WO2000052163A1

WO2000052163A1 - Cloning of cdna of mage's 5, 8, 9 and 11 and their uses in diagnosis of cancer

Info

Publication number: WO2000052163A1
Application number: PCT/US2000/005346
Authority: WO
Inventors: Alfonso Serrano; Bernard Lethe; Christophe Lurquin; Etienne De Plaen; Donata Rimoldi; Thierry Boon-Falleur
Original assignee: Ludwig Institute For Cancer Research
Priority date: 1999-03-02
Filing date: 2000-03-01
Publication date: 2000-09-08
Also published as: NZ513739A; EP1194542A1; AU3389500A; CA2366059A1; KR20020011967A; JP2003512814A

Abstract

The invention relates to cDNA molecules which were isolated and identified in accordance with a method which was developed to facilitate the level of gene expression. Also described are proteins and peptides based upon these cDNA molecules, as well as various diagnostic and therapeutic uses of these materials.

Description

CLONING OF CDNA OF MAGE'S 5, 8, 9 AND 11 AND THEIR USES IN DIAGNOSIS OF

CANCER

RELATED APPLICATION

This is a continuation in part application of Serial No. 09/260,978, filed March 2,

1999, incorporated by reference.

FIELD OF THE INVENTION

This invention relates to nucleic acid molecules which are members of the MAGE

family, uses thereof, and a method for determining and quantifying their expression. Also

a part of the invention are fragments of these nucleic acid molecules, proteins encoded by

both the whole nucleic acid molecules and the fragments, peptides based thereon which

form complexes with MHC or HLA molecules, fusion proteins, polytopes, and so forth.

BACKGROUND AND PRIOR ART

It was shown by Van der Bruggen, et al., Science 254: 1643-1647 (1991 ), that

there is a family of tumor rejection antigens which complex to human leukocyte antigens

("HLAs"), and provoke response by autologous, cytolytic T cells. In addition to Van der

Bruggen et al., supra, see U.S. Patent No. 5,342,774 to Boon, et al., incorporated by

reference. These references also describe the isolation of genes which encode proteins

that are then processed to these tumor lrejection antigens. Further investigations led to

the discovery of twelve closely related genes. These genes have been found to be located

in region q28 of the X chromosome. While first named genes MAGE-1 through MAGE-

12, these genes are now referred to as MAGE-A1 through MAGE-A12, in view of

subsequent discoveries. To elaborate, four additional related genes have been located in

region p21 of the X chromosome, and are referred to as the MAGE-B cluster of genes. Additional MAGE family members have been located at region q26, and have been named

MAGE-C1 and MAGE-C2.

For obvious reasons, it was and is desirable to analyze expression of MAGE genes.

There has been extensive work in this area, with patterns of MAGE- A expression having

been analyzed by reverse transcription-polymerase chain reaction ("RT-PCR"), in various

tumor samples, tumor cell lines, and normal tissues. Essentially, the level of transcription

and expression is established, semi-quantitatively, via RT-PCR. This entails evaluating

intensity of bands on agarose gels, and comparing these to standard dilutions of material

from a reference or control. This research has established that the genes MAGE-A1 , A2,

A3, A4, A6 and Al 2 are transcribed, at high levels, in many tumors. Gene MAGE-A8

was expressed at a high level in one tumor, while MAGE-A5, A9, A10 and Al 1 appeared

to be transcribed weakly in positive tumors. Collectively, M AGE- A genes were not found

to be expressed by any normal tissues except in testis and, in a few cases, placenta. See

Brasseur, et al, Int. J. Cancer 52:839-841 (1992); Brasseur, etal., Int. J. Cane 63: 375-380

(1995); De Plaen, et al, Immunogenetics 40: 360-369 (1994); van der Bruggen, et al.,

supra, and Weynants, et al., Int. J. Cane 56: 826-829 (1994). The expression of MAGE- A

genes in testis was restricted to germ line cells in the early phases of spermatogenesis.

See Takahashi et al., Cane. Res. 55: 3478-3482 (1995). Testis expresses all MAGE-A

genes, except MAGE-A7. MAGE-A4, A8, A9, A10 and Al 1 have been found to be

transcribed in placenta.

For CTLs to recognize complexes of TRAs and HLAs, a certain level of

expression of the relevant MAGE-A gene appears to be required. DePlaen, et al. Methods 12:125-142 (1997); Lethe, et al., Melanoma Res 7: Suppl 2: S83-8 (1997) have

shown that in melanoma, the level of expression of MAGE-Al must exceed 10% of the

level found in reference cell line MZ2-MEL in order to observe recognition of a MAGE-

Al peptide presented by HLA-A1. The level of expression of MAGE-A2, A3, A4, A6

and A12 genes has been shown, via semi-quantitative RT-PCR, to be similar to MAGE-

Al, suggesting that these genes can be processed into TRAs which are presented for

recognition by CTLs. Several peptides from MAGE-Al and A3 have, in fact, been found

to be presented by HLAs, and then recognized by autologous CTLs derived from mixed

lymphocyte-tumor cell culture.

Recently, it was reported that a monoclonal antibody which was reactive with

MAGE-Al cross reacted with another protein expressed in melanoma. See allowed U.S.

Patent Application, Serial No. 08/724,774 filed on October 3, 1996 and Carrel, et al. Int.

J. Cane 67:417-422 (1996), both of which are incorporated by reference. Subsequently,

it was found that this cross-reactive protein was encoded by MAGE-Al 0. In MZ2-MEL,

the abundance of this protein was similar to that of the MAGE-Al protein. These results

were surprising, since very low levels of expression of MAGE-Al 0 had been found in

MZ2-MEL via RT-PCR. This suggested that the primers used to amplify MAGE-Al 0 in

the RT-PCR were not very efficient. As a result, investigations were undertaken to

develop a method for evaluating frequency of expression of a gene which is independent

of aberrations due to primers. Application of the method, described herein, led to the

isolation of nucleic acid molecules which are described herein, and are a feature of the

invention. BRIEF DESCRIPTION OF THE FIGURE

Figure 1 presents exon/intron structures of MAGE genes, including for MAGE-

A5, A8, A9 and Al l (SEQ ID NOS: 17, 18, 19 and 20).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

EXAMPLE 1

Experiments were carried out to determine whether the choice of primer

influenced values obtained when quantifying frequency of expression via RT-PCR. The

frequency of expression of MAGE-Al 0 was determined using cell line MZ2-MEL, and

one of two pairs of primers. The first pair is described by De Plaen, et al.,

Immunogenetics 40: 360-369 (1994); i.e.:

CACAGAGCAG CACTGAAGGA G (SEQIDNO:l) and

CTGGGTAAAG ACTCACTGTC TGG (SEQ ID NO: 2), or

AGCAGCCAAA AGGAGGAGAG TC (SEQ ID NO: 3)

TGACCTCCTC AGGGGTGCAG TA (SEQ ID NO: 4).

