WO2001062917A1 - Sequences genomiques isolees codant pour le cancer ny-eso-1/antigene de la tumeur des testicules et leurs utilisations - Google Patents

Sequences genomiques isolees codant pour le cancer ny-eso-1/antigene de la tumeur des testicules et leurs utilisations Download PDF

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WO2001062917A1
WO2001062917A1 PCT/US2001/002126 US0102126W WO0162917A1 WO 2001062917 A1 WO2001062917 A1 WO 2001062917A1 US 0102126 W US0102126 W US 0102126W WO 0162917 A1 WO0162917 A1 WO 0162917A1
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eso
nucleic acid
seq
acid molecule
sequence
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PCT/US2001/002126
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Bernard Lethe
Thierry Boon-Falleur
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Ludwig Institute For Cancer Research
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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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

Definitions

  • This invention relates to genomic sequences of NY-ESO-1 (also called LAGE-2), a molecule that is processed to at least one HLA binding peptide which is an antigen associated with cancer. These peptides bind to Class I and to Class II MHC molecules.
  • Antige ic peptides are identified after they bind to empty MHC-class I molecules of mutant cell lines, defective in antigen processing, and induce specific reactions with cytotoxic T-lymphocytes. These reactions include induction of CTL proliferation, TNF release, and lysis of target cells, measurable in an MTT assay, or a 51 Cr release assay.
  • the methodologies described rely on the availability of established, permanent cell lines of the cancer type under consideration. It is very difficult to establish cell lines from certain cancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg. Clin. North. Am. 10: 607-637 (1990). It is also known that some epithelial cell type cancers are poorly susceptible to CTLs in vitro, precluding routine analysis. These problems have stimulated the art to develop additional methodologies for identifying cancer associated antigens.
  • NY-ESO-1 The cDNA of NY-ESO-1 is disclosed in US Patent No. 5,804,381. Analysis of NY-ESO- 1 indicated that it is expressed in the testis and in a high percentage of a wide range of different human tumor types (see, e.g., Table 4). This cancer/testis expression pattern is shared by other members of CTL-recognized antigens such as MAGE, BAGE, GAGE, and SSX. NY-ESO-1, also referred to as LAGE 2, is related to LAGE 1 (described in Lethe et al, Int. J. Cancer 76:903- 908 (1998)).
  • the genomic sequence of NY-ESO-1 /LAGE-2 has now been identified.
  • the newly sequenced genomic nucleic acid molecules contain 5' and 3' untranslated sequences as well as two introns that are previously unl ⁇ iown. Further, using these introns, new RNA transcripts of these antigens have been uncovered.
  • the features of the NY-ESO-1 /LAGE-2 genomic sequence are seen in the disclosure which follows.
  • Figure 1 depicts the expression pattern of RNA for the NY-ESO-1 antigen, in various tissue types.
  • Figure 2 depicts Northern Blot analysis of NY-ESO-1 mRNA, which was found in testis and cell line SK-MEL-19, but not in various other cell and tissue samples.
  • Figure 3 depicts potential sites for modification of the deduced amino acid sequence of
  • Figure 4 depicts a hydrophilicity plot of NY-ESO-1, showing hydrophilic domains in the amino terminus and a long, hydrophobic stretch close to the carboxyl end.
  • Figure 5 depicts the results of CTL lysis studies using various cells which are HLA-A2 positive, NY-ESO-1 positive, positive for both, or positive for neither.
  • Figure 6 depicts data establishing that HLA- A2 is the presenting molecule for presentation of SEQ ID NO: 1 derived peptides.
  • Human NY-ESO-1 gene contains three exons and two introns, spanning more than 2 kb of genomic DNA. The location and size of each exon and intron of
  • NY-ESO-1 are listed below. Referring now to SEQ ID NO:l 1, the 5' untranslated sequence of NY-ESO-1 corresponds to nucleotide 1 to 971; exon 1 corresponds to nucleotide 908 to 1240 intron 1 corresponds to nucleotide 1241 to 1912; exon 2 corresponds to nucleotide 1913 to 2047; intron 2 corresponds to nucleotide 2048 to 2286; exon 3 corresponds to nucleotide 2287 to 2577 and 3' untranslated sequence corresponds to 2423 to 2630 of SEQ ID NO:l l.
  • the initiation codon refers to the "ATG" sequence located at nucleotides 972-974 of SEQ ID NO:l 1.
  • the termination codon of NY-ESO-1 refers to the "TAA" sequence located at nucleotides 2423-2425 of SEQ ID NO:l l.
  • the exon-intron boundaries were determined by comparing the sequence of the genomic DNA described herein in SEQ ID NO: 11 to the cDNA sequence described in SEQ ID NO:l. The exon-intron boundaries were identified as the positions where the genomic DNA sequence diverged from that of the cDNA.
  • One embodiment of the invention is directed to an isolated or substantially pure nucleic acid molecule that is capable of hybridizing to SEQ ID NO: 11 under Southern or Northern hybridization conditions.
  • the isolated nucleic acid molecule hybridizes to SEQ ID NO: 11 under stringent hybridization conditions.
  • the isolated nucleic acid molecule hybridizes to the introns presented in SEQ ID NO:l 1 (1241 to 1912 or base 2048 to 2286 of SEQ ID NO: 11), or the 5' and 3' untranslated sequence (base 1 to base 971 of SEQ ID NO:
  • Southern hybridization conditions are known and available in many standard molecular biology manuals. For example, in Molecular Cloning, A Laboratory Manual (Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989, incorporated herein by reference), a number of stringent Southern hybridization conditions are listed for genomic Southern blots made from agarose gels using 10 to 30 ⁇ g of digested genomic DNA molecules per lane.
