CA2366059A1 - Cloning of cdna of mage's 5, 8, 9 and 11 and their uses in diagnosis of cancer - Google Patents
Cloning of cdna of mage's 5, 8, 9 and 11 and their uses in diagnosis of cancer Download PDFInfo
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- CA2366059A1 CA2366059A1 CA002366059A CA2366059A CA2366059A1 CA 2366059 A1 CA2366059 A1 CA 2366059A1 CA 002366059 A CA002366059 A CA 002366059A CA 2366059 A CA2366059 A CA 2366059A CA 2366059 A1 CA2366059 A1 CA 2366059A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
Abstract
The invention relates to cDNA molecules which were isolated and identified i n 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-C 1 and MAGE-C2.
For obvious reasons, it was and is desirable to analyze expression ofMAGE
genes.
There has been extensive work in this area, with patterns of MAGE-A expression having been analyzed by reverse transcription-polymerise 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-Al, A2, A3, A4, A6 and A12 are transcribed, at high levels, in many tumors. Gene MAGE-was expressed at a high level in one tumor, while MAGE-A5, A9, A10 and A11 appeared to be transcribed weakly in positive tumors. Collectively, MAGE-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, et al., Int. J.
Canc 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. Canc 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., Canc. Res. 55: 3478-3482 (1995). Testis expresses all MAGE-A
genes, except MAGE-A7. MAGE-A4, A8, A9, A10 and Al l 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.
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-C 1 and MAGE-C2.
For obvious reasons, it was and is desirable to analyze expression ofMAGE
genes.
There has been extensive work in this area, with patterns of MAGE-A expression having been analyzed by reverse transcription-polymerise 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-Al, A2, A3, A4, A6 and A12 are transcribed, at high levels, in many tumors. Gene MAGE-was expressed at a high level in one tumor, while MAGE-A5, A9, A10 and A11 appeared to be transcribed weakly in positive tumors. Collectively, MAGE-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, et al., Int. J.
Canc 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. Canc 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., Canc. Res. 55: 3478-3482 (1995). Testis expresses all MAGE-A
genes, except MAGE-A7. MAGE-A4, A8, A9, A10 and Al l 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-A1 peptide presented by HLA-Al. 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-A1, 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-A1 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. Canc 67:417-422 (1996), both of which are incorporated by reference.
Subsequently, it was found that this cross-reactive protein was encoded by MAGE-A10. 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-A10 had been found in MZ2-MEL via RT-PCR. This suggested that the primers used to amplify MAGE-A10 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 1VIAGE-A5, A8, A9 and Al 1 (SEQ ID NOS: 17, 18, 19 and 20).
DETAILED DESCRIPTION
OF PREFERRED EMBODIMENTS
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-A10 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 (SEQ ID NO: 1) 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 ~.g samples were used for reverse transcription, following Weynants, et al., su ra. The PCR was then carned out using 1/10 of total cDNA, supplemented with 2.5 q 1 of 10 x PCR
buffer, 2.5 ~ 1 of l OmM of dNTP, 10 pmoles of the primers, and 0.5 units of polymerise, plus water to a volume of 25 ~ 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 carned out using 5ql 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-A10 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.
Recently, it was reported that a monoclonal antibody which was reactive with MAGE-A1 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. Canc 67:417-422 (1996), both of which are incorporated by reference.
Subsequently, it was found that this cross-reactive protein was encoded by MAGE-A10. 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-A10 had been found in MZ2-MEL via RT-PCR. This suggested that the primers used to amplify MAGE-A10 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 1VIAGE-A5, A8, A9 and Al 1 (SEQ ID NOS: 17, 18, 19 and 20).
DETAILED DESCRIPTION
OF PREFERRED EMBODIMENTS
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-A10 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 (SEQ ID NO: 1) 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 ~.g samples were used for reverse transcription, following Weynants, et al., su ra. The PCR was then carned out using 1/10 of total cDNA, supplemented with 2.5 q 1 of 10 x PCR
buffer, 2.5 ~ 1 of l OmM of dNTP, 10 pmoles of the primers, and 0.5 units of polymerise, plus water to a volume of 25 ~ 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 carned out using 5ql 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-A10 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-A10, it was possible that contaminating genomic DNA had also been amplified.
To test this possibility, amplification was carned 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-A10.
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 MAGE-A5, A9 and A11. Hence, it was suspected that changing primers might resolve this.
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 CsCI 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 NotI
To test this possibility, amplification was carned 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-A10.
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 MAGE-A5, A9 and A11. Hence, it was suspected that changing primers might resolve this.
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 CsCI 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 NotI
restriction site. The resulting cDNA was ligated to BstXI adaptors, digested with NotI, and then inserted into the BstXI and NotI sites of expression vector pcDNAI/Amp. The resulting recombinant plasmids were introduced into E. coli DHS«, using standard electroporation techniques, followed by selection with ampicillin (25 ~.g/ml).
All libraries were then diluted in LB medium, supplemented with ampicillin, to obtain 3-6 clones/~1. Following this, 9.6 mls of each dilution were seeded in a 96 microwell plate (100 ~ls 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 ql 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 gl of living bacteria were supplemented with 2.5 ql of 10 x PCR
buffer, 2.5 ~,l of lOmM dNTP, 10 pmoles of each primer, 0.5 units of polymerase, and water to a volume of 25 ~l .
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 _7_ dilution could be carned 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.
_g_ N 7, O a~
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+
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+ N
+ ~ + N C O O
+
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.
~ U N
y Cv d ~ ~' o ~ , o O o '1 O O O U
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+
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o + O + O O te + + G
+ 00 N ..
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+ O n ( U.. O O ~+ N O+ Q- + N N
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i c7 O O + O ~r~ o+ N ~'~, ~.
g+ + + ~ 3 o+
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.
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W o=- , o.+.. ov o~ o+ or. s.o v,+
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+ +
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+ + O
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a The results were compared to the results obtained in RT-PCR assays, as set forth in the table supra. MAGE-A1, A9 and A10 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-A10 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-A1 in several of the lines. In other lines, while the level decreased, it was still comparable to levels for A1, A2 or A12.
The calculated frequencies for A1, 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: S and 6 are more efficient at determining transcript present, but SEQ ID NOS: 7 and 8 are better at determining expression levels.
One aspect of the results of the experiments described su ra, 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 GCCTCTTTGAAGAGAGCAGTC (sense) SEQ ID NO.: 12 CAAAGAAGCAAAAACATACACATA (anti-sense) SEQ ID NO.: 13 CACTCTGTTTGAAGAAAATAGTC (sense) SEQ ID NO.: 14 AGTATCTTTTAATTTATCTCACCTA (anti-sense) SEQ ID NO.: 15 AGCATGTTGGGTGTGAGGGA (sense) SEQ ID NO.: 16 AGGGTACACTAAGAGGTACAG (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 carned 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-A1, levels of A2, A3, A8, A9 and A11 were low, and no cDNA for A6, A10 or A12 was found. These results are in accordance with those provided by Carrel et al. supra, for MAGE-Al AND MAGE-A10.
With respect to placenta, MAGE-A10 showed the highest level of expression, while A1-A7 A9 and A12 were not found at all among 230,000 clones analyzed.
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-A1.
The approach described in example 2, supra, led to isolation of cDNA for MAGE-A5, A8, A9, A10 and A11. 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 l 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-AS cDNA led to some interesting observations, as it consisted of the first two exons of MAGE-A10, 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 determining 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 arrayed 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 "A1", "B2", "C3", "D4", etc.
Ifboth 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-AS and two exons for MAGE-A10, separated by a nucleotide sequence between the MAGE-AS and MAGE-A10 sequences as well as nucleic acid molecules which comprise portions that hybridize to both the MAGE-AS portion, and the MAGE-A10 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 MACE 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:
can be used to determine MAGE-A1, 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, 10 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 a part 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 carned 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 N0:21 is that for MAGE A5, SEQ ID N0:22 is that for MAGE A8, SEQ ID N0:23 is that for MAGE A9, and SEQ ID N0:24 is that for MAGE Al l:
MAGE A5: HLA-A1 Binders MAGE A5: HLA-A2 Binders MAGE A5: HLA-A3 Binders MAGE A5: HLA-A24 Binders MAGE A5: HLA-B7 Binders MAGE A5: HLA-B8 Binders MAGE A5: HLA-B35 Binders MAGE A5: HLA-B44 Binders MAGE A5: HLA-B52 Binders MAGE A5: HLA-CW6 Binders MAGE Al l: HLA-A1 Binders MAGE A11: HLA-A2 Binders MAGE Al 1: HLA-A3 Binders MAGE Al l: HLA-A24 Binders MAGE Al l: HLA-B7 Binders MAGE Al 1: HLA-B8 Binders MAGE A 11: HLA-B 5 Binders MAGE A11: HLA-B44 Binders MAGE Al l: HLA-B52 Binders MAGE A1 l: HLA-CW6 Binders MAGE A9: HLA-Al Binders 2(4~-255TQDWVQENY
MAGE A9: HLA-A2 Binders ~l-166 MQVIFGTDV
MAGE A9: HLA-A3 Binders 1L~#-143MLESVIKNY
?1'~-279FLWGSKAHA
MAGE A9: HLA-A24 Binders ~-238 VYVGKEHMF
236-245MFYGEPRKT.
