Abstract:
The invention relates to new members of the GAGE family referred to as GAGE-7B and GAGE-8. There are differences between these two molecules and the previously described members of the GAGE family on the genomic DNA, complementary DNA, and amino acid level.

Description:
RELATED APPLICATIONS  
       [0001]     This application is a continuation application of U.S. Ser. No. 10/271,617 filed Oct. 16, 2002, incorporated by reference in its entirety, which is a divisional application of U.S. Ser. No. 09/163,748 filed Sep. 30, 1998 (now U.S. Pat. No. 6,509,172). 
     
    
     FIELD OF THE INVENTION  
       [0002]     This invention relates to a nucleic acid molecule which codes for a tumor rejection antigen precursor. More particularly, the invention concerns genes, whose tumor rejection antigen precursor is processed, inter alia, into at least one tumor rejection antigen that is presented by MHC molecules. The genes in question are members of the GAGE family of genes.  
       BACKGROUND AND PRIOR ART  
       [0003]     The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is a complex one. An important faced of the system is the T lymphocyte, or “T cell” response. This response requires that T cells recognize and interact with complexes of cell surface molecules, referred to as human leukocyte antigens (“HLA”), or major histocompatibility complexes (“MHCs”), and peptides. The peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecule. See in this regard Male et al., Advanced Immunology (J. P. Lipincott Company, 1987), especially chapters 6-10. The interaction of T cells and HLA/peptide complexes is restricted, requiring a T cell specific for a particular combination of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. This mechanism is involved in the immune system&#39;s response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities. Much work has focused on the mechanisms by which proteins are processed into the HLA binding peptides. See, in this regard, Barinaga, Science 257: 880 (1992); Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964 (1992). Also see Engelhard, Ann. Rev. Immunol. 12: 181-207 (1994).  
         [0004]     The mechanism by which T cells recognize cellular abnormalities has also been implicated in cancer. For example, in PCT application PCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, and incorporated by reference, a family of genes is disclosed, which are processed into peptides which, in turn, are expressed on cell surfaces, which can lead to lysis of the tumor cells by specific CTLs cytolytic T lymphocytes, or “CTLs” hereafter. The genes are said to code for “tumor rejection antigen precursors” or “TRAP” molecules, and the peptides derived therefrom are referred to as “tumor rejection antigens” or “TRAs”. See Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et al., Science 254: 1643 (1991), for further information on this family of genes. Also, see U.S. Pat. No. 5,342,774.  
         [0005]     In U.S. Pat. No. 5,405,940, the disclosure of which is incorporated by reference, it is explained that the MAGE genes code for proteins which are processed to nonapeptides which are then presented by an HLA-A1 molecule. The reference teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to preferably bind to one HLA molecule. This is important, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.  
         [0006]     In U.S. Pat. No. 5,629,166 incorporated by reference, the fact that the MAGE-1 expression product is processed to a second TRA is disclosed. This disclosure shows that a given TRAP can yield a plurality of TRAs. Also, see U.S. Pat. No. 5,554,506, incorporated by reference, teaching peptides which bind to HLA-A2.  
         [0007]     U.S. Pat. Nos. 5,530,096 and 5,487,934 incorporated by reference herein teach that tyrosinase, a molecule which is produced by some normal cells (e.g., melanocytes), is processed in tumor cells to yield peptides presented by HLA-A2 molecules.  
         [0008]     In U.S. patent application Ser. No. 08/032,978, filed Mar. 18, 1993 (now U.S. Pat. No. 5,620,886), and incorporated by reference in its entirety, a second TRA, not derived from tyrosinase is taught to be presented by HLA-A2 molecule. The TRA is derived from a TRAP, but is coded for by a non-MAGE gene. This disclosure shows that a particular HLA molecule may present TRAs derived from different sources.  
         [0009]     In U.S. Pat. No. 5,571,711, filed Jun. 17, 1993 and incorporated by reference herein, an unrelated tumor rejection antigen precursor, the so-called “BAGE” precursor, is described. The BAGE precursor is not related to the MAGE family.  
         [0010]     A further family of genes which are processed into tumor rejection antigens is taught by U.S. Pat. Nos. 5,610,013 and 5,648,226, as well as patent application Ser. Nos. 08/531,662 and 08/602,039, filed on Sep. 21, 1995 and Feb. 15, 1996 respectively, now U.S. Pat. Nos. 5,858,689 and 6,069,001 respectively, and U.S. patent application Ser. Nos. 08/669,161 (now U.S. Pat. No. 6,013,481) and 09/012,818, filed on Jun. 24, 1996 and Jan. 23, 1998, respectively. All of these applications are incorporated by reference. They reveal that there is a family of genes, the “GAGE” genes, which are related to each other. Six members of the GAGE family are described in these references.  
         [0011]     It has now been found that there are at least two further members of the GAGE family, referred to hereafter as GAGE-7 and GAGE-8. These genes, as well as other aspects of the inventions, will be described in detail in the disclosure which follows. 
     