SEQ ID NOS: 3&4 correspond to sequences complementary to the last exon of

MAGE-A 10.

The frequency of expression of MAGE-Al was determined using cell line LB 11 -

OC1, and two pairs of primers , i.e.:

CGGCCGAAGG AACCTGACCC AG (SEQ ID NO: 5) and

GCTGGAACCC TCACTGGGTT GCC (SEQ ID NO: 6)

or

TCAGGGGACA GGCCAACCC (SEQ ID NO: 7)

and

CTTGCACTGA CCTTGATCAC ATA (SEQ ID NO: 8)

In the experiments, total RNA was extracted from cells. Then, 2 μg samples were

used for reverse transcription, following Weynants, et al., supra. The PCR was then

carried out using 1/10 of total cDNA, supplemented with 2.5 μ 1 of 10 x PCR buffer, 2.5

μ 1 of lOmM of dNTP, 10 pmoles of the primers, and 0.5 units of polymerase, plus water

to a volume of 25 μ 1. Each mixture was heated to 94°C for 5 minutes, followed by

amplification for 30 cycles. In the case of SEQ ID NOS: 1-4, 7 & 8, a cycle was 1 minute

at 94°C, two minutes at 65°C, and three minutes at 72°C. For SEQ ID NOS: 5 & 6, a

cycle was 1 minute at 94°C, and 3 minutes at 72°C. Cycling was concluded with a final

extension at 72°C for 5 minutes. Analysis was carried out using 5μl samples, each of

which was run on a 1% agarose gel, and visualized via standard ethidium bromide

staining.

When SEQ ID NOS: 1 & 2 were used, a low level of expression of MAGE-Al 0

was found, whereas use of SEQ ID NOS: 3 & 4 showed a level of expression equivalent

to that of MAGE-Al. This result is in agreement with the Western blotting work of

Carrel, et al., supra, which showed equivalent levels of expression. Given that SEQ ID NOS: 3 & 4 corresponded to regions of the last exon of

MAGE-Al 0, it was possible that contaminating genomic DNA had also been amplified.

To test this possibility, amplification was carried out with omission of the reverse

transcription step. No amplification was observed, however, indicating that there

probably was no contamination. A number of PCR products were sequenced, where SEQ

ID NOS: 3 & 4 had been used as primers. All corresponded to MAGE-Al 0.

When results obtained using the primers for MAGE-Al were compared, different

levels of expression were observed, with SEQ ID NOS: 7 & 8 showing higher levels than

SEQ ID NOS: 5 & 6.

Very low expression levels had been observed previously for M AGE- A5 , A9 and

Al 1. Hence, it was suspected that changing primers might resolve this.

EXAMPLE 2

The results obtained supra suggested that a better method for determining

frequency of expression of genes, MAGE genes for example, was needed. The method

developed is described in this example.

A cDNA library was prepared from a MAGE-A positive sample, following

standard procedures; see De Plaen, et al., Methods 12: 125-142 (1997), incorporated by

reference, and was maintained as recombinant plasmids in E. coli bacteria.

Specifically, samples were homogenized in guanidine thiocyanate to form a lysate,

which was then loaded on top of a CsCl cushion. Then, poly(A)+ RNA was isolated by

processing total RNA through two successive oligo(dT) cellulose columns. The isolated

poly(A)+ RNA was converted to cDNA with an oligo(dT) primer which contained a Notl restriction site. The resulting cDNA was ligated to BstXI adaptors, digested with Notl,

and then inserted into the BstXI and Notl sites of expression vector pcDNAI/Amp. The

resulting recombinant plasmids were introduced into E. coli DH5°_% using standard

electroporation techniques, followed by selection with ampicillin (25 μg/ml).

All libraries were then diluted in LB medium, supplemented with ampicillin, to

obtain 3-6 clones/μl. Following this, 9.6 mis of each dilution were seeded in a 96

microwell plate (100 μls per microwell). Two or three plates were seeded from every

library in order to obtain about 100,000 independent clones spread over the plates. Plates

were then incubated overnight at 37°C, after which 10 μl from every microwell were

pooled, to obtain 20 different pools from every plate (i.e., 8 pools from lines, and 12 pools

from columns). Plates and pools were duplicated, the masters frozen (-70°C, LB medium

containing 20% glycerol), and duplicates were maintained at 4°C for PCR assays.

The PCR assays were carried out on both the living and frozen bacteria, with the

first assays being carried out on pools from lines and columns. Positive microwells were

found at the intersection of a positive line, and a positive column. To carry out the

amplification assay, 3 μl of living bacteria were supplemented with 2.5 μl of 10 x PCR

buffer, 2.5 μl of lOmM dNTP, 10 pmoles of each primer, 0.5 units of polymerase, and

water to a volume of 25 μl .

In most cases, the number of positive wells in a plate was less than 20%. In

accordance with Poisson distribution if the percent of positive clones was less than 20%,

the likelihood of having a single clone in a well should be 90% or greater. Limiting dilution could be carried out to the point where less than 10% of the wells are positive,

with a presumed accuracy of 95%.

In these experiments, as indicated, the number of positives was less than 20%. It

was then possible to calculate the abundance of the different MAGE-A cDNAs in the

library, taking the number of independent clones in each well into account.

The experiments were repeated for seven cDNA libraries (five tumor cell lines,

one testis library, one placenta library) for all twelve MAGE-A genes. The results are

presented in the Table, which follows.

Table 1. Level of expression of MAGE-A genes in tumor cell lines, testis and placenta

MAGE-Al MAGE-A2 MAGE-A3 MAGE-A4 MAGE-A5 MAGE-A6 MAGE-A7 MAGE-A8 MAGE-A9 MAGE-A 10 MAGE-Al 1 MAGE-A 12

MZ2-MEL43 1/5,000 ( 18) 1/6 200 (16) 1/5,000 (20) 0/100 000 1/500 000 1/5,000 (20) 0/100,000 0/100 000 0/100,000 1/6 600 (15) 0/100 000 0/ 100 000

- (-) +/-

LB373-MEL 1/ ,600 (20) 1/2,400 (36) 1/4 000 (23) 1/600 (105) 0 98 000 1/3,500 (25) 0/96,000 0/96,000 1/250,000 1/6,250 (16) 1/20,000 (5) 1/4,800 (19)

+++ (++) +/- (+) +/-

AVL3-MEL 1/34 200 (4) 1/20,800 (6) 1/10,400 (12) 1/3 900 (30) 0/124 000 1/7,000 (18) 0/124,000 0/124,000 0/124,000 1/20,800 (6) 1/124,000 1/20,800 (6)