MAGE A9: HLA-B7 Binders l~-178 DPAGHSYIL
ll9T7-206KAALLIIVL
WO 00/52163 PCT/iJS00/05346 -MAGE A9: HLA-B8 Binders 1 ~i-202HSMPKAALL
MAGE A9: HLA-B52 Binders 21~-209LLIIVLGVI
~9-178 DPAGHSYIL
MAGE A9: HLA-CW6 Binders MAGE A9: HLA-B44 Binders ~'b-SS EEVSAAGSS
~-286 AETSYEKVI
MAGE A9: HLA-B35 Binders MAGE A8: HLA-Al Binders MAGE A8: HLA-B52 Binders MAGE A8: HLA-A3 Binders MAGE A8: HLA-B7 Binders MAGE A8: HLA-B8 Binders MAGE A8: HLA-A2 Binders MAGE A8: HLA-A24 Binders MAGE A8: HLA-CW6 Binders 240-249 SVYWKLR,KT_.
MAGE A8: HLA-B35 Binders MAGE A8: HLA-B44 Binders 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-2,IL-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 forparticular 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 [3galactosidase, 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 rej ection 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., tumorrejection antigens) produced following expression ofthe 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 ep r 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. E~.
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 rej ection 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., 5' Cr 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: $59 or by FACS analysis of fluorogenic tetramer complexes of MHC Class I/peptide (Dunbar et al., (1998), Current Biolo~y 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.
<110> Serrano, Alfonso Lethe, Bernard Lurquin, Christophe DePlaen, Etienne Rimoldi, Donati Boon-Falleur, Thierry <120> Isolated Nucleic Acid Molecules Encoding MACE Genes, Proteins Encoded, Peptides Derived Therefrom, And Uses The reof <130> LUD 5566.1 <140>
<141>
<150> US 09/260,975 <151> 1999-03-02 <160> 24 <210> 1 <211> 21 <212> DNA
<213> Homo sapiens <220>
<400> 1 cacagagcag cactgaagga g 21 <210> 2 <211> 23 <212> DNA
<213> Homo Sapiens <220>
<400> 2 ctgggtaaag actcactgtc tgg 23 <210> 3 <211> 22 <212> DNA
<213> Homo Sapiens <220>
<400> 3 agcagccaaa aggaggagag tc 22 <210> 4 <211> 22 <212> DNA
<213> Homo Sapiens <220>
<400> 4 tgacctcctc aggggtgcag to 22 <210> 5 <211> 22 <212> DNA
<213> Homo Sapiens <220>
<400> 5 cggccgaagg aacctgaccc ag 22 <210> 6 <211> 23 <212> DNA
<213> Homo Sapiens <220>
<400> 6 gctggaaccc tcactgggtt gcc 23 <210> 7 <211> 19 <212> DNA
<213> Homo Sapiens <220>
<400> 7 tcaggggaca ggccaaccc 19 <210> 8 <211> 23 <212> DNA
<213> Homo Sapiens <220>
<400> 8 cttgcactga ccttgatcac ata 23 <210> 9 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<400> 9 gaagagagcg gtcagtgttc 20 <210> 10 <211> 24 <212> DNA
<213> Homo Sapiens <220>
<400> 10 aatccaggta tgcatatatc ttta 24 <210> 11 <211> 21 <212> DNA
<213> Homo Sapiens <220>
<400> 11 gcctctttga agagagcagt c 21 <210> 12 <211> 24 <212> DNA
<213> Homo sapiens <220>
<400> 12 caaagaagca aaaacataca cata 24 <210> 13 <211> 23 <212> DNA
<213> Homo sapiens <220>
<400> 13 cactctgttt gaagaaaata gtc 23 <210> 14 <211> 25 <212> DNA
<213> Homo Sapiens <220>
<400> 14 agtatctttt aatttatctc accta 25 <210> 15 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<400> 15 agcatgttgg gtgtgaggga 20 <210> 16 <211> 21 <212> DNA
<213> Homo Sapiens <220>
<400> 16 agggtacact aagaggtaca g 21 <210> 17 <211> 1916 <212> DNA
<213> Homo Sapiens <220>
<400> 17 gcttgagatc ggctgaagag agcgggccca ggctctgtga ggaggcaagg gag gtgagaa 60 ccttgctctc agagggtgac tcaagtcaac acagggaacc cctcttttct aca gacacag 120 tgggtcgcag gatctgacaa gagtccagca tcatgcacta tcctgttggg agc atcctca 180 cctccaagac actgtttggg cctgaggaga aggagtctgc agtgaccctg tcg tggtatt 240 ttccacaaga attctgaaat gaagcaagca caggttctca ggggacaggc tga ccaggat 300 caccaggaag ctccagagga tccccaggag gccctagagg agcaccaaag gag aagatct 360 gccagtgggt ctccattgcc cagctcctgc ccacactcct gcctgttgcg gtg accagag 420 tcgtcatgtc tcttgagcag aagagtcagc actgcaagcc tgaggaaggc ctt gacaccc 480 aagaagaggc cctgggcctg gtgggtgtgc aggctgccac tactgaggag cag gaggctg 540 tgtcctcctc ctctcctctg gtcccaggca ccctggggga ggtgcctgct acs gggtcac 600 caggtcctct caagagtcct caaggagcct ccgccatccc cactaccatc aat tcactc 66r r _ tatggaggca atccattaag ggctccagca accaagaaga ggaggggcca agc acctccc 720 ctgacccaga gtctgtgttc cgagcagcac tcagtaagaa ggtggctgac ttg attcatt 780 ttctgctcct caagtattaa gtcaaggagc tggtcacaaa ggcagaaatg ctg gagagcg 840 tcatcaaaaa ttacaagcgc tgctttcctg agatcttcgg caaagcctcc gag tccttgc 900 agctggtctt tggcattgac gtgaaggaag cggaccccac cagcaacacc tac acccttg 960 tcacctgcct gggactccta tgatggcctg ctggttgata ataatcagat cat gcccaag 1020 acgggcctcc tgataatcgt cttgggcatg attgcaatgg agggcaaatg cgt ccctgag 1080 gagaaaatct gggaggagct gagtgtgatg aaggtgtatg ttgggaggga gca cagtgtc 1140 tgtggggagc ccaggaagct gctcacccaa gatttggtgc aggaaaacta cct ggagtac 1200 cggcaggtgc ccagcagtga tcccatatgc tatgagttac tgtggggtcc aag ggcactc 1260 gctgcttgaa agtactggag cacgtggtca gggtcaatgc aagagttctc att tcctacc 1320 catccctgcg tgaagcagct ttgagagagg aggaagaggg agtctgagca tga gctgcag 1380 ccagggccac tgcgaggggg gctgggccag tgcaccttcc agggctccgt cca gtagttt 1440 cccctgcctt aatgtgacat gaggcccatt cttctctctt tgaagagagc agt caacatt 1500 cttagtagtg ggtttctgtt ctattggatg actttgagat ttgtctttgt ttc cttttgg 1560 aattgttcaa atgtttcttt taatgggtgg ttgaatgaac ttcagcattc aaa tttatga 1620 atgacagtag tcacacatag tgctgtttat atagtttagg agtaagagtc ttg tttttta 1680 ttcagattgg gaaatccatt ccattttgtg aattgggaca tagttacagc agt ggaataa 1740 ' gtattcattt agaaatgtga atgagcagta aaactgatga cataaagaaa tta aaagata 1800 tttaattctt gcttatactc agtctattcg gtaaaatttt ttttaaaaaa tat gcatacc 1860 tggatttcct tggcttcttt gagaatgtaa gacaaattaa atctcraataa atc att 1916 <210> 18 <211> 1765 <212> DNA
<213> Homo Sapiens <220>
<400> 18 c-~ggt~ctga gggg~cgg== -.. gGtcgg~ -~gagggaa4~ gggc~caggg t~.~