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
     Example 1  
       [0012]     Melanoma cell line MZ2-MEL and cell lines derived therefrom are known. See, e.g., U.S. Pat. No. 5,342,774, incorporated by reference. One subclone, i.e., MZ2-MEL 3.0 was obtained by limiting dilution, and is described in the &#39;774 patent. A subline, i.e., MZ2-ME2.43 was derived by limiting dilution of MZ2-MEL 3.0 cells which had survived mutagen treatment. See Herin, et al., Int. J. Canc. 39:390-396 (1987); Van den Eynde, et al., Int J. Canc. 44:634-640 (1980). This subline had been used as a source of cDNA from which nucleic acid molecules encoding GAGE 1-6 were isolated. See U.S. Pat. Nos. 5,610,013; 5,648,226; application Ser. Nos. 08/531,662; 08/602,039; 08/669,661; and 09/012,818 cited supra, and Van den Eynde, et al., J. Exp. Med. 182:689-698 (1995), all of which are incorporated by reference.  
         [0013]     The cDNA library from MZ2-MEL.43 was rescreened, using the same protocols as are set forth in the above referenced patents and 1995 paper. Two additional positive clones were identified. These molecules were named GAGE-7B and GAGE-8. They are discussed further, infra. The nucleotide sequences for cDNA for these molecules are set forth as SEQ ID NO: 1 (GAGE-8), and SEQ ID NOS: 2 and 3 (GAGE-7B).  
       Example 2  
       [0014]     These experiments describe the isolation of genomic DNA molecules encoding GAGE-7B.  
         [0015]     Peripheral blood lymphocytes (PBLs) were isolated, and grown, using standard methodologies. The genomic DNA was then isolated from the PBLS, partially digested with endonuclease 5au3A1, size fractionated using NaCl density gradient centrifugation, and then ligates into GEM-11 cloning vector, which had been digested with BamHI and EcoRI.  
         [0016]     The phage library was screened, using a probe labeled with α 32 P dCTP, consisting of nucleotides 18-309 of cDNA for GAGE-1. Conditions for this Southern hybridization was standard, as described by Sambrook et al.: Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 1989), incorporated by reference. The washing conditions were 0.2×SSC, 0.1% SDS, at 65° C.  
         [0017]     One of the positive clones was analyzed, and found to contain an insert corresponding to GAGE-7B. The sequence, set forth at SEQ ID NO: 3, contains 5 exons, including an open reading frame over exons 2 to 5, which encodes a 117 amino acid product.  
         [0018]     The fourth intron of this sequence includes two regions which show strong homology with a region found only in GAGE-1. There is a 561 base pair segment positioned in between these regions at nucleotides 7109-7659, which corresponds to a truncated, L1 retroposon which belongs to the family of long interspersed repeated elements, or “LINE”; as described by Hutchinson, et al., in Berg, et al., eds, “Mobile DNA” (Am. Soc. Microbiol. 1989), incorporated by reference. The LINE element is flanked by a perfect 13 base pair target site duplication, and contains part of the reverse transcriptase coding region, the 3′-untranslated region, and the poly-A tail of the original retroposon.  
       Example 3  
       [0019]     A cosmid library was prepared using genomic DNA from renal cell carcinoma cell line LE9211-RCC, following the methodologies described by Lurquin, et al., Cell 58: 293-303 (1989), and screened using the Southern hybridization method set forth in example 2, using the same probe.  
         [0020]     A cosmid was identified which contained genomic DNA for GAGE-8. Its structure was the same as that of GAGE-7B, including the LINE insertion discussed supra.  
       Example 4  
       [0021]     These experiments describe how the chromosomal location of the GAGE genes was determined. Southern blot analysis was carried out on a panel of hamster or mouse x human somatic cell hybrids, obtained from the Human Genetic Mutant Cell Repository. The DNA from these somatic cell hybrids was isolated, digested with EcoRI, and used to prepare Southern blots, in accordance with Arden, et al, Cytogenet. Cell Genet. 53:161-165 (1990), incorporated by reference. The GAGE-1 probe, labeled with α 32 P dCTP, as described supra, was used. A single, EcoRI band of 4.3 kilobases was detected, indicating that the EcoRI sites defining the fragment are conserved in all GAGE genes. The only hybridization signal came from a hybrid containing the human X chromosome. No signal came from hybrids containing human autosomes, or the Y chromosome.  