+++ (+) - (-) +/-

LBl l -SCSC 1/124,000 1/5 000 (24) 1/5,600 (22) 0/124,000 O'l 24,000 1/5,000 (24) 0/124 000 0/124,000 0/124,000 0/124 000 1/124,000 1/8 300 (15) - 0 '-) - (-) +/-

TT-THYR 1/21 600 (5) 1/ 12 000 (9) 1/15,400 (7) 1/6 300 (17) 0/108 000 1/18,000 (6) 0/108 000 1/12,000 (9) 0/800 000 0/108,000 0/108,000 1/12,000 (9)

4 +4-4- - +++ I- - - (-) - (-)

Testis 1/50,000 (2) 1/100000 1/33 000 (3) 1/7,000 (14) 0/100 000 0/100,000 0/100,000 1/100,000 1/100,000 0/100,000 1/50,000 (2) 0/100,000

Placenta 0/227,000 0/227 000 0/227 000 0'227,000 0 227 000 0'227 000 0/227,000 1/75 600 (3) 1/227,000 1/12,600 (18 1/227,000 0/227,000 - (-) +/-

Facing the name of the tumor cell line or of the tissue, the first line gives the abundance of cloned MAGE-A cDNAs in the library calculated, according to the Poisson distribution, from 10 the number of microwel Is scored positive by the PCR assay (number in brackets) One microwell contained 350 to 860 different clones. The second line gives the results of PCR assays performed on uncloned cDNAs The level of expression evaluated by the Intensity of the band obtained by separating PCR products in agarose gels is represented +++, +++, ++, + or +/- Absence of product is indicated by - The first estimation was obtained with previously described primers (De Plaen et al , 1994) A second one presented in brackets for genes MAGE-Al , 9 and 10 was obtained with recently developed pairs of primers

The results were compared to the results obtained in RT-PCR assays, as set forth in

the table supra. MAGE-Al, A9 and AlO were evaluated twice, with different pairs of

primers, and a level of expression was estimated based upon intensity of banding on an

agarose gel.

The limiting dilution assay revealed a level of expression of MAGE- A 10 which was

comparable to that obtained with primers SEQ ID NOS: 3 and 4, and higher than that

obtained with SEQ ID NOS: 1 and 2. These higher values are in agreement with Western

blotting work reported by Carrel, et al., supra, and in allowed U.S. patent application Serial

No. 0/724,774 filed October 3, 1996, incorporated by reference. The frequency was

comparable to that of MAGE-Al in several of the lines. In other lines, while the level

decreased, it was still comparable to levels for Al, A2 or A12.

The calculated frequencies for Al, A2, A3, A4, A6 and A12 were essentially

consistent with results obtained by RT-PCR. In one library, the results (one clone in 124,000

analyzed), was consistent with the results obtained with SEQ ID NOS: 7 and 8, but not SEQ

ID NOS: 5 and 6, suggesting that SEQ ID NOS: 5 and 6 are more efficient at determining

transcript present, but SEQ ID NOS: 7 and 8 are better at determining expression levels.

EXAMPLE 3

One aspect of the results of the experiments described supra, which provoked further

investigation was expression of MAGE- A8. Weak expression was always observed, with the

exception of the cell line TT-THYR, which showed high levels of expression in a semi-

quantitative PCR assay.

The average size of an insert in the TT-THYR library was only 0.9kb, so primers were

designed which would amplify a segment of the last 450 nucleotides of MAGE-A8 mRNA.

Similar primers were designed for MAGE-Al, A2, A4 and A8 as well, i.e.:

SEQ ID NO.: 9 GAAGAGAGCGGTCAGTGTTC-3 (sense)

SEQ ID NO.: 10 AATCCAGGTATGCATATATCTTTA (anti-sense)

SEQ ID NO. : 11 GCCTCTTTGAAGAGAGC AGTC (sense)

SEQ ID NO. : 12 C AAAGAAGC AAAAAC AT AC AC ATA (anti-sense)

SEQ ID NO. : 13 CACTCTGTTTGAAGAAAATAGTC (sense)

SEQ ID NO. : 14 AGTATCTTTT AATTTATCTCACCTA (anti-sense)

SEQ ID NO.: 15 AGCATGTTGGGTGTGAGGGA (sense)

SEQ ID NO. : 16 AGGGTAC ACTAAGAGGT AC AG (anti-sense) (SEQ ID NOS: 9 and 10 amplify Al, NOS: 11 and 12 amplify A2, NOS: 13 and 14

amplify A4, and NOS: 15 and 16 amplify A8)

RT-PCR was carried out as described, supra, using these primers. When completed, the

frequency of MAGE- A8 expression was found to be much higher; i.e., on a par with MAGE-

A2.

When the results for testis were analyzed, the level of expression of MAGE- A4 was

found to be higher than MAGE-Al, levels of A2, A3, A8, A9 and Al l were low, and no

cDNA for A6, AlO or A12 was found. These results are in accordance with those provided

by Canel et al. supra, for MAGE-Al AND MAGE-A10.

With respect to placenta, MAGE-A 10 showed the highest level of expression, while

A1-A7 A9 and A12 were not found at all among 230,000 clones analyzed.

EXAMPLE 4

While the literature on the MAGE-A genes is substantial, cDNA for several members

of the family has never been isolated, notwithstanding inferences regarding their structure,

based upon the structure of MAGE-Al .

The approach described in example 2, supra, led to isolation of cDNA for MAGE-A5,

A8, A9, AlO and Al l. The cDNA for MAGE-A10 was described in e.g., U.S. patent

application Serial No. 08/724,774, filed October 3, 1996 and incorporated by reference, but

the others were not known. The cDNA for A5, A8, A9 and Al 1 is presented as SEQ ID

NOS: 17 through 20, respectively. Further, knowledge of the sequences of cDNA led to an

ability to complete exon/intron structures of the genes, as shown in figure 1. The sequencing of MAGE-A5 cDNA led to some interesting observations, as it

consisted of the first two exons of MAGE-A 10, followed by a sequence corresponding to a

previously unknown exon, and two exons of MAGE- A5.

The foregoing examples describe the invention, which in addition to nucleic acid

molecules as described herein includes a method for deteraiining the frequency of expression

of a particular gene or gene of interest. The method involves preparing cDNA from a sample,

and then transforming or transfecting the cDNA into cells, to create a library of

transformants/transfectants. These cells are then divided into a plurality of samples of

approximately equal size, after which each sample is assayed for presence (as compared to

quantity), of cDNA. The number of positive samples should be less than or equal to 20% of

the total number of and, more preferably, equal or less than 10% of the number of samples

being tested. If the number of positives is greater than the chosen value, then the library is

diluted, divided into samples and the assay is repeated. When the positive value is below the

chosen value, the frequency of each MAGE cDNA is determined.