gtgagga 60 ggcaaggttc gcagagaaca ggccagccag gaggtcagga ggccccagag aag cactgaa 120 gaagacctgc ctgtgggtct caattgccca gctccggccc acactctcct get gccctga 180 cctgagtcat catgcttctt gggcagaaga gtcagcgcta caaggctgag gaa ggccttc 240 aggcccaagg agaggcacca gggcttatgg atgtgcagat tcccacagct gag gagcaga 300 aggctgcatc ctcctcctct actctgatca tgggaaccct tgaggaggtg act gattctg 360 ggtcaccaag tcctccccag agtcctgagg gtgcctcctc ttccctgact gtc accgaca 420 gcactctgtg gagccaatcc gatgagggtt ccagcagcaa tgaagaggag ggg ccaagca 480 cctccccgga cccagctcac ctggagtccc tgttccggga agcacttgat gag aaagtgg 540 ctgagttagt tcgtttcctg ctccgcaaat atcaaattaa ggag.~.cggtc aca aaggcag 600 aaatgcttga gagtgtcatc aaaaattaca agaaccactt tcctgatatc ttc agcaaag 660 cctctgagtg catgcaggtg atctttggca ttgatgtgaa ggaagtggac cct gccggcc 720 actcctacat ccttgtcacc tgcctgggcc tctcctatga tggcctgctg ggt gatgatc 780 agagtacgcc caagaccggc cLCCtgataa tcgtcctggg catgatctta atg gagggca 840 gccgcgcccc ggaggaggca atctgggaag cattgagtgt gatggggctg tat gatggga 900 gggagcacag tgtctattgg aagctcagga agctgctcac ccaagagtgg gtg caggaga 960 actacctgga gtaccgccag gcgcccggca gtgatcctgt gcgctacgag ttc ctgtggg 1020 gtccaagggc ccttgctgaa accagctatg tgaaagtcct ggaacatgtg gtc agggtca 1080 atgcaagagt tcgcatttcc tacccatccc tgcatgaaga ggc-~~tggga gag gagaaag 1140 gagtttgagc aggagttgca gctagggcca gtggggcagg ttgtgggagg gcc tgggcca 1200 gtgcacgttc cagggccaca tccaccactt tccctgctct gttacatgag gcc cattctt 1260 cactctgtgt ttgaagagag cagtcacagt tctcagtagt ggggagcatg ttg ggtgtga 1320 gggaacacag tgtggacca~ ctctcagttc ctgttctatt gggcgatttg gag atttatc 1380 tttgtttcct tttggaattg ttccaatgtt ccttctaatg gatggtgtaa tga acttcaa 1440 cattcatttt atgtatgaca gtagacagac ttactgcttt ttatatagtt tag gagtaag 1500 agtcttgctt ttcatttata ctgggaaacc catgttattt cttgaattca gac actacaa 1560 gagcagagg.~ t taa J g ~ t ' ~ _ ~ 'agaGa ~ J ~~, ~~a'.~. ~.agca.~, ~aau a"
catgaga 1620 taaagacata aagaaattaa acaatagtta attcttgcct tacctgtacc tct tagtgta 1680 ccctatgtac ctgaatttgc ttggcttctt tgagaatgaa attgaattaa ata tgaataa 1740 ataagtcaaa aaaaaaaaaa aaaaa <210> 19 <211> 1831 <212> DNA
<213> Homo Sapiens <220>
<400> 19 gaggcctcct tctgaggggc ggcttgatac cggtggagga gctccaggaa gca ggcaggc 60 cttggtctga gacagtgtcc tcaggtcgca gagcagagga gacccaggca gtg tcagcag 120 tgaaggttct cgggacaggc taaccaggag gacaggagcc ccaagaggcc cca gagcagc 180 actgacgaag acctgcctgt gggtctccat cgcccagctc ctgcccacgc tcc tgactgc 240 tgccctgacc agagtcatca tgtctctcga gcagaggagt ccgcactgca agc ctgatga 300 agaccttgaa gcccaaggag aggacttggg cctgatgggt gcacaggaac cca caggcga 360 ggaggaggag actacctcct cctctgacag caaggaggag gaggtgtctg ctg ctgggtc 420 atcaagtcct ccccagagtc ctcagggagg cgcttcctcc tccatttccg tct actacac 480 tttatggagc caattcgatg agggctccag cagtcaagaa gaggaagagc caa gctcctc 540 ggtcgaccca gctcagctgg agttcatgtt ccaagaagca ctgaaattga agg tggctga 600 gttggttcat ttcctgctcc acaaatatcg agtcaaggag ccggtcacaa agg cagaaat 660 gctggagagc gtcatcaaaa attacaagcg ctactttcct gtgatcttcg gca aagcctc 720 cgagttcatg caggtgatct ttggcactga tgtgaaggag gtggaccccg ccg accactc 780 ctacatcctt gtcactgctc ttggcctctcc gtgcgatagc atgctgggtg atg a~catag 840 catgcccaag gccgccctcc tgatcatta~ ~~tgggtgtg atcctaacca aag acaacta 900 cgcccctgaa gaggttatct gggaagcgtt gagtgtgatg ggggtgtatg ttg ggaagga 960 gcacatgttc tacggggagc ccaggaagct gctcacccaa gattgggtgc agg aaaacta 1020 cctggagtac cggcaggtgc ccgq_caata_a tcctgcgcac tacgagttcc ta_t ggggtt~ 1080 caaggcccac gctgaaacca gctatgagaa ggtcataaat tatttggtca tgc tcaatgc 1140 aagagagccc atctgctacc catcccttta tgaagaggtt ttgggagagg agc aagaggg 1200 agtctgagca ccagccgcag ccggggccaa agtttgtggg gtcagggccc cat ccagcag 1260 ctgccctgcc ccatgtgaca tgaggcccat tcttcgctct gtgtttgaag aga gcaatca 1320 gtgttctcag tggcagtggg tggaagtgag cacactgtat gtcatctctg ggt tccttgt 1380 ctattgggtg atttggagat ttatccttgc tcccttttgg aattgttcaa atg ttctttt 1440 aatggtcagt ttaatgaact tcaccatcga agttaatgaa tgacagtagt cac acatatt 1500 gctgtttatg ttatttagga gtaagattct tgcttttgag tcacatgggg aaa tccctgt 1560 tattttgtga attgggacaa gataacatag cagaggaatt aataattttt ttg aaacttg 1620 aacttagcag caaaatagag ctcataaaga aatagtgaaa tgaaaatgta gtt aattctt 1680 gccttatacc tctttctctc tcctgtaaaa ttaaaacata tacatgtata cct ggatttg 1740 cttggcttct ttgagcatgt aagagaaata aaaattgaaa gaataaaaaa aaa aaaaaaa 1800 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a <210>20 <211>1886 <212>DNA
<213>Homo Sapiens <220>
<400>20 ctctgggatc tgagagaagc gaaagcgtct ttctgagggg tgtcttgaga gtg gcagagg 60 gcagcgggtc caggctccat gaggaggcaa gccttgggaa tctgagggat gga gactcag 120 ttccgcagag ggggtctggg gtgcagccct gccagcatca agaggaagaa as gagggag 180 gactcaggag actttggact ccaggtgagc actatgttct cagaggacga ct~
ccagtca 240 acagaaagag ccccatatgg tccacaac:~ cagtggtccc aggatctgcc as agtccag 300 gtttttagag aacaggccaa c~tggaaga~ aggagtccca ggagaaccca gao gatcac~ 360 ggaggagaac aagtgctgtg gggccccatc acccagatat ttcccacagt tcg gcctgct 420 gacctaacca gagtcatcat gcctcttgag caaagaagtc agcactgcaa gcc tgaggaa 480 ggccttcagg cccaagaagG ~. ~ctgggc ctggtgggt~ cacagJ~tc~
c~.a agctgag 540 gagcaggagg ctgccttctt c~cctctact ctgaatgtgg gcac~ctaga gga gttgcct 600 gctgctgagt caccaagtcc tccccagagt cctcaggaag agtccttctc tcc cactgcc 660 atggatgcca tctttgggag cctatctgat gagggctctg gcagccaaga aaa ggagggg 720 ccaagtacct cgcctgacct gatagaccct gagtcctttt cccaagatat act acatgac 780 aagataattg atttggttca tttattgctc cgcaagtatc gagtcaaggg get gatcaca 840 aaggcagaaa tgctggggag tgtcatcaaa aattatgagg actactttcc tga gatattt 900 agggaagcct ctgtatgcat gcaactgctc tttggcattg atgtgaagga agt ggacccc 960 actagccact cctatgtcct tgtcacctcc ctcaacctct cttatgatgg cat acagtgt 1020 aatgagcaga gcatgcccaa gtctggcctc ctgataatag tcctgggtgt aat cttcatg 1080 gaggggaact gcatccctga agaggttatg tgggaagtcc tgagcattat ggg ggtgtat 1140 gctggaaggg agcacttcct ctttggggag cccaagaggc tccttaccca aaa ttgggtg 1200 caggaaaagt acctggtgta ccggcaggtg cccggcactg atcctgcatg cta tgagttc 1260 ctgtggggtc caagggccca cgctgagacc agcaagatga aagt~cttga gta catagcc 1320 aatgccaatg ggagggatcc cacttcttac ccatccctgt atgaagatgc ttt gagagag 1380 gagggagagg gagtctgagc a'gagatgca accagggcca gcgggcaggg aaa tgggcca 1440 atgcatgctt cagggccaca cccagcagtt tccctgtcct gtgtgaaatc agg cccattc 1500 ttccctctgt gtttgatgag agaagtcagt gttctcagta gtagaaggca cag tgaatgg 1560 aagggaacac attgtatact gcctttaggt ttctcttcca tcgga~gact tgg agatttg 1620 tttttgtttc cctttggtaa ttt~caaata ttgttcctgt aataaaagtt tta gttagct 1680 tcaacatcta agtgtatgga tgatactgac cacacatgtt gttt~gctta tcc atttcaa 1740 gtgcaagtgt ttgccatttt gtaaaacatt ttgggaaatc ttccatcttg ctg tgatttg 1800 caataggtat tttcttggag aa~gtaagaa cttaacaata aagc~gaact ggt gttgtga 1860 aacagagaaa aaaaaaaaaa aaaaaa <210> 21 <211> 124 <212> PRT