  • one set of stringent Southern blot conditions includes two-hours of prehybridization in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, and 100 ⁇ g/ml denatured salmon sperm DNA, followed by hybridization in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, and 100 ⁇ g/ml denatured salmon sperm DNA and 50% formamide using 10-20 ng of denatured labeled DNA molecule (in 20 ml of hybridization solution) with a specific activity of 10 9 cpm/ ⁇ g for 6-16 hours at 42 °C.
  • Washing may be performed, for example, by three washes at room temperature with 2 X SSC, 0.1% SDS for 15 minutes each followed by two washes with 0.2 X SSC, 0.1 % SDS at 37 °C for 45 minutes each, followed by one wash with 0.2 X SSC, 0.1 % SDS at 60°C for 20 minutes.
  • Southern hybridization may be performed using probes made from DNA molecules with SEQ ID NO: 11 or any segments thereof of appropriate length. It is known that oligonucleotide probes as short as 17 nucleotides in length may be used successfully in Southern Blots (Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989). Thus, any oligonucleotide molecule comprising 17 or more basepairs of contiguous sequence from SEQ
  • the oligonucleotide molecule is greater than about 25 bp in length. More preferably, the oligonucleotide molecule is greater than about 35 bp in length. Most preferably, the oligonucleotide molecule is greater than about 50 bp in length. In a preferred embodiment, the oligonucleotide molecule comprises a contiguous sequence from base 1241 to 1912 or base 2048 to 2286 of SEQ ID NO:ll.
  • the time for the final wash such as the final wash referred to supra, may be reduced to for example, to about 5 minutes, preferably to about 4 minutes, and most preferably to about 3 minutes.
  • the final washing temperature may be reduced to about 50 °C, more preferably to about 45 °C and most preferably to about 42 °C.
  • one preferred final washing condition may be 3 minutes at 42 °C.
  • the isolated nucleic acid molecules of the invention may be labeled with a detectable label.
  • a detectable label refers to a material which, when covalently attached to the isolated nucleic acid molecules of this invention, permit detection of the isolated nucleic acid molecules.
  • Suitable detectable labels are well known in the art and include, by way of example, radioisotopes (e.g., 32 P), fluorescent labels (e.g., fluorescein), biotinylation and the like.
  • the particular detectable label employed is not critical and the selection of detectable labels is well within the skill of the art.
  • the labeled isolated nucleic acid molecules may be used, for example, to detect the expression of a NY-ESO-1 gene in a tissue sample of a patient.
  • Another embodiment of the invention is directed to a recombinant expression vector molecule comprising the isolated nucleic acid molecules of the invention, operably linked to a promoter.
  • Recombinant expression vectors are available from many commercial sources such as the American Tissue Type Collection, Stratagene, or New England Biolabs.
  • the recombinant expression vector may be a eukaryotic, or a wide host range (shuttle) vector. It is understood that expression vectors may also be selected from the group consisting of signal transduction vectors, gene targeting vectors and yeast shuttle vectors.
  • a gene targeting vector may cause the replacement of an endogenous gene with a replacement gene under a constitutive (or inducible) promoter.
  • Suitable vectors include vectors such as pCMV-Script, pPbac, pMbac, pDual, pCMVLacI and pESC which are capable of expression in eukaryotic cells. It is understood that recombinant expression vectors also encompass in vitro expression vectors such as pSPUTK which can express in a cell- free environment such as a rabbit reticulocyte lysate or a wheat germ extract. It is understood that the promoter may be constitutive or inducible. Constitutive promoters may include, for example, the RSV, SV40, or CMV promoter. Inducible promoters include the GAL 1 , GAL 10, and MMTV promoters.
  • promoters, vectors, host cells, and expression systems are available commercially from many suppliers. These commercially available promoters, vectors, host cells, and expression systems, are listed in the catalogs of American Tissue Type Collection, Stratagene or New England Biolabs; incorporated herein by reference. Promoters and control elements may be extracted from a commercially available nucleic acid molecule (i.e., vectors and the like) by standard methods such as PCR. The vectors of the invention may also comprise synthesized elements.
  • Both strands of any expression vector element may be synthesized by an oligonucleotide synthesizer, hybridized to a double stranded nucleic acid molecule, and cloned.
  • Another embodiment of the invention is directed to a prokaryotic or eukaryotic cell line transformed or transfected with the isolated nucleic acid molecule of the invention. This isolated nucleic acid molecule may be cloned in an expression vector such as the vectors listed above.
  • the cell line may be any cell line used in molecular biology such as, for example, mammalian, insect, bacteria, or plant cell lines.
  • Another embodiment of the invention is directed to a method of producing a tumor rejection antigen precursor protein, such as NY-ESO-1, by culturing a host cell transformed or transfected with a construct of the invention.
  • a tumor rejection antigen precursor protein such as NY-ESO-1
  • the NY-ESO-1 genomic DNA molecule is cloned into an expression vector and transfected into a host cell (transfection is the introduction of a foreign gene(s)/nucleic acid into a target cell)
  • the host cell may be cultured under appropriate in vitro conditions for the expression of NY-ESO-1.
