100 (MR-SR) :15 where ER is observed, experimental S~Cr release, SR is spontaneous release measured by incubating 103 labeled cells in 200 ul of medium alone, and MR is maximum release, obtained by adding 100 ul 0.3% Tritori~X-100 to target cells.
:20 Those mononuclear blood samples which showed high CTL
activity were expanded and clamed via limiting dilution, and were screened again, using the same methodology. The CTL
clone MZ2-CTL 82/82 was thus isolated. The clone is referred to as "82/82" hereafter.
a5 The same method was used to test target K562 cells, as well as a melanoma cell line. These results, presented in Figure lA, show that this CTL clone recognizes and ,lyses the melanoma cell line, but not K562. The CTL clone, 82/82, was then tested against melanoma cell lines in the same manner :30 described ,~,g,~. Figure 1 shows that, while MZ2-MEL.43 is lysed by CTL clone 82/82, the cell line M22-M~L 2.2.5, a variant which has lost expression of HLA-A29, HLA-B44, and HLA-C clone 10 is not, suggesting that the TRA is presented by one of these HLA molecules. When cell line MZ2-MEL 2.2.5 was :35 transfected with DNA coding for HLA-C clone 10, using well known techniques, the cells became sensitive to lysis by CTL
clone 82/82, thus demonstrating that the antigen recognized by CTL clone 82/82 is presented by HLA-C clone 10.
Trade-mark Example 2 Further studies were carried out to determine if 82/82 also produced tumor necrosis factor ("TNF") when contacted with target cells. The method used was that described by Traversari et al., Immunogenetics 35: 145-152 (1992) Eriefly, samples of the CTL line were combined with samples of a target cell of interests in culture medium. After 24 hours, supernatant from the cultures was removed, and then tested on TNF sensitive WEHI cells. A panel of fourteen different 1!i cell lines were tested, as shown in Figure 2.
The results, presented i.n terms of the percentage of WEHI
cells which died upon exposure to the supernatant, are shown in Figure 2. The results demonstrate that three melanoma cell.
lines present this antigen. As a result of the strong response by MZ2 MEL43, it was used in the experiments which follow.
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 align-dT binding kit, following well recognized techniques. Once the mRNA was secured, it was transcribed into cDNA, again using standard methodologies.
The cDNA was then ligated to EcoRI adaptors and cloned into the EcoRI site of plasmid pcD-SRa, in accordance with the manufacturer's instructions. The recombinant plasmids were then electroporated into 0'M101 ~. coli (electroporation conditions: 1 pulse at 25 farads, 2500 V).
The transfected bacteria were selected with ampicillin -(50 ~g/ml), and then divided into 87 pools of 400 bacteria and 297 pools of 200 bacteria. Each pool represented either about 280 or about 140 cDNAs, as analysis showed that about 70% of plasmids contained an insert. Each pool was amplified to saturation, and plasmid DNA was isolated via alkaline °

r I
:5 lysis, potassium acetate precipitation without phenol extraction, following Maniatis et al., in Molecular Cloning:
A Laboratory Manual (Cold Spring Harbor, N.Y., 1982).
sample 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 duplicate. Samples of COS-7 cells were seeded, at 15,000 cells/well into tissue culture flat bottom microwells, in Dulbeco~s modified Eagles Medium ("DMEM") supplemented with 1!5 l0% fetal calf serum. The cells were incubated overnight at 37°C, medium was removed and then replaced by 50 ~cl/well of DMEM medium containing 10% Nu~serum, 400 ~Cg/ml DEAF-dextran;
and 100 ~uM chloroquine, plus 100 ng of subject plasmids.
These plasmids were the plasmids of the various pools described supra, and 100 ng of plasmids containing DNA coding for HLA-C clone 10 in plasmid pcD-SRa. Following four hours of incubation at 3T°C, the medium was removed, and replaced by 50 ~l of PBS containing 10% DMSO. This medium was removed after two minutes and replaced by 200 ~1 of DMEM supplemented 2!5 with 10% FCS.
Following this change in medium, COS cells were incubated for 24-48 hours at 37°C. Medium was then discarded, and 1500 cells of CTL clone 82/82 were added, in 100 ~C1 of Iscove medium containing 10% pooled human serum supplemented with 20 3~D U/ml of IL-2. Supernatant was removed after 24 hours, and TNF
content was determined in an assay on WEHI cells, as described by Traversari et al., Immunogenetics 35: 145-152 (1992), 3!5 Of the 384 pools tested, 99% stimulated TNF, at a ' concentration below 5 pg/ml. Two pools gave concentrations above 40 pg/ml, with duplicate wells giving equivalent results. Figure 3 shows this. The bacteria from one of these pools, i.e., pool .19, were selected for further experiments.