<2i3> Ho~r,oSapiens <220>
<400> 21 Met Ser Glu Gln Lys Ser GlnHis Cys Lys Pro Glu Glu G1 Leu y Leu Asp Thr Glu Glu Ala Leu GlyLeu Val Gly Val Gln Ala A1 Gln a Thr Thr Glu Gln Glu Ala Val SerSer Ser Ser Pro Leu Val Pr Glu o Gly Thr Leu Glu Val Pro Ala AlaGly Ser Pro Gly Pro Leu Ly Gly s Ser Pro Gln Ala Ser Ala Ile ProThr Ala Ile Asp_Phe Thr Le Gly a Trp Arg Gln Ser Ile Lys Gly Ser Ser Asn Gln Glu Glu Glu Gly Pr o Ser Thr Ser Pro Asp Pro Glu Ser Val Phe Arg Ala Ala Leu Ser Ly s Lys Val Ala Asp Leu Ile His Phe Leu Leu Leu Lys Tyr <210> 22 <211> 318 <212> PRT
<213> Homo Sapiens <220>
<400> 22 Met Leu Gly Gln Lys Ser Gln Arg Tyr Lys Ala Glu Glu Leu G1 y Leu G=in Ala Gly Glu Ala Pro Gly Leu Met Asp Va- Gln Ile Gln Pr o Thr Ala Glu Gln Lys Ala Ala SerSer Ser Ser Thr Leu Ile Glu Me t Gly Thr Glu Glu Val Thr Asp SerGiy Ser Pro Ser Pro Pro Leu G1 n Ser Pro Gly Ala Ser Ser Ser LeuThr Val Thr Asp Ser Thr Glu Le a Trp Ser Gln Ser Asp Glu Gly Ser Ser Ser Asn Glu Glu Glu Gly Pr o Ser Thr Ser Pro Asp Pro Ala His Leu Glu Ser Leu Phe Arg Glu Al a Leu Asp Glu Lys Val Ala Glu Leu Val Arg Phe Leu Leu Arg Lys Ty r Gln Ile Lys Glu Pro Val Thr Lys Ala Glu Met Leu Glu Ser Val I1 a Lys Asn Tyr Lys Asn His Phe Pro Asp Ile Phe Ser Lys Ala Ser G1 a Cys Met Gln Val Iie Phe Gly Ile Asp Val Lys Glu Val Asp Pro Al a Gly His Ser Tyr Ile Leu Val Thr Cys Leu Gly Leu Ser Tyr Asp G1 y Leu Leu Gly Asp Asp Gln Ser Thr Pro Lys Thr Gly Leu Leu Ile I1 a Val Leu Gly Met Ile Leu Met Glu Gly Ser Arg Ala Pro Glu Glu Al a Ile Trp Glu Ala Leu Met Gly Ser Leu Val Tyr Asp Gly Arg Glu Hi s Ser Val Tyr Trp Lys LeuArg Lys Leu Leu Thr Gln GluTrp Val G1 n Glu Asn Tyr Leu Glu TyrArg Gln Ala Pro Gly Ser AspPro Val Ar g Tyr Glu Phe Leu Trp GlyPro Arg Ala Leu Ala Glu ThrSer Tyr Va 1 Lys Val Leu Glu His ValVal Arg Val Asn Ala Arg ValArg Ile Se r Tyr Pro Ser Leu His GluGlu Ala Leu Gly Glu Glu LysGly Val <210> 23 <211> 315 <212> PRT
<213> Homo Sapiens <220>
<400> 23 Met Ser Leu Glu GlnArg Ser Pro His Cys Lys ProAsp Glu As p Leu Glu Ala Gln Gly GluAsp Leu Gly Leu Met Gly AlaGln Glu P-r o Thr Gly Glu Glu Glu GluThr Thr Ser Ser Ser Asp SerLys Glu G-a Glu Val Ser Ala Ala GlySer Ser Ser Pro Pro Gln SerPro Gln G1 y Gly Ala Ser Ser Ser IleSer Val Tyr Tyr Thr Leu TrpSer Gln P_~
a Asp Glu Gly Ser SerSer Gln Glu Glu Glu GluPro Ser Ser Ser Va 1 Asp t'~~ Aia G1?~.i~Cl.._.., ilc~~ v._?'.Gnu' - _ _ _ . P ~ .L ~: L 1 ._ _ 1 a y E i 1 a ~, s ~
; .
s Val . .. , ~
Ala Glu Leu ValHis Phe Leu Leu His LysTyr Arg Val Lys G1 a Pro Val Thr Lys AlaGlu Met Leu Glu Ser ValIle Lys Asn Tyr Ly s Arg Tyr Pro Val Ile Phe Gly LysAla Ser Glu Phe Met Gln Va Phe 1 Ile Phe Thr Asp Val Lys Glu ValAsp Pro Ala Gly His Ser Ty Gly r Ile 165 1~0 1~
Leu Thr Ala Leu Gly Leu SerCys Asp Ser Met Leu Gly As Val p Gly His Met Pro Lys A1a Ala LeuLeu Ile Ile Val Leu Gly Va Ser 1 Ile Leu Lys Asp Asn Cys Ala ProGlu Glu Val Ile Trp Glu A1 Thr a Leu Ser Val Met Gly Val Tyr Val Gly Lys Glu His Met Phe Tyr Gl y Glu Pro Arg Lys Leu Leu Thr Gln Asp Trp Val Gln Glu Asn Tyr Le a Glu Tyr Arg Gln Val Pro G-y Ser Asp Pro Ala His Tyr Glu Phe Le a Trn -Gly Ser Lys AlaHis Ala Glu Thr Ser TyrGlu LysVal Ile As n Tyr Leu Val Met LeuAsn Ala Arg Glu Pro IleCys TyrPro Ser Le a Tyr Glu Glu Val LeuGly Glu Glu Gln Glu GlyVal <210> 24 <211> 429 <212> PRT
<213> Homo Sapiens <220>
<400> 24 Met Glu Thr GlnPhe Arg Arg Gly Gly LeuGly CysSer Pro A1 a Ser Ile Lys Arg LysLys Lys Arg Glu Asp SerGly AspPhe Gly Le a Gln Val Ser Thr MetPhe Ser Glu Asp Asp PheGln SerThr Glu Ar g Ala Pro Tyr Gly ProGln Leu Gln Trp Ser GlnAsp LeuPro Arg Va 1 Gln Val Phe Arg GluGln Ala Asn Leu Glu AspArg SerPro Arg Ar g Thr Gln Arg Ile Thr Gly Gly Glu Gln Val Leu Trp Gly Pro Ile Th r Gln Ile Phe Pro Thr Val Arg Pro Ala Asp Leu Thr Arg Val Ile Me t Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Giu Glu Gly Leu G1 n Ala -Gln Glu Glu Asp Leu GlyLeu Val Gly Ala Gln Ala LeuGln A1 a Glu Glu G~~:~ Glu Ala Aia Ph Pr:eSer So_ Tn:_Leu Asn VaiG1~ 1::
r Leu Glu Glu Leu Pro Ala AlaGlu Ser Pro Ser Pro Pro GlnSer Pr o Gln Glu Glu Ser Phe Ser ProThr Ala Met Asp Ala Ile PheGly Se r Leu Ser Asp Glu Gly Ser GlySer Gln Glu Lys Glu Gly ProSer Th r Ser Pro Asp Leu Ile Asp ProGlu Ser Phe Ser Gln Asp IleLeu Hi s Asp Lys Ile Ile Asp Leu ValHis Leu Leu Leu Arg Lys TyrArg Va 1 Lys Gly Leu Ile Thr Lys AlaGlu Met Leu Gly Ser Val IleLys As n Tyr Glu Asp Tyr Phe Pro GluIle Phe Arg Giu Ala Ser ValCys Me t Gln Leu Leu Phe Gly Ile AspVal Lys Glu Val Asp Pro ThrSer Hi s Ser Tyr Val Leu Val Thr SerLeu Asn Leu Ser Tyr Asp GlyIle Gl n Cys Asn Glu Gln Ser Met ProLys Ser Gly Leu Leu Ile IleVal Le a Gly -Val Ile Phe Met Glu GlyAsn Cys Ile Pro Glu GluVal MetTr p Glu J
Val Leu Ser Ile Met GlyVal Tyr Ala Gly Arg GluHis PheLe a Phe Gly Glu Pro Lys Arg LeuLeu Thr Gln Asn Trp ValGln GluLy s Tyr Leu Val Tyr Arg Gln ValPro Gly Thr Asp Pro AlaCys TyrG1 a Phe Leu Trp Gly Pro Arg AlaHis Ala Glu Thr Ser LysMet LysVa 1 Leu Glu Tyr Ile Ala Asn AlaAsn Gly Arg Asp Pro ThrSer TyrPr o Ser Leu Tyr Glu Asp Ala LeuArg Glu Glu Gly Glu GlyVal
All libraries were then diluted in LB medium, supplemented with ampicillin, to obtain 3-6 clones/~1. Following this, 9.6 mls of each dilution were seeded in a 96 microwell plate (100 ~ls 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 ql 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 gl of living bacteria were supplemented with 2.5 ql of 10 x PCR
buffer, 2.5 ~,l of lOmM dNTP, 10 pmoles of each primer, 0.5 units of polymerase, and water to a volume of 25 ~l .