  • a functional foreign gene, cloned into an expression vector can produce the RNA and protein product it encodes.
  • Many transfection methods have been developed but all can be classified as either direct or indirect methods.
  • Indirect methods a genetic engineer injects the foreign gene into individual target cells using a microcapillary or microprojectile.
  • Indirect methods involve the target cells passively or actively taking up the foreign gene.
  • Indirect methods are diverse and include, for example, pinocytotic uptake of DNA-calcium phosphate (calcium phosphate transfection) and fusion of liposomes with the plasma membrane of the target cell (lipofection).
  • Another method of transfection involves the use of viral particles to infect the target cell.
  • Viral particles that have been used successfully for transfection include DNA viruses, such as SV40, polyoma, adenovirus, Epstein-Barr, vaccinia, herpes simplex and baculo virus, and RNA viruses, such as tobacco mosaic virus, cucumber mosaic virus, brome mosaic virus and retro virus.
  • DNA viruses such as SV40, polyoma, adenovirus, Epstein-Barr, vaccinia, herpes simplex and baculo virus
  • RNA viruses such as tobacco mosaic virus, cucumber mosaic virus, brome mosaic virus and retro virus.
  • a promoter present in an expression vector can be regulated by the presence of a repressor or the absence of an inducer. This regulation can be effected by suppressing the activity of the promoter at so that a large cell population can be produced with only minimal impairment of the vitality of the cells. Subsequently, the promoter is stimulated, by the removal of the repressor or addition of an inducer, so that the synthesis of the tumor rejection antigen precursor protein protein can take place.
  • the expression of the tumor rej ection antigen precursor protein may be basically divided into a growth phase and a production phase.
  • an inducible promoter is the Mouse Mammary Tumor Virus Long Terminal Repeat (MMTV-LTR) promoter. This promoter express at a relatively low level until a cell population is stimulated with glucocorticoid. In the presence of glucocorticoid, the promoter is expressed at a high level.
  • MMTV-LTR Mouse Mammary Tumor Virus Long Terminal Repeat
  • nucleic acid molecule from the subject is hybridized to a nucleic acid molecule of the invention.
  • the nucleic acid molecule from the subj ect may be, for example, a cDNA or an RNA from the subject.
  • cDNA may be obtained from a sample using standard methods such as, for example, purification of RNA away from DNA (e.g., CsCl sedimentation) followed by reverse transcription of the mRNA with oligo dT primers. It is well known that mRNA from a subject may be amplified by PCR before detection.
  • Amplification may be performed, for example, using a non-specific primer (oligo dT primer) in combination with a specific primer or using two primers specific for the mRNA.
  • the isolated nucleic acid molecule of the invention may be a substantially pure genomic DNA molecule which is capable of hybridizing to SEQ ID NO: 11 under Southern or Northern hybridization conditions.
  • the isolated nucleic acid molecule of the invention comprise the intronic sequence of SEQ ID NO: 11.
  • the DNA probes of the invention may be a sense strand probe, an antisense strand probe, or a double stranded probe. Further, it is known that both strands of a double stranded probe may be labeled for detecting a single stranded nucleic acid molecule. For example, in Northern hybridization, it is common practice to label a double stranded DNA fragment for detection even though only one strand would be expected to hybridize to the target mRNA. Such use of the probes of the invention is also contemplated.
  • the disorder to be detected may be any disorder which is associated with the expression of the tumor antigen precursor. Such disorders include neoplasia, hypertrophy and hyperplasia disorders.
  • the disorder may be, for example, a neoplastic disorder such as an esophageal cancer or a melanoma.
  • the method of detection may be a Northern blot of RNA isolated from a subject. Other detection methods known in the art such as polymerase chain reaction (U.S. Pat. Nos.4,683,195 and 4,683,202); ligase chain reaction (LCR; sometimes referred to as "Ligase Amplification Reaction" or LAR; described by Barany, Proc. Natl. Acad.
  • RNA including mRNA by the incorporation a reverse transcription step using a specific or nonspecific primer.
  • a nonspecific primer may be, for example, an oligo dT or an oligo dl (inosine) primer. Oligo dT primers would be expected to reverse transcribe mRNAs which comprise poly A tails. Inosine hybridizes to all bases with roughly the same affinity and thus, oligo dl serves as an ideal random primer for nonspecific reverse transcription of all RNA species.
  • the depleted serum was then diluted, and mixed with nitrocellulose membranes containing phage plaques.
  • the plaques were incubated overnight, at room temperature. Washing followed, and then the filters were incubated with alkaline phosphatase conjugated goat anti human FC ⁇ secondary antibodies, and reactive phage plaques were visualized by incubating with 5-bromo-4-chloro-indolyl phosphate and nitroblue tetrazolium. A total of 13 positive clones were found.
  • the reactive clones were subcloned to monoclonality via dilution cloning and testing with human serum. These clones were then purified, excised in vitro, and converted into pBK-CMV plasmid forms, using the manufacturer's instructions. The inserted DNA was then evaluated using EcoRI-Xbal restriction mapping to determine different inserts.
  • Table 1 summarizes the results.
  • One gene was represented by four overlapping clones, a second by three overlapping clones, and the remaining six by one clone only.
  • a homology search revealed that the clones referred to as NY-ESO-2, 3, 6, 7 were already known. See Elisei, et al., J. Endocrin. Invest. 16: 533-540 (1993); Spritz, et al, Nucl. Acids Res. 15: 10373-10391 (1987); Rabbits, et al., Nature Genetics 4: 175-180 (1993); Crozat, et al., Nature 363: 640-644 (1993); GenBankH18368 andD25606.
