EP0769053A2 - Produits et procedes relatifs au diagnostic et au traitement prophylactique et therapeutique du synoviosarcome - Google Patents

Produits et procedes relatifs au diagnostic et au traitement prophylactique et therapeutique du synoviosarcome

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Publication number
EP0769053A2
EP0769053A2 EP95925919A EP95925919A EP0769053A2 EP 0769053 A2 EP0769053 A2 EP 0769053A2 EP 95925919 A EP95925919 A EP 95925919A EP 95925919 A EP95925919 A EP 95925919A EP 0769053 A2 EP0769053 A2 EP 0769053A2
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Prior art keywords
sequence
syt
polypeptide
ssx2
oligo
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German (de)
English (en)
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Colin Stephen Cooper
Barry Austin Gusterson
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Cancer Research Campaign Technology Ltd
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Cancer Research Campaign Technology Ltd
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Publication of EP0769053A2 publication Critical patent/EP0769053A2/fr
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention concerns materials and methods relating to the diagnosis and treatment (prophylactic and therapeutic) of synovial sarcomas.
  • the present invention relates to diagnostic, prophylactic and therapeutic materials and methods based upon polynucleotides and polypeptides which are characteristic of synovial sarcoma and specific binding members therefor.
  • the present invention relates to diagnostic materials and methods relating to the use of amplification techniques such as the polymerase chain reaction (PCR) to identify certain polynucleotide sequences characteristic of synovial sarcoma.
  • PCR polymerase chain reaction
  • Synovial sarcomas which account for approximately 10% of soft tissue tumours, occur most commonly in young adults and are found at the extremities in the vicinity of the large joints 1 . Both biphasic and monophasic patterns of tumour histology can be distinguished. In the biphasic pattern, both epithelial-like cells and spindle cells are observed, whilst monophasic tumours contain only spindle cells. Cytogenetic studies have shown that approximately 70% of both biphasic and monophasic synovial sarcomas contain a characteristic chromosomal translocation t (X;18) (pll.2;qll.2) 2 - 8 . The presence of this translocation as the sole cytogenetic abnormality in some primary tumours indicates that its formation is a key molecular event in synovial sarcoma development .
  • Somatic cell hybrids containing the derivative X chromosome that is formed by the t(X;18) translocation have been used to map markers and representative genes relative to the breakpoint. These studies allowed the applicants to delimit the breakpoint as follows: Xpter- DXS228- ( UBE1 - OATL1 - TIMP-OXS226 ) - (DXS255- TFE3 -ELK1 - DXS146) -OATL2-X;18- (DXS14-DXS422-DXS423-DXS674-DXS679) - Xcen 9"11 .
  • a number of candidate genes from this region including ELK1 , TFE3 , TIMP, UBE1 , ARAF1 and SYN, which map to Xpll.2-p22.3, have also been assessed for involvement in the translocation, but no evidence of abnormal transcripts or rearrangement of these genes was found 6 .
  • FISH fluorescence in si tu hybridization
  • YAC Yeast Artificial Chromosome
  • the applicants have used the YAC referred to above and repeat-free subclones derived from it, in strategies to isolate cDNA clones corresponding to the genes involved in the t(X,*18) translocation which is specific for synovial sarcomas.
  • Chromosome X comprises two breakpoints within two respective ornithine- ⁇ -aminotransferase regions designated OATL1 and OATL2. Both OAT regions locate within the pll.2 region of chromosome X, with OATL2 locating closer to the centromere.
  • SSX1 (standing for synovial sarcoma X chromosome break point 1)
  • SSX2 (standing for synovial sarcoma X chromosome break point 2)
  • Chromosome 18 has a breakpoint in the qll.2 region within the nucleotide sequence for a gene newly identified and characterized by the applicants and which they have called the SYT gene (standing for synovial sarcoma translocation) .
  • the translocations have the effect of creating a new polynucleotide sequence which codes for a new fusion polypeptide/protein (which is coded for jointly by the two unrelated sequences brought together as a consequence of the translocation) which is absent from normal individuals in whom the translocation event has not occurred.
  • PCR analysis demonstrates the presence of SYT- SSX1 or SYT-SSX2 fusion transcripts in 29 of 32 synovial sarcomas examined, demonstrating detection of transcripts to be a very useful diagnostic method.
  • the normal pattern for expression of SSXl, SSX2 and SYT nucleic acid and polypeptide sequences appears to be tissue specific. Thus in a normal individual, SSX sequences are only expressed at high levels in the testis.
  • the specific description relates the use of SYT probes to detect rearrangements of the SYT gene by eg Southern analysis.
  • the present invention provides the diagnosis of synovial sarcoma by use of specific binding members such as (a) nucleic acids hybridizable with a nucleic acid comprising either an SYT, SSXl or SSX2 polynucleotide sequence originating from a body site which does not normally show substantial expression of SYT or SSXl or SSX2 polynucleotide sequences; (b) a nucleic acid which hybridizes to both SYT and SSX (SSXl or SSX2) originating portions of a new polynucleotide sequence resulting from a translocation event; (c) substances comprising an antibody binding domain with specificity for either (i) one or more epitopes or sequences characteristic of either the SYT, SSXl or SSX2 polypeptide or (ii) one or more epitopes or sequences characteristic of a new polypeptide sequence of a fusion protein and not of the SYT, SSXl or SSX2
  • the antibody binding domain has a specificity for one or more epitopes characteristic of either the SYT, SSXl or SSX2 polypeptide it may be used to identify the presence or absence of such a polypeptide in a body site which does not normally substantially express that particular polypeptide.