4 ~~
~' Trade-mark WO 95/00159 , PCT/LTS94/06534 Examule 5 The bacteria of pool 19 were cloned, and 800 bacteria were tested. Plasmid DNA was extracted therefrom, transfected into a new sample of COS cells in the same manner as described supra, and the cells were again tested for stimulation of CTL
clone 82/82. Twelve positive clones were found. One of these, referred to as cDNA clone AD5 was tested further. In a comparative test COS cells were transfected with cDNA clone AD5 and the HLA-C clone 10. HLA-C clone 10 and MAGE-1, AD5 alone, or HLA-C clone 10 alone. Control cell lines MZ2-MEL
2.2.5 and MZ2-MEL.43 were also used. TNF release in CTL
supernatant was measured by testing it on WEHI cells, as referred to supra. The optical density of the surviving WEHI
cells was measured using MTT. Figure 4 shows that only the COS cells transfected with HLA-C clone 10 and cDNA-ADS, and the original cell line MZ2-MEL 43 stimulated TNF release from CTL clone 82/82.
Example 6 The cDNA AD5 was sequenced following art known techniques. A sequence search revealed that the plasmid insert showed no homology to known genes or proteins.
SEQUENCE ID NO: 1 presents cDNA nucleotide information for the identified gene, referred to hereafter as "BAGE-1". A
putative open reading frame is located at bases 201-332 of the molecule.
Example 7 Following the sequencing of the cDNA, as per example 6, experiments were carried out to determine if cells of normal tissues expressed the gene. To determine this, RNA isolated from normal tissues was reverse transcribed, using oligo-dT as primer. The resulting cDNA was then amplified, using primers:

5' CAG AAG ATG AAG CAC AGA G-3' (SEQ ID NO: 2) and 5'-GAG CGG TTT TTC TGG CAT TG-3~ (SEQ ID NO: 3) and standard PCR methodologies. Radioactive nucleotides were WO 95/00159 216 5 4 3 5 ~T~S94/06534 added so that the amount of amplification product could be determined via phosphor imaging.
The amount of product was expressed as a percentage of the product secured from cell line MZ2-MEL 3.0, which was shown to express the gene. The results are as follows:
MZ2-MEL 3.0 100%
Lung < 0.5%
Breast "
Stomach "
Skin "
Brain Prostate "
Kidney "
Testis 8%
In additional experiments not elaborated upon herein, the DNA of cell line MZ2-MEL was digested with EcoRI, and then hybridized with a PCR probe corresponding to the first 300 nucleotides of the cDNA described herein. Following standard Southern blotting, four bands, corresponding to sizes of approximately 5.8, 7.5, 8.5 and 11 kilobases were identified, suggesting the existence of a family of Bage genes.
Example 8 Expression of the gene by tumor samples and tumor cell lines was also determined. cDNA was secured just as in example 4, and then nested primer methodologies were carried out to amplify the pertinent sequences. First, twenty cycles of amplification were carried out, using primers:
5'-CGG CTT AGA GGA CCA GGA GAA-3' (SEQ ID NO: 4) and 5'CAG AAG ATG AAG CAC AGA G-3' (SEQ ID NO: 5) This was followed by twenty additional cycles using primers:
5'-GGC TCC AAC CTC CAG CTC AC-3' (SEQ ID NO: 6) AND
5'-TTA GAG GAC CAG GAG AAG G-3' (SEQ ID NO: 7) The results are presented below. The first figure is the 5 number of positive samples; the second is the total number of samples tested:

Melanoma 12/20 Breast Carcinoma 2/5 Small Cell lung carcinoma 2/8 10 Non-small cell lung carcinoma 2/5 Sarcoma 1/4 Head and neck tumors 1/6 Colon carcinoma 0/4 Kidney tumor 0/5 Leukemia/Lymphoma 0/3 Example 9 The experiments set forth supra showed that cytolytic T
cell clone CTL 82/82 recognized an antigen encoded by a GAGE
gene and presented by HLA-C-clone 10. The work described in this example details how the amino acid sequence of the presented antigen was determined.
The cDNA clone identified as coding for BAGE, i.e., ADS, was used to generate a large number of incomplete cDNA
molecules. The cDNA clone was inserted into expression vector pcDNAI/Amp, and digested with NotI and SphI restriction endonucleases, followed by treatment with exonuclease III, in accordance with the manufacturer's instructions. By using exonuclease III for varying lengths of time, progressive deletions of AD5 at its 3' end were obtained. The truncated variants were religated into pcDNAI/Amp, electroporated into _E. co i strain DHSaF'IQ, and selected via ampicillin (50 ~g/ml). Four hundred clones were obtained in this way.
The plasmid DNA was obtained from these 400 cones, and transfected into COS-7 cells together with HLA-C-clone 10 coding cDNA. The transfectants were then tested in a TNF
release assay, as described supra. Positive clones were those which stimulated TNF release by CTL 82/82.
Once cells were divided into positive and negative transfectants, the sequences of plasmid DNA from 10 positives and 10 negatives was determined. Clone 19C2, a positive WO 95100159 216 5 4 3 5 ' PCT/US94/06534 clone, contained part of the open reading frame for the BAGE
gene described.supra,, from nucleotide 1 to nucleotide 67. In contrast, clone 17612, a negative transfectant, contained nucleotides 1-6 of the gene.
By comparing the inserts of positive and negative clones, a region of 22 amino acids was identified as probably containing the sequence of the presented peptide, i.e.:
Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln Ala Arg Leu Met Lys Glu (SEQ ID NO: 8) Synthetic peptides were then prepared, based upon this sequence, and were tested for their ability to render COS-7 cells transfected with HLA-C-clone l0 capable of stimulating TNF release. The first positive peptide was a 16-mer:
Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln (SEQ ID NO: 9) Testing of smaller peptides led to the identification of peptide Ala Ala Arg Ala Val Phe Leu Ala Leu (SEQ ID NO: 10) This peptide effectively stimulated CTL 82/82, with half maximal lysis being reached at a peptide concentration of 80 nM, which is shown in Figure 5.
The foregoing examples show the isolation of a nucleic acid molecule which codes for a tumor rejection antigen precursor. This "TRAP" coding molecule, however, is not homologous with any of the previously disclosed MAGE 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 sequence set forth in SEQ ID NO: 1. This sequence is not a MAGE coding sequence, as will be seen by comparing it to the sequence of any of the MAGE genes described in the references. Also a part of the invention are those nucleic acid sequences which also code for a non-MAGE tumor rejection antigen precursor but which hybridize to a nucleic acid molecule containing the described nucleotide sequence, under stringent conditions. The term "stringent conditions" as used herein refers to parameters ~~~5435 WO 95/00159 PCT/(1S94/06534 with which the art is familiar. More specifically, stringent conditions, as used herein, refers to hybridization in 3.5xSSC, ixDenhardt~s solution, 25 mM sodium phosphate buffer (pH 7.0), 0.5% SDS, and 2 mM EDTA for 18 hours at 65°C. This is followed by four washes of the filter, at 65°C for 20 minutes, in 2xSSC, 0. 1 % SDS, and one wash for up to 20 minutes in 0.3xSSC, 0.1% SDS. 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.
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.g., E. coli), or eukaryotic (e.g., CHO or COS
cells). 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 human leukocyte antigen HLA-C clone 10 presents a tumor rejection antigen derived from these genes, the expression vector may also include a nucleic acid sequence coding for HLA-C clone 10. 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 tumor rejection antigen precursor coding sequence may be used alone, when, e.g., the host cell already expresses HLA-C
clone 10. 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-C clone 10 presenting cells if desired, and the gene for tumor rejection antigen precursor can be used in host cells which do not express HLA-C
clone 10.
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 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 family, the invention shall be referred to as the BAGE family of genes and TRAPS. Hence, whenever "BAGE" is used herein, it refers to the tumor rejection antigen precursors coded for by the previously described sequences. "BAGE coding molecule" and similar terms, are used to describe the nucleic acid molecules themselves.
Also a part of the invention are the peptides of SEQ ID
NO: 8, 9 and 10 which are set forth in Example 9. These peptides can be used, for example, to identify those cells which present MHC molecule HLA-C-clone 10. Administration of the peptides, carrying a detectable signal, e.g., followed by the identification of cells to which the peptide has bound is one way to accomplish this, as is the use of solid phase bound peptides, to which the HLA-C-clone 10 presenting cells bind, thus removing them from the sample being assayed.
Additionally, 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 the TRA presented by HLA-C clone 10. 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.
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 SEQ ID NO: 1.
These isolated molecules when presented as the TRA, or as complexes of TRA and HLA, such as HLA-C clone 10, 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, non-proliferative 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 prove a CTL response, or be cells which express both molecules without transfection. Further, 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.
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.
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-c clone l0 cells. One such approach is the administration of 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. Specifically, a sample of cells, such as blood cells, are contacted to a cell presenting the complex and capable of provoking a specific CTL 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 on 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.
To detail the therapeutic methodology, referred to as adoptive transfer (Greenberg, J. Immunol. 136 (5) : 1917 (1986) ;
Reddel 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 WO 95/00159 2 ~ 6 5 4 ~ 5 PCT/US94/06534 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 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 BAGE
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 BAGE
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 available in accordance with the invention. CTLs can also be provoked in vivo, using a number of approaches. One approach, i.e., the use of non-proliferative 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 melanoma 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. In these systems, the gene of interest is carried by, e.g., a Vaccinia virus 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 tumor rejection antigen or the precursor itself with an adjuvant to facilitate incorporation into HLP.-C clone 10 presenting cells which present the HLA molecule 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 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 the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