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 _7_ dilution could be carned 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.
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a The results were compared to the results obtained in RT-PCR assays, as set forth in the table supra. MAGE-A1, A9 and A10 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-A10 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-A1 in several of the lines. In other lines, while the level decreased, it was still comparable to levels for A1, A2 or A12.
The calculated frequencies for A1, 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: S and 6 are more efficient at determining transcript present, but SEQ ID NOS: 7 and 8 are better at determining expression levels.
One aspect of the results of the experiments described su ra, 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 GCCTCTTTGAAGAGAGCAGTC (sense) SEQ ID NO.: 12 CAAAGAAGCAAAAACATACACATA (anti-sense) SEQ ID NO.: 13 CACTCTGTTTGAAGAAAATAGTC (sense) SEQ ID NO.: 14 AGTATCTTTTAATTTATCTCACCTA (anti-sense) SEQ ID NO.: 15 AGCATGTTGGGTGTGAGGGA (sense) SEQ ID NO.: 16 AGGGTACACTAAGAGGTACAG (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 carned 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-A1, levels of A2, A3, A8, A9 and A11 were low, and no cDNA for A6, A10 or A12 was found. These results are in accordance with those provided by Carrel et al. supra, for MAGE-Al AND MAGE-A10.
With respect to placenta, MAGE-A10 showed the highest level of expression, while A1-A7 A9 and A12 were not found at all among 230,000 clones analyzed.
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-A1.
The approach described in example 2, supra, led to isolation of cDNA for MAGE-A5, A8, A9, A10 and A11. 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 l 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-AS cDNA led to some interesting observations, as it consisted of the first two exons of MAGE-A10, 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 determining 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 arrayed 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 "A1", "B2", "C3", "D4", etc.
Ifboth 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-AS and two exons for MAGE-A10, separated by a nucleotide sequence between the MAGE-AS and MAGE-A10 sequences as well as nucleic acid molecules which comprise portions that hybridize to both the MAGE-AS portion, and the MAGE-A10 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 MACE 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:
can be used to determine MAGE-A1, 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, 10 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 a part 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 carned 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 N0:21 is that for MAGE A5, SEQ ID N0:22 is that for MAGE A8, SEQ ID N0:23 is that for MAGE A9, and SEQ ID N0:24 is that for MAGE Al l:
MAGE A5: HLA-A1 Binders MAGE A5: HLA-A2 Binders MAGE A5: HLA-A3 Binders MAGE A5: HLA-A24 Binders MAGE A5: HLA-B7 Binders MAGE A5: HLA-B8 Binders MAGE A5: HLA-B35 Binders MAGE A5: HLA-B44 Binders MAGE A5: HLA-B52 Binders MAGE A5: HLA-CW6 Binders MAGE Al l: HLA-A1 Binders MAGE A11: HLA-A2 Binders MAGE Al 1: HLA-A3 Binders MAGE Al l: HLA-A24 Binders MAGE Al l: HLA-B7 Binders MAGE Al 1: HLA-B8 Binders MAGE A 11: HLA-B 5 Binders MAGE A11: HLA-B44 Binders MAGE Al l: HLA-B52 Binders MAGE A1 l: HLA-CW6 Binders MAGE A9: HLA-Al Binders 2(4~-255TQDWVQENY
MAGE A9: HLA-A2 Binders ~l-166 MQVIFGTDV
MAGE A9: HLA-A3 Binders 1L~#-143MLESVIKNY
?1'~-279FLWGSKAHA
MAGE A9: HLA-A24 Binders ~-238 VYVGKEHMF
236-245MFYGEPRKT.
MAGE A9: HLA-B7 Binders l~-178 DPAGHSYIL
ll9T7-206KAALLIIVL
WO 00/52163 PCT/iJS00/05346 -MAGE A9: HLA-B8 Binders 1 ~i-202HSMPKAALL
MAGE A9: HLA-B52 Binders 21~-209LLIIVLGVI
~9-178 DPAGHSYIL
MAGE A9: HLA-CW6 Binders MAGE A9: HLA-B44 Binders ~'b-SS EEVSAAGSS
~-286 AETSYEKVI
MAGE A9: HLA-B35 Binders MAGE A8: HLA-Al Binders MAGE A8: HLA-B52 Binders MAGE A8: HLA-A3 Binders MAGE A8: HLA-B7 Binders MAGE A8: HLA-B8 Binders MAGE A8: HLA-A2 Binders MAGE A8: HLA-A24 Binders MAGE A8: HLA-CW6 Binders 240-249 SVYWKLR,KT_.
MAGE A8: HLA-B35 Binders MAGE A8: HLA-B44 Binders 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-2,IL-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 forparticular 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 [3galactosidase, 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 rej ection 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., tumorrejection antigens) produced following expression ofthe 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 ep r 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. E~.
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 rej ection 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., 5' Cr 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: $59 or by FACS analysis of fluorogenic tetramer complexes of MHC Class I/peptide (Dunbar et al., (1998), Current Biolo~y 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.