  • SSX polypeptides normally, SSX polypeptides
  • body sites which do not normally express the particular sequence include not only normal tissues, but also abnormal tissues such as tumours which do not express the particular sequence.
  • SYT, SSXl or SSX2 sequence although characteristic of one tumour eg synovial sarcoma, may not be characteristic of another.
  • SSXl or SSX2 sequence which is associated with synovial sarcoma, may allow one to distinguish between different sorts of tumours eg to distinguish a synovial sarcoma from another sort of tumour. This is important, as the treatment method of choice is often dependent upon the particular type of tumour a patient has.
  • the specific binding member comprises nucleic acid
  • the member may simply be used as a specific probe in accordance with standard techniques and procedures.
  • the specific binding member may comprise a pair of oligo- or polynucleotide sequences for use in an amplification technique such as PCR.
  • a new fusion protein resulting from a translocation event is detected as being "foreign" (ie other than native) by the body, there is the likelihood that the body will mount an immune response against it, generating antibodies with specificity for one or more epitopes of the new fusion protein.
  • an SYT or SSX sequence is expressed at an abnormal body site the body may also mount an immune response, such that tnere may exist antibodies with specificity for one or more epitopes characteristic of the SYT or SSX polypeptide.
  • the present invention provides the diagnosis of synovial sarcoma by use of a specific binding member comprising one or more epitopes or sequences characteristic of either the SYT, SSXl or SSX2 polypeptide or of a new polypeptide sequence of a fusion protein and not of the SYT, SSXl or SSX2 polypeptides, in a specific binding assay to detect the presence or absence of such antibodies in a suitable sample obtained from an individual .
  • specific binding pair is used to describe a pair of molecules comprising a specific binding member (sbm) and a binding partner (bp) therefor which have particular specificity for each other and which in normal conditions bind to each other in preference to binding to other molecules .
  • specific binding pairs are antigens and antibodies, hormones and receptors and complementary nucleotide sequences. The skilled person will be able to think of many other examples and they do not need to be listed here. Further, the term "specific binding pair” is also applicable where either or both of the specific binding member and binding partner comprise just the binding part of a larger molecule.
  • a specific binding member may comprise just a domain of an antibody (antibody binding domain) which is able to bind to either an epitope of an antigen or a short sequence which although unique to or characteristic of an antigen, is unable to stimulate an antibody response except when conjugated to a carrier protein.
  • the present invention provides a binding member which is either: (i) specific for a new polynucleotide sequence resulting from a translocation event which is characteristic of synovial sarcoma (preferably where the specific binding member is for only part of the new polynucleotide sequence, it should be specific for a part which is characteristic of the new sequence and not of SYT, SSXl or SSX2) ; (ii) specific for a new polypeptide sequence of a fusion protein resulting from a translocation event which is characteristic of synovial sarcoma (preferably where the specific binding member is for only part of the new polypeptide, it should be specific for a part which is characteristic of the new polypeptide and not of the constitutive polypeptides SYT, SSXl or SSX2) ; (iii) specific for an antibody for either a new fusion protein produced by the patient as a result of a translocation event which is characteristic of synovial sarcoma or an SYT or SS
  • the present invention provides oligonucleotide primer pairs for amplification of polynucleotide sequences (be they in the form of DNA, RNA, single-stranded or double-stranded) which comprise part or all of a SYT, SSXl and/or SSX2 polynucleotide sequence, or a polynucleotide sequence spanning the breakpoint (which spanning polynucleotide sequence may comprise part or all of the new polynucleotide sequence created by the translocation event) .
  • the oligonucleotide primer pairs are designed to hybridize target regions of the new polynucleotide sequence, which are spaced apart from one another with the breakpoint locating between them.
  • the primer pairs may be designed by use of the sequence information herein provided. Having increased the copy number of an SYT, SSXl or SSX2 polynucleotide sequence, or of a new polynucleotide sequence (or part of it spanning the breakpoint) , the amplified sequences may be readily detected by standard methods such as the provision of radioactive nucleotides for inclusion in the sequences being copied, ethidium bromide staining, sequencing and hybridization probing. Of course, no amplification would occur if polynucleotide sequence comprising targets for the primers is absent from the sample selected for testing.
  • amplification of eg the SSX polynucleotide sequence would be indicative of synovial sarcoma, as SSX sequences (polynucleotide or polypeptide) are not normally present at this site.
  • no amplification would be indicative of the absence of synovial sarcoma.
  • any amplification would be indicative of synovial sarcoma, whereas no amplification would be indicative of the absence of synovial sarcoma.