SEQUENCE LISTIN~3 (1) GENERAL INFORMATION:
(i) APPLICANT: van der Bruggen, Pierre Boon-Falleur, Thierry (ii) TITLE OF INVENTION: ISOLATED PEPTIDES WHICH FORM COMPLEXES WITH

(iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Felfe & Lynch (B) STREET: 805 Third Avenue (C) CITY: New York City (D) STATE: New York (E) COUNTRY: USA
(F) ZIP: 10022 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 5.25 inch, 360 kb storage (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: Wordperfect (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/196,630 (B) FILING DATE: 15-FEB-1994 (C) CLASSIFICATION: 435 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/079,110 (B) FILING DATE: 17-JUN-1993 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hanson, Norman D.
(B) REGISTRATION NUMBER: 30,946 (C) REFERENCE/DOCRET NUMBER: LUD 310.1 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 688-9200 (B) TELEFAX: (212) 838-3884 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1032 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ZD NO: 2:
CAGAAGATGA AGCACAGAG
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

WO 95/00159 ~ PCT/US94/06534 (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: single (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln Ala Arg Leu Met Lys Glu (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:

WO 95/00159 2 ~ b 5 4 3 5 PCT/US94/06534 (A) LENGTH: 16 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: single (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: single (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Ala Ala Arg Ala Val Phe Leu Ala Leu

Claims (41)

CLAIMS:

1. An isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 1.

2. An isolated nucleic acid molecule which hybridizes, under stringent conditions, to the nucleic acid sequence set forth in SEQ ID NO: 1, and codes for a tumor rejection antigen precursor, with the proviso that said isolated nucleic acid molecule does not code for a MAGE tumor rejection antigen precursor, said stringent conditions being 3.5×SSC, 1×Denhardt's solution, 25 mM sodium phosphate buffer (pH 7.0), 0.5% SDS, and 2 mM EDTA for 18 hours at 65°C.

3. An isolated mRNA molecule which is complementary to the nucleic acid molecule of claim 1.

4. A host cell transfected with the nucleic acid molecule of claim 1.

5. A host cell transfected with the nucleic acid molecule of claim 2.

6. An expression vector comprising the isolated nucleic acid molecule of claim 1 operably linked to a promoter.

7. An expression vector comprising the isolated nucleic acid molecule of claim 2 operably linked to a promoter.

8. The host cell of claim 4, wherein said host cell is a mammalian cell which expresses HLA-C clone 10.

9. The host cell of claim 5, wherein said host cell is a mammalian cell which expresses HLA-C clone 10.

10. The expression vector of claim 6, further comprising a nucleic acid molecule which codes for HLA-C clone 10.

11. The expression vector of claim 7, further comprising a nucleic acid molecule which codes for HLA-C clone 10.

12. Expression kit comprising a separate portion of each of:
(i) the isolated nucleic acid molecule of claim 1, and (ii) a nucleic acid molecule which codes for HLA-C
clone 10.