Preferably, the method is carried out by distributing the samples in a predetermined

array, so that different portions of samples can be pooled. When the samples are anayed in

this way, one can determine which samples do contain the cDNA of interest, by determining

where the two sample pools intersect. For example, consider a rectangular array of samples,

arranged in vertical and horizontal lines. If the horizontal lines are represented by letters, i.e.,

"A", "B", "C", etc., and vertical lines by numbers, i.e., "1", "2", "3", etc., then one can create

pools "A", "B", "2", "3", etc. Each vertical and horizontal line will intersect at one point, these points being represented by codes such as "Al", "B2", "C3", "D4", etc. If both pool "B"

and pool "7" are positive, then one can conclude that the sample at point "B7" is positive. By

doing this, one can identify a well containing the sample of interest.

In addition to quantifying expression, the method permits the artisan to identify cDNA

molecules of interest, especially those which are present at low frequency. As was described

herein, practice of the invention led to isolation of cDNA for various MAGE genes. Such

cDNA had not been isolated previously, and is a further feature of the invention, i.e., isolated

cDNA molecules which encode MAGE-A8, MAGE-A9, and MAGE-11 proteins, such as

cDNA molecules which encode proteins whose amino acid sequence is that of the protein

encoded by any of SEQ ID NOS : 18, 19 or 20.

Also a part of the invention are newly isolated nucleic acid molecules, such as the one

set forth in SEQ ID NO: 17 or other similar molecules i.e., those comprising two exons for

MAGE-A5 and two exons for MAGE-Al 0, separated by a nucleotide sequence between the

MAGE-A5 and MAGE-Al 0 sequences as well as nucleic acid molecules which comprise

portions that hybridize to both the MAGE-A5 portion, and the MAGE-Al 0 portion. These

nucleic acid molecules, i.e., all of the nucleic acid molecules described herein, can be used

to make expression vectors which comprise the nucleic acid molecule operably linked to a

promoter. These vectors, as well as the isolated nucleic acid molecules themselves, can be

used to transform or to transfect cells, to produce recombinant cells thereby.

These nucleic acid molecules can also be used both diagnostically and therapeutically.

In the diagnostic area, one can examine a sample, such as a cell containing sample, a cell lysate, etc., for expression of the nucleic acid molecules described herein, using oligomer

probes in connection with standard methods, such as polymerase chain reactions, and so forth.

One can also assay such samples by determining presence of the proteins encoded thereby.

Also a part of the invention are methods based upon these newly identified and

isolated molecules. For example, one can determine expression of a MAGE gene by

contacting a sample with one or more oligonucleotides which hybridize specifically to a

MAGE nucleic acid molecule of interest. For example SEQ ID NO: 9 and or SEQ ID NO:

10 can be used to determine MAGE-Al , SEQ ID NO: 11 and/or 12 can be used to determine

MAGE-A2, and so forth. Any form of hybridization based assay can be used, as exemplified,

but not limited to PCR. One can also assay for the MAGE proteins, using standard

methodologies such as antibody assays, or other systems for determining proteins.

Also apart of the invention are peptides consisting of from about 8 to about 25 amino

acids concatenated to each other along the amino acid sequence of the MAGE proteins which

are a part of the invention. Such peptides are specific binders for MHC molecules, such as

MHC Class I or Class II molecules, including HLA molecules such as HLA-Al, A2, A3,

A24, B7, B8, B35, B44, B52, and CW6. Determination of relevant sequences can be carried

out using, e.g., Parker, et al, J. Immunol 152:163 (1994), D'Amaro, et al Hum. Immunol

43:13-18 (1995), Drijfhout, et al, Hum. Immunol 43:1-12 or Sturniolo, et al, Nat. Biotechnol

17(6):555-61 (1999) all of which are incorporated by reference, or websites such the NIH

worldwide web site, found at URLhttp:\\bimas. dcrt.nih.gov., and http://www.uni-

tuebingen.de/uni/kxc, which are incorporated by reference. The tables which follow list some of these peptides, with reference to the relevant MAGE amino acid sequence. The complete

amino acid sequences are set out at SEQ ID NOS:21-24, where SEQ ID NO:21 is that for

MAGE A5, SEQ ID NO:22 is that for MAGE A8, SEQ ID NO:23 is that for MAGE A9, and

SEQ ID NO:24 is that for MAGE Al 1 :