  • the present invention also provides a method for diagnosing synovial sarcoma by taking a suitable sample from a patient, and detecting the presence or absence of a new polynucleotide sequence resulting from a translocation event which is characteristic of synovial sarcoma by adding to the sample suitable oligonucleotide primer pairs (see above) , and other standard ingredients for carrying out a polynucleotide sequence amplification (an amplification based on a DNA template or an RNA template) , and applying standard hybridization, elongation and denaturation or strand separation conditions to amplify any new polynucleotide sequence positioned between the two primers and looking for the presence or absence of an amplification product or products, to determine the presence or absence of the t(X,*18) translocation.
  • a polynucleotide sequence amplification an amplification based on a DNA template or an RNA template
  • the primers may be designed from any part of the polynucleotide sequences for SYT, SSXl and SSX2 or from any part of a new polynucleotide sequence created by a translocation event (eg as now provided by the applicants) .
  • the length of the primers should be such that they efficiently hybridize with good specificity.
  • the primers may be upwards of about 14 nucleotides.
  • the primers may be 18-20 nucleotides.
  • the primers may comprise one or more non- complementary bases. All that is necessary is that the primer and target sufficiently correspond for specific hybridization to allow the desired amplification reaction to proceed.
  • the present invention also provides nucleic acid which comprises an oligo- or polynucleotide sequence hybridizable to either: (i) a polynucleotide sequence which codes for part or all of either the SYT amino acid sequence shown in Fig. 4a or the SSXl or the SSX2 amino acid shown in Fig. 7; or (ii) a polynucleotide sequence which codes for part or all of a polypeptide having part or all of the amino acid sequence shown in Fig. 4b; or (iii) a polynucleotide sequence which codes for part or all of a polypeptide having part or all of the amino acid sequence shown in Fig. 5.
  • the present invention also provides a nucleic acid which comprises an oligo- or polynucleotide which comprises part or all of a polynucleotide sequence as shown in either Fig. 4a, Fig. 4b or Fig. 5 or Fig. 7 or a sequence complementary thereto.
  • the present invention also provides a nucleic acid which comprises a polynucleotide having part or all of a polynucleotide sequence which comprises the SSX2 polynucleotide sequence of Fig. 4b or the SSXl polynucleotide sequence of Fig. 5.
  • the polynucleotide may comprise a part of the SSX2 polynucleotide, or a part of the SSXl polynucleotide not given by Figs . 4b and 5.
  • the nucleic acid, oligo- or polynucleotide may be substantially free of other substances ie isolated and substantially pure.
  • the present invention also provides recombinant transfer vectors and expression vectors which contain a nucleic acid or oligo- or polynucleotide as described above.
  • the nucleotide sequences may be present in association with suitable control sequences such as promoters .
  • the present invention also provides recombinant host cells which comprise such a transfer vector or expression vector. The recombinant host cells may be used to prepare polypeptides homologous to the polypeptides coded for by the SYT, SSXl or SSX2 polynucleotide sequences, or to part or all of a fusion protein coded for by a new polynucleotide sequence created by the translocation.
  • Polypeptide homologues may be used in diagnostic tests to test patient samples for the presence or absence of antibodies with specificity for one or more epitopes characteristic of the SYT, SSXl or SSX2 proteins, or of the new fusion protein (see earlier discussion) .
  • Polypeptide homologues may also be used to raise antibodies (monoclonal or polyclonal) with specificity for one or more sequences or epitopes characteristic of the native SYT, SSXl, SSX2 or fusion proteins.
  • polynucleotides as described above may be used as specific hybridization probes to determine the presence or absence in samples from patients of either the SYT, SSXl or SSX2 polynucleotide sequences, or of a new polynucleotide sequence resulting from a translocation.
  • the probe is for a new polynucleotide sequence resulting from a translocation, preferably it may bind to a part of the new polynucleotide sequence which comprises nucleotides to either side of the breakpoint, in which case the probe would bind to sequences deriving from both SYT and SSXl or SSX2.
  • Probes may be directed to DNA or mRNA complementary thereto.
  • the probes maybe either cDNA probes, RNA probes or oligonucleotides.
  • the probes may be suitably labelled in accordance with standard procedures to aid detection of hybridization.
  • the labels maybe radio-, fluoro or enzyme- labels.
  • Hybridization may be carried out in accordance with well known methodologies.
  • the use of high stringency conditions will serve to minimise non-specific binding and the occurrence of false positives.
  • the present invention also provides a polypeptide having
  • polypeptides may be immunologically cross-reactive with a native SYT, SSXl, SSX2 or fusion protein. They may be immunologically cross-reactive with a polypeptide having an amino acid sequence as shown in either Fig. 4(a) , Fig 4(b) , Fig 5. or Fig. 7.
  • the polypeptide may comprise a part of the SSX2 polypeptide or a part of the SSXl polypeptide not given by Figs . 4b and 5.
  • polypeptides may be substantially free of other substances ie isolated and substantially pure. Derivative polypeptides retaining the cross-reactivity are also provided.
  • the polypeptides above may be prepared by recombinant methodologies or by methods of standard chemical synthesis.
  • polypeptides may be used in diagnostic assays, or to raise antibodies.