13. Expression kit comprising a separate portion of each of:
(i) the isolated nucleic acid molecule of claim 2, and (ii) a nucleic acid molecule which codes for HLA-C
clone 10.

14. An isolated tumor rejection antigen precursor coded for by the nucleic acid molecule of claim 1.

15. Use of cytolytic T-cells which are specific for complexes of BAGE derived tumor rejection antigen as set forth in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 and HLA-C
clone 10 molecules, and which lyse cells presenting said complexes for treating a subject with a cancer characterized by expression by a BAGE tumor rejection antigen precursor which is processed to a tumor rejection antigen presented by a HLA-C clone 10 molecules, said BAGE
tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2.

16. Use of cytolytic T-cells specific to complexes of an HLA molecule and a tumor rejection antigen derived from a tumor rejection antigen precursor for treating a subject with a cancer characterized by expression of a tumor rejection antigen precursor coded for by a nucleic acid molecule and comprising the nucleotide sequence of SEQ ID
NO:1.

17. Use of an agent which provokes an immune response to complexes of BAGE derived tumor rejection antigen as set forth in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 and HLA-C
clone 10 molecules for treating a subject with a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a tumor rejection antigen presented by HLA-C clone l0 molecules, said BAGE
tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2, said agent being a cytolytic T-cell.

18. Use of an agent which provokes an immune response to complexes of an HLA molecule and a tumor rejection antigen precursor for treating a subject with a cancer characterized by expression of said tumor rejection antigen precursor coded for by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, said agent being a cytolytic T-cell.

19. Use of an agent specific for a complex for diagnosing a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a BAGE
derived tumor rejection antigen as set forth in SEQ ID
NO:8, SEQ ID NO:9 or SEQ ID NO:10, which forms said complex with HLA-C clone 10 molecules. said BAGE tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2, said agent being a cytolytic T-cell.

20. Use of an antigen encoded by SEQ ID NO:1 for diagnosing a cancer characterized by expression of a tumor rejection antigen precursor coded for by a nucleic acid molecule having the sequence set forth in SEQ ID NO:1.

21. Isolated peptide selected from the group consisting of:
SEQ ID NO:8, SEQ ID NO: 9, and SEQ ID NO:10.

22. Use of an HLA-C clone 10 presenting cell for provoking a cytolytic T-cell response, wherein said cell presents complexes of HLA-C clone 10 molecules and a peptide of claim 21 on its surface.

23. Use of cytolytic T-cells which are specific for complexes of BAGE derived tumor rejection antigen as set forth in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 and HLA-C
clone 10 molecules, and which lyse cells presenting said complexes, for treating a subject with a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a tumor rejection antigen consisting of the amino acid sequence of SEQ ID
NO:10.

24. Use of cytolytic T-cells specific to complexes of an HLA molecule and a tumor rejection antigen consisting of the amino acid sequence of SEQ ID NO:10 for treating a subject with a cancer characterized by expression of a tumor rejection antigen precursor coded for by a nucleic acid molecule and comprising the nucleotide sequence of SEQ
ID NO:1.

25. Use of an agent which provokes an immune response to complexes of BAGE derived tumor rejection antigen as set forth in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 and HLA-C
clone 10 molecules for treating a subject with a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a tumor rejection antigen consisting of the amino acid sequence of SEQ ID
NO:10, said BAGE tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2, said agent being a cytolytic T-cell.

26. Use of an agent which provokes an immune response to complexes of HLA-C clone and a peptide consisting of the amino acid sequence of SEQ ID NO:10 for treating a subject with a cancer characterized by expression of a tumor rejection antigen precursor coded for by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, said agent being a cytolytic T-cell.

27. Use of an agent specific for a complex, for diagnosing a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a BAGE
derived tumor rejection consisting of the amino acid sequence of SEQ ID NO:10 which forms said complex with HLA-C clone 10 molecules, said BAGE tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2, said agent being a cytolytic T-cell.

28. Use of an agent specific for a tumor rejection antigen derived from a tumor rejection antigen precursor and consisting of the amino acid sequence of SEQ ID NO:10 for diagnosing a cancer characterized by expression of said tumor rejection antigen precursor coded for by a nucleic acid molecule having the sequence set forth in SEQ ID NO: 1, said agent being a cytolytic T-cell.