  • NY-ESO-3 and NY-ESO-6 Two of the clones (NY-ESO-3 and NY-ESO-6), have previously been shown to be expressed in various normal human tissues. No evidence of lineage restriction has been found. NY-ESO-6 (cDNA), appears to be the 3'- untranslated portion of the FUS/TLS gene. In experiments not reported here, sequencing and Southern Blot analysis of NY-ESO-6 showed no evidence of translocation or point mutations in the cancer.
  • Four of the clones, i.e., NY-ESO-1, 4, 5 and 8 showed no strong homology to sequences in the databases examined, and were thus studied further.
  • NY-ESO-7 ElB-20f -l.Okb human Ul -70k sn RNP different from NY-ESO-2
  • Example 3 Studies were carried out to evaluate mRNA expression of the NY-ESO 1, 4, 5 and 8 clones. To do this, specific oligonucleotide primers were designed for each sequence, such that cDNA segments of 300-400 base pairs could be amplified, and so that the primer melting temperature would be in the range of 65-70 ° C. Reverse transcription-PCR was then carried out using commercially available materials and standard protocols. A variety of normal and tumor cell types were tested. The clones NY-ESO-4 and NY-ESO-8 were ubiquitous, and were not studied further. NY-ESO-5 showed high level expression in the original tumor, and in normal esophageal tissue, suggesting that it was a differentiation marker.
  • NY-ESO-1 was found to be expressed in tumor mRNA and in testis, but not normal colon, kidney, liver or brain tissue. This pattern of expression is consistent with other tumor rejection antigen precursors.
  • ESO-1 was found to be highly expressed in normal testis and ovary cells. Small amounts of RT- PCR production were found in normal uterine myometrium, and not endometrium, but the positive showing was not consistent. Squamous epithelium of various cell types, including normal esophagus and skin, were also negative. When tumors of unrelated cell lineage were tested, 2 of 11 melanomas cell lines showed strong expression, as did 16 of 67 melanoma specimens, 6 of 33 breast cancer specimens and 4 of 4 bladder cancer. There was sporadic expression in other tumor types.
  • Table 3 mRNA distribution of NY-ESO-1 in melanoma and breast cancer cell lines:
  • Cancer patients melanoma 12/127 9.4 ovarian cancer 4/32 12.5 lung, cancer 1/24 4.0 breast cancer 2/26 7.7
  • Northern blot analysis was then carried out to investigate the size of the NY-ESO-1 transcript, and to confirm tissue expression patterns.
  • the methodology of Ausubel, et al., Current Protocols In Molecular Biology (John Wiley & Sons, 1995) was used. To be specific, 20 ⁇ g of total RNA per lane were dissolved in a formamide and formaldehyde containing buffer, heated to 65 °C, and then separated on a 1.2% agarose gel, with 3%> formaldehyde, followed by transfer to nitrocellulose paper. Hybridization was then carried out using a 32 P labeled probe, followed by high stringency washing. The final wash was at O.lxSSC, 0.1% SDS, 60°C, for 15 minutes.
  • An esophageal carcinoma specimen showed a smear in the 0.4-0.9 kb range, reflecting partial degradation.
  • RNA from additional tissues or cell lines tested showed no transcript.
  • the esophageal cDNA library was rescreened, using plaque hybridization, and the original cDNA clone as the hybridization probe. When 3x10 5 clones were screened, six positives were found. The three longest clones were sequenced.
  • a melanoma cell line "NW-MEL-38" was established, in 1995, from a patient who suffered from malignant melanoma. Serum samples, peripheral blood lymphocytes, and tumor samples, were taken from the subj ect and frozen, until the work described herein was carried out. In anticipation of evaluating antitumor T cell response in this patient, the patient was HLA typed as HLA-A1 and HLA-A2.
  • These primers should amplify a segment of SEQ ID NO:l which spans nucleotides 271 to 599.
  • Amplification was carried out over 35 cycles, using an annealing temperature of 60°C.
  • the PCR products were visualized via ethidium bromide staining, on a 1.5 % agarose gel.
  • the isolated cDNA molecule discussed supra. was then used to make recombinant protein. Specifically, the cDNA was PCR amplified, using standard techniques, and was then cloned into a commercially available plasmid vector, i.e., pQE9, which contains His tags. In work not elaborated upon herein, a second vector, pQE9K was also used. This differs from
  • PQE9 in that kanamycin resistance is imparted by pQE9K, rather than ampicillin resistance.
  • the plasmid vector was transformed into E. coli strain XLl-Blue, and positive transformants were identified via restriction mapping and DNA sequencing. Production of recombinant protein was induced using isopropyl ⁇ -D-thiogalactoside, and the protein was purified on an Ni 2+ ion chromatography column, following well known procedures. The protein when analyzed via 15%o SDS-PAGE and silver staining, was identified as a protein with a molecular weight of about 22 kilodaltons. This is consistent with the anticipated size of the protein from its sequence. Two other forms of the recombinant protein were also identified. These consisted of amino acids 10-180, and 10-121 of the amino acid sequence reported in SEQ ID NO : 1. They have molecular weights of about 14 kD and 20 kD, respectively, on SDS-PAGE, as carried out supra.
  • the recombinant protein produced in this vector carries an His-tag, so it was purified on Ni 2+ affinity columns, also as described, supra.