  • the present invention also provides specific binding members comprising an antibody binding domain with specificity for one or more epitopes characteristic of the SYT, SSXl or SSX2 proteins, or of the new fusion protein and not of SYT, SSXl or SSX2 proteins.
  • the specific binding members may comprise antibodies, either monoclonal or polyclonal . Alternatively they may comprise derivatives, synthetic analogues or fragments of such antibodies which retain an antibody binding domain with the specificity described above.
  • the present invention also provides methods of diagnosing synovial sarcoma by detecting the presence or absence of the new fusion protein, or the new nucleotide sequence by use of a specific binding member as described above, or by detecting the presence or absence of SYT, SSXl or SSX2 proteins or polynucleotide sequences in samples from tissues which do not normally express these proteins.
  • Diagnostic kits are also be provided for each of the above mentioned embodiments which comprise a specific binding member as described above, along with other reagents required to conduct the diagnostic test.
  • the cytogenetically defined translocation t(X,*18) found in human synovial sarcomas involves the joining of the chromosome 18SYT gene to either of two genes SSXl or SSX2 at Xpll 2.
  • the fusion genes are then transcribed to produce SYT-SSX1 and SYT- SSX2 hybrid transcripts that are in turn translated to form SYT-SSX1 or SYT-SSX2 fusion proteins.
  • Antisense oligonucleotides can be used against native SYT, SSXl or SSX2 sequences as a therapeutic treatment. Alternatively the SYT-SSX1 and SYT-SSX2 sequences or junctions can be targeted with antisense oligonucleotides.
  • antisense RNA technology can be applied to prevent or inhibit mRNA expression of a sequence characteristically associated with synovial sarcoma.
  • the invention provides a polynucleotide sequence that can be transcribed to produce RNA that is at least in part complementary to a SYT, SSXl, SSX2, SYT-SSXl or SYT-SSX2 mRNA and wherein the complementary portion of said transcribed RNA is of sufficient length to inhibit translation of said mRNA in order to inhibit production of a polypeptide encoded by an SYT, SSXl, SSX2, SYT-SSXl or SYT-SSX2 polynucleotide sequence.
  • the complementary (or "antisense") RNA is homologous with, or complementary to, at least a portion of the nucleic acid shown in either Figure 4 (a) , Fig 4(b) , Fig. 5 or Fig. 7 or a variant or allele thereof and which is effective in blocking expression of DNA.
  • SSX2, SYT-SSXl or SYT-SSX2) can be blocked at the protein level . This may be achieved by administering a specific binding member comprising an antibody binding domain as earlier described.
  • the specific binding member may be an antibody and suitably monoclonal.
  • Antibodies can be raised in accordance with conventional methods known in the art (see earlier) .
  • the use of the sequences provided herein and variants, derivatives or fragments thereof which are antigenically cross-reactive in the generation of antibodies forms a further aspect of the invention.
  • the antibodies where for use in therapy, may be "humanised” for example using methods known in the art, in order to reduce the possibility of harmful hypersensitivity reactions occurring in human patients.
  • the tail region of a non-human antibody with the desired specificity may be exchanged for that of a human antibody.
  • a more complete humanisation may be effected by exchanging further framework regions as is known in the art. This may be done at the DNA level using recombinant techniques as known and used in the art.
  • the invention further provides polynucleotide eg cDNA which encodes the above specific binding members with an antibody binding domain.
  • nucleic acid, oligo and polynucleotide sequences provided herein may also be inserted into suitable vectors for vaccine use eg pox virus vectors such as vaccinia.
  • the vectors may also include appropriate control elements .
  • Such vectors may be used for prophylactic or therapeutic vaccination in order to express in a controlled fusion SYT, SSX or SYT-SSX polypeptide sequences and hence stimulate an immune response thereagainst to prevent or mitigate any effects of sarcoma.
  • the invention relates to the therapeutic application of various agents including anti-sense RNA constructs, substances comprising an antibody binding domain and vaccine vectors in the treatment of synovial sarcoma.
  • these agents will be administered in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier or diluent.
  • Suitable agents may be combined, conjugated or complexed to a targeting agent, such as an antibody or antibody binding domain, which binds a synovial sarcoma antigen.
  • a targeting agent such as an antibody or antibody binding domain
  • the present invention provides the diagnosis of synovial sarcoma by use of specific binding members such as (a) nucleic acids hybridizable with a nucleic acid comprising an insert sequence as above (b) substances comprising an antibody binding domain with specificity for one or more epitopes or a sequence characteristic of an insert amino acid sequence.
  • specific binding members such as (a) nucleic acids hybridizable with a nucleic acid comprising an insert sequence as above (b) substances comprising an antibody binding domain with specificity for one or more epitopes or a sequence characteristic of an insert amino acid sequence.
  • Fig. 1 Restriction map of a 32kb region of the 450kb YAC OATL2.7. This 32kb region is present in cosmids designated e and 52 of C3 described in ref 12. The repeat-free region was identified by hybridization of restriction enzyme digested cosmid DNA to a probe prepared from human Cotl DNA.
  • the "*" indicates the position of the putative ligand that may bind to SH3 domains and "•” indicates the position of the putative ligands that may bind to SH2 domains.
