Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
  • C07K4/12—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof from animals; from humans
• • 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
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to a nucleic acid molecule which codes for a tumor rejection
• the invention concerns genes, whose tumor rejection
• antigen precursor is processed, inter aha, into at least one tumor rejection antigen that is
• the genes in question do not appear to be related to other known tumor rejection antigen precursor coding sequences.
• the invention also relates to
• peptides presented by the HLA-Cw6 molecules and uses thereof. Also a part of the inventions are peptides presented by HLA-A29 molecules, and uses thereof.
• T lymphocyte T cell response. This response requires that T cells recognize and interact with
• HLAs human leukocyte antigens
• MHCs major histocompatibility complexes
• CTLs tumor rejection antigen precursors
• HLA phenotype HLA phenotype
• targeted therapy requires some knowledge of the phenotype of the
• This second TR is disclosed. This second TRA is presented by HLA-C clone 10 molecules.
• a given TRAP can yield a plurality of TRAs.
• TRA derived from tyrosinase
• HLA-A2 molecules HLA-A2 molecules.
• the TRA is derived from a TRAP, but is coded for
• BAGE precursor is described.
• the BAGE precursor is not related to the MAGE family.
• GAGE tumor rejection antigen precursors
• antigen precursors do not show homology to either the MAGE family of genes or the
• the present invention relates to genes encoding such TRAPs, the tumor
• amino acids long and comprise the sequence:
• Xaa is any amino acid and Xaa 2) means that 1 or 2 amino acids may be N-terminal
• Figure 1 sets forth lysis studies using CTL clone 76/6.
• FIG. 1 shows tumor necrosis factor (“TNF”) release assays obtained with various tumor necrosis factor (“TNF”) release assays obtained with various tumor necrosis factor (“TNF”) release assays obtained with various tumor necrosis factor ("TNF”) release assays obtained with various tumor necrosis factor ("TNF) release assays obtained with various tumor necrosis factor ("TNF) release assays obtained with various tumor necrosis factor ("TNF) release assays obtained with various tumor necrosis factor
• Figure 3 compares lysis induced by cytolytic T lymphocytes of clone CTL 76/6.
• Figure 4 presents an alignment of the cDNAs of the six GAGE genes discussed herein.
• Figure 5 sets forth the alignment of deduced amino acid sequences for the members of the
• Figure 6 shows the results obtained when each ofthe GAGE cDNAs was transfected into COS
• Figure 7 compares the stimulation of CTL 22/23 by COS-7 cells, transfected with HLA-A29 cDNA, a MAGE, BAGE or GAGE sequence, as shown. Control values are provided by
• MZ2-MEL.43 and COS cells as stimulators.
• Figure 8 presents results obtained by 51 Cr release studies, using various peptides including
• Example 1 A melanoma cell line, MZ2-MEL was established from melanoma cells taken from
• CTLs cytolytic T cell clones
• PBMCs peripheral blood mononuclear cells
• the lysis assay employed was a chromium release assay following Herin et a , Int.
• clone MZ2-CTL 76/6 was thus isolated.
• the clone is referred to as "76/6" hereafter.
• FIG. 1 shows that this CTL clone recognizes and lyses the melanoma cell line, i.e. , MZ2-
• MZ2-MEL 3.0 was lysed by CTL clone 76/6, the cell line MZ2-MEL.4F, a variant which
• the lysed cell line i.e., MZ2-MEL, is known to express HLA-A1, HLA-A29, HLA-
• the presenting molecule should be one of Al , B37 or Cw6.
• TNF tumor necrosis factor
• Cell line MZ2-MEL.43 a subclone of the
• RNA was isolated from the cell line. The mRNA was isolated using an oligo-dT
• cDNA was then ligated to a BstXI adaptor, digested with NotI, size fractionated by a Sephacryl S-500 HR column, and then cloned, undirectionaUy, into the BstXI and NotI sites of pcDNA I/Amp.
• bacteria contained an insert.
• Each pool was amplified to saturation and plasmid DNA was extracted by alkaline lysis
• transfected into eukaryotic cells The transfections, described herein, were carried out in
• DMEM Dulbecco's modified Eagles Medium
• transfectants could be tested in single weUs. Following four hours of incubation at 37° C, the
• COS cells were incubated for 24-48 hours at 37°C.
• Iscove's medium containing 10% pooled human serum supplemented with 20-30 U/ml of
• the 1500 pools transfected with HLA-A1 , and the 1500 pools transfected with HLA-
• B37 stimulated TNF release to a concentration of 15-20 pg/ml, or 2-6 pg/ml, respectively.
• Plasmid The bacteria of the selected pool were cloned, and 600 clones were tested. Plasmid
• cDNA clone 2D6 four positive clones were found. One of these, referred to as cDNA clone 2D6, was tested
• the cDNA 2D6 was sequenced following art known techniques. A sequence search
• NO: 1 presents cDNA nucleotide information for the identified gene, referred to hereafter as
• GAGE A putative open reading frame is located at bases 51 -467 of the molecule. The first
• PCR polymerase chain reaction
• SEQ ID NOS: 2 and 3 were used.