<110> Serrano, Alfonso Lethe, Bernard Lurquin, Christophe DePlaen, Etienne Rimoldi, Donati Boon-Falleur, Thierry <120> Isolated Nucleic Acid Molecules Encoding MACE Genes, Proteins Encoded, Peptides Derived Therefrom, And Uses The reof <130> LUD 5566.1 <140>
<141>
<150> US 09/260,975 <151> 1999-03-02 <160> 24 <210> 1 <211> 21 <212> DNA
<213> Homo sapiens <220>
<400> 1 cacagagcag cactgaagga g 21 <210> 2 <211> 23 <212> DNA
<213> Homo Sapiens <220>
<400> 2 ctgggtaaag actcactgtc tgg 23 <210> 3 <211> 22 <212> DNA
<213> Homo Sapiens <220>
<400> 3 agcagccaaa aggaggagag tc 22 <210> 4 <211> 22 <212> DNA
<213> Homo Sapiens <220>
<400> 4 tgacctcctc aggggtgcag to 22 <210> 5 <211> 22 <212> DNA
<213> Homo Sapiens <220>
<400> 5 cggccgaagg aacctgaccc ag 22 <210> 6 <211> 23 <212> DNA
<213> Homo Sapiens <220>
<400> 6 gctggaaccc tcactgggtt gcc 23 <210> 7 <211> 19 <212> DNA
<213> Homo Sapiens <220>
<400> 7 tcaggggaca ggccaaccc 19 <210> 8 <211> 23 <212> DNA
<213> Homo Sapiens <220>
<400> 8 cttgcactga ccttgatcac ata 23 <210> 9 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<400> 9 gaagagagcg gtcagtgttc 20 <210> 10 <211> 24 <212> DNA
<213> Homo Sapiens <220>
<400> 10 aatccaggta tgcatatatc ttta 24 <210> 11 <211> 21 <212> DNA
<213> Homo Sapiens <220>
<400> 11 gcctctttga agagagcagt c 21 <210> 12 <211> 24 <212> DNA
<213> Homo sapiens <220>
<400> 12 caaagaagca aaaacataca cata 24 <210> 13 <211> 23 <212> DNA
<213> Homo sapiens <220>
<400> 13 cactctgttt gaagaaaata gtc 23 <210> 14 <211> 25 <212> DNA
<213> Homo Sapiens <220>
<400> 14 agtatctttt aatttatctc accta 25 <210> 15 <211> 20 <212> DNA
<213> Homo Sapiens <220>
<400> 15 agcatgttgg gtgtgaggga 20 <210> 16 <211> 21 <212> DNA
<213> Homo Sapiens <220>
<400> 16 agggtacact aagaggtaca g 21 <210> 17 <211> 1916 <212> DNA
<213> Homo Sapiens <220>
<400> 17 gcttgagatc ggctgaagag agcgggccca ggctctgtga ggaggcaagg gag gtgagaa 60 ccttgctctc agagggtgac tcaagtcaac acagggaacc cctcttttct aca gacacag 120 tgggtcgcag gatctgacaa gagtccagca tcatgcacta tcctgttggg agc atcctca 180 cctccaagac actgtttggg cctgaggaga aggagtctgc agtgaccctg tcg tggtatt 240 ttccacaaga attctgaaat gaagcaagca caggttctca ggggacaggc tga ccaggat 300 caccaggaag ctccagagga tccccaggag gccctagagg agcaccaaag gag aagatct 360 gccagtgggt ctccattgcc cagctcctgc ccacactcct gcctgttgcg gtg accagag 420 tcgtcatgtc tcttgagcag aagagtcagc actgcaagcc tgaggaaggc ctt gacaccc 480 aagaagaggc cctgggcctg gtgggtgtgc aggctgccac tactgaggag cag gaggctg 540 tgtcctcctc ctctcctctg gtcccaggca ccctggggga ggtgcctgct acs gggtcac 600 caggtcctct caagagtcct caaggagcct ccgccatccc cactaccatc aat tcactc 66r r _ tatggaggca atccattaag ggctccagca accaagaaga ggaggggcca agc acctccc 720 ctgacccaga gtctgtgttc cgagcagcac tcagtaagaa ggtggctgac ttg attcatt 780 ttctgctcct caagtattaa gtcaaggagc tggtcacaaa ggcagaaatg ctg gagagcg 840 tcatcaaaaa ttacaagcgc tgctttcctg agatcttcgg caaagcctcc gag tccttgc 900 agctggtctt tggcattgac gtgaaggaag cggaccccac cagcaacacc tac acccttg 960 tcacctgcct gggactccta tgatggcctg ctggttgata ataatcagat cat gcccaag 1020 acgggcctcc tgataatcgt cttgggcatg attgcaatgg agggcaaatg cgt ccctgag 1080 gagaaaatct gggaggagct gagtgtgatg aaggtgtatg ttgggaggga gca cagtgtc 1140 tgtggggagc ccaggaagct gctcacccaa gatttggtgc aggaaaacta cct ggagtac 1200 cggcaggtgc ccagcagtga tcccatatgc tatgagttac tgtggggtcc aag ggcactc 1260 gctgcttgaa agtactggag cacgtggtca gggtcaatgc aagagttctc att tcctacc 1320 catccctgcg tgaagcagct ttgagagagg aggaagaggg agtctgagca tga gctgcag 1380 ccagggccac tgcgaggggg gctgggccag tgcaccttcc agggctccgt cca gtagttt 1440 cccctgcctt aatgtgacat gaggcccatt cttctctctt tgaagagagc agt caacatt 1500 cttagtagtg ggtttctgtt ctattggatg actttgagat ttgtctttgt ttc cttttgg 1560 aattgttcaa atgtttcttt taatgggtgg ttgaatgaac ttcagcattc aaa tttatga 1620 atgacagtag tcacacatag tgctgtttat atagtttagg agtaagagtc ttg tttttta 1680 ttcagattgg gaaatccatt ccattttgtg aattgggaca tagttacagc agt ggaataa 1740 ' gtattcattt agaaatgtga atgagcagta aaactgatga cataaagaaa tta aaagata 1800 tttaattctt gcttatactc agtctattcg gtaaaatttt ttttaaaaaa tat gcatacc 1860 tggatttcct tggcttcttt gagaatgtaa gacaaattaa atctcraataa atc att 1916 <210> 18 <211> 1765 <212> DNA
<213> Homo Sapiens <220>
<400> 18 c-~ggt~ctga gggg~cgg== -.. gGtcgg~ -~gagggaa4~ gggc~caggg t~.~
gtgagga 60 ggcaaggttc gcagagaaca ggccagccag gaggtcagga ggccccagag aag cactgaa 120 gaagacctgc ctgtgggtct caattgccca gctccggccc acactctcct get gccctga 180 cctgagtcat catgcttctt gggcagaaga gtcagcgcta caaggctgag gaa ggccttc 240 aggcccaagg agaggcacca gggcttatgg atgtgcagat tcccacagct gag gagcaga 300 aggctgcatc ctcctcctct actctgatca tgggaaccct tgaggaggtg act gattctg 360 ggtcaccaag tcctccccag agtcctgagg gtgcctcctc ttccctgact gtc accgaca 420 gcactctgtg gagccaatcc gatgagggtt ccagcagcaa tgaagaggag ggg ccaagca 480 cctccccgga cccagctcac ctggagtccc tgttccggga agcacttgat gag aaagtgg 540 ctgagttagt tcgtttcctg ctccgcaaat atcaaattaa ggag.~.cggtc aca aaggcag 600 aaatgcttga gagtgtcatc aaaaattaca agaaccactt tcctgatatc ttc agcaaag 660 cctctgagtg catgcaggtg atctttggca ttgatgtgaa ggaagtggac cct gccggcc 720 actcctacat ccttgtcacc tgcctgggcc tctcctatga tggcctgctg ggt gatgatc 780 agagtacgcc caagaccggc cLCCtgataa tcgtcctggg catgatctta atg gagggca 840 gccgcgcccc ggaggaggca atctgggaag cattgagtgt gatggggctg tat gatggga 900 gggagcacag tgtctattgg aagctcagga agctgctcac ccaagagtgg gtg caggaga 960 actacctgga gtaccgccag gcgcccggca gtgatcctgt gcgctacgag ttc ctgtggg 1020 gtccaagggc ccttgctgaa accagctatg tgaaagtcct ggaacatgtg gtc agggtca 1080 atgcaagagt tcgcatttcc tacccatccc tgcatgaaga ggc-~~tggga gag gagaaag 1140 gagtttgagc aggagttgca gctagggcca gtggggcagg ttgtgggagg gcc tgggcca 1200 gtgcacgttc cagggccaca tccaccactt tccctgctct gttacatgag gcc cattctt 1260 cactctgtgt ttgaagagag cagtcacagt tctcagtagt ggggagcatg ttg ggtgtga 1320 gggaacacag tgtggacca~ ctctcagttc ctgttctatt gggcgatttg gag atttatc 1380 tttgtttcct tttggaattg ttccaatgtt ccttctaatg gatggtgtaa tga acttcaa 1440 cattcatttt atgtatgaca gtagacagac ttactgcttt ttatatagtt tag gagtaag 1500 agtcttgctt ttcatttata ctgggaaacc catgttattt cttgaattca gac actacaa 1560 gagcagagg.~ t taa J g ~ t ' ~ _ ~ 'agaGa ~ J ~~, ~~a'.~. ~.agca.~, ~aau a"
catgaga 1620 taaagacata aagaaattaa acaatagtta attcttgcct tacctgtacc tct tagtgta 1680 ccctatgtac ctgaatttgc ttggcttctt tgagaatgaa attgaattaa ata tgaataa 1740 ataagtcaaa aaaaaaaaaa aaaaa <210> 19 <211> 1831 <212> DNA
<213> Homo Sapiens <220>