  • RNA was from: 255 and 2243; the SS255 and A2243 synovial sarcoma cell lines; RB6 and RB15, the Rag/225/6 and Rag/255/15 human-mouse somatic cell hybrid lines containing the der(X) chromosome formed by the t(X;18) translocation; R, the Rag mouse cell line; and MH, the MNNG-HOS chemically-transformed human osteosarcoma cell line.
  • the 2.3-2.4kb SSX2 transcript was not detected in MNNG-HOS cells, A431 cells, A673 cells and HT1080 human fibrosarcoma cells.
  • a 1.6kb SSX2 transcript was, however, detected in HT1080 cells (Jb) Detection of SYT-SSX2 hybrid transcripts by RT-PCR.
  • PCR was performed using SYT (5' -CAACAGCAAGATGCATACCA-3 ' ) and SSX2 (5'- CACTTGCTATGCACCTGATG-3' ) primers to amplify reverse transcribed total RNA from synovial sarcomas (2243, 124, 498, 114, 159 and 125) and the following human tumour and leukaemia samples: CEM, ALL cell line; HTB86, Ewing sarcoma cell line; A431, epidermoid carcinoma cell line; 229 biphasic leukaemia; 82, neuroepithelioma; and 364, haemangioma. Control experiments using actin primers demonstrated that each sample of reverse transcribed RNA could yield PCR products.
  • Fig. 4. The human chromosome 18 SYT cDNA nucleotide sequence and predicted amino acid sequence (Accession Number X79201) .
  • the open reading frame extends to the 5' end of the nucleotide sequence and the protein is numbered from the first methionine.
  • the arrows show the position at which rearrangement occurs in synovial sarcomas.
  • the eight C-terminal amino acids shown in bold type are removed by rearrangement with SSX2 sequences.
  • Jb The human SYT-SSX2 cDNA nucleotide sequence and predicted amino acid sequence (Accession Number X79200) .
  • the vertical arrow shows the position of the transition between SYT and SSX2 sequences .
  • Amino acids encoded by the SSX sequences are shown in italics. The numbering of nucleotide and protein sequences is consistent with that used for the SYT cDNAs. The nucleotide sequence obtained for SYT-SSX2 cDNAs extends 5' to position 3 of the SYT cDNA. 5' to the breakpoint SYT and SYT-SSX2 cDNA sequences were identical. For both SYT and SYT-SSX2 protein sequences the position of the putative ligand that may bind to SH3 domains is underlined once and the putative ligands that may bind to SH2 domains are double underlined.
  • Fig. 5 The human SYT-SSXl cDNA nucleotide sequence and predicted amino acid sequence.
  • the vertical arrow shows the position of the transition between SYT and SSXl sequences .
  • FIG. 6 Schematic representation of the SYT-SSXl protein junctions together with nucleic acid and protein sequences.
  • A The SYT-SSXl junction originally identified in the HS-SY-II cell line
  • B The SYT-SSX2 junctions originally identified in the A2243 cell line.
  • C The SYT-SSXl junction present in STS498.
  • D Representation of a complex fusion that is found in tumours STS 159 and STS253. In these two tumours an 87bp sequence shown by PCR and Southern hybridization to map to the X chromosome was found between previously identified SYT and SSX2 sequences. Hatched boxes denote SYT protein and the filled boxes represent SSXl and SSX2 sequences. Numbers above the boxes indicate the amino acids at the position of the junction. The number of tumours which contain each type of junction are listed.
  • Fig. 7 The human SSXl and SSX2 cDNA nucleotide and predicted amino acid sequences.
  • the amino acid sequence is numbered from the methionine predicted to act as the site of translational initiation.
  • the position of the SSXl and SSX2 breakpoint found in the majority of synovial sarcomas is indicated.
  • the sequences 3' to this breakpoint which are present in the SYT-SSXl and SYT-SSX2 hybrid transcript are shown in bold type.
  • a polymorphism (T ⁇ C) is found at position 634 in approx 50% of the sarcomas with translocations involving SSXl.
  • the SSXl and SSX2 sequences can be distinguished by digestion with Smal and Lspl . Smal only digests SSX2 while Lspl only digests SSX2. The Smal and Lspl restriction sites are shown.
  • Two methods have been used to select clones from a cDNA library that was prepared using RNA from the A2243 synovial sarcoma cell line.
  • the library was plated at low density and screened using a probe prepared from the 450kb YAC 0ATL2.7.
  • 25% also hybridized to an OAT cDNA probe indicating that they had been located by hybridization to the OAT pseudogene sequence present in the YAC.
  • Based on the presence of common restriction maps and cross hybridization many of the remaining cDNA clones could be assigned to two groups. The applicants found that one group hybridized to an expressed sequence designated SB1 .
  • restriction fragments appeared to correspond to sequences present in YACs spanning the 0ATL2 regions and were present in a cell line (C12D) which contains the X chromosome as its only human component (Fig. 2a, lane X) . There was no hybridization to DNA from a human-hamster hybrid cell line containing chromosome 18 as its only human component (Fig. 2a, lane 18) . When taken together these observations demonstrate that this 3' probe detects sequences that map to the X chromosome. These 3' cDNA sequences mapping to chromosome X have been designated SSX2 (for synovial sarcoma X chromosome breakpoint) .