         [0022]     Experiments were than carried out to refine the localization of the GAGE locus. Somatic cell hybrids containing only a portion of the X chromosome were analyzed via Southern hybridization, as described supra, as well as by PCR.  
         [0023]     For PCR, primers corresponding to nucleotides 453-470 of GAGE-1 cDNA (sense), and nucleotides 613-630 of GAGE-1 cDNA (antisense), were used. These Should amplify a 0.7 kb fragment of genomic DNA, and a fragment consisting of nucleotides 453-630 of GAGE-1 cDNA, as set forth in U.S. Pat. No. 5,610,013 at SEQ ID NO: 1. Thirty five cycles of amplification were carried Out, each cycle consisting of denaturation at 94° C. (1 minute), annealing at 50° C. (1 minute), and extension at 72° C. for 1 minute. The PCR was preceded by 3 minutes of incubation at 94° C., and was followed by a soak at 72° C. for 10 minutes. Amplified products were electrophoresed on 2% agarose gels, and were visualized by ethidium bromide staining. The analysis revealed that the GAGE genes are located in chromosomal region Xp21-Xq13.  
       Example 5  
       [0024]     A further set of experiments were carried out to find the location of the GAGE locus, using fluorescence in situ hybridization, or “FISH”. To accomplish this, PBLs were stimulated with PHA, and cultured for 72 hours. Banded chromosomes were obtained by inoculating some cultures with 5-bromodeoxyuridine, in accordance with Lemieux, et al., Cytogenet. Cell Genet 59:311-312 (1992). Cytogenetic harvests, and slide preparations were prepared using standard methods. Slides were stored at −80° C. until used.  
         [0025]     FISH hybridization to metaphase chromosomes was carried out following Pinkel, et al., Proc. Natl. Acad. Sci USA 83:2934-2938 (1986). Briefly, slides were denatured for 2 minutes in 70% formamide/2×SSC (pH 7.0), and then dehydrated in ice cold ethanol. A cosmid which contained gDNA for GAGE-7B was used as a probe. The probe (100 ng) was labeled with digoxigenin, preannealed with 100 mg of COT-1 DNA, dissolved in buffer (50% formamide, 2×SSC), denatured at 75° C. for 5 minutes, and then applied to slides. The probes were hybridized to the material on the slides, overnight at 37° C., in a humid chamber.  
         [0026]     After the incubation, the slides were washed using standard procedures, and then analyzed using standard FITC-digoxigenin detection methods, together with an amplification protocol for dual color FISH. The slides were counterstained by mounting in an antifade solution containing 1 mg/ml phenylenediamine and 0.3 mg/ml propidium iodide. Spreads were examined, and photographed. A signal was deemed to be specific only if detected on each chromatid of a single chromosome. Chromosome identification was performed via simultaneous hybridization with the satellite repeat probe, or by R-banding, using 5-bromodeoxyuridine in accordance with Lemieux, et al, supra.  
         [0027]     These experiments indicated that the GAGE locus is in the p11.2-p11.4 region of the X chromosome.  
       Example 6  
       [0028]     These experiments were designed to determine expression of GAGE genes in various cell and tumor types. For each type of cell assayed, total RNA was extracted, using standard guanidium-isothiocyanate procedures, as taught by e.g., Davis, et al. in Basic Methods In Molecular Biology, Elsevier Science Publishing Co., N.Y. (1986), pp. 130-135. Reverse transcription was carried out on 2 ug samples of the total RNA, using 2 mM of Oligo(dT) 15  primer, in a reaction volume of 20 ul. Portions of the resulting DNA ( 1/20 of the product), were used in the PCR amplification. In order to amplify GAGE-1, 2, and 8, the primers used were:  
                                       5′-GACCAAGACG CTACGTAG-3′               (sense, SEQ ID NO:4)           and                       5′-CCATCAGGAC CATCTTCA-3′           (antisense, SEQ ID NO:5)          
 