  • the protein consists of amino acids 10-180, and has a molecular weight of 20 kD via SDS-PAGE.
  • eukaryotic tiansfectants were then produced.
  • the NY-ESO-1 coding sequence was isolated from the pQE9 vector described supra, and then cloned into BaniHI-Hindlll sites of eukaryotic expression vector pcDNA 3.1.
  • COS-7 cells were transfected with this vector, by contacting cell samples with 150 ng of the plasmid discussed supra, and 150 ng of plasmid pcDNA 1 Amp, which contained either cDNA for HLA-A2.1 or cDNA for HLA-A1.
  • the well known DEAE-dextran chloroquine method was used. The cells were then incubated at 37 °C, for 48 hours, after which they were tested in a CTL stimulation assay.
  • RPMI RPMI supplemented with 100%> human serum
  • 25 U/ml of recombinant IL-2 were added to microwells containing COS-7 tiansfectants (20,000 cells/well).
  • 50 ⁇ l of supernatant were collected from each well, and TNF- ⁇ levels were determined in a standard assay, i.e., one where cytotoxicity against WEHI 164 clone 13 cells were tested, using MTT. Positive cells were used in the Western Blot analysis, described in the example which follows.
  • the CTLs used were CTL NW38-IVS- 1 , prepared in accordance with Knuth et al. , Proc.
  • mixed lymphocyte T cell cultures were set up, by combining 10 5 autologous NW38 MEL- 1 tumor cells, and 10 6 peripheral blood lymphocytes, taken from the subject. The cytokine IL-2 was added, and the mixed culture was incubated for one week at 37 °C. Tumor cells were removed, and a new aliquot of 5x10 4 tumor cells were added together with IL-2. This process was repeated weekly, until a strong response was seen when tested against 51 Cr labeled NW-MEL-38 cells. The responder T cells were collected and frozen until used in further experiments.
  • Example 8 Western Blot analysis was then carried out, using the serum samples described supra, as well as cell lysates taken from the cell line NW-MEL-38, described supra, and the COS-7 tiansfectants, described supra, and the purified recombinant protein, also described supra. Serum samples were taken from various points of the patient's therapy. There was no difference in the results.
  • BALB/C mice were immunized via five subcutaneous injections of recombinant NY-ESO-1 protein, at 2-3 week intervals.
  • the immunizing formulation included 50 ⁇ g of recombinant protein in adjuvant.
  • the first injection used Complete Freund's Adjuvant, and Incomplete Freund's Adjuvant was used thereafter.
  • Spleen cells were taken from the iimnunized mice, and fused with mouse myeloma cell line SP2/0, to generate hybridomas. Representative hybridoma E978 was used for generation of mAbs.
  • hybridomas were generated, they were cloned, and their supernatants were screened against recombinant protein, using a standard solid phase ELIS A on microtiter plates.
  • the assay was in accordance with Dippold et al, Proc. Natl. Acad. Sci. USA 77: 6114-6118 (1980), incorporated by reference. A series of negative controls were also run, using recombinant NY- ESO-1.
  • Serum antibodies whichbound to recombinant protein, produced by E. coli as described, supra were visualized using goat anti-human IgG, labeled with alkaline phosphatase at 1 : 10,000 dilution, and were then visualized with NBT-phosphate.
  • Untransfected COS-7 cells were also used as a control. Serum from a healthy individual was also used as a control.
  • NY-ESO-1 Four different forms of NY-ESO-1 are described supra, i.e., the form produced by SEQ ID NO:l in E. coli, as well as one consisting of amino acids 10-180, one consisting of amino acids 10-121, and a form, expressed in the baculovirus vector system discussed supra which consisted of amino acids 10-180. Each form was used in ELISAs, following the above described protocols. All forms of the protein were found to be equally reactive with antibodies taken from various patients, as well as the murine monoclonal antibodies discussed, supra.
  • Example 10 In the testing of the COS-7 tiansfectants, supra, and the assays discussed in this example, a cytolytic T cell line "NW38-IVS-1" was used. This "CTL” was generated, via in vitro stimulation of the peripheral blood lymphocytes mentioned supra, using the tumor cell line NW- MEL-38. This was done using standard techniques.
  • the CTL was used in a cytotoxicity assay with NW-MEL-38 (which was HLA-A1, A2 positive, and NY-ESO-1 positive), along with two allogeneic cell lines which were NY-ESO-1 and HLA-A2 positive (SK-MEL-37 and MZ-MEL-19), a cell line which is MHC Class I negative (SK-MEL-19), a cell line which is HLA-A2 positive, but NY-ESO-1 negative (NW- MEL-145), along with control cell lines K562 and autologous phytohemagglutinin stimulated blasts.
  • Various effector/target ratios were used, and lysis of 51 Cr labeled target cells was the parameter measured. Figure 5 shows this.
  • T2 cell line CEMX721.174.T2
  • T2 cell line CEMX721.174.T2
  • T2 cell line CEMX721.174.T2
  • Samples of T2 cells were labeled with 100 uCi of Na( 51 Cr)O 4 , using standard methods, and were then washed three times, followed by incubation with 10 ⁇ g/ml peptide and 2.5 ⁇ g/ml of B2-micro globulin. Incubation was for one hour, at room temperature.