• the reagents included 30.5 ul water, 5 ul
• cDNA ampUfied using the primers set forth supra yields a 238 base pair fragment.
• any one of these tumors can be assayed for expression of the GAGE
• deletion products were Ugated back into pcDNAI/AMP, and then electroporated
• Plasmid DNA was extracted from each recombinant clone and was then transfected into
• the smallest positive clone contained the first 170 nucleotides of SEQ ID NO: 1.
• this fragment contains a sequence which encodes the first 40 amino acids of the GAGE
• PCR Polymerase chain reaction
• the first primer was a 22-mer complementary to a
• second primer was a 29-mer containing at the 3' end nucleotides 102-119 of SEQ ID NO: 1 ,
• the PCR product was digested by BamHI and Xbal, and
• TNF release was observed, indicating that the "minigene” was processed to a TRA.
• the minigene i.e. , nucleotides 1-119 of SEQ ID NO: 1 , the coding region of which runs
• nucleotides 51-119 encoded the first 23 amino acids of the cDNA of SEQ ID NO: 1.
• CTLs were added in 100 microliters of medium, containing 25 units/ml of IL-2. Eighteen
• the second peptide (SEQ ID NO: 13) was found to induce more than 30 pg/ml of TNF,
• HLA-Cw6 positive were incubated with one of the foUowing peptides:
• effector: target ratio was 10:1.
• 51 Cr release was determined after four hours of incubation
• RNA from MZ2-MEL.43 (the same Ubrary that was used for the cloning of GAGE) was
• the probe was a PCR fragment of
• GAGE-2 19 from but highly related to GAGE were identified. They are caUed GAGE-2, 3, 4, 5 and 6
• region of the GAGE-3 cDNA contains a long repeat and a hai ⁇ in structure.
• the deduced GAGE-1 protein corresponding to a tumor rejection antigen precursor is
• SEQ ID NO: 4 The sequence of the peptide is modified in GAGE-3, 4 5 and 6 so that
• VDE24 5'-CCA TCA GGA CCA TCT TCA-3' (SEQ ID NO: 10)
• RNA with primers VDE43 and VDE24 detecting all GAGE genes ( Figure 7) . Absence of PCR product is indicated by - and presence by +.
• HLA-A29 was the first molecule tested. To do so, poly A + RNA was extracted
• the mRNA was then converted to cDNA, using
• the plasmids were electroplated into E. coU strain
• DH5 ⁇ 5'IQ DH5 ⁇ 5'IQ
• ampiciUin 50 ⁇ g/ml
• nitroceUulose filters were prepared, and hybridized overnight in
• the probe is a sequence which surrounds the start codon of most HLA sequences.
• transfectants were then tested for their abiUty to stimulate TNF production by
• GAGE-1 and GAGE-2 in contrast, do not stimulate CTL clone 22/23, thus leading to the
• HLA-A29 molecules and recognized by CTL 22/23.
• the peptide was synthesized, lyophilized, and then dissolved in 1 volume
• the peptide (SEQ ID NO: 21) was tested in a 51 Cr release experiment, foUowing the
• HLA-A29 As is shown herein, such is not the case.
• tumor rejection antigen precursors and tumor rejection antigens.
• tumor rejection antigen precursors and tumor rejection antigens.
• these molecules are not homologous with any of the previously disclosed MAGE and BAGE coding sequences described in the references set forth supra.
• one aspect of the invention is an isolated
• nucleic acid molecule which comprises the nucleotide sequences set forth in any of SEQ ID NOs:
• SEQ ID NOS: 1-6 are neither MAGE nor BAGE coding sequences, as will be seen by
• nucleic acid molecules which also code for a non-MAGE
• stringent conditions refers to parameters with which
• stringent conditions refers to
• skiUed artisan wUl be famiUar with such conditions and, thus, they are not given here.
• the expression vector may also
• the vector contains both coding sequences, it can be used to transfect a ceU which does
• the tumor rejection antigen precursor coding sequence may
• HLA-Cw6 HLA-Cw6
• vectors which contain the two coding sequences may be used in HLA-A29 or HLA-Cw6 presenting cells if desired, and the gene for tumor rejection antigen precursor can be used in
• the invention also embraces so caUed expression kits, which aUow the artisan to
• Such expression kits include at least separate
• nucleic acid molecules and the TRAPs of the invention To distinguish the nucleic acid molecules and the TRAPs of the invention from the
• GAGE family of genes and TRAPs. Hence, whenever “GAGE” is used herein, it refers to
• GAGE coding molecule and simUar terms are used to describe the nucleic acid molecules
• the invention permits the artisan to diagnose a disorder such as melanoma,
• TRAP tumor rejection antigen
• determinations can be carried out via any standard nucleic acid determination assay, including the polymerase chain reaction, or assaying with labelled hybridization probes. In the latter situation, assaying with binding partners for complexes of TRA and HLA, such as antibodies,
• An alternate method for determination is a TNF release assay, of the
• testis ceUs are of type described supra. To carry out the assay, it is preferred to make sure that testis ceUs are of type described supra.