MAGE A5: HLA-Al Binders

98-107 SPDPESVFR

69-78 SAIPTAIDF

32-41 TTEEQEAVS

116-125 LIHFLLLKY 113-122 VADLIHFLL

115-124 DLIHFLLLK

2-11 SLEQKSQHC

77-86 FTLWRQSIK

73-82 TAIDFTLWR 74-83 AIDFTLWRQ

15-24 GLDTQEEAL

MAGE A5: HL A- A2 Binders

112-123 KVADLIHFL

108-117 ALSKKVADL

24-33 GLVGVQAAT

70-79 AIPTAIDFT 38-47 AVSSSSPLV

22-31 ALGLVGVQA

15-24 GLDTQEEAL

45-54 LVPGTLGEV

31-40 ATTEEQEAV

71-80 IPTAIDFTL

113-122 VADLIHFLL

25-34 LVGVQAATT

78-87 TLWRQSIKG

48-57 GTLGEVPAA

18-27 TQEEALGLV

MAGE A5: HLA- A3 Binders

115-124 DLIHFLLLK

103-112 SVFRAALSK

108-116 ALSKKVADL

15-23 GLDTQEEAL

77-85 FTLWRQSIK

116-124 LIHFLLLKY

24-32 GLVGVQAAT

73-81 TAIDFTLWR

22-30 ALGLVGVQA

MAGE A5: HLA- A24 Binders 112-120 KVADLIHFL

42-50 SSPLVPGTL

17-25 DTQEEALGL

37-45 EAVSSSSPL

76-84 DFTLWRQSI

113-121 VADLIHFLL

71-79 IPTAIDFTL

15-23 GLDTQEEAL

108-116 ALSKKVADL

69-77 SAIPTAIDF

97-105 TSPDPESVF

63-71 KSPQGASAI

109-117 LSKKVADLI

67-75 GASAIPTAI

114-122 ADLIHFLLL

111-119 KKVADLIHF

MAGE A5: HLA-B7 Binders

71-79 IPTAIDFTL

112-123 KVADLIHFL

108-116 ALSKKVADL

37-45 EAVSSSSPL

17-25 DTQEEALGL

42-50 SSPLVPGTL

113-121 VADLIHFLL

J8- 38-47 AVSSSSPLV

60-68 GPLKSPQGA

54-62 PAAGSPGPL

114-122 ADLIHFLLL

67-75 GASAIPTAI

15-23 GLDTQEEAL

45-53 LVPGTLGEV

30-38 AATTEEQEA

31-39 ATTEEQEAV

100-108 DPESVFRAA

8-16 QHCKPEEGL

25-33 LVGVQAATT

109-117 LSKKVADLI

MAGE A5: HLA-B 8 Binders

108-117 ALSKKVADL

37-46 EAVSSSSPL

109-118 LSKKVADLI

71-80 IPTAIDFTL

67-75 GASAIPTAI

42-50 SSPLVPGTL

102-110 ESVFRAALS

MAGE A5: HLA-B35 Binders 71-79 IPTAIDFTL

97-105 TSPDPESVF

109-118 LSKKVADLI

42-50 SSPLVPGTL

63-71 KSPQGASAI

112-120 KVADLIHFL

37-46 EAVSSSSPL

69-77 SAIPTAIDF

17-25 DTQEEALGL

60-68 GPLKSPQGA

116-124 LIHFLLLKY

67-75 GASAIPTAI

108-116 ALSKKVADL

113-121 VADLIHFLL

100-107 DPESVFRAA

31-39 ATTEEQEAV

106-114 RAALSKKVA

41-49 SSSPLVPGT

102-110 ESVFRAALS

30-38 AATTEEQEA

MAGE A5: HLA-B44 Binders

69-77 SAIPTAIDF

34-42 EEQEAVSSS

20-28 EEALGLVGV 33-41 TEEQEAVSS

90-98 QEEEGPSTS

51-59 GEVPAAGSP

114-122 ADLIHFLLL

41-49 SSSPLVPGT

92-100 EEGPSTSPD

97-105 TSPDPESVF

48-56 GTLGEVPAA

91-99 EEEGPSTSP

36-44 QEAVSSSSP

116-124 LIHFLLLKY

56-64 AGSPGPLKS

23-31 LGLVGVQAA

13-23 EEGLDTQEE

75-83 IDFTLWRQS

100-108 DPESVFRAA

17-25 DTQEEALGL

MAGE A5: HLA-B 52 Binders

18-26 TQEEALGLV

97-105 TSPDPESVF

45-53 LVPGTLGEV

109-117 LSKKVADLI

71-79 IPTAIDFTL

63-71 KSPQGASAI 23-31 LGLVGVQAA

67-75 GASAIPTAI

20-28 EEALGLVGV

69-77 SAIPTAIDF

46-54 VPGTLGEVP

14-22 EGLDTQEEA

106-114 RAALSKKVA

113-121 VADLIHFLL

31-39 ATTEEQEAV

60-68 GPLKSPQGA

76-84 DFTLWRQSI

96-104 STSPDPESV

89-107 NQEEEGPST

112-120 KVADLIHFL

MAGE A5: HLA-CW6 Binders

112-120 KVADLIHFL

108-116 ALSKKVADL

71-79 IPTAIDFTL

113-121 VADLIHFLL

116-124 LIHFLLLKY

105-113 FRAALSKKV

67-75 GASAIPTAI

18-26 TQEEALGLV

37-45 EAVSSSSPL 114-122 ADLIHFLLL

45-53 LVPGTLGEV

42-50 SSPLVPGTL

15-23 GLDTQEEAL

8-16 QHCKPEEGL

20-28 EEALGLVGV

17-25 DTQEEALGL

41-49 SSSPLVPGT

63-71 KSPQGASAI

76-84 DFTLWRQSI

109-117 LSKKVADLI

MAGE Al l: HLA-Al Binders

376-384 GTDPACYEF

281-290 EVDPTSHSY

211-220 LIDPESFSQ

71-80 NLEDRSPRR

142-150 QAEEQEAAF

352-360 FGEPKRLLT

MAGE Al l : HL A- A2 Binders

313-321 GLLIIVLGV 350-358 FLFGEPKRL 221-229 ILHDKIIDL

89-97 VLWGPITQI

333-341 VMWEVLSIM

384-392 FLWGPRAHA

271-279 MQLLFGIDV

225-233 KIIDLVHLL

398-406 KVLEYIANA

337-345 VLSIMGVYA

289-297 YVLVTSLNL

316-324 IIVLGVIFM

335-353 WEVLSIMGV

MAGE Al 1 : HLA- A3 Binders

272-280 QLLFGIDVK

228-236 DLVHLLLRK 89-97 VLWGPITQI

359-367 LTQNWVQEK

313-321 GLLIIVLGV

MAGE Al l: HLA-A24 Binders

343-351 VYAGREHFL 255-263 NYEDYFPEI 351-359 LFGEPKRLL

225-234 KIIDLVHLL

288-296 SYVLVTSLN

236-244 KYRVKGLIT 82-90 RITGGEQVL