  • responder cells 100 ⁇ l of a suspension of CTL NW38-IVS-1) were added, at an effector/target ratio of 90:1, and incubated for four hours in a water saturated atmosphere, with 5%> CO 2 , at 37 °C. Then, plates were centrifuged at 200xg for five minutes, 100 ⁇ l of supernatant was removed, and radioactivity was measured. The percentage of 51 Cr release was determined in accordance with known strategies. It was found that the peptides SLLMWITQCFL (SEQ ID NO:4), SLLMWITQC (SEQ ID NO:5), and QLSLLMWIT (SEQ ID NO:6), were the three best stimulators of CTLs. Comparable results were found when NW-MEL-38 and cell lines SK-MEL-37 and MZ-MEL-19 were used as targets, as is shown, supra.
  • the amino acid sequence of the protein encoded by SEQ ID NO:l was analyzed for peptide sequences which correspond to HLA binding motifs. This was done using the algorithm taught by Parker et al., J. Immunol. 142: 163 (1994), incorporated by reference. In the Table which follows, the amino acid sequence, the HLA molecule to which it presumably binds, and the positions in SEQ ID NO:l are given. The resulting complexes should provoke a cytolytic T cell response. This could be determined by one skilled in the art following methods taught by, e.g., van der Bruggen, et al., J. Eur. J. Immunol. 24: 3038-3043 (1994), incorporated by reference.
  • Example 14 To isolate the human NY-ESO-1 gene, genomic DNA from three human cell lines (LB373-MEL tumor cell line, an autologous lymphoblastoid cell line, and an allogeneic lymphoblastoid cell line) was screened by polymerase chain reaction (PCR) using two primers made from published cDNA sequences. The two primers used were BLE 73 (5'- ccccaccgcttcccgtg-3': SEQ IDNO:12) andBLE 71 (5'-ctggccactcgtgctggga-3': SEQIDNO:13) described in Lethe et al. (Int. J. Cancer, 76, 903-908 (1998)).
  • the primer pair is specific for coding (exon) sequences; however, if an intron existed between the exons, a PCR of the genomic DNA using the primers would be expected to amplify a fragment of DNA comprising the intron.
  • PCR was performed as follows. About 100 ng of about 700 ng of genomic DNA template was used per reaction with about 0.4 ⁇ M of each primer. The reaction was performed in a 50 ⁇ l volume in 1 x PCR buffer (e.g., DyNAzymeTM assay buffer: 10 mM Tris-HCl (pH 8.8 at25°C), 1.5mMMgCl 2 , 50mMKCl, 0.1%tritonX-100) with 200 ⁇ M of each dNTP.
  • DyNAzymeTM assay buffer 10 mM Tris-HCl (pH 8.8 at25°C), 1.5mMMgCl 2 , 50mMKCl, 0.1%tritonX-100
  • PCR was initated by a first denaturation at 94 °C for 3 minutes followed by 33 cycles of: 95° C for 30 seconds, 62 °C for 1 minute and 72 °C for 2 minutes. After 33 thermocycles, the reaction was incubated at 70°C for 15 minutes for complete extensions of any PCR products. Atotal of three
  • PCR assays were performed using the different genomic DNA templates.
  • the PCR reaction products were assayed by agarose gel electrophoresis followed by ethidium bromide staining. Only one band, corresponding to an amplified PCR product, was observed from the PCR amplification of each template. Furthermore, the PCR amplification product from each of the three genomic DNA templates had the same gel migration speed. Thus, it can be concluded that PCR resulted in three amplified DNA products of the same size. To confirm this result, the three PCR products were sequenced. When the sequences were compared to each other, it was found that the three sequences were identical. This sequence is shown as SEQ ID NO:8. These data indicate that a genomic sequence that is conserved among the three cell lines was amplified.
  • a comparison of the sequence of the PCR amplified product with the cDNA sequence of the LAGE-2 cDNA clone revealed that the PCR amplified product contains a 239 bp sequence, an intron, that is not present in LAGE-2 cDNA, the nucleotide sequence of which is set forth in SEQ ID NO: 1.
  • This 239 intron sequence shares 84%o identity with an intron of LAGE- lb cDNA.
  • the PCR amplified product also contained a partial exon 2 sequence of NY-ESO-1 at nucleotides 1 to 39 and a partial exon 3 sequence at nucleotides 279 to 458.
  • a cDNA library derived from a thyrocarcinoma cell line was screened by colony hybridization.
  • a thyrocarcinoma cDNA library already seeded in microplates, was screened using the PCR product of Y-ESO-1 described supra. Colonies from the positive wells of the screen were subplated for a second round of screening. The subscreening was repeated as necessary until a homogeneous population of positive clones was obtained. The cDNA from the positive clone was isolated and sequenced. The sequence is set forth as SEQ ID NO:9.
  • SEQ ID NO:9 can be characterized as comprising the following parts: (1) first exon sequence from nucleotides 1 to 191; (2) second exon sequence in nucleotides 192 to 326; (3) second intron sequence in nucleotides 327 to 565; and (4) third exon sequence in nucleotides 566 to 873.
  • the sequence of SEQ ID NO: 9 indicates that there are at least two species of mRNA for NY-ESO-1 because U.S. Patent No. 5,804,381 describes intron free NY-ESO-1 cDNA.
  • One species designated LAGE-2a, i.e., the cDNA of 5,804,381
  • the second species designated LAGE-2b, SEQ ID NO:9 contains a 239 base intron.
  • the cDNA library described supra was analyzed to determine the relative abundance of LA GE-2a and LAGE-2 b containing clones in the library.
