Nucleic acid molecules encoding the MAGE-1 tumor rejection antigen precursor

Abstract

The invention relates to an isolated DNA sequence which codes for an antigen expressed by tumor cells which is recognized by cytotoxic T cells, leading to lysis of the tumor which expresses it. Also described are cells transfected by the DNA sequence, and various therapeutic and diagnostic uses arising out of the properties of the DNA and the antigen for which it codes.

Description

This application is a (SEQ ID NOS: 1, 2, and 3)(SEQ ID NO: 17), and CHO10: (GAAGAGGAGGGGCCAAG) (SEQ ID NO: 18). These oligos correspond to regions of exon 3 that are common to previously described mage 1, 2 and 3.

To do this, 1 μg of RNA was diluted to a total volume of 20 μl, using 2 μl of 10×PCR buffer, 2 μl of each of 10 mM dNTP, 1.2 μl of 25 mM MgCl₂, 1 μl of an 80 mM solution of CHO9, described supra, 20 units of RNAs in, and 200 units of M-MLV reverse transciptase. This was followed by incubation for 40 minutes at 42° C. PCR amplification followed, using 8 μl of 10×PCR buffer, 4.8 μl of 25 mM MgCl₂, 1 μl of CHO10, 2.5 units of Thermus acquaticus (“Taq”) polymerase, and water to a total volume of 100 μl. Amplification was then carried out for 30 cycles (1 minute 94° C.; 2 minutes at 52° C., 3 minutes at 72° C.). Ten μl of each reaction were then size fractionated on agarose gel, followed by nitrocellulose blotting. The product was found to hybridize with oligonucleotide probe CHO18 (TCTTGTATCCTGGAGTCC) (SEQ ID NO: 19). This probe identified mage 1 but not mage 2 or 3. However, the product did not hybridize to probe SEQ 4 (TTGCCAAGATCTCAGGAA) (SEQ ID NO: 20). This probe also binds mage 1 but not 2 and 3. This indicated that the PCR product contained a sequence that differed from mage 1, 2 and 3. Sequencing of this fragment also indicated differences with respect to mage 4 and 5. These results indicate a sequence differing from previously identified mage 1, 2, 3, 4 and 5, and is named mage 6.

EXAMPLE 33

In additional experiments using cosmid libraries from PHA-activated lymphocytes of MZ2, the 2.4 kb mage 1 fragment was used as a probe and isolated a complementary fragment. This clone, however, did not bind to oligonucleotides specific for mage 1, 2, 3 or 4. The sequence obtained shows some homology to exon 3 of mage 1, and differs from mages 1-6. It is referred to as mage 7 hereafter.

EXAMPLE 34

The usefulness of the TRAPs, as well as TRAs derived therefrom, was exemplified by the following.

Exon 3 of mage 1 was shown to transfer expression of antigen E. As a result, it was decided to test whether synthetic peptide derived from this exon 3 could be used to confer sensitivity to anti-E CTL.

To do this, and using standard protocols, cells normally insensitive to anti-E/CTLs were incubated with the synthetic peptide derived from:

Asp-Val-Lys-Glu-Ala-Asp-Pro-Thr-Gly-His-Ser-Tyr-Val-Leu-Val. Using the CTL lytic assays described supra on P815A, and a peptide concentration of 3 mM, the assay showed lysis of 30%, including conferring of sensitivity to the anti-E CTL.

The foregoing disclosure, including the examples, places many tools of extreme value in the hands of the skilled artisan. To begin, the examples identify and provide a methodology for isolating nucleic acid molecules which code for tumor rejection antigen precursors as well as the nucleic acid molecules complementary thereto. It is known that DNA exists in double stranded form, and that each of the two strands is complementary to the other. Nucleic acid hybridization technology had developed to the point where, given a strand of DNA, the skilled artisan can isolate its complement, or synthesize it.