311-319 KSGLLIIVL

413-421 SYPSLYEDA

MAGE Al 1 : HLA-B7 Binders

98-106 FPTVRPADL 414-422 YPSLYEDAL

283-291 DPTSHSYVL

64-73 QVFREQANL

76-84 SPRRTQRIT

289-297 YVLVTSLNL 127-135 QAQEEDLGL

307-315 QSMPKSGLL

266-279 EASVCMQLL

147-155 EAAFFSSTL

MAGE Al 1 : HLA-B8 Binders

98-106 FPTVRPADL

221-229 ILHDKIIDL 241-250 GLITKAEML

234-242 LRKYRNKGL

2-10 ETQFRRGGL

309-317 MPKSGLLII 307-315 QSMPKSGLL

MAGE Al 1: HLA-B5 Binders

374-382 VPGTDPACY

410-418 DPTSYPSLY

102-110 RPADLTRVI

394-402 TSKMKVLEY

309-317 MPKSGLLII

283-291 DPTSHSYVL

181-190 SPTAMDAIF

414-422 YPSLYEDAL

98-106 FPTVRPADL

389-497 RAHAETSKM

48-56 APYGPQLQW

311-319 KSGLLIIVL

378-386 DPACYEFLW

MAGE Al 1 : HLA-B44 Binders 143-151 AEEQEAAFF

58-66 QDLPRVQVF

256-269 YEDYFPEIF

392-400 AETSKMKVL 166-174 AESPSPPQS

144-152 EEQEAAFFS

394-402 TSKMKVLEY

280-288 KEVDPTSHS

265-273 REASVCMQL 406-414 ANGRDPTSY

410-418 DPTSYPSLY

335-343 WEVLSIMGV

146-154 QEAAFFSST

331-339 EEVMWEVLS

MAGE Al l : HLA-B52 Binders

309-318 MPKSGLLII

102-110 RPADLTRVI

314-322 LLIIVLGVI

333-341 VMWEVLSIM

271-279 MQLLFGIDV

218-226 SQDILHDKI

181-189 SPTAMDAIF

315-323 LIIVLGVIF

29-37 FGLQVSTMF 219-227 QDILHDKII

57-65 SQDLPRVQV

90-98 LWGPITQIF

245-254 KAEMLGSVI

329-338 IPEEVMWEV

256-264 YEDYFPEIF

58-66 QDLPRVQVF

128-136 AQEEDLGLV

283-291 DPTSHSYVL

87-95 EQVLWGPIT

325-334 EGNCIPEEV

MAGE Al 1 : HLA-CW6 Binders

225-233 KIIDLVHLL

311-319 KSGLLIIVL

287-295 HSYVLVTSL

221-229 ILHDKIIDL

147-155 EAAFFSSTL

229-237 LVHLLLRKY

269-277 VCMQLLFGI

244-252 TKAEMLGSV

313-321 GLLIIVLGV

222-230 LHDKIIDLV

184-192 AMDAIFGSL MAGE A9: HLA-Al Binders

94-103 SVDPAQLEF

167-176 EVDPAGHSY

262-271 GSDPAHYEF

1354-143 MLESVIKNY

153-162 ASEFMQVIF

189-198 LGDGHSMPK

238-247 YGEPRKLLT

112-121 VAELVHFLL

24B-255 TQDWVQENY

280-289 TSYEKVINY

249-258 WVQENYLEY

MAGE A9: HLA-A2 Binders

199-208 ALLIIVLGV

233-232 ALSVMGVYV

102-111 FMFQEALKL

307-316 VLGEEQEGV

270-279 FLWGSKAHA

175-184 YILVTALGL

E3D7-166 MQVIFGTDV

140-149 KNYKRYFPV

219-228 VIWEALSVM

290-299 VMLNAREPI

284-293 KVINYLVML 221-230 WEALSVMGV

24-33 GLMGAQEPT

187-196 SMLGDGHSM

MAGE A9: HLA-A3 Binders

2355-244 HMFYGEPRK

114-123 ELVHFLLHK

203-212 IVLGVILTK

225-234 SVMGVYVGK

102-111 FMFQEALKL

B3M-143 MLESVIKNY

158-167 QVIFGTDVK

118-127 FLLHKYRVK

199-208 ALLIIVLGV

107-116 ALKLKVAEL

27D-279 FLWGSKAHA

148-157 VIFGKASEF

MAGE A9: HLA-A24 Binders

281-290 SYEKVINYL

237-246 FYGEPRKLL

2ZP-238 VYVGKEHMF

71-80 VYYTLWSQF 141-150 NYKRYFPVI

236-245 MFYGEPRKL

284-293 KVINYLVML

MAGE A9: HLA-B7 Binders ιω-ns DPAGHSYIL

300-309 YPSLYEEVL

127-136 EPVTKAEML

284-293 KVINYLVML

111-120 KVAELVHFL

1HS7-206 KAALLIIVL

17-26 EAQGEDLGL

107-116 ALKLKVAEL

193-202 HSMPKAALL

195-204 MPKAALLII

228-237 GVYVGKEHM

181-190 LGLSCDSML

201-210 LIIVLGVIL

173-182 HSYILVTAL

175-184 YILVTALGL

2D- 101 SSSVDPAQL

67-76 SSISVYYTL

102-111 FMFQEALKL

112-121 VAELVHFLL

180-189 ALGLSCDSM MAGE A9: HLA-B8 Binders

107-116 ALKLKVAEL

127-136 EPNTKAEML

195-204 MPKAALLII

153-202 HSMPKAALL

169-178 DPAGHSYIL

300-309 YPSLYEEVL

MAGE A9: HLA-B52 Binders

195-204 MPKAALLII

2⁽10-209 LLIIVLGVI

219-228 VIWEALSVM

157-166 MQVIFGTDV

104-113 FQEALKLKV

131-140 KAEMLESVI

DS2-161 KASEFMQVI

96-105 DPAQLEFMF

201-210 LIIVLGVIL

300-309 YPSLYEEVL

212-221 DΝCAPEEVI

QSP-178 DPAGHSYIL

278-287 AETSYEKVI 141-150 NYKRYFPVI

MAGE A9: HLA-CW6 Binders

197-206 KAALLIIVL

111-120 KVAELVHFL

173-182 HSYILVTAL

107-116 ALKLKVAEL

199-208 ALLIIVLGV

100-109 LEFMFQEAL

130-139 TKAEMLESV

10 115-124 LVHFLLHKY

201-210 LIIVLGVIL

MAGE A9: HLA-B44 Binders

105-113 QEALKLKVA 21-229 WEALSVMGV 75-55 EEVSAAGSS 96-304 EPICYPSLY 80-288 TSYEKVINY 17-225 EEVIWEALS 55-263 LEYRQVPGS X6-286 AETSYEKVI