         [0029]     For GAGE-3, 4, 5, 6 &amp; 7B, the primers were:  
                                       5′-GACCAAGGCG CTATGTAC-3′               (sense, SEQ ID NO:6)           and                       SEQ ID NO:5          
 
         [0030]     For all amplifications, the denaturation step was 94° C. for 5 minutes, then 30 cycles of amplification (1 minute at 94° C., 2 minutes at 58° C., 2 minutes at 72° C.), then a final extension step of 72° C. for 15 minutes. The products were analyzed by agarose gel electrophoresis, with RNA integrity being checked by reverse transcription and amplification of β-actin mRNA.  
         [0031]     When these primers are used, SEQ ID NOS: 4 and 5 produce a fragment consisting of nucleotides 107-350 of SEQ ID NO: 3. SEQ ID NOS: 5 and 6 produce a fragment consisting of nucleotides 92-335 of SEQ ID NO: 2.  
         [0032]     Table 1, which follows, shows the results. The highest fraction of positive tumors were found in melanoma, esophageal and lung carcinomas. GAGE 1, 2 and 8 was found in prostate carcinomas, breast carcinomas, and sarcomas. GAGE 3, 4, 5, 6 and 7B were not found in this tumors. No expression of GAGE was found in colorectal and renal carcinoma.  
                                                                                                 TABLE 1                           Expression of the GAGE genes in tumors                Number of samples   % of samples                    Expression of GAGE-1, 2, 7*   +   +   −   −   expressing GAGE-1, 2, 7       Tumor   tested   Expression of GAGE-3, 4, 5, 6, 8   +   −   +   −   and/or GAGE-3, 4, 5, 6, 8                    Cutaneous melanoma (primaries)   79       22   1   10   46   42%       Cutaneous melanoma (metastases)   211       79   8   26   98   54%       Esophageal squamous cell carcinoma   18       7   1   1   9   50%       Esophageal adenocarcinoma   5       1   0   0   4   20       Lung squamous cell carcinoma   83       28   4   7   44   47       Lung adenocarcinoma   42       13   2   6   21   50       Head &amp; neck carcinoma   92       21   3   5   63   31       Bladder carcinoma (superficial)   35       1   0   0   34   3       Bladder carcinoma (infiltrating)   40       8   2   6   24   40       Leukemia   76       0   0   3   773   4                  
 
       Example 7  
       [0033]     In order to determine if expression of GAGE genes could be induced by demethylation, samples of cultured tumor and normal cells were incubated for 72 hours in culture medium containing 1 uM 5-aza-2′-deoxycytidine. SEQ ID NOS: 4 and 5, supra, were used in the amplification protocol. GAGE 1, 2, and 8 were found to have been induced in sarcoma and melanoma cell lines. All GAGE genes were found to be expressed following treatment of PHA stimulated PBLs.  
         [0034]     The foregoing examples set forth the invention, which includes isolated nucleic acid molecules which encode proteins GAGE 7B and GAGE 8. These may be, e.g., those set forth at SEQ ID NO: 1, 2 or 3, as well as all nucleic acid molecules which encode the proteins encoded by theses sequences. When GAGE-7B and GAGE-8 are compared to the other members of the GAGE family, cDNA for GAGE-8 is found to be identical to cDNA for GAGE-2 but for a single nucleotide, at nucleotide 268 (“C” in GAGE-2, versus “G” in GAGE-8). This leads to a change in the amino acid at position 74 (His in GAGE-2, Asp in GAGE-8). GAGE-7B is identical to GAGE-4, but for two nucleotides at positions 268 and 548. This first difference (“G” in GAGE-4, “C” in GAGE-8), results in a change at amino acid 74 as well (Asp in GAGE-4, His in GAGE-7B).  
         [0035]     There are further differences in the organization of the genomic DNA, as explained supra. Specifically, GAGE-8 and GAGE-7B differ from GAGEs 2-6 in that they contain two inserts in the fourth intron. These inserts are found in GAGE-1 genomic DNA; however, GAGE-8 and 7B also contain a 561 base pair insert positioned in between these two inserts, which is not found in the genomic DNA of GAGE-1.  
         [0036]     In addition to the nucleic acid molecules discussed supra, other features of the invention include expression vectors which include the nucleic acid molecules of the invention, operably linked to a promoter. Both cDNA and genomic DNA can be used, in expression vectors of various types. These, as well as the isolated nucleic acid molecules of the invention, can be used to make recombinant eukaryotic and prokaryotic cells, which contain either the isolated nucleic acid molecules or the expression vectors of the invention. The choice of which nucleic acid molecule or which expression vector to use will be up to the skilled artisan, depending upon the application of interest.  
         [0037]     The nucleic acid molecules of the invention include segments which correspond to peptides presented by HLA-Cw6 and HLA-A29, i.e., YRPRPRRY (SEQ ID NO: 26) (GAGE 1, 2 and 8), and YYWPRPRRY (SEQ ID NO: 27) (GAGE 3, 4, 5, 6 and 7B). Hence, a further aspect of the invention are recombinant cells which, in addition to including molecules which encode GAGE-7B and GAGE-8, also include one or more nucleic acid molecules which encode MHC molecules, such as HLA-Cw6 and/or HLA-A29. It is to be understood that additional genes which are processed to presented antigens may be used as well the GAGE 7B and 8 genes.  
         [0038]     Also a feature of the invention are the proteins encoded by the nucleic acid molecules of the invention. As explained, supra, these proteins are similar, but not identical to other GAGE proteins. Also, part of the invention are fragments of the proteins of the invention. In particular, these fragments compare at least the first 74 amino acids encoded by the SEQ ID NO: 1, 2 or 3, and no more than the entire molecule encoded by these sequences. These proteins are set forth at SEQ ID NOS.: 6 and 7. Also a part of the invention are those peptides, derived form GAGE 7B and/or GAGE 8, which complex to MHC molecules, thereby identify a particular molecule, and also in at least some cases, facilitating the proliferation of cytolytic T cells which recognize complexes of the peptide and the MHC molecule to which it binds. One or more of these peptides can be combined in compositions, which may also include one or more adjuvants, such as GM-CSF, an interleukin, an emulsifying oil such as Vitamin E, a saponin, etc.  
         [0039]     “Minigenes” can also be produced which are nucleic acid molecules that consent of nucleotides that encode these peptides. Constructs can also be prepared, such as expression vectors, which encode one or more of these peptides.  
         [0040]     An exemplary list of such peptides, with the partner MHC molecule, follows.  
                                                                       GAGE 7B                Position   Sequence   HLA Molecule                             43-51   EGEPATQRQ   A1                             9-17   YYWPRPRRY   A24                        16-24   RYVQPPEMI   A24                        24-32   IGPMRPEQF   A24                        11-19   WPRPRRYVQ   B7                        19-27   QPPEMIGPM   B7                        11-19   WPRPRRYVQ   B8                         1-9   MSWRGRSTY   B3501                        19-27   QPPEMIGPM   B3501                        28-36   RPEQFSDEV   B3501                         1-9   MSWRGRSTY   B4403                        33-41   DEVEPATPE   B4403                        56-64   QEGEDEGAS   B4403                       108-116   EEGEKQSQC   B4403                        16-24   RYVQPPEMI   B5201                        19-27   QPPEMIGPM   B5201                        24-32   IGPMRPEQF   B5201                        28-36   RPEQFSDEV   B5201                        97-105   MDPPNPEEV   B5201                        19-27   QPPEMIGPM   Cw0602                        28-36   RPEQFSDEV   Cw0602.                      
 