“Nucleic acid molecule” as used herein refers to all species of DNA and RNA which posses the properties discussed supra. Genomic and complementary DNA, or “cDNA” both code for particular proteins, and as the examples directed to isolation of MAGE coding sequences show, this disclosure teaches the artisan how to secure both of these.

Similarly, RNA molecules, such as mRNA can be secured. Again, with reference to the skilled artisan, once one has a coding sequence in hand, mRNA can be isolated or synthesized.

Complementary sequences which do not code for TRAP, such as “antisense DNA” or mRNA are useful, e.g., in probing for the coding sequence as well as in methodologies for blocking its expression.

It will also be clear that the examples show the manufacture of biologically pure cultures of cell lines which have been transfected with nucleic acid sequences which code for or express the TRAP molecules. Such cultures can be used as a source for tumor rejection antigens, e.g., or as therapeutics. This aspect of the invention is discussed infra.

Cells transfected with the TRAP coding sequences may also be transfected with other coding sequences. Examples of other coding sequences include cytokine genes, such as interleukins (e.g., IL-2 or IL-4), or major histocombatibility complex (MHC) or human leukocyte antigen (HLA) molecules. Cytokine gene transfection is of value because expression of these is expected to enhance the therapeutic efficacy of the biologically pure culture of the cells in vivo. The art is well aware of therapies where interleukin transfectants have been administered to subjects for treating cancerous conditions.

Transfection with an MHC/HLA coding sequence is desirable because certain of the TRAs may be preferentially or specifically presented only by particular MHC/HLA molecules. Thus, where a recipient cell already expresses the MHC/HLA molecule associated with presentation of a TRA, additional transfection is not necessary. On the other hand, it may be desirable to transfect with a second sequence when the recipient cell does not normally express the relevant MHC/HLAmolecule. It is to be understood, of course, that transfection with one additional sequence does not preclude further transfection with other sequences.

The term “biologically pure” as used in connection with the cell line described herein simply means that these are essentially free of other cells. Strictly speaking, a “cell line” by definition is “biologically pure” but the recitation will establish this fully.

Transfection of cells requires that an appropriate vector be used. Thus, the invention encompasses expression vectors where a coding sequence for the TRAP of interest is operably linked to a promoter. The promoter may be a strong promoter, such as those well known to the art, or a differential promoter, i.e., one which is operative only in specific cell types.

The expression vectors may incorporate several coding sequences, as long as the TRAP sequence is contained therein. The cytokine and/or MHC/HLA genes discussed supra may be included in a single vector with the TRAP sequence. Where this is not desired, then an expression system may be provided, where two or more separate vectors are used where each coding sequence is operably linked to a promoter. Again, the promoter may be a strong or differential promoter. Co-transfection is a well know technique, and the artisan in this field is expected to have this technology available for utilization.

As the foregoing discussion makes clear, the sequences code for “tumor rejection antigen precursors” (“TRAPs”) which, in turn, are processed into tumor rejection antigens (“TRAs”). Isolated forms of both of these categories are descried herein, including specific examples of each. Perhaps their most noteworthy aspect is as vaccines for treating various cancerous conditions. The evidence points to presentation of TRAs on tumor cells, followed by the development of an immune response and deletion of the cells. The examples show that when various TRAs are administered to cells, a CTL response is mounted and presenting cells are deleted. This is behavior characteristic of vaccines, and hence TRAPs, which are processed into TRAs, and the TRAs themselves may be used, either alone or in pharmaceutically appropriate compositions, as vaccines. Similarly, present cells may be used in the same manner, either alone or as combined with ingredients to yield pharmaceutical compositions.