166-174 KEVDPAGHS 33-41 GEEEETTSS

96-104 DPAQLEFMF

222-230 EALSVMGVY

MAGE A9: HLA-B35 Binders

5 260-269 VPGSDPAHY

296-305 EPICYPSLY

195-204 MPKAALLII

300-309 YPSLYEEVL

127-136 EPVTKAEML

10 96-105 DPAQLEFMF

169-178 DPAGHSYIL

280-289 TSYEKVINY

65-74 ASSSISVYY

264-273 DPAHYEFLW

15 92-101 SSSVDPAQL

18-27 AQGEDLGLM

222-231 EALSVMGVY

64-73 GASSSISVY

197-206 KAALLIIVL

20 MAGE A8: HLA-Al Binders 171-180 EVDPAGHSY

266-275 GSDPVRYEF

138-147 MLESVIKNY

157-166 ASECMQVIF

250-259 TQEWVQENY

98-107 SPDPAHLES

116-125 VAELVRFLL

253-262 WVQENYLEY

193-202 LGDDQSTPK

181-190 LVTCLGLSY

MAGE A8: HLA-B52 Binders

199-208 TPKTGLLII

223-232 AIWEALSVM

161-170 MQVIFGIDV

286-295 YVKVLEHVV

204-213 LLIIVLGMI

135-144 KAEMLESVI

262-271 RQAPGSDPV

205-214 LIIVLGMIL

156-165 KASECMQVI

173-182 DPAGHSYIL

MAGE A8: HLA- A3 Binders 280-289 ALAETSYVK

118-127 ELVRFLLRK

122-131 FLLRKYQIK

1-10 MLLGQKSQR

203-212 GLLIIVLGM

210-219 GMILMEGSR

162-171 QVIFGIDVK

138-147 MLESVIKNY

2-11 LLGQKSQRY

111-120 ALDEKVAEL

274-283 FLWGPRALA

29-38 QIPTAEEQK

24-33 GLMDVQIPT

253-262 WVQENYLEY

MAGE A8 : HLA-B7 Binders

299-308 RVRISYPSL

304-313 YPSLHEEAL

173-182 DPAGHSYIL

22-31 APGLMDVQI 64-73 SPEGASSSL

240-249 SVYWKLRKL

115-124 KVAELVRFL

37-46 KAASSSSTL

17-26 QAQGEAPGL 199-208 TPKTGLLII

216-225 GSRAPEEAI

196-205 DQSTPKTGL

116-125 VAELVRFLL

MAGE A8: HLA-B8 Binders

197-206 QSTPKTGLL

199-208 TPKTGLLII

240-249 SVYWKLRKL

297-306 NARVRISYP

111-120 ALDEKVAEL

299-308 RVRISYPSL

MAGE A8: HLA-A2 Binders

288-297 KVLEHVVRV

274-283 FLWGPRALA

24-33 GLMDVQIPT

111-120 ALDEKVAEL

115-124 KVAELVRFL

45-54 LIMGTLEEV

179-188 YILVTCLGL

161-170 MQVIFGIDV

203-212 GLLIIVLGM 191-200 GLLGDDQST

223-232 AIWEALSVM

71-80 SLTVTDSTL

279-288 RALAETSYV 251-260 QEWVQENYL

184-193 CLGLSYDGL

MAGE A8: HLA-A24 Binders

241-250 VYWKLRKLL

145-154 NYKNHFPDI

273-282 EFLWGPRAL

121-130 RFLLRKYQI

126-135 KYQIKEPVT

115-124 KVAELVRFL

285-294 SYVKVLEHV

303-312 SYPSLHEEA

201-210 KTGLLIIVL

116-125 VAELVRFLL

299-308 RVRISYPSL

37-46 KAASSSSTL

205-214 LIIVLGMIL

17-26 QAQGEAPGL

64-73 SPEGASSSL

131-140 EPVTKAEML

179-188 YILVTCLGL 185-194 LGLSYDGLL

42-51 SSTLIMGTL

111-120 ALDEKVAEL

MAGE A8: HLA-CW6 Binders

115-1241 KVAELVRFL

201-210 KTGLLIIVL

177-186 HSYILVTCL

240-249 SVYWKLRKL

111-120 ALDEKVAEL

241-250 VYWKLRKLL

119-128 LVRFLLRKY

205-214 LIIVLGMIL

104-113 LESLFREAL

42-51 SSTLIMGTL

134-143 TKAEMLESV

MAGE A8: HLA-B35 Binders

264-273 APGSDPVRY

199-208 TPKTGLLII

304-313 YPSLHEEAL 131-140 EPVTKAEML

173-182 DPAGHSYIL 100-109 DPAHLESLF

39-48 ASSSSTLIM

268-277 DPVRYEFLW

MAGE A8: HLA-B44 Binders

282-291 AETSYVKVL

20-29 GEAPGLMDV

225-234 WEALSVMGL

33-42 AEEQKAASS

234-243 YDGREHSVY 109-118 REALDEKVA

221-230 EEAIWEALS

170-179 KEVDPAGHS

34-43 EEQKAASSS

296-305 VNARVRISY 226-235 EALSVMGLY

237-246 REHSVYWKL

Compositions based upon these molecules are also a part of the invention, such as

compositions containing a MAGE protein in accordance with the invention, and a

pharmaceutically acceptable adjuvant such as a cytokine, an interleukin (e.g., IL-2JL-4, IL-

12, etc.), or GM-CSF. Similarly, compositions containing one or more of the peptides discussed supra and an adjuvant, complexes of HLA or MHC molecules and the peptides plus

adjuvant are also a part of the invention.

These complexes can be combined per se, or on antigen presenting cells, such as

dendritic cells, which may be treated to be rendered non-proliferative, etc.

The skilled artisan will also recognize that nucleic acid molecules encoding the

peptides or proteins may be used in the form of appropriate compositions, such as in liposome

based compositions. Also a part of the invention are isolated cytolytic T cell lines which are

specific for complexes of these peptides and their MHC binding partner, i.e., an HLA

molecule.

The ability of these peptides to bind to HLA molecules makes them useful as agents

for determining presence of cells positive for particular HLA molecules such as HLA- A*0201

positive cells, by determining whether or not the peptides bind to cells in a sample. This

"ligand/receptor" type of reaction is well known in the art, and various methodologies are

available for determining it.

A further aspect of the invention are so-called "mini genes" which carry information

necessary to direct synthesis of modified decapeptides via cells into which the mini genes are

transfected. Mini genes can be designed which encode one or more antigenic peptides, and

are then transferred to host cell genomes via transfection with plasmids, or via cloning into

vaccinia or adenoviruses. See, e.g., Zajac, et al, Int. J. Cancer 71 : 496 (1997), incorporated

by reference These recombinant vectors, such as recombinant vaccinia virus vectors, can be

constructed so as to produce fusion proteins. For example, fusion proteins can be constructed where one portion of the fusion protein is the desired tumor rejection antigen precursor, or

tumor rejection antigen, and additional protein or peptide segments can be included.

Exemplary, but by no means the only types of additional protein or peptide segments which

can be added to the fusion proteins, are reporter proteins or peptides, i.e., proteins or peptides

which give an observable signal so as to indicate that expression has occurred, such as green

fluoresence protein. Additional reporter proteins include, but are by no means limited to,

proteins such as βgalactosidase, luciferase, dhfr, and "eGFP", or enhanced green fluorescent

protein, as described by Cheng, et al., Nature Biotechnology 14:606 (1996), incorporated by

reference, and so forth. The various reporter proteins available to the skilled artisan can be,

and are used, in different ways. For example, "GFP" and "eGFP" can be used to visualize

infected cells, thereby facilitating tracking when flow cytometry is used, and the isolation of

the cells so infected. Other reporter proteins are useful when methods such as western

blotting, immunoprecipitation, and so forth are used. These techniques are standard in the

art and need not be reiterated here. Protein or peptide segments which facilitate the cleavage

of the tumor rejection antigen precursor or tumor rejection antigen from the fusion peptide

may also be included. The fusion protein can include more than one tumor rejection antigen,

as described, supra , and can also include proteins or peptides which facilitate the delivery of

the tumor rejection antigen or antigens to a relevant MHC molecule. Such proteins and

peptides are well known to the art, and need not be elaborated herein.

Also a part of the invention are recombinant cells which have been transfected with

the recombinant vectors described herein. Such cells may be, e.g., any type of eukaryotic cell, with human cells being especially preferred. Such cells can then be used, e.g., to

produce tumor rejection antigen precursors or tumor rejection antigens. They can also be

used, in an ex vivo context, to generate cytolytic T cells specific for particular complexes of

MHC molecules and tumor rejection antigens. This can be done simply by contacting the

transfected cells to a source of T cells, such as a blood sample, so as to provoke the

proliferation of any cells in the sample specific to the complexes of MHC molecules and

TRAs (i.e., tumor rejection antigens) produced following expression of the fusion protein, and

processing of the TRA. Such cells, when rendered non-proliferative, can also be used as

vaccine materials, as they will present the relevant complexes on their surface, and provoke

the same type of T cell response in vivo, as is shown herein. Similarly, the vectors can be

used as vaccine materials per se, and can be administered to a patient in need of a T cell

response against complexes of MHC molecules and peptide on cell surfaces. Of course, T

cells generated ex vivo can also be used to treat patients.