         [0041]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
               
               
                 GAGE 8 
               
             
          
           
               
                   
                 Position 
                 Sequence 
                 HLA Molecule 
               
               
                   
                   
               
             
          
           
               
                   
                  16-24 
                 YVEPPEMIG 
                 A1 
                   
               
               
                   
                   
               
               
                   
                  42-50 
                 EGEPATQRQ 
                 A1 
               
               
                   
                   
               
               
                   
                   8-16 
                 TYRPRPRRY 
                 A24 
               
               
                   
                   
               
               
                   
                  15-23 
                 RYVEPPEMI 
                 A24 
               
               
                   
                   
               
               
                   
                  23-31 
                 IGPMRPEQF 
                 A24 
               
               
                   
                   
               
               
                   
                  10-18 
                 RPRPRRYVE 
                 B7 
               
               
                   
                   
               
               
                   
                  18-26 
                 EPPEMIGPM 
                 B7 
               
               
                   
                   
               
               
                   
                   1-9 
                 MSWRGRSTY 
                 B3501 
               
               
                   
                   
               
               
                   
                  18-26 
                 EPPEMIGPM 
                 B3501 
               
               
                   
                   
               
               
                   
                  27-35 
                 RPEQFSDEV 
                 B3501 
               
               
                   
                   
               
               
                   
                   1-9 
                 MSWRGRSTY 
                 B4403 
               
               
                   
                   
               
               
                   
                  33-41 
                 DEVEPATPE 
                 B4403 
               
               
                   
                   
               
               
                   
                  56-64 
                 QEGEDEGAS 
                 B4403 
               
               
                   
                   
               
               
                   
                 108-116 
                 EEGEKQSQC 
                 B4403 
               
               
                   
                   
               
               
                   
                  18-26 
                 EPPEMIGPM 
                 Cw0602 
               
               
                   
                   
               
               
                   
                  27-35 
                 RPEQFSDEV 
                 Cw0602 
               
               
                   
                   
               
             
          
         
       
     
         [0042]     Other features of the invention will be clear to the skilled artisan, and will not be set forth here.  
         [0043]     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.