  • SSX2 for synovial sarcoma X chromosome breakpoint
  • the 9, 5.5 and 3kb fragments were retained in the human-hamster hybrid line containing chromosome 18 as its only human component (Fig. 2Jb, lane 18) but were not present either in the OATL pseudogene YACs or in the C12D cell line.
  • a probe from the SYT gene hybridized to a large restriction fragment of ⁇ 50kb and to a smaller fragment of llkb in BamHl digested human DNA (Fig. 2c) .
  • additional restriction fragments in the size range 17-23kb were detected in BamHl digested synovial sarcoma DNA.
  • Fig. 2c lanes 125 and 114
  • An abnormal BamHl fragment was detected in 10/13 synovial sarcoma tumour samples examined.
  • Fig. 4 The results obtained when Northern blots of tumour and cell line RNA were hybridized to chromosome X and chromosome 18 probes are shown in Fig. 4.
  • the chromosome 18 SYT probe detected a 3.7kb transcript both in synovial sarcomas and in a variety of other human cell lines (Fig. 3a) .
  • a unique major 2.3-2.4kb transcript was detected in synovial sarcoma cell lines.
  • a SYT gene probe (probe III, Fig. IJb) was used to isolate cDNAs from a library prepared using RNA isolated from the M426 fibroblast cell line. Clones ranging in size from 2.2kb to 3.8 kb were isolated. Sequencing of these clones generated a continuous sequence of 3071bp that contained an open reading frame encoding a protein of 404 amino acids (Fig. 4a) .
  • the SYT protein is rich in glutamine (19%) , proline (16%) and glycine (14%) .
  • SYT protein and nucleic acid sequences failed to exhibit homology to sequences in the EMBL, GeneBank and SWISS PROT databases except for the detection of weak homologies (20-25%) to regions of a variety of proteins that were also rich in glutamine and/or proline.
  • the SYT protein contains no motifs suggestive of biochemical or functional properties such as putative membrane spanning helices, DNA binding domains or nucleotide binding sites with the exception of the presence of several regions that were candidate ligands for binding to SH3 and SH2 domains.
  • the consensus sequence for ligands of SH3 domains is p ⁇ PpXP where P represents proline, p represents residues that are usually proline and ⁇ represents a hydrophobic residue 13 .
  • proline at positions -5 and +4 there is also a preference for proline at positions -5 and +4 relative to the central P.
  • the SH3 binding regions are in general highly proline rich. This residue promotes the formation of a left-handed type II polyproline helix conformation that is adopted by ligands that bind to SH3 domains. Sequences in SYT that were examined centred around prolines at positions 93, 101, 112, 121, 269, 360 and 383 but the best match occurred with amino acids 378-387 (-5) PTQPGPPQPP (+4) which contains prolines at all of the positions mentioned above (Fig. 4a) .
  • the SYT protein sequence also contains a consensus sequence for binding to the SH2 domain of phosphatidylinositol 3-kinase (YXXM) at position 67 and two consensus sequences for binding to the SH2 domains of Grb2 (YXNX) at positions 362 and 399 (ref. 14) .
  • YXXM phosphatidylinositol 3-kinase
  • YXNX phosphatidylinositol 3-kinase
  • Sequencing of the hybrid SYT-SSX2 cDNAs indicates that the transcript produced by the rearranged SYT gene encodes a SYT-SSX2 fusion protein in which the C-terminal 8 amino acids of the SYT protein are replaced by 78 amino acids encoded by SSX2 sequences (Fig. 4Jb) .
  • the C-terminal 8 amino acids of SYT that are removed during formation of SYT-SSX2 contain one of the consensus sequences for binding to the SH2 domain of Grb2.
  • the SSX2 protein and nucleic acid sequences also failed to exhibit significant homology to sequences present in the EMBL, GeneBank and SWISS PROT databases.
  • the SYT-SSX2 sequence extends to nucleotide 3 in the normal SYT cDNA sequence shown in Fig. 4a and an exact match was observed between SYT-SSX2 and normal SYT sequences 5' to the breakpoint.
  • the open reading frame for SYT and SYT-SSX2 extends to the 5' end of the cDNA sequence. This may indicate that the SYT and SYT-SSX2 proteins are slightly larger than shown in Fig. 5 and that additional 5' sequences remain to be cloned. It is also possible that translational initiation may occur at methionines within the sequences shown in Fig. 4a. In analyses of an additional 36 SYT and SYT-SSX2 cDNAs that were identified using probe III we failed to isolate sequences that extended further 5' . We also found that a significant proportion of these clones (25%) ended within a 40bp region corresponding to the 5' end of the SYT sequence.
  • SYT and SSX2 allows the development of PCR-based techniques for detecting the t(X,*18) translocation.
  • SYT and SSX2 primers were used to amplify sequences from reverse transcribed tumour RNA, PCR products of the predicted size were detected in 4/6 synovial sarcomas but not in analyses of other tumour types (Fig. 3Jb) .