The generation of an immune response, be it T-cell or B-cell related, is characteristic of the effect of the presented tumor rejection antigen. With respect to the B-cell response, this involves, inter alia, the generation of antibodies to the TRA, i.e., which specifically bind thereto. In addition, the TRAP molecules are of sufficient size to render them immunogenic, and antibodies which specifically bind thereto are a part of this invention. These antibodies may be polyclonal or monclonal, the latter being prepared by any of the well recognized methodologies for their preparation which need not be repeated here. For example, mAbs may be prepared using an animal model, e.g., a Balb/C mouse or in a test tube, using, e.g., EBV transfomants. In addition, antiserum may be isolated from a subject afflicted with a cancerous condition where certain cells present a TRA.

Review of the foregoing disclosure will show that there are a number of facets to the system which may be referred to as “tumor rejection antigen presentation and recognition”. Recognition of these phenomena has diagnostic consequences. For example, the existence of specific CTL clones, or antibodies to the TRA makes it possible to diagnose or monitor cancerous conditions (explained infra), by monitoring the CTLs in a sample from a subject, binding of antibodies to TRAs, or the activity of anti-TRA CTLs in connection with subject samples. Similarly, the expression of nucleic acid molecules for TRAPs can be monitored via amplification (e.g., “polymerase chain reaction”), anti-sense hybridization, probe technologies, and so forth. Various subject samples, including body fluids (blood, serum, and other exudates, e.g.), tissues and tumors may be so assayed.

The term “cancerous condition” is used herein to embrace all physiological events that commence with the initiation of the cancer and result in final clinical manifestation. Tumors do not spring up “ab initio” as visible tumors; rather there are various events associated with the transformation of a normal cell to malignancy, followed by development of a growth of biomass, such as a tumor, metastasis, etc. In addition, remission may be conceived of as part of “a cancerous condition” as tumors seldom spontaneously disappear. The diagnostic aspects of this invention include all events involved in carcinogenesis, from the first transformation to malignancy of a single cell, through tumor development and metastasis, as well as remission. All are embraced herein.

Where “subject” is used, the term embraces any species which can be afflicted with a cancerous condition. This includes humans and non-humans, such as domesticated animals, breeding stock, and so forth.

There are therapeutic aspects of this invention as well. The efficacy of administration of effective amounts of TRAPs and TRAs as vaccines has already been discussed supra. Similarly, one may develop the specific CTLs in vitro and then administer these to the subject. Antibodies may be administered, either polyclonal or moncolonal, which specifically bind to cells presenting the TRA of interest. These antibodies may be coupled to specific anti tumor agents, including, but not being limited to, methotrexate radio-iodinated compounds, toxins such as ricin, and so forth. Thus, “targeted” antibody therapy is included herein, as is the application of deletion of the cancerous cells by the use of CTLs.

The terms and expressions which have been employed are used as terms of description and not of limitation, and thrre is no intensoin in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

Claims

1. An isolated nucleic acid molecule which codes for the MAGE tumor rejection antigen precursor as set forth in SEQ. ID. NO. 8.

2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid is a DNA cDNA molecule which comprises nucleotides 3881 to 4711 of SEQ. ID. NO. 8.

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

4. An isolated nucleic acid molecule which hybridizes to the nucleic acid molecule which codes for the MAGE-1 tumor rejection antigen precursor as set forth in SEQ ID No: 8 under stringent conditions and which codes for a tumor rejection precursor.