The peptides may be combined with peptides from other tumor rejection antigens to

form 'polytopes'. Exemplary peptides include those listed in U.S. Patent Application Serial

Numbers 08/672,351, 08/718,964, now U.S. Patent No. , 08/487,135 now U.S.

Patent No. 08/530,569 and 08/880,963 all of which are incorporated by

reference.

Additional peptides which can be used are those described in the following references,

all of which are incorporated by reference: U.S. Patent Nos. 5,405,940; 5,487,974;

5,519,117; 5,530,096; 5,554,506; 5,554,724; 5,558,995; 5,585,461; 5,589,334; 5,648,226; and 5,683,886; PCT International Publication Nos. 92/20356; 94/14459; 96/10577; 96/21673;

97/10837; 97/26535; and 97/31017 as well as pending U.S. Application Serial No.

08/713,354.

Polytopes are groups of two or more potentially immunogenic or immune stimulating

peptides, which can be joined together in various ways, to determine if this type of molecule

will stimulate and/or provoke an immune response.

These peptides can be joined together directly, or via the use of flanking sequences.

See Thompson et al., Proc. Natl. Acad. Sci. USA 92(13): 5845-5849 (1995), teaching the

direct linkage of relevant epitopic sequences. The use of polytopes as vaccines is well

known. See, e.g., Gilbert et al.. Nat. Biotechnol. 15(12): 1280-1284 (1997); Thomson et al,

supra; Thomson et al, J. Immunol. 157(2): 822-826 (1996); Tam et al., J. Exp. Med. 171(1):

299-306 (1990), all of which are incorporated by reference. The Tam reference in particular

shows that polytopes, when used in a mouse model, are useful in generating both antibody

and protective immunity. Further, the reference shows that the polytopes, when digested,

yield peptides which can be and are presented by MHCs. Tam shows this by showing

recognition of individual epitopes processed from polytope 'strings' via CTLs. This approach

can be used, e.g., in determining how many epitopes can be joined in a polytope and still

provoke recognition and also to determine the efficacy of different combinations of epitopes.

Different combinations may be 'tailor-made' for the patients expressing particular subsets of

tumor rejection antigens. These polytopes can be introduced as polypeptide structures, or via

the use of nucleic acid delivery systems. To elaborate, the art has many different ways available to introduce DNA encoding an individual epitope, or a polytope such as is discussed

supra. See, e.g., Allsopp et al., Eur. J. Immunol. 26(8); 1951-1959 (1996), incorporated by

reference. Adenovirus, pox-virus, Ty-virus like particles, plasmids, bacteria, etc., can be

used. One can test these systems in mouse models to determine which system seems most

appropriate for a given, parallel situation in humans. They can also be tested in human

clinical trials.

Also, a feature of the invention is the use of these peptides to determine the presence

of cytolytic T cells in a sample. It was shown, supra, that CTLs in a sample will react with

peptide/MHC complexes. Hence, if one knows that CTLs are in a sample, cells positive for

particular HLA molecules can be "lysed" by adding the peptides of the invention to positive

cells, such as HLA-A2 positive cells, and then determining, e.g., radioactive chromium

release, TNF production, etc. or any other of the methods by which T cell activity is

determined. Similarly, one can determine whether or not specific tumor infiltrating

lymphocytes ("TILs") are present in a sample, by adding one of the claimed peptides with

HLA positive cells to a sample, and determining lysis of the HLA positive cells via, e.g., ⁵¹Cr

release, TNF presence and so forth. In addition, CTL may be detected by ELISPOT analysis.

See for example Schmittel et al., (1997). J. Immunol. Methods 210: 167-174 and Lalvani et

al., (1997). J. Exp. Med. 126: 859 or by FACS analysis of fluorogenic tetramer complexes

of MHC Class I/peptide (Dunbar et al., (1998), Current Biology 8: 413-416, Romero, et al.,

J. Exp. Med. 188: 1641-1650 (1998). All are incorporated by reference. Of course, the peptides may also be used to provoke production of CTLs. As was

shown, supra, CTL precursors develop into CTLs when confronted with appropriate

complexes. By causing such a "confrontation" as it were, one may generate CTLs. This is

useful in an in vivo context, as well as ex vivo, for generating such CTLs.

Other aspects of the inventions will be clear to the skilled artisan and will not be

restricted herein.

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.

Claims

WE CLAIM

1. An isolated, complementary DNA molecule which encodes a protein

encoded by a nucleic acid molecule consisting of the nucleotide sequence set

forth in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

2. The isolated, complementary DNA molecule of claim 1, which encodes the

protein encoded by SEQ ID NO: 18.

3. The isolated, complementary DNA molecule of claim 1, which encodes the protein encoded by SEQ ID NO: 19.

4. The isolated nucleic acid molecule of claim 1, which encodes the protein

encoded by SEQ ID NO: 20.

5. The isolated complementary DNA molecule of claim 1, consisting of the

nucleotide sequence of SEQ ID NO: 18.

6. The isolated complementary DNA molecule of claim 1 , consisting of the

nucleotide sequence of SEQ ID NO: 19.

7. The isolated nucleic acid molecule of claim 1 , consisting of the nucleotide

sequence of SEQ ID NO: 20.

8. Expression vector comprising the complementary DNA molecule of claim 1 ,

operably linked to promoter.

9. Recombinant cell comprising the isolated complementary DNA molecule of claim 1.

10. Recombinant cell comprising the expression vector of claim 7.

11. An isolated nucleic acid molecule which comprises (i) a nucleotide sequence

which hybridizes to an isolated nucleic acid molecule which encodes MAGE-

AlO, under stringent conditions, (ii) a second nucleotide sequence which

hybridizes to an isolated nucleic acid molecule which encodes MAGE-A5,

under stringent condition, and (iii) a third nucleotide sequence which is

interposed between (i) and (ii).

12. The isolated nucleic acid molecule of claim 11, comprising the nucleotide

sequence set forth at SEQ ID NO: 17.

13. Expression vector comprising the isolated nucleic acid molecule of claim 11,

operably linked to a promoter.

14. Recombinant cell comprising the isolated nucleic acid molecule of claim 11.

15. Recombinant cell comprising the isolated nucleic acid molecule of claim 12.

16. A method for screening for cancer in a sample, comprising determining

presence of a nucleic acid molecule comprising the nucleotide sequence of

SEQ ID NO: 17, 18, 19 or 20 in said sample, presence of said nucleic acid molecule being indicative of cancer in said sample.

17. The method of claim 16, comprising determining presence of said nucleic acid

molecule via polymerase chain reaction.