  • This observation confirms the presence of SYT-SSX2 hybrid transcripts in synovial sarcomas and provides a new method of detection of this translocation which may be of use in tumour diagnosis.
  • OATL1 that maps at least 2 megabases telomeric to 0ATL2 at chromosome Xpll.2 15 .
  • the SYT-SSX2 transcripts characterized in the present study were isolated from a synovial sarcoma containing a breakpoint at the 0ATL2 region.
  • the SSX gene probe isolated from this region also detected SSX2- related sequences within a YAC spanning the OATLl locus (Fig. 2a, lane LI.2) suggesting that t(X;18) translo ⁇ cations occurring at the OATLl locus also involve SSX sequences.
  • synovial sarcoma cell line SS255 The origin and karyotype of the synovial sarcoma cell line SS255 has been described previously 9 .
  • the A2243 synovial sarcoma line was provided by S. A. Aaronson. Both these cell lines have a t(X,*18) translocation with the breakpoint in the 0ATL2 region.
  • the HS-SY-II cell line characterized as described 25 , has a t(X;18) translocation with the breakpoint in the OATLl region.
  • the human-mouse somatic cell lines Rag/255/6 and Rag/255/15 which were found by fusion synovial sarcoma
  • mice Rag cells carry the Xqter-pll.2;18qll.2-qter chromosome and not the reciprocal derivative or other material from chromosomes X and 18 9 .
  • Each of these hybrid cell lines does, however, contain a selection of other human chromosomes.
  • Tumour and blood samples from cancer patients were collected from the Royal Marsden Hospital, London and the Royal Orthopaedic Hospital, Birmingham.
  • C12D is a human-hamster hybrid cell line containing a single human X chromosome 27 . DNA from two human-hamster cell lines containing a single copy a human chromosome
  • YAC clones YAC clones were isolated and characterized as described previously 11 .
  • Clones OATL2.7 and OATL2.10 corresponding to the OATL2 locus contain inserts of 450kb and 300kb
  • An SSXl polynucleotide sequence may be distinguished from an SSX2 polynucleotide sequence by use of the restriction enzymes Lsp and Smal.
  • Lspl cuts only SSXl polynucleotide originating sequences and not SSX2 polynucleotide originating sequences.
  • Smal cuts only SSX2
  • SUBSTITUTE SHEET (RULE 2 polynucleotide originating sequences and not SSXl polynucleotide originating sequences.
  • cDNA and genomic DNA libraries were made from poly A* RNA from A2243 cells in the XCEV29 vector as described previously 31,32 . Screening of this cDNA library was carried out as described by Snell et al . 33 following plating the library at low density (16,000 plaques per 22 x 22 cm plate) and allowing growth to continue until the plaques were almost confluent. The efficiency of detection of cDNA clones using YACs as probes was high because using the 450kb YAC OATL2.7 which contains OAT pseudogene sequences it was possible to detect 80% of the clones that were detected using an OAT cDNA probe.
  • a cDNA library prepared from M426 human fibroblast cells was obtained from S.A. Aaronson.
  • Genomic DNA libraries were made by inserting DNA from the Rag/225/15 somatic cell hybrid cell line that had been partially digested with Sau3A into the BamHl site of the EMBL4 vector as described previously 31 . Genomic DNA libraries were also screened as described previously 31 .
  • DNA sequencing cDNA inserts were sequenced by the dideoxy method 34 using the Sequenase version 2 sequencing kit (Amersham) after subcloning and exonuclease III deletion of selected restriction fragments in the phagemids KS * , KS " , SK * and
  • Figs. 4b and 5 are respectively in respect of the human SYT-SSX2 and SYT-SSXl cDNA nucleotide and predicted amino acid sequences .
  • part of the SSX sequences have been substituted with SYT sequences.
  • provision by the present applicants of a substantial portion of the SSXl and SSX2 sequences allows the skilled person to identify, obtain and sequence entire SSXl and SSX2 sequences by the use of standard methodologies and procedures eg by designing probes based on the sequence information provided herein to screen a suitable library eg a testis tissue library (see also below) .
  • Figure 7 gives the SSXl and SSX2 cDNA nucleotide and predicted amino acid sequences.
  • RNA was extracted from a selection of human tissues and tumour cell lines and examined by Northern analyses using an SSX probe under hybridization conditions that would detect both SSXl and SSX2 transcripts. These studies identified discrete transcripts of 1.6 kb in normal testis and in the HT1080 fibrosarcoma cell line. In addition a more diffuse band in the size range 1.4-1.6 kb was detected in A673 rhabdomyosarcoma cells and at low levels in normal thyroid.
  • a 3 ' SSX2 gene probe was used to screen cDNA libraries prepared from RNA extracted from the HT1080 cell line and from human testis. Sequencing of the clones isolated from the HT1080 cDNA library produced a continuous sequence of 766 bp that contained an open reading frame of 188 amino acids. The 3' region of this sequence exactly matched the SSXl sequences identified in the SYT-SSXl fusion transcript from the HS-SY-II cell line indicating that the HT1080 cDNA clones correspond to transcripts of the normal SSXl gene.