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

6. The expression vector of claim 5, wherein said promoter is an incurable promoter.

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

8. The expression vector of claim 7, wherein said promoter is an inducible promoter.

9. An expression vector comprising the isolated nucleic acid molecule of claim 4 operably linked to a promoter.

10. The expression vector of claim 9, wherein said promoter is an inducible promoter.

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

12. The host cell of claim 11, wherein said host cell is a mammalian cell.

13. The host cell of claim 12, wherein said cell line is a fibroblast cell line.

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

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

16. The hose cell of claim 15, wherein MAGE 1 has the nucleic acid sequence as follows:

1	GGATCCAGGC	CCTGCCAGGA	AAAATATAAG	GGCCCTGCGT	GAGAACAGAG	GGGGTCATCC	60
61	ACTGCATGAG	AGTGGGGATG	TCACAGAGTC	CAGCCCACCC	TCCTGGTACG	ACTGAGAAGC	120
121	CAGGGCTGTG	CTTGCGGTCT	GCACCCTGAG	GGCCCGTGGA	TTCCTCTTCC	TGGAGCTCCA	180
181	GGAACCAGGC	AGTGAGGCCT	TGGTCTGAGA	CAGTATCCTC	AGGTCACAGA	GCAGAGGATG	240
241	CACAGGGTGT	GCCAGCAGTG	AATGTTTGCC	CTGAATGCAC	ACCAAGGGCC	CCACCTGCCA	300
301	CAGGACACAT	AGGACTCCAC	AGAGTCTGGC	CTCACCTCCC	TACTGTCAGT	CCTGTACAAT	360
361	CGACCTCTGC	TGGCCGGCTG	TACCCTGAGT	ACCCTCTCAC	TTCCTCCTTC	AGGTTTTCAG	420
421	GGGACAGGCC	AACCCAGAGG	ACAGGATTCC	CTGGAGGCCA	CAGAGGAGCA	CCAAGGAGAA	480
481	GATCTGTAAG	TAGGCCTTTG	TTAGAGTCTC	CAAGGTTCAG	TTCTCAGCTG	AGGCCTCTCA	540
541	CACACTCCCT	CTCTCCCCAG	GCCTGTGGGT	CTTCATTGCC	CAGCTCCTGC	CCACACTCCT	600
601	GCCTGCTGCC	CTGACGAGAG	TCATCATGTC	TCTTGAGCAG	AGGAGTCTGC	ACTGCAAGCC	660
661	TGAGGAAGCC	CTTGAGGCCC	AACAAGAGGC	CCTGGGCTGG	TGTGTGTGCA	GGCTGCCACC	720
721	TCCTCCTCCT	CTCCTCTGGT	CCTGGGCACC	CTGGAGGAGG	TGCCCACTGC	TGGGTCAACA	780
781	GATCCTCCCC	AGAGTCCTCA	GGGAGCCTCC	GCCTTTCCCA	CTACCATCAA	CTTCACTCGA	840
841	CAGAGGCAAC	CCAGTGAGGG	TTCCAGCAGC	CGTGAAGAGG	AGGGGCCAAG	CACCTCTTGT	900
901	ATCCTGGAGT	CCTTGTTCCG	AGCAGTAATC	ACTAAGAAGG	TGGCTGATTT	GGTTGGTTTT	960
961	CTGCTCCTCA	AATATCGAGC	CAGGGAGCCA	GTCACAAAGG	CAGAAATGCT	GGAGAGTGTC	1020
1021	ATCAAAAATT	ACAAGCACTG	TTTTCCTGAG	ATCTTCGGCA	AAGCCTCTGA	GTCCTTGCAG	1080
1081	CTGGTCTTTG	GCATTGACGT	GAAGGAAGCA	GACCCCACCG	GCCACTCCTA	TGTCCTTGTC	1140
1141	ACCTGCCTAG	GTCTCTCCTA	TGATGGCCTG	CTGGGTGATA	ATCAGATCAT	GCCCAAGACA	1200
1201	GGCTTCCTGA	TAATTGTCCT	GGTCATGATT	GCAATGGAGG	GCGGCCATGC	TCCTGAGGAG	1260
1261	GAAATCTGGG	AGGAGCTGAG	TGTGATGGAG	GTGTATGATG	GGAGGGAGCA	CAGTGCCTAT	1320