<400> 19 gaggcctcct tctgaggggc ggcttgatac cggtggagga gctccaggaa gca ggcaggc 60 cttggtctga gacagtgtcc tcaggtcgca gagcagagga gacccaggca gtg tcagcag 120 tgaaggttct cgggacaggc taaccaggag gacaggagcc ccaagaggcc cca gagcagc 180 actgacgaag acctgcctgt gggtctccat cgcccagctc ctgcccacgc tcc tgactgc 240 tgccctgacc agagtcatca tgtctctcga gcagaggagt ccgcactgca agc ctgatga 300 agaccttgaa gcccaaggag aggacttggg cctgatgggt gcacaggaac cca caggcga 360 ggaggaggag actacctcct cctctgacag caaggaggag gaggtgtctg ctg ctgggtc 420 atcaagtcct ccccagagtc ctcagggagg cgcttcctcc tccatttccg tct actacac 480 tttatggagc caattcgatg agggctccag cagtcaagaa gaggaagagc caa gctcctc 540 ggtcgaccca gctcagctgg agttcatgtt ccaagaagca ctgaaattga agg tggctga 600 gttggttcat ttcctgctcc acaaatatcg agtcaaggag ccggtcacaa agg cagaaat 660 gctggagagc gtcatcaaaa attacaagcg ctactttcct gtgatcttcg gca aagcctc 720 cgagttcatg caggtgatct ttggcactga tgtgaaggag gtggaccccg ccg accactc 780 ctacatcctt gtcactgctc ttggcctctcc gtgcgatagc atgctgggtg atg a~catag 840 catgcccaag gccgccctcc tgatcatta~ ~~tgggtgtg atcctaacca aag acaacta 900 cgcccctgaa gaggttatct gggaagcgtt gagtgtgatg ggggtgtatg ttg ggaagga 960 gcacatgttc tacggggagc ccaggaagct gctcacccaa gattgggtgc agg aaaacta 1020 cctggagtac cggcaggtgc ccgq_caata_a tcctgcgcac tacgagttcc ta_t ggggtt~ 1080 caaggcccac gctgaaacca gctatgagaa ggtcataaat tatttggtca tgc tcaatgc 1140 aagagagccc atctgctacc catcccttta tgaagaggtt ttgggagagg agc aagaggg 1200 agtctgagca ccagccgcag ccggggccaa agtttgtggg gtcagggccc cat ccagcag 1260 ctgccctgcc ccatgtgaca tgaggcccat tcttcgctct gtgtttgaag aga gcaatca 1320 gtgttctcag tggcagtggg tggaagtgag cacactgtat gtcatctctg ggt tccttgt 1380 ctattgggtg atttggagat ttatccttgc tcccttttgg aattgttcaa atg ttctttt 1440 aatggtcagt ttaatgaact tcaccatcga agttaatgaa tgacagtagt cac acatatt 1500 gctgtttatg ttatttagga gtaagattct tgcttttgag tcacatgggg aaa tccctgt 1560 tattttgtga attgggacaa gataacatag cagaggaatt aataattttt ttg aaacttg 1620 aacttagcag caaaatagag ctcataaaga aatagtgaaa tgaaaatgta gtt aattctt 1680 gccttatacc tctttctctc tcctgtaaaa ttaaaacata tacatgtata cct ggatttg 1740 cttggcttct ttgagcatgt aagagaaata aaaattgaaa gaataaaaaa aaa aaaaaaa 1800 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a <210>20 <211>1886 <212>DNA
<213>Homo Sapiens <220>
<400>20 ctctgggatc tgagagaagc gaaagcgtct ttctgagggg tgtcttgaga gtg gcagagg 60 gcagcgggtc caggctccat gaggaggcaa gccttgggaa tctgagggat gga gactcag 120 ttccgcagag ggggtctggg gtgcagccct gccagcatca agaggaagaa as gagggag 180 gactcaggag actttggact ccaggtgagc actatgttct cagaggacga ct~
ccagtca 240 acagaaagag ccccatatgg tccacaac:~ cagtggtccc aggatctgcc as agtccag 300 gtttttagag aacaggccaa c~tggaaga~ aggagtccca ggagaaccca gao gatcac~ 360 ggaggagaac aagtgctgtg gggccccatc acccagatat ttcccacagt tcg gcctgct 420 gacctaacca gagtcatcat gcctcttgag caaagaagtc agcactgcaa gcc tgaggaa 480 ggccttcagg cccaagaagG ~. ~ctgggc ctggtgggt~ cacagJ~tc~
c~.a agctgag 540 gagcaggagg ctgccttctt c~cctctact ctgaatgtgg gcac~ctaga gga gttgcct 600 gctgctgagt caccaagtcc tccccagagt cctcaggaag agtccttctc tcc cactgcc 660 atggatgcca tctttgggag cctatctgat gagggctctg gcagccaaga aaa ggagggg 720 ccaagtacct cgcctgacct gatagaccct gagtcctttt cccaagatat act acatgac 780 aagataattg atttggttca tttattgctc cgcaagtatc gagtcaaggg get gatcaca 840 aaggcagaaa tgctggggag tgtcatcaaa aattatgagg actactttcc tga gatattt 900 agggaagcct ctgtatgcat gcaactgctc tttggcattg atgtgaagga agt ggacccc 960 actagccact cctatgtcct tgtcacctcc ctcaacctct cttatgatgg cat acagtgt 1020 aatgagcaga gcatgcccaa gtctggcctc ctgataatag tcctgggtgt aat cttcatg 1080 gaggggaact gcatccctga agaggttatg tgggaagtcc tgagcattat ggg ggtgtat 1140 gctggaaggg agcacttcct ctttggggag cccaagaggc tccttaccca aaa ttgggtg 1200 caggaaaagt acctggtgta ccggcaggtg cccggcactg atcctgcatg cta tgagttc 1260 ctgtggggtc caagggccca cgctgagacc agcaagatga aagt~cttga gta catagcc 1320 aatgccaatg ggagggatcc cacttcttac ccatccctgt atgaagatgc ttt gagagag 1380 gagggagagg gagtctgagc a'gagatgca accagggcca gcgggcaggg aaa tgggcca 1440 atgcatgctt cagggccaca cccagcagtt tccctgtcct gtgtgaaatc agg cccattc 1500 ttccctctgt gtttgatgag agaagtcagt gttctcagta gtagaaggca cag tgaatgg 1560 aagggaacac attgtatact gcctttaggt ttctcttcca tcgga~gact tgg agatttg 1620 tttttgtttc cctttggtaa ttt~caaata ttgttcctgt aataaaagtt tta gttagct 1680 tcaacatcta agtgtatgga tgatactgac cacacatgtt gttt~gctta tcc atttcaa 1740 gtgcaagtgt ttgccatttt gtaaaacatt ttgggaaatc ttccatcttg ctg tgatttg 1800 caataggtat tttcttggag aa~gtaagaa cttaacaata aagc~gaact ggt gttgtga 1860 aacagagaaa aaaaaaaaaa aaaaaa <210> 21 <211> 124 <212> PRT
<2i3> Ho~r,oSapiens <220>
<400> 21 Met Ser Glu Gln Lys Ser GlnHis Cys Lys Pro Glu Glu G1 Leu y Leu Asp Thr Glu Glu Ala Leu GlyLeu Val Gly Val Gln Ala A1 Gln a Thr Thr Glu Gln Glu Ala Val SerSer Ser Ser Pro Leu Val Pr Glu o Gly Thr Leu Glu Val Pro Ala AlaGly Ser Pro Gly Pro Leu Ly Gly s Ser Pro Gln Ala Ser Ala Ile ProThr Ala Ile Asp_Phe Thr Le Gly a Trp Arg Gln Ser Ile Lys Gly Ser Ser Asn Gln Glu Glu Glu Gly Pr o Ser Thr Ser Pro Asp Pro Glu Ser Val Phe Arg Ala Ala Leu Ser Ly s Lys Val Ala Asp Leu Ile His Phe Leu Leu Leu Lys Tyr <210> 22 <211> 318 <212> PRT
<213> Homo Sapiens <220>
<400> 22 Met Leu Gly Gln Lys Ser Gln Arg Tyr Lys Ala Glu Glu Leu G1 y Leu G=in Ala Gly Glu Ala Pro Gly Leu Met Asp Va- Gln Ile Gln Pr o Thr Ala Glu Gln Lys Ala Ala SerSer Ser Ser Thr Leu Ile Glu Me t Gly Thr Glu Glu Val Thr Asp SerGiy Ser Pro Ser Pro Pro Leu G1 n Ser Pro Gly Ala Ser Ser Ser LeuThr Val Thr Asp Ser Thr Glu Le a Trp Ser Gln Ser Asp Glu Gly Ser Ser Ser Asn Glu Glu Glu Gly Pr o Ser Thr Ser Pro Asp Pro Ala His Leu Glu Ser Leu Phe Arg Glu Al a Leu Asp Glu Lys Val Ala Glu Leu Val Arg Phe Leu Leu Arg Lys Ty r Gln Ile Lys Glu Pro Val Thr Lys Ala Glu Met Leu Glu Ser Val I1 a Lys Asn Tyr Lys Asn His Phe Pro Asp Ile Phe Ser Lys Ala Ser G1 a Cys Met Gln Val Iie Phe Gly Ile Asp Val Lys Glu Val Asp Pro Al a Gly His Ser Tyr Ile Leu Val Thr Cys Leu Gly Leu Ser Tyr Asp G1 y Leu Leu Gly Asp Asp Gln Ser Thr Pro Lys Thr Gly Leu Leu Ile I1 a Val Leu Gly Met Ile Leu Met Glu Gly Ser Arg Ala Pro Glu Glu Al a Ile Trp Glu Ala Leu Met Gly Ser Leu Val Tyr Asp Gly Arg Glu Hi s Ser Val Tyr Trp Lys LeuArg Lys Leu Leu Thr Gln GluTrp Val G1 n Glu Asn Tyr Leu Glu TyrArg Gln Ala Pro Gly Ser AspPro Val Ar g Tyr Glu Phe Leu Trp GlyPro Arg Ala Leu Ala Glu ThrSer Tyr Va 1 Lys Val Leu Glu His ValVal Arg Val Asn Ala Arg ValArg Ile Se r Tyr Pro Ser Leu His GluGlu Ala Leu Gly Glu Glu LysGly Val <210> 23 <211> 315 <212> PRT
<213> Homo Sapiens <220>
<400> 23 Met Ser Leu Glu GlnArg Ser Pro His Cys Lys ProAsp Glu As p Leu Glu Ala Gln Gly GluAsp Leu Gly Leu Met Gly AlaGln Glu P-r o Thr Gly Glu Glu Glu GluThr Thr Ser Ser Ser Asp SerLys Glu G-a Glu Val Ser Ala Ala GlySer Ser Ser Pro Pro Gln SerPro Gln G1 y Gly Ala Ser Ser Ser IleSer Val Tyr Tyr Thr Leu TrpSer Gln P_~
a Asp Glu Gly Ser SerSer Gln Glu Glu Glu GluPro Ser Ser Ser Va 1 Asp t'~~ Aia G1?~.i~Cl.._.., ilc~~ v._?'.Gnu' - _ _ _ . P ~ .L ~: L 1 ._ _ 1 a y E i 1 a ~, s ~
; .