29. Use of cytolytic T-cells which are specific for complexes of BAGE derived tumor rejection antigen as set forth in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 and HLA-C
clone 10 molecules, and which lyse cells presenting said complexes for the manufacture of a medicament for treating a subject with a cancer characterized by expression by a BAGE tumor rejection antigen precursor which is processed to a tumor rejection antigen presented by a HLA-C clone 10 molecules, said BAGE tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2.

30. Use of cytolytic T-cells specific to complexes of an HLA molecule and a tumor rejection antigen derived from a tumor rejection antigen precursor for the manufacture of a medicament for treating a subject with a cancer characterized by expression of a tumor rejection antigen precursor coded for by a nucleic acid molecule and comprising the nucleotide sequence of SEQ ID NO:1.

31. Use of an agent which provokes an immune response to complexes of BAGE derived tumor rejection antigen as set forth in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 and HLA-C
clone 10 molecules for the manufacture of a medicament for treating a subject with a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a tumor rejection antigen presented by HLA-C clone 10 molecules, said BAGE tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2, said agent being a cytolytic T-cell.

32. Use of an agent which provokes an immune response to complexes of an HLA molecule and a tumor rejection antigen precursor for the manufacture of a medicament for treating a subject with a cancer characterized by expression of said tumor rejection antigen precursor coded for by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO:1, said agent being a cytolytic T-cell.

33. Use of an agent specific for a complex for the manufacture of a medicament for diagnosing a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a BAGE derived tumor rejection antigen as set forth in SEQ ID NO:8, SEQ ID
NO:9 OR SEQ ID NO:10 which forms said complex with HLA-C
clone 10 molecules, said BAGE tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2, said agent being a cytolytic T-cell.

34. Use of an antigen encoded by SEQ ID NO:1 for the manufacture of a medicament for diagnosing a cancer characterized by expression of a tumor rejection antigen precursor coded for by a nucleic acid molecule having the sequence set forth in SEQ ID NO:1.

35. Use of an HLA-C clone 10 presenting cell for the manufacture of a medicament for provoking a cytolytic T-cell response, wherein said cell presents complexes of HLA-C clone 10 molecules and a peptide of claim 21 on its surface.

36. Use of cytolytic T-cells which are specific for complexes of BAGE derived tumor rejection antigen as set forth in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 and HLA-C
clone 10 molecules, and which lyse cells presenting said complexes, for the manufacture of a medicament for treating a subject with a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a tumor rejection antigen consisting of the amino acid sequence of SEQ ID NO:10, said BAGE tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2.

37. Use of cytolytic T-cells specific to complexes of an HLA molecule and a tumor rejection antigen consisting of the amino acid sequence of SEQ ID NO:10 for the manufacture of a medicament for treating a subject with a cancer characterized by expression of a tumor rejection antigen precursor coded for by a nucleic acid molecule and comprising the nucleotide sequence of SEQ ID NO:1.

38. Use of an agent which provokes an immune response to complexes of BAGE derived tumor rejection antigen and HLA-C
clone 10 molecules for the manufacture of a medicament for treating a subject with a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a tumor rejection antigen consisting of the amino acid sequence of SEQ ID NO:10, said BAGE tumor rejection antigen precursor being encoded by the nucleotide sequence of claim 2, said agent being a cytolytic T-cell.

39. Use of an agent which provokes an immune response to complexes of HLA-C clone and a peptide consisting of the amino acid sequence of SEQ ID NO:10 for the manufacture of a medicament for treating a subject with a cancer characterized by expression of a tumor rejection antigen precursor coded for by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, said agent being a cytolytic T-cell.

40. Use of an agent specific for a complex for the manufacture of a medicament for diagnosing a cancer characterized by expression of a BAGE tumor rejection antigen precursor which is processed to a BAGE derived tumor rejection consisting of the amino acid sequence of SEQ ID NO:10 which forms said complex with HLA-C clone 10 molecules, said agent being a cytolytic T-cell.

41. Use of an agent specific for a tumor rejection antigen derived from a tumor rejection antigen precursor and consisting of the amino acid sequence of SEQ ID NO:10 for the manufacture of a medicament for diagnosing a cancer characterized by expression of said tumor rejection antigen precursor coded for by a nucleic acid molecule having the sequence set forth in SEQ ID NO:1, said agent being a cytolytic T-cell.