1321	GGGGAGCCCA	GGAAGCTGCT	CACCCAAGAT	TTGGTGCAGG	AAAAGTACCT	GGAGTACGGC	1380
1381	AGGTGCCGGA	CAGTGATCCC	GCACGCTATG	AGTTCCTGTG	GGGTCCAAGG	GCCCTCGCTG	1440
1441	AAACCAGCTA	TGTGAAAGTC	CTTGAGTATG	TGATCAAGGT	CAGTGCAAGA	GTTCGCTTTT	1500
1501	TCTTCCCATC	CCTGCGTGAA	GCAGCTTTGA	GAGAGGAGGA	AGAGGGAGTC	TGAGCATGAG	1560
1561	TTGCAGCCAA	GGCCAGTGGG	AGGGGGACTG	GGCCAGTGCA	CCTTCCAGGG	CCGCGTCCAG	1620
1621	CAGCTTCCCC	TGCCTCGTGT	GACATGAGGC	CCATTCTTCA	CTCTGAAGAG	AGCGGTCAGT	1680
1681	GTTCTCAGTA	GTAGGTTTCT	GTTCTATTGG	GTGACTTGGA	GATTTATCTT	TGTTCTCTTT	1740
1741	TGGAATTGTT	CAAATGTTTT	TTTTTAAGGG	ATGGTTGAAT	GAACTTCAGC	ATCCAAGTTT	1800
1801	ATGAATGACA	GCAGTCACAC	AGTTCTGTGT	ATATAGTTTA	AGGGTAAGAG	TCTTGTGTTT	1860
1861	TATTCAGATT	GGGAAATCCA	TTCTATTTTG	TGAATTGGGA	TAATAACAGC	AGTGGAATAA	1920
1921	GTACTTAGAA	ATGTGAAAAA	TGAGCAGTAA	AATAGATGAG	ATAAAGAACT	AAAGAAATTA	1980
1981	AGAGATAGTC	AATTCTTGCC	TTATACCTCA	GTCTATTCTG	TAAAATTTTT	AAAGATATAT	2040
2041	GCATACCTGG	ATTTCCTTGG	CTTCTTTGAG	AATGTAAGAG	AAATTAAATC	TGAATAAAGA	2100
2101	ATTCTTCCTG	TTCACTGGCT	CTTTTCTTCT	CCATGCACTG	AGCATCTGCT	TTTTGGAAGG	2160
2161	CCCTGGGTTA	GTAGTGGAGA	TGCTAAGGTA	AGCCAGACTC	ATACCCACCC	ATAGGGTCGT	2220
2221	AGAGTCTAGG	AGCTGCAGTC	ACGTAATCGA	GGTGGCAAGA	TGTCCTCTAA	AGATCTAGGG	2280
2281	AAAAGTGAGA	GAGGGGTGAG	GGTGTGGGGC	TCCGGGTGAG	AGTGGTGGAG	TGTCAATGCC	2340
2341	CTGAGCTGGG	GCATTTTGGG	CTTTGGGAAA	CTGCAGTTCC	TTCTGGGGGA	GCTGATTGTA	2400
2401	ATGATCTTGG	GTGGATCC					2418

17. The host cell of claim 14, wherein said host cell is a mammalian cell.

18. The host cell of claim 15, wherein said host cell is a mammalian cell.

19. An isolated nucleic acid molecule which hybridizes to the nucleic acid molecule which codes for the MAGE-1 tumor rejection antigen precursor as set forth in SEQ ID NO: 8 at 0.1×SSC, 0.1% SDS, and which codes for a tumor rejection antigen precursor.

20. An expression vector comprising the isolated nucleic acid molecule of claim 19, operably linked to a promoter.

21. The expression vector of claim 20, wherein said promoter is an inducible promoter.

22. A host cell transfected with the insolated nucleic acid molecule of claim 19.

23. The host cell of claim 22, wherein said isolated nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 7.

24. The host cell of claim 22, wherein said host cell is a mammalian cell.

25. A host cell transfected with an isolated nucleic acid molecule which hybridizes to the nucleic acid molecule which codes for the MAGE-1 tumor rejection antigen precursor as set forth in SEQ ID NO: 8 at 0.1×SSC, 0.1% SDS and which codes for a tumor rejection antigen precursor.