s Val . .. , ~
Ala Glu Leu ValHis Phe Leu Leu His LysTyr Arg Val Lys G1 a Pro Val Thr Lys AlaGlu Met Leu Glu Ser ValIle Lys Asn Tyr Ly s Arg Tyr Pro Val Ile Phe Gly LysAla Ser Glu Phe Met Gln Va Phe 1 Ile Phe Thr Asp Val Lys Glu ValAsp Pro Ala Gly His Ser Ty Gly r Ile 165 1~0 1~
Leu Thr Ala Leu Gly Leu SerCys Asp Ser Met Leu Gly As Val p Gly His Met Pro Lys A1a Ala LeuLeu Ile Ile Val Leu Gly Va Ser 1 Ile Leu Lys Asp Asn Cys Ala ProGlu Glu Val Ile Trp Glu A1 Thr a Leu Ser Val Met Gly Val Tyr Val Gly Lys Glu His Met Phe Tyr Gl y Glu Pro Arg Lys Leu Leu Thr Gln Asp Trp Val Gln Glu Asn Tyr Le a Glu Tyr Arg Gln Val Pro G-y Ser Asp Pro Ala His Tyr Glu Phe Le a Trn -Gly Ser Lys AlaHis Ala Glu Thr Ser TyrGlu LysVal Ile As n Tyr Leu Val Met LeuAsn Ala Arg Glu Pro IleCys TyrPro Ser Le a Tyr Glu Glu Val LeuGly Glu Glu Gln Glu GlyVal <210> 24 <211> 429 <212> PRT
<213> Homo Sapiens <220>
<400> 24 Met Glu Thr GlnPhe Arg Arg Gly Gly LeuGly CysSer Pro A1 a Ser Ile Lys Arg LysLys Lys Arg Glu Asp SerGly AspPhe Gly Le a Gln Val Ser Thr MetPhe Ser Glu Asp Asp PheGln SerThr Glu Ar g Ala Pro Tyr Gly ProGln Leu Gln Trp Ser GlnAsp LeuPro Arg Va 1 Gln Val Phe Arg GluGln Ala Asn Leu Glu AspArg SerPro Arg Ar g Thr Gln Arg Ile Thr Gly Gly Glu Gln Val Leu Trp Gly Pro Ile Th r Gln Ile Phe Pro Thr Val Arg Pro Ala Asp Leu Thr Arg Val Ile Me t Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Giu Glu Gly Leu G1 n Ala -Gln Glu Glu Asp Leu GlyLeu Val Gly Ala Gln Ala LeuGln A1 a Glu Glu G~~:~ Glu Ala Aia Ph Pr:eSer So_ Tn:_Leu Asn VaiG1~ 1::
r Leu Glu Glu Leu Pro Ala AlaGlu Ser Pro Ser Pro Pro GlnSer Pr o Gln Glu Glu Ser Phe Ser ProThr Ala Met Asp Ala Ile PheGly Se r Leu Ser Asp Glu Gly Ser GlySer Gln Glu Lys Glu Gly ProSer Th r Ser Pro Asp Leu Ile Asp ProGlu Ser Phe Ser Gln Asp IleLeu Hi s Asp Lys Ile Ile Asp Leu ValHis Leu Leu Leu Arg Lys TyrArg Va 1 Lys Gly Leu Ile Thr Lys AlaGlu Met Leu Gly Ser Val IleLys As n Tyr Glu Asp Tyr Phe Pro GluIle Phe Arg Giu Ala Ser ValCys Me t Gln Leu Leu Phe Gly Ile AspVal Lys Glu Val Asp Pro ThrSer Hi s Ser Tyr Val Leu Val Thr SerLeu Asn Leu Ser Tyr Asp GlyIle Gl n Cys Asn Glu Gln Ser Met ProLys Ser Gly Leu Leu Ile IleVal Le a Gly -Val Ile Phe Met Glu GlyAsn Cys Ile Pro Glu GluVal MetTr p Glu J
Val Leu Ser Ile Met GlyVal Tyr Ala Gly Arg GluHis PheLe a Phe Gly Glu Pro Lys Arg LeuLeu Thr Gln Asn Trp ValGln GluLy s Tyr Leu Val Tyr Arg Gln ValPro Gly Thr Asp Pro AlaCys TyrG1 a Phe Leu Trp Gly Pro Arg AlaHis Ala Glu Thr Ser LysMet LysVa 1 Leu Glu Tyr Ile Ala Asn AlaAsn Gly Arg Asp Pro ThrSer TyrPr o Ser Leu Tyr Glu Asp Ala LeuArg Glu Glu Gly Glu GlyVal
Claims (17)
1. An isolated, compl63ementary 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-A10, 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.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US26097899A | 1999-03-02 | 1999-03-02 | |
| US09/260,978 | 1999-03-02 | ||
| PCT/US2000/005346 WO2000052163A1 (en) | 1999-03-02 | 2000-03-01 | Cloning of cdna of mage's 5, 8, 9 and 11 and their uses in diagnosis of cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2366059A1 true CA2366059A1 (en) | 2000-09-08 |
Family
ID=22991462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002366059A Abandoned CA2366059A1 (en) | 1999-03-02 | 2000-03-01 | Cloning of cdna of mage's 5, 8, 9 and 11 and their uses in diagnosis of cancer |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1194542A1 (en) |
| JP (1) | JP2003512814A (en) |
| KR (1) | KR20020011967A (en) |
| AU (1) | AU3389500A (en) |
| CA (1) | CA2366059A1 (en) |
| NZ (1) | NZ513739A (en) |
| WO (1) | WO2000052163A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090028888A1 (en) | 2005-11-14 | 2009-01-29 | Alain Bergeron | Cancer Antigen Mage-A9 and Uses Thereof |
| US10800823B2 (en) | 2017-07-07 | 2020-10-13 | Immatics Biotechnologies Gmbh | Peptides and combination of peptides for use in immunotherapy against lung cancer, including NSCLC, SCLC and other cancers |
| EP4316597A2 (en) | 2017-07-07 | 2024-02-07 | immatics biotechnologies GmbH | Novel peptides and combination of peptides for use in immunotherapy against lung cancer, including nsclc, sclc and other cancers |
| FR3087448B1 (en) | 2018-10-23 | 2023-10-13 | Pdc Line Pharma | PDC LINE MODIFIED TO SECRET A CYTOKINE |
| TW202039535A (en) | 2018-12-18 | 2020-11-01 | 德商英麥提克生物技術股份有限公司 | Immunotherapy with b*08 restricted peptides and combination of peptides against cancers and related methods |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5342774A (en) * | 1991-05-23 | 1994-08-30 | Ludwig Institute For Cancer Research | Nucleotide sequence encoding the tumor rejection antigen precursor, MAGE-1 |
| US5612201A (en) * | 1991-05-23 | 1997-03-18 | Ludwig Institute For Cancer Research | Isolated nucleic acid molecules useful in determining expression of a tumor rejection antigen precursor |
-
2000
- 2000-03-01 NZ NZ513739A patent/NZ513739A/en not_active Application Discontinuation
- 2000-03-01 CA CA002366059A patent/CA2366059A1/en not_active Abandoned
- 2000-03-01 WO PCT/US2000/005346 patent/WO2000052163A1/en not_active Application Discontinuation
- 2000-03-01 AU AU33895/00A patent/AU3389500A/en not_active Abandoned
- 2000-03-01 JP JP2000602775A patent/JP2003512814A/en active Pending
- 2000-03-01 KR KR1020017011090A patent/KR20020011967A/en not_active Application Discontinuation
- 2000-03-01 EP EP00912112A patent/EP1194542A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| JP2003512814A (en) | 2003-04-08 |
| KR20020011967A (en) | 2002-02-09 |
| WO2000052163A1 (en) | 2000-09-08 |
| NZ513739A (en) | 2001-09-28 |
| EP1194542A1 (en) | 2002-04-10 |
| AU3389500A (en) | 2000-09-21 |
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