  • the technique used to determine relative abundance is described by Application Serial No. 09/260,978, incorporated herein by reference. Briefly, the thyrocarcinoma library was diluted in LB medium, supplemented with ampicillin, to obtain about 3 to 6 clones per microliter. Following this, 9.6 mis of the dilution were seeded into a 96-well microtiter plate at 100 ⁇ l per well.
  • microtiter plates Three plates were seeded in order to obtain a total ofabout 100,000 independent clones spread over the microtiterplates.
  • the microtiter plates were then incubated overnight at 37°C, after which 10 ⁇ l from every well in the microtiter plate were pooled to obtain 20 different pools from every plate (i.e., 8 pools from 8 rows, and 12 pools from 12 columns in an 8 by 12 (96) well microtiter plate).
  • a fraction of the samples from the microtiter plates and the pooled samples were used for analysis (stored at 4 ° C) while the rest was frozen at -70 °C in a cold storage medium comprised of LB medium with 20% glycerol.
  • PCR assays were performed on the pools (row pools and columnpools) to determine how many pools contain a LAGE 2a or LAGE 2b clone.
  • the PCR amplification products were assayed by agarose gel electrophoresis to determine the presence of an amplified product. Positive microwells showing the presence of an amplified product (i.e., expression of LAGE-2a or LA GE-2b) were found at the intersection of a positive row and a positive column.
  • the PCR amplification was performed as follows.
  • genomic NY-ESO-1 The structure of genomic NY-ESO-1 was analyzed by PCR. To determine if the genomic structure of LAGE- 1 and NY-ESO-1 are similar, genomic DNA was amplified with primers SEQ ID NO: 1
  • Primers SEQ ID NO: 15 and SEQ ID NO: 13 are expected to amplify, under PCR, a region of the LAGE-1 genomic sequence that is 1250 bp in size. If the structure of LAGE-1 and NY-ESO-1 are similar, then PCR performed with primers SEQ ID NO: 14 and SEQ ID NO: 13 would be expected to amplify a region in NY-ESO-1 that is also about 1250 bp in size. This was indeed the case. PCR using the BLE72 and BLE 71 produced a single product of 1250 in size. Similarly, PCR using BLE 74 and BLE 71 primers also produced a product of about 1250 in size. Thus, it appears that NY- ESO-1 and LAGE-1 genomic structures are similar.
  • genomic DNA molecules were amplified with primers BLE 70 (5'- gccatgcaggccgaaggc -3': SEQ ID NO:16) and BLE 71 (5'- ctggccactcgtgctggga-3': SEQ ID NO: 13), which are located near the 5' and 3' ends of the cDNAs ofbothJ ⁇ 4GE-i an.dNY-ESO-1.
  • BLE 70 5'- gccatgcaggccgaaggc -3': SEQ ID NO:16
  • BLE 71 5'- ctggccactcgtgctggga-3': SEQ ID NO: 13
  • PCR cloning was performed to isolate and determine the sequence of the promoter region of NY-ESO-1 gene.
  • Two different human genomic DNA samples were used for the PCR analysis.
  • the antisense primer used, SEQ ID NO: 17 (BLE77), is specific for exon II of NY- ESO-1. This antisense primer was chosen because NY-ESO-1 and LAGE-1 show the most difference in sequence in this region. Thus, this antisense primer would have the most hybridization specificity for NY-ESO-1.
  • This antisense primer was used with a sense primer, discussed below, for the cloning and sequencing of the 5' untranslated region of genomic NY- ESO-1 gene.
  • the sense primer used, SEQ ID NO: 18 was derived from LAGE-1 5' untranslated sequence. If the LAGE-1 5' untranslated sequence is similar to NY-ESO-1 5' untranslated sequence, then PCR of genomic DNA using the sense primer (SEQ ID NO: 18) and antisense primer (SEQ ID NO: 17) should produce an amplified product containing the 5' untranslated sequence of NY-ESO-1. Genomic DNA from the LB373-MEL cell line was used as the template for PCR amplification. A LAGE-1 cosmid was used as a negative control template for the PCR amplification. The LAGE-1 cosmid is expected to hybridize to SEQ ID NO: 18 (BLE87) was derived from LAGE-1 5' untranslated sequence. If the LAGE-1 5' untranslated sequence is similar to NY-ESO-1 5' untranslated sequence, then PCR of genomic DNA using the sense primer (SEQ ID NO: 18) and antisense primer (SEQ ID NO: 17) should produce an ampl
  • SEQ ID NO: 10 comprises the following parts: (1) 17 bp primer sequence listed as bases 1 to 17 of SEQ ID NO : 10; (2) a region from the promoter listed as bases 18 to 657; (3) exon 1 listed as bases 658 to 979; (4) intron 1 listed as bases 980 to 1651; (5) partial exon 2 sequence listed as bases 1652 to 1751.
  • NY-ESO-1 and LAGE-1 differ only by only one base. To ensure that the sequence of the NY- ESO-1 promoter region was accurate, the sequence was verified two additional times by direct sequencing of PCR products amplified from genomic DNA from two different sources. These additional sequences did not differ from the original sequence and confirmed the reported sequence of the promoter upon 400 nt from the 5'-end of the LAGE-2/NY-ESO-1 cDNA molecules.
  • a commercial genomic library in PAC vectors was screened by filter hybridization using a genomic NY-ESO-1 probe obtained by PCR with primers SEQ ID NO.T2 and SEQ ID NO.T3.