  • the cDNA clones isolated from the human testis cDNA library correspond exactly to the SSX2 sequence present in the SYT-SSX2 fusion transcript from the A2243 cell line. Since these clones were not full length, 5' RACE and PCR amplification were used to isolate the remaining 5' SSX2 sequences from reverse-transcribed testis RNA.
  • the normal SSX2 sequence obtained in these studies has an open reading frame encoding a protein of 188 amino acids which exhibits a high extent of homology (81% identify) to the 188 amino acid, SSXl protein ( Figure 7) .
  • SSXl and SSX2 proteins include the abundance of charged amino acids (40-41%) and the presence of consensus sequences for N-glycosylation and tyrosine phosphorylation. It is also notable that both the SSXl and SSX2 proteins contain acidic C-terminal tails in common with several nuclear proteins such as the HMG proteins, and yeast S1N1.
  • RNA from a series of 32 synovial sarcomas and cell lines was reverse-transcribed and subjected to PCR with SYT and SSX primers .
  • PCR products were detected in 29 of the tumours.
  • the applicants failed to detect either abnormal SSX transcripts by Northern analysis or rearrangement of the SYT gene by Southern analysis.
  • Tumours STS 159 and STS255 contained a second variant transcript in which there was an insert of 87 bp between previously identified SYT and SSX sequences (see Fig. 6) .
  • Mapping by PCR and Southern analyses using OTAL1 and OATL2 YACs and somatic cell hybrid lines containing individual human chromosomes demonstrated that this sequence is derived from the X chromosome (data not shown) .
  • the 87 bp sequence did not, however, match the sequences present in the normal SSX2 transcripts and could not be detected by RT-PCR in cell lines and tissues that expressed normal SSXl and SSX2 transcripts suggesting that it may represent a cryptic exon that is derived from SSX2 intronic sequences.
  • the generation of a cryptic exon from intronic sequences has also been documented for the EWS-FLII gene rearrangement observed in Ewings sarcoma.

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Abstract

L'invention se rapporte à un procédé visant à diagnostiquer la présence ou l'absence de synoviosarcome et consistant à obtenir un échantillon prélevé dans un tissu d'un patient; mettre l'échantillon en contact avec un élément de liaison spécifique (sbm) qui est spécifique d'un partenaire de liaison (bp), ce dernier étant caractéristique du synoviosarcome, de sorte que la présence de bp dans l'échantillon révèle la présence du synoviosarcome, bp étant absent d'un échantillon équivalent prélevé chez un patient ne souffrant pas de synoviosarcome; et détecter toute liaison de sbm avec son bp. Les caractéristiques des partenaires de liaison du synoviosarcome sont les séquences SSX et SYT et les séquences de fusion de celles-ci. L'invention se rapporte également à des séquences, des réactifs et des procédés thérapeutiques ainsi qu'à des produits pharmaceutiques.
EP95925919A 1994-07-19 1995-07-19 Produits et procedes relatifs au diagnostic et au traitement prophylactique et therapeutique du synoviosarcome Withdrawn EP0769053A2 (fr)

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US6291658B1 (en) 1997-05-05 2001-09-18 Ludwig Institute For Cancer Research Isolated nucleic acid molecules encoding SSX family members and thereof
US6287756B1 (en) * 1997-05-05 2001-09-11 Ludwig Institute For Cancer Research Methods for determining presence of cancer in a sample by determining expression of an SSX gene
US6548064B1 (en) * 1997-05-05 2003-04-15 Ludwig Institute For Cancer Research Isolated peptides consisting of amino acid sequences found in SSX or NY-ESO-1 molecules, which bind to HLA molecules
US6800730B1 (en) * 1998-10-02 2004-10-05 Ludwig Institute For Cancer Research Isolated peptides which bind to MHC class II molecules, and uses thereof
US7803370B2 (en) 2002-08-30 2010-09-28 Oncotherapy Science, Inc. Method for treating synovial sarcoma
WO2004020668A2 (fr) * 2002-08-30 2004-03-11 Oncotherapy Science, Inc. Methode de traitement du synovialome
ATE545658T1 (de) * 2003-07-11 2012-03-15 Oncotherapy Science Inc Verfahren zur behandlung von synovialsarkom
WO2005010190A1 (fr) 2003-07-22 2005-02-03 Ludwig Institute For Cancer Research Peptides ssx-2 presentes par des molecules hla de classe ii
EP2044123B1 (fr) 2006-06-21 2013-03-13 Oncotherapy Science, Inc. Anticorps monoclonaux de ciblage tumoral dirigés contre la fzd10 et leurs utilisations
WO2009018018A2 (fr) * 2007-07-31 2009-02-05 University Of Utah Research Foundation Modèle animal d'un sarcome synovial
CN101970456B (zh) * 2008-01-31 2014-11-26 国立大学法人大阪大学 标签肽及其应用
TWI762516B (zh) 2016-10-06 2022-05-01 日商腫瘤療法 科學股份有限公司 針對ｆｚｄ１０之單株抗體及其用途
CN110660451B (zh) * 2018-06-13 2023-04-28 广州华大基因医学检验所有限公司 确定生物样本中是否存在融合基因的方法、设备及应用

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