• testis cells present in non-testicular sample.
• the isolation of the TRAP gene also makes it possible to isolate the TRAP molecule
• TRAP molecules containing the amino acid sequence coded for by any of
• TRA and HLA such as HLA-Cw6 or HLA-A29, may be combined with materials such as
• Exemplary adjuvants include Freund's complete and incomplete adjuvant, kiUed R
• BCG BacUle Calmente-Guerin, Al(OH) 3 , muramyl dipeptide
• MPL monophosphoryl lipid A
• KLH keyhole limpet hemocyanin
• Patent No. 5,057,540 to Kensil, et al. inco ⁇ orated by reference, MTP-MF59, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), the cationic
• amphiphile DOTMA the neutral phospholipids such as DOPE, and combinations of these. This listing is by no means comprehensive, and the artisan of ordinary skill wUl be able to augment this Usting. AU additional adjuvants are encompassed herein.
• vaccines can be prepared from ceUs which present the TRA/HLA
• ceUs transfectants, et cetera.
• these can be ceUs
• TRAP molecule its associated TRAs, as weU as complexes of TRA and HLA, may be used
• disorder refers to any pathological condition where the tumor
• rejection antigen precursor is expressed.
• An example of such a disorder is cancer, melanoma
• Melanoma is well known as a cancer of pigment producing ceUs.
• tumor rejection antigens such as the one presented in SEQ ID NO:
• polypeptides such as
• polypeptides molecules containing from 8 to 16 amino acids, where the polypeptides contain the amino acid
• SEQ ID NO: 21 are processed to the appropriate TRA, and are presented by cancer cells,
• amino acids long and comprise the sequence:
• Xaa in each case is any amino acid.
• amino acids long and which include SEQ ID NO: 23, 24 or 25 subject to the above preferred
• minigenes are isolated nucleic acid molecules which encode any of SEQ ID NOS: 21 , 22, 23, 24 or 25 all of the especiaUy
• SEQ ID NO: 23, 24 or 25 being included. There are only a limited
• nucleic acid molecules which can encode, e.g., SEQ ID NO: 21 or 22, and they
• pathological conditions such as cancer, melanoma in particular.
• pathological conditions such as cancer, melanoma in particular.
• the investigator may study antigens shed into blood or urine, observe physiological changes,
• peptides listed supra preferably in soluble form.
• Such soluble complexes can be used, e.g. ,
• the complexes are preferably
• peptides in accordance with the invention may be used to carry out
• HLA-typing assays It is weU known that when a skin graft, organ transplant, etc., is
• the peptides of the invention may be used to determine whether or not an individual is HLA-
• peptides ofthe invention are contacted to a sample of interest, and
• binding to ceUs in that sample indicates whether or not the individual from which the sample
• HLA-Cw6 or HLA-A29 positive.
• TRA presenting cells such as HLA-A29 orHLA-
• ceUs such as blood ceUs
• target ceU can be a transfectant, such as COS cell of the type described supra. These transfectants present the desired complex on their surface and, when combined with a CTL of interest, stimulate its proliferation.
• COS ceUs such as those used herein, are widely
• the proliferated CTLs are then administered to a subject with a ceUular abnormality
• ceUs presenting the relevant complex are identified via the foregoing
• Adoptive transfer is not the only form of therapy that is avaUable in accordance with
• CTLs can also be provoked jn vivo, using a number of approaches.
• ceUs used in this approach may be those that normally express the complex
• HPV E7 peptides in a therapeutic regime in a therapeutic regime.
• Various ceU types may be used.
• the gene of interest is carried by, e.g. , a Vaccina virus
• the TRAP is processed to yield the
• CTGCCGTCCG GACTCTTTTT CCTCTACTGA GATTCATCTG TGTGAAATAT 50
• CTCATATTTC ACACAGATGA GTTGGCGAGG AAGATCGACC TATTATTGGT 50
• Each Xaa may be any amino acid (xi) SEQUENCE DESCRIP ⁇ ON: SEQ ID NO: 23:
• Each Xaa may be any amino acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
• Each Xaa may be any amino acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

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• 1999
  • 1999-01-12 AU AU23189/99A patent/AU2318999A/en not_active Abandoned
  • 1999-01-12 WO PCT/US1999/000775 patent/WO1999037665A1/en not_active Application Discontinuation
  • 1999-01-12 KR KR1020007008054A patent/KR20010024877A/ko not_active Application Discontinuation
  • 1999-01-12 EP EP99903082A patent/EP1047707A1/de not_active Withdrawn
  • 1999-01-12 CN CN99803245A patent/CN1291992A/zh active Pending
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