  • a 2.6 kb Sph I fragment was subcloned from this isolated clone. After this 2.6 kb Sph I subclone was determined by hybridization to be recognized by the NY-ESO-1 probe, it was sequenced to determine the 5' structure of the NY- ESO-1 gene.
  • Sequencing of the isolated clone provided additional sequence information of the 5' and 3' flanking sequence of the NY-ESO-1 gene. The sequence also confirmed the sequence of the introns. Further, the additional sequence information from this clone also defined a 3' flanking sequence downstream to the NY-ESO-1 polyadenylation site. A complete sequence of the genomic NY-ESO-1 gene, base on the experiments described above, is presented as SEQ ID NO: 11.
  • SEQ ID NO: 11 comprises: (1) a 5' untranscribed sequence at nucleotides 1 to 918; (2) an exon at nucleotides 919 to 1240 with initiation codon "ATG" at nucleotides 972-974; (3) intron 1 at nucleotides 1241 to 1912; (4) a second exon at nucleotides 1913 to 2047; (5) a second intron at nucleotides 2048 to 2286; (6) a third exon at nucleotides 2287 to 2577 with a termination codon "TAA" at nucleotides 2423 to 2425; (7) 3' untranscribed sequence at nucleotides 2578 to 2630.
  • the open reading frame of the mRNA would correspond to nucleotides 972 to 1240, 1913 to 2047, and 2287 to 2422 of SEQ ID NO:l 1, concatenated to each other in a 5' to 3' order.
  • NY-ESO-1 and LAGE-1 display 83% identity in the sequenced regions and both genes are highly similar at the 3' and 5' ends. There is 97% identity at the 1200 bps of the 5' end and 93% identity in about 600 bps at the 3' end. In the central region, there is 61 % homology in a region straddling intron 1 and exon 2 which is 800 bp in length.
  • the promoter of NY-ESO-1 is identical to LAGE-1. Both promoters contain two Spl sites and a consensus core Ets site embedded in a CpG island extending into exon 1.
  • intron 1 the 5' and 3' splicing sites of LAGE-1 and NY-ESO-1 are conserved for more than 20 bp. Near the donor site, a "gttct" sequence motif is repeated in NY-ESO-1. Deletions and additions in intron 1 account for most of the differences betweenJ ⁇ GE-i andNY-ESO-1.
  • this intron the distribution of nucleotides is largely non-random since guanidine (g) accounts for 53.9%o of the nucleotides while total purine content is at 71.7%, a feature already observed for LAGE-1. Purines in this intron are frequently arranged in stretches of 10-12 nt, the size of one helix turn. As a consequence of high guanidine (g) content, this intron also displays an unusually high GC content of 65%>.
  • the expression pattern of NY-ESO-1 is different than LAGE-1.
  • the rare retention of intron 2 in NY-ESO-1 nature transcripts contrasts with its frequent presence in LAGE-1 transcripts of both tumors and testes.
  • This difference may be caused by a putative branch point which is better preserved in NY-ESO-1 than in LAGE-1.
  • This putative branch point lies 29 nucleotides upstream of the acceptor site of intron 2, next to the unique difference between both genes at the 3' ends of this intron.
  • the difference in LAGE transcripts may be caused by sequence differences existing at the 5' end of intron 2 or in adjacent exons. These differences may affect the splicing process (Dominski, Kole (1994) J. Biol. Chem. 269, 23590-
  • nucleic acid molecule which encodes an esophageal cancer associated antigen. "Associated” is used herein because while it is clear that the relevant molecule was expressed by esophageal cancer, other cancers, such as melanoma, breast, prostate and lung also express the antigen.

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Abstract

L'invention concerne la séquence génomique du gène NY-ESO-1, qui comprend une séquence 5' non traduite, une séquence 3' non traduite, et des séquences intron. Des molécules d'acide nucléique contenant une séquence de molécule d'ADN génomique NY-ESO-1 peuvent servir dans le diagnostic de troubles et d'expression in vitro de la protéine NY-ESO-1.
PCT/US2001/002126 2000-02-22 2001-01-22 Sequences genomiques isolees codant pour le cancer ny-eso-1/antigene de la tumeur des testicules et leurs utilisations WO2001062917A1 (fr)

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WO2002026778A2 (fr) * 2000-09-26 2002-04-04 Ludwig Institute For Cancer Research Peptides isoles se liant a des molecules hla-c et utilisations de ceux-ci
WO2003082916A2 (fr) * 2002-03-27 2003-10-09 Isis Innovation Limited Antigenes associes a une tumeur
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002026778A2 (fr) * 2000-09-26 2002-04-04 Ludwig Institute For Cancer Research Peptides isoles se liant a des molecules hla-c et utilisations de ceux-ci
WO2002026778A3 (fr) * 2000-09-26 2003-02-13 Ludwig Inst Cancer Res Peptides isoles se liant a des molecules hla-c et utilisations de ceux-ci
AU2008229722B2 (en) * 2001-11-07 2012-09-06 Mannkind Corporation Expression vectors encoding epitopes of target-associated antigens and methods for their design
WO2003082916A2 (fr) * 2002-03-27 2003-10-09 Isis Innovation Limited Antigenes associes a une tumeur
WO2003082916A3 (fr) * 2002-03-27 2004-03-18 Isis Innovation Antigenes associes a une tumeur

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